Mutations within the membrane domain of HMG-CoA reductase confer resistance to sterol-accelerated degradation

In all eukaryotic cells that have been examined, specific membrane arrays are induced in response to increased levels of the ER membrane protein, HMG-CoA reductase. Analysis of these inducible membranes has the potential to reveal basic insights into general membrane assembly. Yeast express two HMG-CoA reductase isozymes, and each isozyme induces a morphologically distinct proliferation of the endoplasmic reticulum. The isozyme encoded by HMG1 induces karmellae, which are long stacks of membranes that partially enclose the nucleus. In contrast, the isozyme encoded by HMG2 induces short stacks of membrane that may be associated with the nucleus, but are frequently present at the cell periphery. To understand the molecular nature of the different cellular responses to Hmg1p and Hmg2p, we mapped the region of Hmg1p that is needed for karmellae assembly. For this analysis, a series of exchange alleles was examined in which a portion of the Hmg2p membrane domain was replaced with the corresponding Hmg1p sequences. Results of this analysis indicated that the ER lumenal loop between predicted transmembrane domains 6 and 7 was both necessary and sufficient for karmellae assembly, when present in the context of an HMG-CoA reductase membrane domain. Immunoblotting experiments ruled out the simple possibility that differences in the amounts of the various chimeric HMG-CoA reductase proteins was responsible for the altered cellular responses. Our results are consistent with the hypothesis that each yeast isozyme induces or organizes a qualitatively different organization of ER membrane.

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

10.1101/391789 ◽

2018 ◽

Author(s):

Sam A. Menzies ◽

Norbert Volkmar ◽

Dick J. van den Boomen ◽

Richard T. Timms ◽

Anna S. Dickson ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Critical Role ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Hmg Coa Reductase ◽

Accelerated Degradation ◽

Genome Wide ◽

Cholesterol Biosynthetic Pathway ◽

Coa Reductase ◽

Rate Limiting

ABSTRACTHMG-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.

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Faculty Opinions recommendation of Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1008556.184831 ◽

2003 ◽

Author(s):

Tom Rapoport

Keyword(s):

Hmg Coa Reductase ◽

Accelerated Degradation ◽

Coa Reductase

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Faculty Opinions recommendation of Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1008556.183215 ◽

2003 ◽

Author(s):

Ron Prywes

Keyword(s):

Hmg Coa Reductase ◽

Accelerated Degradation ◽

Coa Reductase

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Mutation in the Lumenal Part of the Membrane Domain of HMG-CoA Reductase Alters Its Regulated Degradation

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1995.1026 ◽

1995 ◽

Vol 206 (1) ◽

pp. 186-193 ◽

Cited By ~ 6

Author(s):

M.S. Sekler ◽

R.D. Simoni

Keyword(s):

Membrane Domain ◽

Hmg Coa Reductase ◽

Coa Reductase

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The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.10.10.3409 ◽

1999 ◽

Vol 10 (10) ◽

pp. 3409-3423 ◽

Cited By ~ 19

Author(s):

Deborah A. Profant ◽

Christopher J. Roberts ◽

Ann J. Koning ◽

Robin L. Wright

Keyword(s):

Coenzyme A ◽

Tertiary Structure ◽

Catalytic Domain ◽

Carboxyl Terminus ◽

Membrane Domain ◽

Hmg Coa Reductase ◽

Cytosolic Domain ◽

Coa Reductase ◽

Er Membrane

In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using β-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain.

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Differential regulation of HMG-CoA reductase and Insig-1 by enzymes of the ubiquitin-proteasome system

Molecular Biology of the Cell ◽

10.1091/mbc.e12-08-0631 ◽

2012 ◽

Vol 23 (23) ◽

pp. 4484-4494 ◽

Cited By ~ 40

Author(s):

Yien Che Tsai ◽

Gil S. Leichner ◽

Margaret M. P. Pearce ◽

Gaye Lynn Wilson ◽

Richard J. H. Wojcikiewicz ◽

...

Keyword(s):

Fibroblast Cell ◽

Ubiquitin Proteasome System ◽

Hmg Coa Reductase ◽

Accelerated Degradation ◽

Rate Limiting Step ◽

Ubiquitin Ligase E3 ◽

Ubiquitin Proteasome ◽

Dependent Protein ◽

Regulatory Loop ◽

Coa Reductase

The endoplasmic reticulum (ER)–resident enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the rate-limiting step in sterol production and is the therapeutic target of statins. Understanding HMG-CoA reductase regulation has tremendous implications for atherosclerosis. HMG-CoA reductase levels are regulated in response to sterols both transcriptionally, through a complex regulatory loop involving the ER Insig proteins, and posttranslationally, by Insig-dependent protein degradation by the ubiquitin-proteasome system. The ubiquitin ligase (E3) gp78 has been implicated in the sterol-regulated degradation of HMG-CoA reductase and Insig-1 through ER-associated degradation (ERAD). More recently, a second ERAD E3, TRC8, has also been reported to play a role in the sterol-accelerated degradation of HMG-CoA reductase. We interrogated this network in gp78−/− mouse embryonic fibroblasts and also assessed two fibroblast cell lines using RNA interference. Although we consistently observe involvement of gp78 in Insig-1 degradation, we find no substantive evidence to support roles for either gp78 or TRC8 in the robust sterol-accelerated degradation of HMG-CoA reductase. We discuss factors that might lead to such discrepant findings. Our results suggest a need for additional studies before definitive mechanistic conclusions are drawn that might set the stage for development of drugs to manipulate gp78 function in metabolic disorders.

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Bisphosphonate esters interact with HMG-CoA reductase membrane domain to induce its degradation

Bioorganic & Medicinal Chemistry ◽

10.1016/j.bmc.2020.115576 ◽

2020 ◽

Vol 28 (14) ◽

pp. 115576 ◽

Cited By ~ 1

Author(s):

Yosuke Toyota ◽

Hiromasa Yoshioka ◽

Ikuya Sagimori ◽

Yuichi Hashimoto ◽

Kenji Ohgane

Keyword(s):

Membrane Domain ◽

Hmg Coa Reductase ◽

Coa Reductase

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Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum

The Journal of Cell Biology ◽

10.1083/jcb.117.5.959 ◽

1992 ◽

Vol 117 (5) ◽

pp. 959-973 ◽

Cited By ~ 128

Author(s):

J Roitelman ◽

EH Olender ◽

S Bar-Nun ◽

WA Dunn ◽

RD Simoni

Keyword(s):

Endoplasmic Reticulum ◽

Coenzyme A ◽

Cultured Cells ◽

Critical Role ◽

Transmembrane Helix ◽

Chimeric Protein ◽

Membrane Domain ◽

Hmg Coa Reductase ◽

Coa Reductase ◽

Peptide G

We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. Finer-Moore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.

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