title: cis and trans sites of the TOM complex of mitochondria in unfolding and initial translocation of preproteins.
abstract: Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. Our previous studies led to the concept of two preprotein binding sites acting in series, the surface-exposed cis site and the trans site exposed to the intermembrane space. We report here that preproteins are bound to the cis site in a labile fashion even at low ionic strength, whereas intermediates arrested at the trans site remained firmly bound at higher salt concentration. The stability of the trans site intermediate results from interactions of both the presequence and unfolded parts of the mature part of the preprotein with the TOM complex. Binding to the trans site proceeded at rates comparable with those of unfolding of the mature domain and appeared to be kinetically limited by the unfolding reaction. Efficient binding to the trans site and unfolding were observed with both outer membrane vesicles and intact mitochondria whose membrane potential, DeltaPsi, was dissipated. Upon re-establishing DeltaPsi, trans site-bound preprotein resumed translocation into the matrix. The rates of unfolding and binding to the trans site were the same as those for translocation into intact energized mitochondria. We conclude that preprotein unfolding in intact mitochondria can take place without the involvement of the translocation machinery of the inner membrane and, in particular, the matrix Hsp70 chaperone. Further, preprotein unfolding at the outer membrane can be a rate-limiting step for formation of the trans site intermediate and for the entire translocation reaction.
authors.affiliation: Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universität München, Goethestrabetae 33, 80336 München, Federal Republic of Germany.
authors.forename: D
authors.initials: D
authors.lastname: Rapaport
authors.forename: A
authors.initials: A
authors.lastname: Mayer
authors.forename: W
authors.initials: W
authors.lastname: Neupert
authors.forename: R
authors.initials: R
authors.lastname: Lill
mesh_terms: D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D003575:Cytochromes c1 / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009492:Neurospora crassa / Q000378:metabolism*; D009994:Osmolar Concentration; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
chemical_list: D046911:Macromolecular Substances; D008565:Membrane Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D014508:Urea; D003575:Cytochromes c1; D013762:Tetrahydrofolate Dehydrogenase; D006180:Proton-Translocating ATPases; D008727:Methotrexate