Interaction between the human mitochondrial import receptors Tom20 and Tom70 in vitro suggests a chaperone displacement mechanism.

The mitochondrial import receptor Tom70 contains a tetratricopeptide repeat (TPR) clamp domain, which allows the receptor to interact with the molecular chaperones, Hsc70/Hsp70 and Hsp90. Preprotein recognition by Tom70, a critical step to initiate import, is dependent on these cytosolic chaperones. Preproteins are subsequently released from the receptor for translocation across the outer membrane, yet the mechanism of this step is unknown.

Function of cytosolic chaperones in Tom70-mediated mitochondrial import.

The great majority of mitochondrial proteins are synthesized by cytosolic ribosomes and then imported into the organelle post-translationally. The translocase of the outer membrane (TOM) is a proteinaceous machinery that contains surface receptors for preprotein recognition and also serves as the main entry gateway into mitochondria. Mitochondrial targeting requires various cytosolic factors, in particular the molecular chaperones Hsc70/Hsp70 and Hsp90.

Human mitochondrial import receptor Tom70 functions as a monomer.

The mitochondrial import receptor Tom70 (translocase of the mitochondrial outer membrane 70) interacts with chaperone-preprotein complexes through two domains: one that binds Hsp70 (heat-shock protein 70)/Hsc70 (heat-shock cognate 70) and Hsp90, and a second that binds preproteins. The oligomeric state of Tom70 has been controversial, with evidence for both monomeric and homodimeric forms. In the present paper, we report that the functional state of human Tom70 appears to be a monomer with mechanistic implications for its function in mitochondrial protein import.

Multiple 40-kDa heat-shock protein chaperones function in Tom70-dependent mitochondrial import.

Mitochondrial preproteins that are imported via the translocase of the mitochondrial outer membrane (Tom)70 receptor are complexed with cytosolic chaperones before targeting to the mitochondrial outer membrane. The adenine nucleotide transporter (ANT) follows this pathway, and its purified mature form is identical to the preprotein. Purified ANT was reconstituted with chaperones in reticulocyte lysate, and bound proteins were identified by mass spectrometry.

Hsp90 functions in the targeting and outer membrane translocation steps of Tom70-mediated mitochondrial import.

The Tom70 import receptor on the mitochondrial outer membrane specifically recognizes Hsp90 and Hsc70, a critical step for the import of mitochondrial preproteins, the targeting of which depends on these cytosolic chaperones. To analyze the role of Hsp90 in mitochondrial import, the effects of the Hsp90 inhibitors geldanamycin and novobiocin were compared. Geldanamycin occludes the N-terminal ATP-binding site of Hsp90, whereas novobiocin targets the C-terminal region of the chaperone.