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.

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