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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

Model for the cytopathic effects of dysregulated GKS proteins. a Regulatory immunity-related GTPase (IRG) GMS proteins localize to distinct cellular endomembranes including the endoplasmic reticulum (ER), Golgi, lipid droplets, and lysosomes and maintain effector IRG GKS proteins, which diffuse transiently onto these compartments, in an inactive GDP-bound state. b When T. gondii enters the host cell, GKS proteins diffuse onto this new membrane-bound compartment and activate at the parasitophorous vacuole membranes (PVM) because of the absence of GMS proteins. Activated GKS proteins form GTP-dependent oligomers and disrupt the membrane. c In Irgm1-deficient cells, lysosomal membranes lack any GMS proteins on the lysosomal membrane, allowing GKS proteins to activate and accumulate forming GTP-bound oligomers. Acidification of these lysosomes is impaired and with it lysosomal processing is not functional. These lysosomes cannot properly process autophagosomes after lysosome/autophagosome fusion. Hence, autophagic flux of IFN-γ-induced Irgm1−/− cells is impaired. d In T. gondii-infected Irgm1−/− cells, GKS proteins are accumulated at the lysosomes and therefore cannot properly accumulate and activate at the PVM. Disruption of the PVM does not happen and T. gondii can further proliferate. e In Irgm3-deficient cells it is the ER cisternae that are not protected by GMS proteins. Activated GKS proteins accumulate in local aggregates on the ER, possibly causing local ER deformation, but without such severe consequences for the cell as lysosomal impairment in Irgm1−/− cells. f In T. gondii-infected Irgm3−/− cells, GKS proteins are accumulated at the ER and therefore cannot properly accumulate and activate at the PVM. Disruption of the PVM does not happen and T. gondii can proliferate. g In Irgm1/Irgm3−/− cells, lysosomes, ER, and lipid droplets are GMS-free. For unknown reasons, activated GMS proteins accumulate only at the lipid droplets and not at the other GMS-free compartments. h Upon T. gondii infection of Irgm1/Irgm3−/− cells, GKS proteins activated at the lipid droplets cannot properly load to the PVM and inhibit T. gondii proliferation
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Fig9: Model for the cytopathic effects of dysregulated GKS proteins. a Regulatory immunity-related GTPase (IRG) GMS proteins localize to distinct cellular endomembranes including the endoplasmic reticulum (ER), Golgi, lipid droplets, and lysosomes and maintain effector IRG GKS proteins, which diffuse transiently onto these compartments, in an inactive GDP-bound state. b When T. gondii enters the host cell, GKS proteins diffuse onto this new membrane-bound compartment and activate at the parasitophorous vacuole membranes (PVM) because of the absence of GMS proteins. Activated GKS proteins form GTP-dependent oligomers and disrupt the membrane. c In Irgm1-deficient cells, lysosomal membranes lack any GMS proteins on the lysosomal membrane, allowing GKS proteins to activate and accumulate forming GTP-bound oligomers. Acidification of these lysosomes is impaired and with it lysosomal processing is not functional. These lysosomes cannot properly process autophagosomes after lysosome/autophagosome fusion. Hence, autophagic flux of IFN-γ-induced Irgm1−/− cells is impaired. d In T. gondii-infected Irgm1−/− cells, GKS proteins are accumulated at the lysosomes and therefore cannot properly accumulate and activate at the PVM. Disruption of the PVM does not happen and T. gondii can further proliferate. e In Irgm3-deficient cells it is the ER cisternae that are not protected by GMS proteins. Activated GKS proteins accumulate in local aggregates on the ER, possibly causing local ER deformation, but without such severe consequences for the cell as lysosomal impairment in Irgm1−/− cells. f In T. gondii-infected Irgm3−/− cells, GKS proteins are accumulated at the ER and therefore cannot properly accumulate and activate at the PVM. Disruption of the PVM does not happen and T. gondii can proliferate. g In Irgm1/Irgm3−/− cells, lysosomes, ER, and lipid droplets are GMS-free. For unknown reasons, activated GMS proteins accumulate only at the lipid droplets and not at the other GMS-free compartments. h Upon T. gondii infection of Irgm1/Irgm3−/− cells, GKS proteins activated at the lipid droplets cannot properly load to the PVM and inhibit T. gondii proliferation

Mentions: In this study, we have investigated the possibility proposed in our earlier publications [16, 20] that the contrasting phenotypes of Irgm1, Irgm3, and the Irgm1/Irgm3 double deficient mice might be caused by off-target activation of the GKS proteins at different cellular endomembranes. We have shown that the localization of activated GKS proteins to specific compartments is indeed dependent on the absence of specific GMS proteins (Fig. 9). In IFN-γ induces cells in the absence of Irgm1, activated GKS proteins accumulate at the lysosomes (Fig. 2a, b), to which Irgm1 normally localizes, while in the absence of Irgm3, activated GKS proteins accumulate at the ER (Fig. 4), to which Irgm3 normally localizes. In Irgm1/Irgm3−/− cells, in which lysosomes, ER, and lipid droplets are all GMS-free, GKS proteins activate exclusively at lipid droplets. Thus, the activation of GKS proteins on specific endomembrane systems is determined by the absence of specific GMS proteins (Fig. 5) [21]. However, the targeting specificity rules are evidently complex. Firstly, when single membrane systems, e.g., lysosomes alone or ER alone, are GMS-free, GKS activation occurs only on the GMS-free membranes. However, when multiple membrane systems are GMS-free, GKS activation occurs selectively on one set of GMS-free membranes and not on another. Thus, GKS proteins may show different preferences for different endocellular membranes when given a choice, perhaps on the basis of their lipid composition. To be clear, in the case of Irgm1/Irgm3−/−, we do not argue that Irgm3 normally interacts directly with Irgm1, or is in any sense a regulator of the localization of Irgm1, only that the additional absence of Irgm3 releases further compartments for access by GKS proteins. Secondly, in gs3T3 cells expressing Irga6 under an inducible promoter, but not transfected with GMS proteins, all endocellular membranes must be essentially GMS-free (Fig. 2c, d). If GKS proteins load randomly onto any GMS-free membrane, activated Irga6 should be found on lysosomes, ER, Golgi, and lipid droplets, but this is not the case. Instead, activated Irga6 localizes to a still unidentified punctate compartment which may, indeed, consist of freely cytosolic aggregates (Figs. 2d, 5d, and Additional file 6: Figure S6). Thirdly, when these gs3T3 Irga6 cells are transfected with Irgm3 only, about 35 % of Irga6 aggregates accumulate at the lysosomes. The Golgi complex, which is normally coated with Irgm1 and Irgm2, is also GMS-free in these cells. Irga6 would therefore also be expected to accumulate at Golgi membranes, but this does not happen. Indeed, the Golgi complex seems to be immune to GKS activation independently of GMS loading. Finally, none of the endocellular GMS proteins localizes to the plasma membrane, but this structure is never coated with GKS proteins, even in WT cells. The basis for this protection is not known [14]. However, whatever the critical properties of the plasma membrane are, they must change rapidly during pathogen entry because the new pathogen-containing vacuolar membrane immediately becomes vulnerable to attack by GKS proteins [19]. In summary, the activation of GKS proteins occurs only on GMS-free membranes, but not necessarily on all. Furthermore, although the location of activated GKS proteins depends upon which membranes are GMS-free, the rules of selectivity between potential target membranes remain unclear.Fig. 9


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)

Model for the cytopathic effects of dysregulated GKS proteins. a Regulatory immunity-related GTPase (IRG) GMS proteins localize to distinct cellular endomembranes including the endoplasmic reticulum (ER), Golgi, lipid droplets, and lysosomes and maintain effector IRG GKS proteins, which diffuse transiently onto these compartments, in an inactive GDP-bound state. b When T. gondii enters the host cell, GKS proteins diffuse onto this new membrane-bound compartment and activate at the parasitophorous vacuole membranes (PVM) because of the absence of GMS proteins. Activated GKS proteins form GTP-dependent oligomers and disrupt the membrane. c In Irgm1-deficient cells, lysosomal membranes lack any GMS proteins on the lysosomal membrane, allowing GKS proteins to activate and accumulate forming GTP-bound oligomers. Acidification of these lysosomes is impaired and with it lysosomal processing is not functional. These lysosomes cannot properly process autophagosomes after lysosome/autophagosome fusion. Hence, autophagic flux of IFN-γ-induced Irgm1−/− cells is impaired. d In T. gondii-infected Irgm1−/− cells, GKS proteins are accumulated at the lysosomes and therefore cannot properly accumulate and activate at the PVM. Disruption of the PVM does not happen and T. gondii can further proliferate. e In Irgm3-deficient cells it is the ER cisternae that are not protected by GMS proteins. Activated GKS proteins accumulate in local aggregates on the ER, possibly causing local ER deformation, but without such severe consequences for the cell as lysosomal impairment in Irgm1−/− cells. f In T. gondii-infected Irgm3−/− cells, GKS proteins are accumulated at the ER and therefore cannot properly accumulate and activate at the PVM. Disruption of the PVM does not happen and T. gondii can proliferate. g In Irgm1/Irgm3−/− cells, lysosomes, ER, and lipid droplets are GMS-free. For unknown reasons, activated GMS proteins accumulate only at the lipid droplets and not at the other GMS-free compartments. h Upon T. gondii infection of Irgm1/Irgm3−/− cells, GKS proteins activated at the lipid droplets cannot properly load to the PVM and inhibit T. gondii proliferation
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Fig9: Model for the cytopathic effects of dysregulated GKS proteins. a Regulatory immunity-related GTPase (IRG) GMS proteins localize to distinct cellular endomembranes including the endoplasmic reticulum (ER), Golgi, lipid droplets, and lysosomes and maintain effector IRG GKS proteins, which diffuse transiently onto these compartments, in an inactive GDP-bound state. b When T. gondii enters the host cell, GKS proteins diffuse onto this new membrane-bound compartment and activate at the parasitophorous vacuole membranes (PVM) because of the absence of GMS proteins. Activated GKS proteins form GTP-dependent oligomers and disrupt the membrane. c In Irgm1-deficient cells, lysosomal membranes lack any GMS proteins on the lysosomal membrane, allowing GKS proteins to activate and accumulate forming GTP-bound oligomers. Acidification of these lysosomes is impaired and with it lysosomal processing is not functional. These lysosomes cannot properly process autophagosomes after lysosome/autophagosome fusion. Hence, autophagic flux of IFN-γ-induced Irgm1−/− cells is impaired. d In T. gondii-infected Irgm1−/− cells, GKS proteins are accumulated at the lysosomes and therefore cannot properly accumulate and activate at the PVM. Disruption of the PVM does not happen and T. gondii can further proliferate. e In Irgm3-deficient cells it is the ER cisternae that are not protected by GMS proteins. Activated GKS proteins accumulate in local aggregates on the ER, possibly causing local ER deformation, but without such severe consequences for the cell as lysosomal impairment in Irgm1−/− cells. f In T. gondii-infected Irgm3−/− cells, GKS proteins are accumulated at the ER and therefore cannot properly accumulate and activate at the PVM. Disruption of the PVM does not happen and T. gondii can proliferate. g In Irgm1/Irgm3−/− cells, lysosomes, ER, and lipid droplets are GMS-free. For unknown reasons, activated GMS proteins accumulate only at the lipid droplets and not at the other GMS-free compartments. h Upon T. gondii infection of Irgm1/Irgm3−/− cells, GKS proteins activated at the lipid droplets cannot properly load to the PVM and inhibit T. gondii proliferation
Mentions: In this study, we have investigated the possibility proposed in our earlier publications [16, 20] that the contrasting phenotypes of Irgm1, Irgm3, and the Irgm1/Irgm3 double deficient mice might be caused by off-target activation of the GKS proteins at different cellular endomembranes. We have shown that the localization of activated GKS proteins to specific compartments is indeed dependent on the absence of specific GMS proteins (Fig. 9). In IFN-γ induces cells in the absence of Irgm1, activated GKS proteins accumulate at the lysosomes (Fig. 2a, b), to which Irgm1 normally localizes, while in the absence of Irgm3, activated GKS proteins accumulate at the ER (Fig. 4), to which Irgm3 normally localizes. In Irgm1/Irgm3−/− cells, in which lysosomes, ER, and lipid droplets are all GMS-free, GKS proteins activate exclusively at lipid droplets. Thus, the activation of GKS proteins on specific endomembrane systems is determined by the absence of specific GMS proteins (Fig. 5) [21]. However, the targeting specificity rules are evidently complex. Firstly, when single membrane systems, e.g., lysosomes alone or ER alone, are GMS-free, GKS activation occurs only on the GMS-free membranes. However, when multiple membrane systems are GMS-free, GKS activation occurs selectively on one set of GMS-free membranes and not on another. Thus, GKS proteins may show different preferences for different endocellular membranes when given a choice, perhaps on the basis of their lipid composition. To be clear, in the case of Irgm1/Irgm3−/−, we do not argue that Irgm3 normally interacts directly with Irgm1, or is in any sense a regulator of the localization of Irgm1, only that the additional absence of Irgm3 releases further compartments for access by GKS proteins. Secondly, in gs3T3 cells expressing Irga6 under an inducible promoter, but not transfected with GMS proteins, all endocellular membranes must be essentially GMS-free (Fig. 2c, d). If GKS proteins load randomly onto any GMS-free membrane, activated Irga6 should be found on lysosomes, ER, Golgi, and lipid droplets, but this is not the case. Instead, activated Irga6 localizes to a still unidentified punctate compartment which may, indeed, consist of freely cytosolic aggregates (Figs. 2d, 5d, and Additional file 6: Figure S6). Thirdly, when these gs3T3 Irga6 cells are transfected with Irgm3 only, about 35 % of Irga6 aggregates accumulate at the lysosomes. The Golgi complex, which is normally coated with Irgm1 and Irgm2, is also GMS-free in these cells. Irga6 would therefore also be expected to accumulate at Golgi membranes, but this does not happen. Indeed, the Golgi complex seems to be immune to GKS activation independently of GMS loading. Finally, none of the endocellular GMS proteins localizes to the plasma membrane, but this structure is never coated with GKS proteins, even in WT cells. The basis for this protection is not known [14]. However, whatever the critical properties of the plasma membrane are, they must change rapidly during pathogen entry because the new pathogen-containing vacuolar membrane immediately becomes vulnerable to attack by GKS proteins [19]. In summary, the activation of GKS proteins occurs only on GMS-free membranes, but not necessarily on all. Furthermore, although the location of activated GKS proteins depends upon which membranes are GMS-free, the rules of selectivity between potential target membranes remain unclear.Fig. 9

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