INAUGURAL ARTICLE by a Recently Elected Academy Member:Structure and function of the yeast U-box-containing ubiquitin ligase Ufd2p

In eukaryotes, short-lived proteins are degraded by the ubiquitin (Ub) proteasome system (reviewed in ref. 1). Substrates of the proteasome include transcription factors, cell cycle regulators, signal transducers, and misfolded proteins generated under stress conditions. Protein ubiquitination is achieved by a multistep mechanism involving a cascade of enzymes. Ub-activating enzyme (E1) hydrolyzes ATP to form a high-energy thioester bond between its catalytic cysteine residue and the C terminus of Ub. Activated Ub is subsequently passed to a distinct Ub-conjugating enzyme (E2) by transthiolation. Finally, Ub is transferred to the e-amino group of an internal lysine residue of a target protein by a Ub ligase (E3). Polyubiquitin chains can be assembled when additional Ub molecules are transferred, one at a time, to a lysine residue in the substrate-bound Ub molecule via an isopeptide bond linkage. In addition, with the assistance of an E3 ligase, certain Ub-conjugating enzymes can form Lys-48-linked Ub chains linked to its catalytic cysteine before transferring the assembled Ub chains to a substrate (2, 3). The essential components involved in the degradation of certain short-lived proteins have been identified in Saccharomyces cerevisiae by using a model proteasomal substrate consisting of a Ub moiety fused to the N terminus of a reporter protein. These components, which were designated as UFD1–5, represent the so-called UFD (Ub fusion degradation) pathway (4). Interestingly, with the exception of Ufd5p, a transcriptional regulator of the proteasome (5), and of Ufd4p, a HECT (homologous to E6-associated protein C terminus) domain Ub ligase (6), the other UFD proteins all interact with a conserved AAA ATPase (ATPase associated with various activities) named Cdc48p in yeast or p97 in mammals to regulate its activities in a subset of proteasome-dependent degradation pathways (7). The role of Cdc48p/p97 in protein turnover is best characterized for the degradation of misfolded endoplasmic reticulum (ER) proteins, which occurs through a pathway termed ER-associated protein degradation or retrotranslocation. During this process, Cdc48p/p97 associates with the ER membrane to extract its client proteins (misfolded polypeptides undergoing retrotranslocation) out of the ER membrane and subsequently target them for degradation by the proteasome. The Cdc48p/p97-dependent retrotranslocation requires the function of at least two UFD proteins, Ufd1p and Ufd2p. Ufd1p associates with Npl4p to form a heterodimeric cofactor complex, which promotes substrate recognition by Cdc48p/p97 (8–10). Ufd2p appears to act downstream of Cdc48p/p97 to facilitate the transfer of ER-associated protein degradation substrates to Rad23p, a proteasome-associated Ub receptor (11). Although Ufd2p is not essential for cell viability under normal conditions, its activity becomes critical under stress conditions in yeast (6). Likewise, the Ufd2a+/− mice lacking one functional copy of the Ufd2a gene, a homologue of Ufd2, develop a neurological disorder as a result of axonal dystrophy induced by ER stress. Mice deficient in UFD2a die in utero because of marked apoptosis in the developing heart (12). Taken together, the major function of Ufd2 is probably to cooperate with Cdc48/p97 to maintain a stress-free environment for cells. Ufd2p contains a U-box domain that is structurally related to the RING finger domain that is found in certain E3 Ub ligases (13, 14). Ufd2 is capable of elongating Ub chains, an activity that is essential for its function in ER-associated protein degradation. In the presence of an E1, Ubc4p (E2), and Ufd4p (E3), a short Ub chain attached to a substrate can be further extended by Ufd2p. For this reason, Ufd2p is often referred to as an E4 enzyme (6). It is unclear how Ufd2p promotes Ub chain elongation in conjunction with the HECT domain containing E3 ligase Ufd4p. A HECT domain E3 ligase usually accepts Ub from its cognate E2 and forms a thioester-linked Ub–E3 intermediate before transferring the attached Ub to a substrate. In contrast, RING finger E3 ligases usually associate directly with cognate E2 enzymes to promote the transfer of Ub directly from E2 to a substrate. RING finger E3 ligases can also mediate the discharge of Ub from the E2 active cysteine in the absence of a substrate. When excess free Ub molecules are present, the released Ub molecules can be linked to a lysine residue in these free Ub molecules, forming an isopeptide bond-linked diubiquitin molecule (15). Whether Ufd2p also contains these activities is unknown. Here we present the crystal structure of the full-length yeast Ufd2p enzyme. The structure has an elongated shape with a flexible U-box domain attached to it. The core region of Ufd2p consists of multiple irregular Armadillo-like repeats with two pronounced helical hairpin protrusions. The core region of Ufd2p is structurally reminiscent of importin α, a nuclear transport protein. Based on the CHIP/E2 complex crystal structure (16), we built a model of the Ufd2p/E2 complex that predicts interactions between E2 and the U-box domain as well as the core of Ufd2p. Biochemical experiments confirmed that Ufd2p binds directly to the E2 enzyme Ubc4p and furthermore revealed that its U-box domain is involved in the transfer of an E2-conjugated Ub to a free Ub and to Ufd2p itself. Thus, Ufd2p has the characteristics of a typical RING finger E3 ligase.