Figure 3.
A positively charged surface area mediates Faa1 membrane binding. (A) Alphafold model of scFaa1. Electropositively and electronegatively charged areas are colored in blue and red, respectively. Neutral residues are in white. Mutated amino acids are highlighted with dotted circles. (B) Model of Faa1 membrane interaction with Faa1 in yellow and the membrane bilayer in dark and light teal. The model was generated with CHARMM-GUI HMMM Builder (Jo et al., 2008; Qi et al., 2015). (n) refers to the N-terminus and (c) to the C-terminus of Faa1. (C) Left, cosedimentation assay showing that mutations of positively charged residues in the plane area (Faa1-4D, Faa1-6D) significantly reduce membrane binding compared with Faa1-WT. Right, respective quantification. Data are means ± SD (n = 3). One-way ANOVA: * < 0.05, ** < 0.01. LP = liposome, SN = supernatant, P = pellet. (D) Microscopy-based membrane recruitment assay with different versions of Faa1. Faa1 is immobilized on GFP-trap beads, incubated with 1 mM of rhodamine labelled liposomes and imaged by microscopy. Liposome compositions for C and D resemble Atg9 vesicles + PI3P (44% POPC, 6% POPS, 41.5% liver PI, 5.5% POPE, 2.5% PI3P and 0.5% lissamine rhodamine-DHPE). Data are means ± SD (n = 3). Two-sided t test: * < 0.05, ** < 0.01. (E) Top, Faa1-WT, Faa1-4D, and Faa1-6D localization in cells coexpressing mCherry-Atg8. Indicated strains were imaged via fluorescence microscopy after rapamycin treatment (1 h), scale bars, 1 µm. Bottom, respective quantification of Atg8 structures positive for Faa1 (n = 3). Source data are available for this figure: SourceData F3.

A positively charged surface area mediates Faa1 membrane binding. (A) Alphafold model of scFaa1. Electropositively and electronegatively charged areas are colored in blue and red, respectively. Neutral residues are in white. Mutated amino acids are highlighted with dotted circles. (B) Model of Faa1 membrane interaction with Faa1 in yellow and the membrane bilayer in dark and light teal. The model was generated with CHARMM-GUI HMMM Builder (Jo et al., 2008; Qi et al., 2015). (n) refers to the N-terminus and (c) to the C-terminus of Faa1. (C) Left, cosedimentation assay showing that mutations of positively charged residues in the plane area (Faa1-4D, Faa1-6D) significantly reduce membrane binding compared with Faa1-WT. Right, respective quantification. Data are means ± SD (n = 3). One-way ANOVA: * < 0.05, ** < 0.01. LP = liposome, SN = supernatant, P = pellet. (D) Microscopy-based membrane recruitment assay with different versions of Faa1. Faa1 is immobilized on GFP-trap beads, incubated with 1 mM of rhodamine labelled liposomes and imaged by microscopy. Liposome compositions for C and D resemble Atg9 vesicles + PI3P (44% POPC, 6% POPS, 41.5% liver PI, 5.5% POPE, 2.5% PI3P and 0.5% lissamine rhodamine-DHPE). Data are means ± SD (n = 3). Two-sided t test: * < 0.05, ** < 0.01. (E) Top, Faa1-WT, Faa1-4D, and Faa1-6D localization in cells coexpressing mCherry-Atg8. Indicated strains were imaged via fluorescence microscopy after rapamycin treatment (1 h), scale bars, 1 µm. Bottom, respective quantification of Atg8 structures positive for Faa1 (n = 3). Source data are available for this figure: SourceData F3.

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