Article

Cue1p is an activator of Ubc7p E2 activity in vitro and in vivo

Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 06/2008; 283(19):12797-810. DOI: 10.1074/jbc.M801122200
Source: PubMed

ABSTRACT Ubc7p is a ubiquitin-conjugating enzyme (E2) that functions with endoplasmic reticulum (ER)-resident ubiquitin ligases (E3s) to promote endoplasmic reticulum-associated degradation (ERAD). Ubc7p only functions in ERAD if bound to the ER surface by Cue1p, a membrane-anchored ER protein. The role of Cue1p was thought to involve passive concentration of Ubc7p at the surface of the ER. However, our biochemical studies of Ubc7p suggested that Cue1p may, in addition, stimulate Ubc7p E2 activity. We have tested this idea and found it to be true both in vitro and in vivo. Ubc7p bound to the soluble domain of Cue1p showed strongly enhanced in vitro ubiquitination activity, both in the presence and absence of E3. Cue1p also enhanced Ubc7p function in vivo, and this activation was separable from the established ER-anchoring role of Cue1p. Finally, we tested in vivo activation of Ubc7p by Cue1p in an assay independent of the ER membrane and ERAD. A chimeric E2 linking Ubc7p to the Cdc34p/Ubc3p localization domain complemented the cdc34-2 TS phenotype, and co-expression of the soluble Cue1p domain enhanced complementation by this chimeric Ubc7p E2. These studies reveal a previously unobserved stimulation of Ubc7p E2 activity by Cue1p that is critical for full ERAD and that functions independently of the well known Cue1p anchoring function. Moreover, it suggests a previously unappreciated mode for regulation of E2s by Cue1p-like interacting partners.

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    • "One strategy is to assemble ubiquitin chains on the active site of an E2 enzyme, and then transfer these preassembled ubiquitin chains to substrates (Li et al, 2007). Ubiquitin chain assembly on E2 active site has been reported for several E2s (Haldeman et al, 1997; Cao et al, 2007; Ravid & Hochstrasser, 2007; Bazirgan & Hampton, 2008), but the best characterized example is the ER-associated E2 Ube2g2 (Li et al, 2007, 2009), which acts in conjugation with the RING (Really Interesting New Gene) domain E3 gp78 to ubiquitinate and degrade many misfolded ER proteins (Fang et al, 2001; Song et al, 2005; Christianson et al, 2011). gp78 can rapidly assemble ubiquitin chains on the active site of Ube2g2 and transfer preassembled chains to a substrate (Li et al, 2007). "
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    • "Consistently, we found that this linkage specificity, mediated by Ubc7p, is required for degradation of the ERAD model substrate CPY* in vivo. As previously published, the ligase-associated factor Cue1p is not only required for recruitment of Ubc7p to the ER membrane in vivo but also stimulates polyubiquitin chain formation in vitro (Bazirgan and Hampton, 2008; Kostova et al., 2009). A small C-terminal Ubc7p-binding region in Cue1p promotes a basic activation of Ubc7p-dependent ubiquitin conjugation. "
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    • "The identification of mammalian orthologues of two yeast E2 conjugases, UBC6 and UBC7 (Tiwari and Weissman, 2001), made possible for more detailed studies on the proteins involved in the degradation of D2 via the ERAD pathway. Both UBCs are found close or in association with the ER; where UBC6 is physically anchored to the ER membrane via a transmembrane domain and UBC7 is activated and held close to the ER via interaction with the ER membrane protein Cuelp (Bazirgan and Hampton, 2008). More recently, the crystal structure of yeast and mammalian UBC7 has been solved (Arai et al., 2006, Briggman et al., 2005, Cook et al., 1997), while it also has been shown to functionally interact with the E3 ligases Parkin (Imai et al., 2001), HRD1 (Kikkert et al., 2004) and TEB4 (Hassink et al., 2005) on ERAD pathways. "
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