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J Biol Chem, Vol. 273, Issue 26, 16339-16345, June 26, 1998
From the Steroidogenic acute regulatory protein (StAR)
plays an essential role in steroidogenesis, facilitating delivery of
cholesterol to cytochrome P450scc on the inner
mitochondrial membrane. StAR is synthesized in the cytoplasm and is
subsequently imported by mitochondria and processed to a mature form by
cleavage of the NH2-terminal mitochondrial targeting
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 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. In vitro import assays
demonstrated that wild-type StAR preprotein was imported and processed
to mature protein that was protected from subsequent trypsin treatment.
In contrast, His-tag StAR was not imported and protein associated with
mitochondria was sensitive to trypsin. Using metabolically labeled
COS-1 cells transfected with wild-type or His-tag StAR constructs, we
confirmed that wild-type StAR preprotein was imported and
processed by mitochondria, whereas His-tag StAR remained largely
cytosolic and unprocessed. To determine whether cytosolic factors are
required for StAR action, we developed an assay system using washed
mitochondria isolated from bovine corpora lutea and purified
recombinant His-tag StAR proteins expressed in Escherichia
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, and in the absence of cytosol. The ability to produce bioactive
recombinant StAR protein paves the way for refined structural studies
of StAR and StAR mutants.
The Mechanism of Action of Steroidogenic Acute Regulatory
Protein (StAR)
StAR ACTS ON THE OUTSIDE OF MITOCHONDRIA TO STIMULATE
STEROIDOGENESIS
,
,
,
,
, and
§
Center for Research on Reproduction and
Women's Health and the Department of Obstetrics and Gynecology,
§ Physiology, and ¶ Biochemistry and Biophysics,
University of Pennsylvania Medical Center, Philadelphia,
Pennsylvania 19104
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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