Increase in membrane cholesterol: A possible trigger for degradation of HMG CoA reductase and crystalloid endoplasmic reticulum in UT-1 cells

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.672-680.1990 ◽

1990 ◽

Vol 10 (2) ◽

pp. 672-680

Author(s):

C Sengstag ◽

C Stirling ◽

R Schekman ◽

J Rine

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Coenzyme A ◽

Transmembrane Domains ◽

Protein Domain ◽

Yeast Cells ◽

Endoplasmic Reticulum Membrane ◽

Hmg Coa Reductase ◽

Coa Reductase ◽

Histidinol Dehydrogenase

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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Abstract 365: Inhibiting Hmg Coa Reductase Reduces Palmitate-Induced Inflammation in Adipocytes

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.365 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Asha K Pathak ◽

Chang Yeop Han ◽

Mohamed Omer ◽

Shari Wang ◽

Alan Chait

Keyword(s):

Gene Expression ◽

Plasma Membrane ◽

Er Stress ◽

Lipid Raft ◽

Cholesterol Content ◽

Membrane Cholesterol ◽

Hmg Coa Reductase ◽

Pre Treatment ◽

Coa Reductase ◽

Plasma Membrane Cholesterol

Adipose tissue inflammation associates with insulin resistance and increased cardiovascular disease risk. We previously observed that 3T3-L1 adipocytes exposed to palmitate become inflamed and demonstrate increased plasma membrane cholesterol and lipid raft content. It is known that palmitate induces translocation of NAPH oxidase and toll-like receptor 4 into lipid rafts, increasing adipocyte inflammation. However, it is unclear (1) how palmitate alters plasma membrane cholesterol content; and (2) whether increased cholesterol content in the plasma membrane is related to adipocyte inflammation induced by palmitate exposure. We hypothesize that mechanisms involved in increasing plasma membrane cholesterol content after palmitate treatment could be related to cholesterol synthesis and/or ER stress, and that increased cholesterol in lipid rafts is essential for induction of inflammation in adipocytes. To test these hypotheses, differentiated murine 3T3-L1 adipocytes were exposed to palmitate for 24 hours, with and without pre-treatment with HMG-CoA reductase inhibitors (statins) or HDL. RT-PCR was used to evaluate gene expression of inflammation ( Saa3 , Ccl2 ), ER stress ( Bip , Chop ), and HMG-CoA reductase ( Hmgcr ). Cholera toxin subunit β staining and flow cytometry were used to evaluate plasma membrane lipid raft content. In differentiated adipocytes, palmitate-induced inflammation neither increased expression of ER stress genes nor HMG-CoA reductase gene expression. However, treatment with 3 different statins (simvastatin, lovastatin, atorvastatin) significantly reduced palmitate-induced adipocyte inflammation as indicated by decreased gene expression of Saa3 and Ccl2 ( P <0.05). A similar effect was seen with pre-treatment with HDL. Lipid raft content induced by palmitate was decreased by HMG-CoA reductase inhibitors (difference in mean fluorescence intensity P <0.05) and also by pre-treatment with HDL. These findings indicate that ER stress was not involved in increased plasma membrane cholesterol after palmitate-induced inflammation in adipocytes. However, regulating cholesterol content in lipid rafts plays an important role in adipocyte inflammation induced by palmitate.

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Posttranslational Regulation of HMG CoA Reductase, the Rate-Limiting Enzyme in Synthesis of Cholesterol

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-081820-101010 ◽

2021 ◽

Vol 90 (1) ◽

pp. 659-679

Author(s):

Marc M. Schumacher ◽

Russell A. DeBose-Boyd

Keyword(s):

Endoplasmic Reticulum ◽

Cholesterol Metabolism ◽

Posttranslational Regulation ◽

Hmg Coa Reductase ◽

Feedback Mechanisms ◽

Genetically Manipulated ◽

Coa Reductase ◽

Rate Limiting

The polytopic, endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, the key intermediate in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). Transcriptional, translational, and posttranslational feedback mechanisms converge on this reductase to ensure cells maintain a sufficient supply of essential nonsterol isoprenoids but avoid overaccumulation of cholesterol and other sterols. The focus of this review is mechanisms for the posttranslational regulation of HMG CoA reductase, which include sterol-accelerated ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We discuss how GGpp-induced ER-to-Golgi trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain–containing protein-1 (UBIAD1) modulates HMG CoA reductase ERAD to balance the synthesis of sterol and nonsterol isoprenoids. We also summarize the characterization of genetically manipulated mice, which established that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMG CoA reductase and cholesterol metabolism in vivo.

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Biogenesis of the crystalloid endoplasmic reticulum in UT-1 cells: evidence that newly formed endoplasmic reticulum emerges from the nuclear envelope.

The Journal of Cell Biology ◽

10.1083/jcb.102.6.2158 ◽

1986 ◽

Vol 102 (6) ◽

pp. 2158-2168 ◽

Cited By ~ 83

Author(s):

R K Pathak ◽

K L Luskey ◽

R G Anderson

Keyword(s):

Endoplasmic Reticulum ◽

Nuclear Envelope ◽

Nuclear Membrane ◽

Cholesterol Biosynthesis ◽

Integral Membrane Protein ◽

Enzyme Synthesis ◽

Hmg Coa Reductase ◽

Outer Nuclear Membrane ◽

Sequence Of Events ◽

Coa Reductase

The crystalloid endoplasmic reticulum (ER), a specialized smooth ER of the compactin-resistant UT-1 cell, is composed of multiple membrane tubules packed together in a hexagonal pattern. This membrane contains large amounts of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, an integral membrane protein that enzymatically regulates endogenous cholesterol biosynthesis. Using morphological and immunocytochemical techniques, we have traced the sequence of events in the biogenesis of this ER when compactin-withdrawn UT-1 cells, which do not have a crystalloid ER, are incubated in the presence of compactin. After 15 h of incubation in the presence of compactin, many cells had profiles of ER cisternae that were juxtaposed to the nuclear envelope and studded with ribosomes on their outer membrane. Both the outer nuclear membrane and the ER membrane contained HMG CoA reductase; however, there was little or no detectable enzyme in rough ER that was free in the cytoplasm. With longer times of incubation in the presence of compactin, these cells had lamellar stacks of smooth ER next to the nuclear envelope that contained HMG CoA reductase. Coordinate with the appearance of the smooth ER, crystalloid ER appeared in the same cell. Often regions of continuity were found between the membrane of the smooth ER and the membrane of the crystalloid ER tubules. These studies suggest that HMG CoA reductase is synthesized along the outer nuclear membrane and in response to increased enzyme synthesis, a membrane emerges from the outer nuclear membrane as smooth ER cisternae, which then transforms into crystalloid ER tubules.

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