Other roles of Rsp5p in the internalization process

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Several observations have suggested that, in addition to its function in the ubiquitylation of plasma membrane cargoes, Rsp5p may have other functions in internalization. WW1 and WW3 domains appear to be important in fluid-phase endocytosis [96, 97]. Strikingly, mutations in the WW1 domain strongly inhibit fluid-phase endocytosis, but have no effect on Fur4p ubiquitylation and internalization ([97], and Marchal and Urban-Grimal, unpublished observations). Hence, the WW1 domain of Rsp5p may have a substrate/partner that is essential for fluid phase endocytosis. The C2 domain of Rsp5p appears to be the critical element controlling the location of the protein in both the plasma membrane and endosomal compartments [106, 107]. Although deletion of the C2 domain has no effect on a-factor internalization it has been reported to inhibit fluid phase endocytosis [96],  and to lead to a marked decrease in the rates of internalization of Fur4p [106] and Gap1p [108], with no apparent effect on the ubiquitylation of these proteins. These findings indicate that, in addition to its role in the ubiquitylation of these plasma membrane proteins, Rsp5p is involved in their internalization, via a process dependent on the C2 domain. This led to the suggestion that the Rsp5p-dependent ubiquitylation of a trans-acting protein might be required for the internalization step of endocytosis [109]. Support for this hypothesis was provided by the observation that temperature-sensitive rsp5 mutant cells are defective in the internalization of a-factor by a Ste2p-ubiquitin chimera, a receptor that does not require posttranslational ubiquitylation for internalization. Similarly, a modified version of Ste2p bearing a NPFXD linear peptide sequence as its only internalization signal (ubiquitin-independent) was not internalized in rsp5 cells. The internalization of these variant receptors and fluid-phase endocytosis were found to be dependent on the catalytic cysteine residue of Rsp5p [109].

The substrate(s) of Rsp5p critical for efficient internalization remains to be identified. Proteins playing an important role in organization of the actin cytoskeleton are potential candidates. Synthetic lethality was observed between mutations in RSP5, and mutations in several genes encoding proteins important for cytoskeleton organization, including Vrp1p/End5p, Pan1p and End3p [110, 111] or proteins colocalized with the actin cytoskeleton, such as Ede1p, the homolog of Eps15 [61]. Mutants with impaired actin cytoskeleton organization have been shown to display dislocalization of Rsp5p [106, 111]. Moreover, point mutations in the WW1 domain of Rsp5p result in resistance to latrunculin, a drug that sequesters actin monomers [111]. The link between Rsp5p and the actin cytoskeleton was further underlined by systematic genomic approaches. Large-scale analysis of protein complexes has revealed that Rsp5p interacts with actin, and with Las17p (Bee1p), a member of the Wiskott-Aldrich Syndrome protein (WASP) family of actin-assembly proteins. Affinity precipitation and two hybrid analysis, respectively, have shown an interaction between Rsp5p and two Las17p partners, Lsb1, and Lsb7p/Bzz1p required for the recruitment of actin polymerization machinery [112]. Affinity precipitation also evidenced an interaction between Rsp5p and the two amphiphysin homologs, Rvs161p and Rvs167p. Interestingly, Lsb1p and Rvs167p both display PPXY motif potentially recognized by Rsp5p WW domains. The interaction between Rsp5p and the amphiphysin homolog Rvs167p was indeed documented by two hybrid and biochemical technics [107]. Furthermore, Rvs167p was demonstrated to undergo Rsp5p-dependent monoubiquitylation on a target Lys (Lys481) within Rvs167p SH3 domain. However, mutation of this Lys to Arg did not impair fluid phase endocytosis nor a-factor internalization [107]. Although these data provide the first identification of an Rsp5p substrate among proteins required for endocytosis and actin cytoskeleton organization, the potential role of Rsp5p on the endocytic machinery and/or actin cytoskeleton organization remains to be defined. Whether the mammalian amphiphysin also undergo ubiquitylation also remains an open question.