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.

  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    The EMBO Journal 01/2014; 33(1). DOI:10.1002/embj.201385315 · 10.75 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ubiquitin-binding domains (UBDs) differentially recognize ubiquitin (ub) modifications. Some of them specifically bind mono-ub, as has been shown for the CUE domain. Interestingly, so far no significant ubiquitin binding has been observed for the CUE domain of yeast Cue1p. Cue1p is receptor and activator of the ubiquitin-conjugating enzyme Ubc7p. It integrates Ubc7p into endoplasmic reticulum (ER) membrane-bound ubiquitin ligase complexes, and thus, it is crucial for ER-associated protein degradation (ERAD). Here we show that the CUE domain of Cue1p binds ubiquitin chains, which is pivotal for the efficient formation of K48-linked polyubiquitin chains in vitro. Mutations that abolish ubiquitin binding by Cue1p affect the turnover of ERAD substrates in vivo. Our data strongly imply that the CUE domain facilitates substrate ubiquitylation by stabilizing growing ubiquitin chains at the ERAD ubiquitin ligases. Hence, we demonstrate an unexpected function of a UBD in the regulation of ubiquitin chain synthesis.
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Thyroid hormone action can be customized on a cell-specific fashion through the controlled action of the deiodinase group of enzymes, which are homodimeric thioredoxin fold containing selenoproteins. Whereas the type II deiodinase (D2) initiates thyroid hormone signaling by activating the pro-hormone thyroxine (T4) to the biologically active T3 molecule, the type III deiodinase (D3) terminates thyroid hormone action by catalyzing the inactivation of both T4 and T3 molecules. Deiodinases play a role in thyroid hormone homeostasis, development, growth and metabolic control by affecting the intracellular levels of T3 and thus gene expression on a cell-specific basis. Whereas both Dio2 and Dio3 are transcriptionally regulated, ubiquitination of D2 is a switch mechanism that controls D2 activity and intracellular T3 production. The hedgehog-inducible WSB-1 and the yeast Doa10 mammalian ortholog TEB4 are two E3 ligases that inactivate D2 via ubiquitination. Inactivation involves disruption of the D2:D2 dimer and can be reversed via two ubiquitin-specific proteases, USP20 and USP33, rescuing catalytic activity and T3 production. The ubiquitin-based switch mechanism that controls D2 activity illustrates how different cell types fine-tune thyroid hormone signaling, making D2 a suitable target for pharmacological intervention. This article reviews the cellular and molecular aspects of D2 regulation and the current models of D2-mediated thyroid hormone signaling.
    The international journal of biochemistry & cell biology 06/2011; 43(10):1432-41. DOI:10.1016/j.biocel.2011.05.016 · 4.24 Impact Factor
Show more