Studies of the mitochondrial import receptor Tom70, preprotein targeting and import.

摘要

Most mitochondrial proteins are synthesized as preproteins in the cytosol and post-translationally imported into the organelle. Mitochondrial targeting requires various cytosolic factors, in particular the molecular chaperones Hsc70 and Hsp90, and recognition by the receptors, Tom20 or Tom70, of the translocase of the outer membrane (TOM). The activity of Hsc70 and Hsp90 is dependent on ATP cycling and regulated by a number of co-chaperone proteins, including Tom70. Tom70 contains a TPR clamp domain that binds a conserved C-terminal EEVD motif present on both chaperones. This interaction is critical for the transfer of the preprotein from the chaperones to Tom70 and subsequently to the TOM complex. To gain further insight into the role of chaperones in this process, the effects of small molecule inhibitors of Hsp90 were compared. Geldanamycin, a competitive inhibitor of the ATP binding site of Hsp90, acted after chaperone docking and blocked formation of preprotein import intermediates. These results provide evidence for an active role of Hsp90 on the outer membrane. To understand how Tom70 coordinates the interaction between the preprotein and chaperones, we determined the oligomeric state of the receptor, which had been controversial with evidence for both monomeric and homodimeric forms. Our biophysical and cross-linking studies show that the cytosolic fragment of human Tom70 is in a monomer-dimer equilibrium. A point mutation that shifts the equilibrium to the monomeric form was significantly more active in preprotein targeting. Cross-linking of endogenous Tom70 on the mitochondrial membrane isolated from HeLa cells showed little evidence of homodimers. Therefore, we conclude that Tom70 functions as a monomer. Cross-linking studies further showed that Tom70 forms a stable complex with second import receptor, Tom20, on the outer membrane. Using mutagenesis, we show that the TPR clamp domain of Tom70 binds the C-terminal DDVE motif of Tom20. Although the Tom20-Tom70 interaction competes with the chaperone docking, our mitochondrial import assays suggest that it is a functional interaction required for Tom70-mediated import. Taken together, our results lead to a new model of Tom70 structure and function in the preprotein mitochondrial import pathway.