Abstract
The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A's activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.
Highlights
The kinesin-3 motor protein KIF1A is a neuronal transport motor responsible for the anterograde transport of synaptic vesicle precursors and other vesicular cargo along microtubules (Mt) [1,2,3,4]
We found that the microtubule off-rates in the strong-binding apo state and AMPPNP state are more than an order of magnitude faster in KIF1A than in kinesin-1 (Fig. 6C) [50]
Fast speed and superprocessivity provide competing constraints because each head must cyclically detach from the microtubule, whereas the dimeric motor remains associated over hundreds of steps
Summary
The kinesin-3 motor protein KIF1A is a neuronal transport motor responsible for the anterograde transport of synaptic vesicle precursors and other vesicular cargo along microtubules (Mt) [1,2,3,4]. Optical trapping studies and mixed motor assays have revealed that, despite the enhanced electrostatic association to the microtubule, kinesin-3 has a surprisingly low resistance to force and detaches under load [8, 15, 17, 29,30,31]. DP D binds to the bound head (state 4) and is hydrolyzed to ADP-Pi (state 5), triggering full neck linker docking, which positions the tethered head forward and puts the motor in a weakly bound state [27] From this vulnerable state 5, the bound head can detach from the microtubule and terminate the processive run (state 1). Processivity requires that the rate of tethered-head attachment be considerably faster than the rate of bound head dissociation from the microtubule
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