Self-association and precursor protein binding of Saccharomyces cerevisiaeTom40p, 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 chann el, of which the core component is Tom40p. Nuclear-encoded mitochondrial pr ecursor proteins are first recognized by surface receptors and then pass th rough the import channel. The Tom complex has been purified. however, the p rotein-protein interactions that drive its assembly and maintain its stabil ity have been difficult to study. Here we show that Saccharomyces cerevisia e Tom40p expressed in bacteria and purified to homogeneity associates effic iently with itself. The self-association is very strong and can withstand u p 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 r ecognizes the targeting sequence of mitochondrial precursor proteins. Altho ugh the binding of the targeting sequence to Tom40p is inhibited by urea co ncentrations in excess of 1 M, it is moderately resistant to 1 M salt. Simu ltaneous self-assembly and precursor protein binding suggest that Tom40p co ntains at least two different domains mediating these processes. The experi mental approach described here should be useful for analysing protein-prote in interactions involving individual or groups of components of the mitocho ndrial import machinery.