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.
Highlights
Kar3 is one of six kinesin-related proteins found in the budding yeast Saccharomyces cerevisiae [1,2,3]
Fluorescence imaging and genetic studies have implicated Kar3 in multiple microtubule functions during the life cycle of budding yeast [2, 3]. These diverse functions are modulated in part by Cik1 and Vik1 for cytoplasmic or nuclear localization because the nuclear envelope remains intact during conjugation, meiosis, and mitosis
Our results show that GSTKar3, like dimeric Ncd and dimeric kinesin, releases ADP upon microtubule association, and the motor domain returns to a conformation that is more likely to bind ATP rather than rebind ADP
Summary
Kar is one of six kinesin-related proteins found in the budding yeast Saccharomyces cerevisiae [1,2,3]. Kar interacts with either Cik or Vik forming a stable parallel coiledcoil [7,8,9,10,11] These non-motor polypeptides only show 24% sequence identity, but each has a centrally located ␣-helical domain of ϳ300 amino acids that results in dimerization with Kar3 [7]. In response to mating pheromone, budding yeast initiate polarized cell growth to form a rounded mating projection or “shmoo” tip In these cells, Cik appears to target Kar to cytoplasmic or astral microtubules [7,8,9,10]. Kar3Vik is thought to antagonize the function of Cin and
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