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

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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Partial deletion of membrane-bound domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase eliminates sterol-enhanced degradation and prevents formation of crystalloid endoplasmic reticulum.

The Journal of Cell Biology ◽

10.1083/jcb.104.6.1693 ◽

1987 ◽

Vol 104 (6) ◽

pp. 1693-1704 ◽

Cited By ~ 80

Author(s):

H Jingami ◽

M S Brown ◽

J L Goldstein ◽

R G Anderson ◽

K L Luskey

Keyword(s):

Endoplasmic Reticulum ◽

Half Life ◽

Coenzyme A ◽

Catalytic Domain ◽

Carbohydrate Chain ◽

Hmg Coa Reductase ◽

Membrane Bound ◽

Membrane Spanning ◽

Coa Reductase ◽

Er Membrane

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic NH2-terminal domain that contains seven apparent membrane-spanning regions and a single N-linked carbohydrate chain. The catalytic domain, which includes the COOH-terminal two-thirds of the protein, extends into the cytoplasm. The enzyme is normally degraded with a rapid half-life (2 h), but when cells are depleted of cholesterol, its half-life is prolonged to 11 h. Addition of sterols accelerates degradation by fivefold. To explore the requirements for regulated degradation, we prepared expressible reductase cDNAs from which we either deleted two contiguous membrane-spanning regions (numbers 4 and 5) or abolished the single site for N-linked glycosylation. When expressed in hamster cells after transfection, both enzymes retained catalytic activity. The deletion-bearing enzyme continued to be degraded with a rapid half-life in the presence of sterols, but it no longer was stabilized when sterols were depleted. The glycosylation-minus enzyme was degraded at a normal rate and was stabilized normally by sterol deprivation. When cells were induced to overexpress the deletion-bearing enzyme, they did not incorporate it into neatly arranged crystalloid ER tubules, as occurred with the normal and carbohydrate-minus enzymes. Rather, the deletion-bearing enzyme was incorporated into hypertrophied but disordered sheets of ER membrane. We conclude that the carbohydrate component of HMG CoA reductase is not required for proper subcellular localization or regulated degradation. In contrast, the native structure of the transmembrane component is required to form a normal crystalloid ER and to allow the enzyme to undergo regulated degradation by sterols.

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Role of Hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase 1 in Nodule Development of Soybean

Journal of Plant Physiology ◽

10.1016/j.jplph.2021.153543 ◽

2021 ◽

pp. 153543

Author(s):

Ali Izadi-Darbandi ◽

Peter M. Gresshoff

Keyword(s):

Coenzyme A ◽

Nodule Development ◽

Hmg Coa Reductase ◽

Coa Reductase

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Identification of the sequences in HMG-CoA reductase required for karmellae assembly.

Molecular Biology of the Cell ◽

10.1091/mbc.6.11.1535 ◽

1995 ◽

Vol 6 (11) ◽

pp. 1535-1547 ◽

Cited By ~ 37

Author(s):

M L Parrish ◽

C Sengstag ◽

J D Rine ◽

R L Wright

Keyword(s):

Cellular Responses ◽

Cell Periphery ◽

Membrane Domain ◽

Hmg Coa Reductase ◽

Simple Possibility ◽

Coa Reductase ◽

Necessary And Sufficient ◽

Specific Membrane ◽

Molecular Nature ◽

Er Membrane

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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3-Hydroxymethyl coenzyme A reductase inhibition attenuates spontaneous smooth muscle tone via RhoA/ROCK pathway regulated by RhoA prenylation

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00034.2010 ◽

2010 ◽

Vol 298 (6) ◽

pp. G962-G969 ◽

Cited By ~ 11

Author(s):

Satish Rattan

Keyword(s):

Smooth Muscle ◽

Coenzyme A ◽

Muscle Tone ◽

Cholesterol Biosynthesis ◽

Rho Kinase ◽

Hmg Coa Reductase ◽

Smooth Muscle Tone ◽

Tonic Smooth Muscle ◽

Coa Reductase

RhoA prenylation may play an important step in the translocation of RhoA in the basal internal anal sphincter (IAS) smooth muscle tone. Statins inhibit downstream posttranslational RhoA prenylation by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition (HMGCRI). The role of statins in relation to RhoA prenylation in the pathophysiology of the spontaneously tonic smooth muscle has not been investigated. In the present studies, we determined the effect of classical HMGCRI simvastatin on the basal IAS tone and RhoA prenylation and in the levels of RhoA/Rho kinase (ROCK) in the cytosolic vs. membrane fractions of the smooth muscle. Simvastatin produced concentration-dependent decrease in the IAS tone (via direct actions at the smooth muscle cells). The decrease in the IAS tone by simvastatin was associated with the decrease in the prenylation of RhoA, as well as RhoA/ROCK in the membrane fractions of the IAS, in the basal state. The inhibitory effects of the HMGCRI were completely reversible by geranylgeranyltransferase substrate geranylgeranyl pyrophosphate. Relaxation of the IAS smooth muscle via HMGCRI simvastatin is mediated via the downstream decrease in the levels of RhoA prenylation and ROCK activity. Studies support the concept that RhoA prenylation leading to RhoA/ROCK translocation followed by activation is important for the basal tone in the IAS. Data suggest that the role of HMG-CoA reductase may go beyond cholesterol biosynthesis, such as the regulation of the smooth muscle tone. The studies have important implications in the pathophysiological mechanisms and in the novel therapeutic approaches for anorectal motility disorders.

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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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The role of N-linked glycosylation in the regulation of activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and proliferation of SV40-transformed 3T3 cells

Biochemical Journal ◽

10.1042/bj2600597 ◽

1989 ◽

Vol 260 (2) ◽

pp. 597-600 ◽

Cited By ~ 4

Author(s):

O Larsson ◽

W Engström

Keyword(s):

Cell Proliferation ◽

Dna Synthesis ◽

Coenzyme A ◽

Inhibitory Effects ◽

3T3 Cells ◽

Hmg Coa Reductase ◽

Regulation Of Activity ◽

Coa Reductase

The effects of glycosylation inhibitors on the proliferation of SV40-transformed 3T3 cells (SV-3T3) were examined in vitro. Whereas swainsonine and castanospermine, which inhibit distal steps in the glycosylational processing, exerted marginal or no effects on cell proliferation, a proximal inhibitor, tunicamycin, efficiently decreased the rate of DNA synthesis and also inhibited the activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. The inhibitory effects of tunicamycin on cell proliferation could be partially reversed by addition of dolichol, a metabolite in the pathway regulated by HMG-CoA reductase. This finding suggests that tunicamycin exerts at least one of its effects on cell proliferation by modulating the activity of HMG-CoA reductase.

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