EFFECT OF TRUNCATED FORMS OF THE STEROIDOGENIC ACUTE REGULATORY PROTEIN ON INTRAMITOCHONDRIAL CHOLESTEROL TRANSFER

It has been proposed that the steroidogenic acute regulatory (StAR) pr otein controls hormone-stimulated steroid production by mediating chol esterol transfer to the mitochondrial inner membrane. This study was c onducted to determine the effect of wild-type StAR and several modifie d forms of StAR on intramitochondrial cholesterol transfer. Forty-seve n N-terminal or 28 C-terminal amino acids of the StAR protein were rem oved, and COS-1 cells were transfected with pCMV vector only, wild-typ e StAR, N-47, or the C-28 constructs. Lysates from the transfected COS -1 cells were then incubated with mitochondria from MA-10 mouse Leydig tumor cells that were preloaded with [H-3]cholesterol. After incubati on, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [H-3]cholesterol content was determined in each membrane fraction. In cubation of MA-10 mitochondria with wild-type StAR containing cell lys ate resulted in a significant 34.9% increase in [H-3]cholesterol conte nt in contact sites and a significant 32.8% increase in inner mitochon drial membranes. Incubations with cell lysate containing N-47 StAR pro tein also resulted in a 16.4% increase in [H-3]cholesterol in contact sites and a significant 26.1% increase in the inner membrane fraction. In contrast, incubation with the C-28 StAR protein had no effect on c holesterol transfer. The cholesterol-transferring activity of the N-47 truncation, in contrast to that of the C-28 mutant, was corroborated when COS-1 cells were cotransfected with F2 vector (containing cytochr ome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin reduct ase) and either pCMV empty vector or the complementary DNAs of wild-ty pe StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was signific antly increased in both wild-type and N-47-transfected cells, whereas that in C-28-transfected cells was similar to the control value. Final ly, immunolocalization studies with confocal image and electron micros copy were performed to determine the cellular location of StAR and its truncated forms in transfected COS-1 cells. The results showed that w ild-type and most of the C-28 StAR protein were imported into the mito chondria, whereas most of N-47 protein remained in the cytosol. These studies demonstrate a direct effect of StAR protein on cholesterol tra nsfer to the inner mitochondrial membrane, that StAR need not enter th e mitochondria to produce this transfer, and the importance of the C-t erminus of StAR in this process.