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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions
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K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions

机译：通过针对膜蛋白相互作用的系统研究而优化的遗传系统检测到的K +通道相互作用

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摘要

Organization of proteins into complexes is crucial for many cellular functions. However, most proteomic approaches primarily detect protein interactions for soluble proteins but are less suitable for membrane-associated complexes. Here we describe a mating-based split ubiquitin system (mbSUS) for systematic identification of interactions between membrane proteins as well as between membrane and soluble proteins. mbSUS allows in vivo cloning of PCR products into a vector set, detection of interactions via mating, regulated expression of baits, and improved selection of interacting proteins. Cloning is simplified by introduction of A attachment sites for GATEWAY. Homo- and heteromeric interactions between Arabidopsis K+ channels KAT1, AKT1, and AKT2 were identified. Tests with deletion mutants demonstrate that the C terminus of KAT1 and AKT1 is necessary for physical assembly of complexes. Screening of a sorted collection of 84 plant proteins with K+ channels as bait revealed differences in oligomerization between KAT1, AKT1, and AtKC1, and allowed detection of putative interacting partners of KAT1 and AtKC1. These results show that mbSUS is suited for systematic analysis of membrane protein interactions.
机译：将蛋白质组织成复合物对于许多细胞功能至关重要。但是，大多数蛋白质组学方法主要检测可溶性蛋白质的蛋白质相互作用，但不适用于膜相关复合物。在这里，我们描述了基于交配的分裂泛素系统（mbSUS），用于系统鉴定膜蛋白之间以及膜与可溶性蛋白之间的相互作用。 mbSUS允许将PCR产物体内克隆到载体中，通过交配检测相互作用，调节诱饵的表达，并改善相互作用蛋白的选择。通过为GATEWAY引入A附件站点，简化了克隆过程。鉴定了拟南芥K +通道KAT1，AKT1和AKT2之间的同源和异源相互作用。用缺失突变体进行的测试表明，KAT1和AKT1的C末端对于复合物的物理组装是必需的。筛选带有K +通道作为诱饵的84种植物蛋白的排序集合，揭示了KAT1，AKT1和AtKC1之间的寡聚差异，并允许检测到假定的KAT1和AtKC1相互作用伴侣。这些结果表明，mbSUS适用于膜蛋白相互作用的系统分析。

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