Characterization of the MIRO-independent mitochondrial association activity of TRAK proteins

Abstract Current models suggest that MIRO GTPases anchor cytoskeletal motors to the mitochondrial outer membrane (MOM). However, our previous findings indicate that the unconventional myosin, MYO19, interacts with MIRO weakly but that a MIRO-independent MOM-localizing domain interacts more tightly with the MOM. To test the model that MIRO proteins serve as weaker, initial recruiters of cytoskeletal motors to mitochondria, we examined interactions between TRAK proteins (microtubule motor-mitochondria adapter proteins) and the MOM via quantitative fluorescence microscopy and steady-state kinetic approaches. Using GFP-TRAK truncations expressed in MIRO1-2 double knockout mouse embryonic fibroblasts, we identified a MIRO-independent mitochondrial binding domain in the C-terminus of TRAK1 and TRAK2, sufficient for localization to the MOM--similar to what we observed for full length GFP-TRAK proteins. The MIRO-binding domains (MBD) of the TRAK proteins were only able to localize to mitochondria in the presence of ectopic expression of MIRO. Importantly, fluorescence recovery after photobleaching (FRAP) demonstrated that the steady-state kinetics of TRAK MBD /MIRO2 interaction were faster-exchanging than for either full-length TRAK or the TRAK C-terminal MOM-binding domain expressed alone. These data support the model that MIRO supports weak associations of cytoskeletal motors to the MOM, while MIRO-independent binding contributes significantly to tighter association of such motors to the MOM.