R-SNARE K-channel interactions in coordinating vesicle trafficking and ion transport

囊泡与靶膜的融合是分泌途径的最后一步。这种融合是由特定的尾部锚定的膜蛋白促进的，即所谓的SNARS。除了这一重要功能，SNARs还调节膜蛋白的活性，如钾通道。在这个Emmy Noether项目的背景下，证明了R-SNARE VAMP721的Long in结构域中的一个保守的酪氨酸基序负责拟南芥中K+通道Kc1和KAT1的结合。这种相互作用导致该通道失活，而不是先前描述的VAMP721在膜融合中的伙伴Qa-Snare SYP121对细胞钾内流的正向调节。与拟南芥密切相关的VAMP723既不与同源SNARE伙伴SYP121(或SYP111)相互作用，也不与钾通道相互作用。有趣的是，在大多数A.thaliana生态型中，酪氨酸基序在VAMP723的Long in结构域中是保守的。这似乎对于与QA-SNARE合作伙伴的互动至关重要(例如，SYP121)，而且还用于VAMP的亚细胞定位和蛋白质稳定性。将VAMP723中的天冬氨酸突变为酪氨酸导致VAMP723D57Y与Qa区SYP121和SYP111(Knolle)相互作用。然而，这个VAMP723D57Y和突变的VAMP721Y57D都不能弥补VAMP721vamp722系的胞质分裂缺陷--至少如果表达是由天然的VAMP721启动子控制的话是不是这样。在接下来的12个月里，有四个要点需要澄清：(1)通过雌二醇诱导的过表达能否补充幼苗的致死表型？(2)VAMP723是否完全保留在ER中？-为了测试这一点，我们有交叉标记线，也将使用Brefeldin A的应用。(3)蛋白质的稳定性是否依赖于启动子？-除了使用雌激素诱导和VAMP721启动子？我们计划分析VAMP723启动子控制下的蛋白质表达。(4)对这些品系进行生化分析：利用GFP-TRAP和质谱仪鉴定和比较不同鞋面的相互作用组。

The fusion of vesicle with target membrane is the final step of the secretory pathway. This fusion is facilitated by specific, tail-anchored membrane proteins, so-called SNAREs. Besides this crucial function SNAREs also regulate the activity of membrane proteins such as potassium channels. In the context of this Emmy Noether project it was demonstrated that a conserved tyrosine motif within the longin domain of the R-SNARE VAMP721 is responsible for the binding of the K+-channel KC1 and KAT1 in Arabidopsis. This interaction leads to a deactivation of the channel contrary to a previously described positive regulation of cellular potassium influx by the Qa-SNARE SYP121, partner of VAMP721 in membrane fusion. The closely related, Arabidopsis-specific VAMP723 does not interact with either the cognate SNARE partner SYP121 (or SYP111) or the potassium channels. Interestingly, the tyrosine motif is conserved within the longin domain of VAMP723 in most A. thaliana ecotypes. This seems crucial for interaction with Qa-SNARE partners (eg. SYP121) but also for subcellular localisation and protein stability of the VAMP. Mutating the aspartate in Col-0 VAMP723 to tyrosine leads to an interaction of VAMP723D57Y with the Qa-SNAREs SYP121 and SYP111 (Knolle). Yet, neither this VAMP723D57Y nor the mutated VAMP721Y57D can complement the cytokinesis defect of the vamp721vamp722 line – at least not if expression is controlled by the native VAMP721 promoter.Four main points require clarification in the following 12 months: (1) Can overexpression via estradiol induction complement the seedling lethal phenotype? (2) Is VAMP723 completely retained in the ER? – To test this we have crossed marker lines and will also use the application of Brefeldin A. (3) Does the protein stability depend on the promoter? – In addition to using estradiol-induction and the VAMP721 promoter we plan to analyse protein expression under control of the VAMP723 promoter. (4) Lastly, the lines should be analysed biochemically: using GFP-trap and mass spectrometry we would like to identify and compare the interactome of the different VAMPs.