Inner mitochondrial translocase Tim50 is central in adrenal and testicular steroid synthesis.

Adrenal and gonadal mitochondrial metabolic activity requires electrons from cofactors, cholesterol and a substrate for rapid steroid synthesis, an essential requirement for mammalian survival. Substrate activity depends on its environment, which is regulated by chaperones and mitochondrial translocases. Cytochrome P450 side chain cleavage enzyme (Side Chain Cleavage enzyme/SCC or CYP11A1) catalyzes cholesterol to pregnenolone conversion although its mechanism of action is not well understood. We find that SCC is directly imported into the mitochondrial matrix, where its N-terminal sequence is cleaved sequentially, after which it becomes activated following the second cleavage that is dependent on the folding of the protein. Following integration of the SCC C-terminus into the TIM23 complex, amino acids 141-146 interact with the intermembrane-exposed Tim50 protein, forming a large complex. The absence of Tim50 or its mutation reduced enzymatic activity. For the first time, we report that a protein activated at the matrix remains mostly unfolded and is transported back to the IMS to integrate with the TIM23 translocase complex and align with the Tim50 protein. Amino acid changes that suppress the association of Tim50 with SCC ablate metabolic activity. Thus, the TIM23 complex is the central regulator of metabolism guided by Tim50.