title: 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 pulse-chase 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.
authors.affiliation: Department of Biology and the Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180 and.
authors.forename: Chun Ju
authors.initials: CJ
authors.lastname: Chen
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Ivan
authors.initials: I
authors.lastname: Rayment
authors.affiliation: Department of Biology and the Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180 and. Electronic address: sgilbert@rpi.edu.
authors.forename: Susan P
authors.initials: SP
authors.lastname: Gilbert
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D008868:Microtubule Proteins / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000201:enzymology* / Q000235:genetics; D008954:Models, Biological; D011485:Protein Binding / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000201:enzymology / Q000235:genetics
chemical_list: C076025:CIK1 protein, S cerevisiae; C063504:KAR3 protein, S cerevisiae; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases