title: Steroidogenic acute regulatory protein (StAR) acts on the outside of mitochondria to stimulate steroidogenesis.
abstract: Steroidogenic acute regulatory protein (StAR) facilitates delivery of cholesterol to the inner mitochondrial membranes. StAR is imported into mitochondria and processed to a mature form by cleavage of the N-terminal mitochondrial targeting sequence. We produced His-tagged (His-tag StAR) constructs lacking the N-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected 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 StAR protein to mitochondrial membranes. We established an assay system using mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in E. coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent 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 steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import.
authors.affiliation: Center for Research on Reproduction and Women's Health and the Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
authors.forename: F
authors.initials: F
authors.lastname: Arakane
authors.forename: C B
authors.initials: CB
authors.lastname: Kallen
authors.forename: H
authors.initials: H
authors.lastname: Watari
authors.forename: S E
authors.initials: SE
authors.lastname: Stayrook
authors.forename: M
authors.initials: M
authors.lastname: Lewis
authors.forename: J F
authors.initials: JF
authors.lastname: Strauss
mesh_terms: D000818:Animals; D019556:COS Cells; D002417:Cattle; D003338:Corpus Luteum / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects*; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism / Q000494:pharmacology*; D011284:Pregnenolone / Q000096:biosynthesis*; D011994:Recombinant Proteins; D016324:Sequence Tagged Sites; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D010750:Phosphoproteins; D011994:Recombinant Proteins; D000098682:Steroidogenic Acute Regulatory Protein; D011284:Pregnenolone