Tetra-glutamic acid residues adjacent to Lys248 in HMG-CoA reductase are critical for the ubiquitination mediated by gp78 and UBE2G2.
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 tetraarginine 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 tetraglutamic acid residues as a critical motif in HMGCR for the ubiquitination reaction mediated by gp78 and UBE2G2.
Miao
Honghua
H
The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Jiang
Wei
W
Ge
Liang
L
Li
Boliang
B
Song
Baoliang
B
D000214:Acyl Coenzyme A; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology*
D000214:Acyl Coenzyme A; D018121:Receptors, Cytokine; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D018698:Glutamic Acid; D006903:Hydroxymethylglutaryl CoA Reductases; C471617:UBE2G2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C555000:AMFR protein, human; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases; D008239:Lysine