Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanism.
Cut7, the sole kinesin-5 in Schizosaccharomyces 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 neighbors under crowded conditions, whereas its plus end-directed action, being less space-hungry, is not. In support of this idea, 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 through which collisions of Cut7 motor domains with their neighbors 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.
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom.
Mishan
M
Britto
Institute of Structural and Molecular Biology, Birkbeck College, London WC1E 7HX, United Kingdom.
Adeline
A
Goulet
Institute of Structural and Molecular Biology, Birkbeck College, London WC1E 7HX, United Kingdom.
Syeda
S
Rizvi
Institute of Structural and Molecular Biology, Birkbeck College, London WC1E 7HX, United Kingdom.
Ottilie
O
von Loeffelholz
Institute of Structural and Molecular Biology, Birkbeck College, London WC1E 7HX, United Kingdom.
Carolyn A
CA
Moores
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom; r.a.cross@warwick.ac.uk.
Robert A
0000-0002-0004-7832
RA
Cross
D001665:Binding Sites; D020090:Chromosome Segregation; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D000072417:Protein Domains; D012568:Schizosaccharomyces / Q000737:chemistry / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000378:metabolism*
C104159:Cut7 protein, S pombe; C502171:Klp2 protein, S pombe; D008869:Microtubule-Associated Proteins; D029702:Schizosaccharomyces pombe Proteins; D016547:Kinesins
Cut7; bidirectional kinesin; kinesin crowding; kinesin-5; mitotic kinesin