Determination of Structural Regions Important for Ca2+ Uptake Activity in Arabidopsis MCA1 and MCA2 Expressed in Yeast
Determination of Structural Regions Important for Ca2+ Uptake Activity in Arabidopsis MCA1 and MCA2 Expressed in Yeast
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DOI：
10.1093/pcp/pcr131
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发表时间：
2011-11-01
影响因子：
4.9
通讯作者：
Iida, Hidetoshi
中科院分区：
文献类型：
--
作者：
Nakano, Masataka;Iida, Kazuko;Iida, Hidetoshi
MCA 1是拟南芥中与Ca 2+内流和机械感应相关的质膜蛋白。MCA 2与MCA 1同源性为72.7%，具有几个共同的结构特征，包括跨膜（TM）片段、N端的EF手样区、中间的卷曲螺旋基序和C端的PLAC 8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了MCA 1和MCA 2的截短形式的活性。具有卷曲螺旋基序的MCA 1的N-末端的一半（MCA 1（1-237））不具有Ca 2+摄取活性，而MCA 2（1-237）具有Ca 2+摄取活性。没有卷曲螺旋基序的MCA 1的N-末端一半（MCA 1（1-185））显示出Ca 2+摄取活性，MCA 2（1-186）也是如此。具有EF手样区域的MCA 1（1-173）和MCA 2（1-173）都具有Ca 2+摄取活性。MCA 1和MCA 2中缺失推定的TM片段（Ile 11-Ala 33）以及Asp 21突变为天冬酰胺，消除了Ca 2+吸收活性。最后，缺少N-末端一半的MCA 1（173-421）和MCA 2（173-416）没有Ca 2+摄取活性。这些结果表明，这两种蛋白质的N-末端一半与EF手样区域是必要的和足够的钙摄取和卷曲螺旋基序调节MCA 1负和MCA 2正。
MCA1 is a plasma membrane protein that correlates Ca2+ 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 Ca2+ 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 Ca2+ uptake activity, while MCA2(1-237) did. The N-terminal half of MCA1 without the coiled-coil motif (MCA1(1-185)) showed Ca2+ uptake activity, as did MCA2(1-186). Both MCA1(1-173) and MCA2(1-173) having the EF hand-like region had Ca2+ uptake activity. Deletion of a putative TM segment (Ile11-Ala33) and the Asp21 to asparagine mutation in MCA1 and MCA2 abolished Ca2+ uptake activity. Finally, MCA1(173-421) and MCA2(173-416) lacking the N-terminal half had no Ca2+ uptake activity. These results suggest that the N-terminal half of both proteins with the EF hand-like region is necessary and sufficient for Ca2+ uptake and that the coiled-coil motif regulates MCA1 negatively and MCA2 positively.