Steroidogenic Activity of StAR Requires Contact with Mitochondrial VDAC1 and Phosphate Carrier Protein

Mahuya Bose,Randy M Whittal,Walter L Miller,Himangshu S Bose

doi:10.1074/jbc.m709221200

Abstract

The steroidogenic acute regulatory protein (StAR) is required for adrenal and gonadal steroidogenesis and for male sexual differentiation. StAR acts on the outer mitochondrial membrane (OMM) to facilitate movement of cholesterol from the OMM to the inner mitochondrial membrane to be converted to pregnenolone, the precursor of all steroid hormones. The mechanisms of the action of StAR remain unclear; the peripheral benzodiazepine receptor, an OMM protein, appears to be involved, but the identity of OMM proteins that interact with StAR remain unknown. Here we demonstrate that phosphorylated StAR interacts with voltage-dependent anion channel 1 (VDAC1) on the OMM, which then facilitates processing of the 37-kDa phospho-StAR to the 32-kDa intermediate. In the absence of VDAC1, phospho-StAR is degraded by cysteine proteases prior to mitochondrial import. Phosphorylation of StAR by protein kinase A requires phosphate carrier protein on the OMM, which appears to interact with StAR before it interacts with VDAC1. VDAC1 and phosphate carrier protein are the first OMM proteins shown to contact StAR.

Highlights

Ation of Ser195 [4], processed to a 32-kDa intermediate, imported into mitochondria, and cleaved to a 30-kDa protein [5]
Identification of voltage-dependent anion channel 1 (VDAC1)—steroidogenic acute regulatory protein (StAR) activity is maximal when it is affixed to the outer mitochondrial membrane (OMM) [3], suggesting that StAR interacts with OMM proteins. [35S]StAR synthesized in a cell-free system was chemically cross-linked to OMM proteins on freshly prepared sheep adrenal mitochondria using disuccinimidyl suberate
The proposal that StAR acts through an OMM receptor was challenged by data showing it had activity with mitochondria subjected to proteolysis [34]

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—Antibodies against VDAC1, PCP, glyceraldehyde-3-phosphate dehydrogenase, PBR and COX IV were from Chemicon, Sigma, Calbiochem, and Santa Cruz Biotechnology, respectively. Bioactivity in Isolated Mitochondria—Mitochondria (2 ␮g of protein) from control or VDAC1 knockdown MA-10 cells or from adrenal tissues were resuspended in a final volume of 100 ␮l of bioassay buffer (125 mM sucrose, 80 mM KCl, 5 mM MgCl2, 10 mM NaH2PO4, 10 mM isocitrate, 25 mM HEPES, 0.1 mM ATP, and 10 mg/ml cholesterol, pH 7.4), incubated with full-length, cell-free synthesized or biosynthetic StAR under the conditions. Antiserum (2 ml) was incubated with the column on a rocker for 30 min, the unbound antibodies were removed, and the column was washed three times with phosphate-buffered saline containing 100 mM NaCl and eluted with 100 mM glycine at pH 3.0, which was immediately titrated by addition of 65 ␮l of 1.0 M Tris, pH 9.5. Cellular ATP content was determined using a kit according to the manufacturer’s instructions (ENLITEN ATP Assay system, Promega)

RESULTS

LTLSALLDGK VTQSNFAVGYKa LTFDSSFSPNTGKa

DISCUSSION