Muscle wasting is a debilitating consequence of fasting, inactivity, cancer, and other systemic diseases that results primarily from accelerated protein degradation by the ubiquitin-proteasome pathway. To identify key factors in this process, we have used cDNA microarrays to compare normal and atrophying muscles and found a unique gene fragment that is induced more than ninefold in muscles of fasted mice. We cloned this gene, which is expressed specifically in striated muscles. Because this mRNA also markedly increases in muscles atrophying because of diabetes, cancer, and renal failure, we named it atrogin-1. It contains a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the other components of SCF-type Ub-protein ligases (E3s), as well as a nuclear localization sequence and PDZ-binding domain. On fasting, atrogin-1 mRNA levels increase specifically in skeletal muscle and before atrophy occurs. Atrogin-1 is one of the few examples of an F-box protein or Ub-protein ligase (E3) expressed in a tissuespecific manner and appears to be a critical component in the enhanced proteolysis leading to muscle atrophy in diverse diseases.

more than 40% identity with the six ATPases in the 19S complex and thus appears to be the evolutionary precursor to the 19S base and functioned before prote-Summary olysis became linked to ubiquitination in eukaryotes (Zwickl et al., 2000). These enzymes are all members of The 19S component of the 26S proteasome contains the AAA superfamily of multimeric ATPases, which also six ATPase subunits. To clarify how they unfold and transincludes the ATP-dependent proteases, Lon and FtsH, locate proteins into the 20S proteasome for degradaand the regulatory components of the bacterial ATPtion, we studied the homologous archaebacterial prodependent proteases, ClpAP, ClpXP, and HslUV (Bauteasome-regulatory ATPase complex PAN and the meister et al., 1998; Larsen and Finley, 1997). PAN apglobular substrate GFP-SsrA. When we attached a pears to be composed of one or two hexameric rings small (Biotin) or large (Biotin-Avidin) moiety near its N surrounding a central pore (our unpublished data), and terminus or a Biotin near its C terminus, GFP-SsrA thus its general structure resembles that of other AAA was unfolded and degraded. However, attaching Avifamily members. In the case of PAN and the other ATPdin near its C terminus blocked passage through PAN dependent proteases, it seems likely that protein suband prevented GFP-SsrA degradation. Though not strates are translocated into the proteolytic component translocated, GFP-Avidin still underwent ATP-depenby transfer through this narrow opening in the center of dent unfolding. Moreover, it remained bound to PAN the ATPase ring, and strong evidence in support of this and inhibited further proteolysis. Therefore, (1) transconclusion is presented in this paper. location and degradation of this substrate require Like the base of the eukaryotic 19S particle (Braun et threading through the ATPase in a C to N direction al., 1999; Strickland et al., 2000), PAN exhibits several and (2) translocation does not cause but follows ATPchaperone-like properties including the ability to bind dependent unfolding, which occurs on the surface of selectively unfolded polypeptides and prevent their agthe ATPase ring. gregation (Benaroudj and Goldberg, 2000). In addition, PAN has been shown to catalyze the ATP-dependent conformational changes that cause unfolding of pro-

The regulated degradation of proteins within eukaryotes and bacterial cells is catalyzed primarily by large multimeric proteases in ATP-dependent manner. In eukaryotes, the 26 S proteasome is essential for the rapid destruction of key regulatory proteins, such as cell cycle regulators and transcription factors, whose fast and tuned elimination is necessary for the proper control of the fundamental cell processes they regulate. In addition, the 26 S proteasome is responsible for cell quality control by eliminating defective proteins from the cytosol and endoplasmic reticulum. These defective proteins can be misfolded proteins, nascent prematurely terminated polypeptides, or proteins that fail to assemble into complexes. These diverse activities and its central role in apoptosis have made the proteasome an important target for drug development, in particular to combat malignancies. Marking Proteins for DegradationTargeting of most substrates to the 26 S proteasome requires their prior marking by a covalently linked polyubiquitin chain(s). During association with the proteasome, the substrate is directed into the catalytic core, where it is digested, whereas most of the ubiquitin molecules are recycled.Protein ubiquitination is a multistep process orchestrated by the concerted action of three enzymes. The reaction begins with E1, 2 which initially adenylates the C-terminal glycine of ubiquitin and then forms a thioester bond between the activated glycine residue and a cysteine residue on the E1 catalytic site. Next, E2 acquires the activated ubiquitin through a transthioesterification reaction to form a similar thioester bond between the E2 active-site cysteine and the activated ubiquitin. Finally, E3 recruits the target protein and guides the transfer of the activated ubiquitin from the E2 enzyme to the substrate. In most cases, an ⑀-NH 2 group of a lysine residue on the substrate attacks the thioester bond between the ubiquitin and E2, and an isopeptide bond is formed, linking the activated C-terminal glycine of ubiquitin to the amino group in the attacking lysine of the target substrate (1). Ubiquitin transfer from the E2 enzyme to the substrate is catalyzed directly by RING (really interesting new gene) finger-containing E3 enzymes and indirectly when a HECT (homologous to E6-AP carboxyl terminus) domain-containing E3 is mediating the transfer. The process is repeated in a cyclic manner where, in each step, a new moiety of ubiquitin is conjugated to an internal lysine residue (typically Lys 48 ) of the previously conjugated molecule. This generated polyubiquitin chain is regarded as the targeting signal for the downstream 26 S proteasome. However, in view of recent findings, several alternative mechanisms have been proposed (for a recent review, see Ref. 2). Li et al. (3) demonstrated in a reconstitutedcell-free system that a preformed polyubiquitin chain can be initially assembled on the active-site cysteine of E2 (UBE2G2), presumably by the action of an "exogenous" E2 acting in trans. Once assembled, an E3 en...

The ubiquitin-proteasome pathway plays a crucial role in many cellular processes by degrading substrates tagged by polyubiquitin chains, linked mostly through lysine 48 of ubiquitin. Although polymerization of ubiquitin via its six other lysine residues exists in vivo as part of various physiological pathways, the molecular mechanisms that determine the type of polyubiquitin chains remained largely unknown. We undertook a systematic, in vitro, approach to evaluate the role of E2 enzymes in determining the topology of polyubiquitin. Because this study was performed in the absence of an E3 enzyme, our data indicate that the E2 enzymes are capable of directing the ubiquitination process to distinct subsets of ubiquitin lysines, depending on the specific E2 utilized. Moreover, our findings are in complete agreement with prior analyses of lysine preference assigned to certain E2s in the context of E3 (in vitro and in vivo). Finally, our findings support the rising notion that the functional unit of E2 is a dimer. To our knowledge, this is the first systematic indication for the involvement of E2 enzymes in specifying polyubiquitin chain assembly.In eukaryotic cells, most proteins are degraded by the 26 S proteasome, which hydrolyzes in an ATP-dependant manner, both ubiquitin-conjugated and certain non-ubiquitinated proteins. In addition to its role in the turnover of damaged or misfolded proteins, the proteasome controls the cell cycle and other processes through the degradation of critical regulatory components and transcription factors (1-3). Upon association of ubiquitinated targets with the proteasome, ubiquitin molecules are proteolytically removed for reuse, whereas the unfolded substrates are fed into the 20 S catalytic core, where they are digested into small peptides (4, 5).Protein ubiquitination is a multistep process orchestrated by the concerted action of three enzymes. The chain reaction begins with a ubiquitin-activating enzyme (E1), which initially adenylates the C-terminal glycine of ubiquitin. Next, a thioester bond is formed between the activated C terminus of ubiquitin and a cysteine residue of the E1. A ubiquitin-conjugating enzyme (E2) acquires the activated ubiquitin through a transthioesterification reaction. Finally, a RING ubiquitin-protein ligase (E3) recruits the substrate and guides the transfer of the ubiquitin from the E2 active site cysteine to the substrate. An ⑀-amine of a lysine residue on the substrate (or of additional ubiquitin) attacks the thioester bond between the ubiquitin and the E2 enzyme, forming an isopeptide bond with the C-terminal glycine of the ubiquitin (6 -8). Alternatively, when a HECT E3 catalyzes the transfer of the ubiquitin from the E2 to the target, an intermediate complex, of the activated ubiquitin and the active site cysteine of the HECT domain E3, is formed (9).