Applying Large Scale Genomic and Proteomic Analysis to Identify Potential Determinants of Yeast Pseudohyphal Growth.

[摘要] The budding yeast Saccharomyces cerevisiae can respond to nutritional and environmental stress by implementing a morphogenetic change in growth form where cells elongate and interconnect, forming multicellular pseudohyphal filaments. Classic studies have identified core signaling pathways regulating pseudohyphal growth in yeast; however, the scope of regulatory networks mediating this morphological transformation is broad and incompletely defined. Here, we applied large scale genomic and proteomic approaches to identify additional determinants that are related to filamentation in yeast. The genomes of S. cerevisiae strains competent to undergo filamentous growth exhibit many small genetic differences as compared to the genomes of non-filamentous strains, raising an open question as to which of the observed allelic differences are significant determinants of the filamentous growth phenotype. To address this, we implemented a linkage study incorporating pooled segregant analysis and next-generation DNA sequencing technology. Each of two filamentous strains of the Σ1278b and SK1 genetic backgrounds was crossed with an S288c-derived non-filamentous strain. We subsequently pooled spores resulting from complete meiosis according to invasive growth phenotype. After next-generation sequencing of the phenotypically pooled spores, we identified 522 allelic differences within 201 genes with linkage to invasive growth in yeast. Subsequent studies address a function for the cell polarisome protein Pea2p in regulating filamentous growth by altering the cell budding pattern along with Spa2p. Further studies of Pea2p suggest that it acts downstream of the MAPK pathway. Our results indicate that allelic differences in PEA2 result in protein-protein interaction differences between Pea2p and Spa2p.In a parallel linkage analysis study of another strain capable of undergoing invasive growth, we identified that allelic differences in the MDM32 gene encoding a mitochondrial structural protein are linked with filamentation phenotypes through the regulation of mitochondrial function. In addition, we generated quantitative proteomic profiles of plasma membrane proteins that may contribute to the altered cell-cell adhesion observed during pseudohyphal growth. By two-dimensional liquid chromatography-tandem mass spectrometry of isobaric tagged strains under different conditions, we discovered 11 differentially abundant proteins localized to the yeast cell periphery. Within we identified Ylr414cp enriched in the plasma membrane compartment of Can1 (MCC) under nitrogen stress.

[发布机构] University of Michigan

[效力级别] Filamentous Growth

[关键词] Linkage Analysis;Filamentous Growth;Pea2p;Mdm32p;Pun1p;Molecular;Cellular and Developmental Biology;Science;Molecular, Cellular, and Developmental Biology