A Novel Motif Essential for SNARE Interaction with the K+ Channel KC1 and Channel Gating in Arabidopsis
A Novel Motif Essential for SNARE Interaction with the K+ Channel KC1 and Channel Gating in Arabidopsis
复制标题
DOI：
10.1105/tpc.110.077768
复制
发表时间：
2010-09-01
期刊：
影响因子：
11.6
通讯作者：
Blatt, Michael R.
中科院分区：
文献类型：
--
作者：
Grefen, Christopher;Chen, Zhonghua;Blatt, Michael R.
拟南芥的SNARE（可溶性n -乙基丙烯酰亚胺敏感因子蛋白附着蛋白受体）蛋白SYP121 （=SYR1/PEN1）促进囊泡交通，向质膜输送离子通道和其他货物，有助于植物细胞的扩增和防御。最近，我们报道了SYP121也直接与K+通道亚基KC1相互作用，并与第二个K+通道亚基AKT1形成三方复合物，以控制通道门控和K+转运。在这里，我们报道了SYP121与KC1相互作用的先决条件是分离出一个最小序列基序。我们利用酵母交配分裂泛素和体内双分子荧光互补法进行蛋白-蛋白相互作用、表达和电生理分析。结果表明，SYP121与KC1的相互作用与一个新的FxRF基序相关，该基序独特地位于SNARE序列的前12个残基中，该基序是在异源表达时依赖于SNARE的K+通道门控改变的最小要求，并且在SYP121突变体拟南芥植物中，KC1相关的根表皮K+电流的恢复依赖于含有该基序的SNARE构建体的表达。这些结果确定了FxRF序列是先前未被识别的陷阱离子通道相互作用所需的基序，并使我们提出了理解囊泡运输与跨膜离子运输协调的机制框架。
The SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) protein SYP121 (=SYR1/PEN1) of Arabidopsis thaliana facilitates vesicle traffic, delivering ion channels and other cargo to the plasma membrane, and contributing to plant cell expansion and defense. Recently, we reported that SYP121 also interacts directly with the K+ channel subunit KC1 and forms a tripartite complex with a second K+ channel subunit, AKT1, to control channel gating and K+ transport. Here, we report isolating a minimal sequence motif of SYP121 prerequisite for its interaction with KC1. We made use of yeast mating-based split-ubiquitin and in vivo bimolecular fluorescence complementation assays for protein-protein interaction and of expression and electrophysiological analysis. The results show that interaction of SYP121 with KC1 is associated with a novel FxRF motif uniquely situated within the first 12 residues of the SNARE sequence, that this motif is the minimal requirement for SNARE-dependent alterations in K+ channel gating when heterologously expressed, and that rescue of KC1-associated K+ current of the root epidermis in syp121 mutant Arabidopsis plants depends on expression of SNARE constructs incorporating this motif. These results establish the FxRF sequence as a previously unidentified motif required for SNARE-ion channel interactions and lead us to suggest a mechanistic framework for understanding the coordination of vesicle traffic with transmembrane ion transport.