Recognition of preproteins by the isolated TOM complex of mitochondria.
A multisubunit complex in the mitochondrial outer membrane, the TOM complex, mediates targeting and membrane translocation of nuclear-encoded preproteins. We have isolated the TOM holo complex, containing the preprotein receptor components Tom70 and Tom20, and the TOM core complex, which lacks these receptors. The interaction of recombinant mitochondrial preproteins with both types of soluble TOM complex was analyzed. Preproteins bound efficiently in a specific manner to the isolated complexes in the absence of chaperones and lipids in a bilayer structure. Using fluorescence correlation spectroscopy, a dissociation constant in the nanomolar range was determined. The affinity was lower when the preprotein was stabilized in its folded conformation. Following the initial binding, the presequence was transferred into the translocation pore in a step that required unfolding of the mature part of the preprotein. This translocation step was also mediated by protease-treated TOM holo complex, which contains almost exclusively Tom40. Thus, the TOM core complex, consisting of Tom40, Tom22, Tom6 and Tom7, is a molecular machine that can recognize and partially translocate mitochondrial precursor proteins.
Stan
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Institut für Physiologische Chemie der Universität München, Goethestrabetae 33, D-80336 München, Germany.
Ahting
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Dembowski
M
M
Künkele
K P
KP
Nussberger
S
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Neupert
W
W
Rapaport
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D
D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009492:Neurospora crassa / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism*; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence; D013395:Sucrose / Q000737:chemistry; D013696:Temperature
D005656:Fungal Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C489350:TOM20 protein, S cerevisiae; C090857:TOM22 protein, S cerevisiae; C099634:TOM7 protein, S cerevisiae; C489039:TOM70 protein, S cerevisiae; C066555:Tom40 protein, S cerevisiae; D013395:Sucrose