Structural characterization of the mechanosensitive channel candidate MCA2 from Arabidopsis thaliana

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. © 2014 Shigematsu et al.