Steroid biosynthesis begins with cholesterol and proceeds through well-described pathways. The rapid induction of steroidogenesis requires the steroidogenic acute regulatory protein (StAR), which facilitates the movement of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane (IMM). We proposed the molten globule model of StAR's action, which states that StAR undergoes an acid-induced conformational change on the OMM, where it should exert its activity. We have established that StAR acts exclusively on the outer mitochondrial membrane, that only the carboxyl-terminal a-helix of StAR interacts with the membrane, that movement of this C-terminal helix is an essential component of StAR's molten globule transition, and that this movement is essential for StAR's activity. We now propose further experiments to dissect the complex interaction of StAR with cholesterol and other components of the OMM.
Aim 1 is to 'Determine how StAR 'pauses' on the OMM.' StAR is imported into mitochondria more slowly than other proteins, and its level of activity is proportional to the time it resides on the OMM. We shall investigate the mechanism of this 'paused', slow mitochondrial entry by identifying the site(s) at which StAR is cleaved during this entry, and by using deletional scanning mutagenesis to identify leader domains that exert pause activity.
Aim 2 is to 'Identify proteins on the OMM that interact with StAR.' Substantial data implicate the peripheral benzodiazepine receptor (PER), an 18kDa OMM protein, in the cholesterol-import process. We shall investigate the functional role of PBR and its potential physical interaction with StAR, as well as performing experiments to identify other potential StAR-interacting proteins, irrespective of their identity.
Aim 3 is to 'Characterize the role of the peripheral benzodiazepine receptor (PBR) in StAR's action.' PBR is required for cholesterol's entry into steroidogenic mitochondria, and StAR triggers this entry, but the potential interactions between these two essential proteins remain unclear. We shall determine the membrane topology of PBR by incorporating PBR into liposomes and identifying the domains accessible for proteolysis, and we shall determine if the cholesterol bound to the carboxyl-terminus of PBR constitutes the kinetically distinct pool of 'labile' steroidogenic cholesterol. Fulfilling these aims will substantially advance our understanding of this indispensable initial step in the production of all adrenal and gonadal steroid hormones. ? ? ?
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