Ubx2 links the Cdc48 complex to ER-associated protein degradation

Max-Delbrück Center for Molecular Medicine, Robert-Rössle Strasse 10, 13092 Berlin, Germany.
Nature Cell Biology (Impact Factor: 19.68). 11/2005; 7(10):993-8. DOI: 10.1038/ncb1298
Source: PubMed


Endoplasmic reticulum (ER)-associated protein degradation requires the dislocation of selected substrates from the ER to the cytosol for proteolysis via the ubiquitin-proteasome system. The AAA ATPase Cdc48 (known as p97 or VCP in mammals) has a crucial, but poorly understood role in this transport step. Here, we show that Ubx2 (Sel1) mediates interaction of the Cdc48 complex with the ER membrane-bound ubiquitin ligases Hrd1 (Der3) and Doa10. The membrane protein Ubx2 contains a UBX domain that interacts with Cdc48 and an additional UBA domain. Absence of Ubx2 abrogates breakdown of ER proteins but also that of a cytosolic protein, which is ubiquitinated by Doa10. Intriguingly, our results suggest that recruitment of Cdc48 by Ubx2 is essential for turnover of both ER and non-ER substrates, whereas the UBA domain of Ubx2 is specifically required for ER proteins only. Thus, a complex comprising the AAA ATPase, a ubiquitin ligase and the recruitment factor Ubx2 has a central role in ER-associated proteolysis.

17 Reads
  • Source
    • "Ubiquitylation of Hrd1 substrates is catalyzed by the ubiquitin conjugating enzyme Ubc7 that is recruited to the ligase by its activating factor Cue1 (Biederer et al., 1997; Bagola et al., 2013). An adaptor protein Ubx2 brings the cytoplasmic Cdc48/Npl4/Ufd1 complex to the ligase, which targets ubiquitylated substrates from the ER to the proteasome (Neuber et al., 2005; Schuberth and Buchberger, 2005). Hrd1 forms oligomers upon binding to the scaffolding protein Usa1 (Horn et al., 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase) and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR, which is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70 type chaperone Kar2 and the Hsp40 type cochaperone Scj1 bind to Hrd3KR. Noteworthy, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream acting components like Der1. © 2014 by The American Society for Cell Biology.
    Molecular Biology of the Cell 11/2014; 26(2). DOI:10.1091/mbc.E14-07-1202 · 4.47 Impact Factor
  • Source
    • "Taken together, our results show that polyubiquitinated Hrd1p recruits the Cdc48p complex. Ubx2p stimulates or stabilizes the association but is not essential, consistent with in vivo data (Neuber et al., 2005; Schuberth and Buchberger, 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome, a process called ER-associated protein degradation (ERAD). Here, we use purified components from Saccharomyces cerevisiae to analyze the mechanism of retrotranslocation of luminal substrates (ERAD-L), recapitulating key steps in a basic process in which the ubiquitin ligase Hrd1p is the only required membrane protein. We show that Hrd1p interacts with substrate through its membrane-spanning domain and discriminates misfolded from folded polypeptides. Both Hrd1p and substrate are polyubiquitinated, resulting in the binding of Cdc48p ATPase complex. Subsequently, ATP hydrolysis by Cdc48p releases substrate from Hrd1p. Finally, ubiquitin chains are trimmed by the deubiquitinating enzyme Otu1p, which is recruited and activated by the Cdc48p complex. Cdc48p-dependent membrane extraction of polyubiquitinated proteins can be reproduced with reconstituted proteoliposomes. Our results suggest a model for retrotranslocation in which Hrd1p forms a membrane conduit for misfolded proteins.
    Cell 09/2014; 158(6):1375-88. DOI:10.1016/j.cell.2014.07.050 · 32.24 Impact Factor
  • Source
    • "In yeast, the Cdc48 ATPase binds to the Hrd1 E3 ligase in a RING-dependent manner (Hampton and Sommer, 2012), and the transmembrane Ubx2 (Sel1) protein acts as an adapter using a UBA domain (Neuber et al., 2005; Schuberth and Buchberger, 2005). Several other ubiquitin ligases bind p97 directly or through cofactors (Alexandru et al., 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Endoplasmic reticulum (ER)-associated degradation (ERAD) is a universally important process among eukaryotic cells. ERAD is necessary to preserve cell integrity since the accumulation of defective proteins results in diseases associated with neurological dysfunction, cancer, and infections. This process involves recognition of misfolded or misassembled proteins that have been translated in association with ER membranes. Recognition of ERAD substrates leads to their extraction through the ER membrane (retrotranslocation or dislocation), ubiquitination, and destruction by cytosolic proteasomes. This review focuses on ERAD and its components as well as how viruses use this process to promote their replication and to avoid the immune response.
    Frontiers in Microbiology 07/2014; 5:330. DOI:10.3389/fmicb.2014.00330 · 3.99 Impact Factor
Show more