Steroidogenic acute regulatory protein-binding protein cloned by a yeast two-hybrid system.
Steroidogenic acute regulatory (StAR) protein plays a key role in the transport of cholesterol from the outer mitochondrial membrane to the inner membrane. A StAR mutant protein lacking the first 62 amino acids (N-62 StAR protein) has been reported to be as effective as wild-type StAR protein. In the present study, we examined the mechanism by which StAR protein stimulates steroidogenesis. A Gal4-based yeast two-hybrid system was used to identify proteins interacting with N-62 StAR protein. Nine positive clones were obtained from screening 1 x 106 clones. The results of pull-down assays and mammalian two-hybrid assays confirmed interaction between N-62 StAR protein and the clone 4 translated product. The clone 4 translated product was named StAR-binding protein (SBP). We prepared an expression plasmid (pSBP) by inserting SBP cDNA into the pTarget vector. After cotransfection with the human cytochrome P450scc system, StAR expression vector, and pSBP, the amount of pregnenolone produced by COS-1 cells was increased. The amount of steroid hormones produced by steroidogenic cells subjected to small interfering RNA treatment was less than that produced by control cells. In conclusion, SBP binds StAR protein in cells and enhances the ability of StAR protein to promote syntheses of steroid hormones.
Sugawara
Teruo
T
Department of Biochemistry, Hokkaido University Graduate School of Medicine, Kita-ku, Kita 15, Nishi 7, Sapporo 060-8638, Japan. terusuga@med.hokudai.ac.jp
Shimizu
Hiroshi
H
Hoshi
Nobuhiko
N
Nakajima
Ayako
A
Fujimoto
Seiichiro
S
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011284:Pregnenolone / Q000032:analysis / Q000096:biosynthesis*; D011485:Protein Binding; D013256:Steroids / Q000032:analysis / Q000096:biosynthesis; D013737:Testis; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000098682:Steroidogenic Acute Regulatory Protein
D002352:Carrier Proteins; D018076:DNA, Complementary; D010750:Phosphoproteins; D013256:Steroids; D000098682:Steroidogenic Acute Regulatory Protein; D011284:Pregnenolone; D002786:Cholesterol Side-Chain Cleavage Enzyme