title: Interaction between the human mitochondrial import receptors Tom20 and Tom70 in vitro suggests a chaperone displacement mechanism.
abstract: 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. Here, we report that Tom20 interacts with the TPR clamp domain of Tom70 via a conserved C-terminal DDVE motif. This interaction was observed by cross-linking endogenous proteins on the outer membrane of mitochondria from HeLa cells and in co-precipitation and NMR titrations with purified proteins. Upon mutation of the TPR clamp domain or deletion of the DDVE motif, the interaction was impaired. In co-precipitation experiments, the Tom20-Tom70 interaction was inhibited by C-terminal peptides from Tom20, as well as from Hsc70 and Hsp90. The Hsp90-Tom70 interaction was measured with surface plasmon resonance, and the same peptides inhibited the interaction. Thus, Tom20 competes with the chaperones for Tom70 binding. Interestingly, antibody blocking of Tom20 did not increase the efficiency of Tom70-dependent preprotein import; instead, it impaired the Tom70 import pathway in addition to the Tom20 pathway. The functional interaction between Tom20 and Tom70 may be required at a later step of the Tom70-mediated import, after chaperone docking. We suggest a novel model in which Tom20 binds Tom70 to facilitate preprotein release from the chaperones by competition.
authors.affiliation: Department of Biochemistry and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, Quebec H3G 0B1, Canada.
authors.forename: Anna C Y
authors.initials: AC
authors.lastname: Fan
authors.forename: Guennadi
authors.initials: G
authors.lastname: Kozlov
authors.forename: Annabelle
authors.initials: A
authors.lastname: Hoegl
authors.forename: Richard C
authors.initials: RC
authors.lastname: Marcellus
authors.forename: Michael J H
authors.initials: MJ
authors.lastname: Wong
authors.forename: Kalle
authors.initials: K
authors.lastname: Gehring
authors.forename: Jason C
authors.initials: JC
authors.lastname: Young
mesh_terms: D020816:Amino Acid Motifs; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D006360:Heat-Shock Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010455:Peptides; D011498:Protein Precursors; D011956:Receptors, Cell Surface; C084008:TOMM20 protein, human; C402118:TOMM70 protein, human