Kristie Wrasman, Salvatore L Alioto, Yorke Zhang, Kyle Hoban, Marjon Khairy, Bruce L Goode, Beverly Wendland

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 inSaccharomyces cerevisiaeexpressing the methionine permease Mup1 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 Mup1-pHl cargo to the vacuole, and further assessed subcellular localization of Mup1-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 componentsSLA2, SLA1,andEDE1, and theARP3gene, 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, thearp3R346Hmutation showed reduced actin nucleation activityin vitroFinally, whole genome sequencing of two general endocytic mutants identified mutations in conserved genes not previously implicated in endocytosis,KRE33andIQG1, demonstrating that our screening approach can be used to identify new components involved in endocytosis.