Steroidogenic Acute Regulatory Protein-binding Protein Cloned by a Yeast Two-hybrid System

Teruo Sugawara,Hiroshi Shimizu,Nobuhiko Hoshi,Ayako Nakajima,Seiichiro Fujimoto

doi:10.1074/jbc.m302291200

Abstract

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.

Highlights

Steroidogenic acute regulatory (StAR) protein plays a key role in the transport of cholesterol from the outer mitochondrial membrane to the inner membrane
StAR mutant protein lacking the first 62 amino acids (N-62 StAR protein), which contain the mitochondrial targeting sequence, has been reported to be as effective as wild-type StAR protein in stimulating steroidogenesis [23]. This led to the conclusion that the C terminus of StAR protein encodes its biological function for steroidogenesis and that the StAR protein acts on the outer mitochondrial membrane to stimulate cholesterol translocation
We screened a human testis cDNA library to identify proteins that interact with the StAR protein in the cytoplasm or outer mitochondrial membrane

Summary

Introduction

Steroidogenic acute regulatory (StAR) protein plays a key role in the transport of cholesterol from the outer mitochondrial membrane to the inner membrane. Increased progesterone synthesis prior to an increase in the level of StAR mRNA expression in response to cAMP induction has been revealed by analysis of hyperacetylation in the StAR gene promoter [20] This increased steroidogenesis is thought to be the result of phosphorylation of the StAR protein by protein kinase A [8], the mechanism is still not clear. StAR mutant protein lacking the first 62 amino acids (N-62 StAR protein), which contain the mitochondrial targeting sequence, has been reported to be as effective as wild-type StAR protein in stimulating steroidogenesis [23]. We used a yeasttwo hybrid system to screen for proteins that interact with StAR protein and modulate its steroidogenic action

Methods

Results

Conclusion