Ring finger protein 145 (RNF145) is a ubiquitin ligase for sterol-induced degradation of HMG-CoA reductase

Abstract

Cholesterol biosynthesis is tightly regulated in the cell. For example, high sterol concentrations can stimulate degradation of the rate-limiting cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, HMGCR). HMGCR is broken down by the endoplasmic reticulum membrane-associated protein complexes consisting of insulin-induced genes (Insigs) and the E3 ubiquitin ligase gp78. Here we found that HMGCR degradation is partially blunted in Chinese hamster ovary (CHO) cells lacking gp78 (gp78-KO). To identify other ubiquitin ligase(s) that may function together with gp78 in triggering HMGCR degradation, we performed a small-scale short hairpin RNA-based screening targeting endoplasmic reticulum-localized E3s. We found that knockdown of both ring finger protein 145 (Rnf145) and gp78 genes abrogates sterol-induced degradation of HMGCR in CHO cells. We also observed that RNF145 interacts with Insig-1 and -2 proteins and ubiquitinates HMGCR. Moreover, the tetrapeptide sequence YLYF in the sterol-sensing domain and the Cys-537 residue in the RING finger domain were essential for RNF145 binding to Insigs and RNF145 E3 activity, respectively. Of note, amino acid substitutions in the YLYF or of Cys-537 completely abolished RNF145-mediated HMGCR degradation. In summary, our study reveals that RNF145, along with gp78, promotes HMGCR degradation in response to elevated sterol levels and identifies residues essential for RNF145 function.

Keywords: ER-associated degradation; HMG-CoA reductase; Insig; RNF145; cholesterol metabolism; gp78; lipid; ubiquitin ligase; ubiquitylation (ubiquitination).

Figure 1.

RNF145 is involved in sterol-regulated HMGCR degradation. A, WT CHO and gp78-KO CHO cells were depleted of sterol in medium C for 16 h. Cells were then treated with medium C supplemented with indicated concentrations of 25-HC plus 10 mm mevalonate for 5 h. Cells were harvested and subjected to SDS-PAGE, followed by immunoblot (IB) analysis. B, quantification of the HMGCR protein in A. C, the gp78-KO CHO cells were transfected with plasmids encoding HMGCR-T7, Insig-1-Myc, and the shRNA targeting RNF145. After 48 h, cells were depleted of sterol and then treated with or without 0.3 μg/ml 25-HC plus 10 mm mevalonate for 5 h as described in A. Cells were harvested and subjected to SDS-PAGE, followed by immunoblot analysis. Results shown are representative of three independent experiments. D, WT, gp78-KO, Rnf145-KO, and double KO CHO cells were depleted of sterol and then treated with indicated concentrations of 25-HC plus 10 mm mevalonate for 5 h as described in A. Cells were harvested and subjected to SDS-PAGE, followed by immunoblot analysis. Asterisks indicate nonspecific bands. Results shown are representative of two independent experiments. E, WT, gp78-KO, Rnf145-KO, and double KO CHO cells were depleted of sterol and then treated with 10 μm MG132 in the presence or absence of 1 μg/ml 25-HC and 10 mm mevalonate for 2 h. Cells were harvested, and lysates were immunoprecipitated with protein A/G beads coupled with the anti-HMGCR antibody. Pellet fractions were immunoblotted with anti-ubiquitin (P4D1) and polyclonal anti-HMGCR antibodies.

Figure 2.

RNF145 is an ER-localized ubiquitin ligase. A, predicted topology of RNF145. YLYF, the amino acids from 81 to 84 of RNF145. B, subcellular localization of RNF145. HeLa cells were transfected with a plasmid encoding RNF145-FLAG and stained with anti-FLAG and anti-calnexin antibodies. Scale bar = 10 μm. C, in vitro ubiquitination assay showing that RNF145 (511–663) possesses E3 activity. Recombinant proteins, including E1, E2 (Ubc7), FLAG-ubiquitin (FLAG-Ub), RNF145 (511–663), and gp78 (309–643), were added to the reaction system as indicated. After incubation at 37 °C for 15 min, samples were subjected to SDS-PAGE, followed by immunoblot (IB) analysis. D, in vitro ubiquitination assay comparing RNF145 (511–663) and RNF145 (511–663) (C537A). Experiments were carried out as described in C.

Figure 3.

The E3 activity–deficient RNF145 blocks sterol-induced ubiquitination and degradation of HMGCR. A, CHO cells were transfected with indicated plasmids, depleted of sterol, and treated with 10 μm MG132 in the presence or absence of 1 μg/ml 25-HC and 10 mm mevalonate for 3 h. Cells were harvested, and lysates were immunoprecipitated with anti-FLAG beads. Input and pellet fractions were immunoblotted (IB) with anti-HA, anti-FLAG, and polyclonal anti-RNF145 antibodies. Results shown are representative of two independent experiments. B, CHO cells were transfected with indicated plasmids, depleted of sterol, and treated with or without 1 μg/ml 25-HC plus 10 mm mevalonate for 5 h. Cells were harvested and subjected to SDS-PAGE and immunoblot analysis. Results shown are representative of two independent experiments.

Figure 4.

Insig is required for RNF145-mediated degradation of HMGCR. A, CHO, SRD15 (deficient in Insig-1 and -2), and 4KO (deficient in Insig-1, Insig-2, gp78, and RNF145) cells were incubated in indicated media for 16 h. Cells were harvested and subjected to SDS-PAGE, followed by immunoblot (IB) analysis. B, sequence alignment of the guide RNA targeting region showing that Rnf145 is knocked out. CDS, coding sequence. C–E, 4KO cells were transfected with indicated plasmids, depleted of sterol, and treated with or without 1 μg/ml 25-HC plus 10 mm mevalonate for 5 h. Cells were harvested and subjected to SDS-PAGE and immunoblot analysis.

Figure 5.

RNF145 interacts with Insigs through its transmembrane domain. A, CHO cells were transfected with indicated plasmids, depleted of sterol, and treated with or without 1 μg/ml 25-HC plus 10 mm mevalonate for 2 h. The cell lysates were immunoprecipitated with anti-Myc beads. Pellets and input were immunoblotted (IB) with indicated antibodies. Results shown are representative of two independent experiments. B, CHO cells were transfected with indicated plasmids and cultured in medium B. 48 h later, cells were harvested, and lysates were immunoprecipitated with anti-Myc beads. Pellets and input were blotted with indicated antibodies. C, CHO cells were transfected with indicated plasmids, depleted of sterol, and treated with or without 1 μg/ml 25-HC plus 10 mm mevalonate for 5 h. Cells were harvested and subjected to SDS-PAGE, followed by immunoblot analysis. Results shown are representative of two independent experiments.

Figure 6.

The sterol-sensing domain of RNF145 is essential for Insig binding. A, sequence alignment of human RNF145, HMGCR, and SCAP. Invariant amino acids are shaded in gray. The conserved motif YI(L)YF is boxed. B and C, 4KO cells (B) and CHO cells (C) were transfected with indicated plasmids, depleted of sterol, and treated with or without 1 μg/ml 25-HC plus 10 mm Mevalonate for 5 h. Cells were harvested and subjected to SDS-PAGE, followed by immunoblot (IB) analysis. D, CHO cells were transfected with indicated plasmids and cultured in medium B. 48 h later, cells were harvested, and lysates were immunoprecipitated with anti-Myc beads. Pellets and input were blotted with indicated antibodies.