To confirm the role of IncE in direct SNX5 protein recruitment to the chlamydial inclusion we examined the localisation of GFP-tagged SNX5 in HeLa cells infected with C. trachomatis L2 (CTL2) for 24 hr (MOI ~0.5). Cells expressing the GFP-SNX5 protein showed clear and uniform recruitment to the limiting membrane of the inclusion as defined by mCherry-Rab25 (Figure 6A), which is consistent with the localisation observed by others (Aeberhard et al., 2015; Mirrashidi et al., 2015). In contrast, the GFP-SNX5 (F136A) mutant protein showed no recruitment to the chlamydial inclusion. The change in relative distribution of these GFP-SNX5 proteins on the inclusion was quantified for wildtype SNX5 (Mander’s coefficient 0.67 ± 0.14) and GFP-SNX5 (F136A) (0.041 ± 0.051) (Figure 6—figure supplement 1A). Like wild-type GFP-SNX5 the GFP-SNX5 (F136A) mutant was recruited to punctate endosomal structures throughout the cytoplasm of these cells, and in addition was able to co-immunoprecipate endogenous SNX1 in heterodimeric complexes identically to the wild-type GFP-SNX5 protein (Figure 6—figure supplement 1B). This implies that BAR-domain mediated heterodimer formation with SNX1 or SNX2 is required for endosomal recruitment, and is not perturbed by the IncE-binding mutation in the PX domain. Lastly, we tested the importance of IncE residues for SNX interaction in situ by expressing the GFP-tagged IncE C-terminal domain. The wild-type GFP-IncE(91-132) was recruited to endosomal structures via its interaction with SNX5-related proteins in both uninfected and infected HeLa cells (Figure 6B; Figure 6—figure supplement 1C). In contrast however, GFP-IncE(91-132)(F116D), a SNX5-binding mutant, was exclusively cytosolic. Note that neither IncE construct is localised to the inclusion, as expected due to lack of signal peptides and transmembrane domains (Figure 6—figure supplement 1C).10.7554/eLife.22311.017Figure 6.Mutations in the SNX5 and IncE proteins prevents their interaction in cells.(A) Single amino-acid mutation in the PX domain of the SNX5 (F136A) abolishes recruitment to the chlamydial inclusion. HeLa cells stably expressing mCherry-Rab25 (red) were transfected transiently with GFP-SNX5 or GFP-SNX5 (F136A) (green) and infected with Chlamydia trachomatis L2 for 18–24 hr. The cells were fixed and the nucleic materials were counter-stained with DAPI (blue). (B) HeLa cells were transfected transiently with GFP-IncE(91-132) or GFP-IncE(91-132)(F116D) (green) and co-labelled for the early endosomal marker EEA1 (red). Mutation in the SNX5 binding IncE peptide (F116D) abolishes recruitment to endosomal structures. *Represents the inclusion. Scale bar 20 μm.DOI: http://dx.doi.org/10.7554/eLife.22311.01710.7554/eLife.22311.018Figure 6—figure supplement 1.GFP-IncE C-terminal domain is localised to endosomes but not inclusions.(A) Quantitation of the degree of overlap between the GFP-SNX5 constructs and the mCherry-Rab25 inclusion membrane marker from Figure 6A. Mander’s correlation coefficient of mChRab25 signal over GFP-SNX5 signals (10 cells per group; error bars, S.D). (B) Co-immunoprecipitation of GFP-SNX5 from HeLa cells shows that both the wild-type and mutant protein (F136A) interact equally with endogenous SNX1. This indicates that both proteins are correctly folded and otherwise functional. (C) HeLa cells stably expressing mCherry-Rab25 (red) were transfected transiently with GFP-IncE(91-132) or GFP-IncE(91-132)(F116D) (green) and infected with C. trachomatis L2 for 18–24 hr. Mutation in the SNX5 binding IncE peptide (F116D) abolishes recruitment to endosomal structures. Neither construct is recruited to inclusions, which is consistent with the lack of transmembrane regions. *Represents the inclusion. Scale bar 20 μm.DOI: http://dx.doi.org/10.7554/eLife.22311.018