A Flow Cytometry-Based Phenotypic Screen To Identify Novel Endocytic Factors in Saccharomyces cerevisiae

摘要

Endocytosis is a fundamental process for internalizing material from the plasma membrane, including many transmembrane proteins that are selectively internalized depending on environmental conditions. In most cells, the main route of entry is clathrin-mediated endocytosis (CME), a process that involves the coordinated activity of over 60 proteins; however, there are likely as-yet unidentified proteins involved in cargo selection and/or regulation of endocytosis. We performed a mutagenic screen to identify novel endocytic genes in Saccharomyces cerevisiae expressing the methionine permease tagged with pHluorin (pHl), a pH-sensitive GFP variant whose fluorescence is quenched upon delivery to the acidic vacuole lumen. We used fluorescence-activated cell sorting to isolate mutagenized cells with elevated fluorescence, resulting from failure to traffic -pHl cargo to the vacuole, and further assessed subcellular localization of -pHl to characterize the endocytic defects in 256 mutants. A subset of mutant strains was classified as having general endocytic defects based on mislocalization of additional cargo proteins. Within this group, we identified mutations in four genes encoding proteins with known roles in endocytosis: the endocytic coat components , , and , and the gene, whose product is involved in nucleating actin filaments to form branched networks. All four mutants demonstrated aberrant dynamics of the endocytic machinery at sites of CME; moreover, the ^R346H mutation showed reduced actin nucleation activity in vitro. Finally, whole genome sequencing of two general endocytic mutants identified mutations in conserved genes not previously implicated in endocytosis, and , demonstrating that our screening approach can be used to identify new components involved in endocytosis.