Kinesin Kar3Cik1 ATPase pathway for microtubule cross-linking

Abstract

Kar3Cik1 is a Saccharomyces cerevisiae kinesin-14 that functions to shorten cytoplasmic microtubules (MTs) during yeast mating yet maintains mitotic spindle stability by cross-linking anti-parallel interpolar MTs. Kar3 contains both an ATP- and a MT-binding site, yet there is no evidence of a nucleotide-binding site in Cik1. Presteady-state and steady-state kinetic experiments were pursued to define the regulation of Kar3Cik1 interactions with the MT lattice expected during interpolar MT cross-linking. The results reveal that association of Kar3Cik1 with the MT occurs at 4.9 μM-1 s-1, followed by a 5-s-1 structural transition that limits ADP release from the Kar3 head. Mant-ATP binding occurred at 2.1 μM-1 s-1, and the pulsechase experiments revealed an ATP-promoted isomerization at 69 s-1. ATP hydrolysis was observed as a rapid step at 26 s-1 and was required for the Kar3Cik1 motor to detach from MT. The conformational change at 5 s-1 that occurred after Kar3Cik1 MT association and prior to ADP release was hypothesized to be the rate-limiting step for steady-state ATP turnover. We propose a model in which Kar3Cik1 interacts with the MT lattice through an alternating cycle of Cik1 MT collision followed by Kar3 MT binding with head-head communication between Kar3 and Cik1 modulated by the Kar3 nucleotide state and intramolecular strain. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.