Binding of steroidogenic acute regulatory protein to synthetic membranes suggests an active molten globule

被引:57
作者
Christensen, K
Bose, HS
Harris, FM
Miller, WL [1 ]
Bell, JD
机构
[1] Brigham Young Univ, Dept Zool, Provo, UT 84602 USA
[2] Univ Calif San Francisco, Metab Res Unit, San Francisco, CA 94143 USA
[3] Univ Calif San Francisco, Dept Pediat, San Francisco, CA 94143 USA
关键词
D O I
10.1074/jbc.M100903200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Steroidogenic acute regulatory protein (StAR) mediates cholesterol transport from the outer to the inner mitochondrial membrane during steroid biosynthesis, The mechanism of StAR's action is not established, To address mechanistic issues, we assessed the binding of StAR to artificial membranes by fluorescence resonance energy transfer using endogenous StAR tryptophan residues as the donor and dansyl-phosphatidylethanolamine in the bilayer as the acceptor. Mixing StAR with dansyl-labeled vesicles composed of phosphatidylcholine increased the fluorescence intensity of dansyl emission excited at 280 nm by 10-40%, This interaction was dependent on pH, with a maximum at pH 3.0-3.5 and essentially no change above pH 5, Binding experiments at different temperatures and various combinations of phosphatidylcholine, phosphatidylglycerol, cardiolipin, and cholesterol showed that binding involves an electrostatic step and one or more other steps. Although binding prefers a thermodynamically ordered bilayer, the rate-limiting step occurs either when the bilayer is in a fluid state or when there is cholesterol-induced membrane heterogeneity, Experiments with fluorescence and light scattering indicate that StAR binding promotes ordering and aggregation of anionic membranes. The inactive StAR mutant R182L had lower affinity for the membrane, and the partially active mutant L275P had intermediate affinity. Far-UV CD spectroscopy of StAR in PC membranes show more p-structure than in aqueous buffers, and the presence of cardiolipin or cholesterol in the membrane fosters a molten globule state. Our data suggest that StAR binds to membranes in a partially unfolded molten globule state that is relevant to the activity of the protein.
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页码:17044 / 17051
页数:8
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