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: 4.57). 06/2008; 283(19):12797-810. DOI: 10.1074/jbc.M801122200
Source: PubMed


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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    • "In recent years examples of additional, non-canonical ubiquitination factors have been described, comprising e.g. additional E3 enzymes (E4) like Ufd2p [4] or E2-activators like Cue1p [5], [6]. "
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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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    ABSTRACT: Cellular adaptation to proteotoxic stress at the endoplasmic reticulum (ER) depends on Lys48-linked polyubiquitination by ER-associated ubiquitin ligases (E3s) and subsequent elimination of ubiquitinated retrotranslocation products by the proteasome. The ER-associated E3 gp78 ubiquitinates misfolded proteins by transferring preformed Lys48-linked ubiquitin chains from the cognate E2 Ube2g2 to substrates. Here we demonstrate that Ube2g2 synthesizes linkage specific ubiquitin chains by forming an unprecedented homodimer: The dimerization of Ube2g2, mediated primarily by electrostatic interactions between two Ube2g2s, is also facilitated by the charged ubiquitin molecules. Mutagenesis studies show that Ube2g2 dimerization is required for ER-associated degradation (ERAD). In addition to E2 dimerization, we show that a highly conserved arginine residue in the donor Ube2g2 senses the presence of an aspartate in the acceptor ubiquitin to position only Lys48 of ubiquitin in proximity to the donor E2 active site. These results reveal an unanticipated mode of E2 self-association that allows the E2 to effectively engage two ubiquitins to specifically synthesize Lys48-linked ubiquitin chains.
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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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