Self-association and precursor protein binding of Saccharomyces cerevisiae Tom40p, the core component of the protein translocation channel of the mitochondrial outer membrane.
The precursor protein translocase of the mitochondrial outer membrane (Tom) is a multi-subunit complex containing receptors and a general import channel, of which the core component is Tom40p. Nuclear-encoded mitochondrial precursor proteins are first recognized by surface receptors and then pass through the import channel. The Tom complex has been purified; however, the protein-protein interactions that drive its assembly and maintain its stability have been difficult to study. Here we show that Saccharomyces cerevisiae Tom40p expressed in bacteria and purified to homogeneity associates efficiently with itself. The self-association is very strong and can withstand up to 4 M urea or 1 M salt. The tight self-association does not require the N-terminal segment of Tom40p. Furthermore, purified Tom40p preferentially recognizes the targeting sequence of mitochondrial precursor proteins. Although the binding of the targeting sequence to Tom40p is inhibited by urea concentrations in excess of 1 M, it is moderately resistant to 1 M salt. Simultaneous self-assembly and precursor protein binding suggest that Tom40p contains at least two different domains mediating these processes. The experimental approach described here should be useful for analysing protein-protein interactions involving individual or groups of components of the mitochondrial import machinery.
Gordon
D M
DM
Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, D403 Richards Building, Philadelphia, PA 19104-6085, USA.
Wang
J
J
Amutha
B
B
Pain
D
D
D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D033862:Iron-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D018832:Molecular Chaperones / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D000096923:Frataxin
D005656:Fungal Proteins; D033862:Iron-Binding Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D017853:Phosphotransferases (Alcohol Group Acceptor); D011498:Protein Precursors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000096923:Frataxin; C066555:Tom40 protein, S cerevisiae