Steroidogenic acute regulatory protein (StAR) retains activity in the absence of its mitochondrial import sequence: Implications for the mechanism of StAR action

Abstract

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone biosynthesis, presumably by facilitating the delivery of cholesterol to P450scc in the inner mitochondrial membranes. StAR is synthesized as a 37-kDa preprotein that is processed to a 30-kDa mature form by cleavage of an N-terminal mitochondrial import sequence. To identify structural features required for StAR biological activity, we mutated the human StAR cDNA, including the deletion of N-and C-terminal sequences, and examined the ability of the mutants to promote steroidogenesis and enter the mitochondria of transfected COS-1 cells. Deletion of up to 62 residues from the N terminus (N-62) did not significantly affect steroidogenesis-enhancing activity. The N-terminal deletion mutants were associated with mitochondria-enriched fractions, but import and processing were progressively impaired with increasing length of the deletion. Immunogold electron microscopy and in vitro import assays showed that the active N-62 mutant was not imported into the mitochondria. Removal of the 28 C-terminal amino acids (C-28) inactivated StAR. Deletion of the Cterminal 10 amino acids (C-10) reduced steroidogenic activity by 53%, while truncation of the last 4 amino acids had no effect. The C-28 mutant StAR was not efficiently imported into mitochondria or processed, whereas some of the C-10 mutant was processed, indicating that import had occurred. We conclude that in the COS-1 cell system used, StAR does not need to enter into mitochondria to stimulate steroidogenesis and that residues in the C terminus are essential for steroidogenesis-enhancing activity. These findings imply that StAR acts via C-terminal domains on the outside of the mitochondria.

Isolation, Characterization, and cDNA Sequence of a Carotenoid Binding Protein from the Silk Gland of Bombyx mori Larvae

“…7, boldfaced sequences). This domain plays a role in binding cholesterol to StAR protein (28,29). START domain is not unique to cholesterol transport proteins, but it occurs in proteins involved in lipid metabolism, signal transduction, and transcriptional regulation (30).…”

Impact of Aging on Steroid Hormone Biosynthesis and Secretion

“…StAR is synthesized as a short-lived cytoplasmic 37-kDa protein with a mitochondrial leader peptide that is cleaved upon mitochondrial import to yield the long-lived intramitochondrial 30-kDa form [58]. It is now wellestablished that StAR functions as a sterol transfer protein [70], binds cholesterol [71,72], mediates the acute steroidogenic response by moving cholesterol from the outer to the inner mitochondrial membrane [73], acts on the outer mitochondrial membrane [73][74][75], and requires the structural change previously described as a pH-dependent molten globule [76]. StAR is also a prototype of a family of proteins that contain START (StAR-related lipid transfer) domains (StarD proteins) [77], of which StarD3/MLN64, StarD4, StarD5 and StarD6 exhibit steroidogenic potential [78,79].…”

A pH-dependent Molten Globule Transition Is Required for Activity of the Steroidogenic Acute Regulatory Protein, StAR

“…3B). The carboxylterminal residue of StAR, Cys-285, was changed to Phe to reduce reactivity; deletion of Cys-285 has no effect on StAR's activity (19). Each mutant migrated more rapidly than the wild type, but when reduced with DTT, each had the same migration as the wild type, suggesting that disulfide bonds had been formed.…”