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 membrane is coupled with the translocation of the tightly folded heme-binding domain across the outer membrane and requires a membrane potential DeltaPsi and the functions of mitochondrial Hsp70 (mHsp70) in the matrix. Once the heme-binding domain has passed the outer membrane, the translocation of the rest of the polypeptide chain across the outer membrane becomes independent of DeltaPsi and mHsp70. Here we analyzed the late DeltaPsi- and mHsp70-independent step in the transport of cytochrome b(2) fusion proteins into the intermembrane 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 outer membrane, along the pathway that is consistent with the stop-transfer model. The mature-size form of the translocation intermediate could move across the outer membrane in both directions, and the stabilization of the protein domain in the IMS promoted the forward translocation. On the other hand, the intermediate-size form of the translocation intermediate, which retains the anchorage to the inner membrane, was transported into the IMS independently of the stability of the protein domain in the IMS. These results suggest that two distinct mechanisms, the Brownian ratchet and the anchor diffusion mechanisms, can operate for the transmembrane movement of the mature-size form and the intermediate-size form, respectively, of cytochrome b(2) species.
Esaki
M
M
Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
Kanamori
T
T
Nishikawa
S i
Si
Endo
T
T
D000818:Animals; D001692:Biological Transport; D002474:Cell-Free System; D019286:Endopeptidase K / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D013762:Tetrahydrofolate Dehydrogenase; D019286:Endopeptidase K; D008727:Methotrexate