title: Steroidogenic acute regulatory protein binds cholesterol and modulates mitochondrial membrane sterol domain dynamics.
abstract: The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step of steroidogenesis, delivery of cholesterol to the inner mitochondrial membrane. However, the mechanism whereby cholesterol translocation is accomplished has not been resolved. Recombinant StAR proteins lacking the first N-terminal 62 amino acids comprising the mitochondrial-targeting sequence were used to determine if StAR binds cholesterol and alters mitochondrial membrane cholesterol domains to enhance sterol transfer. First, a fluorescent NBD-cholesterol binding assay revealed 2 sterol binding sites (K(d) values near 32 nm), whereas the inactive A218V N-62 StAR mutant had only a single binding site with 8-fold lower affinity. Second, NBD-cholesterol spectral shifts and fluorescence resonance energy transfer from StAR Trp residues to NBD-cholesterol showed (i) close molecular interaction between these molecules (R(2/3) = 33 A) and (ii) sensitized NBD-cholesterol emission from only one of the two sterol binding sites. Third, circular dichroism showed that cholesterol binding induced a change in StAR secondary structure. Fourth, a fluorescent sterol transfer assay that did not require separation of donor and acceptor mitochondrial membranes demonstrated that StAR enhanced mitochondrial sterol transfer as much as 100-fold and induced/increased the formation of rapidly transferable cholesterol domains in isolated mitochondrial membranes. StAR was 67-fold more effective in transferring cholesterol from mitochondria of steroidogenic MA-10 cells than from human fibroblast mitochondria. In contrast, sterol carrier protein-2 (SCP-2) was only 2.2-fold more effective in mediating sterol transfer from steroidogenic cell mitochondria. Taken together these data showed that StAR is a cholesterol-binding protein, preferentially enhances sterol transfer from steroidogenic cell mitochondria, and interacts with mitochondrial membranes to alter their sterol domain structure and dynamics.
authors.affiliation: Department of Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466 and the Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104.
authors.forename: A D
authors.initials: AD
authors.lastname: Petrescu
authors.forename: A M
authors.initials: AM
authors.lastname: Gallegos
authors.forename: Y
authors.initials: Y
authors.lastname: Okamura
authors.forename: J F
authors.initials: JF
authors.lastname: Strauss
authors.forename: F
authors.initials: F
authors.lastname: Schroeder
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D004735:Energy Transfer; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013261:Sterols / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D014407:Tumor Cells, Cultured; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: C077527:7-nitrobenz-2-oxa-1,3-diazol-4-ylcholesterol; D002352:Carrier Proteins; D010750:Phosphoproteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D013261:Sterols; D000098682:Steroidogenic Acute Regulatory Protein; C072087:sterol carrier proteins; D014364:Tryptophan; D002784:Cholesterol; D009327:4-Chloro-7-nitrobenzofurazan