Why are Kinesin-2 KIF3AB and KIF3AC so Processive?

Mammalian KIF3AC is a heterotrimeric kinesin-2 that is best known for roles in organelle transport and remodeling of the microtubule cytoskeleton in neuronal tissue, yet in vitro studies to characterize its single molecule behavior are lacking. We report that a bacterially expressed native sequence of KIF3AC is highly processive with run lengths matching those exhibited by conventional kinesin-1 K560 in comparative experiments. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck linker sequence that has been reported to be responsible for shorter run lengths observed for kinesin-2 KIF3AB. However, a comparative KIF3AB engineered with native sequence also exhibited long run lengths suggesting that neck linker length is not the sole determinant of run length for native kinesin-2 motors. KIF3C contains a long extension in surface loop L11 not present in KIF3A or KIF3B, or other processive kinesins. L11 is a component of the kinesin microtubule interface and has been implicated in activation of ADP release upon microtubule collision. KIF3AC encoding a truncation in KIF3C L11 (KIF3AC∆L11) is even more processive than wildtype KIF3AC and more similar to KIF3AB, suggesting that L11 also plays a role in determining run length. Steady-state ATPase experiments show that shortening L11 does not alter the kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening L11 does significantly increase microtubule affinity in these dimers. Analysis of homodimeric KIF3CC and KIF3CC∆L11 reveal that both are processive but exceedingly slow. These results reveal that processivity can be regulated in part by L11, consistent with the interpretation that L11 becomes ordered upon microtubule collision to activate ADP release. These studies also expand our understanding of motor processivity and point to alternative structural and mechanistic modulators of processivity. Supported by NIH R37-GM54141.