Mitochondrial processing of bovine adrenal steroidogenic acute regulatory protein.
Steroidogenic acute regulatory (StAR) protein is an important regulatory protein in steroidogenesis and rapidly undergoes proteolysis after import into the mitochondria. In this study, we determined the proteolytic cleavage sites and investigated the effects on the stimulation of steroidogenic activity of the blockage of these sites by mutation. The cleaved StAR proteins, which were purified using an anti-StAR immobilized column, reacted with antiserum against the StAR C-terminal oligopeptide. The molecular weights of the purified proteins were determined by MALDI-TOF mass spectrometry, and were found to be identical to those of the 40-285 and 55-285 amino-acid-regions of the StAR protein. To confirm the identification of the cleavage sites, we constructed site-directed mutants of bovine StAR cDNA, which contained the amino acids R37A/R38A/L40A and/or R53A/R54A/R55A. These mutant StAR proteins expressed in COS-1 cells were not cleaved at positions 39-40 and 54-55, and were processed at sites different from those in the wild-type StAR protein. These mutant proteins stimulated pregnenolone formation at almost the same rate as the wild-type StAR protein in COS-1 cells, which suggests that the cholesterol transfer activity was not affected by the mutation.
Yamazaki
Takeshi
T
Graduate School of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashihitroshima,739-8524, Japan. takey@hiroshima-u.ac.jp
Matsuoka
Chisa
C
Gendou
Miho
M
Izumi
Shunsuke
S
Zhao
Dong
D
Artemenko
Irina
I
Jefcoate
Colin R
CR
Kominami
Shiro
S
D000311:Adrenal Glands / Q000378:metabolism / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011284:Pregnenolone / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000098682:Steroidogenic Acute Regulatory Protein
D010750:Phosphoproteins; D000098682:Steroidogenic Acute Regulatory Protein; D011284:Pregnenolone