Fig. 6: SPX domain-dependent phospho-metabolite sensing by Pho81 regulates pexophagy.
From: A metabolite sensor subunit of the Atg1/ULK complex regulates selective autophagy
a, Schematics of the Pho81 SPX domain variants; Pho81∆SPX (C-terminus 173–1,178); Pho81YKK: Y24A, K28A and K154A indicate mutated residues involved in inositol phosphate sensing. b, Sequence alignment of SPX domain containing proteins from Sc, Saccharomyces cerevisiae; At, Arabidopsis thaliana; Mm, Mus musculus; Hs, Homo sapiens. c, Enrichment of AKC subunits after GFP–Atg11-based CoIP–MS from cells expressing plasmid-encoded PHO81–, pho81YKK– or pho81∆SPX–mCherry. Control is GFP. n = 4 biologically independent experiments. Statistical analysis is presented in Supplementary Table 2. FC, fold change. d, Pex11–GFP turnover in strains harboring empty vector (ev) or expressing indicated plasmid-encoded PHO81 variants in the respective PEX11–GFP-expressing deletion cells upon 24 h of P-S. Data are mean ± s.d. (n = 4 biologically independent experiments). e, Fluorescence imaging of cells expressing genomic PHO81–GFP variants and ATG1–mCherry during growth (0 h) and PS (4 h). Arrows indicate Pho81–GFP positive (white) or negative (purple) Atg1–mCherry foci. Data are representative of three biologically independent experiments. Scale bar, 2 µm. f, Y2H of cells expressing pGAD-PHO81 or indicated variants in combination with pGBDU-empty vector (−) or pGBDU-ATG11 grown on +HIS, −HIS, −ADE selection plates. g, Pex11-GFP turnover in cells harboring ev (−) or expressing PHO81 under an ADH1 promoter (OE) and PEX11–GFP upon 24 h P-S. Data are mean ± s.d. (n = 3 biologically independent experiments). h, 2GFP–Atg8 turnover in indicated strains harboring ev (−) or expressing PHO81 under an ADH1 promoter (OE) after P-S (24 h). Data are mean ± s.d. (n = 3 biologically independent experiments). i, Pex11–GFP turnover in WT and ∆pho81 cells after P-S (24 h) ± InsP6. Data are mean ± s.d. (n = 3 biologically independent experiments). j, Pex11–GFP turnover in cells harbouring pRS315 or pRS315–prADH1–KCS1 (OE) after P-S (24 h). Data are mean ± s.d. (n = 4 biologically independent experiments). Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (d and i), two-tailed t-test (g and j) and two-way ANOVA followed by Tukey’s multiple comparisons test (h); P values are relative to WT (g–j), Δatg13 cells (d) or as indicated: ***P < 0.0001 except *P = 0.0096 and **P = 0.0018 (i) and **P = 0.0018 (j). Source numerical data and unprocessed blots are available in Source data.
