Two distinct mechanisms drive protein translocation across the mitochondrial outer membrane in the late step of the cytochrome b(2) import pathway

The import of cytochrome b(2) into mitochondria consists of two steps, The translocation of the first part of the presequence across the inner membran e is coupled with the translocation of the tightly folded heme-binding doma in across the outer membrane and requires a membrane potential Delta Psi an d the functions of mitochondrial Hsp70 (mHsp70) in the matrix, Once the hem e-binding domain has passed the outer membrane, the translocation of the re st of the polypeptide chain across the outer membrane becomes independent o f Delta Psi and mHsp70. Here we analyzed the late Delta Psi- and mHsp70-ind ependent step in the transport of cytochrome bz fusion proteins into the in termembrane space (IMS), The import of the cytochrome b(2) fusion proteins containing two protein domains linked by a spacer segment into mitochondria was arrested at a stage at which one domain folded on each side of the out er membrane, along the pathway that is consistent with the stop-transfer mo del. The mature-size form of the translocation intermediate could move acro ss the outer membrane in both directions, and the stabilization of the prot ein domain in the IMS promoted the forward translocation. On the other hand , the intermediate-size form of the translocation intermediate, which retai ns the anchorage to the inner membrane, was transported into the IMS indepe ndently of the stability of the protein domain in the IMS. These results su ggest that two distinct mechanisms, the Brownian ratchet and the anchor dif fusion mechanisms, can operate for the transmembrane movement of the mature -size form and the intermediate-size form, respectively, of cytochrome bz s pecies.