Protein-protein interactions mediate mitochondrial cholesterol transport and steroid biosynthesis.
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 RIalpha of the cAMP-dependent protein kinase (PKARIalpha); 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, PKARIalpha, 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-5alpha-cholestan-3beta-ol in the samples. Co-expression of Tspo, Pap7, PkarIalpha, 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 PKARIalpha-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.
Liu
Jun
J
Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, D. C. 20057, USA.
Rone
Malena B
MB
Papadopoulos
Vassilios
V
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D004247:DNA; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D013256:Steroids / Q000096:biosynthesis*; D013347:Subcellular Fractions / Q000378:metabolism
D013256:Steroids; D004247:DNA; D002784:Cholesterol