Isolation of Pichia pastoris genes involved in ER-to-Golgi transport.

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.

The Yeast Proteome Database (YPD): a model for the organization and presentation of genome-wide functional data.

The Yeast Proteome Database (YPD) is a model for the organization and presentation of comprehensive protein information. Based on the detailed curation of the scientific literature for the yeast Saccharomyces cerevisiae, YPD contains more than 50 000 annotations lines derived from the review of 8500 research publications. The information concerning each of the approximately 6100 yeast proteins is structured around a convenient one-page format, the Yeast Protein Report, with additional information provided as pop-up windows. Protein classification schema have been revised this year, defining each protein's cellular role, function and pathway, and adding a Functional to the Yeast Protein Report. These changes provide the user with a succinct summary of the protein's function and its place in the biology of the cell, and they enhance the power of YPD Search functions. Precalculated sequence alignments have been added, to provide a crossover point for comparative genomics. The first transcript profiling data has been integrated into the YPD Protein Reports, providing the framework for the presentation of genome-wide functional data. The Yeast Proteome Database can be accessed on the Web at http://www.proteome.com/YPDhome.html

The Yeast Protein Database (YPD): a curated proteome database for Saccharomyces cerevisiae.

The Yeast Protein Database (YPD) is a curated database for the proteome of Saccharomyces cerevisiae . It consists of approximately 6000 Yeast Protein Reports, one for each of the known or predicted yeast proteins. Each Yeast Protein Report is a one-page presentation of protein properties, annotation lines that summarize findings from the literature, and references. In the past year, the number of annotation lines has grown from 25 000 to approximately 35 000, and the number of articles curated has grown from approximately 3500 to >5000. Recently, new data types have been included in YPD: protein-protein interactions, genetic interactions, and regulators of gene expression. Finally, a new layer of information, the YPD Protein Minireviews, has recently been introduced. The Yeast Protein Database can be found on the Web at http://www.proteome.com/YPDhome. html

Proteome studies of Saccharomyces cerevisiae: identification and characterization of abundant proteins.

Two-dimensional (2-D) gel electrophoresis can now be coupled with protein identification techniques and genome sequence information for direct detection, identification, and characterization of large numbers of proteins from microbial organisms. 2-D electrophoresis, and new protein identification techniques such as amino acid composition, are proteome research techniques in that they allow direct characterization of many proteins at the same time. Another new tool important for yeast proteome research is the Yeast Protein Database (YPD), which provides the sequence-derived protein properties needed for spot identification and tabulations of the currently known properties of the yeast proteins. Studies presented here extend the yeast 2-D protein map to 169 identified spots based upon the recent completion of the yeast genome sequence, and they show that methods of spot identification based on predicted isoelectric point, predicted molecular mass, and determination of partial amino acid composition from radiolabeled gels are powerful enough for the identification of at least 80% of the spots representing abundant proteins. Comparison of proteins predicted by YPD to be detectable on 2-D gels based on calculated molecular mass, isoelectric point and codon bias (a predictor of abundance) with proteins identified in this study suggests that many glycoproteins and integral membrane proteins are missing from the 2-D gel patterns. Using the 2-D gel map and the information available in YDP, 2-D gel experiments were analyzed to characterize the yeast proteins associated with: (i) an environmental change (heat shock), (ii) a temperature-sensitive mutation (the prp2 mRNA splicing mutant), (iii) a mutation affecting post-translational modification (N-terminal acetylation), and (iv) a purified subcellular fraction (the ribosomal proteins). The methods used here should allow future extension of these studies to many more proteins of the yeast proteome.

Yeast Protein database (YPD): a database for the complete proteome of Saccharomyces cerevisiae.

The Yeast Protein Database (YPD) is a database for the proteins of the budding yeast,Saccharomyces cerevisiae. YPD is the first annotated database for the complete proteome of any organism. Now that the complete genome sequence of yeast is available, YPD contains entries for each of the characterized proteins and for each of the uncharacterized proteins predicted from the sequence. Contained in YPD are the calculated properties of each protein such as molecular weight and isoelectric point, experimentally determined properties such as subcellular localization and post-translational modifications, and extensive annotations from the yeast literature. YPD contains 25 000 lines of textual annotation that describe the known functions, mutant phenotypes, interactions, and other properties for the approximately 6000 proteins in the yeast proteome. The information in YPD is updated daily, and it is available on the World Wide Web at http://www.proteome.com/YPDhome.html .

An inducible acid phosphatase from the yeast Pichia pastoris: characterization of the gene and its product.

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.

A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation.

We identified a putative Saccharomyces cerevisiae homolog of a phosphoinositide-specific phospholipase C (PI-PLC) gene, PLC1, which encodes a protein most similar to the delta class of PI-PLC enzymes. The PLC1 gene was isolated during a study of yeast strains that exhibit defects in chromosome segregation. plc1-1 cells showed a 10-fold increase in aberrant chromosome segregation compared with the wild type. Molecular analysis revealed that PLC1 encodes a predicted protein of 101 kDa with approximately 50 and 26% identity to the highly conserved X and Y domains of PI-PLC isozymes from humans, bovines, rats, and Drosophila melanogaster. The putative yeast protein also contains a consensus EF-hand domain that is predicted to bind calcium. Interestingly, the temperature-sensitive and chromosome missegregation phenotypes exhibited by plc1-1 cells were partially suppressed by exogenous calcium.

In vivo genomic footprint of a yeast centromere.

We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDEI, CDEII, and CDEIII) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wild-type cells shows that some guanines within the centromere DNA are protected from methylation by dimethyl sulfate. These results are consistent with studies demonstrating that yeast cells contain sequence-specific centromere DNA-binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function.