Mitotic Kinesin Kar3Cik1 Interaction with Microtubules

Abstract

Kar3Cik1 is a S. cerevisae Kinesin-14 motor protein that functions to shorten cytoplasmic microtubules (MTs) during yeast mating yet crosslinks interpolar MTs (ipMTs) during anaphase. Kar3 contains ATP and MT binding sites, yet Cik1 lacks the nucleotide binding site. Presteady-state kinetic and thermodynamic studies were pursued using paclitaxel-stabilized MTs to define the Kar3Cik1 interactions with the MT lattice expected during ipMT crosslinking. The results reveal that Kar3Cik1's association with the MT occurs at 4.9 μM-1s-1 followed by a 5 s-1 structural transition that limits mantADP release to 4-5 s-1. However, the intrinsic rate of mantADP release from Kar3Cik1 was observed at 109 s-1. ATP binding to nucleotide-free MT•Kar3Cik1 occurred at 2.1 μM-1s-1 followed by an ATP-promoted isomerization at 64 s-1. ATP hydrolysis was a fast step observed at 25 s-1, yet the reduced burst amplitude indicates reversals at this step. ATP hydrolysis was required for ATP-promoted Kar3Cik1 detachment from the MT at 12.7 s-1. The rate constant of phosphate release at 10 s-1 was similar to the rate constant of ATP-promoted MT•Kar3Cik1 dissociation, suggesting that these two steps are coupled. The rate-limiting step for steady-state ATP turnover at 5 s-1 is hypothesized to be the conformational change after Cik1 collision with the MT leading to Kar3 association with the MT followed by ADP release. These findings provide a model in which Kar3Cik1 interacts with the MT through an alternating cycle of Cik1 MT association followed by Kar3 binding with regulation through Kar3 nucleotide state and mechanical strain between the Kar3-Cik1 heads. Supported by NIH grant R01-GM54141 to SPG.