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J. Biol. Chem., Vol. 281, Issue 50, 38879-38893, December 15, 2006
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Protein-Protein Interactions Mediate Mitochondrial Cholesterol Transport and Steroid Biosynthesis*
From the Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, D. C. 20057
Transport of cholesterol into the mitochondria is the rate-determining, hormone-sensitive step in steroid biosynthesis. Here we report that the mechanism underlying mitochondrial cholesterol transport involves the formation of a macromolecular signaling complex composed of the outer mitochondrial membrane translocator protein (TSPO), previously known as peripheral-type benzodiazepine receptor; the TSPO-associated protein PAP7, which binds and brings to mitochondria the regulatory subunit RI
of the cAMP-dependent protein kinase (PKARI); and the hormone-induced PKA substrate, steroidogenic acute regulatory protein (StAR). Hormone treatment of MA-10 Leydig cells induced the co-localization of TSPO, PAP7, PKARI, and StAR in mitochondria, visualized by confocal microscopy, and the formation in living cells of a high molecular weight multimeric complex identified using photoactivable amino acids. The hormone-induced recruitment of exogenous TSPO in this complex was found to parallel the increased presence of 7-azi-5-cholestan-3
-ol in the samples. Co-expression of Tspo, Pap7, PkarI, and Star genes resulted in the stimulation of steroid formation in both steroidogenic MA-10 and non-steroidogenic COS-F2-130 cells that were engineered to metabolize cholesterol. Disruption of these protein-protein interactions and specifically the PKARI-PAP7 and PAP7-TSPO interactions, using PAP7 mutants where the N0 area homologous to dual A-kinase-anchoring protein-1 or the acyl-CoA signature motif were deleted or using the peptide Ht31 known to disrupt the anchoring of PKA, inhibited both basal and hormone-induced steroidogenesis. These results suggest that the initiation of cAMP-induced protein-protein interactions results in the formation of a multivalent scaffold in the outer mitochondrial membrane that mediates the effect of hormones on mitochondrial cholesterol transport and steroidogenesis.
Received for publication, September 12, 2006 , and in revised form, October 16, 2006.
* This work was supported by NICHD, National Institutes of Health Grant HD37031. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, 3900 Reservoir Rd., N. W., Washington, D. C. 20057. Tel.: 202-687-8991; Fax: 202-687-7855; E-mail: papadopv{at}georgetown.edu.
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