Luminescence detection of SNARE-SNARE interaction in Arabidopsis protoplasts
Luminescence detection of SNARE-SNARE interaction in Arabidopsis protoplasts
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DOI：
10.1007/s11103-009-9581-z
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发表时间：
2010-03-01
影响因子：
5.1
通讯作者：
Sato, Masa H.
中科院分区：
文献类型：
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作者：
Kato, Naohiro;Fujikawa, Yukichi;Sato, Masa H.
膜相关蛋白SNARE（可溶性N-乙基马来酰亚胺敏感性融合蛋白附着蛋白受体）提供细胞囊泡融合至真核细胞中的靶细胞器膜所需的最小融合机制。尽管融合机制在所有真核生物中具有保守性，但在高等植物中鉴定功能性SNARE对仍然具有挑战性。我们开发了一种基于分裂荧光素酶互补测定的方法，用于通过在96孔板中以生理水平表达重组蛋白的拟南芥原生质体内发光来检测陷阱-陷阱相互作用的变化。通过三个实验证实了测定的可靠性。首先，通过发光的减少来检测由内体定位的AtVAM 3/SYP 22（植物22的突触融合蛋白）中邻近SNARE基序的单个氨基酸取代引起的SNARE-SNARE相互作用的减少。其次，实时检测响应于叠氮化钠的质膜定位SYP 121和VAMP 722之间的相互作用的减少。第三，用这种方法研究的21个SNARE对的结果与以前报道的免疫共沉淀试验的结果基本一致。利用该方法，我们新发现SYP 121和VAMP 722之间的相互作用在光照下培养的原生质体中显著增加。显微镜观察从自身启动子表达GFP-SYP 121（绿色荧光蛋白标记的SYP 121）的转基因拟南芥表明GFP-SYP 121的质膜定位由光维持。这表明SYP 121介导的拟南芥囊泡运输途径可能受光的调控。总的来说，这篇文章展示了一种方法，可以产生新的生物学见解的陷阱蛋白在植物细胞中的相互作用。
Membrane associated proteins SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) provide the minimal fusion machinery necessary for cellular vesicles to fuse to target organelle membranes in eukaryotic cells. Despite the conserved nature of the fusion machinery in all eukaryotes, it still remains challenging to identify functional SNARE pairs in higher plants. We developed a method based on a split-luciferase complementation assay for detecting changes in SNARE-SNARE interaction by luminescence within Arabidopsis protoplasts that express recombinant proteins at physiological levels in 96-well plates. The reliability of the assay was confirmed by three experiments. First, reduction of the SNARE-SNARE interaction caused by a single amino acid substitution adjacent to the SNARE motif in endosome-localized AtVAM3/SYP22 (syntaxin of plant 22) was detected by a reduction of luminescence. Second, reduction of the interaction between plasma-membrane localized SYP121 and VAMP722 in response to sodium azide was detected in real-time. Third, the results of 21 SNARE pairs investigated by this method largely agreed with the results from previously reported co-immunoprecipitation assays. Using the method, we newly identified the interaction between SYP121 and VAMP722 was significantly increased when the protoplasts were incubated in the light. Microscopic observation of transgenic Arabidopsis expressing GFP-SYP121 (green fluorescent protein tagged SYP121) from its own promoter suggested that the plasma-membrane localization of GFP-SYP121 is maintained by light. These suggested that the vesicle trafficking pathway mediated by SYP121 might be regulated by light in Arabidopsis. In general, this article demonstrated the method that can generate new biological insight of the SNARE protein interactions in plant cells.