Monoubiquitylation is a regulatory signal, like phosphorylation, that can alter the activity, location or structure of a protein. Monoubiquitin signals are likely to be recognized by ubiquitin-binding proteins that transmit the regulatory information conferred by monoubiquitylation. To identify monoubiquitin-binding proteins, we used a mutant ubiquitin that lacks the primary site of polyubiquitin chain formation as bait in a two-hybrid screen. The C-terminus of Vps9, a protein required in the yeast endocytic pathway, interacted speci®cally with monoubiquitin. The region required for monoubiquitin binding mapped to the Vps9 CUE domain, a sequence previously identi®ed by database searches as similar to parts of the yeast Cue1 and mammalian Tollip proteins. We demonstrate that CUE domains bind directly to monoubiquitin and we have de®ned crucial interaction surfaces on both binding partners. The Vps9 CUE domain is required to promote monoubiquitylation of Vps9 by the Rsp5 hect domain ubiquitin ligase. Thus, we conclude that the CUE motif is an evolutionarily conserved monoubiquitin-binding domain that mediates intramolecular monoubiquitylation.

Monoubiquitination serves as a regulatory signal in a variety of cellular processes. Monoubiquitin signals are transmitted by binding to a small but rapidly expanding class of ubiquitin binding motifs. Several of these motifs, including the CUE domain, also promote intramolecular monoubiquitination. The solution structure of a CUE domain of the yeast Cue2 protein in complex with ubiquitin reveals intermolecular interactions involving conserved hydrophobic surfaces, including the Leu8-Ile44-Val70 patch on ubiquitin. The contact surface extends beyond this patch and encompasses Lys48, a site of polyubiquitin chain formation. This suggests an occlusion mechanism for inhibiting polyubiquitin chain formation during monoubiquitin signaling. The CUE domain shares a similar overall architecture with the UBA domain, which also contains a conserved hydrophobic patch. Comparative modeling suggests that the UBA domain interacts analogously with ubiquitin. The structure of the CUE-ubiquitin complex may thus serve as a paradigm for ubiquitin recognition and signaling by ubiquitin binding proteins.

SH3 domains are modules of 50-70 amino acids that promote interactions among proteins, often participating in the assembly of large dynamic complexes. These domains bind to peptide ligands, which usually contain a core Pro-X-X-Pro (PXXP) sequence. Here we identify a class of SH3 domains that bind to ubiquitin. The yeast endocytic protein Sla1, as well as the mammalian proteins CIN85 and amphiphysin, carry ubiquitin-binding SH3 domains. Ubiquitin and peptide ligands bind to the same hydrophobic groove on the SH3 domain surface, and ubiquitin and a PXXP-containing protein fragment compete for binding to SH3 domains. We conclude that a subset of SH3 domains constitutes a distinct type of ubiquitin-binding domain and that ubiquitin binding can negatively regulate interaction of SH3 domains with canonical proline-rich ligands.

To elucidate subunit C function, we performed random and site-directed mutagenesis of the yeast VMA5 gene. Site-directed mutations in the most highly conserved region of Vma5p, residues 305-325, decreased catalytic activity of the V-ATPase by up to 48% without affecting assembly. A truncation mutant (K360stop) identified by random mutagenesis suggested a small region near the C terminus of the protein (amino acids 382-388) might be important for subunit stability. Site-directed mutagenesis revealed that three aromatic amino acids in this region (Tyr-382, Phe-385, and Tyr-388) in addition to four other conserved aromatic amino acids (Phe-260, Tyr-262, Phe-296, Phe-300) are essential for stable assembly of V 1 with V 0 , although alanine substitutions at these positions support some activity in vivo. Surprisingly, three mutations (F260A, Y262A, and F385A) greatly decrease the stability of the V-ATPase in vitro but increase its k cat for ATP hydrolysis and proton transport by at least 3-fold. The peripheral stalk of V-ATPases must balance the stability essential for productive catalysis with the dynamic instability involved in regulation; these three mutations may perturb that balance. Vacuolar Hϩ -ATPases (V-ATPases) 1 are found throughout the endomembrane system of all eukaryotes and at the plasma membrane of certain cells (1-4). In all of these locations, VATPases act as electrogenic proton pumps, coupling hydrolysis of cytosolic ATP to proton transport either into membranebound compartments or across the plasma membrane to the outside of the cell. V-ATPases consist of a peripheral complex containing the sites of ATP hydrolysis, the V 1 sector, attached to an integral membrane complex that forms the proton channel, the V 0 sector.Saccharomyces cerevisiae has proven to be an excellent model system for V-ATPases (1, 5-7). The yeast V-ATPase is very similar to those of higher eukaryotes both in its overall structure and in the primary sequence of its subunit genes. To date, 13 subunits have been identified in the yeast V-ATPase and shown to have homologues in other organisms. The V 1 sector contains eight subunits, designated A-H, which are encoded by the yeast VMA1, VMA2, VMA5, VMA8, VMA4, VMA7, VMA10, and VMA13 genes, respectively. The V 0 sector contains five subunits, designated a, c, cЈ, cЉ, and d, which are encoded by the VPH1 (or STV1), VMA3, VMA11, VMA16, and VMA6 genes, respectively (1).The mechanism by which the V-ATPase couples hydrolysis of cytosolic ATP to proton translocation is not fully understood. The F 1 F 0 -ATP synthase (F-ATPase) has been proposed as a model for the catalytic mechanism of V-ATPases (4). V-and F-ATPases are evolutionarily related, based on sequence similarity in the catalytic and regulatory nucleotide binding and the proteolipid subunits (8). Structural data for the enzymes also suggest they are related. Electron microscopic analysis of the V-ATPase demonstrates that, similar to the structure of the F-ATPase, the V 1 sector is attached to the V 0 sector through two stalks (9, 10...

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