Structural characterization of the mechanosensitive channel candidate MCA2 from Arabidopsis thaliana

Hideki Shigematsu; Kazuko Iida; Masataka Nakano; Pratima Chaudhuri; Hidetoshi Iida; Kuniaki Nagayama

doi:10.1371/journal.pone.0087724

Structural characterization of the mechanosensitive channel candidate MCA2 from Arabidopsis thaliana

PLoS One. 2014 Jan 27;9(1):e87724. doi: 10.1371/journal.pone.0087724. eCollection 2014.

Authors

Hideki Shigematsu¹, Kazuko Iida², Masataka Nakano³, Pratima Chaudhuri¹, Hidetoshi Iida⁴, Kuniaki Nagayama⁵

Affiliations

¹ Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan.
² Biomembrane Laboratory, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.
³ Department of Biology, Tokyo Gakugei University, Koganei, Tokyo, Japan.
⁴ Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan ; Department of Biology, Tokyo Gakugei University, Koganei, Tokyo, Japan.
⁵ Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan ; National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi, Japan ; Department of Physiological Sciences, Graduate University for Advanced Studies, Okazaki, Aichi, Japan.

Abstract

Mechanosensing in plants is thought to be governed by sensory complexes containing a Ca²⁺-permeable, mechanosensitive channel. The plasma membrane protein MCA1 and its paralog MCA2 from Arabidopsis thaliana are involved in mechanical stress-induced Ca²⁺ influx and are thus considered as candidates for such channels or their regulators. Both MCA1 and MCA2 were functionally expressed in Sf9 cells using a baculovirus system in order to elucidate their molecular natures. Because of the abundance of protein in these cells, MCA2 was chosen for purification. Purified MCA2 in a detergent-solubilized state formed a tetramer, which was confirmed by chemical cross-linking. Single-particle analysis of cryo-electron microscope images was performed to depict the overall shape of the purified protein. The three-dimensional structure of MCA2 was reconstructed at a resolution of 26 Å from 5,500 particles and appears to comprise a small transmembrane region and large cytoplasmic region.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Arabidopsis / genetics*
Arabidopsis Proteins / chemistry
Arabidopsis Proteins / genetics
Arabidopsis Proteins / metabolism*
Calcium Channels / genetics
Calcium Channels / metabolism*
Cryoelectron Microscopy
Mechanotransduction, Cellular / genetics*
Membrane Proteins / chemistry
Membrane Proteins / genetics
Membrane Proteins / metabolism*
Models, Molecular*
Protein Conformation
Saccharomyces cerevisiae
Sf9 Cells
Spodoptera

Substances

Arabidopsis Proteins
Calcium Channels
MCA1 protein, Arabidopsis
MCA2 protein, Arabidopsis
Membrane Proteins

Grants and funding

This study was partly supported by a Grant-in-Aid for Creative Scienti??c Research (No. 13GS0016 to K.N.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Core Research for Evolutional Science and Technology (CREST) program of the Japan Science and Technology Corporation to K.N. It was also supported by Grants-in-Aid for Scientific Research (B) (No. 21370017 to H.I.) and for Scientific Research on Priority Areas (Nos. 19045009, 21026009, 23120509 and 25120708 to H.I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; a grant from the Novartis Foundation (Japan) for the Promotion of Science (No. KEN20-191 to H.I.); and a grant for the Frontiers of Membrane Protein Research (a Joint Project between the Institute for Protein Research, Osaka University and Okazaki Institute for Integrative Bioscience) to K.N. and H.I. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.