title: Mechanistic analysis of the Saccharomyces cerevisiae kinesin Kar3.
abstract: Kar3 is a minus-end-directed microtubule motor that is implicated in meiotic and mitotic spindle function in Saccharomyces cerevisiae. To date, the only truncated protein of Kar3 that has been reported to promote unidirectional movement in vitro is GSTKar3. This motor contains an NH2-terminal glutathione S-transferase (GST) tag followed by the Kar3 sequence that is predicted to form an extended alpha-helical coiled-coil. The alpha-helical domain leads into the neck linker and COOH-terminal motor domain. Kar3 does not homodimerize with itself but forms a heterodimer with either Cik1 or Vik1, both of which are non-motor polypeptides. We evaluated the microtubule-GSTKar3 complex in comparison to the microtubule-Kar3 motor domain complex to determine the distinctive mechanistic features required for GSTKar3 motility. Our results indicate that ATP binding was significantly faster for GSTKar3 than that observed previously for the Kar3 motor domain. In addition, microtubule-activated ADP release resulted in an intermediate that bound ADP weakly in contrast to the Kar3 motor domain, suggesting that after ADP release, the microtubule-GSTKar3 motor binds ATP in preference to ADP. The kinetics also showed that GST-Kar3 readily detached from the microtubule rather than remaining bound for multiple ATP turnovers. These results indicate that the extended alpha-helical domain NH2-terminal to the catalytic core provides the structural transitions in response to the ATPase cycle that are critical for motility and that dimerization is not specifically required. This study provides the foundation to define the mechanistic contributions of Cik1 and Vik1 for Kar3 force generation and function in vivo.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Mackey
authors.forename: Lisa R
authors.initials: LR
authors.lastname: Sproul
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Sontag
authors.forename: Lisa L
authors.initials: LL
authors.lastname: Satterwhite
authors.forename: John J
authors.initials: JJ
authors.lastname: Correia
authors.forename: Susan P
authors.initials: SP
authors.lastname: Gilbert
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D020134:Catalytic Domain; D019281:Dimerization; D005656:Fungal Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008868:Microtubule Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D013997:Time Factors; D014461:Ultracentrifugation
chemical_list: C076025:CIK1 protein, S cerevisiae; D005656:Fungal Proteins; C063504:KAR3 protein, S cerevisiae; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C119241:VIK1 protein, S cerevisiae; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases