Abstract：
The final step in secretion is membrane fusion facilitated by SNARE proteins that reside in opposite membranes. The formation of a trans-SNARE complex between one R and three Q coiled-coiled SNARE domains drives the final approach of the membranes providing the mechanical energy for fusion. Biological control of this mechanism is exerted by additional domains within some SNAREs. For example, the N-terminal Longin domain (LD) of R-SNAREs (also called Vesicle-associated membrane proteins, VAMPs) can fold back onto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine the subcellular localization via interaction with other trafficking-related proteins. Here, we provide cell-biological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionality of VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instability and subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects of vamp721vamp722 loss-of-function lines in spite of its wildtype-like localization within the secretory pathway and the ability to interact with cognate SNARE partners. Most importantly, it imposes a dominant negative phenotype interfering with root growth, normal secretion and cytokinesis in wildtype plants generating large aggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57F needs higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning that phosphorylation modulates SNARE function. We propose a model where short-lived phosphorylation of Y57 serves as a regulatory step to control VAMP721 activity, favoring its open state and interaction with cognate partners to ultimately drive membrane fusion.
摘要：
分泌的最后一步是由位于相对膜中的SNARE蛋白促进的膜融合。在一个R和三个Q盘绕-盘绕的SNARE结构域之间形成反式-SNARE复合物驱动膜的最终途径,从而提供用于融合的机械能。该机制的生物学控制由一些SNARE内的额外结构域施加。例如,R-SNAREs的N末端Longin结构域（LD）（也称为囊泡相关膜蛋白，VAMP）可以折回SNARE域，阻断与其他同源SNARE的相互作用。LD还可以通过与其他运输相关蛋白的相互作用来确定亚细胞定位。这里,我们提供了细胞生物学和遗传学证据,证明Tyrosine57残基的磷酸化调节VAMP721的功能.我们发现天冬氨酸突变模拟磷酸化，导致蛋白质不稳定并随后在裂解液泡中降解。尽管突变体SNARE在分泌途径中具有类似野生型的定位以及与同源SNARE伴侣相互作用的能力，但突变体SNARE也未能挽救vamp721vamp722功能丧失系的缺陷。最重要的是,它强加了干扰根生长的显性阴性表型，野生型植物的正常分泌和胞质分裂产生主要含有分泌性囊泡的大聚集体。与磷酸化调节SNARE功能的天然VAMP721相比，非磷酸化VAMP721Y57F需要更高的基因剂量来挽救双突变体。我们提出了一个模型，其中Y57的短寿命磷酸化作为控制VAMP721活性的调节步骤，支持其开放状态和与同源伴侣的相互作用，以最终驱动膜融合。
