Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanism

Britto, M. and Goulet, A. and Rizvi, S. and von Loeffelholz, O. and Moores, Carolyn A. and Cross, R.A. (2016) Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanism. Proceedings of the National Academy of Sciences of the United States of America 113 (47), E7483-E7489. ISSN 0027-8424.

Abstract

Cut7, the only kinesin-5 in S. pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. Here we show that for full-length Cut7, the key determinant of stepping direction is the degree of motor crowding on the microtubule lattice, with greater crowding converting the motor from minus end-directed to plus end-directed stepping. To explain how high Cut7 occupancy causes this reversal, we postulate a simple proximity sensing mechanism that operates via steric blocking. We propose that the minus end directed stepping action of Cut7 is selectively inhibited by collisions with neighbours under crowded conditions, whilst its plus end directed action, being less space-hungry, is not. In support, we show that the direction of Cut7-driven microtubule sliding can be reversed by crowding it with non-Cut7 proteins. Thus, crowding by either dynein microtubule binding domain or Klp2, a kinesin-14, converts Cut7 from net minus end-directed to net plus end-directed stepping. Biochemical assays confirm that the Cut7 N-terminus increases Cut7 occupancy by binding directly to microtubules. Direct observation by CryoEM reveals that this occupancy-enhancing N-terminal domain is partially ordered. Overall, our data point to a steric blocking mechanism for directional reversal by which collisions of Cut7 motor domains with their neighbours inhibit their minus end directed stepping action, but not their plus end directed stepping action. Our model can potentially reconcile a number of previous, apparently conflicting, observations and proposals for the reversal mechanism of yeast kinesins-5.

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