The kinesin-binding site on the microtubule has not been identified because of the technical difficulties involved in the mutant analyses of tubulin. Exploiting the budding yeast expression system, we succeeded in replacing the negatively charged residues in the alpha-helix 12 of beta-tubulin with alanine and analyzed their effect on kinesin-microtubule interaction in vitro. The microtubule gliding assay showed that the affinity of the microtubules for kinesin was significantly reduced in E410A, D417A, and E421A, but not in E412A mutant. The unbinding force measurement revealed that in the former three mutants, the kinesin-microtubule interaction in the adenosine 5'-[beta,gamma-imido]triphosphate state (AMP-PNP state) became less stable when a load was imposed towards the microtubule minus end. In parallel with this decreased stability, the stall force of kinesin was reduced. Our results implicate residues E410, D417, and E421 as crucial for the kinesin-microtubule interaction in the strong binding state, thereby governing the size of kinesin stall force.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1698889 | PMC |
| http://dx.doi.org/10.1038/sj.emboj.7601442 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
BimC family proteins are bipolar motor proteins belonging to the kinesin superfamily which promote mitosis by crosslinking and sliding apart antiparallel microtubules. Understanding the binding mechanism between the kinesin and the microtubule is crucial for researchers to make advances in the treatment of cancer and other malignancies. Experimental research has shown that the ion concentration affects the function of BimC significantly.
View Article and Find Full Text PDFTubulin CFEOM mutations both inhibit or activate kinesin motor activity.
Mol Biol Cell
March 2024
Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011.
Kinesin-mediated transport along microtubules is critical for axon development and health. Mutations in the kinesin Kif21a, or the microtubule subunit β-tubulin, inhibit axon growth and/or maintenance resulting in the eye-movement disorder congenital fibrosis of the extraocular muscles (CFEOM). While most examined CFEOM-causing β-tubulin mutations inhibit kinesin-microtubule interactions, Kif21a mutations activate the motor protein.
View Article and Find Full Text PDFCytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions.
Nat Cell Biol
July 2022
Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA.
A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. By contrast, the principal effectors of Hedgehog signalling, the GLI transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How GLI is tethered to microtubules remains unclear.
View Article and Find Full Text PDFSynergistic autoinhibition and activation mechanisms control kinesin-1 motor activity.
Cell Rep
May 2022
Department of Molecular and Cellular Biology, University of California, Davis, 145 Briggs Hall, Davis, CA 95616, USA. Electronic address:
Kinesin-1 activity is regulated by autoinhibition. Intramolecular interactions within the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The KHC also binds to the kinesin light chain (KLC), which has been implicated in both autoinhibition and activation of the motor.
View Article and Find Full Text PDFLinear-Zero Mode Waveguides for Single-Molecule Fluorescence Observation of Nucleotides in Kinesin-Microtubule Motility Assay.
Methods Mol Biol
April 2022
Department of Micro Engineering, Kyoto University, Kyoto, Japan.
Single-molecule fluorescence microscopy is a key tool to investigate the chemo-mechanical coupling of microtubule-associated motor proteins, such as kinesin. However, a major limitation of the implementation of single-molecule observation is the concentration of fluorescently labeled molecules. For example, in total internal reflection fluorescence microscopy, the available concentration is of the order of 10 nM.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!