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Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center (X.W., Z.L., D.M.S.), Lubbock, Texas 79430; the Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem (S.E., R.T., A.M.W., J.O.), Jerusalem 91904, Israel
Address all correspondence and requests for reprints to: Dr. Douglas M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center, Lubbock, Texas 79430.
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 [3H]cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [3H]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 [3H]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 [3H]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.
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L. P. Walsh and D. M. Stocco Effects of Lindane on Steroidogenesis and Steroidogenic Acute Regulatory Protein Expression Biol Reprod, April 1, 2000; 63(4): 1024 - 1033. [Abstract] [Full Text]
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J.-G. Lehoux, D. B. Hales, A. Fleury, N. Brière, D. Martel, and L. Ducharme The in Vivo Effects of Adrenocorticotropin and Sodium Restriction on the Formation of Different Species of Steroidogenic Acute Regulatory Protein in Rat Adrenal Endocrinology, November 1, 1999; 140(11): 5154 - 5164. [Abstract] [Full Text]
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J. Arensburg, A. H. Payne, and J. Orly Expression of Steroidogenic Genes in Maternal and Extraembryonic Cells During Early Pregnancy in Mice Endocrinology, November 1, 1999; 140(11): 5220 - 5232. [Abstract] [Full Text]
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E. Silverman, S. Eimerl, and J. Orly CCAAT Enhancer-binding Protein beta and GATA-4 Binding Regions within the Promoter of the Steroidogenic Acute Regulatory Protein (StAR) Gene Are Required for Transcription in Rat Ovarian Cells J. Biol. Chem., June 18, 1999; 274(25): 17987 - 17996. [Abstract] [Full Text] [PDF]
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M. E. Reyland, R. M. Evans, and E. K. White Lipoproteins Regulate Expression of the Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenocortical Cells J. Biol. Chem., November 17, 2000; 275(47): 36637 - 36644. [Abstract] [Full Text] [PDF]
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