Effect of Truncated Forms of the Steroidogenic Acute Regulatory Protein on Intramitochondrial Cholesterol Transfer

Abstract

It has been proposed that the steroidogenic acute regulatory (StAR) protein controls hormone-stimulated steroid production by mediating cholesterol transfer to the mitochondrial inner membrane. This study was conducted to determine the effect of wild-type StAR and several modified forms of StAR on intramitochondrial cholesterol transfer. Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein were removed, and COS-1 cells were transfected with pCMV vector only, wild-type 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 [ 3 H]cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [ 3 H]cholesterol content was determined in each membrane fraction. Incubation of MA-10 mitochondria with wild-type StAR containing cell lysate resulted in a significant 34.9% increase in [ 3 H]cholesterol content in contact sites and a significant 32.8% increase in inner mitochondrial membranes. Incubations with cell lysate containing N-47 StAR protein also resulted in a 16.4% increase in [ 3 H]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 cholesterol 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 cytochrome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin reductase) and either pCMV empty vector or the complementary DNAs of wild-type StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was significantly increased in both wild-type and N-47-transfected cells, whereas that in C-28-transfected cells was similar to the control value. Finally, immunolocalization studies with confocal image and electron microscopy were performed to determine the cellular location of StAR and its truncated forms in transfected COS-1 cells. The results showed that wild-type and most of the C-28 StAR protein were imported into the mitochondria, whereas most of N-47 protein remained in the cytosol. These studies demonstrate a direct effect of StAR protein on cholesterol transfer to the inner mitochondrial membrane, that StAR need not enter the mitochondria to produce this transfer, and the importance of the C-terminus of StAR in this process. (Endocrinology 139: [3903][3904][3905][3906][3907][3908][3909][3910][3911][3912] 1998)

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].…”

Dimethoate inhibits steroidogenesis by disrupting transcription of the steroidogenic acute regulatory (StAR) gene

“…Mitochondria were isolated by homogenization and differential centrifugation (Clark et al 1994). Then, Western blot analysis of mitochondrial protein was performed as previously described (Wang et al 1998). Following detection of StAR, the membrane was stripped in 62·5 mM Tris-HCl (pH 6·8), 2% SDS, and 100 mM -mercaptoethanol at 70 C for 30 min, washed in 10 mM Tris-HCl (pH 7·4) and 150 mM NaCl twice for 10 min, and then successively probed with P450 scc or 3 -HSD antisera.…”