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. Based on analytical ultracentrifugation, cross-linking, size-exclusion chromatography and multi-angle light scattering, we found that the soluble cytosolic fragment of human Tom70 exists in equilibrium between monomer and dimer. A point mutation introduced at the predicted dimer interface increased the percentage of monomeric Tom70. Although chaperone docking to the mutant was the same as to the wild-type, the mutant was significantly more active in preprotein targeting. Cross-linking also demonstrated that the mutant formed stronger contacts with preprotein. However, cross-linking of full-length wild-type Tom70 on the mitochondrial membrane showed little evidence of homodimers. These results indicate that the Tom70 monomers are the functional form of the receptor, whereas the homodimers appear to be a minor population, and may represent an inactive state.
Fan
Anna C Y
AC
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Gava
Lisandra M
LM
Ramos
Carlos H I
CH
Young
Jason C
JC
D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; C402118:TOMM70 protein, human