RICARDI Martiniano Maria
A Tyrosine Phospho-switch within the Longin Domain of VAMP721 modulates SNARE functionality
MARTINIANO M. RICARDI; NIKLAS WALLMEROTH; CECILIA CERMESONI; DIETMAR GERALD MEHLHORN; SANDRA RICHTER; LEI ZHANG; JOSEPHINE MITTENDORF; INGEBORG GODEHARDT; KENNETH WAYNE BERENDZEN; EDDA VON ROEPENACK-LAHAYE; YORK-DIETER STIERHOF; VOLKER LIPKA; GERD JÜRGENS; CHRISTOPHER GREFEN
Cold Spring Harbor Laboratory
The final stepin secretion is membrane fusion facilitated by SNARE proteins that reside inopposite membranes. The formation of a trans-SNARE complex between one R andthree Q coiled-coiled SNARE domains drives the final approach of the membranesproviding the mechanical energy for fusion. Biological control of thismechanism is exerted by additional domains within some SNAREs. For example, theN-terminal Longin domain (LD) of R-SNAREs (also called Vesicle-associatedmembrane proteins, VAMPs) can fold back onto the SNARE domain blocking interactionwith other cognate SNAREs. The LD may also determine the subcellularlocalization via interaction with other trafficking related proteins. Here, we provide cell-biological and geneticevidence that phosphorylation of the Tyrosine57 residue regulates the functionalityof VAMP721. We found that an aspartate mutation mimics phosphorylation, leadingto protein instability and subsequent degradation in lytic vacuoles. The mutantSNARE also fails to rescue the defects of vamp721vamp722loss-of-function lines in spite of its wildtype-like localization within thesecretory pathway and the ability to interact with cognate SNARE partners. Mostimportantly, it imposes a dominant negative phenotype interfering with rootgrowth, normal secretion and cytokinesis in wildtype plants generating largeaggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57Fneeds higher gene dosage to rescue double mutants in comparison to native VAMP721underpinning that phosphorylation modulates SNARE function. We propose a modelwhere a short-lived phosphorylation of Y57 serves as a regulatory step to controlVAMP721 activity, favouring its open state and interaction with cognatepartners to ultimately drive membrane fusion.