Regulation of membrane scission in yeast endocytosis

Abstract

During clathrin-mediated endocytosis, a flat plasma membrane is shaped into an invagination that undergoes scission to form a vesicle. In mammalian cells, the force that drives the transition from invagination to vesicle is primarily provided by the GTPase dynamin that acts in concert with crescent-shaped BAR domain proteins. In yeast cells, the mechanism of endocytic scission is unclear. The yeast BAR domain protein complex Rvs161/167 (Rvs) nevertheless plays an important role in this process: deletion of Rvs dramatically reduces scission efficiency. A mechanistic understanding of the influence of Rvs on scission however, remains incomplete. We used quantitative live-cell imaging and genetic manipulation to understand the recruitment and function of Rvs and other late-stage proteins at yeast endocytic sites. We found that arrival of Rvs at endocytic sites is timed by interaction of its BAR domain with specific membrane curvature. A second domain of Rvs167- the SH3 domain- affects localization efficiency of Rvs. We show that Myo3, one of the two type-I myosins in Saccharomyces cerevisiae, has a role in recruiting Rvs167 via the SH3 domain. Removal of the SH3 domain also affects assembly and disassembly of actin and impedes membrane invagination. Our results indicate that both BAR and SH3 domains are important for the role of Rvs as a regulator of scission. We tested other proteins implicated in vesicle formation in Saccharomyces cerevisiae , and found that neither synaptojanins nor dynamin contribute directly to membrane scission. We propose that recruitment of Rvs BAR domains delays scission and allows invaginations to grow by stabilizing them. We also propose that vesicle formation is dependent on the force exerted by the actin network component of the endocytic machinery.

Spatio-temporal regulation of endocytic protein assembly by SH3 domains in yeast

“…1). Recruitment of Rvs167 and Myo5 is also mediated by their SH3 domains, as reported previously (Sun et al, 2006; Menon & Kaksonen, 2021). The Myo3 SH3 domain likely acts similarly to the paralogous Myo5, recruiting Myo3.…”

“…Cooperation of Bzz1 and Rvs167 has already been suggested, but in the context of vesicle scission (Kishimoto et al, 2011). Our growth assay suggests a commonality between Bzz1 and Rvs167 in addition to scission control, as rvs167(SH3Δ) causes no scission failure (Menon & Kaksonen, 2021).…”

“…and Rvs167 in addition to scission control, as rvs167(SH3Δ) causes no scission failure (Menon & Kaksonen, 2021).…”

“…Deletion of Rvs167 causes failure of endocytic vesicle scission and cell growth defects. Therefore, we used a deletion of the Rvs167 SH3 domain, rvs167(SH3Δ) , which causes a milder phenotype (Menon & Kaksonen, 2021).…”

“…These data point to a central role for SH3 domains regulating actin network assembly at endocytic sites. Evidence also indicates that the SH3 domains of Myo5, Lsb4 and Rvs167 are involved in the recruitment of these proteins to endocytic sites (Sun et al, 2006; Urbanek et al, 2015; Menon & Kaksonen, 2021). This is in contrast to Sla1, which has an SH3-independent recruitment to endocytic sites (Sun et al, 2015).…”