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 NH 2 -terminal glutathione S-transferase (GST) tag followed by the Kar3 sequence that is predicted to form an extended ␣-helical coiled-coil. The ␣-helical domain leads into the neck linker and COOHterminal 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 GSTKar3 readily detached from the microtubule rather than remaining bound for multiple ATP turnovers. These results indicate that the extended ␣-helical domain NH 2 -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.

Kar3 interaction with Cik1 alters motor structure and function

“…Recently, Mackey et al (2004) reported analytical ultracentrifugation data that they interpreted as showing that GST/Kar3 277–729 is a monomer in vitro . This conclusion was based on the determination of a sedimentation coefficient of 5.36 s for the protein.…”

“…However, a value of 5.36 s is consistent with either a globular monomer or an elongated dimer. The s value alone is not sufficient to calculate the protein molecular mass and determine its association state—the Stokes radius is also required and this was not determined by Mackey et al (2004). Instead, the conclusion that GST/Kar3 277–729 is a monomer was based only on the s value.…”

“…Instead, the conclusion that GST/Kar3 277–729 is a monomer was based only on the s value. Mackey et al (2004) also performed sedimentation equilibrium studies with GST/Kar3 277–729. However, they interpreted their data to show that the protein was a monomer ‘at the early equilibrium times… but was prone to irreversible aggregation to dimmers’ during the centrifugation runs, even at the lowest protein concentration (2 μM), where they reported that the aggregation was the worst.…”

The ATPase Pathway That Drives the Kinesin-14 Kar3Vik1 Powerstroke

“…The rates of the initial exponential burst phase were plotted as a function of increasing ATP concentration (Fig. 3B) (38,39), we conclude that ATP hydrolysis is independent of Vik1 or Cik1, but heterodimerization through the coiled coil does have an effect. Fig.…”

Role of Transcription Factor Kar4 in Regulating Downstream Events in the Saccharomyces cerevisiae Pheromone Response Pathway

“…Upon cell fusion, the cytoplasmic contents of each cell intermingle, facilitating the connection of cytoplasmic microtubules via Kar3, a kinesin-related protein, and Cik1, its associated light chain (33,38). During nuclear congression, movement and microtubule shortening are produced by Kar3 and Cik1 acting on the cytoplasmic microtubules (25,26,33). Finally, the nuclear envelopes fuse at the spindle pole bodies, resulting in the formation of a single diploid nucleus (3).…”