Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, D-06120 Halle, Germany.
J Biol Chem. 2013 Jan 18;288(3):1871-82. doi: 10.1074/jbc.M112.396200. Epub 2012 Nov 30.
Calcium (Ca(2+)) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (K(d) ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca(2+)/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.
钙（Ca(2+))是真核生物中的关键第二信使，调节多种细胞过程，最显著的是通过钙调蛋白（CaM）。在拟南芥中，IQD1（IQ67 结构域 1）是 IQD 家族假定的 CaM 靶标的创始成员。33 个预测的 IQD 蛋白共享一个保守的 67 个氨基酸的结构域，其特征是多个 CaM 募集基序的独特排列，包括所谓的 IQ 基序。虽然 IQD1 已被牵连到防御代谢的调节中，但 IQD 蛋白的生化功能仍有待阐明。在这项研究中，我们表明 IQD1 在体外和酵母双杂交相互作用测定中与多个拟南芥 CaM 和 CaM 样（CML）蛋白结合。CaM 覆盖测定显示 IQD1 与 CaM2 的中等亲和力（K(d)∼0.6 μm）。IQD1 的缺失作图表明 IQ67 结构域对于体外 CaM2 结合的重要性，这得到了最短的 IQD 成员 IQD20 在酵母中与拟南芥 CaM/CMLs 相互作用的证实。cDNA 文库的遗传筛选鉴定出拟南芥驱动蛋白轻链相关蛋白-1（KLCR1）是 IQD1 的相互作用因子。GFP 标记的 IQD1 蛋白在瞬时和稳定转化的植物组织（烟草叶片和拟南芥幼苗）中的微管和细胞核中的亚细胞定位表明 IQD1 和 KLCR1 之间在植物体内的直接相互作用，这得到了 GFP∼IQD1 依赖性招募 RFP∼KLCR1 和 RFP∼CaM2 到微管的支持。总的来说，出现了这样的前景，即 IQD1 和相关蛋白提供了 Ca(2+)/CaM 调节的支架，用于通过驱动蛋白马达蛋白沿微管轨道促进特定货物的细胞运输。
