The Rsp5 Ubiquitin Ligase Binds to and Ubiquitinates Members of the Yeast CIN85-Endophilin Complex, Sla1-Rvs167

Abstract

Sla1 and Rvs167 are yeast proteins required for receptor internalization and organization of the actin cytoskeleton. Here we provide evidence that Sla1 and Rvs167 are orthologues of the mammalian CIN85 and endophilin proteins, respectively, which are required for ligand-stimulated growth factor receptor internalization. Sla1 is similar in domain structure to CIN85 and binds directly to the endophilin-like Rvs167. Akin to CIN85, Sla1 interacts with synaptojanins and a ubiquitin ligase that regulates endocytosis. This ubiquitin ligase, Rsp5, binds directly to both Sla1 and Rvs167. The interaction between Rsp5 and Rvs167 is mediated through Rsp5 WW domains and PXY motifs in the central Gly-Pro-Ala-rich domain of Rvs167. Rvs167 PXY motifs are required for Rsp5-dependent monoubiquitination of Rvs167 on Lys 481 in the Src homology 3 (SH3) domain. Mutation of Lys 481 3 Arg causes cells to grow slowly on medium containing 1 M NaCl, although this phenotype is not due to the defect in ubiquitination caused by the K481R mutation. We propose that Rsp5 interaction with Sla1-Rvs167 promotes Rvs167 ubiquitination and regulates activity of this protein complex. Rvs167 ubiquitination is not required for general function of Rvs167, but may control specific Rvs167 SH3 domain-protein interactions or negatively regulate SH3 domain activity.

Actin regulation in endocytosis

“…Tubulation is then suggested to facilitate the scission process. The yeast amphiphysins have been shown to bind to a number of known endocytic proteins, including Las17p, Sla1p and actin itself (Lombardi and Riezman, 2001;Friesen et al, 2003;Madania et al, 1999;Stamenova et al, 2004). The proteins arrive after actin, remain immobile for a few seconds, then show transient movement into the cell.…”

The HECT E3 ubiquitin ligase Rsp5 is important for ubiquitin homeostasis in yeast

“…Rsp5 could affect endocytosis by interfering with the organization of the actin cytoskeleton. A link between Rsp5 and the actin cytoskeleton is suggested by genetic interactions between rsp5 and several mutants affected in genes encoding actin cytoskeletal components and by the demonstration of direct physical interactions between Rsp5 and components of the actin cytoskeleton [11,12].…”

Rsp5 ubiquitin ligase modulates translation accuracy in yeast Saccharomyces cerevisiae

“…Rsp5p impacts a wide variety of physiological processes, including regulation of endocytosis and lysosomal degradation of plasma membrane permeases such as Fur4p (Galan et al 1996), Gap1p (Springael et al 1999), Tat2p (Beck et al 1999), and receptors such as Ste2p and Ste3p (Dunn and Hicke 2001b). Rsp5p also ubiquitinates a component(s) of the endocytic machinery (Dunn and Hicke 2001a;Gajewska et al 2001;Kamiń ska et al 2002;Stamenova et al 2004). Moreover, Rsp5p-dependent ubiquitination is involved in sorting of amino acid permeases at the Golgi apparatus (Helliwell et al 2001) and in the multivesicular bodies (Katzmann et al 2004;Morvan et al 2004).…”