Gene_locus Report for: yeast-ppme1

The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C-terminus, which in mammalian cells is added by a specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase. We have identified genes in yeast that show significant homology to human carboxyl methyltransferase and methylesterase. Extracts of wild-type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity. Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B' regulatory subunit. Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesterase, yields phenotypes similar to those observed after inactivation of either regulatory subunit. These phenotypes can be reversed by overexpression of the B regulatory subunit. These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme.

We have purified 13 large subunit proteins of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae and determined their partial amino acid sequences. To elucidate the structure and function of these proteins, we searched for their genes by comparing our sequence data with those deduced from the genomic nucleotide sequence data of S. cerevisiae and analyzed them. In addition, we searched for the genes encoding proteins whose N-terminal amino acid sequences we have reported previously [Grohmann, L., Graack, H.-R., Kruft, V., Choli, T., Goldschmidt-Reisin, S. & Kitakawa, M. (1991) FEBS Lett. 284, 51-56]. Thus, we were able to identify and characterize 12 new genes for large subunit proteins of the yeast mitochondrial ribosome. Furthermore, we determined the N-terminal amino acid sequences of seven small subunit proteins and subsequently identified the genes for five of them, three of which were found to be new.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VIII reveals that it contains 269 predicted or known genes (300 base pairs or larger). Fifty-nine of these genes (22 percent) were previously identified. Of the 210 novel genes, 65 are predicted to encode proteins that are similar to other proteins of known or predicted function. Sixteen genes appear to be relatively recently duplicated. On average, there is one gene approximately every 2 kilobases. Although the coding density and base composition across the chromosome are not uniform, no regular pattern of variation is apparent.

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species.

Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (approximately 2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non-Saccharomyces origin. We identified Zygosaccharomyces bailii, a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non-Saccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations.

Many industrial strains of Saccharomyces cerevisiae have been selected primarily for their ability to convert sugars into ethanol efficiently despite exposure to a variety of stresses. To begin investigation of the genetic basis of phenotypic variation in industrial strains of S. cerevisiae, we have sequenced the genome of a wine yeast, AWRI1631, and have compared this sequence with both the laboratory strain S288c and the human pathogenic isolate YJM789. AWRI1631 was found to be substantially different from S288c and YJM789, especially at the level of single-nucleotide polymorphisms, which were present, on average, every 150 bp between all three strains. In addition, there were major differences in the arrangement and number of Ty elements between the strains, as well as several regions of DNA that were specific to AWRI1631 and that were predicted to encode proteins that are unique to this industrial strain.

We sequenced the genome of Saccharomyces cerevisiae strain YJM789, which was derived from a yeast isolated from the lung of an AIDS patient with pneumonia. The strain is used for studies of fungal infections and quantitative genetics because of its extensive phenotypic differences to the laboratory reference strain, including growth at high temperature and deadly virulence in mouse models. Here we show that the approximately 12-Mb genome of YJM789 contains approximately 60,000 SNPs and approximately 6,000 indels with respect to the reference S288c genome, leading to protein polymorphisms with a few known cases of phenotypic changes. Several ORFs are found to be unique to YJM789, some of which might have been acquired through horizontal transfer. Localized regions of high polymorphism density are scattered over the genome, in some cases spanning multiple ORFs and in others concentrated within single genes. The sequence of YJM789 contains clues to pathogenicity and spurs the development of more powerful approaches to dissecting the genetic basis of complex hereditary traits.

To elucidate the specific biological role of the yeast homologues of PTPA (phosphatase 2A phosphatase activator), Ypa1 and Ypa2 (where Ypa stands for yeast phosphatase activator), in the regulation of PP2A (protein phosphatase 2A), we investigated the physical interaction of both Ypa proteins with the catalytic subunit of the different yeast PP2A-like phosphatases. Ypa1 interacts specifically with Pph3, Sit4 and Ppg1, whereas Ypa2 binds to Pph21 and Pph22. The Ypa1 and Ypa2 proteins do not compete with Tap42 (PP2A associating protein) for binding to PP2A family members. The interaction of the Ypa proteins with the catalytic subunit of PP2A-like phosphatases is direct and independent of other regulatory subunits, implicating a specific function for the different PP2A-Ypa complexes. Strikingly, the interaction of Ypa2 with yeast PP2A is promoted by the presence of Ypa1, suggesting a positive role of Ypa1 in the regulation of PP2A association with other interacting proteins. As in the mammalian system, all yeast PP2A-like enzymes associate as an inactive complex with Yme (yeast methyl esterase). Ypa1 as well as Ypa2 can reactivate all these inactive complexes, except Pph22-Yme. Ypa1 is the most potent activator of PP2A activity, suggesting that there is no direct correlation between activation potential and binding capacity.

An analysis of the structurally and catalytically diverse serine hydrolase protein family in the Saccharomyces cerevisiae proteome was undertaken using two independent but complementary, large-scale approaches. The first approach is based on computational analysis of serine hydrolase active site structures; the second utilizes the chemical reactivity of the serine hydrolase active site in complex mixtures. These proteomics approaches share the ability to fractionate the complex proteome into functional subsets. Each method identified a significant number of sequences, but 15 proteins were identified by both methods. Eight of these were unannotated in the Saccharomyces Genome Database at the time of this study and are thus novel serine hydrolase identifications. Three of the previously uncharacterized proteins are members of a eukaryotic serine hydrolase family, designated as Fsh (family of serine hydrolase), identified here for the first time. OVCA2, a potential human tumor suppressor, and DYR-SCHPO, a dihydrofolate reductase from Schizosaccharomyces pombe, are members of this family. Comparing the combined results to results of other proteomic methods showed that only four of the 15 proteins were identified in a recent large-scale, "shotgun" proteomic analysis and eight were identified using a related, but similar, approach (neither identifies function). Only 10 of the 15 were annotated using alternate motif-based computational tools. The results demonstrate the precision derived from combining complementary, function-based approaches to extract biological information from complex proteomes. The chemical proteomics technology indicates that a functional protein is being expressed in the cell, while the computational proteomics technology adds details about the specific type of function and residue that is likely being labeled. The combination of synergistic methods facilitates analysis, enriches true positive results, and increases confidence in novel identifications. This work also highlights the risks inherent in annotation transfer and the use of scoring functions for determination of correct annotations.

Mechanisms that coordinate cell growth with division are thought to determine the timing of initiation of cell division and to limit overall cell proliferation. To identify genes involved in this process in Saccharomyces cerevisiae, we describe a method that does not rely on cell size alterations or resistance to pheromone. Instead, our approach was based on the cell surface deposition of the Flo1p protein in cells having passed START. We found that over-expression of HXT11 (which encodes a plasma membrane transporter), PPE1 (coding for a protein methyl esterase), or SIK1 (which encodes a protein involved in rRNA processing) shortened the duration of the G1 phase of the cell cycle, prior to the initiation of DNA replication. In addition, we found that, although SIK1 was not part of a mitotic checkpoint, SIK1 over-expression caused spindle orientation defects and sensitized G2/M checkpoint mutant cells. Thus, unlike HXT11 and PPE1, SIK1 over-expression is also associated with mitotic functions. Overall, we used a novel enrichment approach and identified genes that were not previously associated with cell cycle progression. This approach can be extended to other organisms.

The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C-terminus, which in mammalian cells is added by a specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase. We have identified genes in yeast that show significant homology to human carboxyl methyltransferase and methylesterase. Extracts of wild-type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity. Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B' regulatory subunit. Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesterase, yields phenotypes similar to those observed after inactivation of either regulatory subunit. These phenotypes can be reversed by overexpression of the B regulatory subunit. These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme.

We have purified 13 large subunit proteins of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae and determined their partial amino acid sequences. To elucidate the structure and function of these proteins, we searched for their genes by comparing our sequence data with those deduced from the genomic nucleotide sequence data of S. cerevisiae and analyzed them. In addition, we searched for the genes encoding proteins whose N-terminal amino acid sequences we have reported previously [Grohmann, L., Graack, H.-R., Kruft, V., Choli, T., Goldschmidt-Reisin, S. & Kitakawa, M. (1991) FEBS Lett. 284, 51-56]. Thus, we were able to identify and characterize 12 new genes for large subunit proteins of the yeast mitochondrial ribosome. Furthermore, we determined the N-terminal amino acid sequences of seven small subunit proteins and subsequently identified the genes for five of them, three of which were found to be new.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VIII reveals that it contains 269 predicted or known genes (300 base pairs or larger). Fifty-nine of these genes (22 percent) were previously identified. Of the 210 novel genes, 65 are predicted to encode proteins that are similar to other proteins of known or predicted function. Sixteen genes appear to be relatively recently duplicated. On average, there is one gene approximately every 2 kilobases. Although the coding density and base composition across the chromosome are not uniform, no regular pattern of variation is apparent.