Membrane-associated Ubiquitin Ligase Complex Containing gp78 Mediates Sterol-accelerated Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase

Youngah Jo,Peter V Sguigna,Russell A Debose-Boyd

doi:10.1074/jbc.m110.211326

Abstract

The endoplasmic reticulum (ER)-associated degradation (ERAD) pathway in the yeast Saccharomyces cerevisiae is mediated by two membrane-bound ubiquitin ligases, Doa10 and Hrd1. These enzymes are found in distinct multiprotein complexes that allow them to recognize and target a variety of substrates for proteasomal degradation. Although multiprotein complexes containing mammalian ERAD ubiquitin ligases likely exist, they have yet to be identified and characterized in detail. Here, we identify two ER membrane proteins, SPFH2 and TMUB1, as associated proteins of mammalian gp78, a membrane-bound ubiquitin ligase that bears significant sequence homology with mammalian Hrd1 and mediates sterol-accelerated ERAD of the cholesterol biosynthetic enzyme HMG-CoA reductase. Co-immunoprecipitation studies indicate that TMUB1 bridges SPFH2 to gp78 in ER membranes. The functional significance of these interactions is revealed by the observation that RNA interference (RNAi)-mediated knockdown of SPFH2 and TMUB1 blunts both the sterol-induced ubiquitination and degradation of endogenous reductase in HEK-293 cells. These studies mark the initial steps in the characterization of the mammalian gp78 ubiquitin ligase complex, the further elucidation of which may yield important insights into mechanisms underlying gp78-mediated ERAD.

Highlights

Ubiquitination is a key step in the endoplasmic reticulum (ER)2-associated degradation (ERAD) pathway, a highly conserved cellular process through which misfolded or unassembled proteins are selectively degraded from ER membranes by 26 S proteasomes [1,2,3,4]
Our results indicate that binding of SPFH2 to gp78 is mediated by a ubiquitin-like (UBL)-domain containing ER membrane protein called TMUB1
To identify proteins that associate with gp78, we began by generating a line of Chinese hamster ovary 7 (CHO-7) cells that stably overexpress the full-length enzyme with a C-terminal Tandem Affinity Purification (TAP) tag

Summary

EXPERIMENTAL PROCEDURES

Materials—We obtained 25-hydroxycholesterol (25-HC) from Steraloids Inc. (Wilton, NH), MG-132 from Boston Biochem (Cambridge, MA) and Peptides International (Osaka, Japan), digitonin from Calbiochem, and horseradish peroxidase-conjugated donkey anti-mouse, anti-rabbit, and anti-goat from Jackson ImmunoResearch Laboratories (West Grove, PA). The cells were subsequently harvested by centrifugation, lysed in Buffer A, and subjected to affinity chromatography with human IgG-conjugated Sepharose beads as described above. At the end of the incubations, the cells were harvested, lysed in PBS containing 1% Nonidet P-40, 1% deoxycholic acid, 5 mM EDTA, 5 mM EGTA, 0.1 mM leupeptin, 10 mM N-ethylmaleimide, and the protease inhibitor mixture, and subjected to centrifugation at 16,000 ϫ g for 15 min at 4 °C. On day 2, the RNAi procedure was repeated as described above, except that the cells were incubated for 16 h at 37 °C in medium B containing 10% LPDS, 50 ␮M compactin, and 50 ␮M mevalonate. Transfection of siRNA duplexes was performed on days 1 and 3 in medium B supplemented with 10% FCS and 10% LPDS plus 10 ␮M compactin and 50 ␮M mevalonate, respectively, as described above. Knockdown efficiency was verified by quantitative real time PCR using each specific primer for human SPFH1, SPFH2, TMUB1, and the control mRNA GAPDH

RESULTS

HMG CoA Reductase

DISCUSSION