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

Roitelman, Joseph; Olender, E H; Bar-Nun, Shoshana; Wa, Dunn; Rd, Simoni

doi:10.1083/jcb.117.5.959

Cited by 171 publications

(138 citation statements)

References 62 publications

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“…Although details concerning the structure and function of the HMG-CoA reductase catalytic domain are beginning to be solved (Darnay et al, 1992;Lawrence et al, 1995;Rogers et al, 1997;Tabernero et al, 1999), the HMG-CoA reductase membrane domain remains much more cryptic. In plants, the membrane domain contains two transmembrane helices (Denbow et al, 1996;Re et al, 1997), whereas in animals and fungi, it contains of a series of seven or eight transmembrane helices (Liscum et al, 1985;Basson et al, 1988;Sengstag et al, 1990;Olender and Simon, 1992;Roitelman et al, 1992). Although there is little obvious primary sequence conservation, the structural conservation of this complex membrane domain over the 1 billion years of evolution since divergence of fungi and animals (Doolittle et al, 1996;Lum et al, 1996;Feng et al, 1997) suggests that the structure has an important functional role.…”

Section: Discussionmentioning

confidence: 99%

“…The structure of HMG-CoA reductase can be divided into two domains: a complex membrane-spanning domain at the amino terminus followed by a cytosolic catalytic domain (Liscum et al, 1985;Basson et al, 1986;Roitelman et al, 1991). The membrane domain of mammalian and yeast HMG-CoA reductases spans the membrane seven or eight times and is connected to the catalytic domain through a flexible linker sequence (Liscum et al, 1985;Lum et al, 1996;Roitelman et al, 1992). The membrane domain is essential for both the proliferation of ER membranes (Jingami et al, 1987;Parrish et al, 1995) and the regulated degradation of both the mammalian and yeast HMG-CoA reductases (Gil et al, 1985;Skalnik et al, 1988;Hampton et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

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

Profant¹,

Roberts

Koning

et al. 1999

MBoC

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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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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Profant¹,

Roberts

Koning

et al. 1999

MBoC

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“…Cite this article as Cold Spring Harb Perspect Biol 2011;3:a004754 1985; Roitelman et al 1992). Accumulation of sterols causes Insigs to bind to the membrane domain of HMG CoA reductase, initiating a series of reactions that lead to the polyubiquitination of the enzyme and its subsequent degradation by proteasomes.…”

Section: Regulation Of Cholesterol and Fatty Acid Synthesismentioning

confidence: 99%

Regulation of Cholesterol and Fatty Acid Synthesis

Jin

DeBose‐Boyd²

2011

Cold Spring Harbor Perspectives in Biology

209

148

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“…Digitonin permeabilizes only the plasma membrane, whereas Triton X-100 permeabilizes intracellular membranes, including the OMM (24,25). We confirmed this differential permeabilization by examining the susceptibility of adenylate kinase to protease digestion.…”

Section: Figmentioning

confidence: 99%

Mitochondrial Glycerol Phosphate Acyltransferase Contains Two Transmembrane Domains with the Active Site in the N-terminal Domain Facing the Cytosol

González-Baró¹,

Granger

Coleman

2001

Journal of Biological Chemistry

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The topography of mitochondrial glycerol-3-phosphate acyltransferase (GPAT) was determined using rat liver mitochondria and mutagenized recombinant rat GPAT (828 aa (amino acids)) expressed in CHO cells. Hydrophobicity analysis of GPAT predicts two transmembrane domains (TMDs), residues 472-493 and 576 -592. Residues 224 -323 correspond to the active site of the enzyme, which is believed to lie on the cytosolic face of the outer mitochondrial membrane. Protease treatment of rat liver mitochondria revealed that GPAT has a membraneprotected segment of 14 kDa that could correspond to the mass of the two predicted TMDs plus a loop between aa 494 and 575. Recombinant GPAT constructs containing tagged epitopes were transiently expressed in Chinese hamster ovary cells and immunolocalized. Both the C and N termini epitope tags could be detected after selective permeabilization of only the plasma membrane, indicating that both termini face the cytosol. A 6 -8-fold increase in GPAT-specific activity in the transfected cells confirmed correct protein folding and orientation. When the C terminus and loop-tagged GPAT construct was immunoassayed, the epitope at the C terminus could be detected when the plasma membrane was permeabilized, but loop-epitope accessibility required disruption of the outer mitochondrial membrane. Similar results were observed when GPAT was truncated before the second TMD, again consistent with an orientation in which the loop faces the mitochondrial intermembrane space. Although protease digestion of the HA-tagged loop resulted in preservation of a 14-kDa fragment, consistent with a membrane protected loop domain, neither the truncated nor loop-tagged enzymes conferred GPAT activity when overexpressed, suggesting that the loop plays a critical structural or regulatory role for GPAT function. Based on these data, we propose a GPAT topography model with two transmembrane domains in which both the N (aa 1-471) and C (aa 593-end) termini face the cytosol and a single loop (aa 494 -575) faces the intermembrane space.Glycerol phosphate acyltransferase (GPAT) 1 (EC 2.3.1.15) catalyzes the first and committed step in de novo cellular glycerolipid synthesis, the formation of 1-acyl-sn-glycerol-3-phosphate (lysophosphatidic acid) from glycerol-3-phosphate and long chain fatty acyl-CoA substrates (1, 2). Mammalian cells contain two GPAT isozymes that have different cellular locations, one in the endoplasmic reticulum and the other in mitochondria, that can be distinguished by sensitivity to inactivation by sulfhydryl reagents. The mitochondrial GPAT is an outer mitochondrial membrane (OMM) protein composing about 10% of the total enzymatic activity in most mammalian tissues. In liver, however, GPAT specific activity is similar in the two subcellular organelles. Only the mitochondrial GPAT isoform has been cloned. Recombinant mouse (3) and rat (4) GPAT open reading frames encode proteins of 827 and 828 amino acids, respectively, with a high degree of homology. Alignment of rat mitochondrial GPAT with other acyl...

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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

Cited by 171 publications

References 62 publications

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

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

Regulation of Cholesterol and Fatty Acid Synthesis

Mitochondrial Glycerol Phosphate Acyltransferase Contains Two Transmembrane Domains with the Active Site in the N-terminal Domain Facing the Cytosol

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