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

RICARDI, MARTINIANO MARIA; WALLMEROTH, NIKLAS; CERMESONI, CECILIA; MEHLHORN, DIETMAR GERALD; RICHTER, SANDRA; ZHANG, LEI; MITTENDORF, JOSEPHINE; GODEHARDT, INGEBORG; BERENDZEN, KENNETH WAYNE; VON ROEPENACK?LAHAYE, EDDA; STIERHOF, YORK?DIETER; LIPKA, VOLKER; JÜRGENS, GERD; GREFEN, CHRISTOPHER

PLANT JOURNAL

WILEY-BLACKWELL PUBLISHING, INC

Año: 2023 vol. 116 p. 1633 - 1651

0960-7412

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 domainsdrives the final approach of the membranes providing the mechanical energy for fusion. Biological controlof this mechanism is exerted by additional domains within some SNAREs. For example, the N-terminalLongin domain (LD) of R-SNAREs (also called Vesicle-associated membrane proteins, VAMPs) can fold backonto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine thesubcellular localization via interaction with other trafficking-related proteins. Here, we provide cellbiological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionalityof VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instabilityand subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects ofvamp721vamp722 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 plantsgenerating large aggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57Fneeds higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning thatphosphorylation modulates SNARE function. We propose a model where short-lived phosphorylation of Y57serves as a regulatory step to control VAMP721 activity, favoring its open state and interaction with cognate partners to ultimately drive membrane fusion.