Department of Biology, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan.
Plant Cell Physiol. 2011 Nov;52(11):1915-30. doi: 10.1093/pcp/pcr131. Epub 2011 Sep 24.
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.
MCA1 是一种质膜蛋白，与拟南芥中的 Ca(2+)内流和机械感应相关。MCA2 是 MCA1 的同源物，两者共享 72.7%的氨基酸序列同一性和几个共同的结构特征，包括假定的跨膜 (TM) 片段、N 端一半的 EF 手样区域、中间的卷曲螺旋基序和 C 端一半的 PLAC8 基序。为了确定对 Ca(2+)摄取活性重要的结构区域，使用酵母表达测定评估 MCA1 和 MCA2 的截断形式的活性。MCA1 的 N 端一半（带有卷曲螺旋基序的 MCA1(1-237)) 没有 Ca(2+)摄取活性，而 MCA2(1-237) 有。没有卷曲螺旋基序的 MCA1 的 N 端一半（MCA1(1-185)) 显示出 Ca(2+)摄取活性，MCA2(1-186) 也是如此。MCA1(1-173) 和 MCA2(1-173) 都具有 EF 手样区域，具有 Ca(2+)摄取活性。MCA1 和 MCA2 中的一个假定 TM 片段（Ile11-Ala33) 的缺失和 Asp21 到天冬酰胺的突变消除了 Ca(2+)摄取活性。最后，缺乏 N 端一半的 MCA1(173-421) 和 MCA2(173-416) 没有 Ca(2+)摄取活性。这些结果表明，两种蛋白的 N 端一半都具有 EF 手样区域，是 Ca(2+)摄取所必需的，卷曲螺旋基序负调控 MCA1，正调控 MCA2。
