Abstract
Scap 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.
Highlights
Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation
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)
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)
Summary
The experiments described in this paper were made possible by the recent production of a monoclonal antibody directed against luminal loop 1 of Scap, hereafter designated anti-loop 1 (Fig. 1A). At the highest added amount of proteinase K (30 g), densitometry analysis of the immunoblot (Fig. 2B, lanes 11 and 12) showed that band L4 decreased by 96%, whereas band L6 increased by 54% These experiments suggest that cholesterol causes a conformational change in Scap that sequesters a cytoplasmically disposed arginine or lysine, leading to a reduction in band L4. We transfected Scap-deficient SRD-13A cells with a plasmid encoding wild-type Scap, treated sealed membrane vesicles with trypsin, performed SDSPAGE, and blotted with anti-loop 1 (top panel) or anti-loop 7 (bottom panel). The result with loop 6 is entirely different Both mutant proteins show cleavage at Arg-505 even in the absence of cholesterol, and there is no further increase when cholesterol is present (Fig. 7, bottom panel, lanes [3,4,5,6])
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