Protein import into mammalian mitochondria - Characterization of the intermediates along the import pathway of the precursor into the matrix

We have characterized several intermediates in the mitochondrial import stimulation factor (MSF)-dependent import into mammalian mitochondria of a matrix-targeted precursor, preadrenodoxin (pAd). In the first step, pAd docks onto the 37-kDa protein of the outer membrane (OM37) as a complex with MSF (stage I intermediate). It is then transferred to the import pore of OM in the presence of ATP, but in the absence of Delta psi across the inner membrane (IM), to form stage II intermediate. Depletion of matrix ATP in the presence of both extramitochondrial ATP and Delta psi, induces accumulation of stage III intermediate, which is a mixture of the precursor with different intramitochondrial localizations: the precursor whose presequence had crossed either OM (IIIa) or both OM and IM (IIIb), but with a bulk portion remaining exposed to the cytosol and the precursor whose presequence had crossed both membranes, but with a residual portion staying within the intermembrane space (IIIc). These intermediates are on the correct import pathway and are characteristic in their protease accessibility, salt extractability, and antibody accessibility, as well as in their energy requirement for the chase reaction.