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R D Simoni

Publications and source records attributed to R D Simoni.

At least 19 recordsLinked to original sources
PubMed1998

Degradation of HMG-CoA reductase in vitro. Cleavage in the membrane domain by a membrane-bound cysteine protease.

We have recently shown that the endoplasmic reticulum (ER) membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is cleaved in isolated membrane fractions enriched for endoplasmic reticulum. Importantly, the cleavage rate is accelerated when the membranes are prepared from cells that have been pretreated with mevalonate or sterols, physiological regulators of the degradation process in vivo (McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1996) J. Biol. Chem. 271, 25630-25638). In the current study, we further characterize this in vitro cleavage of HMG-CoA reductase. E64, a specific inhibitor of cysteine-proteases, inhibits HMG-CoA reductase cleavage in vitro. In contrast, lactacystin, an inhibitor of the proteasome, inhibits HMG-CoA reductase degradation in vivo but does not inhibit the in vitro cleavage. Purified ER fractions contain lactacystin-sensitive and E64-insensitive proteasome activity as measured by succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin hydrolysis. We removed the proteasome from purified ER fractions by solubilization with heptylthioglucoside and observed that the detergent extracted, proteasome-depleted membrane fractions retain regulated cleavage of HMG-CoA reductase. This indicates that ER-associated proteasome is not involved in degradation of HMG-CoA reductase in vitro. In order to determine the site(s) of proteolysis of HMG-CoA reductase in vitro, four antisera were prepared against peptide sequences representing various domains of HMG-CoA reductase and used for detection of proteolytic intermediates. The sizes and antibody reactivity of the intermediates suggest that HMG-CoA reductase is cleaved in the in vitro degradation system near the span 8 membrane region, which links the N-terminal membrane domain to the C-terminal catalytic domain of the protein. We conclude that HMG-CoA reductase can be cleaved in the membrane-span 8 region by a cysteine protease(s) tightly associated with ER membranes.

Acetylcysteine↗
PubMed1997

Farnesol as a regulator of HMG-CoA reductase degradation: characterization and role of farnesyl pyrophosphatase.

We have recently reported that the isoprenoid compound farnesol accelerates degradation of the cholesterologenic enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, when added to cultured cells. We have thus proposed that farnesol is a required nonsterol regulator of this degradation event (T. E. Meigs, D. S. Roseman, and R. D. Simoni, 1996, J. Biol. Chem. 271, 7916-7922). In this report, we have studied the enzyme farnesyl pyrophosphatase (FPPase) in Chinese hamster ovary cells. We demonstrate that FPPase activity increases under conditions of increased metabolic flow through the isoprenoid pathway. Also, we show that a nonhydrolyzable analog of farnesyl pyrophosphate, an isoprenoid (phosphinylmethyl)phosphonate, inhibits FPPase in vitro, and when added to cells this inhibitor blocks the mevalonate-dependent, sterol-induced degradation of HMG-CoA reductase. Furthermore, exogenous farnesol overcomes the effect of this inhibitor. These results suggest an isoprenoid-mediated regulatory mechanism governing intracellular farnesol production and support the hypothesis that farnesol is a nonsterol regulator of reductase degradation.

Animals↗
PubMed1996

Degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in endoplasmic reticulum membranes is accelerated as a result of increased susceptibility to proteolysis.

The endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to regulated degradation when cells are presented with an excess of sterols or mevalonate. In this report, we demonstrate the degradation of HMG-CoA reductase in ER membranes prepared from cells which have been pretreated with mevalonate or sterols prior to membrane purification. Degradation of HMG-CoA reductase in membranes prepared from pretreated cells is more rapid than in membranes prepared from cells which have received no regulatory molecules. In vitro degradation is blocked by protease inhibitors previously shown to inhibit reductase degradation in vivo and is specific for intact HMG-CoA reductase. The lumenal contents of the ER membranes are dispensible for the regulated proteolysis and the proteases responsible for reductase degradation are stably associated with the ER membrane. Regulated proteolysis of HMG-CoA reductase is inhibited by lactacystin, a newly defined inhibitor of the multicatalytic protease, the proteasome, and in vitro degradation of reductase correlates with the presence of proteasome subunits in purified ER membranes. The ubiquitin system for protein degradation, which has recently been shown to be required for the degradation of several ER membrane proteins, is not required for the degradation of HMG-CoA reductase. Finally, we conclude that the regulated proteolysis of HMG-CoA reductase in response to regulatory molecules such as mevalonate or sterols is mediated by increased susceptibility of the reductase to ER proteases, rather than the induction of a new proteolytic activity.

Acetylcysteine↗
PubMed1996

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation by the nonsterol mevalonate metabolite farnesol in vivo.

We have previously reported that degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in the isoprenoid pathway leading to cholesterol production, can be accelerated in cultured cells by the addition of farnesyl compounds, which are thought to mimic a natural, nonsterol mevalonate metabolite(s). In this paper we report accelerated reductase degradation by the addition of farnesol, a natural product of mevalonate metabolism, to intact cells. We demonstrate that this regulation is physiologically meaningful, shown by its blockage by several inhibitory conditions that are known to block the degradation induced by mevalonate addition. We further show that intracellular farnesol levels increase significantly after mevalonate addition. Based on these results, we conclude that farnesol is a nonsterol, mevalonate-derived product that plays a role in accelerated reductase degradation. Our conclusion is in agreement with a previous report (Correll, C. C., Ng, L., and Edwards, P. A. (1994) J. Biol. Chem. 269, 17390-17393), in which an in vitro system was used to study the effect of farnesol on reductase degradation. However, the apparent stimulation of degradation in vitro appears to be due to nonphysiological processes. Our findings demonstrate that in vitro, farnesol causes reductase to become detergent insoluble and thus lost from immunoprecipitation experiments, yielding apparent degradation. We further show that another resident endoplasmic reticulum protein, calnexin, similarly gives the appearance of protein degradation after farnesol addition in vitro. However, after the addition of farnesol to cells in vivo, calnexin remains stable, whereas reductase is degraded, providing further evidence that the in vivo effects of farnesol are physiologically meaningful and specific for reductase, whereas the in vitro effects are not.

Animals↗
PubMed1995

Molecular dissection of the role of the membrane domain in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We have previously shown that the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from hamster contains all of the sequences required for both localization to the endoplasmic reticulum and regulated degradation of the enzyme. It has been reported that the enzymatic activity and mRNA levels of HMG-CoA reductase from sea urchin embryos cultured in the presence of regulators were unchanged compared to levels in control embryos (Woodward, H.D., Allen, M.C., and Lennarz, W.J. (1988) J. Biol. Chem. 263, 18411-18418). This observation led us to investigate the possibility that the sea urchin enzyme is not subject to regulated protein turnover. Interestingly, the sea urchin enzyme shares 62% amino acid sequence identity with the hamster enzyme in the membrane domain and shares similar predicted topological features. In the current studies we have compared the degradation phenotypes of the sea urchin HMG-CoA reductase and the hamster HMG-CoA reductase in Chinese hamster ovary cells to further elucidate the role of the membrane domain in enzyme degradation in response to physiological regulators. To accomplish this, we constructed sea urchin HMGal (uHMGal), the structural equivalent of hamster HMGal (httMGal), which has the sea urchin HMG-CoA reductase membrane domain fused to Escherichia coli beta-galactosidase. The uHMGal was stably expressed in CHO cells, and we found that the degradation of uHMGal is not accelerated by sterols, and even in the absence of sterols, it is less stable than hHMGal. We also constructed chimeric hamster/sea urchin HMGal molecules to investigate which amino acid sequences from the hamster enzyme are sufficient to confer sterol-regulated degradation upon the sea urchin enzyme. Our results identify the second membrane-spanning domain of hamster enzyme as important for the regulated degradation of HMG-CoA reductase.

Amino Acid Sequence↗
PubMed1995

Mutation in the lumenal part of the membrane domain of HMG-CoA reductase alters its regulated degradation.

The involvement of ER lumenal domains of HMG-CoA reductase in the regulated degradation process was examined. For this purpose we studied three cell lines expressing HMG-CoA reductase molecules with introduced functional N-glycosylation sites located in the linker segments between transmembrane spans 1 and 2 (HMGal/Bins(-)), 3 and 4 (HMGal/Dins(-)) and 5 and 6 (HMGal/Fins(-)), all facing the ER lumen (Olender, E. H. and Simoni, R. D. (1992) J. Biol. Chem. 267, 4223-4235. The glycosylation insertion between spons 5 and 6 (HMGal/Fins(-)) is the only one of these mutations which eliminates regulated degradation of the enzyme. The half lives of the HMGal/Fins(-) in the presence or absence of regulatory molecules are indistinguishable. In contrast the HMGal/Bins(-) and HMGal/Dins(-) mutants show a normal pattern of regulated degradation. Tunicamycin treatment of cells expressing the HMGal/Fins(-) mutant does not significantly alter the regulation defect indicating that it is the mutation per se not the glycosylation that alters the degradation response. These results suggest that the linker segments between transmembrane spans 5 and 6 (loop F) are involved in the process of regulated degradation of HMG-CoA reductase and that the regulated degradation process may occur on the lumenal side of the ER membrane.

Animals↗
PubMed1995

Farnesyl acetate, a derivative of an isoprenoid of the mevalonate pathway, inhibits DNA replication in hamster and human cells.

Cells treated with compactin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme which catalyzes the rate-limiting step of the mevalonate pathway, are arrested prior to the DNA synthesis (S) phase of the cell cycle. Identification of a specific pathway product or products with a role in DNA replication, however, has remained elusive. In this report we demonstrate that farnesyl acetate, a derivative of the key isoprenoid pathway intermediate farnesyl pyrophosphate, inhibits DNA replication in both Chinese hamster ovary cells and human (HeLa) cells. This effect is revealed by measurement of DNA content using fluorescence-activated cell sorter analysis and by measurement of [3H]thymidine incorporation. We show that cells treated with farnesyl acetate retain protein synthesis capacity as DNA replication is inhibited and remain intact as viewed with the vital stain propidium iodide. The inhibition of DNA replication by farnesyl acetate occurs in cells treated with high levels of compactin and in cells lacking HMG-CoA reductase. These results indicate that farnesyl acetate action is not dependent on metabolism through the isoprenoid pathway and is not the result of the loss of a metabolite required for replication nor the accumulation of a metabolite which is inhibitory. In addition, cells treated with farnesyl acetate for over 6 h are irreversibly blocked from progressing through S phase, a phenomenon which differs sharply from the results with compactin, removal of which results in synchronous progression through S phase. Farnesyl acetate also blocks protein prenylation in cells, to a degree comparable to a known farnesylation inhibitor, BZA-5B. We propose that farnesyl acetate is acting in a manner quite different from the metabolic block caused by compactin, causing a rapid and irreversible block of DNA replication.

Animals↗
PubMed1994

Non-sterol compounds that regulate cholesterogenesis. Analogues of farnesyl pyrophosphate reduce 3-hydroxy-3-methylglutaryl-coenzyme A reductase levels.

Farnesyl acetate and ethyl farnesyl ether, two analogues of farnesyl pyrophosphate, stimulate post-transcriptional down-regulation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the biosynthesis of cholesterol and isoprenoids. Farnesyl acetate and ethyl farnesyl ether reduce translation of HMG-CoA reductase mRNA and enhance degradation of the enzyme, the same regulatory effects attributed to the putative non-sterol regulatory metabolite (Goldstein, J.L., and Brown, M.S. (1990) Nature 343, 425-430). HMGal, a fusion protein consisting of the membrane domain of HMG-CoA reductase linked to Escherichia coli beta-galactosidase, is subject to the same regulated degradation as HMG-CoA reductase (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). At 10 micrograms/ml (37.8 microM), farnesyl acetate and ethyl farnesyl ether trigger a 50-80% reduction in HMGal activity. Farnesyl acetate reduces the synthesis of HMG-CoA reductase and HM-Gal by 60-80%, but neither farnesyl compound affects HMG-CoA reductase mRNA levels. Farnesyl acetate and ethyl farnesyl ether stimulated the degradation of HMG-CoA reductase and HMGal, reducing the half-lives of the enzymes by 40-70%. In addition to their regulatory effects on HMG-CoA reductase, these farnesyl compounds also directly disrupt sterol synthesis.

Animals↗
PubMed1993

In vivo inhibition of cyclin B degradation and induction of cell-cycle arrest in mammalian cells by the neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal.

The cytotoxic neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal (ALLN) inhibits cell-cycle progression in CHO cells, affecting the G1/S and metaphase-anaphase transition points, as well as S phase. Mitotic arrest induced by ALLN is associated with the inhibition of cyclin B degradation. At mitosis-inhibiting concentrations of ALLN, cells undergo nuclear-envelope breakdown, spindle formation, chromosome condensation, and congression to the metaphase plate. However, normal anaphase events do not occur, and cells arrest in a metaphase configuration for a prolonged period. Steady-state levels of cyclin B increase to greater than normal mitotic levels, and cyclin B is not degraded for an extended period. Histone H1 kinase activity remains elevated during mitotic arrest. Duration of mitotic arrest depends on ALLN concentration; high concentrations (> 50 micrograms/ml) produce a prolonged mitotic arrest, whereas at lower concentrations, cells are transiently delayed through mitosis (up to 4-12 hr), after which they undergo aberrant cell division resulting in randomly sized daughter cells containing variable amounts of DNA. Cyclin B degradation fails to occur, and histone H1 kinase remains activated for the duration of mitotic arrest at all ALLN concentrations.

Animals↗
PubMed1993

Cellular detoxification of tripeptidyl aldehydes by an aldo-keto reductase.

Calpain inhibitor I, N-acetyl-leucyl-leucyl-norleucinal (ALLN), a cell-permeable synthetic tripeptide with an aldehyde at its C terminus specifically inhibits the activity of cysteine proteases. Since the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in Chinese hamster ovary (CHO) cells is blocked by ALLN and ALLN has a cytotoxic effect on cells, we attempted to isolate ALLN-resistant cells that overproduce an ALLN-sensitive protease(s). However, we obtained an ALLN-resistant cell line that overproduced P-glycoprotein (Sharma, R. C., Inoue, S., Roitelman, J., Schimke, R. T., and Simoni, R. D. (1992) J. Biol. Chem. 267, 5731-5734). To circumvent the multidrug resistance (MDR) phenotype during selection, we have stepwise selected an ALLN-resistant cell line of CHO cells in the presence of verapamil, a competitive inhibitor of P-glycoprotein. These non-MDR ALLN-resistant cells overexpress a 35-kDa protein and have increased aldo-keto reductase activity. Partial amino acid sequences of the 35-kDa protein are highly homologous to members of the aldo-keto reductase superfamily. The aldo-keto reductases are NADPH-dependent oxidoreductases and catalyze reduction of a wide range of carbonyl compounds such as aldehydes, sugars, and ketones. Our findings support the concept that a physiological function for aldo-keto reductases may be detoxification.

Alcohol Oxidoreductases↗
PubMed1992

Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase.

The in vivo turnover rate of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate (MVA) pathway, is accelerated when excess MVA or sterols are added to the growth medium of cells. As we have shown recently (Roitelman, J., Bar-Nun, S., Inoue, S., and Simoni, R. D. (1991) J. Biol. Chem. 266, 16085-16091), perturbation of cellular Ca2+ homeostasis abrogates the MVA-accelerated degradation of HMG-CoA reductase and HMGal. Here we show that, in contrast, the sterol-accelerated degradation of HMG-CoA reductase is unaffected by Ca2+ perturbation achieved either by Ca2+ ionophore or by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. The differential effects of Ca2+ perturbation can be attributed neither to global alteration in protein synthesis nor to inhibition of MVA conversion to sterols. Yet, such manipulations markedly reduce the incorporation of MVA into cellular macromolecules, including prenylated proteins. Furthermore, we directly demonstrate that MVA gives rise to at least two distinct signals, one that is essential to support the effect of sterols and another that operates independently of sterols. Our results indicate that the cellular signals operating in the MVA-accelerated turnover of HMG-CoA reductase are distinct from those involved in the sterol-regulated degradation. A working model for the degradation pathway is proposed.

Animals↗
PubMed1992

Squalene synthase-deficient mutant of Chinese hamster ovary cells.

Squalene synthase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) converts farnesyl pyrophosphate to squalene, the first metabolic step committed solely to the biosynthesis of sterols. Using a fluorescence-activated cell sorting technique designed to screen for cells defective in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, we isolated a squalene synthase-deficient mutant of Chinese hamster ovary cells. The mutant cell line, designated SSD, exhibits less than 7% of the squalene synthase activity of the parental cell line, CHO-HMGal. Both the SSD and the parental cells stably express HMGal, a model protein for studying the regulated degradation of HMG-CoA reductase, which consists of the membrane domain of HMG-CoA reductase fused to bacterial beta-galactosidase (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this study, the regulatory effects of mevalonate and compactin on the activity levels of HMGal are substantially reduced in SSD cells as compared to the parental cell line. In lipid-poor medium, SSD cell growth is arrested. The rate of [3H]acetate incorporation into cholesterol for the mutant SSD cells is less than 2% of the rate for the parental cells. However, the incorporation of [3H] squalene into sterols is essentially wild type for SSD cells. When the mutant SSD cells are fed [3H]acetate, radioactivity accumulates in farnesol, much of which is secreted into the medium. By growing SSD cells in lipid-poor medium, a revertant cell type, designated SSR, was isolated. In every assay performed the revertant SSR cells exhibited a phenotype that was essentially wild type, demonstrating that the SSD mutant phenotype was the result of a single mutation.

Acetates↗
PubMed1992

Regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in permeabilized cells.

We have studied the regulated degradation of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase within the endoplasmic reticulum in cells permeabilized with digitonin. Using Chinese hamster ovary cells transfected with a plasmid encoding HMGal, a chimeric protein containing the membrane domain of HMG-CoA reductase coupled to beta-galactosidase, we have demonstrated mevalonate and sterol-stimulated loss of beta-galactosidase activity. In pulse-chase experiments we have demonstrated mevalonate-stimulated degradation of both HMGal and HMG-CoA reductase. The rate of mevalonate-stimulated degradation observed in permeabilized cells tends to be slightly slower than that observed in intact cells treated with mevalonate and is dependent upon incubation of cells with mevalonate prior to permeabilization. The degradation process measured in this report extends a previous report of HMG-CoA reductase degradation in digitonin-permeabilized cells (Leonard, D. A., and Chen, H. W. (1987) J. Biol. Chem. 262, 7914-7919) by mimicking key physiological features of the in vivo process, including: stimulation by regulatory molecules, specifically mevalonate and sterols; inhibition by cycloheximide; and inhibition by an inhibitor of neutral cysteine proteases.

Adenosine Triphosphate↗
PubMed1992

Influence of subunit-specific antibodies on the activity of the F0 complex of the ATP synthase of Escherichia coli. I. Effects of subunit b-specific polyclonal antibodies.

Incubation of F1-stripped everted membrane vesicles with antibodies against subunit b of the ATP synthase from Escherichia coli resulted in an inhibition of the binding of F1 to F0, whereas the proton translocation remained unaffected. Incubation of unstripped everted membrane vesicles with anti-b antibodies resulted in a partial loss of F1, and the remaining membrane-bound ATP-hydrolyzing activity is uncoupled from proton translocation. Similar results were obtained when F(ab')2 or Fab fragments were used. The immunoblot analysis of truncated b' subunits different in length showed that the antigenic determinants are located in the carboxyl-terminal half of the polypeptide chain.

Adenosine Triphosphatases↗
PubMed1992

3-Hydroxy-3-methylglutaryl-coenzyme A reductase and T cell receptor alpha subunit are differentially degraded in the endoplasmic reticulum.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is located in the endoplasmic reticulum (ER) and responds to rapid degradation which is regulated by mevalonate or sterols. T cell antigen receptor alpha chain (TCR alpha) is also known to be rapidly degraded within the ER. In both cases, the membrane domains of the proteins have a crucial role in their rapid degradation. In order to investigate protein degradation in the ER, we compared the degradation of HMG-CoA reductase and TCR alpha in the same Chinese hamster ovary cells. Among the protease inhibitors tested, N-acetyl-leucyl-leucyl-methioninal blocks the degradation of HMG-CoA reductase and also inhibits the degradation of TCR alpha. On the other hand, N-tosyl-L-phenylalanine chloromethyl ketone and N-carbobenzoxy-L-phenylalanine chloromethyl ketone inhibit the degradation of TCR alpha but have no effect on the degradation of HMG-CoA reductase. Diamide, a thiol-oxidizing agent, blocks the degradation of both HMG-CoA reductase and TCR alpha. Perturbation of cellular Ca2+ attenuates the rapid degradation of HMG-CoA reductase but does not affect the degradation of TCR alpha. Furthermore, thapsigargin, a selective ER Ca(2+)-ATPase inhibitor, and Co2+, a potent Ca2+ antagonist, increase the half-life of HMG-CoA reductase but not that of TCR alpha. Energy inhibitors diminish the rapid degradation of HMG-CoA reductase but not that of TCR alpha. These results suggest that although HMG-CoA reductase and TCR alpha appear to be degraded in the same subcellular compartment, the mechanisms responsible for degradation differ.

1-Deoxynojirimycin↗
PubMed1992

Peptide transport by the multidrug resistance pump.

The membrane P-glycoprotein (P170) is an ATP-hydrolyzing transmembrane pump, and elevated levels of P170, due to higher expression with or without amplification of the multidrug resistance gene (mdr1), result in resistance to a variety of chemotherapeutic agents in mammalian cells. The function of the P170 pump has been proposed as a protection against toxic substances present in animal diets. Here we describe a Chinese hamster ovary cell line that was selected for resistance to a synthetic tripeptide, N-acetyl-leucyl-leucyl-norleucinal (ALLN). This ALLN-resistant variant shows the classical multidrug resistance (MDR) phenotype, including overexpression and amplification of the mdr1 gene. Additionally, a mouse embryo cell line overexpressing the transfected mdr1 gene is likewise resistant to ALLN. Our results demonstrate that P170 is capable of transporting peptides and raise the possibility that the mdr1 gene product or other MDR-like genes, present in the genome of mammalian cells, may be involved in secretion of peptides or cellular proteins as is the case with the structurally similar hylB and ste6 gene products of Escherichia coli and yeast, respectively.

3T3 Cells↗
PubMed1992

The role of the membrane domain in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We have constructed a series of mutations in the membrane and linker domains of Syrian hamster 3-hydroxy-3-methylglutaryl-(HMG) CoA reductase in order to determine the regions critical for the regulated degradation of the enzyme. In transfected Chinese hamster ovary cells, we have expressed a fusion protein, HMGal, which consists of the membrane and linker domains of the Syrian hamster HMG-CoA reductase fused to beta-galactosidase. Using this fusion protein, we have determined that a deletion of 64 amino acids from the central region of the membrane domain causes the protein to be degraded extremely rapidly. In addition, deletion of PEST sequences has little effect on degradation, but deletion of the linker domain makes the protein's degradation insensitive to sterols and mevalonate. In addition to deletion mutations, we have systematically replaced each hydrophobic, putative membrane spanning region of the membrane domain with the first transmembrane sequence from bacteriorhodopsin. Replacement of span 4 has no effect on degradation. Replacements of spans 5 or 6 result in a protein which has a normal basal rate of degradation, but this rate of degradation is not accelerated by mevalonate, low density lipoprotein, or 25-hydroxycholesterol. Replacement of span 3 results in a protein whose degradation is similarly not accelerated by sterols or mevalonate, but since this protein might be mislocalized, these results are inconclusive. Replacement of span 7 yields a short-lived protein which is degraded more rapidly in response to mevalonate but not in response to exogenous sterols. Replacement of span 8 extends both the basal and mevalonate-accelerated half-life about 5-fold. This work begins to define the critical regions for regulated degradation within the membrane domain of HMG-CoA reductase.

Amino Acid Sequence↗