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.
Highlights
The interferon-inducible immunity-related GTPases (IRG proteins, previously named p47 GTPases) are resistance factors against intracellular pathogens in mice
We previously reported that IFNc-induced Irgm1 (LRG-47) is exclusively membrane-bound and localises in large part to the Golgi apparatus in L929 fibroblasts, TIB-75 hepatocytes and Raw 264.7 macrophages
By using TGN38 as a trans-Golgi and trans-Golgi network (TGN) marker, the Golgi localisation domain of Irgm1 is extended to these compartments (Figure 1A)
Summary
The interferon-inducible immunity-related GTPases (IRG proteins, previously named p47 GTPases) are resistance factors against intracellular pathogens in mice. Alone of the IRG proteins assayed, Irgm has never been found at the parasitophorous vacuole, it is clear that this IRG protein is not directly involved in the observed vesiculation and disruption of the parasitophorous vacuole membrane It has, been shown that Irgm, with its closest homologues Irgm and Irgm, is required for regulating the GTPase cycle of the other IRG proteins in the cytoplasm [8]. Been shown that Irgm, with its closest homologues Irgm and Irgm, is required for regulating the GTPase cycle of the other IRG proteins in the cytoplasm [8] It follows that the general susceptibility of Irgm1-deficient mice to protozoal infection may be accounted for as a regulatory failure that disables the IRG-dependent resistance mechanism. Been proposed for the role of Irgm in these and other aspects of immunity, especially in the case of resistance to Mycobacteria, which is lost in Irgm deficiency: these include accelerated lysosome fusion and/or acidification of phagosomes [12,13], and induction of autophagy [14,15]
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