Abstract
In this study, we analyzed intracellular functions and motile properties of neck-linker (NL) variants of the bi-directional S. cerevisiae kinesin-5 motor, Cin8. We also examined - by modeling - the configuration of H-bonds during NL docking. Decreasing the number of stabilizing H-bonds resulted in partially functional variants, as long as a conserved backbone H-bond at the N-latch position (proposed to stabilize the docked conformation of the NL) remained intact. Elimination of this conserved H-bond resulted in production of a non-functional Cin8 variant. Surprisingly, additional H-bond stabilization of the N-latch position, generated by replacement of the NL of Cin8 by sequences of the plus-end directed kinesin-5 Eg5, also produced a nonfunctional variant. In that variant, a single replacement of N-latch asparagine with glycine, as present in Cin8, eliminated the additional H-bond stabilization and rescued the functional defects. We conclude that exact N-latch stabilization during NL docking is critical for the function of bi-directional kinesin-5 Cin8.
Highlights
Kinesin-5 motor proteins perform essential mitotic functions by providing the force that separates the spindle poles apart during spindle assembly, maintenance and elongation [reviewed in 1-6]
Cin[8] containing NL sequences from plus-end directed kinesin motors is not functional in cells To assess the intracellular functionality of the NL variants, we first examined their ability to support yeast viability as the sole source of kinesin-5 function
The current study is the first to demonstrate that mutations in the NL modulate the motile properties and intracellular functions of a bi-directional Nterminal kinesin motor
Summary
Kinesin-5 motor proteins perform essential mitotic functions by providing the force that separates the spindle poles apart during spindle assembly, maintenance and elongation [reviewed in 1-6]. These motors are homotetramers, with two pairs of catalytic domains located on opposite sides of a central minifilament 7-10. This unique structure enables kinesin-5 motors to crosslink the antiparallel microtubules (MTs) of the spindle and to slide them apart by moving in the plus-end direction of the two MTs that they crosslink 11-13. The above-described body of work notwithstanding, the molecular mechanism and physiological implications of such bi-directional motility remain elusive
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