Unless otherwise stated, all reagents were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO). Enhanced chemi-luminescence Western blotting detection kit was purchased from Amer-sham (Arlington Heights, IL). Polyclonal antiprohibitin antibody and preimmune rabbit serum were purchased from Neomarkers (Fremont, CA), monoclonal antiproliferating cell nuclear antigen (anti-PCNA) antibody from Santa Cruz Biotechnology (Santa Cruz, CA), and polyclonal anticholesterol side-chain cleavage cytochrome P450 (P450scc) was from Chemicon International, Inc. (Temecula, CA). Goat anti-mouse immunoglobulin (Ig) G (H+L) conjugated to Alexa Fluor 488, goat anti-rabbit IgG conjugated to Alexa Fluor 594, and 4′,6′-diamidino-2-phenylindole were purchased from Molecular Probes (Eugene, OR).
A growing body of evidence has implicated prohibitin in mitochondrial structure, function, and inheritance. Prohibitin is predominantly localized to the inner mitochondrial membrane of rat granulosa cells. Both rat and human prohibitin possesses a short transmembrane helix near their N-termini that may be integrated into the mitochondrial membranes. It has been speculated that pro-hibitin, as an inner mitochondrial membrane protein, may control ion transport and calcium-dependent ATP production. If this hypothesis is correct, then prohibitin may play a similar role in regulating the granulosa cell steroidogenic machinery, because the rate-limiting step of steroidogenesis is also located in the inner mitochondrial membrane.
In humans, the prohibitin gene is located on chromosome 17q21, close to the ovarian and breast carcinoma susceptibility gene (BRAC1) locus. Prohibitin is a highly conserved protein that is thought to play a role in cell-cycle control, differentiation, senescence, and antiproliferative activity. The rat and mouse protein sequences are identical and differ from the human sequence by a single amino acid.