A novel VPS33A interaction motif is a master-switch controlling the inhibition and promotion of mammalian Stx17 interactions with SNAP29 and VAMP7 during autophagy
Autophagosome clearance is accomplished by SNARE-mediated fusion of the vesicle membrane with the endolysosome. This must be carefully regulated to maintain the organisation of the membrane system and prevent mistargeted degradation. Here, we dissect the autophagosomal SNARE complex pathway and its regulation using FLIM-FRET as a readout of protein interaction in situ. We show that within the spatio-temporal framework of the cell, autophagosomal Stx17 preferentially heterodimerises with SNAP29, subsequently associating with VAMP7, not VAMP8 as currently believed. Additionally, we identify for the first time multi-modal regulation of SNARE assembly by the SM protein VPS33A, finding parallels and differences with other syntaxin-SM interactions and suggesting a unifying model of SM regulation. Contrary to current theory, the Stx17 N-peptide interacts in a positionally conserved, but mechanistically divergent manner with VPS33A, providing a late go, no-go step for autophagic fusion via a phosphoserine master-switch.