A growing number of cellular regulatory mechanisms are being linked to protein modification by the polypeptide ubiquitin. These include key transitions in the cell cycle, class I antigen processing, signal transduction pathways, and receptor-mediated endocytosis. In most, but not all, of these examples, ubiquitination of a protein leads to its degradation by the 26S proteasome. Following attachment of ubiquitin to a substrate and binding of the ubiquitinated protein to the proteasome, the bound substrate must be unfolded (and eventually deubiquitinated) and translocated through a narrow set of channels that leads to the proteasome interior, where the polypeptide is cleaved into short peptides. Protein ubiquitination and deubiquitination are both mediated by large enzyme families, and the proteasome itself comprises a family of related but functionally distinct particles. This diversity underlies both the high substrate specificity of the ubiquitin system and the variety of regulatory mechanisms that it serves.

Following the discovery of protein modification by the small, highly conserved ubiquitin polypeptide, a number of distinct ubiquitin-like proteins (Ubls) have been found to function as protein modifiers as well. These Ubls, which include SUMO, ISG15, Nedd8, and Atg8, function as critical regulators of many cellular processes, including transcription, DNA repair, signal transduction, autophagy, and cell-cycle control. A growing body of data also implicates the dysregulation of Ubl-substrate modification and mutations in the Ubl-conjugation machinery in the etiology and progression of a number of human diseases. The primary aim of this review is to summarize the latest developments in our understanding of the different Ubl-protein modification systems, including the shared and unique features of these related pathways.

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Summary All seven lysine residues in ubiquitin contribute to the synthesis of polyubiquitin chains on protein substrates. Whereas K48-linked chains are well established as mediators of proteasomal degradation, and K63-linked chains act in nonproteolytic events, the roles of unconventional polyubiquitin chains linked through K6, K11, K27, K29, or K33 are not well understood. Here we report that the unconventional linkages are abundant in vivo, and all non-K63 linkages may target proteins for degradation. Ubiquitin with K48 as the single lysine cannot support yeast viability, and different linkages have partially redundant functions. By profiling both the entire yeast proteome and ubiquitinated proteins in wild-type and ubiquitin K11R mutant strains using mass spectrometry, we identified K11 linkage-specific substrates, including Ubc6, a ubiquitin conjugating enzyme involved in endoplasmic reticulum-associated degradation (ERAD). Ubc6 primarily synthesizes K11-linked chains, and K11 linkages function in the ERAD pathway. Thus, unconventional polyubiquitin chains are critical for ubiquitin-proteasome system function.

Attachment of ubiquitin to proteins is a crucial step in many cellular regulatory mechanisms and contributes to numerous biological processes, including embryonic development, the cell cycle, growth control, and prevention of neurodegeneration. In these diverse regulatory settings, the most widespread mechanism of ubiquitin action is probably in the context of protein degradation. Polyubiquitin attachment targets many intracellular proteins for degradation by the proteasome, and (mono)ubiquitination is often required for down-regulating plasma membrane proteins by targeting them to the vacuole (lysosome). Ubiquitin-protein conjugates are highly dynamic structures. While an array of enzymes directs the conjugation of ubiquitin to substrates, there are also dozens of deubiquitinating enzymes (DUBs) that can reverse the process. Several lines of evidence indicate that DUBs are important regulators of the ubiquitin system. These enzymes are responsible for processing inactive ubiquitin precursors, proofreading ubiquitin-protein conjugates, removing ubiquitin from cellular adducts, and keeping the 26S proteasome free of inhibitory ubiquitin chains. The present review focuses on recent discoveries that have led to a better understanding the mechanisms and physiological roles of this diverse and still poorly understood group of enzymes. We also discuss briefly some of the proteases that act on ubiquitin-like protein (UBL) conjugates and compare them to DUBs.

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In eukaryotes, protein function can be modulated by ligation to ubiquitin or to ubiquitin-like proteins (Ubl proteins). The vertebrate Ubl protein SUMO-1 is only 18% identical to ubiquitin but is 48% identical to the yeast protein Smt3. Both SUMO-1 and Smt3 are ligated to cellular proteins, and protein conjugation to SUMO-1/Smt3 is involved in many physiological processes. It remained unknown, however, whether deconjugation of SUMO-1/Smt3 from proteins is also essential. Here we describe a yeast Ubl-specific protease, Ulp1, which cleaves proteins from Smt3 and SUMO-1 but not from ubiquitin. Ulp1 is unrelated to any known deubiquitinating enzyme but shows distant similarity to certain viral proteases, indicating the existence of a widely conserved protease fold. Proteins related to Ulp1 are present in many organisms, including several human pathogens. The pattern of Smt3-coupled proteins in yeast changes markedly throughout the cell cycle, and specific conjugates accumulate in ulp1 mutants. Ulp1 has several functions, including an essential role in the G2/M phase of the cell cycle.

PrefaceThe ubiquitin-proteasome system degrades an enormous variety of proteins, which are targeted by specific degradation signals (degrons). Besides the degradation of regulatory proteins, virtually every protein suffers from sporadic biosynthetic errors or misfolding, and cells can recognize such aberrant proteins and rapidly degrade them. Structural and functional data on a handful of degrons allows some generalizations about their mechanism of action. We focus on different strategies of degron recognition by the ubiquitin system, and contrast regulatory degrons subject to signalling-dependent modification and those controlled by protein folding or assembly, as frequently occurs during protein quality control. IntroductionIntracellular protein degradation has been studied for more than half a century, and it became clear early on that such degradation is highly selective, with individual protein half-lives ranging from minutes to years (for reviews of the early literature, see refs. 1-2). Moreover, much of this degradation was found to be energy-dependent despite the exergonic nature of peptide-bond cleavage. This energy dependence derives from the dual requirements of high substrate specificity and substrate protein unfolding to make the polypeptide backbone fully accessible for proteolytic cleavage. The vast majority of regulated protein degradation in eukaryotes is executed by the ubiquitin-proteasome system 3-5. Polyubiquitin tagging of substrates by specific enzymes provides the major source of selectivity in the system (Box 1), whereas the 26S proteasome complex performs the protein unfolding necessary for processive cleavage of the tagged proteins into short peptides (Box 2). In addition, ubiquitin ligation can function independently of the proteasome by directing certain -usually membrane-proteins to the lysosome/vacuole for proteolysis. Conversely, proteasomes can degrade some proteins without their prior modification by ubiquitin.A fundamental question about intracellular proteolysis is how specific proteins are recognized by the proteolytic machinery, resulting in proteins being degraded only under specific conditions with highly characteristic degradation rates. Early work had suggested that global structural features determine the metabolic stability of individual proteins. For instance, mutant proteins or proteins that had incorporated amino acid analogues during their synthesis were found to have shorter half-lives in vivo than their wild-type counterparts 6,7. Moreover, protein degradation rates appeared to correlate with gross protein physicochemical properties such as molecular mass or isoelectric point 8,9. However, later analyses revealed that correlations with gross protein properties did not generally hold true, and though abnormal proteins were frequently short-lived, this need not reflect a global change in their structure. NIH Public Access Author ManuscriptNat Rev Mol Cell Biol. Author manuscript; available in PMC 2009 March 1. Published in final edited form as:Nat Rev Mol Cell Bio...