Localisation and mislocalisation of the interferon-inducible immunity-related GTPase, Irgm1 (LRG-47) in mouse cells

Abstract

Irgm1 (LRG-47) is an interferon-inducible Golgi membrane associated GTPase of the mouse whose disruption causes susceptibility to many different intracellular pathogens. Irgm1 has been variously interpreted as a regulator of homologous effector GTPases of the IRG family, a regulator of phagosome maturation and as an initiator of autophagy in interferon-induced cells. We find that endogenous Irgm1 localises to late endosomal and lysosomal compartments in addition to the Golgi membranes. The targeting motif known to be required for Golgi localisation is surprisingly also required for endolysosomal localisation. However, unlike Golgi localisation, localisation to the endolysosomal system also requires the functional integrity of the nucleotide binding site, and thus probably reflects transient activation. Golgi localisation is lost when Irgm1 is tagged at either N- or C-termini with EGFP, while localisation to the endolysosomal system is relatively favoured. N-terminally tagged Irgm1 localises predominantly to early endosomes, while C-terminally tagged Irgm1 localises to late endosomes and lysosomes. Both these anomalous distributions are reversed by inactivation of the nucleotide binding site, and the tagged proteins both revert to Golgi membrane localisation. Irgm1 is the first IRG protein to be found associated with the endolysosomal membrane system in addition to either Golgi (Irgm1 and Irgm2) or ER (Irgm3) membranes, and we interpret the result to be in favour of a regulatory function of IRGM proteins at cellular membrane systems. In future analyses it should be borne in mind that tagging of Irgm1 leads to loss of Golgi localisation and enhanced localisation on endolysosomal membranes, probably as a result of constitutive activation.

Figure 1. Localisation of IFNγ-induced endogenous Irgm1.

(A) MEFs were treated with 200 U/ml IFNγ for 24 hours, fixed and stained for Irgm1 with goat antiserum P20 (a–c, g–l) or mouse monoclonal antibody 1B2 (d–f) (both directed against Irgm1) and the indicated marker proteins. Irgm1 accurately co-localised with GM130 (a–c) and TGN38 (d–e), and localised adjacent to, or partially co-localised with, the CI-M6PR positive compartments (g–i). (B) MEFs were treated with 200 U/ml IFNγ for 24 hours. Cells were either fixed and stained for Irgm1, LAMP1 and Irgm2 using 1B2, 1D4B, and H53 immunoreagents, respectively (a–d), or further incubated with 50 nM LysoTracker Red DND-99 for 30 minutes in complete medium, and stained for Irgm1 with 1B2 antibody (e–g). Irgm1 was found to associate with LAMP1 positive compartments (a–d) and accumulate around the LysoTracker enriched compartments (e–g) in addition to Golgi localisation. Irgm2 (c) localises exclusively to the Golgi apparatus. (C) After treatment with 200 U/ml IFNγ for 24 hours, MEFs were incubated with Alexa-Fluor-546-labelled transferrin for 5 minutes (a–c) or pulsed with labelled transferrin for 10 minutes, then chased for 30 minutes (d–f). Cells were then fixed and stained for Irgm1 with P20 goat antiserum. The granular signals from Irgm1 were not found convincingly associated with transferrin-positive compartments.

Figure 2. Amphipathic αK helix is responsible for lysosomal targeting of full length Irgm1 protein.

MEFs were transfected with plasmids encoding either Irgm1 wild type (a–c) or the ins 362, 367E mutant (d–f) for 24 hours in the absence of IFNγ. Cells were then fixed and stained for Irgm1 and LAMP1. Wild type Irgm1 is strongly associated with the LAMP1 positive compartment, while Irgm1 ins 362, 367E mutant showed granular signals throughout cytoplasm which do not overlap with LAMP1 signals. Nuclei were labelled with DAPI.

Figure 3. EGFP-Irgm1 αK localises to both Golgi and lysosomes.

MEFs were transfected with an expression plasmid encoding EGFP-Irgm1 αK in the absence of IFNγ (20). 24 hours later cells were fixed and stained for either GM130 (a–i) or LAMP1 (j–o). Transfected cells stained with GM130 showed 3 rather different staining patterns for EGFP-Irgm1 αK: clear Golgi staining with only diffuse staining elsewhere (a–c), clear Golgi staining and punctate staining elsewhere (d–f), and only punctate staining without significant Golgi staining (g–i). When stained for LAMP1, cytoplasmic dots of EGFP-Irgm1 αK, without (j–l) or with (m–o) Golgi localisation were more or less strongly associated with LAMP1-positive vesicles. Nuclei were labelled with DAPI.

Figure 4. EGFP-Irgm1 αK is not recruited to phagosomes.

EGFP-Irgm1 αK was transiently transfected into MEFs for 24 hours in the absence of IFNγ. During transfection, cells were incubated with 2-µm latex beads overnight. Cells were then fixed and stained for LAMP1. EGFP-Irgm1 αK was not associated with latex bead phagosomes (arrowheads), whether the association with other cellular compartments was Golgi only (a–d), lysosomal only (e–h) or both Golgi and lysosomal (i–l). Despite not associating with latex bead phagosomes, EGFP-Irgm1 αK associated normally with the LAMP1 compartment elsewhere (arrows). Nuclei were labelled with DAPI.

Figure 5. Alanine mutagenesis scan of EGFP-Irgm1 αK.

EGFP-Irgm1 αK quadruple mutant (F362A F363A L365A L366A; Figure 6A), single mutants of the hydrophilic residues (N360A;R364A;R367A; Figure 6B including enlargements (for d, e, f)), or combined mutants of more than one hydrophilic residues (N360A, R364A; R364A, R367A; N360A, R364A, R367A; Figure 6C) were transfected into MEFs for 24 hours. Cells were then fixed and staining for GM130 or LAMP1. Only when more than one hydrophilic residue was mutated to alanine, the EGFP-Irgm1 αK no longer localised to Golgi or lysosomes. These mutants localised to unidentified endomembrane-like structures or are delocalized and possibly cytosolic. Nuclei were labelled with DAPI.

Figure 6. Synthetic αK amphipathic peptide mimics the localisation of endogenous Irgm1.

MEFs were treated with 200 U/ml IFNγ for 24 hours and then fixed with 3% PFA in PBS. Cy3-streptavidin-peptide solutions (0.33 µM streptavidin) were prepared as described in Materials and Methods. Antibodies against the indicated proteins were added to the streptavidin-peptide solution and the mixtures used as primary reagents in immunofluorescence staining. Streptavidin (0.33 µM final) alone (a) was used as control. Image (a) and (b) were taken at the same exposure time. Colocalisation was seen between streptavidin-Irgm1 αK peptide and endogenous Irgm1 at both Golgi apparatus and lysosomes.

Figure 7. Localisation of EGFP-tagged Irgm1.

(A) N-terminally EGFP-tagged Irgm1 (EGFP-Irgm1) or (B) C-terminally EGFP-tagged Irgm1 (Irgm1-EGFP, pF25) were transfected into MEFs. 24 hours later, cells were fixed and stained for GM130 (a–c) and TGN38 (d–f). Both constructs were absent from the Golgi apparatus. Nuclei were labelled with DAPI. (C) EGFP-Irgm1 (a–f) or Irgm1-EGFP (pF25, g–l) were transfected into MEFs. 24 hours later cells were incubated with Alexa-Fluor-546-labelled transferrin for 5 minutes (a–c, g–i) or pulsed with labelled transferrin for 10 minutes, then chased for 30 minutes (d–f, j–l). Cells were then fixed in 3% PFA in PBS. The vesicular and dotty signals from EGFP-Irgm1 and Irgm1-EGFP strongly overlap with transferrin-labelled early and recycling endosomes. (D) EGFP-Irgm1 (a–c) or Irgm1-EGFP (d–f, pF25) were transfected into MEFs. 24 hours later, cells were fixed and stained for LAMP1. Irgm1-EGFP was strongly colocalised with LAMP1 while lysosomal association of EGFP-Irgm1 was largely but not completely abolished. Nuclei were labelled with DAPI.

Figure 8. Mislocalisation of EGFP-tagged Irgm1 is nucleotide-dependent.

EGFP-Irgm1 S90N (a–c) and Irgm1-EGFP S90N (d–f) were transfected into MEFs for 24 hours. Cells were then fixed and stained for TGN38. Both constructs co-localised with TGN38, although a significant proportion of transfected proteins displayed additional cytoplasmic signals. Nuclei were labelled with DAPI.

Figure 9. Phagosomal accumulation of Irgm1 is nucleotide-dependent, but IFNγ-independent.

(A) Irgm1 wild type (a–d) and S90N mutant (e–h) were transfected into MEFs in the absence of IFNγ for 24 hours. During transfection, cells were incubated with 2-µm latex beads overnight. Cells were then fixed and stained for Irgm1 and LAMP1. Irgm1 accumulated around the latex beads phagosomes (seen as refringent spheres) while the loss of the nucleotide-binding site completely abolished the phagosomal accumulation. Arrows indicate the co-localisation of transfected wild type Irgm1 with LAMP1 positive compartments outside latex-bead phagosomes. (B) EGFP-Irgm1 or Irgm1-EGFP were transfected into MEFs for 24 hours in the absence of IFNγ. During transfection, cells were also incubated with 2-µm latex beads overnight. Cells were then fixed and stained for LAMP1. Irgm1-EGFP accumulated around the latex bead phagosomes while EGFP-Irgm1 did not. (C) The same experiments were performed as in (B) but using the nucleotide-binding deficient mutants (S90N) of N- and C-terminally EGFP-tagged Irgm1. Both mutants were absent from latex bead phagosomes. Nuclei were labelled with DAPI.