Steroidogenic acute regulatory protein (StAR) plays a key role in steroid hormone synthesis by enhancing the metabolism of cholesterol into pregnenolone. We determined the organization of the StAR structural gene, mapped to 8pl 1.2. The genespans 8 kb and consists of seven exons interrupted by six introns. The 1.3 kb of DNA upstream from the transcription start site directed expression of a luciferase reporter gene in mouse Yl adrenal cortical tumor cells but not in BeWo choriocarcinoma cells. Reporter gene expression in the Yl cells was increased more than 2-fold by 8-Br-cAMP, indicating that the 1.3 kb DNA fragment contains sequences that confer tissue-specific expressionand cAMP regulation. The sequence of a related StAR pseudogene, mapped to chromosome 13, lacks introns and has an insertion, numerous substitutions, and deletions. Expression of StAR in COS-1 cells cotransfected with cholesterol 27-hydroxylase (P450c27) and adrenodoxin resulted in a 6-fold increase in formation of 3/3-hydroxy-5-cholestenoic acid, demonstrating that StAR’s actions are not specific to steroidogenesis but extendto other mitochondrial cholesterol-metabolizing enzymes.

The rate-limiting step in steroid hormone synthesis is the formation of pregnenolone from cholesterol, catalyzed by the cholesterol side-chain cleavage enzyme (P450scc), which resides with its associated electron transport chain in the inner mitochondrial membranes [for review see Miller (1988)]. This first committed reaction in the biosynthesis of steroid hormones is acutely stimulated by tropic hormones (ACTH in the adrenal cortex; LH in the gonads) acting through the intermediacy of cAMP. It has been known for two decades that the acute steroidogenic response to tropic stimulation involves the translocation of cholesterol from theouter to inner mitochondrial membranes. This translocation process is believed to be mediated by a short-lived, cycloheximidesensitive protein (Ferguson, 1963; Garrenet al., 1968;