Fig. 3: VAMP8 phosphorylation impairs SNARE complex formation.
From: mTOR-mediated phosphorylation of VAMP8 and SCFD1 regulates autophagosome maturation
A Flag-Trap assays testing Flag-VAMP8 precipitation of endogenous STX17 and SNAP29 in Torin-treated or untreated control cells. Cell lysates from HEK293T cells transfected with Flag-VAMP8 were immunoprecipitated by Flag-beads with or without Torin treatment. STX17 and SNAP29 were detected by anti-STX17/SNAP29 antibody. B SNARE complex formation was clearly impeded upon disruption of mTORC1-mediated VAMP8 phosphorylation based on a single (T48D) mutation or a double (T48D, S55D) mutation. Flag-VAMP8WT or VAMP8 variants (VAMP8T48A, VAMP8T48D, VAMP8S55A, VAMP8S55D, VAMP82A, or VAMP82D) were transfected into HEK293T cells. The interactions between transfected Flag-tagged proteins and endogenous STX17 were visualized by Flag co-immunoprecipitation and western blotting. C In vitro GST pull-down analysis using GST, GST-Tagged VAMP8WT, or the indicated GST-tagged VAMP8 variants, and Flag-STX17 recombinant proteins, followed by western blotting. D Markedly enhanced Flag-VAMP82A overlap with GFP-STX17 and endogenous LC3 compared with Flag-VAMP8WT. Immunofluorescence of U2OS cells expressing GFP-STX17 and Flag- VAMP8WT, VAMP82A, or VAMP82D variant proteins. Scale bar, 10 µm. E Quantification for E. One-way ANOVA (Data are shown as the mean ± SEM. ***p < 0.001, n = 20).
