The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1

Abstract

21HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic 22 pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-23 accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and 24 degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR 25 turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide 26 screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the 27 E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that 28 RNF145 and gp78, independently co-ordinate HMGCR ubiquitination and degradation. 29 RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following 30 sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR and Insig-1, 31promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of 32 both RNF145 and gp78, Hrd1, a third UBE2G2-dependent ligase partially regulates HMGCR 33 activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR 34 regulation and elucidate the complexity of sterol-accelerated HMGCR degradation. 35

Differential use of E2 ubiquitin conjugating enzymes for regulated degradation of the rate-limiting enzymes HMGCR and SQLE in cholesterol biosynthesis

“…These ligases stimulate ubiquitylation of their targets in response to a plethora of signals. Importantly, the substrate specificity of some ligases may overlap, as evident in the case of ubiquitylation and degradation of HMGCR for which GP78, TRC8, and more recently RNF145, have been implicated [4,[14][15][16][17][18]. Intriguingly, SQLE seems to be solely recognized by MARCH6 [19].…”

“…Ubiquitylation follows the mandatory sequence of enzymatic reactions consisting of activating ubiquitin by an E1 enzyme, binding of ubiquitin to an E2 ubiquitin conjugating enzyme, and finally the ligation of ubiquitin onto the target protein that is supported by the E3 ubiquitin ligase [13]. Extensive research into the ubiquitylation cascade of HMGCR has implicated the E2 enzyme UBE2G2, as well as the E3 ubiquitin ligases gp78, TRC8 and RNF145 in its degradation [4,[14][15][16][17][18], while the RING-type ubiquitin ligase MARCH6 has been shown to promote the degradation of SQLE [19]. However, the E2 enzyme(s) governing this process remains elusive.…”

A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

“…Cloning of the top 1000 sub-genomic sgRNA library. The top 1000 subgenomic sgRNA library was cloned as detailed previously 67,68 . sgRNA sequences for the top 1000 genes of the genome-wide CRISPR screen were retrieved from the Bassik genome-wide CRISPR library (10 sgRNAs/gene, Supplementary Data 1) 39 .…”

Insights into endoplasmic reticulum‐associated degradation in plants

“…In addition to clearing unfolded proteins under stress, ERAD also functions in the turnover of normal proteins to regulate cell development, such as HMG COENZYME A REDUCTASE (HMGCR) in mammals (Menzies et al , ). ERAD functions in development seem to be conserved from mammals to plants.…”