A tyrosine phospho-switch within the Longin domain of VAMP721 modulates SNARE functionality.

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.

Where R-SNAREs like to roam - the vesicle-associated membrane proteins VAMP721 & VAMP722 in trafficking hotspots.

VAMP721 and VAMP722, play crucial roles in membrane fusion at post-Golgi compartments. They are involved in cell plate formation, recycling, endocytosis, and secretion. While individual SNARE actors and regulators exhibit significant overlap, specificity is achieved through distinct combinations of these components. Cytokinesis-related SNAREs traffic as preformed CIS-complexes, which require disassembly by the NSF/αSNAP chaperoning complex to facilitate subsequent homotypic fusion at the cell plate. Recent findings suggest a similar mechanism may operate during secretion. Regulation of VAMP721 activity involves interactions with tethers, GTPases, and Sec1/Munc18 proteins, along with a newly discovered phosphorylation at Tyrosine residue 57. These advances provide valuable insights into the fascinating world of cellular trafficking and membrane fusion.