MABF2264-100UL Sigma-AldrichAnti-IRGM1 (LRG-47) Antibody, clone 1B2

The Immunity-Related GTPases (IRG) are a family of large GTPases that mediate innate immune responses. Irgm1 is particularly critical for immunity to bacteria and protozoa, and for inflammatory homeostasis in the intestine. Although precise functions for Irgm1 have not been identified, prior studies have suggested roles in autophagy/mitophagy, phagosome remodeling, cell motility, and regulating the activity of other IRG proteins. These functions ostensibly hinge on the ability of Irgm1 to localize to intracellular membranes, such as those of the Golgi apparatus and mitochondria. Previously, it has been shown that an amphipathic helix, the αK helix, in the C-terminal portion of the protein partially mediates membrane binding. However, in absence of αK, there is still substantial binding of Irgm1 to cellular membranes, suggesting the presence of other membrane binding motifs. In the current work, an additional membrane localization motif was found in the form of palmitoylation at a cluster of cysteines near the αK. An Irgm1 mutant possessing alanine to cysteine substitutions at these amino acids demonstrated little residual palmitoylation, yet it displayed only a small decrease in localization to the Golgi and mitochondria. In contrast, a mutant containing the palmitoylation mutations in combination with mutations disrupting the amphipathic character of the αK displayed a complete loss of apparent localization to the Golgi and mitochondria, as well as an overall loss of association with cellular membranes in general. Additionally, Irgm1 was found to promote mitochondrial fission, and this function was undermined in Irgm1 mutants lacking the palmitoylation domain, and to a greater extent in those lacking the αK, or the αK and palmitoylation domains combined. Our data suggest that palmitoylation together with the αK helix firmly anchor Irgm1 in the Golgi and mitochondria, thus facilitating function of the protein.

The IFN-inducible protein Irgm1 (LRG-47) belongs to the family of immunity-related GTPases that function in cell-autonomous resistance against intracellular pathogens in mice. Irgm1 deficiency is associated with a severe immunodeficiency syndrome. The protein has been variously interpreted as a direct effector molecule on bacterial phagosomes or on other organelles or as an inducer of autophagy. In this study, we re-examined one of these claims, namely that Irgm1 targets mycobacterial and listerial phagosomes. We found no colocalization of endogenous Irgm1, using two immunofluorescent staining techniques, either in fibroblasts or in macrophages. We demonstrated the predicted existence of two protein isoforms of Irgm1 derived from differential splicing and described immunological reagents for their detection. Both Irgm1 isoforms localize to the Golgi apparatus and weakly to mitochondria; however, only the long Irgm1 isoforms can be detected on endolysosomal membranes. Together with the previous observation that the general immunodeficiency phenotype of Irgm1(-/-) mice is reversed in Irgm1/Irgm3 double-deficient mice, our results argue against a direct effector function of Irgm1 at the bacterial phagosome. We discuss these findings in the context of evidence that Irgm1 functions as a negative regulator of other members of the immunity-related GTPase protein family.

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.

The cytokine gamma interferon (IFN-gamma) is critical for resistance to Toxoplasma gondii. IFN-gamma strongly activates macrophages and nonphagocytic host cells to limit intracellular growth of T. gondii; however, the cellular factors that are required for this effect are largely unknown. We have shown previously that IGTP and LRG-47, members of the IFN-gamma-regulated family of p47 GTPases, are required for resistance to acute T. gondii infections in vivo. In contrast, IRG-47, another member of this family, is not required. In the present work, we addressed whether these GTPases are required for IFN-gamma-induced suppression of T. gondii growth in macrophages in vitro. Bone marrow macrophages that lacked IGTP or LRG-47 displayed greatly attenuated IFN-gamma-induced inhibition of T. gondii growth, while macrophages that lacked IRG-47 displayed normal inhibition. Thus, the ability of the p47 GTPases to limit acute infection in vivo correlated with their ability to suppress intracellular growth in macrophages in vitro. Using confocal microscopy and sucrose density fractionation, we demonstrated that IGTP largely colocalizes with endoplasmic reticulum markers, while LRG-47 was mainly restricted to the Golgi. Although both IGTP and LRG-47 localized to vacuoles containing latex beads, neither protein localized to vacuoles containing live T. gondii. These results suggest that IGTP and LRG-47 are able to regulate host resistance to acute T. gondii infections through their ability to inhibit parasite growth within the macrophage.