Identifying Signaling Pathways Mediating Filamentous Growth in Saccharomyces Cerevisiae.

Abstract

Multiple fungal species exhibit complex morphological changes in response to environmental conditions. Notably, the ability to transition between a cellular yeast-like form and a filamentous form is linked to virulence in several plant and human pathogens. My research is focused on identifying the molecular mechanisms driving this dimorphism in budding yeast. In S. cerevisiae, nutrient stress can induce morphological changes where unicellular yeast transition to a filamentous state, marked by pseudohyphal filaments of elongated and connected cells. This pseudohyphal differentiation is regulated by core signaling pathways responsive to diverse environmental stimuli, but the complete repertoire of molecular components coordinating these signals is unclear. To identify novel regulators of yeast stress-responsive pseudohyphal growth, I leveraged an innovative bioinformatics tool to discover biologically meaningful relationships from high-throughput data. Specifically, I analyzed publicly available and novel DNA microarray data sets using the Topology Enrichment Analysis framework (TEAK) and identified two previously unreported genes necessary for the yeast nitrogen stress response (DPL1 and LAG1) as well as a key regulator of lipid metabolism (SLC1) that is required for pseudohyphal differentiation. Separately, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen stress. To identify the Sks1p signaling network, we applied mass spectrometry-based phosphoproteomics and identified over 900 phosphosites that exhibited Sks1p kinase-dependent changes. From this analysis, we report a set of novel phosphosites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2. Additionally, the pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans.