Evidence that StAR and MLN64 act on the outer mitochondrial membrane as molten globules

StAR increases the flow of cholesterol from the outer to inner mitochondria l membrane (OMM to IMM), but its mechanism of action remains unclear. MLN64 is a 445 amino acid protein of unknown function that has four N-terminal t ransmembrane domains and whose C-terminal domain from 218-445 is 37% identi cal to StAR. N-62 StAR is as active as wild-type StAR, and N-234 MLN64 has 1/3 to 1/2 of StAR's activity. N-62 StAR lacks a mitochondrial leader and i s confined to the cytoplasm, indicating that it acts on the OMM. Bacteriall y expressed N-62 StAR and N-218 MLN64 are active with isolated MA-IO cell m itochondria, indicating the proteins were properly folded. Far-UV CD spectr oscopy, unfolding in urea, and fluorescence spectroscopy indicate that StAR undergoes a pH-dependent transition to a molten globule (retained secondar y structure, partially lost tertiary structure) and stabilizes in mildly ac id conditions. Far-UV CD data indicate that MLN64 undergoes a much less pro nounced transition. Western blotting shows that normal human placenta has a bundant N-terminally-cleaved 30 kDa MLN64. Partial proteolysis followed by mass spectrometry shows that the C-termini of StAR and MLN64 are sensitive to proteolysis, indicating looser folding. Our model of StAR action is that the protease-resistant domain unfolds slowly during normal mitochondrial e ntry, keeping StAR in contact with the OMM longer, increasing activity. The transition to the molten globule may be related to interaction with the OM M. These data are consistent with the recent crystallographic structure of N-216 MLN64 in which MLN64 binds cholesterol one molecule at a time, but ar e not consistent with the suggestion that StAR/MLN64 must reside in the int ramembraneous space to transfer cholesterol form the OMM to the IMM.