| 1. |
Klionsky DJ,
Cregg JM,
Dunn WA,
Emr SD,
Sakai Y,
Sandoval IV,
Sibirny A,
Subramani S,
Thumm M,
Veenhuis M,
Ohsumi Y,
( 2003 ) A unified nomenclature for yeast autophagy-related genes. PMID : 14536056 : Abstract >>
N/A
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2. |
Alamäe T,
Karp H,
Järviste A,
Kriegel TM,
( 2003 ) Cloning and biochemical characterization of hexokinase from the methylotrophic yeast Hansenula polymorpha. PMID : 14530868 : DOI : 10.1007/s00294-003-0448-6 Abstract >>
We previously showed that, unlike other yeasts, Hansenula polymorpha possesses a glucokinase HPGLK1 that can mediate glucose repression in this yeast, although it cannot replace the regulatory function of hexokinase 2 in Saccharomyces cerevisiae. In the present study, the H. polymorpha hexokinase gene HPHXK1 was cloned by complementation of the glucose growth deficiency of the H. polymorpha double kinase-negative mutant A31-10 with a genomic library. The sequence of the 483-amino acid hexokinase protein deduced from the HPHXK1 gene showed the highest degree of identity (56%) with hexokinase from Schwanniomyces occidentalis, whereas the identity with hexokinase from Kluyveromyces lactis and both hexokinases from Sac. cerevisiae was 55%. The hexokinase protein was purified from crude extracts of H. polymorpha, using ion exchange chromatography and gel filtration. The K(m) values of the purified enzyme for glucose, fructose and ATP were 0.26 mM, 1.1 mM and 0.32 mM, respectively. H. polymorpha hexokinase was inhibited by trehalose-6-phosphate (K(i)=12 microM) and ADP (K(i)=1.6 mM), but not by glucose-6-phosphate. Transformation of a H. polymorpha hexokinase-negative mutant with a plasmid carrying the HPHXK1 gene restored the ability of the mutant to phosphorylate fructose and to repress the synthesis of alcohol oxidase and catalase by fructose. Therefore, hexokinase is specifically needed for the establishment of fructose repression in H. polymorpha.
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3. |
Ubiyvovk VM,
Nazarko TY,
Stasyk OG,
Sohn MJ,
Kang HA,
Sibirny AA,
( 2002 ) GSH2, a gene encoding gamma-glutamylcysteine synthetase in the methylotrophic yeast Hansenula polymorpha. PMID : 12702282 : DOI : 10.1016/S1567-1356(02)00111-3 Abstract >>
The GSH2 gene, encoding Hansenula polymorpha gamma-glutamylcysteine synthetase, was cloned by functional complementation of a glutathione (GSH)-deficient gsh2 mutant of H. polymorpha. The gene was isolated as a 4.3-kb XbaI fragment that was capable of restoring GSH synthesis, heavy-metal resistance and cell proliferation when introduced into gsh2 mutant cells. It possesses 53% identical and 69% similar amino acids compared with the Candida albicans homologue (Gcs1p). In comparison to the Saccharomyces cerevisiae homologue (Gsh1p), it possesses 47% identical and 61% similar amino acids. The GSH2 sequence appears in the GenBank database under accession No. AF435121.
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4. |
Haan GJ,
van Dijk R,
Kiel JA,
Veenhuis M,
( 2002 ) Characterization of the Hansenula polymorpha PUR7 gene and its use as selectable marker for targeted chromosomal integration. PMID : 12702317 : DOI : 10.1111/j.1567-1364.2002.tb00064.x Abstract >>
The Hansenula polymorpha genes encoding the putative functional homologs of the enzymes involved in the seventh and eighth step in purine biosynthesis, HpPUR7 and HpPUR8, were cloned and sequenced. An overexpression vector designated pHIPA4 was constructed, which contains the HpPUR7 gene as selectable marker and allows expression of genes of interest via the strong, inducible alcohol oxidase promoter. An ade11 auxotrophic mutant that is affected in the activity of the HpPUR7 gene product was used to construct strain NCYC495 ade11.1 leu1.1 ura3. This strain grew on methanol at wild-type rates (doubling time of approximately 4 h) and is suitable for independent introduction of four expression cassettes, each using one of the markers for selection, in addition to the zeocin resistance marker. It was subsequently used as a host for overproduction of two endogenous peroxisomal matrix proteins, amine oxidase and catalase. Efficient site-specific integration of pHIPA4 and overproduction of amine oxidase and catalase is demonstrated. The expression cassette appeared to be pre-eminently suited to mediate moderate protein production levels. The advantages of pHIPA4 and the new triple auxotrophic strain in relation to the use of H. polymorpha as a versatile cell factory or as a model organism for fundamental studies on the principles of peroxisome homeostasis is discussed.
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5. |
Komduur JA,
Veenhuis M,
Kiel JA,
( 2003 ) The Hansenula polymorpha PDD7 gene is essential for macropexophagy and microautophagy. PMID : 12702243 : DOI : 10.1016/s1567-1356(02)00135-6 Abstract >>
Hansenula polymorpha PDD genes are involved in the selective degradation of peroxisomes via macropexophagy. We have isolated various novel pdd mutants by a gene-tagging method. Here we describe the isolation and characterisation of PDD7, which encodes a protein with high sequence similarity (40% identity) to Saccharomyces cerevisiae Apg1p/Aut3p, previously described to be involved in random autophagy and the cytoplasm-to-vacuole targeting pathway. Our data indicate that HpPdd7p is essential for two processes that degrade peroxisomes, namely the highly selective process of macropexophagy and microautophagy, which occurs in H. polymorpha upon nitrogen starvation.
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6. |
Leao-Helder AN,
Krikken AM,
van der Klei IJ,
Kiel JA,
Veenhuis M,
( 2003 ) Transcriptional down-regulation of peroxisome numbers affects selective peroxisome degradation in Hansenula polymorpha. PMID : 12902346 : DOI : 10.1074/jbc.M304029200 Abstract >>
We have isolated and characterized a novel transcription factor of Hansenula polymorpha that is involved in the regulation of peroxisomal protein levels. This protein, designated Mpp1p, belongs to the family of Zn(II)2Cys6 proteins. In cells deleted for the function of Mpp1p the levels of various proteins involved in peroxisome biogenesis (peroxins) and function (enzymes) are reduced compared with wild type or, in the case of the matrix protein dihydroxyacetone synthase, fully absent. Also, upon induction of mpp1 cells on methanol, the number of peroxisomes was strongly reduced relative to wild type cells and generally amounted to one organelle per cell. Remarkably, this single organelle was not susceptible to selective peroxisome degradation (pexophagy) and remained unaffected during exposure of methanol-induced cells to excess glucose conditions. We show that this mechanism is a general phenomenon in H. polymorpha in the case of cells that contain only a single peroxisome.
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7. |
Berardi E,
Gambini A,
Bellu AR,
( 2003 ) ALG2, the Hansenula polymorpha isocitrate lyase gene. PMID : 12845606 : DOI : 10.1002/yea.1002 Abstract >>
To set the basis for molecular and cellular studies of the glyoxylate cycle in methylotrophic yeasts, we isolated and characterized ALG2, the Hansenula polymorpha isocitrate lyase gene. Complementation work and sequence analysis revealed an ORF of 1458 nucleotides, encoding a 486 amino acid protein with a predicted molecular mass of 54.9 kDa. This protein is shorter than the Saccharomyces cerevisiae and Candida tropicalis ICLs, lacks a PST1 signal and possesses a PTS2-like signal. The transcriptional regulation of ALG2 mRNA levels by carbon source is mainly achieved by glucose repression-derepression, whereas ethanol induction plays only a minor role. We present evidence indicating that, in H. polymorpha, neither isocitrate lyase activity nor the ALG2 gene product are necessary for C(1)-peroxisome degradation triggered by ethanol. Therefore, the involvement of glyoxylate in degradation, as described by Kulachkovsky et al. (1997) for Pichia methanolica, does not necessarily apply to all methylotrophic yeasts. The relevant nucleotide sequence has been deposited at GenBank (Accession No. AF373067.1).
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8. |
Shalguev VI,
Kil' Y,
Yurchenko LV,
Lantsov VA,
( N/A ) Temperature dependence of HpRad51, the central protein of the homological recombination in the yeast Hansenula polymorpha. PMID : 12577614 : Abstract >>
N/A
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9. |
Ozimek P,
van Dijk R,
Latchev K,
Gancedo C,
Wang DY,
van der Klei IJ,
Veenhuis M,
( 2003 ) Pyruvate carboxylase is an essential protein in the assembly of yeast peroxisomal oligomeric alcohol oxidase. PMID : 12589070 : DOI : 10.1091/mbc.e02-07-0417 PMC : PMC150008 Abstract >>
Hansenula polymorpha ass3 mutants are characterized by the accumulation of inactive alcohol oxidase (AO) monomers in the cytosol, whereas other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. These mutants also have a glutamate or aspartate requirement on minimal media. Cloning of the corresponding gene resulted in the isolation of the H. polymorpha PYC gene that encodes pyruvate carboxylase (HpPyc1p). HpPyc1p is a cytosolic, anapleurotic enzyme that replenishes the tricarboxylic acid cycle with oxaloacetate. The absence of this enzyme can be compensated by addition of aspartate or glutamate to the growth media. We show that HpPyc1p protein but not the enzyme activity is essential for import and assembly of AO. Similar results were obtained in the related yeast Pichia pastoris. In vitro studies revealed that HpPyc1p has affinity for FAD and is capable to physically interact with AO protein. These data suggest that in methylotrophic yeast pyruvate carboxylase plays a dual role in that, besides its well-characterized metabolic function as anapleurotic enzyme, the protein fulfils a specific role in the AO sorting and assembly process, possibly by mediating FAD-binding to AO monomers.
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10. |
Kim MW,
Agaphonov MO,
Kim JY,
Rhee SK,
Kang HA,
( 2002 ) Sequencing and functional analysis of the Hansenula polymorpha genomic fragment containing the YPT1 and PMI40 genes. PMID : 12112240 : DOI : 10.1002/yea.881 Abstract >>
A 6.0 kb genomic DNA segment was isolated by its ability to rescue the temperature-sensitive growth defect and the hypersensitivity to sodium deoxycholate of a spontaneous vanadate-resistant mutant derived from Hansenula polymorpha DL-1. The genomic fragment contains four open reading frames homologous to the Saccharomyces cerevisiae genes YPT1 (which codes for a GTP-binding protein; 75% amino acid identity), PMI40 (encoding phosphomannose isomerase; 61% identity), YLR065c (30% identity) and CST13 (28% identity). The H. polymorpha YPT1 homologue (HpYPT1) was found to be responsible for the complementation of the temperature-sensitive phenotype and the sodium deoxycholate sensitivity of the mutant strain. Disruption of the H. polymorpha PMI40 homologue (HpPMI40) resulted in the auxotrophic requirement for D-mannose. The heterologous expressions of HpYPT1 and HpPMI40 were able to complement the temperature-sensitive phenotype of S. cerevisiae ypt1-1 mutant and the mannose auxotrophy of S. cerevisiae pmi40 null mutant, respectively, indicating that the H. polymorpha genes encode the functional homologues of S. cerevisiae YPT1 and PMI40 proteins. The nucleotide sequence has been submitted to GenBank under Accession No. AF454544.
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11. |
Laht S,
Karp H,
Kotka P,
Järviste A,
Alamäe T,
( 2002 ) Cloning and characterization of glucokinase from a methylotrophic yeast Hansenula polymorpha: different effects on glucose repression in H. polymorpha and Saccharomyces cerevisiae. PMID : 12383517 : DOI : 10.1016/s0378-1119(02)00859-4 Abstract >>
Glucokinase gene (HPGLK1) was cloned from a methylotrophic yeast Hansenula polymorpha by complementation of glucose-phosphorylation deficiency in a H. polymorpha double kinase-negative mutant A31-10 by a genomic library. An open reading frame of 1416 nt encoding a 471-amino-acid protein with calculated molecular weight 51.6 kDa was characterized in the genomic insert of the plasmid pH3. The protein sequence deduced from HPGLK1 exhibited 55 and 46% identity with glucokinases from Saccharomyces cerevisiae and Aspergillus niger, respectively. The enzyme phosphorylated glucose, mannose and 2-deoxyglucose, but not fructose. Transformation of HPGLK1 into A31-10 restored glucose repression of alcohol oxidase and catalase in the mutant. Transformation of HPGLK1 into S. cerevisiae triple kinase-negative mutant DFY632 showed that H. polymorpha glucokinase cannot transmit the glucose repression signal in S. CEREVSIAE: synthesis of invertase and maltase in respective transformants was insensitive to glucose repression similarly to S. cerevisiae DFY568 possessing only glucokinase.
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12. |
Komduur JA,
Leão AN,
Monastyrska I,
Veenhuis M,
Kiel JA,
( 2002 ) Old yellow enzyme confers resistance of Hansenula polymorpha towards allyl alcohol. PMID : 12228809 : DOI : 10.1007/s00294-002-0321-z Abstract >>
In the methylotrophic yeast, Hansenula polymorpha, peroxisomes are formed during growth on methanol as sole carbon and energy source and contain the key enzymes for its metabolism, one of the major enzymes being alcohol oxidase (AO). Upon a shift of these cells to glucose-containing medium, peroxisomes become redundant for growth and are rapidly degraded via a highly selective process designated macropexophagy. H. polymorpha pdd mutants are disturbed in macropexophagy and hence retain high levels of peroxisomal AO activity upon induction of this process. To enable efficient isolation of PDD genes via functional complementation, we make use of the fact that AO can convert allyl alcohol into the highly toxic compound acrolein. When allyl alcohol is added to cells under conditions that induce macropexophagy, pdd mutants die, whereas complemented pdd mutants and wild-type cells survive. Besides isolating bona fide PDD genes, we occasionally obtained pdd transformants that retained high levels of AO activity although their allyl alcohol sensitive phenotype was suppressed. These invariably contained extra copies of a gene cluster encoding homologues of Saccharomyces carlsbergensis old yellow enzyme. Our data suggest that the proteins encoded by these genes detoxify acrolein by converting it into less harmful components.
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13. |
Baerends RJ,
Sulter GJ,
Jeffries TW,
Cregg JM,
Veenhuis M,
( 2002 ) Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase. PMID : 11754481 : DOI : 10.1002/yea.805 Abstract >>
Glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required forthe catabolism of methanol as a carbon source and certain primary amines, such as methylamine as nitrogen sources in methylotrophic yeasts. Here we describe the molecular characterization of the FLD1 gene from the yeast Hansenula polymorpha. Unlike the recently described Pichia pastoris homologue, the H. polymorpha gene does not contain an intron. The predicted FLD1 product (Fld1p) is a protein of 380 amino acids (ca. 41 kDa) with 82% identity to P. pastoris Fld1p, 76% identity to the FLD protein sequence from n-alkane-assimilating yeast Candida maltosa and 63-64% identity to dehydrogenase class III enzymes from humans and other higher eukaryotes. The expression of FLD1 is strictly regulated and can be controlled at two expression levels by manipulation of the growth conditions. The gene is strongly induced under methylotrophic growth conditions; moderate expression is obtained under conditions in which a primary amine, e.g. methylamine, is used as nitrogen source. These properties render the FLD1 promoter of high interest for heterologous gene expression. The availability of the H. polymorpha FLD1 promoter provides an attractive alternative for expression of foreign genes besides the commonly used alcohol oxidase promoter.
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14. |
van Dijk R,
Faber KN,
Hammond AT,
Glick BS,
Veenhuis M,
Kiel JA,
( 2001 ) Tagging Hansenula polymorpha genes by random integration of linear DNA fragments (RALF). PMID : 11810237 : DOI : 10.1007/s004380100584 Abstract >>
We have investigated the feasibility of using gene tagging by restriction enzyme-mediated integration (REMI) to isolate mutants in Hansenula polymorpha. A plasmid that cannot replicate in H. polymorpha and contains a dominant zeocin resistance cassette, pREMI-Z, was used as the integrative/mutagenic plasmid. We observed that high transformation efficiency was primarily dependent on the use of linearised pREMI-Z, and that the addition of restriction endonuclease to linearised pREMI-Z prior to transformation increased the transformation frequency only slightly. Integration of linearised pREMI-Z occurred at random in the H. polymorpha genome. Therefore, we termed this method Random integration of Linear DNA Fragments (RALF). To explore the potential of RALF in H. polymorpha, we screened a collection of pREMI-Z transformants for mutants affected in peroxisome biogenesis (pex) or selective peroxisome degradation (pdd). Many previously described PEX genes were obtained from the mutant collection, as well as a number of new genes, including H. polymorpha PEX12 and genes whose function in peroxisome biogenesis is still unclear. These results demonstrate that RALF is a powerful tool for tagging genes in H. polymorpha that should make it possible to carry out genome-wide mutagenesis screens.
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15. |
van der Heide M,
Hollenberg CP,
van der Klei IJ,
Veenhuis M,
( 2002 ) Overproduction of BiP negatively affects the secretion of Aspergillus niger glucose oxidase by the yeast Hansenula polymorpha. PMID : 11954796 : Abstract >>
We have cloned the Hansenula polymorpha BIP gene from genomic DNA using a PCR-based strategy. H. polymorpha BIP encodes a protein of 665 amino acids, which shows very high homology to Saccharomvces cerevisiae KAR2p. KAR2p belongs to the Hsp70 family of molecular chaperones and resides in the endoplasmic reticulum (ER)-lumen. H. polymorpha BiP contains a putative N-terminal signal sequence of 30 amino acids together with the conserved -HDEL sequence, the typical ER retention signal, at the extreme C-terminus. We have analysed the effect of BIP overexpression, placing the gene under control of the strong alcohol oxidase promoter (P(MOX)) on the secretion of artificially produced Aspergillus niger glucose oxidase (GOX) by H. polymorpha. BiP overproduction did not lead to any growth defects of the cells; at the subcellular level, proliferation of ER-like vesicles was observed. However, artificially enhanced BiP levels strongly affected GOX secretion and led to accumulation of this protein in the ER-like vesicles. This was not simply due to the high BiP overproduction, because it was also observed under conditions of low P(MOX) induction during growth of cells on glycerol. Vacuolar carboxypeptidase Y was properly sorted to its target organelle in the BiP overproducing strains.
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16. |
Kim SY,
Sohn JH,
Kang HA,
Yoo SK,
Pyun YR,
Choi ES,
( 2001 ) Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae MNN9 gene. PMID : 11255253 : DOI : 10.1002/yea.699 Abstract >>
A gene homologous to Saccharomyces cerevisiae MNN9 has been cloned and characterized in the methylotrophic yeast Hansenula polymorpha. This gene was cloned from a H. polymorpha genomic DNA library using the S. cerevisiae MNN9 gene as a probe. The H. polymorpha MNN9 homologue (HpMNN9) contained a 1062 bp open reading frame encoding a predicted protein of 354 amino acids. The deduced amino acid sequence showed 58% and 51% identity, respectively, with the S. cerevisiae and Candida albicans Mnn9 proteins. Disruption of HpMNN9 leads to phenotypic effects suggestive of cell wall defects, including detergent sensitivity and hygromycin B sensitivity. The hygromycin B sensitivity of S. cerevisiae mnn9 null mutant was complemented in the presence of the HpMNN9 gene. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession No. AF264786.
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17. |
Agaphonov MO,
Packeiser AN,
Chechenova MB,
Choi ES,
Ter-Avanesyan MD,
( 2001 ) Mutation of the homologue of GDP-mannose pyrophosphorylase alters cell wall structure, protein glycosylation and secretion in Hansenula polymorpha. PMID : 11255248 : DOI : 10.1002/yea.678 Abstract >>
A Hansenula polymorpha mutant with enhanced ability to secrete a heterologous protein has been isolated. The mutation defines a gene, designated OPU24, which encodes a protein highly homologous to GDP-mannose pyrophosphorylase Psa1p/Srb1p/Vig9p of Saccharomyces cerevisiae and CaSrb1p of Candida albicans. The opu24 mutant manifests phenotypes similar to those of S. cerevisiae mutants depleted for GDP-mannose, such as cell wall fragility and defects in N- and O-glycosylation of secreted proteins. The influence of the opu24 mutation on endoplasmic reticulum-associated protein degradation is discussed. The GenBank Accession No. for the OPU24 sequence is AF234177.
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18. |
Liiv L,
Pärn P,
Alamäe T,
( 2001 ) Cloning of maltase gene from a methylotrophic yeast, Hansenula polymorpha. PMID : 11255010 : DOI : 10.1016/s0378-1119(01)00359-6 Abstract >>
The Hansenula polymorpha maltase structural gene (HPMAL1) was isolated from a genomic library by hybridization of the library clones with maltase-specific gene probe. An open reading frame of 1695 nt encoding a 564 amino-acid protein with calculated molecular weight of 65.3 kD was characterized in the genomic DNA insert of the plasmid p51. The protein sequence deduced from the HPMAL1 exhibited 58 and 47% identity with maltases from Candida albicans and Saccharomyces carlsbergesis encoded by CAMAL2 and MAL62, respectively, and 44% identity with oligo-alpha-1,6-glucosidase from Bacillus cereus. The recombinant Hansenula polymorpha maltase produced in Escherichia coli hydrolyzed p-nitrophenyl-alpha-D-glucopyranoside (PNPG), sucrose, maltose and alpha-methylglucoside and did not act on melibiose, cellobiose, trehalose and o-nitrophenyl-beta-D-galactopyranoside (ONPG). The affinity of the recombinant enzyme for its substrates increased in the order maltose
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19. |
Schwartz B,
Williams NK,
Li R,
Chen Z,
( 2000 ) Crystal structure at 2.5 A resolution of zinc-substituted copper amine oxidase of Hansenula polymorpha expressed in Escherichia coli. PMID : 10933787 : DOI : 10.1021/bi000639f Abstract >>
Copper amine oxidases (CAOs) catalyze the two-electron oxidation of primary amines to aldehydes, utilizing molecular oxygen as a terminal electron acceptor. To accomplish this transformation, CAOs utilize two cofactors: a mononuclear copper, and a unique redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ or TOPA quinone). TPQ is derived via posttranslational modification of a specific tyrosine residue within the protein itself. In this study, the structure of an amine oxidase from Hansenula polymorpha has been solved to 2.5 A resolution, in which the precursor tyrosine is unprocessed to TPQ, and the copper site is occupied by zinc. Significantly, the precursor tyrosine directly ligands the metal, thus providing the closest analogue to date of an intermediate in TPQ production. Besides this result, the rearrangement of other active site residues (relative to the mature enzyme) proposed to be involved in the binding of molecular oxygen may shed light on how CAOs efficiently use their active site to carry out both cofactor formation and catalysis.
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20. |
Cox H,
Mead D,
Sudbery P,
Eland RM,
Mannazzu I,
Evans L,
( 2000 ) Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1 promoter. PMID : 10992283 : DOI : 10.1002/1097-0061(20000930)16:13<1191::AID-YEA589>3.0.CO;2-2 Abstract >>
The methylotrophic yeast H. polymorpha is a popular system for the expression of recombinant proteins using the strong and regulatable methanol oxidase (MOX) promoter. Here we show that the constitutive PMA1 promoter can programme the expression of two heterologous proteins, glucose oxidase and human serum albumin. A constitutive promoter provides a useful additional facility to the H. polymorpha expression system because it allows a simplified fermentation regime, avoids the use of methanol, which is both toxic and an explosive hazard, and allows more flexibility for ectopic gene expression during the course of academic studies. A fragment previously isolated in a promoter screen, using glucose oxidase (GOD) as a reporter gene, was shown to consist of the promoter region and the first 659 bp of the H. polymorpha PMA1 gene, encoding the plasma membrane H(+)-ATPase. When the PMA1 promoter was optimally aligned with the GOD coding region, it produced 185 mg/l glucose oxidase in high cell density fed batch fermentations, whereas in previous experiments using the MOX promoter, a yield of 500 mg/l was recovered. The PMA1 promoter was also used to express recombinant human serum albumin (rHA) in H. polymorpha. In high cell density fermentations the PMA1 promoter produced 460 mg/l rHA, whereas 280 mg/l rHA was obtained using the MOX promoter. Taken together, these experiments show that the HpPMA1 programmes the constitutive expression of recombinant proteins and provides a yield comparable to that from the MOX promoter.
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21. |
Pries R,
Gellissen G,
Krappmann S,
( 2000 ) HARO7 encodes chorismate mutase of the methylotrophic yeast Hansenula polymorpha and is derepressed upon methanol utilization. PMID : 10894726 : DOI : 10.1128/jb.182.15.4188-4197.2000 PMC : PMC101906 Abstract >>
The HARO7 gene of the methylotrophic, thermotolerant yeast Hansenula polymorpha was cloned by functional complementation. HARO7 encodes a monofunctional 280-amino-acid protein with chorismate mutase (EC 5.4. 99.5) activity that catalyzes the conversion of chorismate to prephenate, a key step in the biosynthesis of aromatic amino acids. The HARO7 gene product shows strong similarities to primary sequences of known eukaryotic chorismate mutase enzymes. After homologous overexpression and purification of the 32-kDa protein, its kinetic parameters (k(cat) = 319.1 s(-1), n(H) = 1.56, [S](0.5) = 16.7 mM) as well as its allosteric regulatory properties were determined. Tryptophan acts as heterotropic positive effector; tyrosine is a negative-acting, heterotropic feedback inhibitor of enzyme activity. The influence of temperature on catalytic turnover and the thermal stability of the enzyme were determined and compared to features of the chorismate mutase enzyme of Saccharomyces cerevisiae. Using the Cre-loxP recombination system, we constructed mutant strains carrying a disrupted HARO7 gene that showed tyrosine auxotrophy and severe growth defects. The amount of the 0.9-kb HARO7 mRNA is independent of amino acid starvation conditions but increases twofold in the presence of methanol as the sole carbon source, implying a catabolite repression system acting on HARO7 expression.
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22. |
Veenhuis M,
Cregg JM,
Faber KN,
Salomons FA,
Hilbrands RE,
van der Klei IJ,
Rasmussen SW,
( 1999 ) Hansenula polymorpha Pex1p and Pex6p are peroxisome-associated AAA proteins that functionally and physically interact. PMID : 10455230 : DOI : 10.1002/(SICI)1097-0061(199908)15:11<1059::AID-YEA434>3.0.CO;2-I Abstract >>
We have cloned the Hansenula polymorpha PEX1 and PEX6 genes by functional complementation of the corresponding peroxisome-deficient (pex) mutants. The gene products, HpPex1p and HpPex6p, are ATPases which both belong to the AAA protein family. Cells deleted for either gene (Deltapex1 or Deltapex6) were characterized by the presence of small peroxisomal remnants which contained peroxisomal membrane proteins and minor amounts of matrix proteins. The bulk of the matrix proteins, however, resided in the cytosol. In cell fractionation studies HpPex1p and HpPex6p co-sedimented with the peroxisomal membrane protein HpPex3p in both wild-type cells and in Deltapex4, Deltapex8 or Deltapex14 cells. Both proteins are loosely membrane-bound and face the cytosol. Furthermore, HpPex1p and HpPex6p physically and functionally interact in vivo. Overexpression of PEX6 resulted in defects in peroxisomal matrix protein import. By contrast, overexpression of PEX1 was not detrimental to the cells. Interestingly, co-overproduction of HpPex1p rescued the protein import defect caused by HpPex6p overproduction. Overproduced HpPex1p and HpPex6p remained predominantly membrane-bound, but only partially co-localized with the peroxisomal membrane protein HpPex3p. Our data indicate that HpPex1p and HpPex6p function in a protein complex associated with the peroxisomal membrane and that overproduced, mislocalized HpPex6p prevents HpPex1p from reaching its site of activity.
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23. |
Veenhuis M,
Rechinger KB,
van der Klei IJ,
Salomons FA,
( 1999 ) The Hansenula polymorpha PDD1 gene product, essential for the selective degradation of peroxisomes, is a homologue of Saccharomyces cerevisiae Vps34p. PMID : 10398343 : DOI : 10.1002/(SICI)1097-0061(19990630)15:9<741::AID-YEA416>3.0.CO;2-O Abstract >>
Via functional complementation we have isolated the Hansenula polymorpha PDD1 gene essential for selective, macroautophagic peroxisome degradation. HpPDD1 encodes a 116 kDa protein with high similarity (42% identity) to Saccharomyces cerevisiae Vps34p, which has been implicated in vacuolar protein sorting and endocytosis. Western blotting experiments revealed that HpPDD1 is expressed constitutively. In a H. polymorpha pdd1 disruption strain peroxisome degradation is fully impaired. Sequestered peroxisomes, typical for the first stage of peroxisome degradation in H. polymorpha, were never observed, suggesting that HpPdd1p plays a role in the tagging of redundant peroxisomes and/or sequestration of these organelles from the cytosol. Possibly, HpPdd1p is the functional homologue of ScVps34p, because-like S. cerevisiae vps34 mutants-H. polymorpha pdd1 mutants are temperature-sensitive for growth and are impaired in the sorting of vacuolar carboxypeptidase Y. Moreover, HpPdd1p is associated to membranes, as was also observed for ScVps34p.
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24. |
De Virgilio C,
Wiemken A,
Romano I,
( 1999 ) The thermophilic yeast Hansenula polymorpha does not require trehalose synthesis for growth at high temperatures but does for normal acquisition of thermotolerance. PMID : 10419968 : PMC : PMC103601 Abstract >>
The TPS1 gene from Hansenula polymorpha, which encodes trehalose-6-phosphate (Tre6P) synthase, has been isolated and characterized. The deletion of TPS1 rendered H. polymorpha cells incapable of trehalose synthesis under conditions where wild-type cells normally accumulate high levels of trehalose. Interestingly, the loss of Tre6P synthase did not cause any obvious growth defects on a glucose-containing medium, even at high temperatures, but seriously compromised the cells' ability to acquire thermotolerance.
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25. |
Esteban PF,
Vazquez de Aldana CR,
( 1999 ) Cloning and characterization of 1,3-beta-glucanase-encoding genes from non-conventional yeasts. PMID : 10029988 : DOI : 10.1002/(SICI)1097-0061(19990130)15:2<91::AID-YEA343>3.0.CO;2-# Abstract >>
The molecular cloning of 1,3-beta-glucanase-encoding genes from different yeast species was achieved by screening genomic libraries with DNA probes obtained by PCR-amplification using oligonucleotides designed according to conserved regions in the EXG1, EXG2 and SSG1 genes from Saccharomyces cerevisiae. The nucleotide sequence of the KlEXG1 (Kluyveromyces lactis), HpEXG1 (Hansenula polymorpha) and SoEXG1 (Schwanniomyces occidentalis) genes was determined. K1EXG1 consists of a 1287 bp open reading frame encoding a protein of 429 amino acids (49,815 Da). HpEXG1 specifies a 435-amino acid polypeptide (49,268 Da) which contains two potential N-glycosylation sites. SoEXG1 encodes a protein of 425 residues (49,132 Da) which contains one potential site for N-linked glycosylation. Expression in S. cerevisiae of KlEXG1, SoEXG1 or HpEXG1 under control of their native promoters resulted in the secretion of active 1,3-beta-glucanases. Disruption of KlEXG1 did not result in a phenotype under laboratory conditions. Comparison of the primary translation products encoded by KlEXG1, HpEXG1 and SoEXG1 with the previously characterized exo-1,3-beta-glucanases from S. cerevisiae and C. albicans reveals that enzymes with this type of specificity constitute a family of highly conserved proteins in yeasts. KlExg1p, HpExg1p and SoExg1p contain the invariant amino acid positions which have been shown to be important in the catalytic function of family 5 glycosyl hydrolases.
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26. |
Kiel JA,
Veenhuis M,
Yavuz M,
van der Klei IJ,
( 1999 ) Characterization of the Hansenula polymorpha CPY gene encoding carboxypeptidase Y. PMID : 10077185 : DOI : 10.1002/(SICI)1097-0061(199902)15:3<181::AID-YEA355>3.0.CO;2-Y Abstract >>
We have isolated the Hansenula polymorpha CPY gene encoding carboxypeptidase Y (Hp-CPY). The deduced amino acid sequence revealed that Hp-CPY consists of 541 amino acids and has a calculated Mr of 60,793. The protein is highly similar to Saccharomyces cerevisiae CPY (61.8% identity). At the N-terminus of Hp-CPY signals for the entry into the secretory pathway and subsequent sorting to the vacuole were identified. Immunocytochemically, using monospecific antibodies raised against Hp-CPY, the protein was localized to the vacuole. On Western blots, a diffuse protein band was observed in extracts of H. polymorpha cells, suggesting that the protein is glycosylated. This was confirmed by endoglycosidase H treatment, which resulted in a strong reduction of the apparent Mr of the protein. We have investigated the effect of CPY deletion on the degradation of peroxisomes, an autophagous process that occurs when the organelles become redundant for growth. In deltacpy cells peroxisomal proteins were degraded in the vacuole as efficiently as in wild-type H. polymorpha cells, indicating that CPY is not a major proteinase in this pathway.
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27. |
Prasitchoke P,
Kaneko Y,
Sugiyama M,
Bamba T,
Fukusaki E,
Kobayashi A,
Harashima S,
( 2007 ) Functional analysis of very long-chain fatty acid elongase gene, HpELO2, in the methylotrophic yeast Hansenula polymorpha. PMID : 17520249 : DOI : 10.1007/s00253-007-1012-y Abstract >>
We describe the cloning and functional characterization of the fatty acid elongase gene HpELO2, a homologue of the HpELO1 gene required for the production of C24:0 in the yeast Hansenula polymorpha. The open reading frame (ORF) of HpELO2 consists of 1,035 bp, encoding 344 amino acids, sharing about 65% identity with that of Saccharomyces cerevisiae Elo2. Expression of HpELO2 rescued the lethality of the S. cerevisiae elo2Delta elo3Delta double disruptant. An accumulation of C18:0 and a significant increase and decrease in the levels of C24:0 and C26:0, respectively, were observed in the Hpelo2Delta disruptant. These results supported an idea that HpELO2 encodes a fatty acid elongase involved in the elongation of C18:0 to very long-chain fatty acids. The Hpelo1Delta Hpelo2Delta double disruption was nonviable, suggesting that HpELO1 and HpELO2 are the only two genes necessary for the biosynthesis in H. polymorpha. Interestingly, transcription of HpELO2 and HpELO1 were found to be transiently up-regulated by exogenous long-chain fatty acids; however, this up-regulation was not observed with HpELO1 and HpELO2 genes driven by the constitutively expressed promoter of the HpACT gene, suggesting that exogenous fatty acids specifically trigger the transcriptional induction of HpELO1 and HpELO2 through their promoter regions.
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28. |
Kiel JA,
Titorenko VI,
van der Klei IJ,
Veenhuis M,
( 2007 ) Overproduction of translation elongation factor 1-alpha (eEF1A) suppresses the peroxisome biogenesis defect in a Hansenula polymorpha pex3 mutant via translational read-through. PMID : 17425673 : DOI : 10.1111/j.1567-1364.2007.00232.x Abstract >>
In eukaryotes, elongation factor 1-alpha (eEF1A) is required during the elongation phase of translation. We observed that a portion of the cellular eEF1A colocalizes with purified peroxisomes from the methylotrophic yeast Hansenula polymorpha. We have isolated two genes (TEF1 and TEF2) that encode eEF1A, and which are constitutively expressed. We observed that overproduction of eEF1A suppressed the peroxisome deficient phenotype of an H. polymorpha pex3-1 mutant, which was not observed in a strain deleted for PEX3. The pex3-1 allele contains a UGG to UGA mutation, thereby truncating Pex3p after amino acid 242, suggesting that the suppression effect might be the result of translational read-through. Consistent with this hypothesis, overexpression of the pex3-1 gene itself (including its now untranslated part) partly restored peroxisome biogenesis in a PEX3 null mutant. Subsequent co-overexpression of TEF2 in this strain fully restored its peroxisome biogenesis defect and resulted in the formation of major amounts of full-length Pex3p, presumably via translational read-through.
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29. |
Gidijala L,
van der Klei IJ,
Veenhuis M,
Kiel JA,
( 2007 ) Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl gene. PMID : 17419769 : DOI : 10.1111/j.1567-1364.2007.00228.x Abstract >>
We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host-vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine beta-lyase. With this new host-vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein-PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL-SKL produced in H. polymorpha displays normal enzymatic activities.
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30. |
Stasyk OG,
van Zutphen T,
Ah Kang H,
Stasyk OV,
Veenhuis M,
Sibirny AA,
( 2007 ) The role of Hansenula polymorpha MIG1 homologues in catabolite repression and pexophagy. PMID : 17854468 : DOI : 10.1111/j.1567-1364.2007.00286.x Abstract >>
In the methanol-utilizing yeast Hansenula polymorpha, glucose and ethanol trigger the repression of peroxisomal enzymes at the transcriptional level, and rapid and selective degradation of methanol-induced peroxisomes by means of a process termed pexophagy. In this report we demonstrate that deficiency in the putative H. polymorpha homologues of transcriptional repressors Mig1 (HpMig1 and HpMig2), as well as HpTup1, partially and differentially affects the repression of peroxisomal alcohol oxidase by sugars and ethanol. As reported earlier, deficiency in HpTup1 leads to impairment of glucose- or ethanol-induced macropexophagy. In H. polymorpha mig1mig2 double-deletion cells, macropexophagy was also substantially impaired, whereas micropexophagy became a dominant mode of autophagic degradation. Our findings suggest that homologues of the elements of the Saccharomyces cerevisiae main repression pathway have pleiotropic functions in H. polymorpha.
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31. |
Bystrykh LV,
Dijkhuizen L,
Harder W,
( 1991 ) Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha. PMID : 1770353 : DOI : 10.1099/00221287-137-10-2381 Abstract >>
Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95%, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in Vmax and a significant (about one order) decrease in the Km of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k = 0.78 h-1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (kapp = 0.27 h-1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as D-amino acid oxidase from Candida boidinii.
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32. |
Prasitchoke P,
Kaneko Y,
Bamba T,
Fukusaki E,
Kobayashi A,
Harashima S,
( 2007 ) Identification and characterization of a very long-chain fatty acid elongase gene in the methylotrophic yeast, Hansenula polymorpha. PMID : 17236726 : DOI : 10.1016/j.gene.2006.11.013 Abstract >>
To understand the biosynthetic network of fatty acids in the methylotrophic yeast Hansenula polymorpha, which is able to produce poly-unsaturated fatty acids, we have attempted to identify genes encoding fatty acid elongase. Here we have characterized HpELO1, a fatty acid elongase gene encoding a 319-amino-acid protein containing five predicted membrane-spanning regions that is conserved throughout the yeast Elo protein family. Phylogenetic analysis of the deduced amino acid sequence suggests that HpELO1 is an ortholog of the Saccharomyces cerevisiae ELO3 gene that is involved in the elongation of very long-chain fatty acids (VLCFAs). In the fatty acid profile of the Hpelo1Delta disruptant by gas chromatography/mass spectrometry, the amount of C24:0 and C26:0 decreased to undetectable levels, whereas there was a large accumulation of C22:0, suggesting that the HpELO1 is involved in the elongation of VLCFAs and is essential for the production of C24:0. Expression of HpELO1 suppressed the lethality of the S. cerevisiae elo2Delta elo3Delta double disruptant and recovered the synthesis of VLCFAs. Similar to the S. cerevisiae elo3Delta strain, the Hpelo1Delta disruptant exhibited the extraordinary growth sensitivity to fumonisin B(1), a ceramide synthase inhibitor. Furthermore, cells of the Hpelo1Delta disruptant were more sensitive to Zymolyase and more flocculent than the wild-type cells, clumping together and falling rapidly out of suspension, suggesting that the Hpelo1Delta mutation causes changes in cell wall composition and structure.
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33. |
Meijer WH,
van der Klei IJ,
Veenhuis M,
Kiel JA,
( N/A ) ATG genes involved in non-selective autophagy are conserved from yeast to man, but the selective Cvt and pexophagy pathways also require organism-specific genes. PMID : 17204848 : DOI : 10.4161/auto.3595 Abstract >>
ATG genes encode proteins that are required for macroautophagy, the Cvt pathway and/or pexophagy. Using the published Atg protein sequences, we have screened protein and DNA databases to identify putative functional homologs (orthologs) in 21 fungal species (yeast and filamentous fungi) of which the genome sequences were available. For comparison with Atg proteins in higher eukaryotes, also an analysis of Arabidopsis thaliana and Homo sapiens databases was included. This analysis demonstrated that Atg proteins required for non-selective macroautophagy are conserved from yeast to man, stressing the importance of this process in cell survival and viability. The A. thaliana and human genomes encode multiple proteins highly similar to specific fungal Atg proteins (paralogs), possibly representing cell type-specific isoforms. The Atg proteins specifically involved in the Cvt pathway and/or pexophagy showed poor conservation, and were generally not present in A. thaliana and man. Furthermore, Atg19, the receptor of Cvt cargo, was only detected in Saccharomyces cerevisiae. Nevertheless, Atg11, a protein that links receptor-bound cargo (peroxisomes, the Cvt complex) to the autophagic machinery was identified in all yeast species and filamentous fungi under study. This suggests that in fungi an organism-specific form of selective autophagy may occur, for which specialized Atg proteins have evolved.
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34. |
Otzen M,
Krikken AM,
Ozimek PZ,
Kurbatova E,
Nagotu S,
Veenhuis M,
van der Klei IJ,
( 2006 ) In the yeast Hansenula polymorpha, peroxisome formation from the ER is independent of Pex19p, but involves the function of p24 proteins. PMID : 17156013 : DOI : 10.1111/j.1567-1364.2006.00102.x Abstract >>
The peroxin Pex19p is important for the formation of functional peroxisomal membranes. Here we show that Hansenula polymorpha Pex19p is also required for peroxisome inheritance. Peroxisome inheritance is partly defective when Pex19p farnesylation is blocked, whereas deletion of PEX19 resulted in a severe defect in partitioning of peroxisomal structures. Time lapse imaging revealed that in newly formed buds, which had not inherited a peroxisome from the mother cell, new peroxisomes are formed that derive from the nuclear envelope/endoplasmic reticulum. This process was impaired upon deletion of EMP24 and ERP3, genes that encode p24 proteins. p24 Proteins are components of coated vesicles that mediate trafficking between the endoplasmic reticulum and Golgi apparatus. In an H. polymorpha wild-type background, deletion of EMP24 and ERP3 resulted in a strong reduction of organelle number in conjunction with an increase in the size of individual peroxisomes. This observation suggests that p24 proteins also play a role in peroxisome development in wild-type H. polymorpha cells.
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35. |
Kiel JA,
Veenhuis M,
van der Klei IJ,
( 2006 ) PEX genes in fungal genomes: common, rare or redundant. PMID : 16978390 : DOI : 10.1111/j.1600-0854.2006.00479.x Abstract >>
PEX genes encode proteins, termed peroxins, that are required for the biogenesis and proliferation of microbodies (peroxisomes). We have screened the available protein and DNA databases to identify putative peroxin orthologs in 17 fungal species (yeast and filamentous fungi) and in humans. This analysis demonstrated that most peroxins are present in all fungi under study. Only Pex16p is absent in most yeast species, with the exception of Yarrowia lipolytica, but this peroxin is present in all filamentous fungi. Furthermore, we found that the Y. lipolytica PEX9 gene, a putative orphan gene, might encode a Pex26p ortholog. In addition, in the genomes of Saccharomyces cerevisiae and Candida glabrata, several PEX genes appear to have been duplicated, exemplified by the presence of paralogs of the peroxins Pex5p and Pex21p, which were absent in other organisms. In all organisms, we observed multiple paralogs of the peroxins involved in organelle proliferation. These proteins belong to two groups of peroxins that we propose to designate the Pex11p and Pex23p families. This redundancy may complicate future studies on peroxisome biogenesis and proliferation in fungal species.
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36. |
Aksam EB,
Koek A,
Kiel JA,
Jourdan S,
Veenhuis M,
van der Klei IJ,
( N/A ) A peroxisomal lon protease and peroxisome degradation by autophagy play key roles in vitality of Hansenula polymorpha cells. PMID : 17172804 : DOI : 10.4161/auto.3534 Abstract >>
In eukaryote cells various mechanisms exist that are responsible for the removal of non-functional proteins. Here we show that in the yeast Hansenula polymorpha (H. polymorpha) a peroxisomal Lon protease, Pln, plays a role in degradation of unfolded and non-assembled peroxisomal matrix proteins. In addition, we demonstrate that whole peroxisomes are constitutively degraded by autophagy during normal vegetative growth of WT cells. Deletion of both H. polymorpha PLN and ATG1, required for autophagy, resulted in a significant increase in peroxisome numbers, paralleled by a decrease in cell viability relative to WT cells. Also, in these cells and in cells of PLN and ATG1 single deletion strains, the intracellular levels of reactive oxygen species had increased relative to WT controls. The enhanced generation of reactive oxygen species may be related to an uneven distribution of peroxisomal catalase activities in the mutant cells, as demonstrated by cytochemistry. We speculate that in the absence of HpPln or autophagy unfolded and non-assembled peroxisomal matrix proteins accumulate, which can form aggregates and lead to an imbalance in hydrogen peroxide production and degradation in some of the organelles.
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37. |
Baker RE,
Rogers K,
( 2006 ) Phylogenetic analysis of fungal centromere H3 proteins. PMID : 17028330 : DOI : 10.1534/genetics.106.062794 PMC : PMC1667059 Abstract >>
Centromere H3 proteins (CenH3's) are variants of histone H3 specialized for packaging centromere DNA. Unlike canonical H3, which is among the most conserved of eukaryotic proteins, CenH3's are rapidly evolving, raising questions about orthology and conservation of function across species. To gain insight on CenH3 evolution and function, a phylogenetic analysis was undertaken on CenH3 proteins drawn from a single, ancient lineage, the Fungi. Using maximum-likelihood methods, a credible phylogeny was derived for the conserved histone fold domain (HFD) of 25 fungal CenH3's. The collection consisted mostly of hemiascomycetous yeasts, but also included basidiomycetes, euascomycetes, and an archaeascomycete. The HFD phylogeny closely recapitulated known evolutionary relationships between the species, supporting CenH3 orthology. The fungal CenH3's lacked significant homology in their N termini except for those of the Saccharomyces/Kluyveromyces clade that all contained a region homologous to the essential N-terminal domain found in Saccharomyces cerevisiae Cse4. The ability of several heterologous CenH3's to function in S. cerevisiae was tested and found to correlate with evolutionary distance. Domain swapping between S. cerevisiae Cse4 and the noncomplementing Pichia angusta ortholog showed that species specificity could not be explained by the presence or absence of any recognized secondary structural element of the HFD.
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38. |
Monastyrska I,
Kiel JA,
Krikken AM,
Komduur JA,
Veenhuis M,
van der Klei IJ,
( 2005 ) The Hansenula polymorpha ATG25 gene encodes a novel coiled-coil protein that is required for macropexophagy. PMID : 16874036 : DOI : 10.4161/auto.1.2.1832 Abstract >>
We have isolated the Hansenula polymorpha ATG25 gene, which is required for glucose-induced selective peroxisome degradation by macropexophagy. ATG25 represents a novel gene that encodes a 45 kDa coiled-coil protein. We show that this protein colocalizes with Atg11 on a small structure, which most likely represents the pre-autophagosomal structure (PAS). In cells of a constructed ATG25 deletion strain (atg25) peroxisomes are constitutively degraded by nonselective microautophagy, a process that in WT H. polymorpha is only observed at nitrogen limitation conditions. This suggests that nonselective microautophagy is deregulated in H. polymorpha atg25 cells.
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39. |
Kim MW,
Kim EJ,
Kim JY,
Park JS,
Oh DB,
Shimma Y,
Chiba Y,
Jigami Y,
Rhee SK,
Kang HA,
( 2006 ) Functional characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 genes as members of the yeast OCH1 mannosyltransferase family involved in protein glycosylation. PMID : 16407250 : DOI : 10.1074/jbc.M508507200 Abstract >>
The alpha-1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 (ScOCH1) is responsible for the outer chain initiation of N-linked oligosaccharides. To identify the genes involved in the first step of outer chain biosynthesis in the methylotrophic yeast Hansenula polymorpha, we undertook the functional analysis of three H. polymorpha genes, HpHOC1, HpOCH1, and HpOCR1, that belong to the OCH1 family containing seven members with significant sequence identities to ScOCH1. The deletions of these H. polymorpha genes individually resulted in several phenotypes suggestive of cell wall defects. Whereas the deletion of HpHOC1 (Hphoc1Delta) did not generate any detectable changes in N-glycosylation, the null mutant strains of HpOCH1 (Hpoch1Delta) and HpOCR1 (Hpocr1Delta) displayed a remarkable reduction in hypermannosylation. Although the apparent phenotypes of Hpocr1Delta were most similar to those of S. cerevisiae och1 mutants, the detailed structural analysis of N-glycans revealed that the major defect of Hpocr1Delta is not in the initiation step but rather in the subsequent step of outer chain elongation by alpha-1,2-mannose addition. Most interestingly, Hpocr1Delta showed a severe defect in the O-linked glycosylation of extracellular chitinase, representing HpOCR1 as a novel member of the OCH1 family implicated in both N- and O-linked glycosylation. In contrast, addition of the first alpha-1,6-mannose residue onto the core oligosaccharide Man8GlcNAc2 was completely blocked in Hpoch1Delta despite the comparable growth of its wild type under normal growth conditions. The complementation of the S. cerevisiae och1 null mutation by the expression of HpOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in Hpoch1Delta provided supportive evidence that HpOCH1 is the functional orthologue of ScOCH1. The engineered Hpoch1Delta strain with the targeted expression of Aspergillus saitoi alpha-1,2-mannosidase in the endoplasmic reticulum was shown to produce human-compatible high mannose-type Man5GlcNAc2 oligosaccharide as a major N-glycan.
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40. |
Stevens P,
Monastyrska I,
Leão-Helder AN,
van der Klei IJ,
Veenhuis M,
Kiel JA,
( 2005 ) Hansenula polymorpha Vam7p is required for macropexophagy. PMID : 16269391 : DOI : 10.1016/j.femsyr.2005.02.009 Abstract >>
We have analyzed the functions of two vacuolar t-SNAREs, Vam3p and Vam7p, in peroxisome degradation in the methylotrophic yeast Hansenula polymorpha. A Hp-vam7 mutant was strongly affected in peroxisome degradation by selective macropexophagy as well as non-selective microautophagy. Deletion of Hp-Vam3p function had only a minor effect on peroxisome degradation processes. Both proteins were located at the vacuolar membrane, with Hp-Vam7p also having a partially cytosolic location. Previously, in baker's yeast Vam3p and Vam7p have been demonstrated to be components of a t-SNARE complex essential for vacuole biogenesis. We speculate that the function of this complex in macropexophagy includes a role in membrane fusion processes between the outer membrane layer of sequestered peroxisomes and the vacuolar membrane. Our data suggest that Hp-Vam3p may be functionally redundant in peroxisome degradation. Remarkably, deletion of Hp-VAM7 also significantly affected peroxisome biogenesis and resulted in organelles with multiple, membrane-enclosed compartments. These morphological defects became first visible in cells that were in the mid-exponential growth phase of cultivation on methanol, and were correlated with accumulation of electron-dense extensions that were connected to mitochondria.
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41. |
Agaphonov MO,
Sokolov SS,
Romanova NV,
Sohn JH,
Kim SY,
Kalebina TS,
Choi ES,
Ter-Avanesyan MD,
( 2005 ) Mutation of the protein-O-mannosyltransferase enhances secretion of the human urokinase-type plasminogen activator in Hansenula polymorpha. PMID : 16200504 : DOI : 10.1002/yea.1297 Abstract >>
Human urokinase-type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein-O-mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene (HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O-glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C-terminal KDEL retention signal.
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42. |
Viigand K,
Tammus K,
Alamäe T,
( 2005 ) Clustering of MAL genes in Hansenula polymorpha: cloning of the maltose permease gene and expression from the divergent intergenic region between the maltose permease and maltase genes. PMID : 16103021 : DOI : 10.1016/j.femsyr.2005.06.003 Abstract >>
Hansenula polymorpha uses maltase to grow on maltose and sucrose. Inspection of genomic clones of H. polymorpha showed that the maltase gene HPMAL1 is clustered with genes corresponding to Saccharomyces cerevisiae maltose permeases and MAL activator genes orthologues. We sequenced the H. polymorpha maltose permease gene HPMAL2 of the cluster. The protein (582 amino acids) deduced from the HPMAL2 gene is predicted to have eleven transmembrane domains and shows 39-57% identity with yeast maltose permeases. The identity of the protein is highest with maltose permeases of Debaryomyces hansenii and Candida albicans. Expression of the HPMAL2 in a S. cerevisiae maltose permease-negative mutant CMY1050 proved functionality of the permease protein encoded by the gene. HPMAL1 and HPMAL2 genes are divergently positioned similarly to maltase and maltose permease genes in many yeasts. A two-reporter assay of the expression from the HPMAL1-HPMAL2 intergenic region showed that expression of both genes is coordinately regulated, repressed by glucose, induced by maltose, and that basal expression is higher in the direction of the permease gene.
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43. |
Serrani F,
Berardi E,
( 2005 ) The NII2 gene of Hansenula polymorpha is involved in nitrite assimilation. PMID : 16144777 : DOI : 10.1016/j.femsyr.2005.06.002 Abstract >>
To establish a basis for genetic and molecular studies of nitrite assimilation in the methylotrophic yeast Hansenula polymorpha, we isolated and characterised six nitrite-negative mutants still capable of growing on nitrate. Gene isolation work yielded the NII2 gene, encoding a membrane protein homologous to the Saccharomyces cerevisiae Pho86p. Sequence analysis revealed an ORF of 860 bp encoding a 286-amino-acid protein with a predicted molecular mass of 32.8 kDa. This protein is shorter than its S. cerevisiae homologue, and is predicted to lack an ER-retention signal. Cell suspension work revealed that the null mutant is unable to take up nitrite from the medium.
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44. |
Fischer K,
Barbier GG,
Hecht HJ,
Mendel RR,
Campbell WH,
Schwarz G,
( 2005 ) Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site. PMID : 15772287 : DOI : 10.1105/tpc.104.029694 PMC : PMC1087994 Abstract >>
Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 angstroms. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis.
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45. |
Didion T,
Roggenkamp R,
( 1992 ) Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansenula polymorpha. PMID : 1607006 : DOI : 10.1016/0014-5793(92)80500-g Abstract >>
The methylotrophic yeast, Hansenula polymorpha, harbours a unique catalase (EC 1.11.1.6), which is essential for growth on methanol as a carbon source and is located in peroxisomes. Its corresponding gene has been cloned and the nucleotide sequence determined. The deduced amino acid sequence displayed the tripeptide serine-lysine-isoleucine at the extreme C-terminus, which is similar to sequences of other peroxisomal targeting signals. Exchange of the ultimate amino acid, isoleucine, of catalase for serine revealed a cytosolic enzyme activity and a concomitant loss of peroxisome function. We concluded that the tripeptide is essential for targeting of catalase in H. polymorpha.
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46. |
Otzen M,
Wang D,
Lunenborg MG,
van der Klei IJ,
( 2005 ) Hansenula polymorpha Pex20p is an oligomer that binds the peroxisomal targeting signal 2 (PTS2). PMID : 16079284 : DOI : 10.1242/jcs.02463 Abstract >>
We have cloned and characterized the Hansenula polymorpha PEX20 gene. The HpPEX20 gene encodes a protein of 309 amino acids (HpPex20p) with a calculated molecular mass of approximately 35 kDa. In cells of an HpPEX20 disruption strain, PTS2 proteins were mislocalized to the cytosol, whereas PTS1 matrix protein import proceeded normally. Also, the PTS2 proteins amine oxidase and thiolase were normally assembled and active in these cells, suggesting HpPex20p is not involved in oligomerization/activation of these proteins. Localization studies revealed that HpPex20p is predominantly associated with peroxisomes. Using fluorescence correlation spectroscopy we determined the native molecular mass of purified HpPex20p and binding of a synthetic peptide containing a PTS2 sequence. The data revealed that purified HpPex20p forms oligomers, which specifically bind PTS2-containing peptides.
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47. |
Bae JH,
Sohn JH,
Rhee SK,
Choi ES,
( 2005 ) Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A. PMID : 15593064 : DOI : 10.1002/yea.1193 Abstract >>
The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S. cerevisiae proteinase A. That the cloned H. polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H. polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate. When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium. N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H. polymorpha. The nucleotide sequence of the H. polymorpha PEP4 gene has been submitted to GenBank under Accession No. U67173.
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48. |
Leão-Helder AN,
Krikken AM,
Gellissen G,
van der Klei IJ,
Veenhuis M,
Kiel JA,
( 2004 ) Atg21p is essential for macropexophagy and microautophagy in the yeast Hansenula polymorpha. PMID : 15556634 : DOI : 10.1016/j.febslet.2004.10.055 Abstract >>
ATG genes are required for autophagy-related processes that transport proteins/organelles destined for proteolytic degradation to the vacuole. Here, we describe the identification and characterisation of the Hansenula polymorpha ATG21 gene. Its gene product Hp-Atg21p, fused to eGFP, had a dual location in the cytosol and in peri-vacuolar dots. We demonstrate that Hp-Atg21p is essential for two separate modes of peroxisome degradation, namely glucose-induced macropexophagy and nitrogen limitation-induced microautophagy. In atg21 cells subjected to macropexophagy conditions, sequestration of peroxisomes tagged for degradation is initiated but fails to complete.
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49. |
Neves L,
Oliveira R,
Lucas C,
( 2004 ) Yeast orthologues associated with glycerol transport and metabolism. PMID : 15381122 : DOI : 10.1016/j.femsyr.2004.06.012 Abstract >>
Glycerol is a key compound in the regulation of several metabolic pathways in Saccharomyces cerevisiae. From this yeast most of the genes involved in glycerol consumption, production and transport are now available. Some of the mechanisms involving glycerol metabolism and transport are common to other yeasts. This work presents a search for GPD1/2, GUT1, GUP1/2 and FPS1 orthologues in a series of hemiascomycetous yeasts. All the genes cloned were able to complement S. cerevisiae mutant phenotypes and presented a high degree of similarity to the corresponding genes in this yeast. A phylogenetic analysis is presented. The allocation of GUP genes in the membrane bound O-acyl transferases (MBOAT) family is suggested as more consistent than their inclusion in the TC-DB/glycerol uptake family.
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50. |
Ozimek P,
Lahtchev K,
Kiel JA,
Veenhuis M,
van der Klei IJ,
( 2004 ) Hansenula polymorpha Swi1p and Snf2p are essential for methanol utilisation. PMID : 15093770 : DOI : 10.1016/j.femsyr.2004.01.009 Abstract >>
We have cloned the Hansenula polymorpha SWI1 and SNF2 genes by functional complementation of mutants that are defective in methanol utilisation. These genes encode proteins similar to Saccharomyces cerevisiae Swi1p and Snf2p, which are subunits of the SWI/SNF complex. This complex belongs to the family of nucleosome-remodeling complexes that play a role in transcriptional control of gene expression. Analysis of the phenotypes of constructed H. polymorpha SWI1 and SNF2 disruption strains indicated that these genes are not necessary for growth of cells on glucose, sucrose, or various organic nitrogen sources which involve the activity of peroxisomal oxidases. Both disruption strains showed a moderate growth defect on glycerol and ethanol, but were fully blocked in methanol utilisation. In methanol-induced cells of both disruption strains, two peroxisomal enzymes involved in methanol metabolism, alcohol oxidase and dihydroxyacetone synthase, were hardly detectable, whereas in wild-type cells these proteins were present at very high levels. We show that the reduction in alcohol oxidase protein levels in H. polymorpha SWI1 and SNF2 disruption strains is due to strongly reduced expression of the alcohol oxidase gene. The level of Pex5p, the receptor involved in import of alcohol oxidase and dihydroxyacetone synthase into peroxisomes, was also reduced in both disruption strains compared to that in wild-type cells.
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51. |
Butler G,
Kenny C,
Fagan A,
Kurischko C,
Gaillardin C,
Wolfe KH,
( 2004 ) Evolution of the MAT locus and its Ho endonuclease in yeast species. PMID : 14745027 : DOI : 10.1073/pnas.0304170101 PMC : PMC341799 Abstract >>
The genetics of the mating-type (MAT) locus have been studied extensively in Saccharomyces cerevisiae, but relatively little is known about how this complex system evolved. We compared the organization of MAT and mating-type-like (MTL) loci in nine species spanning the hemiascomycete phylogenetic tree. We inferred that the system evolved in a two-step process in which silent HMR/HML cassettes appeared, followed by acquisition of the Ho endonuclease from a mobile genetic element. Ho-mediated switching between an active MAT locus and silent cassettes exists only in the Saccharomyces sensu stricto group and their closest relatives: Candida glabrata, Kluyveromyces delphensis, and Saccharomyces castellii. We identified C. glabrata MTL1 as the ortholog of the MAT locus of K. delphensis and show that switching between C. glabrata MTL1a and MTL1alpha genotypes occurs in vivo. The more distantly related species Kluyveromyces lactis has silent cassettes but switches mating type without the aid of Ho endonuclease. Very distantly related species such as Candida albicans and Yarrowia lipolytica do not have silent cassettes. In Pichia angusta, a homothallic species, we found MATalpha2, MATalpha1, and MATa1 genes adjacent to each other on the same chromosome. Although some continuity in the chromosomal location of the MAT locus can be traced throughout hemiascomycete evolution and even to Neurospora, the gene content of the locus has changed with the loss of an HMG domain gene (MATa2) from the MATa idiomorph shortly after HO was recruited.
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52. |
Otzen M,
Perband U,
Wang D,
Baerends RJ,
Kunau WH,
Veenhuis M,
Van der Klei IJ,
( 2004 ) Hansenula polymorpha Pex19p is essential for the formation of functional peroxisomal membranes. PMID : 14981078 : DOI : 10.1074/jbc.M314275200 Abstract >>
We have cloned and characterized the Hansenula polymorpha PEX19 gene. In cells of a pex19 disruption strain (Hppex19), induced on methanol, peroxisome structures were not detectable; peroxisomal matrix proteins accumulated in the cytosol, whereas peroxisomal membrane proteins (PMPs) were mislocalized to the cytosol (Pex3p) and mitochondria (Pex14p) or strongly reduced to undetectable levels (Pex10p). The defect in peroxisome formation in Hppex19 cells was largely suppressed upon overproduction of HpPex3p or a fusion protein that consisted of the first 50 N-terminal amino acids of Pex3p and GFP. In these cells PMPs were again correctly sorted to peroxisomal structures, which also harbored peroxisomal matrix proteins. In Saccharomyces cerevisiae pex19 cells overproduction of ScPex3p led to the formation of numerous vesicles that contained PMPs but lacked the major matrix protein thiolase. Taken together, our data are consistent with a function of Pex19p in membrane protein assembly and function.
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53. |
Chechenova MB,
Romanova NV,
Deev AV,
Packeiser AN,
Smirnov VN,
Agaphonov MO,
Ter-Avanesyan MD,
( 2004 ) C-terminal truncation of alpha-COP affects functioning of secretory organelles and calcium homeostasis in Hansenula polymorpha. PMID : 14871936 : DOI : 10.1128/ec.3.1.52-60.2004 PMC : PMC329505 Abstract >>
In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding alpha-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated alpha-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca2+ ions, whereas a lack of alpha-COP expression was lethal. The alpha-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca2+-ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca2+ dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca2+ homeostasis is discussed.
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54. |
Song H,
Li Y,
Fang W,
Geng Y,
Wang X,
Wang M,
Qiu B,
( 2003 ) Development of a set of expression vectors in Hansenula polymorpha. PMID : 14719813 : Abstract >>
Four expression vectors based on formate dehydrogenase promoter (FMDp) and methanol oxidase promoter (MOXp) from Hansenula polymorpha were developed to express heterologous genes in Hansenula polymorpha. A secretion signal sequence of the mating factor-alpha from Saccharomyces cerevisiae was inserted in the secretory expression plasmids for efficient secretion. A modified green fluorescent protein (mGFP5) was used as the marker of expression for the first time in H. polymorpha NCYC495 (leu 1.1) to determine the expression ability of these plasmids. The mGFP5 thus expressed retained its biochemical and physiological properties, such as accumulation inside cells and efficient secretion into the culture media. These results indicated that the four integrative vectors are useful expression systems which could be directly applied for production of heterologous proteins of interests in H. polymorpha.
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55. |
Hansen H,
Didion T,
Thiemann A,
Veenhuis M,
Roggenkamp R,
( 1992 ) Targeting sequences of the two major peroxisomal proteins in the methylotrophic yeast Hansenula polymorpha. PMID : 1465101 : DOI : 10.1007/bf00279370 Abstract >>
Dihydroxyacetone synthase (DAS) and methanol oxidase (MOX) are the major enzyme constituents of the peroxisomal matrix in the methylotrophic yeast Hansenula polymorpha when grown on methanol as a sole carbon source. In order to characterize their topogenic signals the localization of truncated polypeptides and hybrid proteins was analysed in transformed yeast cells by subcellular fractionation and electron microscopy. The C-terminal part of DAS, when fused to the bacterial beta-lactamase or mouse dihydrofolate reductase, directed these hybrid polypeptides to the peroxisome compartment. The targeting signal was further delimited to the extreme C-terminus, comprising the sequence N-K-L-COOH, similar to the recently identified and widely distributed peroxisomal targeting signal (PTS) S-K-L-COOH in firefly luciferase. By an identical approach, the extreme C-terminus of MOX, comprising the tripeptide A-R-F-COOH, was shown to be the PTS of this protein. Furthermore, on fusion of a C-terminal sequence from firefly luciferase including the PTS, beta-lactamase was also imported into the peroxisomes of H. polymorpha. We conclude that, besides the conserved PTS (or described variants), other amino acid sequences with this function have evolved in nature.
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56. |
Ramezani-Rad M,
Hollenberg CP,
Lauber J,
Wedler H,
Griess E,
Wagner C,
Albermann K,
Hani J,
Piontek M,
Dahlems U,
Gellissen G,
( 2003 ) The Hansenula polymorpha (strain CBS4732) genome sequencing and analysis. PMID : 14613885 : DOI : 10.1016/S1567-1356(03)00125-9 Abstract >>
The methylotrophic yeast Hansenula polymorpha is a recognised model system for investigation of peroxisomal function, special metabolic pathways like methanol metabolism, of nitrate assimilation or thermostability. Strain RB11, an odc1 derivative of the particular H. polymorpha isolate CBS4732 (synonymous to ATCC34438, NRRL-Y-5445, CCY38-22-2) has been developed as a platform for heterologous gene expression. The scientific and industrial significance of this organism is now being met by the characterisation of its entire genome. The H. polymorpha RB11 genome consists of approximately 9.5 Mb and is organised as six chromosomes ranging in size from 0.9 to 2.2 Mb. Over 90% of the genome was sequenced with concomitant high accuracy and assembled into 48 contigs organised on eight scaffolds (supercontigs). After manual annotation 4767 out of 5933 open reading frames (ORFs) with significant homologies to a non-redundant protein database were predicted. The remaining 1166 ORFs showed no significant similarity to known proteins. The number of ORFs is comparable to that of other sequenced budding yeasts of similar genome size.
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57. |
Stasyk OV,
Stasyk OG,
Komduur J,
Veenhuis M,
Cregg JM,
Sibirny AA,
( 2004 ) A hexose transporter homologue controls glucose repression in the methylotrophic yeast Hansenula polymorpha. PMID : 14660581 : DOI : 10.1074/jbc.M310960200 Abstract >>
Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser(85)) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker's yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport.
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58. |
Alamäe T,
Pärn P,
Viigand K,
Karp H,
( 2003 ) Regulation of the Hansenula polymorpha maltase gene promoter in H. polymorpha and Saccharomyces cerevisiae1. PMID : 14613881 : DOI : 10.1016/S1567-1356(03)00142-9 Abstract >>
Hansenula polymorpha is an exception among methylotrophic yeasts because it can grow on the disaccharides maltose and sucrose. We disrupted the maltase gene (HPMAL1) in H. polymorpha 201 using homologous recombination. Resulting disruptants HP201HPMAL1Delta failed to grow on maltose and sucrose, showing that maltase is essential for the growth of H. polymorpha on both disaccharides. Expression of HPMAL1 in HP201HPMAL1Delta from the truncated variants of the promoter enabled us to define the 5'-upstream region as sufficient for the induction of maltase by disaccharides and its repression by glucose. Expression of the Saccharomyces cerevisiae maltase gene MAL62 was induced by maltose and sucrose, and repressed by glucose if expressed in HP201HPMAL1Delta from its own promoter. Similarly, the HPMAL1 promoter was recognized and correctly regulated by the carbon source in a S. cerevisiae maltase-negative mutant 100-1B. Therefore we suggest that the transcriptional regulators of S. cerevisiae MAL genes (MAL activator and Mig1 repressor) can affect the expression of the H. polymorpha maltase gene, and that homologues of these proteins may exist in H. polymorpha. Using the HPMAL1 gene as a reporter in a H. polymorpha maltase disruption mutant it was shown that the strength of the HPMAL1 promoter if induced by sucrose is quite comparable to the strength of the H. polymorpha alcohol oxidase promoter under conditions of methanol induction, revealing the biotechnological potential of the HPMAL1 promoter.
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59. |
Suwannarangsee S,
Oh DB,
Seo JW,
Kim CH,
Rhee SK,
Kang HA,
Chulalaksananukul W,
Kwon O,
( 2010 ) Characterization of alcohol dehydrogenase 1 of the thermotolerant methylotrophic yeast Hansenula polymorpha. PMID : 20635082 : DOI : 10.1007/s00253-010-2752-7 Abstract >>
The thermotolerant methylotrophic yeast Hansenula polymorpha has recently been gaining interest as a promising host for bioethanol production due to its ability to ferment xylose, glucose, and cellobiose at elevated temperatures up to 48 degrees C. In this study, we identified and characterized alcohol dehydrogenase 1 of H. polymorpha (HpADH1). HpADH1 seems to be a cytoplasmic protein since no N-terminal mitochondrial targeting extension was detected. Compared to the ADHs of other yeasts, recombinant HpADH1 overexpressed in Escherichia coli exhibited much higher catalytic efficiency for ethanol oxidation along with similar levels of acetaldehyde reduction. HpADH1 showed broad substrate specificity for alcohol oxidation but had an apparent preference for medium chain length alcohols. Both ADH isozyme pattern analysis and ADH activity assay indicated that ADH1 is the major ADH in H. polymorpha DL-1. Moreover, an HpADH1-deleted mutant strain produced less ethanol in glucose or glycerol media compared to wild-type. Interestingly, when the ADH1 mutant was complemented with an HpADH1 expression cassette, the resulting strain produced significantly increased amounts of ethanol compared to wild-type, up to 36.7 g l(-1). Taken together, our results suggest that optimization of ADH1 expression would be an ideal method for developing H. polymorpha into an efficient bioethanol production strain.
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60. |
Biswas D,
Datt M,
Ganesan K,
Mondal AK,
( 2010 ) Cloning and characterization of thermotolerant xylitol dehydrogenases from yeast Pichia angusta. PMID : 20717664 : DOI : 10.1007/s00253-010-2818-6 Abstract >>
Pichia angusta (syn. Hansenula polymorpha) represents one of the rare yeast that can grow and ferment both xylose and glucose at higher temperature (50�XC). However, little is known about the enzymes involved in xylose utilization from this species. Previous studies indicated the presence of one xylose reductase and two xylitol dehydrogenase genes in P. angusta. In this study, we have expressed both xylitol dehydrogenases (PaXdh1p and PaXdh2p) in Escherichia coli and purified them as 6X-Histidine-tagged proteins. Biochemical characterization of the recombinant proteins reveals that both PaXdh1p and PaXdh2p are thermotolerant enzymes. PaXdh2p contains a catalytic and a structural Zn atom. However, the structural Zn atom is not present in PaXdh1p. Both enzymes also differ in their affinity for the substrate as well as in the catalytic efficiency. Through mutagenesis and modeling approaches, we have also identified residues important for catalysis and substrate binding.
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61. |
Kurtzman CP,
Robnett CJ,
( 2010 ) Systematics of methanol assimilating yeasts and neighboring taxa from multigene sequence analysis and the proposal of Peterozyma gen. nov., a new member of the Saccharomycetales. PMID : 20522116 : DOI : 10.1111/j.1567-1364.2010.00625.x Abstract >>
The relatedness among methanol-assimilating yeasts assigned to the genus Ogataea and neighboring taxa (Phylum Ascomycota, Subphylum Saccharomycotina, Class Saccharomycetes, Order Saccharomycetales) was determined from phylogenetic analyses of gene sequences for nuclear large and small subunit (SSU) rRNAs, translation elongation factor-1alpha and mitochondrial SSU rRNA. On the basis of the analyses, Williopsis salicorniae and seven species of Pichia are proposed for transfer to the genus Ogataea, which has been emended, and Pichia angophorae, a nonhyphal species, is proposed for transfer to the mycelium forming genus Ambrosiozyma. Pichia toletana and Pichia xylosa form an independent lineage and are assigned to the genus Peterozyma, which is newly proposed.
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62. |
Song H,
Qian W,
Wang H,
Qiu B,
( 2010 ) Identification and functional characterization of the HpALG11 and the HpRFT1 genes involved in N-linked glycosylation in the methylotrophic yeast Hansenula polymorpha. PMID : 20696712 : DOI : 10.1093/glycob/cwq121 Abstract >>
The initial steps in N-linked glycosylation involve the synthesis of a lipid-linked core oligosaccharide followed by the transfer of the core glycan to nascent polypeptides in the endoplasmic reticulum (ER). In this study, we have identified two genes, HpALG11and HpRFT1, in the metylotrophic yeast Hansenula polymorpha. Detailed analysis of the glycan structures of the N-linked glycans of secreted recombinant glucose oxidase in mutant strains Hpalg3�G, Hpalg11�G, and Hpalg3�Galg11�G with the assistance of over-expression of RFT1 was performed by linkage-specific mannosidase digestion. The results suggest that HpALG11 and HpRFT1 were responsible for catalyzing the sequential transfer of terminal �\-1,2-Man residues to form the Man(5)GlcNAc(2)-PP-Dol intermediate at the cytosolic side of the ER before flipping to the luminal side and encoding an evolutionarily conserved protein required for the translocation of Man(5)GlcNAc(2)-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane, respectively. Deletion of the HpALG11 gene leads to poor growth and temperature-sensitive lethality, whereas over-expression of HpRft1p can improve growth of the Hpalg11�G and Hpalg3�Galg11�G strains. Furthermore, deletion of the HpALG11 gene in the Hpalg3�G strain resulted in the secretion of glycoproteins with a predicted structure mainly containing trimannosyl core N-linked glycans (Man(3)GlcNAc(2)).
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63. |
Saraya R,
Cepi?ska MN,
Kiel JA,
Veenhuis M,
van der Klei IJ,
( 2010 ) A conserved function for Inp2 in peroxisome inheritance. PMID : 20153784 : DOI : 10.1016/j.bbamcr.2010.02.001 Abstract >>
In budding yeast Saccharomyces cerevisiae, the peroxisomal protein Inp2 is required for inheritance of peroxisomes to the bud, by connecting the organelles to the motor protein Myo2 and the actin cytoskeleton. Recent data suggested that the function of Inp2 may not be conserved in other yeast species. Using in silico analyses we have identified a weakly conserved Inp2-related protein in 18 species of budding yeast and analyzed the role of the identified protein in the methylotrophic yeast Hansenula polymorpha in peroxisome inheritance. Our data show that H. polymorpha Inp2 locates to peroxisomes, interacts with Myo2, and is essential for peroxisome inheritance.
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64. |
Cavener DR,
Krasney PA,
( 1991 ) Drosophila glucose dehydrogenase and yeast alcohol oxidase are homologous and share N-terminal homology with other flavoenzymes. PMID : 2002763 : DOI : 10.1093/oxfordjournals.molbev.a040634 Abstract >>
N/A
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65. |
van Zutphen T,
Baerends RJ,
Susanna KA,
de Jong A,
Kuipers OP,
Veenhuis M,
van der Klei IJ,
( 2010 ) Adaptation of Hansenula polymorpha to methanol: a transcriptome analysis. PMID : 20044946 : DOI : 10.1186/1471-2164-11-1 PMC : PMC2827406 Abstract >>
Methylotrophic yeast species (e.g. Hansenula polymorpha, Pichia pastoris) can grow on methanol as sole source of carbon and energy. These organisms are important cell factories for the production of recombinant proteins, but are also used in fundamental research as model organisms to study peroxisome biology. During exponential growth on glucose, cells of H. polymorpha typically contain a single, small peroxisome that is redundant for growth while on methanol multiple, enlarged peroxisomes are present. These organelles are crucial to support growth on methanol, as they contain key enzymes of methanol metabolism.In this study, changes in the transcriptional profiles during adaptation of H. polymorpha cells from glucose- to methanol-containing media were investigated using DNA-microarray analyses. Two hours after the shift of cells from glucose to methanol nearly 20% (1184 genes) of the approximately 6000 annotated H. polymorpha genes were significantly upregulated with at least a two-fold differential expression. Highest upregulation (> 300-fold) was observed for the genes encoding the transcription factor Mpp1 and formate dehydrogenase, an enzyme of the methanol dissimilation pathway. Upregulated genes also included genes encoding other enzymes of methanol metabolism as well as of peroxisomal beta-oxidation.A moderate increase in transcriptional levels (up to 4-fold) was observed for several PEX genes, which are involved in peroxisome biogenesis. Only PEX11 and PEX32 were higher upregulated. In addition, an increase was observed in expression of the several ATG genes, which encode proteins involved in autophagy and autophagy processes. The strongest upregulation was observed for ATG8 and ATG11.Approximately 20% (1246 genes) of the genes were downregulated. These included glycolytic genes as well as genes involved in transcription and translation. Transcriptional profiling of H. polymorpha cells shifted from glucose to methanol showed the expected downregulation of glycolytic genes together with upregulation of the methanol utilisation pathway. This serves as a confirmation and validation of the array data obtained. Consistent with this, also various PEX genes were upregulated. The strong upregulation of ATG genes is possibly due to induction of autophagy processes related to remodeling of the cell architecture required to support growth on methanol. These processes may also be responsible for the enhanced peroxisomal beta-oxidation, as autophagy leads to recycling of membrane lipids. The prominent downregulation of transcription and translation may be explained by the reduced growth rate on methanol (td glucose 1 h vs td methanol 4.5 h).
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66. |
Qian W,
Song H,
Liu Y,
Zhang C,
Niu Z,
Wang H,
Qiu B,
( 2009 ) Improved gene disruption method and Cre-loxP mutant system for multiple gene disruptions in Hansenula polymorpha. PMID : 19765620 : DOI : 10.1016/j.mimet.2009.09.004 Abstract >>
In H. polymorpha, there is still a lack of a highly efficient gene disruption method. To help address this issue, we presented a simple and efficient method for both single and multiple gene disruptions in H. polymorpha. The knockout system combined a variation of sticky-end polymerase chain reaction method (SEP), split marker deletion method, co-transformation of single-stranded DNA and mutant Cre-loxP system. Using a slightly modified LiAc/SS-DNA/PEG procedure, the co-transformation double-stranded split marker constructs together with single-stranded split marker constructs resulted in at least 70% homologous recombination events when the homologous genomic DNA fragment had a size of approximately 500bp. Our evidence suggested that single-stranded DNA may be responsible for the increased gene disruption efficiency. We demonstrated the effectiveness of the method for gene disruption by constructing both single and double gene disruptions at the ALG3 and URA5 loci in the same genetic background. The method described here presents an improved strategy for gene disruption and a potential application for investigation of biological processes in other yeast strains.
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67. |
Aksam EB,
de Vries B,
van der Klei IJ,
Kiel JA,
( 2009 ) Preserving organelle vitality: peroxisomal quality control mechanisms in yeast. PMID : 19538506 : DOI : 10.1111/j.1567-1364.2009.00534.x Abstract >>
Cellular proteins and organelles such as peroxisomes are under continuous quality control. Upon synthesis in the cytosol, peroxisomal proteins are kept in an import-competent state by chaperones or specific proteins with an analogous function to prevent degradation by the ubiquitin-proteasome system. During protein translocation into the organelle, the peroxisomal targeting signal receptors (Pex5, Pex20) are also continuously undergoing quality control to enable efficient functioning of the translocon (RADAR pathway). Even upon maturation of peroxisomes, matrix enzymes and peroxisomal membranes remain subjected to quality control. As a result of their oxidative metabolism, peroxisomes are producers of reactive oxygen species (ROS), which may damage proteins and lipids. To counteract ROS-induced damage, yeast peroxisomes contain two important antioxidant enzymes: catalase and an organelle-specific peroxiredoxin. Additionally, a Lon-type protease has recently been identified in the peroxisomal matrix, which is capable of degrading nonfunctional proteins. Finally, cellular housekeeping processes keep track of the functioning of peroxisomes so that dysfunctional organelles can be quickly removed via selective autophagy (pexophagy). This review provides an overview of the major processes involved in quality control of yeast peroxisomes.
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68. |
Cheon SA,
Choo J,
Ubiyvovk VM,
Park JN,
Kim MW,
Oh DB,
Kwon O,
Sibirny AA,
Kim JY,
Kang HA,
( 2009 ) New selectable host-marker systems for multiple genetic manipulations based on TRP1, MET2 and ADE2 in the methylotrophic yeast Hansenula polymorpha. PMID : 19653331 : DOI : 10.1002/yea.1701 Abstract >>
Interest has been increasing in the thermotolerant methylotrophic yeast Hansenula polymorpha as a useful system for fundamental research and applied purposes. Only a few genetic marker genes and auxotrophic hosts are yet available for this yeast. Here we isolated and developed H. polymorpha TRP1, MET2 and ADE2 genes as selectable markers for multiple genetic manipulations. The H. polymorpha TRP1 (HpTRP1), MET2 (HpMET2) and ADE2 (HpADE2) genes were sequentially disrupted, using an HpURA3 pop-out cassette in H. polymorpha to generate a series of new multiple auxotrophic strains, including up to a quintuple auxotrophic strain. Unexpectedly, the HpTRP1 deletion mutants required additional tryptophan supplementation for their full growth, even on complex media such as YPD. Despite the clearly increased resistance to 5-fluoroanthranilic acid of the HpTRP1 deletion mutants, the HpTRP1 blaster cassette does not appear to be usable as a counter-selection marker in H. polymorpha. Expression vectors carrying HpADE2, HpTRP1 or HpMET2 with their own promoters and terminators as selectable markers were constructed and used to co-transform the quintuple auxotrophic strain for the targeted expression of a heterologous gene, Aspergillus saitoi MsdS, at the ER, the Golgi and the cell surface, respectively.
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69. |
Krikken AM,
Veenhuis M,
van der Klei IJ,
( 2009 ) Hansenula polymorpha pex11 cells are affected in peroxisome retention. PMID : 19187239 : DOI : 10.1111/j.1742-4658.2009.06883.x Abstract >>
We have cloned and characterized the Hansenula polymorpha PEX11 gene. Our morphological data are consistent with previous observations that peroxisome proliferation can be regulated by modulating Pex11p levels. Surprisingly, pex11 cells also showed a defect in peroxisome retention in mother cells during vegetative cell reproduction. Until now, Saccharomyces cerevisiae Inp1p has been the only peroxisomal protein that has been shown to play a role in the organelle retention process. H. polymorpha inp1 cells are also affected in peroxisome retention, like pex11 cells. We show by time-lapse imaging that Inp1-green fluorescent protein localization varies during the cell cycle and that the protein is normally recruited to peroxisomes in pex11 cells. Taken together, our data show that H. polymorpha Pex11p is not only important for peroxisome proliferation but is also required for proper peroxisome segregation during cell division.
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70. |
Nagotu S,
Krikken AM,
Otzen M,
Kiel JA,
Veenhuis M,
van der Klei IJ,
( 2008 ) Peroxisome fission in Hansenula polymorpha requires Mdv1 and Fis1, two proteins also involved in mitochondrial fission. PMID : 18513378 : DOI : 10.1111/j.1600-0854.2008.00772.x Abstract >>
We show that Mdv1 and Caf4, two components of the mitochondrial fission machinery in Saccharomyces cerevisiae, also function in peroxisome proliferation. Deletion of MDV1, CAF4 or both, however, had only a minor effect on peroxisome numbers at peroxisome-inducing growth conditions, most likely related to the fact that Vps1--and not Dnm1--is the key player in peroxisome fission in this organism. In contrast, in Hansenula polymorpha, which has only a Dnm1-dependent peroxisome fission machinery, deletion of MDV1 led to a drastic reduction of peroxisome numbers. This phenotype was accompanied by a strong defect in mitochondrial fission. The MDV1 paralog CAF4 is absent in H. polymorpha. In wild-type H. polymorpha, cells Dnm1-mCherry and green fluorescent protein (GFP)-Mdv1 colocalize in spots that associate with both peroxisomes and mitochondria. Furthermore, Fis1 is essential to recruit Mdv1 to the peroxisomal and mitochondrial membrane. However, formation of GFP-Mdv1 spots--and related to this normal organelle fission--is strictly dependent on the presence of Dnm1. In dnm1 cells, GFP-Mdv1 is dispersed over the surface of peroxisomes and mitochondria. Also, in H. polymorpha mdv1 or fis1 cells, the number of Dnm1-GFP spots is strongly reduced. These spots still associate to organelles but are functionally inactive.
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71. |
van der Klei IJ,
Harder W,
Veenhuis M,
( 1991 ) Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeasts: a review. PMID : 1882546 : DOI : 10.1002/yea.320070302 Abstract >>
Alcohol oxidase (AO) catalyses the first step of methanol metabolism in yeasts. In vivo the enzyme is compartmentalized in special cell compartments, called peroxisomes. The enzyme along with the organelles are induced during growth of methylotrophic yeasts on methanol as the sole carbon source. Like all other peroxisomal matrix proteins, AO is encoded by a nuclear gene. Expression of the protein is regulated by a repression/derepression mechanism, but also by induction. Inactive monomeric precursor protein is synthesized in the cytosol and subsequently imported post-translationally into peroxisomes without further processing. Assembly into the active homo-octameric enzyme and binding of the prosthetic group flavin adenine dinucleotide occurs inside the organelle. When enhanced concentration of octameric alcohol oxidase are present in the organelles, the enzyme may form a crystalloid. Oligomerization is not dependent on translocation of AO precursors into their target organelle since octameric, active AO is detected in the cytosol and nucleus of peroxisome-deficient mutants of Hansenula polymorpha: at high expression rates large cytosolic AO crystalloids are formed, which occasionally are also encountered inside the nucleus of such mutants. This paper summarizes recent findings and views on the mechanisms involved in synthesis, import, assembly and crystallization of this important peroxisomal enzyme.
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72. |
Bener Aksam E,
Jungwirth H,
Kohlwein SD,
Ring J,
Madeo F,
Veenhuis M,
van der Klei IJ,
( 2008 ) Absence of the peroxiredoxin Pmp20 causes peroxisomal protein leakage and necrotic cell death. PMID : 18694816 : DOI : 10.1016/j.freeradbiomed.2008.07.010 Abstract >>
We analyzed the role of the peroxisomal peroxiredoxin Pmp20 of the yeast Hansenula polymorpha. Cells of a PMP20 disruption strain (pmp20) grew normally on substrates that are not metabolized by peroxisomal enzymes, but showed a severe growth defect on methanol, the metabolism of which involves a hydrogen peroxide producing peroxisomal oxidase. This growth defect was paralleled by leakage of peroxisomal matrix proteins into the cytosol. Methanol-induced pmp20 cells accumulated enhanced levels of reactive oxygen species and lipid peroxidation products. Moreover, the fatty acid composition of methanol-induced pmp20 cells differed relative to WT controls, suggesting an effect on fatty acid homeostasis. Plating assays and FACS-based analysis of cell death markers revealed that pmp20 cells show loss of clonogenic efficiency and membrane integrity, when cultured on methanol. We conclude that the absence of the peroxisomal peroxiredoxin leads to loss of peroxisome membrane integrity and necrotic cell death.
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73. |
Oh DB,
Park JS,
Kim MW,
Cheon SA,
Kim EJ,
Moon HY,
Kwon O,
Rhee SK,
Kang HA,
( 2008 ) Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N-glycan by blocking core oligosaccharide assembly. PMID : 18320567 : DOI : 10.1002/biot.200700252 Abstract >>
The initial lipid-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-dolichyl pyrophosphate (Dol-PP) for N-glycan is synthesized and assembled at the membrane of the endoplasmic reticulum (ER) and subsequently transferred to a nascent polypeptide by the oligosaccharide transferase complex. We have identified an ALG3 homolog (HpALG3) coding for a dolichyl-phosphate-mannose dependent alpha-1,3-mannosyltransferase in the methylotrophic yeast Hansenula polymorpha. The detailed analysis of glycan structure by linkage-specific mannosidase digestion showed that HpALG3 is responsible for the conversion of Man5GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP, the first step to attach a mannose to the lipid-linked oligosaccharide in the ER. The N-glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain blocked in the yeast-specific outer mannose chain synthesis and by introducing an ER-targeted Aspergillus saitoi alpha-1,2-mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core backbone of various human-type N-glycans. The results demonstrate the high potential of H. polymorpha to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.
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74. |
Stasyk OG,
Maidan MM,
Stasyk OV,
Van Dijck P,
Thevelein JM,
Sibirny AA,
( 2008 ) Identification of hexose transporter-like sensor HXS1 and functional hexose transporter HXT1 in the methylotrophic yeast Hansenula polymorpha. PMID : 18310355 : DOI : 10.1128/EC.00028-08 PMC : PMC2292620 Abstract >>
We identified in the methylotrophic yeast Hansenula polymorpha (syn. Pichia angusta) a novel hexose transporter homologue gene, HXS1 (hexose sensor), involved in transcriptional regulation in response to hexoses, and a regular hexose carrier gene, HXT1 (hexose transporter). The Hxs1 protein exhibits the highest degree of primary sequence similarity to the Saccharomyces cerevisiae transporter-like glucose sensors, Snf3 and Rgt2. When heterologously overexpressed in an S. cerevisiae hexose transporter-less mutant, Hxt1, but not Hxs1, restores growth on glucose or fructose, suggesting that Hxs1 is nonfunctional as a carrier. In its native host, HXS1 is expressed at moderately low level and is required for glucose induction of the H. polymorpha functional low-affinity glucose transporter Hxt1. Similarly to other yeast sensors, one conserved amino acid substitution in the Hxs1 sequence (R203K) converts the protein into a constitutively signaling form and the C-terminal region of Hxs1 is essential for its function in hexose sensing. Hxs1 is not required for glucose repression or catabolite inactivation that involves autophagic degradation of peroxisomes. However, HXS1 deficiency leads to significantly impaired transient transcriptional repression in response to fructose, probably due to the stronger defect in transport of this hexose in the hxs1Delta deletion strain. Our combined results suggest that in the Crabtree-negative yeast H. polymorpha, the single transporter-like sensor Hxs1 mediates signaling in the hexose induction pathway, whereas the rate of hexose uptake affects the strength of catabolite repression.
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75. |
Nagotu S,
Saraya R,
Otzen M,
Veenhuis M,
van der Klei IJ,
( 2008 ) Peroxisome proliferation in Hansenula polymorpha requires Dnm1p which mediates fission but not de novo formation. PMID : 18060881 : DOI : 10.1016/j.bbamcr.2007.10.018 Abstract >>
We show that the dynamin-like proteins Dnm1p and Vps1p are not required for re-introduction of peroxisomes in Hansenula polymorpha pex3 cells upon complementation with PEX3-GFP. Instead, Dnm1p, but not Vps1p, plays a crucial role in organelle proliferation via fission. In H. polymorpha DNM1 deletion cells (dnm1) a single peroxisome is present that forms long extensions, which protrude into developing buds and divide during cytokinesis. Budding pex11.dnm1 double deletion cells lack these peroxisomal extensions, suggesting that the peroxisomal membrane protein Pex11p is required for their formation. Life cell imaging revealed that fluorescent Dnm1p-GFP spots fluctuate between peroxisomes and mitochondria. On the other hand Pex11p is present over the entire organelle surface, but concentrates during fission at the basis of the organelle extension in dnm1 cells. Our data indicate that peroxisome fission is the major pathway for peroxisome multiplication in H. polymorpha.
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76. |
Klabunde J,
Kleebank S,
Piontek M,
Hollenberg CP,
Hellwig S,
Degelmann A,
( 2007 ) Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorpha. PMID : 17617219 : DOI : 10.1111/j.1567-1364.2007.00271.x PMC : PMC2040192 Abstract >>
The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for super-transformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-gamma and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L(-1) range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MFalpha1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha.
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77. |
Koek A,
Komori M,
Veenhuis M,
van der Klei IJ,
( 2007 ) A comparative study of peroxisomal structures in Hansenula polymorpha pex mutants. PMID : 17608706 : DOI : 10.1111/j.1567-1364.2007.00261.x Abstract >>
In a recent study, we performed a systematic genome analysis for the conservation of genes involved in peroxisome biogenesis (PEX genes) in various fungi. We have now performed a systematic study of the morphology of peroxisome remnants ('ghosts') in Hansenula polymorpha pex mutants (pex1-pex20) and the level of peroxins and matrix proteins in these strains. To this end, all available H. polymorpha pex strains were grown under identical cultivation conditions in glucose-limited chemostat cultures and analyzed in detail. The H. polymorpha pex mutants could be categorized into four distinct groups, namely pex mutants containing: (1) virtually normal peroxisomal structures (pex7, pex17, pex20); (2) small peroxisomal membrane structures with a distinct lumen (pex2, pex4, pex5, pex10, pex12, pex14); (3) multilayered membrane structures lacking apparent matrix protein content (pex1, pex6, pex8, pex13); and (4) no peroxisomal structures (pex3, pex19).
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78. |
Vinothkumar KR,
Montgomery MG,
Liu S,
Walker JE,
( 2016 ) Structure of the mitochondrial ATP synthase from Pichia angusta determined by electron cryo-microscopy. PMID : 27791192 : DOI : 10.1073/pnas.1615902113 PMC : PMC5111644 Abstract >>
The structure of the intact monomeric ATP synthase from the fungus, Pichia angusta, has been solved by electron cryo-microscopy. The structure provides insights into the mechanical coupling of the transmembrane proton motive force across mitochondrial membranes in the synthesis of ATP. This mechanism requires a strong and integral stator, consisting of the catalytic �\3�]3-domain, peripheral stalk, and, in the membrane domain, subunit a and associated supernumerary subunits, kept in contact with the rotor turning at speeds up to 350 Hz. The stator's integrity is ensured by robust attachment of both the oligomycin sensitivity conferral protein (OSCP) to the catalytic domain and the membrane domain of subunit b to subunit a. The ATP8 subunit provides an additional brace between the peripheral stalk and subunit a. At the junction between the OSCP and the apparently stiff, elongated �\-helical b-subunit and associated d- and h-subunits, an elbow or joint allows the stator to bend to accommodate lateral movements during the activity of the catalytic domain. The stator may also apply lateral force to help keep the static a-subunit and rotating c10-ring together. The interface between the c10-ring and the a-subunit contains the transmembrane pathway for protons, and their passage across the membrane generates the turning of the rotor. The pathway has two half-channels containing conserved polar residues provided by a bundle of four �\-helices inclined at ?30�X to the plane of the membrane, similar to those described in other species. The structure provides more insights into the workings of this amazing machine.
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79. |
Ledeboer AM,
Edens L,
Maat J,
Visser C,
Bos JW,
Verrips CT,
Janowicz Z,
Eckart M,
Roggenkamp R,
Hollenberg CP,
( 1985 ) Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. PMID : 2582370 : DOI : 10.1093/nar/13.9.3063 PMC : PMC341221 Abstract >>
The structural gene and the regulatory DNA sequence of the yeast Hansenula polymorpha methanol oxidase have been isolated. According to the nucleotide sequence data obtained, the structural gene encodes a 664 amino acids long protein, contains no intervening sequences, and the 5'- and 3'-non-coding region contains several sequences implicated in transcription initiation and termination in the yeast Saccharomyces cerevisiae. Although the methanol oxidase is translocated to the peroxisomes, no cleavable signal sequence was found at the N-terminus of the protein.
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80. |
Viigand K,
Visnapuu T,
Mardo K,
Aasamets A,
Alamäe T,
( 2016 ) Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to the resurrected ancestor protein ancMALS of yeast maltases and isomaltases. PMID : 26919272 : DOI : 10.1002/yea.3157 PMC : PMC5074314 Abstract >>
Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, �\-methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous �\-glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between Op MAL1 and ancMALS. Op MAL1 represents an �\-glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose-like substrates (�\-1,4-glucosides), confirming the requirement of Thr at the respective position for this function. Differential scanning fluorimetry (DSF) of the catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate-binding pocket of MAL1 has three subsites (-1, +1 and +2) and that binding is strongest at the -1 subsite. The DSF assay results were in good accordance with affinity (Km) and inhibition (Ki) data of the enzyme for tested substrates, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or �\-glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars. ? 2016 The Authors. Yeast published by John Wiley & Sons, Ltd.
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81. |
Liu S,
Charlesworth TJ,
Bason JV,
Montgomery MG,
Harbour ME,
Fearnley IM,
Walker JE,
( 2015 ) The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species. PMID : 25759169 : DOI : 10.1042/BJ20150197 PMC : PMC4422255 Abstract >>
The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-�]-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-�]-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.
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82. |
Bruinenberg PG,
Evers M,
Waterham HR,
Kuipers J,
Arnberg AC,
AB G,
( 1989 ) Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha. PMID : 2500147 : DOI : 10.1016/0167-4781(80)90003-2 Abstract >>
We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.
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83. |
Sohn MJ,
Yoo SJ,
Oh DB,
Kwon O,
Lee SY,
Sibirny AA,
Kang HA,
( 2014 ) Novel cysteine-centered sulfur metabolic pathway in the thermotolerant methylotrophic yeast Hansenula polymorpha. PMID : 24959887 : DOI : 10.1371/journal.pone.0100725 PMC : PMC4069077 Abstract >>
In yeast and filamentous fungi, sulfide can be condensed either with O-acetylhomoserine to generate homocysteine, the precursor of methionine, or with O-acetylserine to directly generate cysteine. The resulting homocysteine and cysteine can be interconverted through transsulfuration pathway. Here, we systematically analyzed the sulfur metabolic pathway of the thermotolerant methylotrophic yeast Hansenula polymorpha, which has attracted much attention as an industrial yeast strain for various biotechnological applications. Quite interestingly, the detailed sulfur metabolic pathway of H. polymorpha, which was reconstructed based on combined analyses of the genome sequences and validation by systematic gene deletion experiments, revealed the absence of de novo synthesis of homocysteine from inorganic sulfur in this yeast. Thus, the direct biosynthesis of cysteine from sulfide is the only pathway of synthesizing sulfur amino acids from inorganic sulfur in H. polymorpha, despite the presence of both directions of transsulfuration pathway Moreover, only cysteine, but no other sulfur amino acid, was able to repress the expression of a subset of sulfur genes, suggesting its central and exclusive role in the control of H. polymorpha sulfur metabolism. 35S-Cys was more efficiently incorporated into intracellular sulfur compounds such as glutathione than 35S-Met in H. polymorpha, further supporting the cysteine-centered sulfur pathway. This is the first report on the novel features of H. polymorpha sulfur metabolic pathway, which are noticeably distinct from those of other yeast and filamentous fungal species.
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84. |
Stoyanov A,
Petrova P,
Lyutskanova D,
Lahtchev K,
( N/A ) Structural and functional analysis of PUR2,5 gene encoding bifunctional enzyme of de novo purine biosynthesis in Ogataea (Hansenula) polymorpha CBS 4732T. PMID : 24135445 : DOI : 10.1016/j.micres.2013.08.008 Abstract >>
We describe the cloning, sequencing and functional characterization of gene PUR2,5, involved in de novo purine biosynthesis of the yeast Ogataea (Hansenula) polymorpha. This gene (2369 bp) was cloned by genetic complementation of adenine requiring mutation. It encodes a bifunctional enzyme of 789 amino acids (85 kDa) that catalyzes the second and the fifth steps of de novo purine biosynthesis pathway and shows dual enzymatic activity - of glycinamide ribotide synthetase (GARS, EC 6.3.4.13) and of aminoimidazole ribotide synthetase (AIRS, EC 6.3.3.1). Nucleotide sequence analysis revealed the presence of putative regulatory elements located in the adjacent 5' region. Canonical motives that function as binding sites for BAS1 transcription activator were found at positions (-593) and (-389). The putative TAATTA-box was located at (-20) to (-14) and AT-rich heteroduplex was found in the 3'-non-translated region. We compared the amino acid sequence of OpPUR2,5p with those of the corresponding enzymes of other yeast species as well as with distant organisms like bacteria Escherichia coli and human Homo sapiens. A successful disruption of OpPUR2,5 gene was done. It was found that OpPUR2,5::LEU2 replacement affects both mating and sporulation processes. OpPUR2,5 sequence is deposited in the GenBank of NCBI with accession no. JF967633.
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85. |
Park JN,
Choo J,
Kang HA,
( 2011 ) Functional analysis of a Hansenula polymorpha MNN2-2 homologue encoding a putative UDP-N-acetylglucosamine transporter localized in the endoplasmic reticulum. PMID : 22203566 : DOI : 10.1007/s12275-011-1520-4 Abstract >>
The Kluyveromyces lactis UDP-GlcNAc transporter (KlMnn2-2p) is responsible for the biosynthesis of N-glycans containing N-acetylglucosamine. A putative gene of Hansenula polymorpha encoding a KlMnn2-2p homologue, HpMNN2-2, was identified and investigated for its function. The deletion mutant strain of HpMNN2-2 (Hpmnn2-2�G) showed increased sensitivity to geneticin, hygromycin B, and tunicamycin. However, the Hpmnn2-2�G strain exhibited increased resistance to Calcofluor white, an inhibitor of chitin biosynthesis, along with a reduced chitin content. The localization of HpMnn2-2p at the endoplasmic reticulum-enriched membrane, different from the Golgi localization of a K. lactis homologue, further supports the involvement of HpMnn2-2p in cell wall chitin biosynthesis.
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86. |
( 1997 ) The Hansenula polymorpha PEX14 gene encodes a novel peroxisomal membrane protein essential for peroxisome biogenesis. PMID : 9009266 : DOI : 10.1093/emboj/16.1.44 PMC : PMC1169612 Abstract >>
We have cloned the Hansenula polymorpha PEX14 gene by functional complementation of the chemically induced pex14-1 mutant, which lacked normal peroxisomes. The sequence of the PEX14 gene predicts a novel protein product (Pex14p) of 39 kDa which showed no similarity to any known protein and lacked either of the two known peroxisomal targeting signals. Biochemical and electron microscopical analysis indicated that Pex14p is a component of the peroxisomal membrane. The synthesis of Pex14p is induced by peroxisome-inducing growth conditions. In cells of both pex14-1 and a PEX14 disruption mutant, peroxisomal membrane remnants were evident; these contained the H.polymorpha peroxisomal membrane protein Pex3p together with a small amount of the major peroxisomal matrix proteins alcohol oxidase, catalase and dihydroxyacetone synthase, the bulk of which resided in the cytosol. Unexpectedly, overproduction of Pex14p in wild-type H. polymorpha cells resulted in a peroxisome-deficient phenotype typified by the presence of numerous small vesicles which lacked matrix proteins; these were localized in the cytosol. Apparently, the stoichiometry of Pex14p relative to one or more other components of the peroxisome biogenesis machinery appears to be critical for protein import.
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87. |
( 1996 ) Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family. PMID : 8840502 : DOI : 10.1002/(SICI)1097-0061(199607)12:9%3C849::AID-YEA985%3E3.0.CO;2-Z Abstract >>
We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was constitutively present. Peptides derived from both Hsp72 and Hsc74 showed sequence homology to the cytosolic Saccharomyces cerevisiae Hsp70s, Ssa1p and Ssa2p. The gene encoding Hsp72 (designated HSA1) was cloned, sequenced and used to construct HSA1 disruption and HSA1 overexpression strains. Comparison of the stress tolerances of these strains with those of wild-type H. polymorpha revealed that HSA1 overexpression negatively affected the tolerance of the cells to killing effects of temperature or ethanol, but enhanced the tolerance to copper and cadmium. The tolerance for other chemicals (arsenite, arsenate, H2O2) or to high osmolarity was unaffected by either deletion or overexpression of HSA1.
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88. |
( 1996 ) Flavin adenine dinucleotide binding is the crucial step in alcohol oxidase assembly in the yeast Hansenula polymorpha. PMID : 8873445 : DOI : 10.1002/(SICI)1097-0061(199608)12:10%3C917::AID-YEA984%3E3.0.CO;2-4 Abstract >>
We have studied the role of flavin adenine dinucleotide (FAD) in the in vivo assembly of peroxisomal alcohol oxidase (AO) in the yeast Hansenula polymorpha. In previous studies, using a riboflavin (Rf) autotrophic mutant, an unequivocal judgement could not be made, since Rf-limitation led to a partial block of AO import in this mutant. This resulted in the accumulation of AO precursors in the cytosol where they remained separated from the putative peroxisomal AO assembly factors. In order to circumvent the peroxisomal membrane barrier, we have now studied AO assembly in a peroxisome-deficient/Rf-autotrophic double mutant (delta per1.rif1) of H. polymorpha. By sucrose density centrifugation and native gel electrophoresis, three conformations of AO were detected in crude extracts of delta per1.rif1 cells grown under Rf-limitation, namely active octameric AO and two inactive, monomeric forms. One of the latter forms lacked FAD; this form was barely detectable in extracts wild-type and delta per1 cells, but had accumulated in the cytosol of rif1 cells. The second form of monomeric AO contained FAD; this form was also present in delta per1 cells but absent/very low in wild-type and rif1 cells. In vivo only these FAD-containing monomers associate into the active, octameric protein. We conclude that in H. polymorpha FAD binding to the AO monomer is mediated by a yet unknown peroxisomal factor and represents the crucial and essential step to enable AO oligomerization; the actual octamerization and the eventual crystallization in peroxisomes most probably occurs spontaneously.
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89. |
( 1997 ) The YNT1 gene encoding the nitrate transporter in the yeast Hansenula polymorpha is clustered with genes YNI1 and YNR1 encoding nitrite reductase and nitrate reductase, and its disruption causes inability to grow in nitrate. PMID : 9020872 : DOI : 10.1042/bj3210397 PMC : PMC1218082 Abstract >>
DNA sequencing in the phage lambda JA13 isolated from a lambda EMBL3 Hansenula polymorpha genomic DNA library containing the nitrate reductase-(YNR1) and nitrite reductase-(YNI1) encoding genes revealed an open reading frame (YNT1) of 1524 nucleotides encoding a putative protein of 508 amino acids with great similarity to the nitrate transporters from Aspergillus nidulans and Chlamydomonas reinhardtii. Disruption of the chromosomal YNT1 copy resulted in incapacity to grow in nitrate and a significant reduction in rate of nitrate uptake. The disrupted strain is still sensitive to chlorate, and, in the presence of 0.1 mM nitrate, the expression of YNR1 and YNI1 and the activity of nitrate reductase and nitrite reductase are significantly reduced compared with the wild-type. Northern-blot analysis showed that YNT1 is expressed when the yeast is grown in nitrate and nitrite but not in ammonium solution.
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90. |
( 1996 ) The Hansenula polymorpha PER9 gene encodes a peroxisomal membrane protein essential for peroxisome assembly and integrity. PMID : 8621531 : DOI : 10.1074/jbc.271.15.8887 Abstract >>
We have cloned and characterized the Hansenula polymorpha PER9 gene by functional complementation of the per9-1 mutant of H. polymorpha, which is defective in peroxisome biogenesis. The predicted product, Per9p, is a polypeptide of 52 kDa with sequence similarity to Pas3p, a protein involved in peroxisome biogenesis in Saccharomyces cerevisiae. In a per9 disruption strain (Deltaper9), peroxisomal matrix and membrane proteins are present at wild-type levels. The matrix proteins accumulated in the cytoplasm. However, the location of the membrane proteins remained obscure; fully induced Deltaper9 cells lacked residual peroxisomal vesicles ("ghosts"). Analysis of the activity of the PER9 promoter revealed that PER9 expression was low in cells grown on glucose, but was enhanced during growth of cells on peroxisome-inducing substrates. The highest expression levels were observed in cells grown on methanol. Localization studies revealed that Per9p is an integral membrane protein of the peroxisome. Targeting studies suggested that Per9p may be sorted to the peroxisome via the endoplasmic reticulum. Overexpression of PER9 induced a significant increase in the number of peroxisomes per cell, a result that suggests that Per9p may be involved in peroxisome proliferation and/or membrane biosynthesis. When PER9 expression was placed under the control of a strongly regulatable promoter and switched off, peroxisomes were observed to disintegrate over time in a manner that suggested that Per9p may be required for maintenance of the peroxisomal membrane.
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91. |
van der Klei IJ,
Hilbrands RE,
Swaving GJ,
Waterham HR,
Vrieling EG,
Titorenko VI,
Cregg JM,
Harder W,
Veenhuis M,
( 1995 ) The Hansenula polymorpha PER3 gene is essential for the import of PTS1 proteins into the peroxisomal matrix. PMID : 7615522 : DOI : 10.1074/jbc.270.29.17229 Abstract >>
PER genes are essential for the assembly of peroxisomes in Hansenula polymorpha. Here we describe the PER3 gene which was cloned by functional complementation of a H. polymorpha per3 mutant. The complementing PER3 gene encodes a protein of 569 amino acids (Per3p) with a calculated mass of 63.9 kDa; Per3p belongs to the tetratricopeptide repeat protein family and is located in both the cytosol and the peroxisomal matrix. Remarkably, Per3p does not contain a known targeting signal (PTS1 or PTS2). The PER3 gene product shows similarity to the Saccharomyces cerevisiae Pas10p (40% identity) and the Pichia pastoris Pas8p (55% identity). However, their function apparently cannot be interchanged since the P. pastoris PAS8 gene failed to functionally complement a H. polymorpha per3 disruption mutant. The per3 disruption mutant contained normal but small peroxisomes in which PTS2 proteins (both homologous and heterologous) were imported. Other matrix proteins (in particular PTS1 proteins) resided in the cytosol where they were normally assembled and active. We argue that Per3p is a component of the peroxisomal import machinery and most probably shuttles matrix proteins from the cytosol to the organellar matrix.
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92. |
Nuttley WM,
Szilard RK,
Smith JJ,
Veenhuis M,
Rachubinski RA,
( 1995 ) The PAH2 gene is required for peroxisome assembly in the methylotrophic yeast Hansenula polymorpha and encodes a member of the tetratricopeptide repeat family of proteins. PMID : 7628714 : DOI : 10.1016/0378-1119(95)00230-4 Abstract >>
Peroxisome assembly mutants in the methylotrophic yeast, Hansenula polymorpha, were selected by a novel procedure involving the inability of mutants to use both oleic acid and methanol as carbon sources. These compounds are both metabolized within peroxisomes through two different enzymatic pathways. 15 mutant strains called mut (methanol non-utilizing) were isolated. These strains were assigned to ten genetic complementation groups. Subcellular fractionation analysis showed that peroxisomal matrix enzymes were mislocalized to the cytoplasm in mut strains. Electron microscopy confirmed that the inability of mut strains to grow on oleic acid and methanol was due to defects in peroxisome assembly. Functional complementation of a mutant strain, mut2, with a plasmid library of H. polymorpha genomic DNA sequences has identified a gene, PAH2, that restores growth on methanol and the correct localization of matrix enzymes to the peroxisome. PAH2 encodes Pah2p, a polypeptide of 569 amino acids that is a member of the tetratricopeptide repeat (TPR) family of proteins. Pah2p shows identity with Pas8p and Pas10p, two proteins required for peroxisome assembly in the yeasts Pichia pastoris and Saccharomyces cerevisiae, respectively, and which have been suggested to be receptors that recognize peroxisomal targeting signal-1 (PTS1) motifs.
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93. |
( 1994 ) The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals. PMID : 7962056 : DOI : 10.1083/jcb.127.3.737 PMC : PMC2120227 Abstract >>
We describe the cloning of the Hansenula polymorpha PER1 gene and the characterization of the gene and its product, PER1p. The gene was cloned by functional complementation of a per1 mutant of H. polymorpha, which was impaired in the import of peroxisomal matrix proteins (Pim- phenotype). The DNA sequence of PER1 predicts that PER1p is a polypeptide of 650 amino acids with no significant sequence similarity to other known proteins. PER1 expression was low but significant in wild-type H. polymorpha growing on glucose and increased during growth on any one of a number of substrates which induce peroxisome proliferation. PER1p contains both a carboxy- (PTS1) and an amino-terminal (PTS2) peroxisomal targeting signal which both were demonstrated to be capable of directing bacterial beta-lactamase to the organelle. In wild-type H. polymorpha PER1p is a protein of low abundance which was demonstrated to be localized in the peroxisomal matrix. Our results suggest that the import of PER1p into peroxisomes is a prerequisite for the import of additional matrix proteins and we suggest a regulatory function of PER1p on peroxisomal protein support.
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94. |
Tan X,
Waterham HR,
Veenhuis M,
Cregg JM,
( 1995 ) The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation. PMID : 7844145 : DOI : 10.1083/jcb.128.3.307 PMC : PMC2120355 Abstract >>
We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis. Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene. In either methanol or methylamine medium, conditions that normally induce the organelles, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol. The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD. Analysis of the primary sequence of Per8p revealed a 39-amino-acid cysteine-rich segment with similarity to the C3HC4 family of zinc-finger motifs. Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers. We show that Per8p is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organelles. We propose that Per8p is a component of the molecular machinery that controls the proliferation of this organelle.
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95. |
Avila J,
Pérez MD,
Brito N,
González C,
Siverio JM,
( 1995 ) Cloning and disruption of the YNR1 gene encoding the nitrate reductase apoenzyme of the yeast Hansenula polymorpha. PMID : 7789531 : DOI : 10.1016/0014-5793(95)00511-7 Abstract >>
The nitrate reductase gene (YNR1) from the yeast H. polymorpha was isolated from a lambda EMBL3 genomic DNA library. As probe a 350 bp DNA fragment synthesized by PCR from H. polymorpha cDNA was used. By DNA sequencing an ORF of 2,577 bp was found. The predicted protein has 859 amino acids and presents high identity with nitrate reductases from other organisms. Chromosomal disruption of YNR1 causes inability to grow in nitrate. Northern blot analysis showed that YNR1 expression is induced by nitrate and repressed by ammonium.
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96. |
( 1994 ) Isolation and characterization of the LEU2 gene of Hansenula polymorpha. PMID : 7941737 : DOI : 10.1002/yea.320100410 Abstract >>
A DNA fragment carrying the LEU2 gene of methylotrophic yeast Hansenula polymorpha was isolated by complementation of the leuB mutation of Escherichia coli. The nucleotide sequence of the isolated DNA fragment contains an open reading frame of 363 codons, coding for a protein 80% identical to the LEU2 gene product of Saccharomyces cerevisiae. Further downstream, there is a partial reading frame with no obvious similarity to known proteins. The LEU2 gene of H. polymorpha cannot complement the leu2 mutation of S. cerevisiae.
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97. |
Merckelbach A,
Gödecke S,
Janowicz ZA,
Hollenberg CP,
( 1993 ) Cloning and sequencing of the ura3 locus of the methylotrophic yeast Hansenula polymorpha and its use for the generation of a deletion by gene replacement. PMID : 7764392 : Abstract >>
The ura3 gene of Hansenula polymorpha was cloned, sequenced and used to generate a ura3 mutant from the wild-type strain of this yeast via integrative mutagenesis. The Tn5 neomycin-resistance marker (neo) under control of the ADH1 promoter from Saccharomyces cerevisiae served as a transformation marker. The results show that gene replacement can be achieved in H. polymorpha, a yeast with a high level of non-homologous integration.
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98. |
Reid GA,
( 1988 ) Anthranilate synthase component II from Hansenula polymorpha. PMID : 3399398 : DOI : 10.1093/nar/16.13.6236 PMC : PMC336871 Abstract >>
N/A
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99. |
Plastino J,
Klinman JP,
( 1995 ) Limited proteolysis of Hansenula polymorpha yeast amine oxidase: isolation of a C-terminal fragment containing both a copper and quino-cofactor. PMID : 7556609 : DOI : 10.1016/0014-5793(95)00907-q Abstract >>
Limited proteolysis of recombinant Hansenula polymorpha yeast amino oxidase produces a 48 kDa fragment which corresponds to the C-terminal two-thirds of the protein. The fragment contains both TOPA (2,4,5-trihydroxyphenylalanine) and copper, as well as the histidine ligands implicated in copper binding. The fragment is proposed to be the domain responsible for cofactor production in yeast amine oxidase.
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100. |
Janowicz ZA,
Eckart MR,
Drewke C,
Roggenkamp RO,
Hollenberg CP,
Maat J,
Ledeboer AM,
Visser C,
Verrips CT,
( 1985 ) Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha. PMID : 2987872 : DOI : 10.1093/nar/13.9.3043 PMC : PMC341220 Abstract >>
A gene library from the methanol utilizing yeast Hansenula polymorpha, constructed in a lambda Charon4A vector, was used to clone the gene encoding a key methanol assimilating enzyme, dihydroxyacetone synthase (DHAS) by differential plaque hybridization. The nucleotide sequence of the 2106 bp structural gene and the 5' and 3' non-coding regions was determined. The deduced amino acid sequence of the protein is in agreement with the apparent molecular weight and amino acid composition of the purified protein. The codon bias is not so pronounced as in some Saccharomyces cerevisiae genes.
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101. |
Groves MR,
Schroer CFE,
Middleton AJ,
Lunev S,
Danda N,
Ali AM,
Marrink SJ,
Williams C,
( 2018 ) Structural insights into K48-linked ubiquitin chain formation by the Pex4p-Pex22p complex. PMID : 29288668 : DOI : 10.1016/j.bbrc.2017.12.150 DOI : 10.1016/j.bbrc.2017.12.150 Abstract >>
Pex4p is a peroxisomal E2 involved in ubiquitinating the conserved cysteine residue of the cycling receptor protein Pex5p. Previously, we demonstrated that Pex4p from the yeast Saccharomyces cerevisiae binds directly to the peroxisomal membrane protein Pex22p and that this interaction is vital for receptor ubiquitination. In addition, Pex22p binding allows Pex4p to specifically produce lysine 48 linked ubiquitin chains in vitro through an unknown mechanism. This activity is likely to play a role in targeting peroxisomal proteins for proteasomal degradation. Here we present the crystal structures of Pex4p alone and in complex with Pex22p from the yeast Hansenula polymorpha. Comparison of the two structures demonstrates significant differences to the active site of Pex4p upon Pex22p binding while molecular dynamics simulations suggest that Pex22p binding facilitates active site remodelling of Pex4p through an allosteric mechanism. Taken together, our data provide insights into how Pex22p binding allows Pex4p to build K48-linked Ub chains.
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102. |
Monastyrska I,
van der Heide M,
Krikken AM,
Kiel JA,
van der Klei IJ,
Veenhuis M,
( 2005 ) Atg8 is essential for macropexophagy in Hansenula polymorpha. PMID : 15569246 : DOI : 10.1111/j.1600-0854.2004.00252.x Abstract >>
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103. |
( N/A ) Cloning and sequencing of the malate synthase gene from Hansenula polymorpha. PMID : 2349836 : DOI : 10.1002/yea.320060309 Abstract >>
We have cloned the MAS gene, encoding the microbody matrix enzyme malate synthase (EC 4.1.3.2.) from the methylotrophic yeast Hansenula polymorpha. The gene was isolated by screening of a genomic library with a mixed-sequence probe, based on the partial amino acid sequence of the purified enzyme. The nucleotide sequence of a 2.4-kilobase stretch of DNA covering the MAS gene was determined. The gene contains an open reading frame of 555 amino acids, amounting to a calculated molecular mass of 63,254 for the encoded protein. Comparison of the amino acid sequence with the malate synthase sequences of Escherichia coli, Brassica napus L. and Cucumis sativus L. clearly establishes the homology of all four proteins. Compared to the soluble enzyme from E. coli, the malate synthases from H. polymorpha and both plant species, which are located in the microbodies, have a short carboxy-terminal extension. In the plant malate synthases, the extension is probably involved in routing to the microbodies, since it contains the potential peroxisomal targeting signal, Ser-Arg/Lys-Leu, at the carboxy terminus. The H. polymorpha enzyme terminates with similar amino acids, but their sequence, Ser-Leu-Lys, does not conform to any of the known peroxisomal targeting signals.
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104. |
( 2013 ) Relationships among genera of the Saccharomycotina (Ascomycota) from multigene phylogenetic analysis of type species. PMID : 22978764 : DOI : 10.1111/1567-1364.12006 Abstract >>
Relationships among ascomycetous yeast genera (subphylum Saccharomycotina, phylum Ascomycota) have been uncertain. In the present study, type species of 70 currently recognized genera are compared from divergence in the nearly entire nuclear gene sequences for large subunit rRNA, small subunit (SSU) rRNA, translation elongation factor-1�\, and RNA polymerase II, subunits 1 (RPB1) and 2 (RPB2). The analysis substantiates earlier proposals that all known ascomycetous yeast genera now assigned to the Saccharomycotina represent a single clade. Maximum likelihood analysis resolved the taxa into eight large multigenus clades and four-one- and two-genus clades. Maximum parsimony and neighbor-joining analyses gave similar results. Genera of the family Saccharomycetaceae remain as one large clade as previously demonstrated, to which the genus Cyniclomyces is now assigned. Pichia, Saturnispora, Kregervanrija, Dekkera, Ogataea and Ambrosiozyma are members of a single large clade, which is separate from the clade that includes Barnettozyma, Cyberlindnera, Phaffomyces, Starmera and Wickerhamomyces. Other clades include Kodamaea, Metschnikowia, Debaryomyces, Cephaloascus and related genera, which are separate from the clade that includes Zygoascus, Trichomonascus, Yarrowia and others. This study once again demonstrates that there is limited congruence between a system of classification based on phenotype and a system determined from DNA sequences.
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105. |
( 1998 ) The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a novel protein, Pak1p, involved in peroxisome integrity. PMID : 9683670 : Abstract >>
The Hansenula polymorpha per6-210 mutant is impaired in respect of growth on methanol (Mut-) and is characterized by aberrant peroxisome formation. The functionally complementing DNA fragment contains two open reading frames. The first encodes dihydroxyacetone kinase (DAK), a cytosolic enzyme essential for formaldehyde assimilation; the second ORF codes for a novel protein (Pak1p). We have demonstrated that per6-210 cells lack DAK activity, causing the Mut- phenotype, and have strongly reduced levels of Pak1p, resulting in peroxisomal defects. Sequence analysis revealed that per6-210 contains a mutation in the 3' end of the DAK coding region, which overlaps with the promoter region of PAK1. Possibly this mutation also negatively affects PAK1 expression.
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106. |
( 1998 ) Cloning and characterization of the gene encoding a repressible acid phosphatase (PHO1) from the methylotrophic yeast Hansenula polymorpha. PMID : 9720203 : Abstract >>
A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M(r) of 49400. The encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.
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107. |
( 1998 ) Copper amine oxidase from Hansenula polymorpha: the crystal structure determined at 2.4 A resolution reveals the active conformation. PMID : 9551552 : Abstract >>
Copper-containing amine oxidases (CAOs) are widespread in nature. These enzymes oxidize primary amine substrates to the aldehyde product, reducing molecular oxygen to hydrogen peroxide in the process. CAOs contain one type 2 copper atom and topaquinone (TPQ), a modified tyrosine sidechain utilized as a redox cofactor. The methylamine oxidase from the yeast Hansenula polymorpha (HPAO) is an isoform of CAO with a preference for small aliphatic amine or phenethylamine substrates. The enzyme is dimeric with a subunit molecular weight of 78 kDa. Structural studies are directed at understanding the basis for cofactor biogenesis and catalytic efficiency. The X-ray crystal structure of HPAO has been solved at 2.4 A resolution by a combination of molecular replacement and single isomorphous replacement followed by refinement using sixfold symmetry averaging. The electron density at the catalytic site shows that the TPQ conformation corresponds to that of the active form of the enzyme. Two channels, one on either side of TPQ, are observed in the structure that provide access between the active site and the bulk solvent. The structure shows TPQ in a position poised for catalysis. This is the first active CAO structure to reveal this conformation and may help further our understanding of the catalytic mechanism. On the substrate side of TPQ a water-containing channel leading to the protein surface can serve as an entrance or exit for substrate and product. On the opposite side of TPQ there is direct access from the bulk solvent of the dimer interface by which molecular oxygen may enter and hydrogen peroxide depart. In addition, a network of conserved water molecules has been identified which may function in the catalytic mechanism.
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108. |
( 1998 ) Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae PMR1 gene. PMID : 9791894 : DOI : 10.1002/(SICI)1097-0061(19980930)14:13<1233::AID-YEA322>3.0.CO;2-Y Abstract >>
A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPase. The cloned gene product exhibits 60.3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevesiae PMR1 gene product, a P-type Ca(2+)-ATPase.
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109. |
( 1998 ) The ubiquitin-conjugating enzyme Pex4p of Hansenula polymorpha is required for efficient functioning of the PTS1 import machinery. PMID : 9649431 : DOI : 10.1093/emboj/17.13.3608 PMC : PMC1170697 Abstract >>
We have cloned the Hansenula polymorpha PEX4 gene by functional complementation of a peroxisome-deficient mutant. The PEX4 translation product, Pex4p, is a member of the ubiquitin-conjugating enzyme family. In H.polymorpha, Pex4p is a constitutive, low abundance protein. Both the original mutant and the pex4 deletion strain (Deltapex4) showed a specific defect in import of peroxisomal matrix proteins containing a C-terminal targeting signal (PTS1) and of malate synthase, whose targeting signal is not yet known. Import of the PTS2 protein amine oxidase and the insertion of the peroxisomal membrane proteins Pex3p and Pex14p was not disturbed in Deltapex4 cells. The PTS1 protein import defect in Deltapex4 cells could be suppressed by overproduction of the PTS1 receptor, Pex5p, in a dose-response related manner. In such cells, Pex5p is localized in the cytosol and in peroxisomes. The peroxisome-bound Pex5p specifically accumulated at the inner surface of the peroxisomal membrane and thus differed from Pex5p in wild-type peroxisomes, which is localized throughout the matrix. We hypothesize that in H. polymorpha Pex4p plays an essential role for normal functioning of Pex5p, possibly in mediating recycling of Pex5p from the peroxisome to the cytosol.
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