The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR)

Abstract

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 processed to a mature form by cleavage of the NH 2 -terminal mitochondrial targeting sequence. To explore the mechanism of StAR action, we produced 6-histidinetagged N-62 StAR (His-tag StAR) constructs lacking the NH 2 -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.

Metastatic lymph node 64 (MLN64), a gene overexpressed in breast cancers, is regulated by Sp/KLF transcription factors

“…Moreover, the region conserved between StAR and MLN64, the StAR-related transfer (START) domain was shown to be a cholesterol-binding domain for both proteins (Tsujishita and Hurley, 2000). While StAR acts as a sterol-carrier directly on the mitochondria (King et al, 1995;Arakane et al, 1998), MLN64 is involved in the cholesterol transport from endosome to cytoplasmic acceptor(s) (Alpy et al, 2001).…”

Targeted Mutation of the MLN64 START Domain Causes Only Modest Alterations in Cellular Sterol Metabolism

“…Recent findings indicate that a family of proteins related to the steroidogenic acute regulatory protein (StAR) 1 perform critical functions in moving lipids within cells (1)(2)(3)(4). StAR, the prototype of the family, promotes the translocation of cholesterol from the outer to the inner mitochondrial membrane and cholesterol side-chain cleavage enzyme in steroidogenic cells (1,5,6). Mutations in the StAR gene cause congenital lipoid adrenal hyperplasia, a cholesterol storage disorder in which synthesis of all gonadal and adrenal cortical steroid hormones is severely impaired; the cholesterol that is not efficiently moved into the mitochondria accumulates in cytoplasmic lipid droplets (7,8).…”

“…It consists of a 210-amino acid residue sequence that forms a compact ␣/␤ structure, a helix-grip fold, with a hydrophobic tunnel that can accommodate a sterol molecule (2, 10 -12). START domains can bind sterol, facilitate the transfer of cholesterol from sterol-rich unilammelar liposomes to acceptor membranes, and stimulate steroidogenesis when expressed in cells co-expressing the cholesterol side-chain cleavage system or when added to isolated steroidogenic mitochondria (5,6,10).…”

ACTH-induced Nucleocytoplasmic Translocation of Salt-inducible Kinase

“…The activated PKA then promptly stimulates the biosynthesis and secretion of steroid hormone. This is achieved through stimulation of several intracellular events; PKA activates cholesterol esterases (4 -7), thus increasing the intracellular cholesterol pool, and elevates the levels of transcription (8,9), mRNA stability (10), translation (8), and posttranslational modification (11)(12)(13)(14)(15) of the steroidogenic acute regulatory (StAR) protein (16). This accelerates the transport of cholesterol from the outer to inner mitochondrial membranes, which activates the side chain cleavage cytochrome P450 (CYP11A) reaction (17)(18)(19)(20)(21).…”