Fig. 4: Functional analysis of the SEAC wing.
a, Structure of the SEAC active site, superimposed on the structure of the active GATOR1-Ragulator-Rag structure. b, Representative thin layer chromatography of in vitro GAP assays (n = 2–3 independent experiments). c, Relative fold change in GAP activity between the wild-type SEAC, Sea1R943A, Npr2R84A and isolated SEAC wing. The comparison between full complex and wing was done with protein purified from Sea1-TAP strains. Independent data points are presented as mean values ±s.d. where appropriate (n = 2 for Sea4-TAP strains and n = 3 for Sea1-TAP strains). d, Growth assays on proline wherein GAP-defective mutants present a slow-growth phenotype. e, SEAC localization, tracked by visualizing endogenously tagged Sea4-GFP, in wild-type (WT), ∆Sea3, ∆Sea3SAP and ∆Sea1 backgrounds. f, SEAC localization in ∆Sea1Cter and ∆Sea1CS strains. g, Growth assays on proline for Sea1 mutant strains. h, Rapamycin growth assays showing that SEA1 mutations are epistatic to SEA3 mutations. i, SEAC localization in the ∆Gtr1∆Gtr2 strain. j, Quantification of vacuole to cytosol ratio (mean ± s.d.) of Sea4-GFP signal in cells from e, f and i (n = 30 cells per strain). k, Model of the SEAC bound to the EGOC in the vacuolar membrane. An alternative anchor is shown as an unknown protein that might interact with the Sea1Cter. Scale bars, 10 μm.
