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

点击翻译按钮获取中文摘要

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.