Torque generation by one of the motor subunits of heterotrimeric kinesin-2

Abstract

Heterotrimeric kinesin-2 motors transport intraflagellar transport (IFT)-particles from the base to the tip of the axoneme to assemble and maintain cilia. These motors are distinct in containing two non-identical motor subunits together with an accessory subunit. We evaluated the significance of this organization by comparing purified wild type kinesin-2 holoenzymes that support IFT in vivo, with mutant trimers containing only one type of motor domain that do not support IFT in vivo. In motility assays, wild type kinesin-2 moved microtubules (MTs) at a rate intermediate between the rates supported by the two mutants. Interestingly, one of the mutants, but not the other mutant or the wild type protein, was observed to drive a persistent counter-clock-wise rotation of the gliding MTs. Thus one of the two motor domains of heterotrimeric kinesin-2 exerts torque as well as axial force as it moves along a MT, which may allow kinesin-2 to control its circumferential position around a MT doublet within the cilium.

Figure 1

Recombinant kinesin-II chimeras form heterotrimeric complexes. (A) Diagram showing the sequences of KLP-11, KLP-20, and the regions of exchange for chimeric KLP-20 motor/KLP-11stalk-tail (KLP-2011) and KLP-11 motor/KLP-20stalk-tail (KLP-1120). (B – E) The components and SDS gels of purified recombinant kinesin-II (B), kinesin-II(homo-11) (C), kinesin-II(homo-20) (D), and kinesin-II(double chimera) (E). (F–H) In vitro motility assay results of recombinant kinesin-II chimeras. (F) Velocities of MT gliding driven by kinesin-II (blue circle), kinesin-II(homo-20) (red), kinesin-II(homo-11) (green), and kinesin-II(double chimera) (yellow) in 5mM [Mg-ATP] and different PIPES concentrations. The kinesin-II chimeras did not bind to microtubules at PIPES concentrations below 40 mM for kinesin-II(homo-20) and kinesin-II(double chimera) or 60 mM for kinesin-II(homo-11). The velocities of gliding driven by kinesin-II were close to the average of the velocities of the kinesin-II(homo-11) and kinesin-II(homo20) motors. (G) Double reciprocal plots of the rates of MT gliding driven by kinesin-II (blue), kinesin-II(homo-20) (red), kinesin-II(homo-11) (green), and kinesin-II(double chimera) (yellow) versus [Mg-ATP]. (H) Rotation of gliding microtubules by kinesin-II(homo-11). The numbers show the time in seconds and the black arrows indicate the direction of translocation of the microtubules. Bar: 2.5 µm.