THE MECHANISM OF ACTION OF STEROIDOGENIC ACUTE REGULATORY PROTEIN (STAR) - STAR ACTS ON THE OUTSIDE OF MITOCHONDRIA TO STIMULATE STEROIDOGENESIS

Steroidogenic acute regulatory protein (StAR) plays an essential role in steroidogenesis, facilitating delivery of cholesterol to cytochrome P450(scc) on the inner mitochondrial membrane. StAR is synthesized in the cytoplasm and is subsequently imported by mitochondria and proces sed to a mature form by cleavage of the NH,terminal mitochondrial targ eting sequence. To explore the mechanism of StAR action, we produced 6 -histidine-tagged N-62 StAR (His-tag StAR) constructs lacking the NH2- terminal 62 amino acids that encode the mitochondrial targeting sequen ce and examined their steroidogenic activity in intact cells and on is olated mitochondria, His-tag StAR proteins stimulated pregnenolone syn thesis to the same extent as wild-type StAR when expressed in COS-1 ce lls transfected with the cholesterol side-chain cleavage system. His-t ag StAR was diffusely distributed in the cytoplasm of transfected COS- 1 cells whereas wild-type StAR was localized to mitochondria, There wa s no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. In v itro import assays demonstrated that wild-type StAR preprotein was imp orted and processed to mature protein that was protected from subseque nt trypsin treatment. In contrast, His-tag StAR was not imported and p rotein associated with mitochondria was sensitive to trypsin, Using me tabolically labeled COS-1 cells transfected with wild-type or His-tag StAR constructs, we confirmed that wild-type StAR preprotein was impor ted and processed by mitochondria, whereas His-tag StAR remained large ly cytosolic and unprocessed To determine whether cytosolic factors ar e required for StAR action, we developed an assay system using washed mitochondria isolated from bovine corpora lutea and purified recombina nt His-tag StAR proteins expressed in Escherichia coli. Recombinant Hi s-tag StAR stimulated pregnenolone production in a dose- and time-depe ndent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenical ly inactive in COS-1 cells and on isolated mitochondria. Our observati ons conclusively document that StAR acts on the outside of mitochondri a, independent of mitochondrial import, and in the absence of cytosol, The ability to produce bioactive recombinant StAR protein paves the w ay for refined structural studies of StAR and StAR mutants.