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 interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0 degrees C) and stage B (accumulated 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 crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.
Kanamori
T
T
Department of Chemistry, Faculty of Science, Nagoya University, Nagoya 464-8602, Japan.
Nishikawa
S
S
Nakai
M
M
Shin
I
I
Schultz
P G
PG
Endo
T
T
D000595:Amino Acid Sequence; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism
D004792:Enzyme Precursors; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C090857:TOM22 protein, S cerevisiae; C066555:Tom40 protein, S cerevisiae; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D013762:Tetrahydrofolate Dehydrogenase; D006180:Proton-Translocating ATPases