Identification of a Dynamic Mitochondrial Protein Complex Driving Cholesterol Import, Trafficking, and Metabolism to Steroid Hormones

Abstract

Steroid hormones are critical for organismal development and health. The rate-limiting step in steroidogenesis is the transport of cholesterol from the outer mitochondrial membrane (OMM) to the cytochrome P450 enzyme CYP11A1 in the inner mitochondrial membrane (IMM). Cholesterol transfer occurs through a complex termed the "transduceosome," in which cytosolic steroidogenic acute regulatory protein interacts with OMM proteins translocator protein and voltage-dependent anion channel (VDAC) to assist with the transfer of cholesterol to OMM. It has been proposed that cholesterol transfer from OMM to IMM occurs at specialized contact sites bridging the two membranes composed of VDAC and IMM adenine nucleotide translocase (ANT). Blue native PAGE of Leydig cell mitochondria identified two protein complexes that were able to bind cholesterol at 66- and 800-kDa. Immunoblot and mass spectrometry analyses revealed that the 800-kDa complex contained the OMM translocator protein (18-kDa) and VDAC along with IMM CYP11A1, ATPase family AAA domain-containing protein 3A (ATAD3A), and optic atrophy type 1 proteins, but not ANT. Knockdown of ATAD3A, but not ANT or optic atrophy type 1, in Leydig cells resulted in a significant decrease in hormone-induced, but not 22R-hydroxycholesterol-supported, steroid production. Using a 22-phenoxazonoxy-5-cholene-3-beta-ol CYP11A1-specific probe, we further demonstrated that the 800-kDa complex offers the microenvironment needed for CYP11A1 activity. Addition of steroidogenic acute regulatory protein to the complex mobilized the cholesterol bound at the 800-kDa complex, leading to increased steroid formation. These results identify a bioactive, multimeric protein complex spanning the OMM and IMM unit that is responsible for the hormone-induced import, segregation, targeting, and metabolism of cholesterol.

Steroidogenesis in MA-10 Mouse Leydig Cells Is Altered via Fatty Acid Import into the Mitochondria1

“…We have previously shown that the primary isoform of VDAC in the mitochondria, VDAC1, is associated with the protein complex that is necessary for cholesterol import in MA-10 cells, and Lee et al demonstrated that VDAC1 is associated with the fatty acid import complex [5,7]. In fatty acid import, VDAC1 is proposed to function in the regulation of the import of activated fatty acids into the inner membrane space.…”

“…Upon the addition of etomoxir (second arrow), we observed a decrease in OCR and an increase in ECAR, as previously seen. The points shown represent the mean 6 SEM of the three experiments, with six higher-molecular-weight complex where TSPO is known to associate with and import cholesterol into the IMM [7]. This observation suggests that VDAC1 might provide the regulatory mechanism through which the import of lipids into the mitochondria could be selected.…”

“…Each BN-PAGE gel was divided into 18 horizontal slices starting at 20 kDa, cut into 3-mm slices, increasing in molecular mass until reaching 1200 kDa, and subjected to in-gel digestion and mass spectrometry, as previously described [7]. Samples identified via mass spectrometry were run through the MitoMiner mitochondrial protein database to identify mitochondrial pathways [17] Normalization of MS data was corrected by calculating the slope of the averaged control versus treated hits, linearity was corrected by multiplying the control hits by 1.32, and the final quantification of total hits was plotted using Excel.…”

“…Proteins separated by BN-PAGE or SDS-PAGE were electrophoretically transferred to a nitrocellulose membrane, as previously described [7]. SDS-PAGE membranes were incubated with primary antibodies against TSPO (1:2500), VDAC (1:2000), PMP70 (1:1000), and PECI (1:1000), and SDS-PAGE and BN-PAGE membranes were probed with ACSL4 (1:500), ACDVL (1:1000), CPT1a (1:1000), and cytochrome c oxidase subunit IV (CoxIV; 1:1000).…”

“…Previous work in our laboratory, utilizing Blue-Native PAGE (BN-PAGE), demonstrated that the import of cholesterol into the mitochondria occurs through an 800-kDa protein complex containing outer mitochondrial membrane (OMM) proteins, including the translocator protein (TSPO; 18 kDa) and the voltage-dependent anion channel (VDAC), along with IMM proteins, including the AAAþ ATPase ATAD3A and the CYP11A1 proteins [7]. The import and metabolism of cholesterol for steroid production has previously been shown to be dependent upon a functioning electron transport chain (ETC) and on intramitochondrial ATP production [8,9].…”