Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM ) complex. By using site-specific photocrosslinking, we have mapped interac tions between TOM proteins and a mitochondrial precursor protein arrested a t two distinct stages, stage A (accumulated at 0 degrees C) and stage B (ac cumulated at 30 degrees C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crossl inking indicate that the presequence of the intermediates at both stage A a nd stage B is already on the trans side of the outer membrane. The mature d omain is unfolded and bound to Tom40 at stage B whereas it remains folded a t stage A. After dissociation from the TOM complex, translocation of the st age B intermediate, but not of the stage A intermediate, across the inner m embrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where tra nslocation of the presequence and unfolding of the mature domain are not ne cessarily coupled.