ABSTRACT
HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78, independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR and Insig-1, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent ligase partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation.
ABBREVIATIONS
- AMFR
- - Autocrine Motility Factor Receptor
- B2M
- - beta-2-microglobulin
- Chol
- - cholesterol
- CHX
- - cycloheximide
- CRISPR
- - clustered regularly interspaced short palindromic repeats
- CTR
- - control
- ER
- - endoplasmic reticulum
- ERAD
- - ER-associated degradation
- FCS
- - fetal calf serum
- Gp78
- - glycoprotein 78
- HMGCR
- - 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase
- Hrd1
- - HMG-CoA Reductase Degradation 1
- IAA
- - iodoacetamide
- Insig-1/2
- - Insulin-induced gene-1/2
- LPDS
- - lipoprotein-deficient serum
- MBCD
- - methyl-β-cyclodextrin
- RNF145
- - RING finger protein 145
- SD
- - sterol-depleted
- SREBP2
- - Sterol Regulatory Element Binding transcription factor 2
- TRC8
- - Translocation in renal carcinoma on chromosome 8
- UBE2G2
- - Ubiquitin-conjugating enzyme E2 G2
- UPS
- - ubiquitin proteasome system
- VCP
- - Valosin-containing protein
- WT
- - wild-type