Topology of SREBP Cleavage-activating Protein, a Polytopic Membrane Protein with a Sterol-sensing Domain

Nohturfft, Axel; Brown, Michael S.; Goldstein, Joseph L.

doi:10.1074/jbc.273.27.17243

Cited by 177 publications

(173 citation statements)

References 36 publications

(61 reference statements)

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“…Such an alignment is not without precedent, as the topography of two enzymes central to cellular cholesterol homeostasis (i.e. sterol regulatory element-binding protein cleavage-activating protein and 3-hydroxy-3-methylglutaryl CoA reductase) features a series of five closely aligned transmembrane domains that together constitute a conserved sterol-sensing domain (35,36).…”

Section: Discussionmentioning

confidence: 99%

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Shields¹,

Lehner²,

Agellon³

et al. 2003

Journal of Biological Chemistry

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In liver, phosphatidylethanolamine is converted to phosphatidylcholine through a series of three sequential methylation reactions. Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes each transmethylation reaction, and S-adenosylmethionine is the methyl group donor. Biochemical analysis of human liver revealed that the methyltransferase activity is primarily localized to the endoplasmic reticulum and mitochondria-associated membranes. Bioinformatic analysis of the predicted amino acid sequence suggested that the enzyme adopts a polytopic conformation in those membranes. To elucidate the precise membrane topography of PEMT and thereby provide the basis for in-depth functional characterization of the enzyme, we performed endoproteinase-protection analysis of epitopetagged, recombinant protein. Our data suggest a topographical model of PEMT in which four transmembrane regions span the membrane such that both the N and C termini of the enzyme are localized external to the ER. Two hydrophilic connecting loops protrude into the luminal space of the microsomes whereas a corresponding loop on the cytosolic side remains proximate to the membrane. Further support for this model was obtained following endoproteinase-protection analysis of mutant recombinant PEMT derivatives in which specific protease cleavage sites had been genetically engineered or ablated.

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Section: Discussionmentioning

confidence: 99%

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Shields¹,

Lehner²,

Agellon³

et al. 2003

Journal of Biological Chemistry

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“…The block in SREBP export is achieved through sterolinduced binding of SCAP to Insigs (1,2,9). The Insig-binding site in SCAP is located within its membrane domain, which comprises eight membrane-spanning segments (10). A segment corresponding to helices 2-6 of the SCAP membrane domain demonstrates significant sequence similarities to the corresponding region in reductase, whose membrane domain also contains eight membrane-spanning segments (11), and this region has become known as the sterol-sensing domain (12)(13)(14).…”

mentioning

confidence: 99%

Isolation of Mutant Cells Lacking Insig-1 through Selection with SR-12813, an Agent That Stimulates Degradation of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase

Sever

Lee

Song

et al. 2004

Journal of Biological Chemistry

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“…1A shows a diagram of the membrane domain of hamster Scap (4). It is comprised of eight transmembrane (TM) segments connected by three loops that are less than 15 amino acids in size and four larger loops-loops 1, 4, 6, and 7.…”

mentioning

confidence: 99%

“…Scap is a polytopic membrane protein with eight transmembrane helices that is synthesized on endoplasmic reticulum (ER) membranes (4). The carboxyl-terminal domain of Scap projects into the cytosol, where it binds to the cytosolic regulatory domain of SREBPs (5).…”

mentioning

confidence: 99%

Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis

Gao¹,

Zhou²,

Goldstein

et al. 2017

Journal of Biological Chemistry

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Edited by George M. CarmanScap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases cholesterol synthesis. Previously, we provided evidence that cholesterol inhibition is initiated when cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of cholesterol synthesis. These insights suggest a new structural model for cholesterol-mediated regulation of Scap activity.A membrane protein called Scap controls cholesterol homeostasis in animal cells. Scap facilitates the proteolytic activation of sterol regulatory element-binding proteins (SREBPs), 6 transcription factors that activate the genes encoding all of the enzymes required for synthesis of cholesterol and unsaturated fatty acids and their uptake from circulating plasma LDL (2). Scap is also the cholesterol sensor that turns off SREBP processing when cellular cholesterol levels are too high. Our laboratory has made recent progress in understanding the mechanism for this cholesterol regulation through a delineation of the conformational changes cholesterol produces when it binds to Scap (1, 3).Scap is a polytopic membrane protein with eight transmembrane helices that is synthesized on endoplasmic reticulum (ER) membranes (4). The carboxyl-terminal domain of Scap projects into the cytosol, where it binds to the cytosolic regulatory domain of SREBPs (5). Like Scap, SREBPs are synthesized on ER-bound ribosomes. Immediately after their synthesis, SREBPs bind to Scap. When ER cholesterol levels are low, Scap serves as the binding site for coat protein complex II (COPII) proteins that incorporate the Scap/SREBP complex into COPIIcoated vesicles that move to the Golgi (6). There the SREBPs are processed proteolytically to release their active transcription ...

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Topology of SREBP Cleavage-activating Protein, a Polytopic Membrane Protein with a Sterol-sensing Domain

Cited by 177 publications

References 36 publications

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Isolation of Mutant Cells Lacking Insig-1 through Selection with SR-12813, an Agent That Stimulates Degradation of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase

Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis

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