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.