Mechanistic Analysis of Kar3Cik1 for Mitotic Function

Abstract

Kar3Cik1 is a S. cerevisae Kinesin-14 motor protein that functions to shorten cytoplasmic microtubule (MT) during yeast mating for nuclear fusion, yet crosslinks interpolar MTs (ipMTs) during anaphase. The Kar3 head contains both an ATP and MT binding site, yet the Cik1 head lacks an ATP catalytic site. Presteady-state and steady state experiments were conducted to define the mechanochemical pathway by which Kar3Cik1 stabilizes anti-parallel ipMTs for its mitotic function. To initiate the cycle, we used a high ADP strategy to promote MT-binding by the Cik1 head at 4.9 ∈1/4M-1s-1. The initial association is then followed by a 4-5 s-1 conformational change to induce Kar3 head binding to the MT with rapid ADP release from the active site at 109 s-1. MantATP binding to the nucleotide free MT•Kar3Cik1 is fast at 2.1 ∈1/4M-1s-1 with koff = 16.6 s-1. Pulse-chase methodology further reveals that MgATP binding to MT•Kar3Cik1 follows a two step process, formation of a collision complex followed by a 64 s-1 isomerization step. ATP hydrolysis occurs at 26 s-1 followed by motor detachment from microtubule at 11.5 s-1. The rate-limiting step for steady-state ATP turnover at 5 s-1 is hypothesized to be the conformational change leading to Kar3 head binding to MT. These initial results suggest a model in which Kar3Cik1 interacts with the MT through an alternating cycle of Cik1 binding followed by Kar3 binding. Because Cik1 does not have a nucleotide binding site, we propose that head-head communication is mediated by a strain-dependent mechanism. Supported by NIH GM54141.