Determination of structural regions important for Ca ²⁺ uptake activity in arabidopsis MCA1 and MCA2 expressed in yeast

MCA1 is a plasma membrane protein that correlates Ca ²⁺ influx and mechanosensing in Arabidopsis. MCA2 is a paralog of MCA1, and both share 72.7 amino acid sequence identity and several common structural features, including putative transmembrane (TM) segments, an EF hand-like region in the N-terminal half, a coiled-coil motif in the middle and a PLAC8 motif in the C-terminal half. To determine structural regions important for Ca ²⁺ uptake activity, the activity of truncated forms of MCA1 and MCA2 was assessed using yeast expression assays. The N-terminal half of MCA1 with a coiled-coil motif (MCA1 ^1-237) did not have Ca ²⁺ uptake activity, while MCA2 ^1-237 did. The N-terminal half of MCA1 without the coiled-coil motif (MCA1 ^1-185) showed Ca ²⁺ uptake activity, as did MCA2 ^1-186. Both MCA1 ^1-173 and MCA2 ^1-173 having the EF hand-like region had Ca ²⁺ uptake activity. Deletion of a putative TM segment (Ile11-Ala33) and the Asp21 to asparagine mutation in MCA1 and MCA2 abolished Ca ²⁺ uptake activity. Finally, MCA1 ^173-421 and MCA2 ^173-416 lacking the N-terminal half had no Ca ²⁺ uptake activity. These results suggest that the N-terminal half of both proteins with the EF hand-like region is necessary and sufficient for Ca ²⁺ uptake and that the coiled-coil motif regulates MCA1 negatively and MCA2 positively.