KIF3A accelerates KIF3C within the kinesin-2 heterodimer to generate symmetrical phosphate release rates for each processive step

Sean M Quinn,Troy Vargason,Nilisha Pokhrel,Edwin Antony,Juergen Hahn,Susan P Gilbert

doi:10.1074/jbc.ra120.015272

Abstract

Heterodimeric KIF3AC is a mammalian kinesin-2 that is highly expressed in the central nervous system and associated with vesicles in neurons. KIF3AC is an intriguing member of the kinesin-2 family because the intrinsic kinetics of KIF3A and KIF3C when expressed as homodimers and analyzed in vitro are distinctively different from each other. For example, the single-molecule velocities of the engineered homodimers KIF3AA and KIF3CC are 293 and 7.5 nm/s, respectively, whereas KIF3AC has a velocity of 186 nm/s. These results led us to hypothesize that heterodimerization alters the intrinsic catalytic properties of the two heads, and an earlier computational analysis predicted that processive steps would alternate between a fast step for KIF3A followed by a slow step for KIF3C resulting in asymmetric stepping. To test this hypothesis directly, we measured the presteady-state kinetics of phosphate release for KIF3AC, KIF3AA, and KIF3CC followed by computational modeling of the KIF3AC phosphate release transients. The results reveal that KIF3A and KIF3C retain their intrinsic ATP-binding and hydrolysis kinetics. Yet within KIF3AC, KIF3A activates the rate of phosphate release for KIF3C such that the coupled steps of phosphate release and dissociation from the microtubule become more similar for KIF3A and KIF3C. These coupled steps are the rate-limiting transition for the ATPase cycle suggesting that within KIF3AC, the stepping kinetics are similar for each head during the processive run. Future work will be directed to define how these properties enable KIF3AC to achieve its physiological functions.

Highlights

Heterodimeric KIF3AC is a mammalian kinesin-2 that is highly expressed in brain and the spinal cord, yet its physiological function and intracellular transport properties are not well understood
We present the kinetics of phosphate release after ATP hydrolysis for KIF3AC, KIF3AA, and KIF3CC and use computational modeling to probe the steps of phosphate release coupled to motor head dissociation from the MT (E5A–E1C or E5C–E1A)
From the modeling and experimental work, the rates for the combined transitions resulting in phosphate release were 45 s−1 for KIF3A and 40 s−1 for KIF3C

Summary

Introduction

Heterodimeric KIF3AC is a mammalian kinesin-2 that is highly expressed in brain and the spinal cord, yet its physiological function and intracellular transport properties are not well understood (reviewed in Refs. [1,2,3,4]). We pursued experiments to measure the presteady-state kinetics of phosphate release because the transitions associated with phosphate release coupled with motor head dissociation from the MT represent the duration of each step of KIF3A and KIF3C in the processive run. The results indicate that phosphate release coupled with motor dissociation from the MT is rate limiting for the ATPase cycle and predicts that within a processive run, KIF3A and KIF3C will exhibit similar step kinetics.

Results

Conclusion