Endocytosis in the budding yeast Saccharomyces cerevisiae involves the ordered recruitment, activity and disassembly of nearly 60 proteins at distinct sites on the plasma membrane. Two-color live-cell fluorescence microscopy has proven to be invaluable for in vivo analysis of endocytic proteins: identifying new components, determining the order of protein arrival and dissociation, and revealing even very subtle mutant phenotypes. Yeast genetics and functional genomics facilitate identification of complex interaction networks between endocytic proteins and their regulators. Quantitive datasets produced by these analyses have made theoretical modeling possible. Here, we discuss recent findings on budding yeast endocytosis that have advanced our knowledge of how ~60 endocytic proteins are recruited, regulated by lipid and protein modifications, and disassembled with remarkable regularity.

ETOC: During yeast endocytic site formation, Ede1p (yeast Eps15), but not clathrin light chain, is important for the recruitment of most other early-arriving proteins to endocytic sites. Cargo and clathrin light chain may play roles in regulating the transition of endocytic sites out of the “intermediate coat” stage of endocytosis.

Summary Background Clathrin-mediated endocytosis in budding yeast requires the regulated recruitment and disassociation of over 60 proteins at discrete plasma membrane punctae. Post-translational modifications such as ubiquitination may play important regulatory roles in this highly processive and ordered process. However, while ubiquitination plays an important role in cargo selection, functions for ubiquitination of the endocytic machinery are not known. Results We identified the deubiquitinase (DUB) Ubp7 as a late arriving endocytic protein. Deletion of the DUBs Ubp2 and Ubp7 resulted in elongation of endocytic coat protein lifetimes at the plasma membrane and recruitment of endocytic proteins to internal membranes. These phenotypes could be replicated by expressing a permanently ubiquitinated version of Ede1, the yeast Eps15 homolog, which is implicated in endocytic site initiation, while EDE1 deletion partially suppressed the deubiquitinase deletion phenotype. Both DUBs are capable of deubiquitinating Ede1 in vitro. Conclusions Deubiquitination regulates formation of endocytic sites and stability of the endocytic coat. This regulation appears to occur through Ede1, since permanently ubiquitinated Ede1 phenocopies deletion of UBP2 and UBP7. Moreover, incomplete suppression of the ubp2Δ ubp7Δ phenotype by ede1Δ indicates that ubiquitination and deubiquitination are likely to regulate additional components of the endocytic machinery.