Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Apr 21;9(4):e95021.
doi: 10.1371/journal.pone.0095021. eCollection 2014.

Palmitoylation of the Immunity Related GTPase, Irgm1: Impact on Membrane Localization and Ability to Promote Mitochondrial Fission

Affiliations
Free PMC article

Palmitoylation of the Immunity Related GTPase, Irgm1: Impact on Membrane Localization and Ability to Promote Mitochondrial Fission

Stanley C Henry et al. PLoS One. .
Free PMC article

Abstract

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Palmitoylation of Irgm1.
WT 3T3 MEF, Irgm1 KO 3T3 MEF, or Irgm1 KO 3T3 MEF stably transduced with the indicated Irgm1 mutants were exposed to 100U/mL IFN-γ for 24 h, and then incubated with 3H-palmitate for 4 h. Lysates were prepared and used for (A) 10% SDS-PAGE and western blotting with anti-Irgm1 antibodies, or (B) immunoprecipitation (IP) with anti-Irgm1 antibodies followed by 10% SDS-PAGE and autoradiography. Shown are representative results selected from 3 separate experiments. The positions of MW markers are shown at the left. Palmitoylation was quantified in each of the three immunoprecipitation studies, expressed as a value for each Irgm1 mutant relative to the value for WT Irgm1, and then average values across the three experiments displayed in (C), with error bars indicating standard error of the mean, and * indicating p<0.05 as determined using a one-sided z-distribution that was corrected for multiple comparisons.
Figure 2
Figure 2. Effect of Irgm1 palmitoylation mutation on Golgi association.
Irgm1 KO MEF were transfected with plasmids expressing wild-type or mutant Irgm1 proteins, as indicated. The cells were exposed to 100 U/ml IFN-γ for 24 h, stained with anti-Irgm1 and anti-GM130 antibodies, and used for immunofluorescence analysis. The experiment was performed 3 times, with at least 20 cells analyzed per group in each experiment. (A) Shown are images from representative cells. The scale bar represents 20 µm. (B) As detailed in the EXPERIMENTAL PROCEDURES, co-localization analysis was performed to quantify overlap between the Irgm1 and GM130 signals. In each experiment, the degree of co-localization was averaged for cells within an experimental group, and these values were then averaged across the three experiments, with error bars representing standard error the mean, and * representing p<0.05 as assessed by Student’s t-test.
Figure 3
Figure 3. Effect of an Irgm1 palmitoylation mutation on mitochondrial association.
Irgm1 KO MEF were transfected with plasmids expressing wild-type or mutant Irgm1 proteins, as indicated. The cells were exposed to 100 U/ml IFN-γ for 24 h, stained with anti-Irgm1 and anti-Tom20 antibodies, and used for immunofluorescence analysis. The experiment was performed 3 times, with at least 20 cells analyzed per group in each experiment. (A) Shown are images from representative cells. The scale bar represents 20 µm. (B) As detailed in the EXPERIMENTAL PROCEDURES, co-localization analysis was performed to quantify overlap between the Irgm1 and Tom20 signals. In each experiment, the degree of co-localization was averaged for cells within an experimental group, and these values were then averaged across the three experiments, with error bars representing standard error the mean, and * representing p<0.05 as assessed by Student’s t-test.
Figure 4
Figure 4. Effect of 2-bromopalmitate on Irgm1 association with the Golgi.
WT MEF were exposed to 100/ml IFN-γ for 24 h, followed by exposure to the palmitoylation inhibitor, 2-bromopalmitate (0.1 mM), for 2 h. The cells were then co-stained with anti-Irgm1, and (A) anti-GM130 or (B) anti-Tom20 antibodies, and used for immunofluorescence analysis. Shown is the overlap between the Irgm1 and GM130 signals as determined by colocalization analysis, and expressed as an average from three (GM130) or four (Tom20) separate experiments, with between 25 and 59 cells analyzed per group in each experiment. The error bars represent standard error of the mean. The effect of 2-BP was significant in (A) (p = 0.009), but not (B) (p = 0.066).
Figure 5
Figure 5. Effect of an Irgm1 palmitoylation mutation on membrane binding.
Irgm1 KO MEF were transfected with plasmids expressing the indicated wild-type or mutant Irgm1 proteins. The cells were exposed to 100 U/ml IFN-γ for 16–18 h, and then used for preparation of detergent-free lysates that were separated into soluble (S) and membrane bound fractions (P) by centrifugation at 100,000×g. These fractions were separated on 10% SDS-PAGE gels that were used for western blotting with anti-Irgm1 and GAPDH antibodies. (A) Shown is a representative western blot, with the positions of MW markers shown at the left. (B) The percentage of the total protein that was detected in the P fraction was determined for three separate experiments. The average values are shown with error bars indicating standard error of the mean, and * indicating p<0.05 as determined using a one-sided z-distribution that was corrected for multiple comparisons.
Figure 6
Figure 6. Effect of an Irgm1 palmitoylation mutation on the ability of the protein to promote formation of punctate mitochondria.
Irgm1 KO MEF were transfected with plasmids expressing wild-type or mutant Irgm1 proteins, as indicated. The cells were exposed to 100 U/ml IFN-γ for 24 h, stained with anti-Irgm1 and anti-Tom20 antibodies, and used for immunofluorescence analysis. Images were collected from cells that expressed the Irgm1 proteins; the mitochondria in these images were scored in a blinded fashion as being punctate, tubular, or mixed phenotype. At least 20 cells were scored per experimental group, and the results displayed as percent of the total. Shown is the average of four separate studies, with error bars representing standard deviation, and * representing p<0.05 as assessed by Student’s t-test.

Similar articles

See all similar articles

Cited by 14 articles

See all "Cited by" articles

References

    1. Howard JC, Hunn JP, Steinfeldt T (2011) The IRG protein-based resistance mechanism in mice and its relation to virulence in Toxoplasma gondii. Curr Opin Microbiol 14: 414–421. - PubMed
    1. Kim BH, Shenoy AR, Kumar P, Bradfield CJ, MacMicking JD (2012) IFN-inducible GTPases in host cell defense. Cell Host Microbe 12: 432–444. - PMC - PubMed
    1. Taylor GA (2007) IRG proteins: key mediators of interferon-regulated host resistance to intracellular pathogens. Cell Microbiol 9: 1099–1107. - PubMed
    1. Bekpen C, Hunn JP, Rohde C, Parvanova I, Guethlein L, et al. (2005) The interferon-inducible p47 (IRG) GTPases in vertebrates: loss of the cell autonomous resistance mechanism in the human lineage. Genome Biol 6: R92. - PMC - PubMed
    1. Collazo CM, Yap GS, Sempowski GD, Lusby KC, Tessarollo L, et al. (2001) Inactivation of LRG-47 and IRG-47 reveals a family of interferon gamma-inducible genes with essential, pathogen-specific roles in resistance to infection. J Exp Med 194: 181–188. - PMC - PubMed

Publication types

Feedback