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Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

Maric-Biresev J, Hunn JP, Krut O, Helms JB, Martens S, Howard JC - BMC Biol. (2016)

Bottom Line: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family).In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes.The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Cologne, Germany.

ABSTRACT

Background: The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse.

Results: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency.

Conclusions: The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.

No MeSH data available.


Related in: MedlinePlus

Irga6 co-localizes with the endoplasmic reticulum in Irgm3−/− cells. aIrgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ and transfected with pEYFP-calreticulin for 24 hours. Cells were fixed and stained with anti-Irga6 antibody (10D7). Representative microscopic images of Irga6 and calreticulin co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: calreticulin, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 4a, showing percent of Irga6 structures co-localizing with calreticulin; 50 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown
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Fig4: Irga6 co-localizes with the endoplasmic reticulum in Irgm3−/− cells. aIrgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ and transfected with pEYFP-calreticulin for 24 hours. Cells were fixed and stained with anti-Irga6 antibody (10D7). Representative microscopic images of Irga6 and calreticulin co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: calreticulin, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 4a, showing percent of Irga6 structures co-localizing with calreticulin; 50 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown

Mentions: Since Irgm3 is the only GMS protein identified as localizing to the ER [31], we asked whether, in the absence of Irgm3, Irga6 accumulates on the ER. WT, Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− MEFs were induced with IFN-γ and simultaneously transfected with an ER marker, pEYFP-calreticulin. The cells were again stained for activated Irga6 with 10D7 antibody (Fig. 4a). The Irga6 structures formed in GMS knock-out (KO) cells consist of activated, GTP-bound Irga6 [16, 33]. More than 85 % of active Irga6 accumulations co-localized with calreticulin in Irgm3−/− cells (Fig. 4b). In contrast, fewer than 5 % of active Irga6 structures co-localized with calreticulin in Irgm1−/− cells. These results strongly support the idea that the positioning of individual GMS proteins on specific endomembrane systems determines the inhibition of activation of GKS effector proteins on the same membranes.Fig. 4


Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

Maric-Biresev J, Hunn JP, Krut O, Helms JB, Martens S, Howard JC - BMC Biol. (2016)

Irga6 co-localizes with the endoplasmic reticulum in Irgm3−/− cells. aIrgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ and transfected with pEYFP-calreticulin for 24 hours. Cells were fixed and stained with anti-Irga6 antibody (10D7). Representative microscopic images of Irga6 and calreticulin co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: calreticulin, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 4a, showing percent of Irga6 structures co-localizing with calreticulin; 50 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown
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Related In: Results  -  Collection

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Fig4: Irga6 co-localizes with the endoplasmic reticulum in Irgm3−/− cells. aIrgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ and transfected with pEYFP-calreticulin for 24 hours. Cells were fixed and stained with anti-Irga6 antibody (10D7). Representative microscopic images of Irga6 and calreticulin co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: calreticulin, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 4a, showing percent of Irga6 structures co-localizing with calreticulin; 50 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown
Mentions: Since Irgm3 is the only GMS protein identified as localizing to the ER [31], we asked whether, in the absence of Irgm3, Irga6 accumulates on the ER. WT, Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− MEFs were induced with IFN-γ and simultaneously transfected with an ER marker, pEYFP-calreticulin. The cells were again stained for activated Irga6 with 10D7 antibody (Fig. 4a). The Irga6 structures formed in GMS knock-out (KO) cells consist of activated, GTP-bound Irga6 [16, 33]. More than 85 % of active Irga6 accumulations co-localized with calreticulin in Irgm3−/− cells (Fig. 4b). In contrast, fewer than 5 % of active Irga6 structures co-localized with calreticulin in Irgm1−/− cells. These results strongly support the idea that the positioning of individual GMS proteins on specific endomembrane systems determines the inhibition of activation of GKS effector proteins on the same membranes.Fig. 4

Bottom Line: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family).In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes.The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Cologne, Germany.

ABSTRACT

Background: The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse.

Results: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency.

Conclusions: The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.

No MeSH data available.


Related in: MedlinePlus