Inner Mitochondrial Translocase Tim50 Interacts with 3β-Hydroxysteroid Dehydrogenase Type 2 to Regulate Adrenal and Gonadal Steroidogenesis

In the adrenals, testes, and ovaries, 3 beta-hydroxysteroid dehydrogenase type 2 (3 beta HSD2) catalyzes the conversion of pregnenolone to progesterone and dehydroepiandrostenedione to androstenedione. Alterations in this pathway can have deleterious effects, including sexual development impairment, spontaneous abortion, and breast cancer. 3 beta HSD2, synthesized in the cytosol, is imported into the inner mitochondrial membrane (IMM) by translocases. Steroidogenesis requires that 3 beta HSD2 acts as both a dehydrogenase and isomerase. To achieve this dual functionality, 3 beta HSD2 must undergo a conformational change; however, what triggers that change remains unknown. We propose that 3 beta HSD2 associates with IMM or outer mitochondrial membrane translocases facing the intermembrane space (IMS) and that this interaction promotes the conformational change needed for full activity. Fractionation assays demonstrate that 3 beta HSD2 associated with the IMM but did not integrate into the membrane. Through mass spectrometry and Western blotting of mitochondrial complexes and density gradient ultracentrifugation, we show that that 3 beta HSD2 formed a transient association with the translocases Tim50 and Tom22 and with Tim23. This association occurred primarily through the interaction of Tim50 with the N terminus of 3 beta HSD2 and contributed to enzymatic activity. Tim50 knockdown inhibited catalysis of dehydroepiandrostenedione to androstenedione and pregnenolone to progesterone. Although Tim50 knock-down decreased 3 beta HSD2 expression, restoration of expression via proteasome and protease inhibition did not rescue activity. In addition, protein fingerprinting and CD spectroscopy reveal the flexibility of 3 beta HSD2, a necessary characteristic for forming multiple associations. In summary, Tim50 regulates 3 beta HSD2 expression and activity, representing a new role for translocases in steroidogenesis.