Steroidogenic acute regulatory protein (StAR) acts on the outside of mitochondria to stimulate steroidogenesis.
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.
Arakane
F
F
Center for Research on Reproduction and Women's Health and the Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
Kallen
C B
CB
Watari
H
H
Stayrook
S E
SE
Lewis
M
M
Strauss
J F
JF
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
D010750:Phosphoproteins; D011994:Recombinant Proteins; D000098682:Steroidogenic Acute Regulatory Protein; D011284:Pregnenolone