Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Both 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase isozymes of the yeast Saccharomyces cerevisiae are predicted to contain seven membrane-spanning domains. Previous work had established the utility of the histidinol dehydrogenase protein domain, encoded by HIS4C, as a topologically sensitive monitor that can be used to distinguish between the lumen of the endoplasmic reticulum and the cytoplasm. This study directly tested the structural predictions for HMG-CoA reductase by fusing the HIS4C domain to specific sites in the HMG-CoA reductase isozymes. Yeast cells containing the HMG-CoA reductase-histidinol dehydrogenase fusion proteins grew on histidinol-containing medium if the HIS4C domain was present on the cytoplasmic side of the endoplasmic reticulum membrane but not if the HIS4C domain was targeted to the endoplasmic reticulum lumen. Systematic exchanges of transmembrane domains between the isozymes confirmed that both isozymes had equivalent membrane topologies. In general, deletion of an even number of putative transmembrane domains did not interfere with the topology of the protein, but deletion or duplication of an odd number of transmembrane domains inverted the orientation of the protein. The data confirmed the earlier proposed topology for yeast HMG-CoA reductase, demonstrated that the yeast enzymes are core glycosylated, and provided in vivo evidence that the properties of transmembrane domains were, in part, dependent upon their context within the protein.

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Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.

Molecular Biology of the Cell ◽

10.1091/mbc.7.5.769 ◽

1996 ◽

Vol 7 (5) ◽

pp. 769-789 ◽

Cited By ~ 81

Author(s):

A J Koning ◽

C J Roberts ◽

R L Wright

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Subcellular Localization ◽

Nuclear Envelope ◽

Fluorescent Protein ◽

Endoplasmic Reticulum Membrane ◽

Hmg Coa Reductase ◽

Endogenous Expression ◽

Coa Reductase ◽

Er Membrane

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.

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The regulated degradation of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase reporter construct occurs in the endoplasmic reticulum

Journal of Cell Science ◽

10.1242/jcs.107.9.2635 ◽

1994 ◽

Vol 107 (9) ◽

pp. 2635-2642

Author(s):

L.W. Lecureux ◽

B.W. Wattenberg

Keyword(s):

Endoplasmic Reticulum ◽

Coenzyme A ◽

Cholesterol Biosynthesis ◽

Mevalonic Acid ◽

Dna Encoding ◽

Hmg Coa Reductase ◽

Steady State Levels ◽

Coa Reductase ◽

Membrane Anchoring ◽

Beta Galactosidase

The rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase, is regulated at a number of levels. One important mechanism is regulation of the half-life of the protein by a controlled proteolytic system. This comes about in response to downstream products of the sterol biosynthetic pathway. Little is known about this system, including where in the cell this regulated degradation occurs. HMG CoA reductase resides in the endoplasmic reticulum. To localize the site of regulated degradation of HMG CoA reductase, we used a construct that fuses the N-terminal membrane-anchoring domain of HMG CoA reductase in-frame with beta-galactosidase as a reporter domain (HM-Gal). HM-Gal has previously been shown to reproduce faithfully the degradative properties of native HMG CoA reductase (Chun et al. (1990) J. Biol. Chem. 265, 22004–22010). CHO cells transfected with DNA encoding HM-Gal were exposed to mevalonic acid, which enhances the rate of HMG CoA reductase degradation several fold, and leads to the reduction of the steady state levels of HM-Gal by 80–90%. To accumulate HMG CoA reductase at the site of degradation, cells were simultaneously treated with N-acetyl-leucyl-leucyl-norleucinal (ALLN), which inhibits the protease responsible for reductase degradation. HM-Gal was localized morphologically by immunofluorescence and biochemically by measuring beta-galactosidase activity in Percoll gradients of cellular homogenates. Using either technique HM-Gal localization was indistinguishable from that of ER markers in both control cells and in cells treated to accumulate HMG CoA reductase at the site of degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

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Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3797-3808.1988 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3797-3808

Author(s):

M E Basson ◽

M Thorsness ◽

J Finer-Moore ◽

R M Stroud ◽

J Rine

Keyword(s):

Coenzyme A ◽

Sterol Biosynthesis ◽

New Drugs ◽

Yeast Cells ◽

Cdna Clones ◽

Hmg Coa Reductase ◽

Functional Conservation ◽

Mammalian Enzyme ◽

Coa Reductase ◽

Rate Limiting

The pathway of sterol biosynthesis is highly conserved in all eucaryotic cells. We demonstrated structural and functional conservation of the rate-limiting enzyme of the mammalian pathway, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), between the yeast Saccharomyces cerevisiae and humans. The amino acid sequence of the two yeast HMG-CoA reductase isozymes was deduced from DNA sequence analysis of the HMG1 and HMG2 genes. Extensive sequence similarity existed between the region of the mammalian enzyme encoding the active site and the corresponding region of the two yeast isozymes. Moreover, each of the yeast isozymes, like the mammalian enzyme, contained seven potential membrane-spanning domains in the NH2-terminal region of the protein. Expression of cDNA clones encoding either hamster or human HMG-CoA reductase rescued the viability of hmg1 hmg2 yeast cells lacking this enzyme. Thus, mammalian HMG-CoA reductase can provide sufficient catalytic function to replace both yeast isozymes in vivo. The availability of yeast cells whose growth depends on human HMG-CoA reductase may provide a microbial screen to identify new drugs that can modulate cholesterol biosynthesis.

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Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

Molecular Biology of the Cell ◽

10.1091/mbc.5.6.655 ◽

1994 ◽

Vol 5 (6) ◽

pp. 655-665 ◽

Cited By ~ 51

Author(s):

D Dimster-Denk ◽

M K Thorsness ◽

J Rine

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Coenzyme A ◽

Mevalonate Pathway ◽

Feedback Regulation ◽

Eukaryotic Cells ◽

Yeast Gene ◽

Hmg Coa Reductase ◽

Common Precursor ◽

Coa Reductase

In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.

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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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Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis.

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3797 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3797-3808 ◽

Cited By ~ 83

Author(s):

M E Basson ◽

M Thorsness ◽

J Finer-Moore ◽

R M Stroud ◽

J Rine

Keyword(s):

Coenzyme A ◽

Sterol Biosynthesis ◽

New Drugs ◽

Yeast Cells ◽

Cdna Clones ◽

Hmg Coa Reductase ◽

Functional Conservation ◽

Mammalian Enzyme ◽

Coa Reductase ◽

Rate Limiting

The pathway of sterol biosynthesis is highly conserved in all eucaryotic cells. We demonstrated structural and functional conservation of the rate-limiting enzyme of the mammalian pathway, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), between the yeast Saccharomyces cerevisiae and humans. The amino acid sequence of the two yeast HMG-CoA reductase isozymes was deduced from DNA sequence analysis of the HMG1 and HMG2 genes. Extensive sequence similarity existed between the region of the mammalian enzyme encoding the active site and the corresponding region of the two yeast isozymes. Moreover, each of the yeast isozymes, like the mammalian enzyme, contained seven potential membrane-spanning domains in the NH2-terminal region of the protein. Expression of cDNA clones encoding either hamster or human HMG-CoA reductase rescued the viability of hmg1 hmg2 yeast cells lacking this enzyme. Thus, mammalian HMG-CoA reductase can provide sufficient catalytic function to replace both yeast isozymes in vivo. The availability of yeast cells whose growth depends on human HMG-CoA reductase may provide a microbial screen to identify new drugs that can modulate cholesterol biosynthesis.

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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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