Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis

Abstract

During endocytosis in S. cerevisiae, actin polymerization is proposed to provide the driving force for invagination against the effects of turgor pressure. In previous studies, Ysc84 was demonstrated to bind actin through a conserved N-terminal domain. However, full length Ysc84 could only bind actin when its C-terminal SH3 domain also bound to the yeast WASP homologue Las17. Live cell-imaging has revealed that Ysc84 localizes to endocytic sites after Las17/WASP but before other known actin binding proteins, suggesting it is likely to function at an early stage of membrane invagination. While there are homologues of Ysc84 in other organisms, including its human homologue SH3yl-1, little is known of its mode of interaction with actin or how this interaction affects actin filament dynamics. Here we identify key residues involved both in Ysc84 actin and lipid binding, and demonstrate that its actin binding activity is negatively regulated by PI(4,5)P2. Ysc84 mutants defective in their lipid or actin-binding interaction were characterized in vivo. The abilities of Ysc84 to bind Las17 through its C-terminal SH3 domain, or to actin and lipid through the N-terminal domain were all shown to be essential in order to rescue temperature sensitive growth in a strain requiring YSC84 expression. Live cell imaging in strains with fluorescently tagged endocytic reporter proteins revealed distinct phenotypes for the mutants indicating the importance of these interactions for regulating key stages of endocytosis.

Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1

“…Coupled with the direct interaction between actin and dynamin, actin polymerization at the site of the invagination then either could directly drive scission or could be associated with subsequent movement of the released vesicle within the cytoplasm ( 24 , 29 ). We have recently demonstrated that overexpression of an early endocytic protein, Ysc84, that binds Las17 at a site overlapping Rvs167 can cause retractions in endocytosis similar to the rvs167 deletion, supporting the in vivo importance of the Rvs167-Las17 interaction ( 55 ).…”

Disruption of the plant-specific CFS1 gene impairs autophagosome turnover and triggers EDS1-dependent cell death

“…Ysc84 has been shown to regulate early steps of endocytosis 33 , and SH3YL1 is localized at dorsal ruffles, actin-rich membrane regions with intense receptor endocytosis 34 . Both proteins appear to contribute to the integration of F-actin and membrane morphology 44 . Accordingly, their SYLF domains have been shown to bind to phospholipids 34 , 44 , and in case of Ysc84 to actin 33 .…”

“…Both proteins appear to contribute to the integration of F-actin and membrane morphology 44 . Accordingly, their SYLF domains have been shown to bind to phospholipids 34 , 44 , and in case of Ysc84 to actin 33 . Biochemically, CFS1 shows similarities to the behavior of Ysc84.…”

“…However, the phospholipid binding specificity differs, reflecting the different localizations in the endomembrane system. Whereas Ysc84 and SH3YL1 bind to plasma membrane enriched PIs 34 , 44 , CFS1 specifically interacts with PI3P (Fig. 5A,B ) that is enriched in the endosomal and autophagosomal membrane.…”

“…In full length Ysc84, actin can only bind when the interaction partner Las17p is associated with the adjacent SH3 domain 33 . Phospholipid binding inhibits the actin interaction 44 . CFS1 shows a similar actin binding activity with the isolated SYLF domain, but not with the complete protein (Figs 4F and S5A ).…”