Interaction between the mitochondrial adaptor MIRO and the motor adaptor TRAK

MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Cat+ -binding EF-hand domains. A Cterminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dyneindynactin. The mechanism by which MIRO interacts with TRAK is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Cat+ and/or GTP binding. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1's EFhands and C -terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394 to 431, immediately C -terminal to the Spindly motif. This sequence is suffi- cient for MIRO1 binding in vitro and is necessary for MIRO1dependent localization of TRAK1 to mitochondria in cells. MIRO1's EF-hands bind Cat+ with dissociation constants (KD) of 3.9 mu M and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Cat+ whereas the other EF-hand binds Cat+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Cat+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C -terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.