The enzyme 3β-hydroxysteroid dehydrogenase type 2 (3βHSD2) catalyzes the conversion of the prohormone pregnenolone to progesterone, a key intermediate in the biosynthetic pathways for mineralocorticoids, glucocorticoids, androgens and estrogens—steroids involved in salt and water reabsorption by the kidneys, stress responses, immune system regulation, development of male and female secondary sex characteristics and other important biological functions. In this article
, Pawlak and his colleagues test their hypothesis that interaction of 3βHSD2 with inner or outer mitochondrial membrane translocases (Tims and Toms, respectively) is required for enzyme activity. They do so by identifying the translocases that interact with 3βHSD2 and examining the effects of reduced translocase levels on 3βHSD2 enzyme activity in cultured cells.
Key to their research was a mitochondrial import assay that used intact mitochondria and 35S-labeled 3βHSD2, which was expressed in a TnT® rabbit reticulocyte lysate system (Promega). The radiolabeled 3βHSD2 substrate allowed the authors to characterize the relationship between 3βHSD2 and the inner mitochondrial membrane (IMM) and detect protein complexes that contained 3βHSD2.
The authors first used the import assay with radiolabeled 3βHSD2 as the substrate, then extracted the imported proteins with Na2CO3, which disrupts protein:protein interactions but not lipid:protein interactions, to show that 3βHSD2 was associated with but not integrated into the IMM. The researchers then performed additional import assays with 35S-labeled 3βHSD2, lysed the mitochondria with digitonin and analyzed the resulting lysates by native gel electrophoresis to reveal three high-molecular-weight 3βHSD2-containing protein complexes. Through Western blot and co-immunoprecipitation analyses of these complexes using antibodies that recognize 3βHSD2, Tom22, Tim50 and Tim23, they determined that 3βHSD2 interacts with these three translocases and participates in a fluid and transient protein network that connects the inner and outer mitochondrial membranes.
To determine which, if any, of these interactions is required for 3βHSD2 enzyme activity, the authors used short interfering RNAs (siRNAs) to knockdown Tim50, Tom22 and Tim23 expression, which resulted in 82%, 63% and 80% reductions in 3βHSD2-mediated conversion of pregnenolone to progesterone, respectively. To identify amino acids necessary for these interactions, they also used the TnT® rabbit reticulocyte lysate system to express deletion mutants of 3βHSD2, which were used as substrates in the mitochondrial import assay. They identified fifteen N-terminal amino acids that were required for association with the translocases and full enzyme activity. Interestingly, these were not the same amino acids required for import into the mitochondria.
Finally, for 3βHSD2 to interact with all three translocases, the protein must be flexible. To examine flexibility, the authors performed protein fingerprinting using Sequencing Grade Modified Trypsin (Promega) to show that there were no protease-resistant domains, indicating that 3βHSD2 is a flexible protein. All of these data support the authors’ hypothesis that 3βHSD2 interacts with three mitochondrial translocases and that these interactions are necessary for normal steroidogenesis, which is essential for survival in all animals.