Determination of regions important for Ca²⁺ uptake activity in Arabidopsis MCA1 and MCA2 expressed in yeast

Abstract

MCA1 and MCA2 are plasma membrane proteins that correlate Ca²⁺ influx and mechanosensing in Arabidopsis. MCA1 and MCA2 share 72.7% amino acid sequence identity and several common structural features, including putative transmembrane segments, an EF hand-like region in the N-half, a coiled-coil motif in the middle, and a PLAC8 motif in the C-half. To determine regions important for Ca²⁺ uptake activity, the activity of truncated forms of MCA1 and MCA2 was assessed using yeast expression assays. MCA1^1-173 and MCA2^1-173 had Ca²⁺ uptake activity, while MCA1^173-421 and MCA2^173-416 lacking the N-half had no Ca²⁺ uptake activity. MCA1^1-173 and MCA2^1-173 have a putative transmembrane region, and helical wheel analysis of the region showed that highly hydrophilic residues were lined up on one side of the helix. To test the possibility that Asp²¹ in the TM1 plays a role in Ca²⁺ uptake, Asp²¹ was replaced with Asn. MCA1^D21N had no Ca²⁺ uptake activity and MCA2^D21N had partial activity, although the levels of the mutant proteins were not reduced. These results suggest that the N-half of both proteins with the EF hand-like region is necessary and sufficient for Ca²⁺ uptake.