Assembly of Tim9 and Tim10 into a Functional Chaperone

Abstract

The TIM10 complex is localized in the mitochondrial intermembrane space and mediates insertion of hydrophobic proteins at the inner membrane. We have characterized TIM10 assembly and analyzed the structural properties of its subunits, Tim9 and Tim10. Both proteins are ␣-helical with a protease-resistant central domain, and each self-associates to form mainly dimers and trimers in solution. Tim9 and Tim10 bound to one another with submicromolar affinity in equimolar amounts and assembled in a stable, significantly extended complex that was indistinguishable from the native mitochondrial TIM10 complex. Importantly, the reconstituted TIM10 complex is functional because it bound to the physiological substrate ADP/ATP carrier and displayed chaperone activity in refolding the model substrate firefly luciferase. These data demonstrate that the individual subunits can exist as independent, dynamically self-associating proteins. Assembly into the thermodynamically stable hexameric complex is necessary for the TIM10 chaperone function.Almost all mitochondrial proteins are synthesized in the cytosol and then imported into the organelle in a process that is dictated by each protein's sequence and that is ensured by the function of specialized translocation machineries in the organelle (1-4). Most import and intramitochondrial sorting pathways are variants of the general "matrix pathway," first described in detail for matrix-targeted proteins (1-4). In this pathway, a mitochondrial precursor, which is usually synthesized with an N-terminal, positively charged, amphiphilic presequence, first interacts with cytosolic chaperones. It is then bound by a hetero-oligomeric receptor system on the surface of mitochondria in a process that requires ATP hydrolysis in the cytosol. The polypeptide chain is then transported across two hetero-oligomeric protein import channels, the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. Translocation is completed by the electrophoretic function of the electrochemical potential across the inner membrane and the ATP-powered import motor attached to the inner side of the TIM23 complex. In this pathway, targeting of the polypeptide chain from one complex to the other appears to be directed by increasing avidity of the positive presequence for a series of acidic receptor domains ("acid chain hypothesis") (5-9).The structural basis of this mechanism is becoming increasingly clear through advances in understanding the structural characteristics of key components at different steps of this pathway. First, the solution structure (determined by NMR) of the cytosolic part of the Tom20 receptor in complex with a synthetic presequence has been solved (10, 11): this showed that the presequence is in ␣-helical conformation and that binding between the receptor and the presequence involves hydrophobic stretches. Second, the existence of a hydrophilic channel of TOM40 as the outer membrane import pore and its dynamic behavior have been established (12-16). Third, Tim23 was shown to f...

The Dynamic Dimerization of the Yeast ADP/ATP Carrier in the Inner Mitochondrial Membrane Is Affected by Conserved Cysteine Residues

“…The TIM10 complex, composed of Tim9 and Tim10 proteins subsequently binds to AAC, mediating the passage of the hydrophobic carrier across the intermembrane space (13)(14)(15)(16)(17)(18). In doing so, the TIM10 complex presumably prevents aggregation in the aqueous intermembrane space in agreement with a recently demonstrated chaperonelike function in vitro (19). This carrier-chaperone complex is then guided to Tim12, which is peripherally attached to the outer surface of the inner membrane (stage IIIb).…”

Juxtaposition of the Two Distal CX3C Motifs via Intrachain Disulfide Bonding Is Essential for the Folding of Tim10

“…It then escorts the hydrophobic carrier across the intermembrane space, thus preventing its aggregation and acting as a chaperone (8,17,20,27,28). Subsequently, the precursor is inserted in the membrane by the 300-kDa TIM22 complex in a membrane potential-dependent manner (29,30).…”

“…The hetero-hexameric TIM10 complex (20,25,28) is made exclusively of Tim9 and Tim10 that are necessary and sufficient to form the complex (20). They bind to each other with 1:1 stoichiometry (three molecules of Tim9 with three molecules of Tim10) and an affinity of 5 ϫ 10 6 M Ϫ1 (28).…”

“…They bind to each other with 1:1 stoichiometry (three molecules of Tim9 with three molecules of Tim10) and an affinity of 5 ϫ 10 6 M Ϫ1 (28). The TIM10 complex has been reconstituted in vitro either by co-expression of its two subunits in bacteria (20,25) or by separately purifying them (28).…”

A cryptic matrix targeting signal of the yeast ADP/ATP carrier normally inserted by the TIM22 complex is recognized by the TIM23 machinery

“…Therefore, interaction with the TIM10 complex in this case does not commit the precursor to the TIM22 pathway. In fact two recent reports [36,37] have shown that the small Tim complexes can interact even with OM β-barrel proteins, thus expanding the idea that TIM10 functions as a low affinity generic chaperone of the mitochondrial IMS [38]. Commitment to one or the other membrane for insertion is probably dependent on specific substrate sequences that are recognized by the membrane-embedded insertion machinery TIM22 in the IM, or the sorting machinery SAM in the OM.…”