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Functional Characterization of Calmodulin-like Proteins, CML13 and CML14, as Novel Light Chains of Arabidopsis Class VIII Myosins

Kyle Symonds, Howard J. Teresinski, Bryan Hau, Einat Sadot, Vikas Dwivedi, Eduard Belausov, Sefi Bar-Sinai, Motoki Tominaga, Takeshi Haraguchi, Kohji Ito, Wayne A. Snedden
doi: https://doi.org/10.1101/2023.05.12.540561
Kyle Symonds
1Department of Biology, Queen’s University, Kingston, ON, Canada
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Howard J. Teresinski
1Department of Biology, Queen’s University, Kingston, ON, Canada
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Bryan Hau
1Department of Biology, Queen’s University, Kingston, ON, Canada
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Einat Sadot
2Institute of Plant Sciences, Volcani Institute, ARO, Rishon LeZion 7528809, Israel
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Vikas Dwivedi
2Institute of Plant Sciences, Volcani Institute, ARO, Rishon LeZion 7528809, Israel
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Eduard Belausov
2Institute of Plant Sciences, Volcani Institute, ARO, Rishon LeZion 7528809, Israel
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Sefi Bar-Sinai
2Institute of Plant Sciences, Volcani Institute, ARO, Rishon LeZion 7528809, Israel
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Motoki Tominaga
3Faculty of Education and Integrated Arts and Sciences, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan
4Major in Integrative Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan
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Takeshi Haraguchi
5Department of Biology, Graduate School of Science, Chiba University, Inage-ku, Chiba, 263-8522, Japan
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Kohji Ito
5Department of Biology, Graduate School of Science, Chiba University, Inage-ku, Chiba, 263-8522, Japan
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Wayne A. Snedden
1Department of Biology, Queen’s University, Kingston, ON, Canada
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  • For correspondence: sneddenw@queensu.ca
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Abstract

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck regions to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins, CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a member of the myosin VIII class. Here, using in vivo and in vitro assays we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among ten CML isoforms tested for in planta binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein-interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. Subcellular localization analysis indicated that CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with RFP-myosin fusion proteins containing IQ- and tail-domains of myosin VIIIs. In addition, in vitro actin-motility assays using recombinant myosin holoenzymes demonstrated that CaM, CML13, and CML14 function as light chains for myosin VIIIs. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.

Highlight Myosins are key proteins in the plant cytoskeleton, but the identity of their light chain components is unknown. Here, we show that calmodulin-like proteins function as novel myosin light chains.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted May 13, 2023.
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Functional Characterization of Calmodulin-like Proteins, CML13 and CML14, as Novel Light Chains of Arabidopsis Class VIII Myosins
Kyle Symonds, Howard J. Teresinski, Bryan Hau, Einat Sadot, Vikas Dwivedi, Eduard Belausov, Sefi Bar-Sinai, Motoki Tominaga, Takeshi Haraguchi, Kohji Ito, Wayne A. Snedden
bioRxiv 2023.05.12.540561; doi: https://doi.org/10.1101/2023.05.12.540561
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Functional Characterization of Calmodulin-like Proteins, CML13 and CML14, as Novel Light Chains of Arabidopsis Class VIII Myosins
Kyle Symonds, Howard J. Teresinski, Bryan Hau, Einat Sadot, Vikas Dwivedi, Eduard Belausov, Sefi Bar-Sinai, Motoki Tominaga, Takeshi Haraguchi, Kohji Ito, Wayne A. Snedden
bioRxiv 2023.05.12.540561; doi: https://doi.org/10.1101/2023.05.12.540561

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