Fig. 4: Rab41 binds to VPS4 and is required for VPS4 localization to xenophagolysosomes.

a Co-immunoprecipitation between FLAG-Rab41 and GFP-ESCRT proteins during GAS infection. HeLa cells expressing GFP tagged ESCRT components and FLAG-Rab41 were infected with GAS for 4 h. Cell extract were immunoprecipitated (IP) with GFP-Trap beads. The resulting samples, IP and whole cell lysate (WCL), were analyzed by immunoblot analysis. Data shown are representative of three independent experiments. b, c The recruitment of ESCRT-I subunit VPS28 to GcAVs in Rab41-knockout cells. HeLa WT and Rab41 knockout (KO) cells were infected with GAS for 4 h, fixed, and immunostained for endogenous VPS28. Cellular and bacterial DNA were stained with DAPI. The proportion of the LC3 vacuole signal (GcAVs) overlapping VPS28 was quantified by Mander’s coefficient M1. Representative confocal images of single slice (b) and quantification of VPS4 recruitment to GcAVs (c). Data are individual values and mean ± SEM (n = 30 GcAVs examined over three independent experiments). Unpaired two-tailed t test. Scale bar, 10 μm. d, e The recruitment of VPS4 to GcAVs in Rab41-knockout cells. HeLa WT and Rab41 knockout cells expressing FLAG-Rab41, -Rab41 T44N, or -Q89L were infected with GAS for 4 h, fixed, and immunostained for endogenous VPS4. Cellular and bacterial DNA were stained with DAPI. The proportion of the LC3 vacuole signal (GcAVs) overlapping VPS4 was quantified by Mander’s coefficient M1. Representative confocal images of single slice (d) and quantification of VPS4 recruitment to GcAVs (e). Scale bar, 10 μm. Data are individual values and mean ± SEM (n = 30 GcAVs examined over three independent experiments). One-way ANOVA, Tukey’s test. f HeLa WT and Rab41 knockout cells expressing FLAG-Rab41, -Rab41 T44N, or -Q89L were infected with GAS JRS4 and the intracellular bacterial CFU was determined at 6 hpi. Data are individual values and mean ± SEM (n = 3 biologically independent experiments). One-way ANOVA, Tukey’s test. g Immunoprecipitation analysis. HeLa cells were infected with GAS for 4 h and then lysed with IP lysis buffer. Cell lysate were immunoprecipitated with anti-Rab41 or control rabbit antibody. The resulting samples were analyzed by immunoblot analysis. Data shown are representative of three independent experiments. h Domain organization and deletion mutants of VPS4. i, j Co-immunoprecipitation analysis. HEK293T were transfected with GFP tagged VPS4 deletion mutants and FLAG tagged Rab41, and then cell lysates were immunoprecipitated with GFP-Trap beads. The resulting samples were analyzed by immunoblot analysis. Data shown are representative of three independent experiments. k HeLa cells transfected with VPS4-EGFP FL, MIT or LA domain and mCherry-LC3 were infected with GAS for 4 h, fixed and stained with DAPI. Shown are representative confocal single-slice images of three independent experiments. Scale bar, 10 μm. l Co-immunoprecipitation analysis. HEK293T were transfected with GFP tagged Rab41 wild-type or Rab41 S94A mutant, and then cell lysates were immunoprecipitated with GFP-Trap beads. The resulting samples were analyzed by immunoblot analysis. Data shown are representative of three independent experiments. m HeLa WT and Rab41 knockout cells expressing GFP-Rab41 constructs were infected with GAS, and intracellular survival bacteria were determined by colony counting. Data are individual values and mean ± SEM (n = 3 biologically independent experiments). One-way ANOVA, Tukey’s test. Source data are provided as a Source Data file.