Functional relevance of an individual amino acidic residue within the longin domain of the R-SNARE proteins VAMP721 and VAMP723.

MARTINIANO MARIA RICARDI

Glasgow

Workshop; 8TH INTERNATIONAL WORKSHOP ON PLANT MEMBRANE BIOLOGY; 2019

University of Glasgow

SNARE proteins facilitate the final step in vesicle traffic ? membrane fusion. Two types of SNAREs can be found on target (Q-SNAREs) and vesicle (R-SNAREs) membranes that together form the core SNARE complex enabling fusion of the lipid bilayers. R-SNAREs are tail-anchored membrane proteins that bear the (R-)SNARE-motif and an N-terminal Longin Domain (LD) which is thought to backfold on the SNARE-motif to prevent promiscuous binding thereby exerting spatio-temporal control on the SNARE activity. The domain has also been implicated in determining subcellular localization of R-SNAREs. We had previously reported that R-SNAREs can bind to plasma membrane potassium channels downregulating their activity. A single point mutation within the LD prevented binding and regulation of the channel. Here we study the effects of this mutation on SNARE capacity to rescue the lethal, cytokinesis-related vamp721-/-vamp722-/- phenotype. Furthermore, we performed a deep characterization of its subcellular localization and SNARE complex formation in vivo. Comparison of the corresponding residue in the closely related, but apparently non-functional VAMP723 demonstrates that a Tyrosine residue at position 57 (VAMP723D57Y) is sufficient to change subcellular localization from the ER to the endocytic pathway and cell plate. Nonetheless, this spatio-temporal shift does not lead to complementation of the seedling lethal phenotype. Similarly, mutating the Tyr57 of VAMP721 to an Aspartate maintains cell plate localization, while completely abolishing rescue of the vamp721-/-vamp722-/- mutant line. Reference: Zhang et al. 2015, Plant Cell, 27:1697-717