Abstract
Kinesin-2 and dynein motors transport intracellular cargo bidirectionally along both axonemal and cytoplasmic microtubules. These motor activities underlie intraflagellar transport, melanosome dynamics and other vital transport functions in cells, but the mechanism by which the activities of these oppositely-directed motors are coordinated is not well understood. One important question is whether the properties of kinesin-2 motors are specifically tuned for bidirectional transport rather than long-distance plus-ended transport. Consistent with this, kinesin-2 motors were found to detach much more readily than kinesin-1 under hindering loads, and also to rapidly rebind and continue stepping following detachment. To test the hypothesis that kinesin-2 motors are specifically tuned for bidirectional transport, we carried out stopped-flow and steady-state biochemical studies of monomeric and dimeric kinesin-1 and kinesin-2. In solution, kinesin-2 motors were found to have a 30-fold higher affinity for mantADP than unmodified ADP, presumably due to the hydrophobic nature of the mant moiety. Extrapolated ADP off-rates in the presence of microtubules indicated that for unlabeled nucleotide, ADP dissociation is not rate limiting for either kinesin-1 or kinesin-2. Microtubule pelleting experiments indicated that in the ADP state, monomeric kinesin-2 motor domains have a nearly 10-fold higher microtubule affinity than kinesin-1 motor domains. However, this increased microtubule affinity does not translate into enhanced processivity of dimeric kinesin-2 when compared to kinesin-1. This result suggests that kinesin-2 spends a larger fraction of its hydrolysis cycle in the ADP state and thus is more prone to detaching under hindering loads, but following detachment it readily rebinds to the microtubule. This behavior results in a more dynamic competition with dynein that avoids the cargo coming to a complete standstill due to motor stalling.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have