Apoplastic calmodulin receptor-like binding proteins in suspension-cultured cells of Arabidopsis thaliana
Apoplastic calmodulin receptor-like binding proteins in suspension-cultured cells of Arabidopsis thaliana
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DOI：
10.1074/jbc.m501349200
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发表时间：
2005-09-09
影响因子：
4.8
通讯作者：
Sun, D
中科院分区：
文献类型：
--
作者：
Cui, SJ;Guo, XQ;Sun, D
钙调素是一种高度保守的蛋白质家族，长期以来一直被认为是细胞内的钙感受器，通过免疫印迹分析发现，CaM存在于拟南芥悬浮培养细胞的培养液和细胞壁中。通过检测核DNA掺入[甲基-H-3]胸腺嘧啶核苷，发现外源性纯化的钙调素对悬浮细胞的增殖有促进作用，而加入钙调素抗血清或钙调素拮抗剂则对悬浮细胞的增殖有抑制作用。用S-35标记的钙调素进行放射配基结合分析表明，拟南芥悬浮培养细胞及其原生质体表面都存在特异的、可逆的和可饱和的钙调蛋白结合位点，其中至少一个位于拟南芥原生质体表面，其K-d接近9.2 nM，两个位于拟南芥悬浮培养细胞外表面，其K-d值接近47.5和830 nM。将S-35标记的钙调蛋白化学交联到原生质体上，发现117kDa和41 kDa的质膜蛋白与钙调蛋白特异结合，而与完整悬浮培养细胞的化学交联证实了更多的钙调蛋白结合蛋白，这些蛋白可能是膜定位的，也可能是细胞壁相关的。综上所述，我们的研究结果首次为拟南芥悬浮培养细胞表面存在质外体钙调素受体样结合蛋白提供了证据，这有力地支持了我们先前的观点，即质外体钙调素作为参与细胞生长发育调控的多肽信号发挥作用。
Calmodulin, a highly conserved protein family that has long been well known as an intracellular calcium sensor, was identified in the culture medium and cell walls of Arabidopsis thaliana suspension-cultured cells by immunoblotting assay. A promotion effect by applying exogenous purified calmodulin and an inhibition effect by the addition of anti-calmodulin anti-serum or calmodulin antagonist to the medium on proliferation of suspension cells were found by monitoring incorporation of [methyl-H-3]thymidine into nuclear DNA. Radioligand binding analysis with S-35-labeled calmodulin indicated the presence of specific, reversible, and saturable calmodulin binding sites on the surface of both A. thaliana suspension-cultured cells and its protoplasts; among them at least one is on the surface of Arabidopsis protoplasts, with the K-d similar to 9.2 nM, and two are on the out-surface of Arabidopsis suspension-cultured cells, with K-d values of similar to 47.5 and 830 nM. Chemical cross-linking of S-35-labeled calmodulin to protoplasts revealed 117- and 41-kDa plasma membrane proteins specifically bound to calmodulin, whereas cross-linking with intact suspension-cultured cells verified more calmodulin binding proteins which might be cell wall-associated in addition to membrane-localized. Taking together, our data provide first evidence for the presence of apoplastic calmodulin receptor-like binding proteins on the cell surface of Arabidopsis suspension-cultured cells, which strongly supports our previous idea that apoplastic calmodulin functions as a peptide signal involved in regulation of cell growth and development.