Requirements and molecular roles of phosphoinositides in yeast external signal-dependent polarized growth – YePIP

The spatio-temporal regulation of short-lived phosphorylated phosphatidylinositol derivatives (such as PI(4)P, PI(4,5)P2, PI(3,4,5)P3) is crucial in processes that require membrane reorganization and membrane-cytoskeleton association, including cell morphology changes. Signal transduction is frequently initiated by the recruitment and assembly of protein complexes at the plasma membrane in response to external signals. This recruitment and assembly is mediated by lipid-protein interactions and a range lipid binding motifs (such as pleckstrin homology-PH, basic rich-BR, epsin/AP180 N-terminal homology-E/ANTH,') have been identified which bind specific phosphoinositides (PIPs). Yeast has proven to be an ideal organism to study the roles of PIPs. Indeed, there are only 6 lipid kinases to generate phosphoinositides in Saccharomyces cerevisiae (PI(3,4,5)P3 and the kinases necessary for its synthesis are not present in this organism). While many of the physiological functions of phosphoinositides have been discovered in this eukaryote, their roles in external signal-mediated polarized growth are largely unknown. The main goal of this project is to identify the requirements, roles, and molecular functions both of phosphatidylinositol 4-phosphate, PI(4)P and phosphatidylinositol 4,5-bisphosphate, PI(4,5)P2, in yeast external signal-mediated polarized growth. We will examine the requirements, roles and functions of these two PIPs in both the baker's yeast S. cerevisiae and the human pathogenic yeast Candida albicans. A combination of mutant strains will be used in which the levels of these PIPs can be manipulated in vivo, such as kinase loss of function mutants and phosphatase ectopic gain of function mutants. In particular, we will focus on the transition from oval budding yeast cells to invasive filamentous yeast cells, which can be triggered by different environmental stimuli, including changes in nutrient levels. Such external signal-dependent polarized growth is likely to be critical for the adaptation of both organisms to their respective environments and for the virulence of the opportunistic pathogen C. albicans. Wild-type and mutant S. cerevisiae and C. albicans strains, in which the PIPs levels can be altered, will be used to investigate in which signaling pathways and at what level in these pathways PI(4)P and PI(4,5)P2 are critical. At least 6 yeast proteins, most conserved in higher eukaryotes, have been shown to bind PI(4)P in vivo and/or in vitro with varying degrees of specificity and affinity, and more than 10 yeast proteins have been shown to bind PI(4,5)P2. These proteins are all involved in processes in which PI(4)P and/or PI(4,5)P2 are critical, such as in lipid homeostasis and vesicle trafficking (PI(4)P), polarized growth (PI(4)P and PI(4,5)P2) and exocytosis or endocytosis (PI(4)P and PI(4,5)P2). Hence, we will examine whether these PIPs are required for the localization, recruitment, increase in local concentration (clustering) and/or activation of a range of proteins involved in these processes in response to external stimuli. The requirements, functions, and roles of these PIPs in two yeasts that have diverged ~900 million years ago, will be compared in order to identify the common versus distinct mechanisms in phospholipid-dependent external signal-mediated growth. Finally, the distribution and levels of PIPs in both organisms (wild-type and mutants) during external signal-dependent polarized growth will be analyzed. In particular, we will address whether the PI(4)P or PI(4,5)P2 level and distribution changes in response to different external stimuli. The levels of phosphoinositides will be determined by precursor labeling and chromatographic separation of lipids. The cellular localization of these PIPs will be followed in wild-type and mutant cells responding to external stimuli, using a range of defined protein domains which bind either PI(4)P or PI(4,5)P2. A major goal will be to determine whether, in response to external stimuli, PI(4,5)P2 becomes asymmetrically or non-uniformly distributed on the inner leaflet of plasma membrane. Our preliminary results indicate that PI(4,5)P2 is required for external signal-mediated polarized growth in both S. cerevisiae and C. albicans. Understanding the regulation of PI(4)P and PI(4,5)P2-dependent processes at a molecular level will be of fundamental importance and should ultimately lead to the identification of novel anti-fungal targets in a major etiological agent of nosocomial infections.