University of Colorado
Structural insights into the mechanism of movement of the heterodimeric kinesin Kar3Vik1.
Degree: PhD, Microbiology, Molecular Biology and Biochemistry, 2011, University of Colorado
The microtubule-associated motor, Kar3 is a minus-end directed kinesin-14 with important roles in mitosis in Saccharomyces cerevisiae. In vivo
Kar3 forms a heterodimer with Vik1, a non-motor protein that regulates Kar3’s localization and function. Unexpectedly, Vik1 has a structure similar to a kinesin motor domain but lacks an active site for ATP hydrolysis. Despite being unable to hydrolyze ATP, Vik1 was shown by co-sedimentation to bind tightly to microtubules supporting a model where both Kar3 and Vik1 interact with the microtubule during Kar3Vik1’s motility cycle. As it is the hydrolysis of ATP that allows kinesins to dissociate from the microtubule, the way Kar3Vik1 is able to facilitate motility was not known. This work aimed to identify a novel motor-microtubule interaction and obtain insight into Kar3Vik1’s mechanism of movement along microtubules. Using cryo-EM I have shown that Kar3Vik1 binds to microtubules in a highly cooperative fashion through only one of its globular domains. Employing helical reconstruction of Kar3Vik1 bound to microtubules in various nucleotide states, I have revealed that Kar3Vik1 undergoes a large conformational change upon uptake of ATP that results in ~90° rotation of Kar3Vik1’s coiled-coil stalk. This stalk rotation likely represents the powerstroke used by Kar3Vik1 for movement. Using a Nanogold® label, I have demonstrated that Kar3 contacts the microtubule to facilitate this powerstroke. Analysis of Kar3Vik1 constructs with crosslinks engineered to constrain the heterodimer at specific locations provided further insight into the conformational changes that take place during Kar3Vik1’s powerstroke. While I have made a significant contribution to our understanding of Kar3Vik1’s force generating mechanism for movement, I was not able to visualize Vik1 binding to microtubules. My findings thus suggest that Kar3Vik1 does not move along microtubules in a way that is novel, but instead provide evidence for conservation of a motility mechanism among minus end directed motors.
Advisors/Committee Members: Gia Voeltz, Andreas Hoenger, Mark Winey.
Subjects/Keywords: cryo-electron microscopy; cryo-electron tomography; helical reconstruction; kinesin-14; Nanogold-labeling; subvolume averaging; Biological and Chemical Physics
to Zotero / EndNote / Reference
APA (6th Edition):
Cope, J. (2011). Structural insights into the mechanism of movement of the heterodimeric kinesin Kar3Vik1. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/mcdb_gradetds/7
Chicago Manual of Style (16th Edition):
Cope, Julia. “Structural insights into the mechanism of movement of the heterodimeric kinesin Kar3Vik1.” 2011. Doctoral Dissertation, University of Colorado. Accessed November 27, 2020.
MLA Handbook (7th Edition):
Cope, Julia. “Structural insights into the mechanism of movement of the heterodimeric kinesin Kar3Vik1.” 2011. Web. 27 Nov 2020.
Cope J. Structural insights into the mechanism of movement of the heterodimeric kinesin Kar3Vik1. [Internet] [Doctoral dissertation]. University of Colorado; 2011. [cited 2020 Nov 27].
Available from: https://scholar.colorado.edu/mcdb_gradetds/7.
Council of Science Editors:
Cope J. Structural insights into the mechanism of movement of the heterodimeric kinesin Kar3Vik1. [Doctoral Dissertation]. University of Colorado; 2011. Available from: https://scholar.colorado.edu/mcdb_gradetds/7