STEROIDOGENIC ACUTE REGULATORY PROTEIN (STAR) RETAINS ACTIVITY IN THEABSENCE OF ITS MITOCHONDRIAL IMPORT SEQUENCE - IMPLICATIONS FOR THE MECHANISM OF STAR ACTION

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 ma ture 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-te rminal sequences, and examined the ability of the mutants to promote s teroidogenesis and enter the mitochondria of transfected COS-1 cells, Deletion of up to 62 residues from the N terminus (N-62) did not signi ficantly affect steroidogenesis-enhancing activity, The N-terminal del etion mutants were associated with mitochondria-enriched fractions, bu t import and processing were progressively impaired with increasing le ngth of the deletion, Immunogold electron microscopy and in vitro impo rt assays showed that the active N-62 mutant was not imported into the mitochondria, Removal of the 28 C-terminal amino acids (C-28) inactiv ated StAR Deletion of the C-terminal 10 amino acids (C-10) reduced ste roidogenic activity by 53%, while truncation of the last 4 amino acids had no effect, The C-28 mutant StAR,vas not efficiently imported into mitochondria or processed, whereas some of the C-10 mutant was proces sed, indicating that import had occurred, We conclude that in the COS- 1 cell system used, StAR does not need to enter into mitochondria to s timulate steroidogenesis and that residues in the C terminus are essen tial for steroidogenesis-enhancing activity. These findings imply that StAR acts via C-terminal domains on the outside of the mitochondria.