Fig. 5: Pho81 interacts with Atg11 via an Atg11/FIP200 interaction motif to promote phosphorylation of Atg11 required for pexophagy.
From: A metabolite sensor subunit of the Atg1/ULK complex regulates selective autophagy
a, Schematics of Pho81 and ankyrin repeat variants; Pho81A (Pho81 ankyrin repeat replaced by Ankyrin repeat of Akr1); Pho81LA1 to Pho81LA4 (Pho81 ankyrin repeat loops 1 to 4 replaced by corresponding loop of Akr1 Ankyrin repeat loops 1 to 4). b, Y2H of cells expressing indicated pGAD-PHO81 variants and pGBDU-empty vector (−) or ATG11 were grown on SD + HIS or SD − HIS medium. c, Consensus motif of Atg11/FIP200 interaction region with homology logo (WebLogo 3.0)50. based on alignment of indicated Atg11/FIP200 interaction regions and corresponding amino acid probability. Sc, Saccharomyces cerevisiae; Pp, Pichia pastoris; Hs, Homo sapiens. d, Schematics of Pho81DD, Pho81T and Pho81DE. e, Y2H of cells expressing indicated pGAD-PHO81 variants and pGBDU-empty vector (−) or ATG11 were grown on SD + HIS or SD − HIS medium. f, Enrichment of AKC subunits after GFP–Atg11-based CoIP–MS from Δpho81 cells expressing plasmid-encoded PHO81–mCherry or pho81DD–mCherry. Control is GFP. Data are mean ± s.d. (n = 3–4 biologically independent experiments). Statistical analysis is presented in Supplementary Table 2. FC, fold change. g,h, Pex11–GFP turnover in indicated strains harboring empty vector (ev) or expressing plasmid-encoded PHO81 variants after P-S (24 h). Data are mean ± s.d. (g, n = 9; h, n = 4 biologically independent experiments). i, Atg11 peptides containing S121, S613 or S935 identified upon phospho-proteomic analysis of data shown in f and volcano plot of identified phosphosites with significant enrichment in red. j, Pex11–GFP turnover in Δatg11 or Δatg11Δpho81 cells harboring ev or expressing plasmid-encoded ATG11 or atg113SA (S121A, S613A, S935A) after P-S (24 h). Data are mean ± s.d. (n = 6 biologically independent experiments). In g, h and j, statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test. P values relative to WT (ev, *) or PHO81 expressing ∆pho81 cells (#) (g and h) or ATG11. P values *** or ###, P < 0.0001 except *P = 0.0492 (h) and **P = 0.0037 (j), non-significant (n.s.). Source numerical data and unprocessed blots are available in Source data.
