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).

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 IP 7 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.The cellular homeostasis of inorganic phosphate is required for optimal growth and efficient metabolism. The response of the model organism Saccharomyces cerevisiae to extracellular phosphate starvation is well characterized and mediated by the phosphate signal transduction (PHO) pathway (20,24). To determine whether the PHO pathway is conserved in other Ascomycota fungal species, we screened for PHO pathway mutants in the evolutionarily distantly related Schizosaccharomyces pombe, which last shared a common ancestor with S. cerevisiae more than 1 billion years ago (7).The PHO pathway in S. cerevisiae often is defined by the regulation of PHO5, which encodes a phosphate starvationregulated acid phosphatase (17,20). PHO5 is highly induced during phosphate starvation. ScPho5 activity is detected using a diazo-coupling assay with 1-napthylphosphate (9). Numerous studies have determined that PHO5 transcription is regulated by the specific transcription factors Pho4 and Pho2 and by more general chromatin remodeling complexes, such as SWI/ SNF, SAGA, and INO80 (1, 16, 31). Pho4 localization and activity is regulated by a cyclin/cyclin-dependent kinase complex (Pho81/Pho80/Pho85) (11,12,25). During high extracellular phosphate conditions, the kinase complex is active and phosphorylates Pho4, leading to nuclear exclusion and little transcription of PHO5 (10, 15). During low extracellular phosphate conditions, Pho81 inhibits the kinase complex through a noncovalent interaction with IP 7 (inositol heptakisphosphate) (18,19). Certain isomers of IP 7 increase in abundance in response to phosphate starvation, although how extracellular phosphate concentration leads to these increases is unclear. However, Vip1 is required to phosphorylate IP 6 to form 4-PP-IP 5 or 6-PP-IP 5 , and Ddp1 is required for dephosphorylation back to IP 6 (19). Increases in IP 7 during extracellular phosphate st...

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 threegene 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.

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.