H Miao, W Jiang, L Ge, B Li and B. Song
Acta Biochimica et Biophysica Sinica, 2010, 42(5), 303-310. DOI: 10.1093/abbs/gmq022
Sterol-regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is a rapid feedback regulatory mechanism by which cells employ to control the cholesterol biosynthesis. This process is initiated by the sterol-induced interaction between HMGCR and Insig-1/gp78, a membrane-bound ubiquitin ligase complex. There are two Lys residues (Lys89 and Lys248) facing cytosol in the membrane domain of HMGCR, and Lys248 is the major ubiquitination site. In this study, we investigated the mechanism of ubiquitination site selection in HMGCR. We find that the distance of Lys248 to membrane is dispensable for its ubiquitination. However, the conserved tetra-glutamic acid residues adjacent to Lys248 in HMGCR are essential. Replacement of these negatively charged residues with tetra-arginine causes the resistance of HMGCR to sterol-induced ubiquitination and degradation, albeit this mutant HMGCR can still binds to Insig-1. We further find that the tetra-glutamic acid residues are necessary but not sufficient for the modification on their adjacent Lys, since they are not functional on Lys89 of HMGCR or in SCAP. UBE2G2, a previously known E2 of gp78, is demonstrated to be involved in the sterol-regulated ubiquitination and degradation of HMGCR. In summary, these results identify the tetra-glutamic acid residues as a critical motif in HMGCR for the ubiquitination reaction mediated by gp78 and UBE2G2.