ABSTRACT
The Target of Rapamycin (TOR) pathway regulates morphogenesis and responses to host cells in the fungal pathogen Candida albicans Eukaryotic Target of Rapamycin complex 1 (TORC1) induces growth and proliferation in response to nitrogen and carbon source availability. Our unbiased genetic approach seeking unknown components of TORC1 signaling in C. albicans revealed that the phosphate transporter Pho84 is required for normal TORC1 activity. We found that mutants in PHO84 are hypersensitive to rapamycin and in response to phosphate feeding, generate less phosphorylated ribosomal protein S6 (P-S6) than the WT. The small GTPase Gtr1, a component of the TORC1-activating EGO complex, links Pho84 to TORC1. Mutants in Gtr1 but not in another TORC1-activating GTPase, Rhb1, are defective in the P-S6 response to phosphate. Overexpression of Gtr1 and a constitutively active Gtr1Q67L mutant suppresses TORC1-related defects. In Saccharomyces cerevisiae pho84 mutants, constitutively active Gtr1 suppresses a TORC1 signaling defect but does not rescue rapamycin hypersensitivity. Hence, connections from phosphate homeostasis (PHO) to TORC1 may differ between C. albicans and S. cerevisiae The converse direction of signaling from TORC1 to the PHO regulon previously observed in S. cerevisiae was genetically shown in C. albicans using conditional TOR1 alleles. A small molecule inhibitor of Pho84, a Food and Drug Administration-approved drug, inhibits TORC1 signaling and potentiates the activity of the antifungals amphotericin B and micafungin. Anabolic TORC1-dependent processes require significant amounts of phosphate. Our study shows that phosphate availability is monitored and also controlled by TORC1 and that TORC1 can be indirectly targeted by inhibiting Pho84.
Subject(s)
Candida albicans/metabolism , Fungal Proteins/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Phosphates/metabolism , Proton-Phosphate Symporters/metabolism , Antifungal Agents/pharmacology , Candida albicans/drug effects , Candida albicans/genetics , Fungal Proteins/antagonists & inhibitors , Fungal Proteins/genetics , Gene Deletion , Genes, Fungal , Hyphae/genetics , Hyphae/growth & development , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1/genetics , Models, Biological , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , Mutation , Proton-Phosphate Symporters/antagonists & inhibitors , Proton-Phosphate Symporters/genetics , Regulon , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Signal TransductionABSTRACT
Phosphatidylinositol is an important membrane lipid in Saccharomyces cerevisiae and other eukaryotes. Phosphatidylinositol and its metabolites (phosphoinositides, inositol polyphosphates, etc.) affect many cellular processes with implications in human diseases. Phosphatidylinositol synthesis in S. cerevisiae requires the essential PIS1 gene. Recent studies reveal that PIS1 expression is regulated at the level of transcription in response to carbon source, oxygen, and zinc. However, the consequence of this regulation on phosphatidylinositol levels and functions has not been thoroughly studied. To investigate this, we created a strain with a galactose-inducible GAL1-PIS1 gene. In this strain, the amount of phosphatidylinositol correlated with PIS1 expression but did not exceed c. 25% of the total phospholipid composition. Interestingly, we found that 4% phosphatidylinositol was sufficient for cell growth. We also found that reduced PIS1 expression yielded derepression of two phospholipid biosynthetic genes (INO1 and CHO1) and the INO2 regulatory gene. Consistent with this derepression, reduced PIS1 expression also yielded an overproduction of inositol (Opi(-)) phenotype. The effect on transcription of the INO1, CHO1, and INO2 genes is consistent with the accepted model that phosphatidic acid (PA) is the signal for regulation of these genes because decreased phosphatidylinositol synthesis would affect PA levels.
Subject(s)
Gene Expression Regulation, Fungal , Phosphatidylinositols/biosynthesis , Saccharomyces cerevisiae/physiology , Transferases (Other Substituted Phosphate Groups)/biosynthesis , Basic Helix-Loop-Helix Transcription Factors/biosynthesis , CDPdiacylglycerol-Serine O-Phosphatidyltransferase/biosynthesis , Gene Expression Profiling , Humans , Myo-Inositol-1-Phosphate Synthase/biosynthesis , Phosphatidic Acids/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/biosynthesis , Transcription, Genetic , Transferases (Other Substituted Phosphate Groups)/geneticsABSTRACT
In Saccharomyces cerevisiae, transcription of most of the phospholipid biosynthetic genes (e.g. INO1, CHO1, CHO2 and OPI3) is repressed by growth in the presence of inositol and choline and derepressed in their absence. This regulation requires the Ino2p and Ino4p activators and the Opi1p repressor. The PIS1 structural gene is required for the synthesis of the essential lipid phosphatidylinositol. Previous reports show that PIS1 expression is uncoupled from inositol/choline regulation, but is regulated by carbon source, hypoxia and zinc. However, in this study we found that the expression of PIS1 is induced twofold by inositol. This regulation did not require Ino2p and Ino4p, although Ino4p was required for full expression. Ino4p is a basic helix-loop-helix protein that requires a binding partner. Curiously, none of the other basic helix-loop-helix proteins affected PIS1 expression. Inositol induction did require another general regulator of phospholipid biosynthesis, Ume6p. Ume6p was found to be a positive regulator of PIS1 gene expression. Ume6p, and several associated factors, were required for inositol-mediated induction and chromatin immunoprecipitation analysis showed that Ume6p directly regulates PIS1 expression. Thus, we demonstrate novel regulation of the PIS1 gene by Ume6p.
