Mechanistic Analysis of Kar3Cik1 for Mitotic Function

Abstract

Kar3Cik1 is a S. cerevisae Kinesin-14 motor protein that promotes microtubule (MT) shortening during karyogamy yet acts to crosslink interpolar MTs (ipMTs) during anaphase. The Kar3 head contains both an ATP and MT binding site, yet there is no nucleotide binding site in Cik1. Presteady-state and steady state experiments have been pursued to define the mechanism by which Kar3Cik1 performs its mitotic function to crosslink and stabilize anti-parallel ipMTs. We have developed an approach to begin our experiments with a homogenous population in which the Cik1 head binds to the MT first followed by the Kar3 motor domain (Kar3MD). The MT association kinetics at 2.6 μM-1s-1 are fast followed by Kar3MD association and rapid ADP release at 26 s-1. ATP binding to the Kar3MD is also a fast step at 4 μM-1s-1 with koff = 12 s-1. Dissociation of the MT-Kar3Cik1 complex occurs as a slow step at 3.8 s-1. These initial results suggest a model in which Kar3Cik1 interacts with the MT through an alternating cycle of Cik1 binding followed by Kar3MD 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 grant GM54141 and NIH Career Development Award K02-AR47841 to SPG.