title: Hsp90 functions in the targeting and outer membrane translocation steps of Tom70-mediated mitochondrial import.
abstract: 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. Here, novobiocin was found to inhibit preprotein import and, in particular, targeting to the purified cytosolic fragment of Tom70. Hsp90 cross-linking to preprotein and coprecipitation of Hsp90 with Tom70 were both impaired by novobiocin. Overall, novobiocin treatment increased preprotein aggregation, contributing to reduced import competence. In contrast, geldanamycin had no apparent effect on preprotein interactions with Hsp90, formation of preprotein-chaperone complexes, Hsp90 docking onto Tom70, or preprotein association with the outer membrane. Instead, geldanamycin impaired formation of preprotein import intermediates at the outer membrane. This suggests a novel active role for Hsp90 in import steps subsequent to Tom70 targeting. Our results outline the mechanisms of Hsp90 function in preprotein targeting and transport.
authors.affiliation: Department of Biochemistry, McGill University, Montréal, Québec H3G 1Y6, Canada.
authors.forename: Anna C Y
authors.initials: AC
authors.lastname: Fan
authors.forename: Melanie K
authors.initials: MK
authors.lastname: Bhangoo
authors.forename: Jason C
authors.initials: JC
authors.lastname: Young
mesh_terms: D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D026901:Membrane Transport Proteins; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009675:Novobiocin / Q000494:pharmacology; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*
chemical_list: D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C402118:TOMM70 protein, human; D009675:Novobiocin; C001277:geldanamycin