Steroidogenic acute regulatory protein (StAR) acts on the outside of mitochondria to stimulate steroidogenesis

Steroidogenic acute regulatory protein (StAR) facilitates delivery of chole sterol to the inner mitochondrial membranes. StAR is imported into mitochon dria and processed to a mature form by cleavage of the N-terminal mitochond rial targeting sequence. We produced His-tagged (His-tag StAR) constructs l acking the N-terminal 62 amino acids that encode the mitochondrial targetin g sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synth esis to the same extent as wild-type StAR when expressed in COS-1 cells tra nsfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells, whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag S tAR protein to mitochondrial membranes. We established an assay system usin g mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in E, coli. Recombinant His-tag StAR stimul ated pregnenolone production in a dose- and time-dependent manner, function ing at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag pr otein. This mutant was steroidogenically inactive in COS-1 cells and on iso lated mitochondria. Our observations conclusively document that StAR acts o n the outside of mitochondria, independent of mitochondrial import.