TRAK adaptors coordinate the recruitment and activation of dynein and kinesin to control mitochondrial transport

In neurons, mitochondria are transported to distal regions for supplying energy and buffer Ca 2+ . Mitochondrial transport is mediated by Miro and TRAK adaptors that recruit kinesin and dynein-dynactin. To understand how mitochondria are transported by these opposing motors and stalled at regions with elevated Ca 2+ , we reconstituted the mitochondrial transport machinery in vitro. We show that the coiled-coil domain of TRAK activates dynein-dynactin motility, but kinesin requires an additional factor to efficiently transport Miro/TRAK. Unexpectedly, TRAK adaptors that recruit both motors move towards kinesin’s direction, whereas kinesin is excluded from binding TRAK transported by dynein-dynactin. The assembly and motility of the transport machinery are not affected by Ca 2+ . Instead, the mitochondrial docking protein syntaphilin is sufficient to oppose the forces generated by kinesin and stall the motility. Our results provide mechanistic insight into how mitochondria are transported by the coordinated action of motors and statically anchored to regions with high neuronal activity.