Abstract
Kar3, a Saccharomyces cerevisiae microtubule minus-end-directed kinesin-14, dimerizes with either Vik1 or Cik1. The C-terminal globular domain of Vik1 exhibits the structure of a kinesin motor domain and binds microtubules independently of Kar3 but lacks a nucleotide binding site. The only known function of Kar3Vik1 is to cross-link parallel microtubules at the spindle poles during mitosis. In contrast, Kar3Cik1 depolymerizes microtubules during mating but cross-links antiparallel microtubules in the spindle overlap zone during mitosis. A recent study showed that Kar3Vik1 binds across adjacent microtubule protofilaments and uses a minus-end-directed powerstroke to drive ATP-dependent motility. The presteady-state experiments presented here extend this study and establish an ATPase model for the powerstroke mechanism. The results incorporated into the model indicate that Kar3Vik1 collides with the microtubule at 2.4 μm(-1) s(-1) through Vik1, promoting microtubule binding by Kar3 followed by ADP release at 14 s(-1). The tight binding of Kar3 to the microtubule destabilizes the Vik1 interaction with the microtubule, positioning Kar3Vik1 for the start of the powerstroke. Rapid ATP binding to Kar3 is associated with rotation of the coiled-coil stalk, and the postpowerstroke ATP hydrolysis at 26 s(-1) is independent of Vik1, providing further evidence that Vik1 rotates with the coiled coil during the powerstroke. Detachment of Kar3Vik1 from the microtubule at 6 s(-1) completes the cycle and allows the motor to return to its initial conformation. The results also reveal key differences in the ATPase cycles of Kar3Vik1 and Kar3Cik1, supporting the fact that these two motors have distinctive biological functions.
Highlights
Kar3Vik1 binds side-by-side microtubule protofilaments and utilizes a minus-end-directed powerstroke
Rapid ATP binding to Kar3 is associated with rotation of the coiled-coil stalk, and the postpowerstroke ATP hydrolysis at 26 s؊1 is independent of Vik1, providing further evidence that Vik1 rotates with the coiled coil during the powerstroke
Because the nonhydrolyzable ATP analog AMPPNP promotes rotation of the Kar3Vik1 coiled-coil stalk [14], we propose that the 55 sϪ1 isomerization measured here represents the rate constant of the ATP-promoted stalk rotation to generate the postpowerstroke intermediate that is poised for ATP hydrolysis
Summary
Kar3Vik binds side-by-side microtubule protofilaments and utilizes a minus-end-directed powerstroke. Unlike the well known N-terminal motor domain kinesins that use an asymmetric hand-over-hand mechanism for MT plus-end-directed processive stepping (8 –11), kinesin-14s use an MT minus-end-directed rotation or bending of the coiled-coil stalk to generate force [12,13,14]. This rotation coupled to ATP turnover is designated the powerstroke and is used to slide one MT relative to another [15,16,17,18,19]. The results provide insights to understand intermolecular communication mediated through the neck coiled coil for Ncd, Kar3Vik, and Kar3Cik
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
