The ubiquitin-associated (UBA) domain occurs frequently in proteins involved in ubiquitin-dependent signaling pathways. Although polyubiquitin chain binding is considered to be a defining feature of the UBA domain family, the generality of this property has not been established. Here we have surveyed the polyubiquitin interaction properties of 30 UBA domains, including 16 of 17 occurrences in budding yeast. The UBA domains sort into four classes that include linkage-selective polyubiquitin binders and domains that bind different chains (and monoubiquitin) in a nondiscriminatory manner; one notable class ( approximately 30%) did not bind any ubiquitin ligand surveyed. The properties of a given UBA domain are conserved from yeast to mammals. Their functional relevance is further suggested by the ability of an ectopic UBA domain to alter the specificity of a deubiquitylating enzyme in a predictable manner. Conversely, non-UBA sequences can modulate the interaction properties of a UBA domain.

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Although functional diversity in polyubiquitin chain signaling has been ascribed to the ability of differently linked chains to bind in a distinctive manner to effector proteins, structural models of such interactions have been lacking. Here, we use NMR to unveil the structural basis of selective recognition of Lys48-linked di- and tetraubiquitin chains by the UBA2 domain of hHR23A. Although the interaction of UBA2 with Lys48-linked diubiquitin involves the same hydrophobic surface on each ubiquitin unit as that utilized in monoubiquitin:UBA complexes, our results show how the "closed" conformation of Lys48-linked diubiquitin is crucial for high-affinity binding. Moreover, recognition of Lys48-linked diubiquitin involves a unique epitope on UBA, which allows the formation of a sandwich-like diubiqutin:UBA complex. Studies of the UBA-tetraubiquitin interaction suggest that this mode of UBA binding to diubiquitin is relevant for longer chains.

FAT10 is a small ubiquitin-like modifier that is encoded in the major histocompatibility complex and is synergistically inducible by tumor necrosis factor alpha and gamma interferon. It is composed of two ubiquitinlike domains and possesses a free C-terminal diglycine motif that is required for the formation of FAT10 conjugates. Here we show that unconjugated FAT10 and a FAT10 conjugate were rapidly degraded by the proteasome at a similar rate. Fusion of FAT10 to the N terminus of very long-lived proteins enhanced their degradation rate as potently as fusion with ubiquitin did. FAT10-green fluorescent protein fusion proteins were not cleaved but entirely degraded, suggesting that FAT10-specific deconjugating enzymes were not present in the analyzed cell lines. Interestingly, the prevention of ubiquitylation of FAT10 by mutation of all lysines or by expression in ubiquitylation-deficient cells did not affect FAT10 degradation. Thus, conjugation with FAT10 is an alternative and ubiquitin-independent targeting mechanism for degradation by the proteasome, which, in contrast to polyubiquitylation, is cytokine inducible and irreversible.The ubiquitin (Ub)-proteasome pathway is the main system for the targeted degradation of intracellular proteins (37). Depending on the metabolic and functional requirement of a cell, regulatory proteins like cell cycle regulators, transcription factors, or key enzymes can be specifically selected for degradation by the 26S proteasome. The basis for selectivity does not lie in the protease itself but rather in the selective covalent modification of target proteins with Lys48-linked polyubiquitin chains. Polyubiquitylation is achieved by an enzymatic cascade of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). The specificity of substrate recognition is afforded by the numerous ubiquitin ligases which selectively bind a substrate as well as an E2 enzyme, thus facilitating the formation of isopeptide bonds between the ε-amino group of lysines in a substrate protein and the diglycine motif at the carboxy terminus of ubiquitin (11). Monomeric ubiquitin is processed from precursor proteins through ubiquitin-specific proteases which recognize the Cterminal diglycine motif along with the ubiquitin domain and which cleave after the diglycine motif irrespective of whether it is isopeptide linked or linked through a conventional peptide bond (38). Before degradation, the ubiquitin chains are removed from the substrate and disassembled into monomeric ubiquitin which can be reused. Ubiquitin levels are hence kept at a steady-state level, and the ubiquitin protein itself is long lived (9, 26).A growing number of proteins which contain domains with significant homology to ubiquitin have been discovered over the past 5 years. These ubiquitin-like proteins can be assigned either to the group of ubiquitin domain proteins which contain a ubiquitin homology domain but which do not become covalently linked to target proteins or to the group of ubiquitinli...

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Ubiquilin/PLIC proteins belong to the family of UBL-UBA proteins implicated in the regulation of the ubiquitin-dependent proteasomal degradation of cellular proteins. A human presenilin-interacting protein, ubiquilin-1, has been suggested as potential therapeutic target for treating Huntington's disease. Ubiquilin's interactions with mono- and polyubiquitins are mediated by its UBA domain, which is one of the tightest ubiquitin binders among known ubiquitin-binding domains. Here we report the three-dimensional structure of the UBA domain of ubiquilin-1 (UQ1-UBA) free in solution and in complex with ubiquitin. UQ1-UBA forms a compact three-helix bundle structurally similar to other known UBAs, and binds to the hydrophobic patch on ubiquitin with a K(d) of 20 microM. To gain structural insights into UQ1-UBA's interactions with polyubiquitin chains, we have mapped the binding interface between UQ1-UBA and Lys48- and Lys63-linked di-ubiquitins and characterized the strength of UQ1-UBA binding to these chains. Our NMR data show that UQ1-UBA interacts with the individual ubiquitin units in both chains in a mode similar to its interaction with mono-ubiquitin, although with an improved binding affinity for the chains. Our results indicate that, in contrast to UBA2 of hHR23A that has strong binding preference for Lys48-linked chains, UQ1-UBA shows little or no binding selectivity toward a particular chain linkage or between the two ubiquitin moieties in the same chain. The structural data obtained in this study provide insights into the possible structural reasons for the diversity of polyubiquitin chain recognition by UBA domains.

FAT10 is a ubiquitin-like protein that is encoded in the major histocompatibility complex class I locus and is synergistically inducible with interferon-␥ and tumor necrosis factor ␣. The molecule consists of two ubiquitin-like domains in tandem arrangement and bears a conserved diglycine motif at its carboxyl terminus commonly used in ubiquitin-like proteins for isopeptide linkage to conjugated proteins. We investigated the function of FAT10 by expressing murine FAT10 in a hemagglutinin-tagged wild type form as well as a diglycine-deficient mutant form in mouse fibroblasts in a tetracycline-repressible manner. FAT10 expression did not affect major histocompatibility complex class I cell surface expression or antigen presentation. However, we found that wild type but not mutant FAT10 caused apoptosis within 24 h of induction in a caspase-dependent manner as indicated by annexin V cell surface staining and DNA fragmentation. Wild type FAT10, but not its diglycine mutant, was covalently conjugated to thus far unidentified proteins, indicating that specific FAT10 activating and conjugating enzymes must be operative in unstimulated fibroblasts. Because FAT10 expression causes apoptosis and is inducible with tumor necrosis factor ␣, it may be functionally involved in the programmed cell death mediated by this cytokine.The covalent posttranslational modification of proteins is a versatile principle of determining the half-life, intracellular localization, and activity of proteins. In addition to modification by small molecules like orthophosphate, acetic acid, lipids, or sugars, the attachment of protein tags via isopeptide linkage to lysine residues in target proteins has recently been recognized as a frequently used and very diverse theme in cell biology. The prototype of such a protein tag is ubiquitin, which, when assembled as a polyubiquitin chain onto substrate proteins, can target these proteins to the 26S proteasome for degradation (1). Recently, it has become clear that the manner in which ubiquitin is linked in polyubiquitin chains defines the fate of the modified protein. The specific targeting signal for the proteasome pathway is a polyubiquitin chain that uses the K48 residue of the proximal ubiquitin as a target for further rounds of ubiquitination. The formation of K63-linked ubiquitin polymers, in contrast, does not lead to degradation but seems to play a role in DNA repair (2) and endocytosis (3). The conjugation of receptors with a single ubiquitin moiety can also serve as a signal for endocytosis (4), whereas in the case of histone H2B, monoubiquitination appears to regulate nucleosome function and cell division (5).The specificity of substrate selection and the mode of ubiquitin conjugation are determined by an enzymatic cascade required for the activation and specific transfer of ubiquitin. It consists of ubiquitin-activating enzyme (E1) 1 , which uses the energy of ATP to form a thioester linkage between a cysteine residue of the E1 enzyme and the carboxyl terminus of ubiquitin consisting of two ...