Figure 1. ATG5 interacts with retromer.
(A) ATG5 functions. X, a postulated additional function. (B) 2D scatter plot (log2 fold changes; color coded p value cutoff matrix for comparisons between samples as per the lookup table) of proximity biotinylation LC/MS/MS datasets: FlpIn-HeLaAPEX2-ATG5-WT cells (X-axis) and FlpIn-HeLaAPEX2-ATG5-K130R (Y-axis) treated with 2 mM LLOMe for 30 min ratioed vs. HeLaAPEX2-ATG5-WT without LLOMe treatment (Ctrl). Coimmunoprecipitation (co-IP) analyses and quantification of VPS26A (C), VPS29 (D), and VPS35 (E) interaction with ATG5 in HeLa cells treated with or without 2 mM LLOMe for 30 min. Data, means ± SE (n = 3); unpaired t-test; p values indicated above the bars.
Figure 1—source data 1. PDF files containing original immunoblots for Figure 1 indicating relevant bands.
elife-100928-fig1-data1.zip (1.7MB, zip)
Figure 1—source data 2. Original files for immunoblots displayed in Figure 1.
elife-100928-fig1-data2.zip (3.9MB, zip)
Figure 1—source data 3. Numerical values for quantification in graphs.
elife-100928-fig1-data3.xlsx (10.2KB, xlsx)
Figure 1—figure supplement 1. Cornell model of M. tuberculosis latent infection in mice and effects of Atg5 loss in myeloid lineage on disease reactivation.
(A) Summary of mice mortality data in infection model of active Mtb infection (aerosol) based on survival curves in Wang et al., 2023 (B) Details and timeline of the Cornell latency model experiments (see narrative in Methods). (C) Effects of Atg5 loss on spontaneous reactivation of M. tuberculosis infection in Atg5fl/fl Lyz2Cre mice (conditional knockout of Atg5 in myeloid lineage) vs. Atg5fl/fl (Lyz2Cre-negative control) mice. Mice were infected with an aerosol of M. tuberculosis (initial deposition, 100–120 CFUs per lung). After a period of 2.5 weeks, initial bacterial growth was assessed by determining lung CFUs (triangles), mice were treated PO with antibiotics (0.1 g/l INH and 0.15 g/l RIF in drinking water) for 8 weeks, bacterial clearance after chemotherapy assessed by determining lung CFUs (open circles), and remaining mice subjected to antibiotic washout for 7 weeks plus spontaneous reactivation period with no treatment of 3 weeks at which time the mice were sacrificed and lung CFUs determined by plating (filled circles). Data and statistics for spontaneous reactivation: means, †p ≥ 0.05, t-test; n = 8 mice per group. (D) Cornell murine model of M. tuberculosis latent infection and dexamethasone (DXM) induced reactivation and effects of Atg5 loss in myeloid lineage of Atg5fl/fl LysM-Cre+ mice (vs. Atg5fl/fl LysM-Cre− control mice). Mice were infected with M. tuberculosis aerosols (initial lung deposition 100–120 CFUs), bacteria allowed to replicate in vivo, mice subjected to antibiotic regimen, followed by antibiotic washout period, after which immunosuppression with DXM was carried out to reactivate infection/bacterial replication (details in Methods). Data, CFU’s per mouse lungs (means ± SE, t-test, n = 10 mice per group).
Figure 1—figure supplement 2. ATG5 interactome analysis.
(A) Volcano plot of proximity biotinylation LC–MS/MS interactome comparing FlpIn-HeLaAPEX2-ATG5-WT treated with or without 2 mM LLOMe for 30 min. Diameter of symbols reflects relative number of unique peptides identified. (B) Volcano plot of proximity biotinylation LC–MS/MS interactome comparing FlpIn-HeLaAPEX2-ATG5-K130R treated with 2 mM LLOMe for 30 min and FlpIn-HeLaAPEX2-ATG5-WT without LLOMe treatment. (C) VPS proteins in the MS DIA data. (D) VPS proteins identified here compared to VPS proteins in Baines et al., 2022. (E) Table: Control MS data (APEX2-SGALS1) for comparison with data in panel C; note no increase in retromer subunits with LLOMe treatment in the APEX1-SGALS1 dataset.