Linda Hicke

Protein regulation by monoubiquitin

Multi-ubiquitin chains at least four subunits long are required for efficient recognition and degradation of ubiquitylated proteins by the proteasome, but other functions of ubiquitin have been discovered that do not involve the proteasome. Some proteins are modified by a single ubiquitin or short ubiquitin chains. Instead of sending proteins to their death through the proteasome, monoubiquitylation regulates processes that range from membrane transport to transcriptional regulation.

Ubiquitin-binding domains

Ubiquitin-binding domains (UBDs) are a collection of modular protein domains that non-covalently bind to ubiquitin. These recently discovered motifs interpret and transmit information conferred by protein ubiquitylation to control various cellular events. Detailed molecular structures are known for a number of UBDs, but to understand their mechanism of action, we also need to know how binding specificity is determined, how ubiquitin binding is regulated, and the function of UBDs in the context of full-length proteins. Such knowledge will be key to our understanding of how ubiquitin regulates cellular proteins and processes.

Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels.

G-protein-coupled receptors and transporters in Saccharomyces cerevisiae are modified with ubiquitin in response to ligand biding. In most cases, the proteasome does not recognize these ubiquitinated proteins. Instead, ubiquitination serves to trigger internalization and degradation of plasma membrane proteins in the lysosome-like vacuole. A number of mammalian receptors and at least one ion channel undergo ubiquitination at the plasma membrane, and this modification is required for their downregulation. Some of these cell-surface proteins appear to be degraded by both the proteasome and lysosomal proteases. Recent evidence indicates that other proteins required for receptor internalization might also be regulated by ubiquitination, suggesting that ubiquitin plays diverse roles in regulating plasma membrane protein activity.

Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis

Ubiquitin functions as a signal for sorting cargo at multiple steps of the endocytic pathway and controls the activity of trans-acting components of the endocytic machinery (reviewed in refs 1, and 2). By contrast to proteasome degradation, which generally requires a polyubiquitin chain that is at least four subunits long, internalization and sorting of endocytic cargo at the late endosome are mediated by mono-ubiquitination. Here, we demonstrate that ubiquitin-interacting motifs (UIMs) found in epsins and Vps27p (ref. 9) from Saccharomyces cerevisiae are required for ubiquitin binding and protein transport. Epsin UIMs are important for the internalization of receptors into vesicles at the plasma membrane. Vps27p UIMs are necessary to sort biosynthetic and endocytic cargo into vesicles that bud into the lumen of a late endosomal compartment, the multivesicular body. We propose that mono-ubiquitin regulates internalization and endosomal sorting by interacting with modular ubiquitin-binding domains in core components of the protein transport machinery. UIM domains are found in a broad spectrum of proteins, consistent with the idea that mono-ubiquitin can function as a regulatory signal to control diverse biological activities.

A Function for Monoubiquitination in the Internalization of a G Protein–Coupled Receptor

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.

A New Ticket for Entry into Budding Vesicles—Ubiquitin

The role of ubiquitin in promoting internalization is not restricted to tagging cargo; ubiquitination appears to regulate the activity of one or more component(s) of and Cell Biology the endocytic apparatus as well. At least one plasma Northwestern University membrane protein, the growth hormone receptor, re-Evanston, Illinois 60208 quires cellular ubiquitinating enzymes to be efficiently internalized even though it does not need to be ubiquiti-Sorting signals determine the location and fate of pro-nated itself (Govers et al., 1999). Similarly, the internal-teins. These signals can be carried within the amino acid ization of a yeast receptor-ubiquitin chimera that does sequence of a protein or they can be posttranslationally not need to be posttranslationally ubiquitinated prior to appended in a regulated manner. With several recent endocytosis still requires ubiquitin-conjugating en-and surprising revelations, it is now clear that the poly-zymes and the Rsp5 ubiquitin ligase (a Nedd4 homolog) peptide ubiquitin can act as a regulated sorting signal (Dunn and Hicke, 2001). In these cases, the target of the at different steps of the endosomal and biosynthetic ubiquitination machinery appears to be a transacting pathways. Ubiquitin is a 76 amino acid protein that be-endocytic protein(s). One candidate is Eps15, a core comes conjugated to proteins through the concerted component of the clathrin-based machinery, that be-action of three enzymes: a ubiquitin-activating enzyme comes monoubiquitinated upon treatment of cells with (E1), a ubiquitin-conjugating enzyme (E2), and a ubiqui-growth factor (van Delft et al., 1997). tin ligase (E3) involved in target recognition. The ubiquiti-Ubiquitin and ubiquitin ligases are required not only nation reaction usually results in the formation of an for vesicle budding into the cell but also play a role in isopeptide bond between the C terminus of ubiquitin budding into the extracellular milieu, specifically in the and lysine residues in substrates. Substrates can be release of enveloped viruses from infected cells (re-modified with monoubiquitin or with a polyubiquitin viewed in Hicke, 2001). The role of ubiquitin in budding chain that is linked through lysines present in ubiquitin is best characterized for retroviruses. The retroviral Gag itself. Ubiquitins well-established function is to target protein carries a short sequence called the late (L) do-proteins for degradation by the 26S proteasome. How-main that is required late in the budding process. L ever, noncanonical functions of ubiquitin are proliferat-domains of most enveloped viruses carry proline-rich ing rapidly and some of the best characterized are in sequences that promote Gag …

Monoubiquitin carries a novel internalization signal that is appended to activated receptors

Ubiquitin modification of signal transducing receptors at the plasma membrane is necessary for rapid receptor internalization and downregulation. We have investigated whether ubiquitylation alters a receptor cytoplasmic tail to reveal a previously masked internalization signal, or whether ubiquitin itself carries an internalization signal. Using an α‐factor receptor–ubiquitin chimeric protein, we demonstrate that monoubiquitin can mediate internalization of an activated receptor that lacks all cytoplasmic tail sequences. Furthermore, fusion of ubiquitin in‐frame to the stable plasma membrane protein Pma1p stimulates endocytosis of this protein. Ubiquitin does not carry a functional tyrosine‐ or di‐leucine‐based internalization signal. Instead, the three‐dimensional structure of the folded ubiquitin polypeptide carries an internalization signal that consists of two surface patches surrounding the critical residues Phe4 and Ile44. We conclude that ubiquitin functions as a novel regulated internalization signal that can be appended to a plasma membrane protein to trigger downregulation.

A ubiquitin‐binding motif required for intramolecular monoubiquitylation, the CUE domain

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 specifically with monoubiquitin. The region required for monoubiquitin binding mapped to the Vps9 CUE domain, a sequence previously identified 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 defined 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.

Solution Structure of a CUE-Ubiquitin Complex Reveals a Conserved Mode of Ubiquitin Binding

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.

Solution structure of Vps27 UIM–ubiquitin complex important for endosomal sorting and receptor downregulation

Monoubiquitylation is a well‐characterized signal for the internalization and sorting of integral membrane proteins to distinct cellular organelles. Recognition and transmission of monoubiquitin signals is mediated by a variety of ubiquitin‐binding motifs such as UIM, UBA, UEV, VHS and CUE in endocytic proteins. The yeast Vps27 protein requires two UIMs for efficient interactions with ubiquitin and for sorting cargo into multivesicular bodies. Here we show that the individual UIMs of Vps27 exist as autonomously folded α‐helices that bind ubiquitin independently, non‐cooperatively and with modest affinity. The Vps27 N‐terminal UIM engages the Leu8–Ile44–Val70 hydrophobic patch of ubiquitin through a helical surface conserved in UIMs of diverse proteins, including that of the S5a proteasomal regulatory subunit. The Leu8–Ile44–Val70 ubiquitin surface is also the site of interaction for CUE and UBA domains in endocytic proteins, consistent with the view that ubiquitin‐ binding endocytic proteins act serially on the same monoubiquitylated cargo during transport from cell surface to the lysosome.