Dennis D. Wykoff

Identification of Sumoylated Proteins by Systematic Immunoprecipitation of the Budding Yeast Proteome

The identification of post-translational modifications to proteins is critical for understanding many important aspects of biology. Utilizing a collection of epitope-tagged yeast strains, we developed a novel approach to determine which proteins are modified by the small ubiquitin-related modifier (SUMO). We crossed traits useful for the detection of SUMO conjugation into 4246 tandem affinity purification-tagged strains and successfully immunoprecipitated and screened 2893 of these proteins for association with SUMO (∼70% of the expressed proteome detectable by immunoblot analysis). We found 82 proteins associated with SUMO, including many of low abundance. Because our screen was performed under non-denaturing conditions, we were able to identify multiple members of four complexes that were associated with SUMO: the RSC chromatin remodeling complex, the mediator complex, the TFIID complex, and the septin complex. In addition, we describe five new direct conjugates of SUMO, and we mutated SUMO conjugation sites in four proteins. This is the first attempt to immunoprecipitate a large fraction of the proteome of a eukaryote, and it demonstrates the utility of this method to identify post-translational modifications in the yeast proteome.

Partially Phosphorylated Pho4 Activates Transcription of a Subset of Phosphate-Responsive Genes

A cells ability to generate different responses to different levels of stimulus is an important component of an adaptive environmental response. Transcriptional responses are frequently controlled by transcription factors regulated by phosphorylation. We demonstrate that differential phosphorylation of the budding yeast transcription factor Pho4 contributes to differential gene expression. When yeast cells are grown in high-phosphate growth medium, Pho4 is phosphorylated on four critical residues by the cyclin–CDK complex Pho80–Pho85 and is inactivated. When yeast cells are starved for phosphate, Pho4 is dephosphorylated and fully active. In intermediate-phosphate conditions, a form of Pho4 preferentially phosphorylated on one of the four sites accumulates and activates transcription of a subset of phosphate-responsive genes. This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression. Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80–Pho85, and one transcription factor, Pho4. Differential phosphorylation of Pho4 by Pho80–Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs.

Construction, verification and experimental use of two epitope-tagged collections of budding yeast strains.

A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein–protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available.

Genome-Wide Characterization of the Phosphate Starvation Response in Schizosaccharomyces pombe

BackgroundInorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway, leading to expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway involving genetically identified positive (pho7+) and negative (csk1+) regulators. The genes induced by phosphate limitation and the molecular mechanism by which pho7+ and csk1+ function are unknown. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response.ResultsWe define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response that contains the ScPHO5 (pho1+), ScPHO84 (SPBC8E4.01c), and ScGIT1 (SPBC1271.09) orthologs. We identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction.ConclusionsWe provide a global analysis of the transcriptional response to phosphate limitation in S. pombe. Our results elucidate the conserved core regulon induced in response to phosphate starvation in this ascomycete distantly related to S. cerevisiae and provide a better understanding of flexibility in environmental stress response networks.

Systematic Screen of Schizosaccharomyces pombe Deletion Collection Uncovers Parallel Evolution of the Phosphate Signal Transduction Pathway in Yeasts

ABSTRACT The phosphate signal transduction (PHO) pathway, which regulates genes in response to phosphate starvation, is well defined in Saccharomyces cerevisiae. We asked whether the PHO pathway was the same in the distantly related fission yeast Schizosaccharomyces pombe. We screened a deletion collection for mutants aberrant in phosphatase activity, which is primarily a consequence of pho1+ transcription. We identified a novel zinc finger-containing protein (encoded by spbc27b12.11c+), which we have named pho7+, that is essential for pho1+ transcriptional induction during phosphate starvation. Few of the S. cerevisiae genes involved in the PHO pathway appear to be involved in the regulation of the phosphate starvation response in S. pombe. Only the most upstream genes in the PHO pathway in S. cerevisiae (ADO1, DDP1, and PPN1) share a similar role in both yeasts. Because ADO1 and DDP1 regulate ATP and IP7 levels, we hypothesize that the ancestor of these yeasts must have sensed similar metabolites in response to phosphate starvation but have evolved distinct mechanisms in parallel to sense these metabolites and induce phosphate starvation genes.

Candida glabrata PHO4 Is Necessary and Sufficient for Pho2-Independent Transcription of Phosphate Starvation Genes

Comparative genomic analyses of Candida glabrata and Saccharomyces cerevisiae suggest many signal transduction pathways are highly conserved. Focusing on the phosphate signal transduction (PHO) pathway of C. glabrata, we demonstrate that components of the pathway are conserved and confirm the role of CgPHO81, CgPHO80, CgPHO4, and CgMSN5 in the PHO pathway through deletion analysis. Unlike S. cerevisiae, C. glabrata shows little dependence on the transcription factor, Pho2, for induction of phosphate-regulated genes during phosphate limitation. We show that the CgPho4 protein is necessary and sufficient for Pho2-independent gene expression; CgPho4 is capable of driving expression of PHO promoters in S. cerevisiae in the absence of ScPHO2. On the basis of the sequences of PHO4 in the hemiascomycetes and complementation analysis, we suggest that Pho2 dependence is a trait only observed in species closely related to S. cerevisiae. Our data are consistent with trans-regulatory changes in the PHO pathway via the transcription factor Pho4 as opposed to cis-regulatory changes (the promoter).

Novel Acid Phosphatase in Candida glabrata Suggests Selective Pressure and Niche Specialization in the Phosphate Signal Transduction Pathway

Evolution through natural selection suggests unnecessary genes are lost. We observed that the yeast Candida glabrata lost the gene encoding a phosphate-repressible acid phosphatase (PHO5) present in many yeasts including Saccharomyces cerevisiae. However, C. glabrata still had phosphate starvation-inducible phosphatase activity. Screening a C. glabrata genomic library, we identified CgPMU2, a member of a three-gene family that contains a phosphomutase-like domain. This small-scale gene duplication event could allow for sub- or neofunctionalization. On the basis of phylogenetic and biochemical characterizations, CgPMU2 has neofunctionalized to become a broad range, phosphate starvation-regulated acid phosphatase, which functionally replaces PHO5 in this pathogenic yeast. We determined that CgPmu2, unlike ScPho5, is not able to hydrolyze phytic acid (inositol hexakisphosphate). Phytic acid is present in fruits and seeds where S. cerevisiae grows, but is not abundant in mammalian tissues where C. glabrata grows. We demonstrated that C. glabrata is limited from an environment where phytic acid is the only source of phosphate. Our work suggests that during evolutionary time, the selection for the ancestral PHO5 was lost and that C. glabrata neofunctionalized a weak phosphatase to replace PHO5. Convergent evolution of a phosphate starvation-inducible acid phosphatase in C. glabrata relative to most yeast species provides an example of how small changes in signal transduction pathways can mediate genetic isolation and uncovers a potential speciation gene.

Dissection of the PHO pathway in Schizosaccharomyces pombe using epistasis and the alternate repressor adenine

In Saccharomyces cerevisiae, intracellular phosphate levels are maintained by the PHO pathway, activation of which is assayed by increased phosphatase activity. The PHO pathway of Schizosaccharomyces pombe upregulates phosphatase activity (encoded by pho1+) during low extracellular phosphate levels, but the underlying mechanism is poorly understood. We utilized an alternate repressor of pho1+ expression (adenine supplementation) along with epistasis analysis to develop a model of how S. pombe PHO pathway components interact. Analyzing Pho1 activity in S. pombe PHO pathway deletion mutants during adenine starvation, we observed most mutants with a phosphatase defect in phosphate starvation also had a defect in adenine starvation. Pho7, a transcription factor in the PHO pathway, is necessary for an adenine starvation-mediated increase in Pho1 activity. Comparing adenine starvation to phosphate starvation, there are differences in the degree to which individual mutants regulate the two responses. Through epistasis studies, we identified two positive regulatory arms and one repressive arm of the PHO pathway. PKA activation is a positive regulator of Pho1 activity under both environmental conditions and is critical for transducing adenine concentrations in the cell. The synthesis of IP7 also appears critical for the induction of Pho1 activity during adenine starvation, but IP7 is not critical during phosphate starvation, which differs from S. cerevisiae. Finally, Csk1 is critical for repression of pho1+ expression during phosphate starvation. We believe all of these regulatory arms converge to increase transcription of pho1+ and some of the regulation acts through pho7+.

The fate of linear DNA in Saccharomyces cerevisiae and Candida glabrata: the role of homologous and non-homologous end joining.

In vivo assembly of plasmids has become an increasingly used process, as high throughput studies in molecular biology seek to examine gene function. In this study, we investigated the plasmid construction technique called gap repair cloning (GRC) in two closely related species of yeast – Saccharomyces cerevisiae and Candida glabrata. GRC utilizes homologous recombination (HR) activity to join a linear vector and a linear piece of DNA that contains base pair homology. We demonstrate that a minimum of 20 bp of homology on each side of the linear DNA is required for GRC to occur with at least 10% efficiency. Between the two species, we determine that S. cerevisiae is slightly more efficient at performing GRC. GRC is less efficient in rad52 deletion mutants, which are defective in HR in both species. In dnl4 deletion mutants, which perform less non-homologous end joining (NHEJ), the frequency of GRC increases in C. glabrata, whereas GRC frequency only minimally increases in S. cerevisiae, suggesting that NHEJ is more prevalent in C. glabrata. Our studies allow for a model of the fate of linear DNA when transformed into yeast cells. This model is not the same for both species. Most significantly, during GRC, C. glabrata performs NHEJ activity at a detectable rate (>5%), while S. cerevisiae does not. Our model suggests that S. cerevisiae is more efficient at HR because NHEJ is less prevalent than in C. glabrata. This work demonstrates the determinants for GRC and that while C. glabrata has a lower efficiency of GRC, this species still provides a viable option for GRC.

De novo generation of a phosphate starvation-regulated promoter in Candida glabrata

What steps are required for a promoter to acquire regulation by an environmental condition? We address this question by examining a promoter in Candida glabrata that is regulated by phosphate starvation and the transcription factor Pho4. The gene PMU2 encodes a secreted acid phosphatase that resulted from gene duplication events not present in other Ascomycetes, and only this gene of the three paralogs has acquired Pho4 regulation. We observe that the PMU2 promoter from C. glabrata is not functional in Saccharomyces cerevisiae, which is surprising because it is regulated by Pho4, and Pho4 is regulated in a similar manner in both species - through phosphorylation and localization. Additionally, we determine that phosphate starvation-regulated promoters in C. glabrata do not require the coactivator Pho2, which is essential to the phosphate starvation response in S. cerevisiae. We define a region of the PMU2 promoter that is important for Pho4 regulation, and this promoter region does not contain the canonical CACGTX sequence that ScPho4 utilizes for phosphate starvation-dependent transcription. However, CgPho4 utilizes CACGTX in the CgPHO84 promoter, as mutation of this sequence decreases transcription. We conclude that the acquisition of PMU2 has expanded the binding specificity of CgPho4 relative to ScPho4.