Ubiquitin regulates protein transport between membrane compartments by serving as a sorting signal on protein cargo and by controlling the activity of trafficking machinery. Monoubiquitin attached to integral plasma membrane proteins or to associated transport modifiers serves as a regulated signal for internalization into the endocytic pathway. Similarly, monoubiquitin attached to biosynthetic and endocytic membrane proteins is a signal for sorting of cargo into vesicles that bud into the late endosome lumen for delivery into the lysosome. Ubiquitination of trans-acting endocytic proteins is also required for transport, and key endocytic proteins are modified by monoubiquitin. Regulatory enzymes of the ubiquitination machinery, ubiquitin ligases, control the timing and specificity of plasma membrane protein downregulation in such diverse biological processes as cell fate specification and neurotransmission. Monoubiquitin signals appended by these ligases are recognized by endocytic proteins carrying ubiquitin-binding motifs, including UBA, UEV, UIM, and CUE domains. The UIM proteins epsins and Hrs are excellent candidates for adaptors that link ubiquitinated cargo to the clathrin-based sorting machinery at appropriate regions of the endosomal or plasma membranes. Other ubiquitin-binding proteins also play crucial roles in cargo transport, although in most cases the role of ubiquitin-binding is not defined. Ubiquitin-binding proteins such as epsins, Hrs, and Vps9 are monoubiquitinated, indicating the general nature of ubiquitin regulation in endocytosis and suggesting new models to explain how recognition of monoubiquitin signals may be regulated.

Modification of an S. cerevisiae G protein-coupled receptor with ubiquitin is required for its ligand-stimulated internalization. We now demonstrate that monoubiquitination on a single lysine residue is sufficient to signal receptor internalization, a modification distinct from that required for proteasome recognition. Formation of a polyubiquitin chain is not necessary, as demonstrated by the ability of mutant ubiquitins that lack lysine residues to serve as efficient internalization signals. Fusion of ubiquitin in-frame to a receptor that lacks cytoplasmic tail lysines also promotes rapid receptor internalization, indicating that ubiquitin itself and not a specific type of linkage to the receptor acts as an internalization signal. Thus, we have defined a cellular function for monoubiquitination in alpha-factor receptor endocytosis.

Ubiquitin ligases of the Nedd4 family regulate membrane protein trafficking by modifying both cargo proteins and the transport machinery with ubiquitin. Here, we investigate the role of the yeast Nedd4 homologue, Rsp5, in protein sorting into vesicles that bud into the multivesicular endosome (MVE) en route to the vacuole. A mutant lacking the Rsp5 C2 domain is unable to ubiquitinate or sort biosynthetic cargo into MVE vesicles, whereas endocytic cargo is ubiquitinated and sorted efficiently. The C2 domain binds specifically to phosphoinositides in vitro and is sufficient for localization to membranes in intact cells. Mutation of a lysine-rich patch on the surface of the C2 domain abolishes membrane interaction and disrupts sorting of biosynthetic cargo. Translational fusion of ubiquitin to a biosynthetic cargo protein alleviates the requirement for the C2 domain in its MVE sorting. These results demonstrate that the C2 domain specifies Rsp5-dependent ubiquitination of endosomal cargo and suggest that Rsp5 function is regulated by membrane phosphoinositides.

Yeast Rsp5p and its mammalian homologue, Nedd4, are hect domain ubiquitin-protein ligases (E3s) required for the ubiquitin-dependent endocytosis of plasma membrane proteins. Because ubiquitination is sufficient to induce internalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis. Rsp5p is an essential, multidomain protein containing an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity. In this study, we demonstrate that Rsp5p is peripherally associated with membranes and provide evidence that Rsp5p functions as part of a multimeric protein complex. We define the function of Rsp5p and its domains in the ubiquitindependent internalization of the yeast ␣-factor receptor, Ste2p. Temperature-sensitive rsp5 mutants were unable to ubiquitinate or to internalize Ste2p at the nonpermissive temperature. Deletion of the entire C2 domain had no effect on ␣-factor internalization; however, point mutations in any of the three WW domains impaired both receptor ubiquitination and internalization. These observations indicate that the WW domains play a role in the important regulatory event of selecting phosphorylated proteins as endocytic cargo. In addition, mutations in the C2 and WW1 domains had more severe defects on transport of fluid-phase markers to the vacuole than on receptor internalization, suggesting that Rsp5p functions at multiple steps in the endocytic pathway. INTRODUCTIONUbiquitin is a 76-amino acid polypeptide that is highly conserved and expressed in all eukaryotic cells. One role of ubiquitin is to tag proteins for degradation by the cytosolic 26S proteasome (reviewed by Hershko and Ciechanover, 1998). Another is to trigger the internalization of cell surface proteins (reviewed by Bonifacino and Weissman, 1998;Hicke, 1999;and Strous and Govers, 1999). Ubiquitin is linked to substrates by a covalent isopeptide bond between the carboxy-terminal glycine of the ubiquitin molecule and the ⑀-amino group of lysines within the substrate protein.Protein ubiquitination is an ATP-dependent reaction catalyzed by the sequential activity of a cascade of three enzymes: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin-protein ligases (E3s). In most ubiquitination reactions, E3s recognize specific substrates. E3s are broadly defined as proteins that bind to a substrate, directly or indirectly, and promote the transfer of ubiquitin from a thiolester intermediate to the protein substrate or a growing polyubiquitin chain on the substrate (Hershko and Ciechanover, 1998). There are two known major classes of E3s. Members of the first class contain a conserved ϳ350-amino acid hect (homologous to E6-AP carboxy terminus) catalytic domain that participates directly in catalysis by forming a thiolester bond with ubiquitin during the ubiquitination reaction (Huibregtse et al., 1995). The second class of ubiquitin-protein ligases are characterized by the presence of a zin...

Ubiquitination of integral plasma membrane proteins triggers their rapid internalization into the endocytic pathway. The yeast ubiquitin ligase Rsp5p, a homologue of mammalian Nedd4 and Itch, is required for the ubiquitination and subsequent internalization of multiple plasma membrane proteins, including the ␣-factor receptor (Ste2p). Here we demonstrate that Rsp5p plays multiple roles at the internalization step of endocytosis. Temperature-sensitive rsp5 mutant cells were defective in the internalization of ␣-factor by a Ste2p-ubiquitin chimera, a receptor that does not require post-translational ubiquitination. Similarly, a modified version of Ste2p bearing a NPFXD linear peptide sequence as its only internalization signal was not internalized in rsp5 cells. Internalization of these variant receptors was dependent on the catalytic cysteine residue of Rsp5p and on ubiquitin-conjugating enzymes that bind Rsp5p. Thus, a Rsp5p-dependent ubiquitination event is required for internalization mediated by ubiquitindependent and -independent endocytosis signals. Constitutive Ste2p-ubiquitin internalization and fluidphase endocytosis also required active ubiquitination machinery, including Rsp5p. These observations indicate that Rsp5p-dependent ubiquitination of a transacting protein component of the endocytosis machinery is required for the internalization step of endocytosis.Ubiquitin is a highly conserved 76-amino acid polypeptide that becomes covalently linked to substrate proteins by an isopeptide bond. Two characterized functions of protein ubiquitination are to target proteins for degradation by the cytosolic 26 S proteasome or to promote the internalization of cell surface proteins into the endocytic pathway. Recognition of cytosolic proteins by the proteasome generally requires modification with a polyubiquitin chain of at least four ubiquitin subunits (1, 2). In contrast, modification of plasma membrane proteins with monoubiquitin or Lys 63 -linked di-ubiquitin chains triggers internalization into the endocytic pathway (3-8), ultimately leading to degradation in the lumen of the lysosome.Protein ubiquitination is catalyzed by a cascade of three enzymes (reviewed in Refs. 9 and 10). Ubiquitin-activating enzymes (E1s) 1 activate ubiquitin in an ATP-dependent reaction, forming a high-energy thiolester bond with ubiquitin. The activated ubiquitin is then passed to a cysteine residue in a ubiquitin-conjugating enzyme (E2). Normally, E2s function together with ubiquitin ligases (E3s) to catalyze isopeptide bond formation between the carboxyl terminus of ubiquitin and ⑀-amino groups in lysine side chains of specific substrates. E3s bind directly to substrates and comprise the major specificity determinants of the ubiquitination machinery. There are two major classes of E3s. One class carries a RING finger domain, and the other carries a hect (homologous to E6-AP carboxyl terminus) domain. RING finger E3s function as adapter proteins, bringing the substrate to the ubiquitin-charged E2, whereas E3s of the hect domain fa...