Protein-Protein Interactions Mediate Mitochondrial Cholesterol Transport and Steroid Biosynthesis

• Published in: The Journal of biological chemistry 2006-12, Vol.281 (50), p.38879-38893
• Main Authors: Liu, Jun, Rone, Malena B., Papadopoulos, Vassilios
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological Transport; Cell Line; Cholesterol - metabolism; DNA; Microscopy, Confocal; Mitochondria - metabolism; Molecular Sequence Data; Protein Binding; Steroids - biosynthesis; Subcellular Fractions - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M608820200

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 RIα of the cAMP-dependent protein kinase (PKARIα); 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, PKARIα, 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-5α-cholestan-3β-ol in the samples. Co-expression of Tspo, Pap7, PkarIα, 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 PKARIα-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.