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Vol. 20, Issue 14, 3330-3341, July 15, 2009

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Dislocation of HMG-CoA Reductase and Insig-1, Two Polytopic Endoplasmic Reticulum Proteins, En Route to Proteasomal Degradation

Gil S. Leichner^*,, Rachel Avner, Dror Harats, and Joseph Roitelman^*,

*Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; and Bert W. Strassburger Lipid Center, Sheba Medical Center, Tel Hashomer 52621, Israel

Submitted September 18, 2008; Revised May 8, 2009; Accepted May 12, 2009
Monitoring Editor: Thomas Sommer

The endoplasmic reticulum (ER) glycoprotein HMG-CoA reductase (HMGR) catalyzes the rate-limiting step in sterols biosynthesis. Mammalian HMGR is ubiquitinated and degraded by the proteasome when sterols accumulate in cells, representing the best example for metabolically controlled ER-associated degradation (ERAD). This regulated degradation involves the short-lived ER protein Insig-1. Here, we investigated the dislocation of these ERAD substrates to the cytosol en route to proteasomal degradation. We show that the tagged HMGR membrane region, HMG₃₅₀-HA, the endogenous HMGR, and Insig-1-Myc, all polytopic membrane proteins, dislocate to the cytosol as intact full-length polypeptides. Dislocation of HMG₃₅₀-HA and Insig-1-Myc requires metabolic energy and involves the AAA-ATPase p97/VCP. Sterols stimulate HMG₃₅₀-HA and HMGR release to the cytosol concurrent with removal of their N-glycan by cytosolic peptide:N-glycanase. Sterols neither accelerate dislocation nor stimulate deglycosylation of ubiquitination-defective HMG₃₅₀-HA^{(K89 + 248R)} mutant. Dislocation of HMG₃₅₀-HA depends on Insig-1-Myc, whose dislocation and degradation are sterol independent. Coimmunoprecipitation experiments demonstrate sterol-stimulated association between HMG₃₅₀-HA and Insig-1-Myc. Sterols do not enhance binding to Insig-1-Myc of HMG₃₅₀-HA mutated in its sterol-sensing domain or of HMG₃₅₀-HA^{(K89 + 248R)}. Wild-type HMG₃₅₀-HA and Insig-1-Myc coimmunoprecipitate from the soluble fraction only when both proteins were coexpressed in the same cell, indicating their encounter before or during dislocation, raising the possibility that they are dislocated as a tightly bound complex.

Address correspondence to: Joseph Roitelman (roitelma{at}post.tau.ac.il)

Abbreviations used: ALLN, N-acetyl-leucyl-leucyl-norleucinal; DOC, deoxycholate; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated protein degradation; EndoH, endoglycosidase H; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; LPDS, lipoprotein-deficient serum; MVA, mevalonate; PAGE, polyacrylamide gel electrophoresis; PNGase, peptide N:glycanase; PBS, phosphate-buffered saline; SREBP, sterol regulatory element-binding protein; SCAP, sterol regulatory element-binding protein cleavage-activating protein; TMs, transmembrane span(s).