A molecular switch for coordinating kinesin and dynein transport of mitochondrial cargo.

The cellular distribution of mitochondria in response to stress and local energy needs is governed by the relative activities of the microtubule-based molecular motors kinesin and dynein. The mechanism for switching between these two opposite-polarity microtubule motors remains unknown. In this study, we coupled a cellular synthetic cargo transport assay with AlphaFold2-guided mutagenesis to identify a regulatory helix in the mitochondrial adaptor protein [trafficking kinesin-binding protein (TRAK)] that mediates switching between kinesin- and dynein-driven transport. Differences in the helix sequence explained why two near-identical TRAK isoforms transported mitochondria in predominantly opposite directions. Phosphorylation of the regulatory helix by stress-activated kinases caused the activation of dynein and dissociation of kinesin. Our results reveal a molecular mechanism for coordinating the directional transport of mitochondria in response to intracellular signals.