| 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. |
Mukaiyama H,
Baba M,
Osumi M,
Aoyagi S,
Kato N,
Ohsumi Y,
Sakai Y,
( 2004 ) Modification of a ubiquitin-like protein Paz2 conducted micropexophagy through formation of a novel membrane structure. PMID : 13679515 : DOI : 10.1091/mbc.e03-05-0340 PMC : PMC307527 Abstract >>
Microautophagy is a versatile process in which vacuolar or lysosomal membranes directly sequester cytosolic targets for degradation. Recent genetic evidence suggested that microautophagy uses molecular machineries essential for macroautophagy, but the details of this process are still unknown. In this study, a ubiquitin-like protein Paz2 essential for micropexophagy in the yeast Pichia pastoris has been shown to receive modification through the function of Paz8 and Gsa7, yielding a modified form Paz2-I, similar to the ubiquitin-like lipidation of Aut7 that is essential for macroautophagy in Saccharomyces cerevisiae. We identified a novel membrane structure formed after the onset of micropexophagy, which we suggest is necessary for the sequestration of peroxisomes by the vacuole. Assembly of this newly formed membrane structure, which is followed by localization of Paz2 to it, was found to require a properly functioning Paz2-modification system. We herein show that Paz2 and its modification system conduct micropexophagy through formation of the membrane structure, which explains the convergence between micropexophagy and macroautophagy with regard to de novo membrane formation.
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3. |
Liu YY,
Woo JH,
Neville DM,
( 2003 ) Targeted introduction of a diphtheria toxin resistant mutation into the chromosomal EF-2 locus of Pichia pastoris and expression of immunotoxin in the EF-2 mutants. PMID : 12880776 : Abstract >>
In an attempt to increase the production of a diphtheria toxin (DT) based immunotoxin by Pichia pastoris, we have created DT-resistant mutants that contain a substitution of arginine for glycine at position 701 in elongation factor 2 (EF-2). To achieve this, we first cloned and characterized the EF-2 gene (PEF1), and then made a construct pBLURA-Delta5'mutEF-2 that efficiently introduces specific mutations into the chromosomal EF-2 gene in P. pastoris by in vivo homologous recombination. pBLURA-Delta5(')mutEF-2 contains a selection marker URA3 and a 5' truncated form of the P. pastoris PEF1 that had been modified in vitro to carry the nucleotide mutations for the Gly(701) to Arg transition. Unlike the non-mutated strains, the EF-2 mutants are resistant to high-level intracellular expression of DT A chain that can catalyze the ADP-ribosylation. When used to express the secreted bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), the EF-2 mutant strains showed increased viability compared to the non-mutated strains. However, they did not show an advantage over the non-mutated expressing strain in the production of the immunotoxin. Western blotting analysis revealed that although the EF-2 mutants did not increase the accumulation of intact A-dmDT390-bisFv(G4S) in the culture medium, they generated larger amounts of degraded products found in both the medium and cell pellets compared to the non-mutant expressing clone. In addition, double copy expression resulted in greater amounts of intact immunotoxin being retained within cellular compartments as well as degraded products. Based on these findings, we suggest that the secretory capacity may be rate limiting for divalent immunotoxin production in P. pastoris.
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4. |
Edelmann A,
Bär J,
( 2002 ) Molecular genetics of 6-phosphofructokinase in Pichia pastoris. PMID : 12125051 : DOI : 10.1002/yea.889 Abstract >>
Previously, studies on glucose-induced microautophagy in the methylotrophic yeast Pichia pastoris provided evidence that the glucose-induced selective autophagy-1-protein is the alpha-subunit of 6-phosphofructokinase (Pfk), a key enzyme in the glycolytic pathway. In our work, we could clearly demonstrate that two types of subunits of Pfk exist in P. pastoris. Investigating the yeast cell-free extract by Western blot analysis, two distinct signals of Pfk were obtained. In addition, we isolated a DNA sequence containing the complete ORF of PpPFK2 encoding the beta-subunit of Pfk from P. pastoris with a deduced molecular mass of 103.7 kDa. On the basis of these results, a hetero-oligomeric structure of Pfk in P. pastoris became obvious. Because the molecular and kinetic properties of a homo-oligomeric yeast Pfk appear to be more similar to those of mammalian Pfk, as described in the literature, our results are of interest for the growing number of studies on P. pastoris as a heterologous production system. Furthermore, the 3'- and 5'-non-coding regions of PpPFK2 were isolated and several putative binding sites for regulatory factors could be identified in the promoter region.
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5. |
Kirchberger J,
Bär J,
Schellenberger W,
Dihazi H,
Kopperschläger G,
( 2002 ) 6-phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme. PMID : 12125050 : DOI : 10.1002/yea.885 Abstract >>
6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower. Aqueous two-phase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species. The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein.
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6. |
Mukaiyama H,
Oku M,
Baba M,
Samizo T,
Hammond AT,
Glick BS,
Kato N,
Sakai Y,
( 2002 ) Paz2 and 13 other PAZ gene products regulate vacuolar engulfment of peroxisomes during micropexophagy. PMID : 11856375 : Abstract >>
In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded through direct engulfment by the vacuole in a process known as micropexophagy, but the mechanism of micropexophagy is not known. To gain molecular insights into micropexophagy, we used fluorescence time-lapse microscopy, coupled with gene-tagging mutagenesis to isolate P. pastoris mutants defective in micropexophagy. The relevant genes have been designated PAZ genes. Morphological and genetic analyses enabled us to postulate a schematic model for micropexophagy. This new model invokes the generation of new vacuolar compartments as an intermediate structure during micropexophagy. Different classes of paz mutants arrest micropexophagy at distinct stages of the process. Most of APG-related paz mutants ceased micropexophagy at Stage 1c and that GCN-family paz mutants ceased micropexophagy at Stage 2. The paz2Delta strain shows a unique phenotype. Paz2 is the homologue of Saccharomyces cerevisiae Apg8, which is necessary for macroautophagy in that yeast. Our analysis revealed that in P. pastoris, Paz2 plays a key role in repressing the engulfment of peroxisomes by the vacuole before the onset of micropexophagy. Paz2 is proteolytically processed by another autophagy-related Paz protein Paz8, but this processing is not required for the ability of Paz2 to suppress aberrant micropexophagy. Micropexophagy has been dissected into a multistep reaction that involves 14 identified Paz gene products. Our studies indicate that Paz2 controls the engulfment of peroxisomes by the vacuole, pointing to a novel early function of this protein.
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7. |
Strømhaug PE,
Bevan A,
Dunn WA,
( 2001 ) GSA11 encodes a unique 208-kDa protein required for pexophagy and autophagy in Pichia pastoris. PMID : 11533052 : DOI : 10.1074/jbc.M104087200 Abstract >>
Cells are capable of adapting to changes in their environment by synthesizing needed proteins and degrading superfluous ones. Pichia pastoris synthesizes peroxisomal enzymes to grow in methanol medium. Upon adapting from methanol medium to one containing glucose, this yeast rapidly and selectively degrades peroxisomes by an autophagic process referred to as pexophagy. In this study, we have utilized a novel approach to identify genes required for this degradative pathway. Our approach involves the random integration of a vector containing the Zeocin resistance gene into the yeast genome by restriction enzyme-mediated integration. Cells unable to degrade peroxisomes during glucose adaptation were isolated, and the genes that were disrupted by the insertion of the vector were determined by sequencing. By using this approach, we have identified a number of genes required for glucose-induced selective autophagy of peroxisomes (GSA genes). We report here the characterization of Gsa11, a unique 208-kDa protein. We found that this protein is required for glucose-induced pexophagy and starvation-induced autophagy. Gsa11 is a cytosolic protein that becomes associated with one or more structures situated near the vacuole during glucose adaptation. The punctate localization of Gsa11 was not observed in gsa10, gsa12, gsa14, and gsa19 mutants. We have previously shown that Gsa9 appears to relocate from a compartment at the vacuole surface to regions between the vacuole and the peroxisomes being sequestered. In the gsa11 mutants, the vacuole only partially surrounded the peroxisomes, but Gsa9 was still distributed around the peroxisome cluster. This suggests that Gsa9 binds to the peroxisomes independent of the vacuole. The data also indicate that Gsa11 is not necessary for Gsa9 to interact with peroxisomes but acts at an intermediate event required for the vacuole to engulf the peroxisomes.
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8. |
Paronetto MP,
Miele R,
Maugliani A,
Borro M,
Bonaccorsi di Patti MC,
( 2001 ) Cloning of Pichia pastoris Fet3: insights into the high affinity iron uptake system. PMID : 11469807 : DOI : 10.1006/abbi.2001.2425 Abstract >>
High-affinity iron uptake by yeast cells appears to require the presence of a complex formed on the plasma membrane by the multicopper oxidase Fet3 and the permease Ftr1 which work together to allow iron to enter safely inside the cell. The Pichia pastoris ferroxidase Fet3 has been cloned and it has been found to display high sequence similarity to other yeast multicopper oxidases, including all the predicted ligands for the catalytic copper atoms and for the iron substrate. P. pastoris appears to possess a high-affinity iron uptake system similar to that of S. cerevisiae, as far as regulation of expression is concerned. However, the P. pastoris high-affinity iron uptake system presents a K(m) value for iron almost ten times higher than that of S. cerevisiae, possibly to control iron fluxes over a wider range of concentrations of this metal, in order to avoid toxic iron overloading.
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9. |
Brummer MH,
Richard P,
Sundqvist L,
Väänänen R,
Keränen S,
( 2001 ) The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae. PMID : 11447595 : DOI : 10.1002/yea.736 Abstract >>
The nucleotide sequences of 2.8 kb and 2.9 kb fragments containing the Kluyveromyces lactis and Pichia pastoris GDI1 genes, respectively, were determined. K. lactis GDI1 was found during sequencing of a genomic library clone, whereas the P. pastoris GDI1 was obtained from a genomic library by complementing a Saccharomyces cerevisiae sec19-1 mutant strain. The sequenced DNA fragments contain open reading frames of 1338 bp (K.lactis) and 1344 bp (P. pastoris), coding for polypeptides of 445 and 447 residues, respectively. Both sequences fully complement the S. cerevisiae sec19-1 mutation. They have high degrees of homology with known GDP dissociation inhibitors from yeast species and other eukaryotes.
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10. |
Warsame A,
Vad R,
Kristensen T,
Oyen TB,
( 2001 ) Characterization of a gene encoding a Pichia pastoris protein disulfide isomerase. PMID : 11243858 : DOI : 10.1006/bbrc.2001.4479 Abstract >>
Protein disulphide isomerases belong to the thioredoxin superfamily of protein-thiol oxidoreductases that have two double-cysteine redox-active sites and take part in protein folding in the endoplasmic reticulum (ER). We report here the cloning of a Pichia pastoris genomic DNA fragment (2919 bp) that encodes the full length of a protein disulphide isomerase (PpPDI). The deduced amino acid sequence of PDI consists of 517 residues and carries the two characteristic PDI-type redox-active domains -CGHC-, separated by 338 residues, and two potential N-glycosylation sites. The N-terminal end forms a putative signal sequence, and an acidic C-terminal region represents a possible calcium-binding domain. Together with the -HDEL ER retrieval sequence at the C-terminus, these features indicate that the gene encodes a redox-active ER-resident protein disulphide isomerase. The nucleotide sequence, which also contains two other open reading frames, has been submitted to the EMBL Nucleotide Sequence Database, Accession No. AJ302014.
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11. |
Lin Cereghino GP,
Lin Cereghino J,
Sunga AJ,
Johnson MA,
Lim M,
Gleeson MA,
Cregg JM,
( 2001 ) New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris. PMID : 11223254 : DOI : 10.1016/s0378-1119(00)00576-x Abstract >>
We describe the isolation and characterization of three new biosynthetic genes-ARG4, ADE1, and URA3-from the methylotrophic yeast Pichia pastoris. The predicted products of the genes share significant sequence similarity to their Saccharomyces cerevisiae counterparts, namely argininosuccinate lyase, PR-aminoimidazolesuccinocarboxamide synthase, and orotidine-5'-phosphate decarboxylase, respectively. Along with the previously described HIS4 gene, each gene was incorporated as the yeast selectable marker into a set of shuttle vectors designed to express foreign genes in P. pastoris. In addition, we have constructed a series of host strains containing all possible combinations of ade1, arg4, his4, and ura3 auxotrophies to be used with these new vectors.
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12. |
Kim J,
Kamada Y,
Stromhaug PE,
Guan J,
Hefner-Gravink A,
Baba M,
Scott SV,
Ohsumi Y,
Dunn WA,
Klionsky DJ,
( 2001 ) Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole. PMID : 11309418 : DOI : 10.1083/jcb.153.2.381 PMC : PMC2169458 Abstract >>
Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.
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13. |
Johnson MA,
Snyder WB,
Cereghino JL,
Veenhuis M,
Subramani S,
Cregg JM,
( 2001 ) Pichia pastoris Pex14p, a phosphorylated peroxisomal membrane protein, is part of a PTS-receptor docking complex and interacts with many peroxins. PMID : 11329173 : DOI : 10.1002/yea.711 Abstract >>
The peroxisomal protein import machinery plays a central role in the assembly of this organelle in all eukaryotes. Genes encoding components of this machinery, termed peroxins or Pex proteins, have been isolated and characterized in several yeast species and in mammals, including humans. Here we report on one of these components, Pex14p, from the methylotrophic yeast Pichia pastoris. Work in other organisms has shown that Pex14p is located on the cytoplasmic surface of the peroxisomal membrane and binds peroxisomal targeting signal (PTS) receptors carrying proteins bound for the peroxisomal matrix, results that have led to the hypothesis that Pex14p is a receptor-docking protein. P. pastoris Pex14p (PpPex14p) behaves like an integral membrane protein, with its C-terminus exposed on the cytosolic side of the peroxisomal membrane. PpPex14p complexes with many peroxins, including Pex3p (Snyder et al., 1999b), Pex5p, Pex7p, Pex13p, Pex17p, itself, and a previously unreported peroxin, Pex8p. A portion of Pex14p is phosphorylated, but both phosphorylated and unphosphorylated forms of Pex14p interact with several peroxins. The interactions between Pex14p and other peroxins provide clues regarding the function of Pex14p in peroxisomal protein import.
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14. |
Kucha JA,
Dooley DM,
( 2001 ) Cloning, sequence analysis, and characterization of the 'lysyl oxidase' from Pichia pastoris. PMID : 11237259 : Abstract >>
Lysyl oxidase from Pichia pastoris has been successfully overexpressed. EPR and resonance Raman experiments have shown that copper and TPQ are present, respectively. Lysyl oxidase from P. pastoris has a similar substrate specificity to the mammalian enzyme (both have been shown to oxidize peptidyl lysine residues) and is 30% identical to the human kidney diamine oxidase (the highest of any non-mammalian source). This enzyme also has a relatively broad substrate specificity compared to other amine oxidases. Molecular modeling data suggest that the substrate channel in lysyl oxidase from P. pastoris permits greater active site access than observed in structurally-characterized amine oxidases. This larger channel may account for the diversity of substrates that are turned over by this enzyme.
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15. |
Huynh TT,
Vad R,
Kristensen T,
Oyen TB,
( 2001 ) The genes of two G-proteins involved in protein transport in Pichia pastoris. PMID : 11162538 : DOI : 10.1006/bbrc.2000.4138 Abstract >>
Members of the Rab protein family play essential roles in vesicle fusion during protein secretion and represent highly conserved GTP binding proteins. The Saccharomyces cerevisiae Sec4p and Ypt1p, promoting vesicle fusion at the plasma membrane and in ER-Golgi transport, respectively, are among the best characterised yeast members. We have here cloned the Pichia pastoris SEC4 homologue using a S. cerevisiae SEC4 probe. In addition we isolated a crosshybridising clone encoding another Rab-/Ypt-like protein. The deduced full-length PpSec4p comprises 204 amino acid residues with an over all identity of 64% to the Sec4p from S. cerevisiae and 72% to the Candida albicans Sec4p. The YPT-like gene encodes a 216 amino acid residue protein showing highest similarity to the S. cerevisiae Ypt10p and Ypt53p. Both PpSec4p and the Ypt-like protein carry a -Cys-Cys C-terminus, indicating that these proteins are targets for geranyl-geranylation by a type II prenyltransferase.
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16. |
Bevis BJ,
Soderholm J,
Fu D,
Kaiser CA,
Payne WE,
( 2000 ) Isolation of Pichia pastoris genes involved in ER-to-Golgi transport. PMID : 10923020 : DOI : 10.1002/1097-0061(200008)16:11<979::AID-YEA594>3.0.CO;2-C Abstract >>
Pichia pastoris has discrete transitional ER sites and coherent Golgi stacks, making this yeast an ideal system for studying the organization of the early secretory pathway. To provide molecular tools for this endeavour, we isolated P. pastoris homologues of the SEC12, SEC13, SEC17, SEC18 and SAR1 genes. The P. pastoris SEC12, SEC13, SEC17 and SEC18 genes were shown to complement the corresponding S. cerevisiae mutants. The SEC17 and SAR1 genes contain introns at the same relative positions in both P. pastoris and S. cerevisiae, whereas the SEC13 gene contains an intron in P. pastoris but not in S. cerevisiae. Intron structure is similar in the two yeasts, although the favoured 5' splice sequence appears to be GTAAGT in P. pastoris vs. GTATGT in S. cerevisiae. The predicted amino acid sequences of Sec13p, Sec17p, Sec18p and Sar1p show strong conservation in the two yeasts. By contrast, the predicted lumenal domain of Sec12p is much larger in P. pastoris, suggesting that this domain may help localize Sec12p to transitional ER sites. A comparison of the SEC12 loci in various budding yeasts indicates that the SEC12-related gene SED4 is probably unique to the Saccharomyces lineage.
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17. |
Ingavale SS,
Sharma KG,
Bachhawat AK,
( 2000 ) Construction of fission yeast vectors with a novel selection strategy that allows their use in wild-type fission yeasts. PMID : 11015731 : DOI : 10.1002/1097-0061(200010)16:14<1345::AID-YEA628>3.0.CO;2-9 Abstract >>
Novel vectors that use the Pichia pastoris INO1 gene as a selectable marker and exploit the natural inositol auxotrophy of the fission yeast are described. These plasmids also contained other features desirable in a plasmid cloning vector. These plasmids were evaluated in other species of Schizosaccharomyces and found to replicate autonomously in another variety of S. pombe, S. pombe var. malidevorans. These plasmids can be used for transformation of any wild-type S. pombe strain without the need for selection by induced auxotrophic mutations, or by selection by drug resistance markers, and should greatly assist genetic and molecular manipulations in these yeasts.
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18. |
van der Klei IJ,
Bellu AR,
Shen S,
Stasyk OV,
( 1999 ) A Pichia pastoris VPS15 homologue is required in selective peroxisome autophagy. PMID : 10591966 : Abstract >>
Methylotrophic yeasts contain large peroxisomes during growth on methanol. Upon exposure to excess glucose or ethanol these organelles are selectively degraded by autophagy. Here we describe the cloning of a Pichia pastoris gene (PpVPS15) involved in peroxisome degradation, which is homologous to Saccharomyces cerevisiae VPS15. In methanol-grown cells of a P. pastoris VPS15 deletion strain, the levels of peroxisomal marker enzymes remained high after addition of excess glucose or ethanol. Electron microscopic studies revealed that the organelles were not taken up by vacuoles, suggesting that PpVPS15 is required at an early stage in peroxisome degradation.
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19. |
Cregg JM,
Rangell L,
Johnson MA,
Choy AJ,
Koller A,
Snyder WB,
( 1999 ) Pex17p is required for import of both peroxisome membrane and lumenal proteins and interacts with Pex19p and the peroxisome targeting signal-receptor docking complex in Pichia pastoris. PMID : 10588639 : DOI : 10.1091/mbc.10.12.4005 PMC : PMC25739 Abstract >>
Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Delta mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Delta mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal-receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs.
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20. |
Chien P,
Osherovich LZ,
Santoso A,
( 2000 ) Molecular basis of a yeast prion species barrier. PMID : 10660050 : DOI : 10.1016/s0092-8674(00)81565-2 Abstract >>
The yeast [PSI+] factor is inherited by a prion mechanism involving self-propagating Sup35p aggregates. We find that Sup35p prion function is conserved among distantly related yeasts. As with mammalian prions, a species barrier inhibits prion induction between Sup35p from different yeast species. This barrier is faithfully reproduced in vitro where, remarkably, ongoing polymerization of one Sup35p species does not affect conversion of another. Chimeric analysis identifies a short domain sufficient to allow foreign Sup35p to cross this barrier. These observations argue that the species barrier results from specificity in the growing aggregate, mediated by a well-defined epitope on the amyloid surface and, together with our identification of a novel yeast prion domain, show that multiple prion-based heritable states can propagate independently within one cell.
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21. |
Spong AP,
Lüers GH,
Subramani S,
( 1999 ) Analysis of the peroxisomal acyl-CoA oxidase gene product from Pichia pastoris and determination of its targeting signal. PMID : 10455228 : DOI : 10.1002/(SICI)1097-0061(199908)15:11<1035::AID-YEA432>3.0.CO;2-1 Abstract >>
Acyl-CoA oxidase (Pox1p) is involved in the beta-oxidation of fatty acids and is targeted to the peroxisomal matrix via the use of different signals in various organisms. In rat, mouse and human, Pox1p contains a canonical peroxisomal targeting signal 1 (PTS1), whereas in the yeasts Candida tropicalis, Saccharomyces cerevisiae, C. maltosa and Yarrowia lipolytica neither a PTS1 nor a PTS2 sequence is present, suggesting that Pox1p might be targeted to the peroxisomes via a third unknown pathway. Alternatively, since proteins lacking a PTS sequence can enter peroxisomes in association with other polypeptides containing a PTS, Pox1p might 'piggy-back' its way into the peroxisomal matrix together with other proteins. To understand the mechanism of peroxisomal targeting of a yeast Pox1p, we cloned the Pichia pastoris POX1 gene to study the pathway of import of PpPox1p into peroxisomes. The gene was cloned by PCR, hybridization and plasmid rescue. The 2157 bp gene encodes a protein with a predicted molecular weight of 80 kDa. Antisera against PpPox1p detected a protein specifically induced on oleate with an apparent molecular weight of 72 kDa. Immunolocalization studies confirmed the peroxisomal localization of PpPox1p. The carboxy-terminus of PpPox1p ends with a PTS1-like sequence, APKI. The sequence PKI was necessary for transport of PpPox1p into peroxisomes and interacted with the PTS1 receptor, Pex5p. Furthermore, addition of the sequence APKI to the C-terminus of the green fluorescent protein directed this fusion protein to the peroxisome. Therefore, PpPox1p uses the PTS1 pathway for its import into peroxisomes.
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22. |
Keller GA,
Rangell L,
Wenzel TJ,
Snyder WB,
Faber KN,
( 1999 ) Pex22p of Pichia pastoris, essential for peroxisomal matrix protein import, anchors the ubiquitin-conjugating enzyme, Pex4p, on the peroxisomal membrane. PMID : 10402463 : DOI : 10.1083/jcb.146.1.99 PMC : PMC2199742 Abstract >>
We isolated a Pichia pastoris mutant that was unable to grow on the peroxisome-requiring media, methanol and oleate. Cloning the gene by complementation revealed that the encoded protein, Pex22p, is a new peroxin. A Deltapex22 strain does not grow on methanol or oleate and is unable to import peroxisomal matrix proteins. However, this strain targets peroxisomal membrane proteins to membranes, most likely peroxisomal remnants, detectable by fluorescence and electron microscopy. Pex22p, composed of 187 amino acids, is an integral peroxisomal membrane protein with its NH2 terminus in the matrix and its COOH terminus in the cytosol. It contains a 25-amino acid peroxisome membrane-targeting signal at its NH2 terminus. Pex22p interacts with the ubiquitin-conjugating enzyme Pex4p, a peripheral peroxisomal membrane protein, in vivo, and in a yeast two-hybrid experiment. Pex22p is required for the peroxisomal localization of Pex4p and in strains lacking Pex22p, the Pex4p is cytosolic and unstable. Therefore, Pex22p anchors Pex4p at the peroxisomal membrane. Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis. The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.
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23. |
Bachhawat AK,
( 1999 ) Restoration of inositol prototrophy in the fission yeast Schizosaccharomyces pombe. PMID : 10463156 : DOI : 10.1099/13500872-145-8-1903 Abstract >>
The biosynthesis of inositol requires only two enzymes, inositol-1-phosphate synthase (encoded by INO1) and an inositol monophosphatase, but the regulation of inositol biosynthesis is under multiple controls and is exquisitely regulated. In the budding yeast Saccharomyces cerevisiae, mutations in any of 26 different genes lead to inositol auxotrophy. The fission yeast Schizosaccharomyces pombe, however, is a natural inositol auxotroph. An investigation has been initiated to examine the possible reasons that might have led to inositol auxotrophy in Sch. pombe. Complementation with a genomic library of an inositol prototrophic yeast indicated that a Pichia pastoris INO1 gene alone could confer inositol prototrophy to Sch. pombe and that the gene was absent in Sch. pombe. To investigate possible reasons for the loss of INO1 gene in Sch. pombe, an attempt was made to disrupt inositol homeostasis in Sch. pombe by overproduction of intracellular inositol, but this did not lead to any discernible adverse effects. The sources of inositol in the natural environment of Sch. pombe were also examined. As the natural environment of Sch. pombe contains significant amounts of phytic acid (inositol hexaphosphate), an investigation was carried out and it was discovered that Sch. pombe can utilize phytic acid as a source of inositol under very specific conditions.
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24. |
Keller GA,
Rangell L,
Lüers GH,
Koller A,
Faber KN,
Wenzel TJ,
( 1999 ) Pex19p interacts with Pex3p and Pex10p and is essential for peroxisome biogenesis in Pichia pastoris. PMID : 10359594 : DOI : 10.1091/mbc.10.6.1745 PMC : PMC25367 Abstract >>
We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective in pex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity to Saccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans and Schizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses of pex19Delta cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of peroxisome biogenesis.
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25. |
Shoumura Y,
( 1999 ) Detection and analysis of translation elongation factor 3 genes from various yeasts. PMID : 10361693 : DOI : 10.1271/bbb.63.769 Abstract >>
Yeast translation requires a unique elongation factor, EF-3. However, information about EF-3 genes has been limited to only a few yeast species. Here, we developed a PCR-based system to detect the EF-3 genes specifically, and identified EF-3 gene fragments from various yeast species in which EF-3 genes have not yet been found.
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26. |
Folkman J,
Pirie-Shepherd S,
Trinh LB,
Shiloach J,
( 1999 ) Disruption of the KEX1 gene in Pichia pastoris allows expression of full-length murine and human endostatin. PMID : 10341419 : DOI : 10.1002/(SICI)1097-0061(199905)15:7<563::AID-YEA398>3.0.CO;2-R Abstract >>
Endostatin is a potent angiogenesis inhibitor. In order to isolate sufficient quantities of soluble protein for in vivo studies in mice, we expressed murine endostatin in Pichia pastoris. Analysis of the expressed protein by mass spectrometry indicated that the protein was truncated. N-terminal sequence analysis determined that the N-terminus was intact, suggesting that the C-terminal lysine was missing. In Saccharomyces cerevisiae, Kex1p can cleave lysine and arginine residues from the C-terminus of peptides and proteins. We hypothesized that the KEX1 homologue in P. pastoris is responsible for the loss of the C-terminal lysine of endostatin. To test this hypothesis, we cloned and disrupted the P. pastoris KEX1 gene. Although the overall amino acid identity between the P. pastoris and the S. cerevisae Kex1p is only 36%, the amino acid residues involved in the catalytic activity or close to the active residues are highly conserved. Disruption of the KEX1 reading frame allowed expression of murine and human endostatin with the C-terminal lysine. The KEX1 disruption strain may be a useful tool for the expression of other proteins with a C-terminal basic amino acid. Addition of a lysine to the C-terminus of recombinant proteins may protect the C-terminus from degradation by other carboxypeptidases.
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27. |
Yuan W,
Stromhaug PE,
( 1999 ) Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein. PMID : 10233149 : DOI : 10.1091/mbc.10.5.1353 PMC : PMC25277 Abstract >>
Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Delta had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(DeltaATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25-30 kDa protein. This conjugate was not observed in cells expressing Gsa7(DeltaATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.
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28. |
Tanneberger K,
Kirchberger J,
Bär J,
Schellenberger W,
Rothemund S,
Kamprad M,
Otto H,
Schöneberg T,
Edelmann A,
( 2007 ) A novel form of 6-phosphofructokinase. Identification and functional relevance of a third type of subunit in Pichia pastoris. PMID : 17522059 : DOI : 10.1074/jbc.M611547200 Abstract >>
Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of alpha subunits and alpha/beta subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, alpha, beta, and gamma. The sequence of the gamma subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the gamma subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the gamma subunit is not required for enzymatic activity, the gamma subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and gamma subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the gamma subunit. Therefore, we suggest that the gamma subunit contributes to adaptation of P. pastoris to energy resources.
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29. |
Kern A,
Hartner FS,
Freigassner M,
Spielhofer J,
Rumpf C,
Leitner L,
Fröhlich KU,
Glieder A,
( 2007 ) Pichia pastoris "just in time" alternative respiration. PMID : 17379734 : DOI : 10.1099/mic.0.2006/001404-0 Abstract >>
Alternative oxidases (Aox or Aod) are present in the mitochondria of plants, fungi and many types of yeast. These enzymes transfer electrons from the ubiquinol pool directly to oxygen without contributing to the proton transfer across the mitochondrial membrane. Alternative oxidases are involved in stress responses, programmed cell death and maintenance of the cellular redox balance. The alternative oxidase gene of the methylotrophic yeast Pichia pastoris was isolated and cloned to study its regulation and the effects of deregulation of the alternative respiration by overexpression or disruption of the gene. Both disruption and overexpression had negative effects on the biomass yield; however, the growth rate and substrate uptake rate of the strain overexpressing the alternative oxidase were slightly increased. These effects were even more pronounced when higher glucose concentrations were used. The occurrence of free intracellular radicals and cell death phenomena was investigated using dihydrorhodamine 123 and the TUNEL test. The results suggest a major contribution of the alternative oxidase to P. pastoris cell viability. The negative effects of deregulated alternative respiration clearly indicated the importance of precise regulation of the alternative oxidase in this yeast.
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30. |
Dux MP,
Inan M,
( 2006 ) Identification and characterization of calcium and manganese transporting ATPase (PMR1) gene of Pichia pastoris. PMID : 16823889 : DOI : 10.1002/yea.1379 Abstract >>
A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Pichia pastoris. The entire P. pastoris PMR1 gene (PpPMR1) codes a protein of 924 amino acids. Sequence analysis of the PpPMR1 cDNA and the genomic DNA revealed that there is no intron in the coding region. The putative gene product contains all of the conserved regions observed in P-type ATPases and exhibits 66.2%, 60.3% and 50.6% identity to Pichia angusta (Hansenula polymorpha), Saccharomyces cerevisiae PMR1 and human ATP2C1 gene products, respectively. A pmr1 null mutant strain of P. pastoris exhibited growth defects in media with the addition of EGTA, but with supplementation of Ca2+ to a calcium-deficient media reversed the growth defects of the mutant strain. Manganese reversed the growth defects of the mutant strain; however, the cell growth was not as profound as the Ca2+ -supplemented media. The results demonstrated that the P. pastoris gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+/Mn2+ -ATPase. The DNA sequence of the P. pastoris PMR1 gene has been submitted to GenBank under Accession No. DQ239958.
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31. |
Ahn J,
Hong J,
Lee H,
Park M,
Lee E,
Kim C,
Choi E,
Jung J,
Lee H,
( 2007 ) Translation elongation factor 1-alpha gene from Pichia pastoris: molecular cloning, sequence, and use of its promoter. PMID : 17124582 : DOI : 10.1007/s00253-006-0698-6 Abstract >>
The gene encoding translation elongation factor 1-alpha from the yeast Pichia pastoris was cloned. The gene revealed an open reading frame of 1,380 bp with the potential to encode a polypeptide of 459 amino acids with a calculated mass of 50.1 kDa. The potential of the promoter (P (TEF1)) in P. pastoris was investigated with comparison to the glyceraldehyde-3-phosphate dehydrogenase promoter (P (GAP)) by using a bacterial lipase gene as a reporter gene. P (TEF1) demonstrated a tighter growth-associated expression mode, improved functioning in the presence of high glucose concentrations, and promoter activities that yielded recombinant protein at levels similar to or in one case greater than P (GAP). The sequence of the gene was deposited in GenBank under accession no. EF014948.
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32. |
Marx H,
Sauer M,
Resina D,
Vai M,
Porro D,
Valero F,
Ferrer P,
Mattanovich D,
( 2006 ) Cloning, disruption and protein secretory phenotype of the GAS1 homologue of Pichia pastoris. PMID : 17020547 : DOI : 10.1111/j.1574-6968.2006.00427.x Abstract >>
The aim of the study was the identification, cloning and disruption of the GAS1 homologue of Pichia pastoris. Gas1p is a glycoprotein anchored to the outer layer of the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. Gas1p is a beta-1,3-glucanosyltransglycosylase (EC 2.4.1.-). This cross-linking enzyme highly affects the structure and permeability of the yeast cell wall. The gene coding for the GAS1 homologue of P. pastoris was cloned by PCR, and its functionality was proven in a Saccharomyces cerevisiae GAS1 null mutant. Based on the nucleotide sequence information of the P. pastoris GAS1 homologue, a disruption cassette was constructed for the knockout of the GAS1 in P. pastoris. The morphology of DeltaGAS1 P. pastoris was identical to that of S. cerevisiae GAS1 mutants. Finally, the impact of GAS1 disruption on secretion of three recombinant model proteins in P. pastoris, human trypsinogen, human serum albumin and Rhizopus oryzae lipase, was evaluated. While the disruption had no effect on the secretion of trypsinogen and albumin, the amount of lipase released from the cells was doubled.
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33. |
Arakawa K,
Abe M,
Noda Y,
Adachi H,
Yoda K,
( 2006 ) Molecular cloning and characterization of a Pichia pastoris ortholog of the yeast Golgi GDP-mannose transporter gene. PMID : 16960330 : Abstract >>
There are two structural profiles in the yeast Golgi. The Golgi of Saccharomyces cerevisiae is composed of a number of vesicular compartments dispersed in the cytoplasm as recognized by a large number of Golgi marker proteins. In contrast, the Golgi of Pichia pastoris was reported to be organized in a small number of stacked cisternae located near the transitional endoplasmic reticulum (tER) sites by electron microscopy and immunofluorescent staining of a few marker proteins. The guanosine diphosphate (GDP)-mannose transporter (GMT) is an essential component in the yeast Golgi apparatus. We isolated an ortholog of the GMT gene of P. pastoris and visualized the gene product by epitope tagging to verify the structural characteristics of the Golgi. The tagged product in P. pastoris cell was observed in rod-like compartments in which Och1 mannosyltransferase was also found and the tER marker Sec12 and Sec13 proteins localized very close to them. The present results add further evidence of the restricted localization of the Golgi in P. pastoris cell.
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34. |
Duff AP,
Cohen AE,
Ellis PJ,
Hilmer K,
Langley DB,
Dooley DM,
Freeman HC,
Guss JM,
( 2006 ) The 1.23 Angstrom structure of Pichia pastoris lysyl oxidase reveals a lysine-lysine cross-link. PMID : 16929109 : DOI : 10.1107/S0907444906026333 Abstract >>
The structure of Pichia pastoris lysyl oxidase (PPLO) in a new crystal form has been refined at 1.23 Angstrom resolution. PPLO, a copper amine oxidase (CuAO) with a 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor, differs from most other members of the CuAO enzyme family in having the ability to oxidize the side chain of lysine residues in a polypeptide. In the asymmetric unit of the crystals, the structure analysis has located residues 43-779 of the polypeptide chain, seven carbohydrate residues, the active-site Cu atom, an imidazole molecule bound at the active site, two buried Ca(2+) ions, five surface Mg(2+) ions, five surface Cl(-) ions and 1045 water molecules. The crystallographic residuals are R = 0.112 and R(free) = 0.146. The TPQ cofactor and several other active-site residues are poorly ordered, in contrast to the surrounding well ordered structure. A covalent cross-link is observed between two lysine residues, Lys778 and Lys66. The cross-link is likely to have been formed by the oxidation of Lys778 followed by a spontaneous reaction with Lys66. The link is modelled as dehydrolysinonorleucine.
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35. |
Esaki M,
Liu Y,
Glick BS,
( 2006 ) The budding yeast Pichia pastoris has a novel Sec23p homolog. PMID : 16962585 : DOI : 10.1016/j.febslet.2006.08.058 DOI : 10.1016/j.febslet.2006.08.058 Abstract >>
In Pichia pastoris, coat protein complex II (COPII) vesicles form at discrete transitional ER (tER) sites. Analyzing COPII coat proteins in this yeast will help to reveal the mechanisms of tER organization. Here, we show that like Saccharomyces cerevisiae, P. pastoris contains essential SEC23 and SEC24 genes, as well as the non-essential SEC24 homolog LST1. In addition, P. pastoris contains a novel non-essential SEC23 homolog that we have designated SHL23. The products of all four genes are concentrated at tER sites. Deletion of SHL23 does not disrupt tER morphology. As judged by two-hybrid analysis, Sec23p associates with both Sec24p and Lst1p, whereas Shl23p associates selectively with Lst1p. These results suggest that P. pastoris COPII vesicles contain an Shl23p/Lst1p complex that is absent in S. cerevisiae.
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36. |
Yamashita S,
Oku M,
Wasada Y,
Ano Y,
Sakai Y,
( 2006 ) PI4P-signaling pathway for the synthesis of a nascent membrane structure in selective autophagy. PMID : 16754956 : DOI : 10.1083/jcb.200512142 PMC : PMC2063888 Abstract >>
Phosphoinositides regulate a wide range of cellular activities, including membrane trafficking and biogenesis, via interaction with various effector proteins that contain phosphoinositide binding motifs. We show that in the yeast Pichia pastoris, phosphatidylinositol 4'-monophosphate (PI4P) initiates de novo membrane synthesis that is required for peroxisome degradation by selective autophagy and that this PI4P signaling is modulated by an ergosterol-converting PpAtg26 (autophagy-related) protein harboring a novel PI4P binding GRAM (glucosyltransferase, Rab-like GTPase activators, and myotubularins) domain. A phosphatidylinositol-4-OH kinase, PpPik1, is the primary source of PI4P. PI4P concentrated in a protein-lipid nucleation complex recruits PpAtg26 through an interaction with the GRAM domain. Sterol conversion by PpAtg26 at the nucleation complex is necessary for elongation and maturation of the membrane structure. This study reveals the role of the PI4P-signaling pathway in selective autophagy, a process comprising multistep molecular events that lead to the de novo membrane formation.
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37. |
Stasyk OV,
Stasyk OG,
Mathewson RD,
Farré JC,
Nazarko VY,
Krasovska OS,
Subramani S,
Cregg JM,
Sibirny AA,
( N/A ) Atg28, a novel coiled-coil protein involved in autophagic degradation of peroxisomes in the methylotrophic yeast Pichia pastoris. PMID : 16874081 : DOI : 10.4161/auto.2226 Abstract >>
In methylotrophic yeasts, peroxisomes are required for methanol utilization, but are dispensable for growth on most other carbon sources. Upon adaptation of cells grown on methanol to glucose or ethanol, redundant peroxisomes are selectively and quickly shipped to, and degraded in, vacuoles via a process termed pexophagy. We identified a novel gene named ATG28 (autophagy-related genes) involved in pexophagy in the yeast Pichia pastoris. This yeast exhibits two morphologically distinct pexophagy pathways, micro- and macropexophagy, induced by glucose or ethanol, respectively. Deficiency in ATG28 impairs both pexophagic mechanisms but not general (bulk turnover) autophagy, a degradation pathway in yeast triggered by nitrogen starvation. It is known that the micro-, macropexophagy, and general autophagy machineries are distinct but share some molecular components. The identification of ATG28 suggests that pexophagy may involve species-specific components, since this gene appears to have only weak homologues in other yeasts.
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38. |
Reggiori F,
Shintani T,
Nair U,
Klionsky DJ,
( 2005 ) Atg9 cycles between mitochondria and the pre-autophagosomal structure in yeasts. PMID : 16874040 : DOI : 10.4161/auto.1.2.1840 PMC : PMC1762033 Abstract >>
Autophagy is a degradative process conserved among eukaryotic cells. It allows the elimination of cytoplasm including aberrant protein aggregates and damaged organelles. Accordingly, it is implicated in normal developmental processes and also serves a protective role in tumor suppression and elimination of invading pathogens, whereas defects in autophagy are associated with various human diseases including cancer and neurodegeneration. Atg proteins mediate the sequestration event that occurs at the preautophagosomal structure (PAS) by catalyzing the formation of double-membrane vesicles, termed autophagosomes. In Saccharomyces cerevisiae, the integral membrane protein Atg9 that is required for autophagy cycles through the PAS. Here, we demonstrate that Atg9 shuttles between this location and mitochondria. These data support a new model where mitochondria may provide at least part of the autophagosomal lipids and suggest a novel cellular function for this well-studied organelle.
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39. |
Lin-Cereghino GP,
Godfrey L,
de la Cruz BJ,
Johnson S,
Khuongsathiene S,
Tolstorukov I,
Yan M,
Lin-Cereghino J,
Veenhuis M,
Subramani S,
Cregg JM,
( 2006 ) Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris. PMID : 16428444 : DOI : 10.1128/MCB.26.3.883-897.2006 PMC : PMC1347016 Abstract >>
Growth of the yeast Pichia pastoris on methanol induces the expression of genes whose products are required for its metabolism. Three of the methanol pathway enzymes are located in an organelle called the peroxisome. As a result, both methanol pathway enzymes and proteins involved in peroxisome biogenesis (PEX proteins) are induced in response to this substrate. The most highly regulated of these genes is AOX1, which encodes alcohol oxidase, the first enzyme of the methanol pathway, and a peroxisomal enzyme. To elucidate the molecular mechanisms responsible for methanol regulation, we identify genes required for the expression of AOX1. Mutations in one gene, named MXR1 (methanol expression regulator 1), result in strains that are unable to (i) grow on the peroxisomal substrates methanol and oleic acid, (ii) induce the transcription of AOX1 and other methanol pathway and PEX genes, and (iii) form normal-appearing peroxisomes in response to methanol. MXR1 encodes a large protein with a zinc finger DNA-binding domain near its N terminus that has similarity to Saccharomyces cerevisiae Adr1p. In addition, Mxr1p is localized to the nucleus in cells grown on methanol or other gluconeogenic substrates. Finally, Mxr1p specifically binds to sequences upstream of AOX1. We conclude that Mxr1p is a transcription factor that is necessary for the activation of many genes in response to methanol. We propose that MXR1 is the P. pastoris homologue of S. cerevisiae ADR1 but that it has gained new functions and lost others through evolution as a result of changes in the spectrum of genes that it controls.
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40. |
Bonaccorsi di Patti MC,
Miele R,
Eugenia Schinin? M,
Barra D,
( 2005 ) The yeast multicopper oxidase Fet3p and the iron permease Ftr1p physically interact. PMID : 15946650 : DOI : 10.1016/j.bbrc.2005.05.121 Abstract >>
High affinity iron uptake in yeast is carried out by a multicomponent system formed by the ferroxidase Fet3p and the iron permease Ftr1p. The currently accepted model predicts that Fet3p and Ftr1p are functionally associated, however, a structural interaction between these two proteins has not been proven yet. The methylotrophic yeast Pichia pastoris has been used to perform cross-linking studies aimed to demonstrate the existence of a Fet3p-Ftr1p complex. Cross-linking of membrane suspensions with the membrane-impermeable reagents DTSSP and BS(3) has evidenced the presence of a high molecular weight band with Fet3p oxidase activity. This band has been purified and subjected to N-terminal sequence analysis. Two sequences were found in the cross-linked species, one of which could be assigned to Fet3p and the other to Ftr1p. This is the first experimental demonstration that Fet3p and Ftr1p are physically associated.
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41. |
Léon S,
Zhang L,
McDonald WH,
Yates J,
Cregg JM,
Subramani S,
( 2006 ) Dynamics of the peroxisomal import cycle of PpPex20p: ubiquitin-dependent localization and regulation. PMID : 16390998 : DOI : 10.1083/jcb.200508096 PMC : PMC2063535 Abstract >>
We characterize the peroxin PpPex20p from Pichia pastoris and show its requirement for translocation of PTS2 cargoes into peroxisomes. PpPex20p docks at the peroxisomal membrane and translocates into peroxisomes. Its peroxisomal localization requires the docking peroxin Pex14p but not the peroxins Pex2p, Pex10p, and Pex12p, whose absence causes peroxisomal accumulation of Pex20p. Similarities between Pex5p and Pex20p were noted in their protein interactions and dynamics during import, and both contain a conserved NH2-terminal domain. In the absence of the E2-like Pex4p or the AAA proteins Pex1p and Pex6p, Pex20p is degraded via polyubiquitylation of residue K19, and the K19R mutation causes accumulation of Pex20p in peroxisome remnants. Finally, either interference with K48-branched polyubiquitylation or removal of the conserved NH2-terminal domain causes accumulation of Pex20p in peroxisomes, mimicking a defect in its recycling to the cytosol. Our data are consistent with a model in which Pex20p enters peroxisomes and recycles back to the cytosol in an ubiquitin-dependent manner.
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42. |
Huang L,
Li DY,
Wang SX,
Zhang SM,
Chen JH,
Wu XF,
( 2005 ) Cloning and identification of methionine synthase gene from Pichia pastoris. PMID : 15944751 : DOI : 10.1111/j.1745-7270.2005.00054.x Abstract >>
Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5' and 3' regions were further cloned by 5'- and 3'-rapid amplification of cDNA ends (RACE), respectively. The assembled sequence reveals an open reading frame encoding a polypeptide of 768 residues, and the deduced product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two domains common to MetEs. The active site is located in the C-terminal domain, in which the residues involved in the interaction of zinc with substrates are conserved. Homologous expression of PpMetE in P. pastoris was achieved, and the heterologous expression of PpMetE in the S. cerevisiae strain XJB3-1D that is MetE-defective restored the growth of the mutant on methionine-free minimal media. The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No. AY601648.
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43. |
Xu Z,
Shih MC,
Poulton JE,
( 2006 ) An extracellular exo-beta-(1,3)-glucanase from Pichia pastoris: purification, characterization, molecular cloning, and functional expression. PMID : 16427312 : DOI : 10.1016/j.pep.2005.11.025 Abstract >>
An extracellular exo-beta-(1,3)-glucanase (designated EXG1) was purified to apparent homogeneity from Pichia pastoris X-33 cultures by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The native enzyme is unglycosylated and monomeric with a molecular mass of approximately 47kDa. At its optimal pH of 6.0, the enzyme shows highest activity among physiological substrates toward laminarin (apparent Km, 3.5 mg/ml; Vmax, 192 micromole glucose produced/min/mg protein) but also hydrolyzes amygdalin and esculin, and the chromogenic substrates p-nitrophenyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-xylopyranoside. The P. pastoris EXG1 gene was cloned by a PCR-based strategy using genomic DNA as template. This intronless gene predicts an ORF that encodes a primary translation product of 414 amino acids. We believe that this preproprotein is processed sequentially by signal peptidase and a Kex2-like endoprotease to yield a mature protein of 392 amino acids (45,376 Da; pI, 4.46) that shares 36-64% amino acid identity with other yeast exo-beta-(1,3)-glucanases belonging to Glycoside Hydrolase Family 5. It also possesses the eight invariant residues and signature pattern [LIV]-[LIVMFYWGA](2)-[DNEQG]-[LIVMGST]-X-N-E-[PV]-[RHDNSTLIVFY] shown by all Family 5 members. Overexpression of the cloned EXG1 gene in Pichia cells, followed by Ni-CAM HC resin chromatography, yielded milligram quantities of homogeneous recombinant EXG1 in active form for further characterization studies.
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44. |
Whittaker MM,
Whittaker JW,
( 2005 ) Construction and characterization of Pichia pastoris strains for labeling aromatic amino acids in recombinant proteins. PMID : 15866712 : DOI : 10.1016/j.pep.2004.11.008 Abstract >>
Strains of the methylotrophic yeast Pichia pastoris auxotrophic for the aromatic amino acids (tyrosine, phenylalanine, and tryptophan) have been constructed by targeted gene disruption for protein labeling applications. Three strains, with defects in ARO1 (coding for a homolog of the arom pentafunctional enzyme), ARO7 (coding for chorismate mutase), and TYR1 (coding for prephenate dehydrogenase), have been engineered in a P. pastoris ura3Delta1 parent strain using standard methods. The nutritional requirements of these auxotrophic strains have been characterized and their utility as expression hosts for labeling recombinant proteins has been demonstrated. All three strains show a surprising sensitivity to rich culture medium and must be grown in supplemented minimal medium. The tyr1::URA3 strain in particular is strongly inhibited by tryptophan, and to a lesser extent by phenylalanine, leucine, and isoleucine. Highly efficient incorporation of exogenously supplied amino acids by these three auxotroph strains has been demonstrated using recombinant galactose oxidase. Stereochemically pure l-amino acids and racemic d,l-mixtures serve nearly equally well to support protein expression and labeling. These strains allow efficient labeling of aromatic amino acids in recombinant proteins, supporting NMR structural biology and a wide range of other biophysical studies.
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45. |
Werten MW,
de Wolf FA,
( 2005 ) Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant. PMID : 15870316 : DOI : 10.1128/AEM.71.5.2310-2317.2005 PMC : PMC1087524 Abstract >>
Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae alpha-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the alpha-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the alpha-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.
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46. |
Connerly PL,
Esaki M,
Montegna EA,
Strongin DE,
Levi S,
Soderholm J,
Glick BS,
( 2005 ) Sec16 is a determinant of transitional ER organization. PMID : 16111939 : DOI : 10.1016/j.cub.2005.06.065 Abstract >>
Proteins are exported from the ER at transitional ER (tER) sites, which produce COPII vesicles. However, little is known about how COPII components are concentrated at tER sites. The budding yeast Pichia pastoris contains discrete tER sites and is, therefore, an ideal system for studying tER organization. We show that the integrity of tER sites in P. pastoris requires the peripheral membrane protein Sec16. P. pastoris Sec16 is an order of magnitude less abundant than a COPII-coat protein at tER sites and seems to show a saturable association with these sites. A temperature-sensitive mutation in Sec16 causes tER fragmentation at elevated temperature. This effect is specific because when COPII assembly is inhibited with a dominant-negative form of the Sar1 GTPase, tER sites remain intact. The tER fragmentation in the sec16 mutant is accompanied by disruption of Golgi stacks. Our data suggest that Sec16 helps to organize patches of COPII-coat proteins into clusters that represent tER sites. The Golgi disruption that occurs in the sec16 mutant provides evidence that Golgi structure in budding yeasts depends on tER organization.
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47. |
Liu YY,
Woo JH,
Neville DM,
( 2005 ) Overexpression of an anti-CD3 immunotoxin increases expression and secretion of molecular chaperone BiP/Kar2p by Pichia pastoris. PMID : 16151122 : DOI : 10.1128/AEM.71.9.5332-5340.2005 PMC : PMC1214624 Abstract >>
We previously reported that the secretory capacity of Pichia pastoris is limited with respect to the secretion of a 96.5-kDa bivalent anti-CD3 immunotoxin; double-copy expression generated more translation products than single-copy expression but did not increase the secretion of the immunotoxin. In Saccharomyces cerevisiae heterologous protein secretion has been reported to increase the expression of molecular chaperones, most prominently BiP/Kar2p. We therefore investigated the relationships between immunotoxin secretion and Kar2p expression in P. pastoris. We found that expression of the immunotoxin in P. pastoris increased the expression of Kar2p to levels that surpassed the retrieval capacity of the cell, leading to secretion of Kar2p into the medium. The level of Kar2p secretion was correlated with the copy number of the immunotoxin gene. Intracellular Kar2p was found to bind exclusively to the unprocessed immunotoxin containing the prosequence of alpha-factor in the endoplasmic reticulum. These results show that Kar2p is intimately involved in immunotoxin secretion in P. pastoris. The limited capacity of P. pastoris to retain a sufficiently high level of intracellular Kar2p may be a factor restricting the production of the immunotoxin.
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48. |
Nett JH,
Hodel N,
Rausch S,
Wildt S,
( 2005 ) Cloning and disruption of the Pichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6 genes and their use as auxotrophic markers. PMID : 15789348 : DOI : 10.1002/yea.1202 Abstract >>
Screening of a partial genomic database of Pichia pastoris allowed us to identify the ARG1, ARG2, ARG3, HIS1, HIS2, HIS5 and HIS6 genes, based on homology to their Saccharomyces cerevisiae counterparts. Based on the cloned sequences, a set of disruption vectors was constructed, using the previously described PpURA5-blaster as a selectable marker, and the cloned genes were individually disrupted. All disruptants exhibited the expected auxotrophic phenotypes, with only the his2 knockouts displaying a bradytroph phenotype. To allow their use as auxotrophic markers, we amplified the open reading frames and respective promoters and terminator regions of PpARG1, PpARG2, PpARG3, PpHIS1, PpHIS2 and PpHIS5. We then designed a set of integration vectors harbouring cassettes of the ARG pathway as selectable markers, to disrupt the genes of the HIS pathway and vice versa. Employing this strategy, we devised a scheme allowing for the rapid and stable introduction of several heterologous genes into the genome of P. pastoris without the need for recyclable markers or strains with multiple auxotrophies. Furthermore, simple replica-plating, instead of cost-consuming and labour-intensive colony PCR or Southern analysis, can be used to identify positive transformants, making this approach amendable for initial high-throughput applications, which can then be followed up by a more careful analysis of the selected transformants.
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49. |
Chang T,
Schroder LA,
Thomson JM,
Klocman AS,
Tomasini AJ,
Strømhaug PE,
Dunn WA,
( 2005 ) PpATG9 encodes a novel membrane protein that traffics to vacuolar membranes, which sequester peroxisomes during pexophagy in Pichia pastoris. PMID : 16079180 : DOI : 10.1091/mbc.e05-02-0143 PMC : PMC1237094 Abstract >>
When Pichia pastoris adapts from methanol to glucose growth, peroxisomes are rapidly sequestered and degraded within the vacuole by micropexophagy. During micropexophagy, sequestering membranes arise from the vacuole to engulf the peroxisomes. Fusion of the sequestering membranes and incorporation of the peroxisomes into the vacuole is mediated by the micropexophagy-specific membrane apparatus (MIPA). In this study, we show the P. pastoris ortholog of Atg9, a novel membrane protein is essential for the formation of the sequestering membranes and assembly of MIPA. During methanol growth, GFP-PpAtg9 localizes to multiple structures situated near the plasma membrane referred as the peripheral compartment (Atg9-PC). On glucose-induced micropexophagy, PpAtg9 traffics from the Atg9-PC to unique perivacuolar structures (PVS) that contain PpAtg11, but lack PpAtg2 and PpAtg8. Afterward, PpAtg9 distributes to the vacuole surface and sequestering membranes. Movement of the PpAtg9 from the Atg9-PC to the PVS requires PpAtg11 and PpVps15. PpAtg2 and PpAtg7 are essential for PpAtg9 trafficking from the PVS to the vacuole and sequestering membranes, whereas trafficking of PpAtg9 proceeds independent of PpAtg1, PpAtg18, and PpVac8. In summary, our data suggest that PpAtg9 transits from the Atg9-PC to the PVS and then to the sequestering membranes that engulf the peroxisomes for degradation.
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50. |
Thor D,
Xiong S,
Orazem CC,
Kwan AC,
Cregg JM,
Lin-Cereghino J,
Lin-Cereghino GP,
( 2005 ) Cloning and characterization of the Pichia pastoris MET2 gene as a selectable marker. PMID : 15996626 : DOI : 10.1016/j.femsyr.2005.03.009 Abstract >>
We describe the isolation and characterization of a new biosynthetic gene, MET2, from the methylotrophic yeast Pichia pastoris. The predicted product of PpMET2 is significantly similar to its Saccharomyces cerevisiae counterpart, ScMET2, which encodes homoserine-O-transacetylase. The ScMET2 was able to complement the P. pastoris met2 strain; however, the converse was not true. Expression vectors based on PpMET2 for the intracellular and secreted production of foreign proteins and corresponding auxotrophic strains were constructed and tested for use in heterologous expression. The expression vectors and corresponding strains provide greater flexibility when using P. pastoris for recombinant protein expression.
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51. |
Ano Y,
Hattori T,
Oku M,
Mukaiyama H,
Baba M,
Ohsumi Y,
Kato N,
Sakai Y,
( 2005 ) A sorting nexin PpAtg24 regulates vacuolar membrane dynamics during pexophagy via binding to phosphatidylinositol-3-phosphate. PMID : 15563611 : DOI : 10.1091/mbc.e04-09-0842 PMC : PMC545876 Abstract >>
Diverse cellular processes such as autophagic protein degradation require phosphoinositide signaling in eukaryotic cells. In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded via two types of pexophagic pathways, macropexophagy and micropexophagy. Both involve membrane fusion events at the vacuolar surface that are characterized by internalization of the boundary domain of the fusion complex, indicating that fusion occurs at the vertex. Here, we show that PpAtg24, a molecule with a phosphatidylinositol 3-phosphate-binding module (PX domain) that is indispensable for pexophagy, functions in membrane fusion at the vacuolar surface. CFP-tagged PpAtg24 localized to the vertex and boundary region of the pexophagosome-vacuole fusion complex during macropexophagy. Depletion of PpAtg24 resulted in the blockage of macropexophagy after pexophagosome formation and before the fusion stage. These and other results suggest that PpAtg24 is involved in the spatiotemporal regulation of membrane fusion at the vacuolar surface during pexophagy via binding to phosphatidylinositol 3-phosphate, rather than the previously suggested function in formation of the pexophagosome.
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52. |
Duff AP,
Trambaiolo DM,
Cohen AE,
Ellis PJ,
Juda GA,
Shepard EM,
Langley DB,
Dooley DM,
Freeman HC,
Guss JM,
( 2004 ) Using xenon as a probe for dioxygen-binding sites in copper amine oxidases. PMID : 15533431 : DOI : 10.1016/j.jmb.2004.09.075 Abstract >>
Potential dioxygen-binding sites in three Cu amine oxidases have been investigated by recording X-ray diffraction data at 1.7-2.2A resolution for crystals under a high pressure of xenon gas. Electron-density difference maps and crystallographic refinement provide unequivocal evidence for a number of Xe-binding sites in each enzyme. Only one of these sites is present in all three Cu amine oxidases studied. Structural changes elsewhere in the protein molecules are insignificant. The results illustrate the use of xenon as a probe for cavities, in which a protein may accommodate a dioxygen molecule. The finding of a potential dioxygen-binding cavity close to the active site of Cu amine oxidases may be relevant to the function of the enzymes, since the formation of a transient protein-dioxygen complex is a likely step in the catalytic mechanism. No evidence was found for xenon binding in a region of the molecule that was previously identified in two other Cu amine oxidases as a potential transient dioxygen-binding site.
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53. |
Bhatnagar A,
Raghavendra PR,
Kranthi BV,
Rangarajan PN,
( 2004 ) Yeast cytochrome c is a sequence-specific DNA-binding protein. PMID : 15358111 : DOI : 10.1016/j.bbrc.2004.07.044 Abstract >>
A protein binding to the alcohol oxidase 2 upstream activation sequence (AOX2UAS) of the methylotropic yeast, Pichia pastoris, has been purified and identified as cytochrome c (cyt c). Cyt c purified from P. pastoris or Saccharomyces cerevisiae binds to AOX2UAS. Specific point mutations in AOX2UAS abolish cyt c binding. We conclude that yeast cyt c is a sequence-specific DNA-binding protein and may have a regulatory role in the nucleus.
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54. |
Miura M,
Hirose M,
Miwa T,
Kuwae S,
Ohi H,
( 2004 ) Cloning and characterization in Pichia pastoris of PNO1 gene required for phosphomannosylation of N-linked oligosaccharides. PMID : 14693378 : DOI : 10.1016/j.gene.2003.09.023 Abstract >>
The yeast Pichia pastoris PNO1 (Phosphomannosylation of N-linked Oligosaccharides) gene, which is involved in phosphomannosylation of N-linked oligosaccharides, was cloned using the Saccharomyces cerevisiae MNN4 gene [Glycobiology 6 (1996) 805] as a probe. The PNO1 open reading frame (ORF) encodes a type II membrane protein composed of 777 amino acid residues. Only in the short region extending from amino acid position 450 to 606 of Pno1p, sequence homology to S. cerevisiae Mnn4p was observed at a level of 45%. The tandem repeat sequence of Lys-Lys-Lys-Lys-Glu-Glu-Glu-Glu characteristic of the C-terminal region of S. cerevisiae Mnn4p is not present in Pno1p. To investigate the function of the PNO1 gene, we constructed a PNO1 gene disruptant by replacement with an expression cassette of human antithrombin (AT), a glycoprotein in plasma. The cell growth and recombinant human antithrombin (rAT) production levels of the disruptant were similar to those of recombinant human antithrombin-expressing wild-type strains. Moreover, the level of alcian blue dye cell staining, which shows the presence of acidic sugar chains on the cell surface, was also similar. However, the phosphomannosylation ratio of N-linked oligosaccharides on recombinant human antithrombin decreased dramatically from 20% in wild-type strains to less than 1% in the PNO1 disruptant. When the PNO1 gene was re-introduced into the disruptant, the phosphomannosylation ratio recovered to the original level. These results suggest that the newly cloned PNO1 gene promotes phosphomannosylation only to core-like oligosaccharides, and not to the hypermannosylated outer chain, and that it has a different function from the MNN4 gene, which promotes the phosphomannosylation of both core and outer sugar chains.
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55. |
Duff AP,
Cohen AE,
Ellis PJ,
Kuchar JA,
Langley DB,
Shepard EM,
Dooley DM,
Freeman HC,
Guss JM,
( 2003 ) The crystal structure of Pichia pastoris lysyl oxidase. PMID : 14690425 : DOI : 10.1021/bi035338v Abstract >>
Pichia pastoris lysyl oxidase (PPLO) is unique among the structurally characterized copper amine oxidases in being able to oxidize the side chain of lysine residues in polypeptides. Remarkably, the yeast PPLO is nearly as effective in oxidizing a mammalian tropoelastin substrate as is a true mammalian lysyl oxidase isolated from bovine aorta. Thus, PPLO is functionally related to the copper-containing lysyl oxidases despite the lack of any significant sequence similarity with these enzymes. The structure of PPLO has been determined at 1.65 A resolution. PPLO is a homodimer in which each subunit contains a Type II copper atom and a topaquinone cofactor (TPQ) formed by the posttranslational modification of a tyrosine residue. While PPLO has tertiary and quaternary topologies similar to those found in other quinone-containing copper amine oxidases, its active site is substantially more exposed and accessible. The structural elements that are responsible for the accessibility of the active site are identified and discussed.
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56. |
Nett JH,
Gerngross TU,
( 2003 ) Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris. PMID : 14618566 : DOI : 10.1002/yea.1049 Abstract >>
A pair of degenerate primers was used for amplification and cloning of a DNA fragment containing parts of the P. pastoris URA5 and SEC65 genes. Using additional information from a partial genomic sequence of P. pastoris, we cloned and sequenced a 1.9 kb chromosomal fragment containing the complete orotate-phosphoribosyltransferase-encoding URA5 gene. A disruption cassette was constructed by replacing a small part of the open reading frame with a kanamycin-resistance gene. The P. pastoris wild-type strain NRRL Y-11430 was transformed with the disruption cassette and an ura5 auxotrophic strain was identified. To generate marker constructs that can be reused in successive transformations of a single strain, we constructed two lacZ-PpURA3-lacZ and lacZ-PpURA5-lacZ cassettes and used them to disrupt PpOCH1. The PpURA3 and PpURA5 genes in the disruptants were then successfully recycled by selecting for resistance to 5'-fluoro-orotic acid. We also assembled a set of modular plasmids that can be used for the stable genetic modification of P. pastoris via a double cross-over event. The sequence presented here has been submitted to the EMBL data library under Accession No. AY303544.
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57. |
Khasa YP,
Conrad S,
Sengul M,
Plautz S,
Meagher MM,
Inan M,
( 2011 ) Isolation of Pichia pastoris PIR genes and their utilization for cell surface display and recombinant protein secretion. PMID : 21360733 : DOI : 10.1002/yea.1832 Abstract >>
Proteins with internal repeats are highly conserved among budding yeasts. In this study, the isolation of two proteins with internal repeats (PIR) genes, i.e. PpPIR1 and PpPIR2, from the methylotrophic yeast Pichia pastoris has been reported. The PIR1 and PIR2 genes' open reading frames were found to contain 1068 and 972 bases, respectively. The sequence homology search showed a homologous conserved repeat of PIR yeast block (SQIGDGQIQATT) in both proteins. The PIR yeast block was present eight times in the PpPir1p and four times in the PpPir2p proteins. Both proteins showed conserved glutamine (Q) and aspartic acid (D) in the repeated sequences, indicating a possible alkali-sensitive �]1,3-glucan ester linkage. The fusion constructs of PpPir1-2p and enhanced green fluorescent protein (EGFP) were developed for yeast cell surface display. The immunofluorescence assay showed uniform localization of EGFP protein on the P. pastoris cell surface in all fusion constructs. Furthermore, new vectors were developed for recombinant protein secretion in P. pastoris, utilizing the pre-pro signal of PpPir1p protein. Efficient processing of the signal sequence was observed from EGFP and human �\1-antitrypsin (AAT) fusion constructs and recombinant protein secretion was obtained in the culture supernatant.
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58. |
Salamin K,
Sriranganadane D,
Léchenne B,
Jousson O,
Monod M,
( 2010 ) Secretion of an endogenous subtilisin by Pichia pastoris strains GS115 and KM71. PMID : 20472730 : DOI : 10.1128/AEM.00412-10 PMC : PMC2897435 Abstract >>
The methylotrophic yeast Pichia pastoris is widely used for the expression of heterologous enzymes. While the purity of the desired expression product is of major importance for many applications, we found that recombinant enzymes produced in methanol medium were contaminated by a 37-kDa endogenous yeast protease. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) but not by 1,10-phenanthroline, EDTA, and pepstatin A, suggesting the nature of a serine protease. Its secretion was abolished in P. pastoris strains GS115 and KM71 by specific mutagenesis of a subtilisin gene (SUB2) but not by inactivation of the gene encoding vacuolar proteinase B (PRB). Bioinformatic comparisons of Sub2 protein with subtilisins from other fungal genomes and phylogenetic analyses indicated that this enzyme is not an orthologue of the vacuolar protease cerevisin generally present in yeasts but is more closely related to another putative subtilisin found in a small number of yeast genomes. During growth of P. pastoris, Sub2 was produced as a secreted enzyme at a concentration of 10 microg/ml of culture supernatant after overexpression of the full-length SUB2 gene. During fermentative production of recombinant enzymes in methanol medium, 1 ml of P. pastoris culture supernatant was found to contain approximately 3 ng of Sub2, while the enzyme was not detected during growth in a medium containing glycerol as a carbon source. The mutant strain GS115-sub2 was subsequently used as a host for the production of recombinant proteases without endogenous subtilisin contamination.
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59. |
Sträter N,
Marek S,
Kuettner EB,
Kloos M,
Keim A,
Brüser A,
Kirchberger J,
Schöneberg T,
( 2011 ) Molecular architecture and structural basis of allosteric regulation of eukaryotic phosphofructokinases. PMID : 20833871 : DOI : 10.1096/fj.10-163865 Abstract >>
Eukaryotic ATP-dependent 6-phosphofructokinases (Pfks) differ from their bacterial counterparts in a much more complex structural organization and allosteric regulation. Pichia pastoris Pfk (PpPfk) is, with ? 1 MDa, the most complex and probably largest eukaryotic Pfk. We have determined the crystal structure of full-length PpPfk to 3.05 ? resolution in the T state. PpPfk forms a (�\�]�^)(4) dodecamer of D(2) symmetry with dimensions of 161 �� 157 �� 233 ? mainly via interactions of the �\ chains. The N-terminal domains of the �\ and �] chains have folds that are distantly related to glyoxalase I, but the active sites are no longer functional. Interestingly, these domains located at the 2 distal ends of this protein along the long 2-fold axis form a (�\�])(2) dimer as does the core Pfk domains; however, the domains are swapped across the tetramerization interface. In PpPfk, the unique �^ subunit participates in oligomerization of the �\�] chains. This modulator protein was acquired from an ancient S-adenosylmethionine-dependent methyltransferase. The identification of novel ATP binding sites, which do not correspond to the bacterial catalytic or effector binding sites, point to marked structural and functional differences between bacterial and eukaryotic Pfks.
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60. |
Bridges HR,
Fearnley IM,
Hirst J,
( 2010 ) The subunit composition of mitochondrial NADH:ubiquinone oxidoreductase (complex I) from Pichia pastoris. PMID : 20610779 : DOI : 10.1074/mcp.M110.001255 PMC : PMC2953923 Abstract >>
Respiratory complex I (NADH:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. It is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. Here we present a comprehensive analysis of the protein composition and post-translational modifications of complex I from Pichia pastoris, using a combination of proteomic and bioinformatic approaches. Forty-one subunits were identified in P. pastoris complex I, comprising the 14 core (conserved) subunits and 27 supernumerary subunits; seven of the core subunits are mitochondrial encoded. Three of the supernumerary subunits (named NUSM, NUTM, and NUUM) have not been observed previously in any species of complex I. However, homologues to all three of them are present in either Yarrowia lipolytica or Pichia angusta complex I. P. pastoris complex I has 39 subunits in common with Y. lipolytica complex I, 37 in common with N. crassa complex I, and 35 in common with the bovine enzyme. The mitochondrial encoded subunits (translated by the mold mitochondrial genetic code) retain their N-�\-formyl methionine residues. At least eight subunits are N-�\-acetylated, but the N-terminal modifications of the nuclear encoded subunits are not well-conserved. A combination of two methods of protein separation (SDS-PAGE and HPLC) and three different mass spectrometry techniques (peptide mass fingerprinting, tandem MS and molecular mass measurements) were required to define the protein complement of P. pastoris complex I. This requirement highlights the need for inclusive and comprehensive strategies for the characterization of challenging membrane-bound protein complexes containing both hydrophilic and hydrophobic components.
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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. |
Zhang P,
Zhang W,
Zhou X,
Bai P,
Cregg JM,
Zhang Y,
( 2010 ) Catabolite repression of Aox in Pichia pastoris is dependent on hexose transporter PpHxt1 and pexophagy. PMID : 20656869 : DOI : 10.1128/AEM.00607-10 PMC : PMC2937511 Abstract >>
In this work, the identification and characterization of two hexose transporter homologs in the methylotrophic yeast Pichia pastoris, P. pastoris Hxt1 (PpHxt1) and PpHxt2, are described. When expressed in a Saccharomyces cerevisiae hxt-null mutant strain that is unable to take up monosaccharides, either protein restored growth on glucose or fructose. Both PpHXT genes are transcriptionally regulated by glucose. Transcript levels of PpHXT1 are induced by high levels of glucose, whereas transcript levels of PpHXT2 are relatively lower and are fully induced by low levels of glucose. In addition, PpHxt2 plays an important role in glycolysis-dependent fermentative growth, since PpHxt2 is essential for growth on glucose or fructose when respiration is inhibited. Notably, we firstly found that the deletion of PpHXT1, but not PpHXT2, leads to the induced expression of the alcohol oxidase I gene (AOX1) in response to glucose or fructose. We also elucidated that a sharp dropping of the sugar-induced expression level of Aox at a later growth phase is caused mainly by pexophagy, a degradation pathway in methylotrophic yeast. The sugar-inducible AOX1 promoter in an Deltahxt1 strain may be promising as a host for the expression of heterologous proteins. The functional analysis of these two hexose transporters is the first step in elucidating the mechanisms of sugar metabolism and catabolite repression in P. pastoris.
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63. |
Benjamin S,
Radermacher M,
Kirchberger J,
Schöneberg T,
Edelmann A,
Ruiz T,
( 2009 ) 3D structure of phosphofructokinase from Pichia pastoris: Localization of the novel gamma-subunits. PMID : 19559794 : DOI : 10.1016/j.jsb.2009.06.014 PMC : PMC3586196 Abstract >>
The largest and one of the most complex ATP-dependent allosteric phosphofructokinase (Pfk) has been found in the methylotrophic yeast, Pichia pastoris. The enzyme is a hetero-oligomer (approximately 1MDa) composed of three distinct subunits (alpha, beta and gamma) with molecular masses of 109, 104 and 41kDa, respectively. While the alpha- and beta-subunits show sequence similarities to other phosphofructokinase subunits, the gamma-subunit does not show high homology to any known protein in the databases. We have determined the first quaternary structure of P. pastoris phosphofructokinase by 3D electron microscopy. Random conical techniques and tomography have been instrumental to ascertain the quality of the sample preparations for structural studies and to obtain a reliable 3D structure. The final reconstruction of P. pastoris Pfk resembles its yeast counterparts with four additional densities, assigned to four gamma-subunits, bridging the N-terminal domains of the four pairs of alpha- and beta-subunits. Our data has evidenced novel interactions between the gamma- and the alpha-subunits comparable in intensity to the interactions, shown by cross-linking and limited proteolytic degradation experiments, between the gamma- and beta-subunits. The structural data provides clear insights into the allosteric fine-tuned regulation of the enzyme by ATP and AMP observed in this yeast species.
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64. |
Yano T,
Yurimoto H,
Sakai Y,
( 2009 ) Activation of the oxidative stress regulator PpYap1 through conserved cysteine residues during methanol metabolism in the yeast Pichia pastoris. PMID : 19502720 : DOI : 10.1271/bbb.90109 Abstract >>
The methylotrophic yeast Pichia pastoris can grow on methanol as sole source of carbon and energy. The first reaction in yeast methanol metabolism, catalyzed by an abundant peroxisomal enzyme, alcohol oxidase, generates high levels of H(2)O(2), but the oxidative stress response during methanol metabolism has not been elucidated. In this study, we isolated the Yap1 homolog of P. pastoris (PpYap1) and analyzed the properties of a PpYAP1-disruption strain. The PpYap1 transcription factor is activated after exposure to various reactive agents, and therefore functions as a regulator of the redox system in P. pastoris. We have also identified PpGPX1, the unique glutathione peroxidase-encoding gene in P. pastoris whose expression is induced by PpYap1. PpGpx1, but not the ScTsa1 or SpTpx1 homolog PpTsa1, functions as a H(2)O(2) sensor and activates PpYap1. This study is the first demonstration of a yeast Yap1 family protein activated during conventional metabolism.
|
65. |
Kurtzman CP,
( 2009 ) Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. PMID : 19760441 : DOI : 10.1007/s10295-009-0638-4 Abstract >>
Pichia pastoris was reassigned earlier to the genus Komagataella following phylogenetic analysis of gene sequences. Since that time, two additional species of Komagataella have been described, K. pseudopastoris and K. phaffii. Because these three species are unlikely to be resolved from the standard fermentation and growth tests used in yeast taxonomy, the identity of biotechnologically important strains of K. pastoris was determined from multigene sequence analyses. Results from this study show that the strain of 'Pichia pastoris' commonly used in gene expression studies is actually K. phaffii.
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66. |
Ahn J,
Hong J,
Park M,
Lee H,
Lee E,
Kim C,
Lee J,
Choi ES,
Jung JK,
Lee H,
( 2009 ) Phosphate-responsive promoter of a Pichia pastoris sodium phosphate symporter. PMID : 19329662 : DOI : 10.1128/AEM.02913-08 PMC : PMC2687300 Abstract >>
To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na(+))-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (P(PHO89)) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P(PHO89) was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1alpha and the glyceraldehyde-3-phosphate dehydrogenase promoter, P(PHO89) exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P(PHO89)-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P(PHO89) for controlled production of recombinant proteins in P. pastoris.
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67. |
Yano T,
Takigami E,
Yurimoto H,
Sakai Y,
( 2009 ) Yap1-regulated glutathione redox system curtails accumulation of formaldehyde and reactive oxygen species in methanol metabolism of Pichia pastoris. PMID : 19252120 : DOI : 10.1128/EC.00007-09 PMC : PMC2669188 Abstract >>
The glutathione redox system, including the glutathione biosynthesis and glutathione regeneration reaction, has been found to play a critical role in the yeast Pichia pastoris during growth on methanol, and this regulation was at least partly executed by the transcription factor PpYap1. During adaptation to methanol medium, PpYap1 transiently localized to the nucleus and activated the expression of the glutathione redox system and upregulated glutathione reductase 1 (Glr1). Glr1 activates the regeneration of the reduced form of glutathione (GSH). Depletion of Glr1 caused a severe growth defect on methanol and hypersensitivity to formaldehyde (HCHO), which could be complemented by addition of GSH to the medium. Disruption of the genes for the HCHO-oxidizing enzymes PpFld1 and PpFgh1 caused a comparable phenotype, but disruption of the downstream gene PpFDH1 did not, demonstrating the importance of maintaining intracellular GSH levels. Absence of the peroxisomal glutathione peroxidase Pmp20 also triggered nuclear localization of PpYap1, and although cells were not sensitive to HCHO, growth on methanol was again severely impaired due to oxidative stress. Thus, the PpYap1-regulated glutathione redox system has two important roles, i.e., HCHO metabolism and detoxification of reactive oxygen species.
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68. |
Prabha L,
Govindappa N,
Adhikary L,
Melarkode R,
Sastry K,
( 2009 ) Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris. PMID : 19028585 : DOI : 10.1016/j.pep.2008.10.021 Abstract >>
Exendin-4 is a naturally occurring 39 amino acid peptide that is useful for the control of Type 2 diabetes. Recombinant Exendin-4, with an extra glycine at the carboxy-terminus (Exdgly), was expressed in the methylotropic yeast Pichia pastoris. A high proportion of the Exdgly molecules secreted into medium were found to be clipped, lacking the first two amino acids (His-Gly) from the N-terminus. Disruption of the P. pastoris homolog of the Saccharomyces cerevisiae dipeptidyl aminopeptidase (STE13) gene in Pichia genome resulted in a clone that expressed N-terminally intact Exdgly. Elimination of N-terminal clipping enhanced the yield and simplified the purification of Exdgly from P. pastoris culture supernatant.
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69. |
Kurtzman CP,
Robnett CJ,
Basehoar-Powers E,
( 2008 ) Phylogenetic relationships among species of Pichia, Issatchenkia and Williopsis determined from multigene sequence analysis, and the proposal of Barnettozyma gen. nov., Lindnera gen. nov. and Wickerhamomyces gen. nov. PMID : 18671746 : DOI : 10.1111/j.1567-1364.2008.00419.x Abstract >>
Relationships among species assigned to the yeast genera Pichia, Issatchenkia and Williopsis, which are characterized by the ubiquinone CoQ-7 and inability to utilize methanol, were phylogenetically analyzed from nucleotide sequence divergence in the genes coding for large and small subunit rRNAs and for translation elongation factor-1alpha. From this analysis, the species separated into five clades. Species of Issatchenkia are members of the Pichia membranifaciens clade and are proposed for transfer to Pichia. Pichia dryadoides and Pichia quercuum are basal members of the genus Starmera. Williopsis species are dispersed among hat-spored taxa in each of the remaining three clades, which are proposed as the new genera Barnettozyma, Lindnera and Wickerhamomyces. Lineages previously classified as varieties of Pichia kluyveri, 'Issatchenkia'scutulata, Starmera amethionina and 'Williopsis'saturnus are elevated to species rank based on sequence comparisons.
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70. |
Farré JC,
Manjithaya R,
Mathewson RD,
Subramani S,
( 2008 ) PpAtg30 tags peroxisomes for turnover by selective autophagy. PMID : 18331717 : DOI : 10.1016/j.devcel.2007.12.011 PMC : PMC3763908 Abstract >>
Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction with peroxins, and during pexophagy it colocalizes transiently at the preautophagosomal structure (PAS) and interacts with the autophagy machinery. PpAtg30 is required for formation of pexophagy intermediates, such as the micropexophagy apparatus (MIPA) and the pexophagosome (Ppg). During pexophagy, PpAtg30 undergoes multiple phosphorylations, at least one of which is required for pexophagy. PpAtg30 overexpression stimulates pexophagy even under peroxisome-induction conditions, impairing peroxisome biogenesis. Therefore, PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy.
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71. |
Yan M,
Rachubinski DA,
Joshi S,
Rachubinski RA,
Subramani S,
( 2008 ) Dysferlin domain-containing proteins, Pex30p and Pex31p, localized to two compartments, control the number and size of oleate-induced peroxisomes in Pichia pastoris. PMID : 18094040 : DOI : 10.1091/mbc.e07-10-1042 PMC : PMC2262989 Abstract >>
Yarrowia lipolytica Pex23p and Saccharomyces cerevisiae Pex30p, Pex31p, and Pex32p comprise a family of dysferlin domain-containing peroxins. We show that the deletion of their Pichia pastoris homologues, PEX30 and PEX31, does not affect the function or division of methanol-induced peroxisomes but results in fewer and enlarged, functional, oleate-induced peroxisomes. Synthesis of Pex30p is constitutive, whereas that of Pex31p is oleate-induced but at a much lower level relative to Pex30p. Pex30p interacts with Pex31p and is required for its stability. At steady state, both Pex30p and Pex31p exhibit a dual localization to the endoplasmic reticulum (ER) and peroxisomes. However, Pex30p is localized mostly to the ER, whereas Pex31p is predominantly on peroxisomes. Consistent with ER-to-peroxisome trafficking of these proteins, Pex30p accumulates on peroxisomes upon overexpression of Pex31p. Additionally, Pex31p colocalizes with Pex30p at the ER in pex19Delta cells and can be chased from the ER to peroxisomes in a Pex19p-dependent manner. The dysferlin domains of Pex30p and Pex31p, which are dispensable for their interaction, stability, and subcellular localization, are essential for normal peroxisome number and size. The growth environment-specific role of these peroxins, their dual localization, and the function of their dysferlin domains provide novel insights into peroxisome morphogenesis.
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72. |
Miele R,
Barra D,
Bonaccorsi di Patti MC,
( 2007 ) A GATA-type transcription factor regulates expression of the high-affinity iron uptake system in the methylotrophic yeast Pichia pastoris. PMID : 17592720 : DOI : 10.1016/j.abb.2007.05.020 Abstract >>
The ferroxidase Fet3 and the permease Ftr1 constitute a well-conserved high-affinity iron uptake system in yeast. We have investigated the mechanism of transcriptional regulation of Fet3 in the methylotrophic yeast Pichia pastoris. Isolation and functional analysis of the Fet3 promoter indicate that a GATA sequence element plays a role in iron-dependent expression of Fet3. A GATA-type transcription factor, which we have named Fep1, has been partially cloned and it is shown to belong to the family of iron-responsive fungal GATA-factors. These factors share the presence of two Cys(2)-Cys(2) zinc-finger motifs and a set of four conserved cysteines, and are involved in the regulation of siderophore biosynthesis and/or high-affinity iron uptake. Disruption of the FEP1 gene in P. pastoris leads to constitutively high expression of Fet3, irrespective of iron levels, indicating that Fep1 is a transcriptional repressor. EMSA analyses evidence that Fep1 binds to DNA only in the presence of iron.
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73. |
Zhang X,
Li M,
Wei D,
Xing L,
( 2008 ) Identification and characterization of a novel yeast omega3-fatty acid desaturase acting on long-chain n-6 fatty acid substrates from Pichia pastoris. PMID : 17914745 : DOI : 10.1002/yea.1546 Abstract >>
A cDNA sequence putatively encoding a omega(3)-fatty acid desaturase gene was isolated from methylotrophic yeast Pichia pastoris GS115. The deduced amino acid sequence of this cloned cDNA showed high identity to known fungal omega(3)-fatty acid desaturases. Functional identification of this gene heterologously in Saccharomyces cerevisiae strain INVScl indicated that the deduced amino acid sequence exhibited omega(3)-fatty acid desaturase activity. The newly identified omega(3)-fatty acid desaturase, named Pp-FAD3, is novel because it showed broad n-6 fatty acid substrate specificity by its ability to convert all the 18-carbon and 20-carbon n-6 substrates examined to the corresponding n-3 fatty acids, with an approximately equivalent high conversion rate. Pp-FAD3 is the first known yeast omega(3)-fatty acid desaturase to act on long-chain n-6 fatty acid substrates. Heterologous expression of the newly identified omega(3) desaturase in different hosts will be an alternative method to increase the flow of n-6 fatty acid intermediates into their n-3 derivatives.
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74. |
Bharucha N,
Liu Y,
Papanikou E,
McMahon C,
Esaki M,
Jeffrey PD,
Hughson FM,
Glick BS,
( 2013 ) Sec16 influences transitional ER sites by regulating rather than organizing COPII. PMID : 24006484 : DOI : 10.1091/mbc.E13-04-0185 PMC : PMC3814151 Abstract >>
During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.
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75. |
( 1997 ) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. PMID : 9047342 : DOI : 10.1016/s0378-1119(96)00675-0 Abstract >>
We report the cloning and sequence of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) from the yeast Pichia pastoris. The gene is predicted to encode a 35.4-kDa protein with significant sequence similarity to glyceraldehyde-3-phosphate dehydrogenases from other organisms. Promoter studies in P. pastoris using bacterial beta-lactamase as a reporter showed that the GAP promoter (P(GAP)) is constitutively expressed, although its strength varies depending on the carbon source used for cell growth. Expression of beta-lactamase under control of P(GAP) in glucose-grown cells was significantly higher than under control of the commonly employed alcohol oxidase 1 promoter (P(AOX1)) in methanol-grown cells. As an example of the use of P(GAP), we showed that beta-lactamase synthesized under transcriptional control of P(GAP) is correctly targeted to peroxisomes by addition of either a carboxy-terminal or an amino-terminal peroxisomal targeting signal. P(GAP) has been successfully utilized for synthesis of heterologous proteins from bacterial, yeast, insect and mammalian origins, and therefore is an attractive alternative to P(AOX1) in P. pastoris.
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76. |
( 1996 ) Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor. PMID : 8858165 : DOI : 10.1083/jcb.135.1.85 PMC : PMC2121023 Abstract >>
Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome-associated Pex5p by approximately 40-fold. Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins. We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.
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77. |
( 1993 ) Cloning and characterization of PAS5: a gene required for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. PMID : 8227124 : DOI : 10.1083/jcb.123.3.535 PMC : PMC2200126 Abstract >>
The biogenesis and maintenance of cellular organelles is of fundamental importance in all eukaryotic cells. One such organelle is the peroxisome. The establishment of a genetic system to study peroxisome biogenesis in the methylotrophic yeast Pichia pastoris has yielded many different complementation groups of peroxisomal assembly (pas) or peroxisome-deficient (per) mutants. Each appears to be deficient in functional peroxisomes. One of these mutants, pas5, has been characterized, complemented, and the gene sequenced. Ultrastructural studies show that normal peroxisomes are not present in pas5, but aberrant peroxisomal structures resembling "membranous ghosts" are frequently observed. The "peroxisome ghosts" appear to be induced and segregated to daughter cells normally. Biochemical fractionation analysis of organelles of the pas5 mutant reveals that peroxisomal matrix enzymes are induced normally but are found mostly in the cytosol. However, purification of peroxisome ghosts from the mutant shows that small amounts (< 5%) of matrix enzymes are imported. The PAS5 gene was cloned and found to encode a 127-kD protein, which contains a 200-amino acid-long region of homology with PAS1, NEM-sensitive factor (NSF), and other related ATPases. Weak homology to a yeast myosin was also observed. The gene is not essential for growth on glucose but is essential for growth on oleic acid and methanol. The role of PAS5 in peroxisome biogenesis is discussed.
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78. |
( 1996 ) Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins. PMID : 8670828 : PMC : PMC451890 Abstract >>
Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity.
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79. |
( 1996 ) Protein translocation into peroxisomes. PMID : 8955066 : DOI : 10.1074/jbc.271.51.32483 Abstract >>
N/A
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80. |
( 1996 ) The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1. PMID : 8628321 : DOI : 10.1128/mcb.16.5.2527 PMC : PMC231242 Abstract >>
We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1.
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81. |
( 1994 ) Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesis. PMID : 7962088 : DOI : 10.1083/jcb.127.5.1259 PMC : PMC2120248 Abstract >>
Several groups have reported the cloning and sequencing of genes involved in the biogenesis of yeast peroxisomes. Yeast strains bearing mutations in these genes are unable to grow on carbon sources whose metabolism requires peroxisomes, and these strains lack morphologically normal peroxisomes. We report the cloning of Pichia pastoris PAS1, the homologue (based on a high level of protein sequence similarity) of the Saccharomyces cerevisiae PAS1. We also describe the creation and characterization of P. pastoris pas1 strains. Electron microscopy on the P. pastoris pas1 cells revealed that they lack morphologically normal peroxisomes, and instead contain membrane-bound structures that appear to be small, mutant peroxisomes, or "peroxisome ghosts." These "ghosts" proliferated in response to induction on peroxisome-requiring carbon sources (oleic acid and methanol), and they were distributed to daughter cells. Biochemical analysis of cell lysates revealed that peroxisomal proteins are induced normally in pas1 cells. Peroxisome ghosts from pas1 cells were purified on sucrose gradients, and biochemical analysis showed that these ghosts, while lacking several peroxisomal proteins, did import varying amounts of several other peroxisomal proteins. The existence of detectable peroxisome ghosts in P. pastoris pas1 cells, and their ability to import some proteins, stands in contrast with the results reported by Erdmann et al. (1991) for the S. cerevisiae pas1 mutant, in which they were unable to detect peroxisome-like structures. We discuss the role of PAS1 in peroxisome biogenesis in light of the new information regarding peroxisome ghosts in pas1 cells.
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82. |
( 1996 ) The gamma subfamily of DNA polymerases: cloning of a developmentally regulated cDNA encoding Xenopus laevis mitochondrial DNA polymerase gamma. PMID : 8628681 : DOI : 10.1093/nar/24.8.1481 PMC : PMC145809 Abstract >>
We used the known sequence of the Saccharomyces cerevisiae DNA polymerase gamma to clone the genes or cDNAs encoding this enzyme in two other yeasts, Pychia pastoris and Schizosaccharomyces pombe, and one higher eukaryote, Xenopus laevis. To confirm the identity of the final X.laevis clone, two antisera raised against peptide sequences were shown to react with DNA polymerase gamma purified from X.laevis oocyte mitochondria. A developmentally regulated 4.6 kb mRNA is recognized on Northern blots of oocyte RNA using the X.laevis cDNA. Comparison of the four DNA polymerase gamma gene sequences revealed several highly conserved sequence blocks, comprising an N-terminal 3'-->5'exonuclease domain and a C-terminal polymerase active center interspersed with gamma-specific gene sequences. The consensus sequences for the DNA polymerase gamma exonuclease and polymerase domains show extensive sequence similarity to DNA polymerase I from Escherichia coli. Sequence conservation is greatest for residues located near the active centers of the exo and pol domains of the E.coli DNA polymerase I structure. The domain separating the exonuclease and polymerase active sites is larger in DNA polymerase gamma than in other members of family A (DNA polymerase I-like) polymerases. The S.cerevisiae DNA polymerase gamma is atypical in that it includes a 240 residue C-terminal extension that is not found in the other members of the DNA polymerase gamma family, or in other family A DNA polymerases.
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83. |
( 1994 ) The Pichia pastoris PAS4 gene encodes a ubiquitin-conjugating enzyme required for peroxisome assembly. PMID : 8063827 : Abstract >>
We report here the cloning and initial characterization of PAS4, a gene required for peroxisome assembly in the yeast Pichia pastoris. The PAS4 gene encodes a 24-kDa protein (Pas4p) that is located on the cytoplasmic surface of peroxisomes and is induced during peroxisome proliferation. Analysis of the Pas4p sequence revealed a high degree of similarity to ubiquitin-conjugating enzymes, particularly in the region surrounding the putative active-site cysteine residue with which ubiquitin forms a thioester bond. As expected for a ubiquitin-conjugating enzyme, substitution of alanine or serine for the conserved active-site cysteine residue abolished PAS4 function. In addition, a small amount of a 32 kDa form of Pas4p (the predicted size of a Pas4p-ubiquitin conjugate) was detected both in vivo and in vitro. This species was eliminated by reducing agents and was not detected in the cysteine to alanine substitution mutant, suggesting that it is a Pas4p-ubiquitin conjugate. Using a yeast strain that overexpresses a Myc-ubiquitin fusion protein, we demonstrate directly that this conjugate contains ubiquitin. We conclude from these observations that PAS4 is a member of the ubiquitin-conjugating enzyme gene family and that one or more ubiquitination reactions are required for peroxisome assembly.
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84. |
( 1993 ) The pas8 mutant of Pichia pastoris exhibits the peroxisomal protein import deficiencies of Zellweger syndrome cells--the PAS8 protein binds to the COOH-terminal tripeptide peroxisomal targeting signal, and is a member of the TPR protein family. PMID : 8098333 : DOI : 10.1083/jcb.121.4.761 PMC : PMC2119792 Abstract >>
We previously described the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas mutants). We describe the characterization of one of these mutants, pas8, and the cloning of the PAS8 gene. The pas8 mutant is deficient for growth, but not for division or segregation of peroxisomes, or for induction of peroxisomal proteins. Two distinct peroxisomal targeting signals, PTS1 and PTS2, have been identified that are sufficient to direct proteins to the peroxisomal matrix. We show that the pas8 mutant is deficient in the import of proteins with the PTS1, but not the PTS2, targeting signal. This is the same import deficiency as that found in cells from patients with the lethal human peroxisomal disorder Zellweger syndrome. Cloning and sequencing of the PAS8 gene reveals that it is a novel member of the tetratricopeptide repeat gene family. Antibodies raised against bacterially expressed PAS8 are used to show that PAS8 is a peroxisomal, membrane-associated protein. Also, we have found that in vitro translated PAS8 protein is capable of binding the PTS1 targeting signal specifically, raising the possibility that PAS8 is a PTS1 receptor.
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85. |
Veenhuis M,
Cregg JM,
Liu H,
Tan X,
Russell KA,
( 1995 ) PER3, a gene required for peroxisome biogenesis in Pichia pastoris, encodes a peroxisomal membrane protein involved in protein import. PMID : 7738036 : DOI : 10.1074/jbc.270.18.10940 Abstract >>
PER genes are essential for the biogenesis of peroxisomes in the yeast Pichia pastoris. Here we describe the cloning of PER3 and functional characterization of its product Per3p. The PER3 sequence predicts that Per3p is a 713-amino acid (81-kDa) hydrophobic protein with at least three potential membrane-spanning domains. We show that Per3p is a membrane protein of the peroxisome. Methanol- or oleate-induced cells of per3-1, a mutant strain generated by chemical mutagenesis, lack normal peroxisomes but contain numerous abnormal vesicular structures. The vesicles contain thiolase, a PTS2 protein, but only a small portion of several other peroxisomal enzymes, including heterologously expressed luciferase, a PTS1 protein. These results suggest that the vesicles in per3-1 cells are peroxisomal remnants similar to those observed in cells of patients with the peroxisomal disorder Zellweger syndrome, and that the mutant is deficient in PTS1 but not PTS2 import. In a strain in which most of PER3 was deleted, peroxisomes as well as peroxisomal remnants appeared to be completely absent, and both PTS1- and PTS2-containing enzymes were located in the cytosol. We propose that Per3p is an essential component of the machinery required for import of all peroxisomal matrix proteins and is composed of independent domains involved in the import of specific PTS groups.
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86. |
Crane DI,
Gould SJ,
( N/A ) The Pichia pastoris HIS4 gene: nucleotide sequence, creation of a non-reverting his4 deletion mutant, and development of HIS4-based replicating and integrating plasmids. PMID : 7874737 : Abstract >>
We have obtained a clone of the Pichia pastoris HIS4 gene and have determined its nucleotide sequence. Based upon its deduced amino-acid sequence, the product of the P. pastoris HIS4 gene has the same structural organization as the Saccharomyces cerevisiae His4 protein and appears to encode a trifunctional enzyme catalyzing the second (phosphoribosyl-ATP pyrophosphohydrolase), third (phosphoribosyl-AMP cyclohydrolase), and tenth (histidinol dehydrogenase) steps in histidine biosynthesis. The chromosomal copy of the HIS4 gene was disrupted by homologous recombination, creating the strain SGY58. The his4 delta deletion mutation in this strain lacks the entire coding region of this gene and has a reversion rate that is undetectable. A set of complementary plasmids that carry the HIS4 gene was also developed. Among these are nine E. coli-P. pastoris shuttle vectors that transform the his4 delta deletion mutant at high efficiency and an integration vector for creating site-specific alterations of the P. pastoris genome.
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87. |
Kumagai MH,
Sverlow GG,
della-Cioppa G,
Grill LK,
( 1993 ) Conversion of starch to ethanol in a recombinant Saccharomyces cerevisiae strain expressing rice alpha-amylase from a novel Pichia pastoris alcohol oxidase promoter. PMID : 7763608 : Abstract >>
A recombinant Saccharomyces cerevisiae, expressing and secreting rice alpha-amylase, converts starch to ethanol. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of the promoter from a newly described Pichia pastoris alcohol oxidase genomic clone. The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed. A highly conserved sequence (TTG-N3-GCTTCCAA-N5-TGGT) was found in the 5' flanking regions of alcohol oxidase, methanol oxidase, and dihydroxyacetone synthase genes in Pichia pastoris, Hansenula polymorpha, and Candida boidinii S2. The yeast strain containing the ZZA1-OS103 fusion secreted biologically active enzyme into the culture media while fermenting soluble starch.
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88. |
Terlecky SR,
Nuttley WM,
McCollum D,
Sock E,
Subramani S,
( 1995 ) The Pichia pastoris peroxisomal protein PAS8p is the receptor for the C-terminal tripeptide peroxisomal targeting signal. PMID : 7641682 : PMC : PMC394437 Abstract >>
The peroxisomal targeting signal 1 (PTS1), consisting of a C-terminal tripeptide (SKL and variants), directs polypeptides to the peroxisome matrix in evolutionarily diverse organisms. Previous studies in the methylotrophic yeast Pichia pastoris identified a 68 kDa protein, PAS8p, as a potential component of the PTS1 import machinery. We now report several new properties of this molecule which, taken together, show that it is the peroxisomal PTS1 receptor. (i) PAS8p is localized to and tightly associated with the cytoplasmic side of the peroxisomal membrane, (ii) peroxisomes of wild-type, but not of pas8 delta (null) mutant, P.pastoris cells bind a PTS1-containing peptide (CRYHLKPLQSKL), (iii) CRYHLKPLQSKL can be cross-linked to PAS8p after binding at the peroxisome membrane and (iv) purified PAS8p binds CRYHLKPLQSKL with high affinity (nanomolar dissociation constant). In addition, the tetratricopeptide repeat (TPR) domain of PAS8p is identified as the PTS1 binding region.
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89. |
Scorer CA,
Clare JJ,
McCombie WR,
Romanos MA,
Sreekrishna K,
( 1994 ) Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. PMID : 7764433 : Abstract >>
Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins. Expression vectors are based on the methanol-inducible AOX1 promoter and are integrated into the host chromosome. In most cases high copy number integration has been shown to be important for high-level expression. Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones. In this paper we report the use of vectors containing the Tn903 kanr gene conferring G418-resistance. Initial experiments demonstrated that copy number showed a tight correlation with drug-resistance. Using a G418 growth inhibition screen, we readily isolated a series of transformants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV-1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels. Northern blot analysis indicated that ENV mRNA levels from a single-copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA. We have also developed protocols for the selection, using G418, of high copy number transformants following spheroplast transformation or electroporation. We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.
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90. |
Kock M,
Brückner S,
Wozniak N,
Maestre-Reyna M,
Veelders M,
Schlereth J,
Mösch HU,
Essen LO,
( 2018 ) Structural and Functional Characterization of PA14/Flo5-Like Adhesins From Komagataella pastoris. PMID : 30425696 : DOI : 10.3389/fmicb.2018.02581 PMC : PMC6218569 Abstract >>
Cell-cell and cell-substrate based adhesion of yeasts are major determinants of their adoption of different life styles. Genome-mining of ascomycetous GPI-anchored cell wall proteins with lectin-like PA14 domains identified a unique class of putative adhesins in the clade of methylotrophic Komagataella yeasts, many of which are known to colonize plants and insects involving yet unknown adhesion mechanisms. Here, we report the functional and structural analysis of two of its members: KpFlo1 (=Cea1), that is highly specific for terminal N-acetylglucosamine moieties, and KpFlo2, which represents an orphan lectin with intact binding site but unknown specificity. Crystal structures of the Cea1 adhesion domain complexed to N-acetylglucosamine and N,N'-diacetylchitobiose reveal a Ca2+-dependent binding mode that differs from other members of the PA14/Flo5 adhesin family. Heterologous expression of Cea1A in Saccharomyces cerevisiae promotes cellular adhesion to non-reducing ends of non-crystalline chitin. Overall, our data suggest that high-affinity recognition of �]-GlcNAc-capped glycans by Cea1 enable Komagataella species to interact with surface cues present in fungi and insects.
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91. |
Kalish JE,
Theda C,
Morrell JC,
Berg JM,
Gould SJ,
( 1995 ) Formation of the peroxisome lumen is abolished by loss of Pichia pastoris Pas7p, a zinc-binding integral membrane protein of the peroxisome. PMID : 7565793 : DOI : 10.1128/mcb.15.11.6406 PMC : PMC230892 Abstract >>
We have cloned and sequenced PAS7, a gene required for peroxisome assembly in the yeast Pichia pastoris. The product of this gene, Pas7p, is a member of the C3HC4 superfamily of zinc-binding proteins. Point mutations that alter conserved residues of the C3HC4 motif abolish PAS7 activity and reduce zinc binding, suggesting that Pas7p binds zinc in vivo and that zinc binding is essential for PAS7 function. As with most pas mutants, pas7 cells exhibit a pronounced deficiency in import of peroxisomal matrix proteins that contain either the type 1 peroxisomal targeting signal (PTS1) or the type 2 PTS (PTS2). However, while other yeast and mammalian pas mutants accumulate ovoid, vesicular peroxisomal intermediates, loss of Pas7p leads to accumulation of membrane sheets and vesicles which lack a recognizable lumen. Thus, Pas7p appears to be essential for protein translocation into peroxisomes as well as formation of the lumen of the organelle. Consistent with these data, we find that Pas7p is an integral peroxisomal membrane protein which is entirely resistant to exogenous protease and thus appears to reside completely within the peroxisome. Our observations suggest that the function of Pas7p defines a previously unrecognized step in peroxisome assembly: formation of the peroxisome lumen. Furthermore, because the peroxisomal intermediates in the pas7 delta mutant proliferate in response to peroxisome-inducing environmental conditions, we conclude that Pas7p is not required for peroxisome proliferation.
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92. |
Payne WE,
Gannon PM,
Kaiser CA,
( 1995 ) An inducible acid phosphatase from the yeast Pichia pastoris: characterization of the gene and its product. PMID : 7557473 : DOI : 10.1016/0378-1119(95)00379-k Abstract >>
To develop the budding yeast Pichia pastoris (Pp) as a model system for the study of protein secretion, we have characterized a secreted acid phosphatase (Pho1p) from this yeast. Pho1p can be induced 100-fold by starvation for phosphate. The enzyme was purified to homogeneity from a cell-wall extract by DEAE-Sepharose chromatography. We selected mutants that lacked extracellular phosphatase activity and the gene (PHO1) encoding Pho1p was isolated from a recombinant plasmid library of Pp DNA by complementation of the mutant defect. PHO1 encodes a protein of 468 amino acids (aa) with homology to repressible acid phosphatases from other yeast species. The sequence contains a 15-aa N-terminal signal sequence and six potential N-linked glycosylation sites. Antiserum to Pho1p was used to show that Pho1p transits the Pp secretory pathway in less than 5 min.
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93. |
( 2013 ) Phosphorylation of mitophagy and pexophagy receptors coordinates their interaction with Atg8 and Atg11. PMID : 23559066 : DOI : 10.1038/embor.2013.40 PMC : PMC3642380 DOI : 10.1038/embor.2013.40 PMC : PMC3642380 Abstract >>
The selective autophagy receptors Atg19 and Atg32 interact with two proteins of the core autophagic machinery: the scaffold protein Atg11 and the ubiquitin-like protein Atg8. We found that the Pichia pastoris pexophagy receptor, Atg30, also interacts with Atg8. Both Atg30 and Atg32 interactions are regulated by phosphorylation close to Atg8-interaction motifs. Extending this finding to Saccharomyces cerevisiae, we confirmed phosphoregulation for the mitophagy and pexophagy receptors, Atg32 and Atg36. Each Atg30 molecule must interact with both Atg8 and Atg11 for full functionality, and these interactions occur independently and not simultaneously, but rather in random order. We present a common model for the phosphoregulation of selective autophagy receptors.
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94. |
Ehara H,
Yokoyama T,
Shigematsu H,
Yokoyama S,
Shirouzu M,
Sekine SI,
( 2017 ) Structure of the complete elongation complex of RNA polymerase II with basal factors. PMID : 28775211 : DOI : 10.1126/science.aan8552 Abstract >>
In the early stage of transcription, eukaryotic RNA polymerase II (Pol II) exchanges initiation factors with elongation factors to form an elongation complex for processive transcription. Here we report the structure of the Pol II elongation complex bound with the basal elongation factors Spt4/5, Elf1, and TFIIS. Spt4/5 (the Spt4/Spt5 complex) and Elf1 modify a wide area of the Pol II surface. Elf1 bridges the Pol II central cleft, completing a "DNA entry tunnel" for downstream DNA. Spt4 and the Spt5 NGN and KOW1 domains encircle the upstream DNA, constituting a "DNA exit tunnel." The Spt5 KOW4 and KOW5 domains augment the "RNA exit tunnel," directing the exiting nascent RNA. Thus, the elongation complex establishes a completely different transcription and regulation platform from that of the initiation complexes.
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95. |
( 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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96. |
( 1998 ) Cloning and sequence analysis of the Pichia pastoris TRP1, IPP1 and HIS3 genes. PMID : 9818724 : DOI : 10.1002/(SICI)1097-0061(19980630)14:9<861::AID-YEA276>3.0.CO;2-N Abstract >>
The Pichia pastoris TRP1 and HIS3 genes were cloned by complementation of the Saccharomyces cerevisiae trip1 and his3 mutants, respectively, and their nucleotide sequence was determined. The P. pastoris TRP1 gene includes an open reading frame (ORF) of 714 nucleotides corresponding to a polypeptide of 237 amino acids whose sequence shares about 40% identity with that of TRP1 encoding proteins in other yeast species. DNA sequencing showed that an ORF of 858 nucleotides, encoding a protein of 285 amino acids with high homology to inorganic pyrophosphatases (IPP1), is located downstream of the P. pastoris TRP1 gene. Both genes converge in this chromosomal region, showing a genetic organization analogous to that found in the Kluyveromyces lactis genome. The P. pastoris HIS3 gene possesses an ORF of 675 nucleotides, encoding a polypeptide of 224 amino acids which shows 74.1% identity to the homologous S. cerevisiae protein. The hexameric consensus GCN4 binding sequence (TGACTC), characteristic of many amino acid biosynthetic genes, is present in the promoter region.
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97. |
( 1998 ) A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. PMID : 9714758 : DOI : 10.1016/s0378-1119(98)00315-1 Abstract >>
In methylotrophic yeasts, glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the metabolism of methanol as a carbon source and certain alkylated amines such as methylamine as nitrogen sources. We describe the isolation and characterization of the FLD1 gene from the yeast Pichia pastoris. The gene contains a single short intron with typical yeast-splicing signals near its 5' end, the first intron to be demonstrated in this yeast. The predicted FLD1 product (Fld1p) is a protein of 379 amino acids (approx. 40 kDa) with 71% identity to the FLD protein sequence from the n-alkane-assimilating yeast Candida maltosa and 61-65% identity with dehydrogenase class III enzymes from humans and other higher eukaryotes. Using beta-lactamase as a reporter, we show that the FLD1 promoter (PFLD1) is strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Furthermore, with either methanol or methylamine induction, levels of beta-lactamase produced under control of PFLD1 are comparable to those obtained with the commonly used alcohol oxidase I gene promoter (PAOX1). Thus, PFLD1 is an attractive alternative to PAOX1 for expression of foreign genes in P. pastoris, allowing the investigator a choice of carbon (methanol) or nitrogen source (methylamine) regulation with the same expression strain.
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98. |
( 1998 ) The Pichia pastoris dihydroxyacetone kinase is a PTS1-containing, but cytosolic, protein that is essential for growth on methanol. PMID : 9675820 : DOI : 10.1002/(SICI)1097-0061(19980615)14:8<759::AID-YEA275>3.0.CO;2-A Abstract >>
Dihydroxyacetone kinase (DAK) is essential for methanol assimilation in methylotrophic yeasts. We have cloned the DAK gene from Pichia pastoris by functional complementation of a mutant that was unable to grow on methanol. An open reading frame of 1824 bp was identified that encodes a 65.3 kDa protein with high homology to DAK from Saccharomyces cerevisiae. Although DAK from P. pastoris contained a C-terminal tripeptide, TKL, which we showed can act as a peroxisomal targeting signal when fused to the green fluorescent protein, the enzyme was primarily cytosolic. The TKL tripeptide was not required for the biochemical function of DAK because a deletion construct lacking the DNA encoding this tripeptide was able to complement the P. pastoris dak delta mutant. Peroxisomes, which are essential for growth of P. pastoris on methanol, were present in the dak delta mutant and the import of peroxisomal proteins was not disturbed. The dak delta mutant grew at normal rates on glycerol and oleate media. However, unlike the wild-type cells, the dak delta mutant was unable to grow on methanol as the sole carbon source but was able to grow on dihydroxyacetone at a much slower rate. The metabolic pathway explaining the reduced growth rate of the dak delta mutant on dihydroxyacetone is discussed. The nucleotide sequence reported in this paper has been submitted to GenBank with Accession Number AF019198.
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99. |
( 1998 ) Isolation of the Pichia pastoris PYC1 gene encoding pyruvate carboxylase and identification of a suppressor of the pyc phenotype. PMID : 9639311 : DOI : 10.1002/(SICI)1097-0061(199805)14:7<647::AID-YEA269>3.0.CO;2-L Abstract >>
We have cloned and characterized a gene encoding pyruvate carboxylase from the methylotrophic yeast Pichia pastoris. Disruption of this gene produced inability to grow in minimal medium with glucose as carbon source and ammonium as nitrogen source. Growth was possible with aspartate or glutamate as nitrogen source. The gene PpPYC1 expressed from its own promoter was able to rescue the phenotype of Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. In a P. pastoris strain carrying a disrupted PpPYC1 gene we have isolated spontaneous mutants able to grow in non-permissive conditions. In a mutant strain grown in glucose several enzymes sensitive to catabolite repression were derepressed. The strain also had elevated levels of glutamate dehydrogenase (NAD) both in repressed and derepressed conditions.
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100. |
( 1998 ) A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris. PMID : 9472033 : DOI : 10.1083/jcb.140.4.807 PMC : PMC2141746 Abstract >>
Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2). Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor. The pex7Delta mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism. In contrast, pex7Delta cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1. Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal. This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes. In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata. The corresponding PpPex7p mutant proteins were stably expressed in P. pastoris, but they failed to complement the pex7Delta mutant and were impaired in binding to the PTS2 sequence.
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