ABSTRACT
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.
Subject(s)
GTP-Binding Proteins/immunology , Interferon-gamma/pharmacology , Phagosomes/immunology , Alternative Splicing , Amino Acid Sequence , Animals , Antibody Specificity , Cell Line , Fibroblasts/chemistry , Fluorescent Antibody Technique, Direct , GTP Phosphohydrolases/classification , GTP-Binding Proteins/analysis , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Golgi Apparatus/chemistry , Humans , Immunization , Immunologic Deficiency Syndromes/enzymology , Immunologic Deficiency Syndromes/genetics , Listeria monocytogenes/immunology , Macrophages/chemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/chemistry , Molecular Sequence Data , Mycobacterium bovis/immunology , Peptide Fragments/immunology , Phagosomes/microbiology , Protein Isoforms/analysis , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/immunologyABSTRACT
We have recently reported that poly-SUMO-2/3 conjugates are subject to a ubiquitin-dependent proteolytic control in human cells. Here we show that arsenic trioxide (ATO) increases SUMO-2/3 modification of promyelocytic leukemia (PML) leading to its subsequent ubiquitylation in vivo. The SUMO-binding ubiquitin ligase RNF4 mediates this modification and causes disruption of PML nuclear bodies upon treatment with ATO. Reconstitution of SUMO-dependent ubiquitylation of PML by RNF4 in vitro and in a yeast trans vivo system revealed a preference of RNF4 for chain forming SUMOs. Polysumoylation of PML in response to ATO thus leads to its recognition and ubiquitylation by RNF4.
