title: Palmitoylation of the immunity related GTPase, Irgm1: impact on membrane localization and ability to promote mitochondrial fission.
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.
authors.affiliation: Geriatric Research, Education, and Clinical Center, VA Medical Center, Durham, North Carolina, United States of America.
authors.forename: Stanley C
authors.initials: SC
authors.lastname: Henry
authors.affiliation: Departments of Medicine; Molecular Genetics and Microbiology; and Immunology; Division of Geriatrics, and Center for the Study of Aging and Human Development, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Elyse A
authors.initials: EA
authors.lastname: Schmidt
authors.affiliation: Laboratory of Respiratory Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Fessler
authors.affiliation: Geriatric Research, Education, and Clinical Center, VA Medical Center, Durham, North Carolina, United States of America; Departments of Medicine; Molecular Genetics and Microbiology; and Immunology; Division of Geriatrics, and Center for the Study of Aging and Human Development, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Taylor
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000172:deficiency / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D007109:Immunity / Q000187:drug effects; D054878:Lipoylation / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D063154:Mitochondrial Dynamics / Q000187:drug effects; D009154:Mutation / Q000235:genetics; D010168:Palmitates / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects
chemical_list: C096000:Ifi1 protein, mouse; D010168:Palmitates; C022776:2-bromopalmitate; D019204:GTP-Binding Proteins
references.citation: Annu Rev Biochem. 1996;65:241-69
references.pmid: 8811180
references.citation: J Biol Chem. 1997 Apr 18;272(16):10639-45
references.pmid: 9099712
references.citation: Cell. 1994 Jul 1;77(7):1063-70
references.pmid: 7912657
references.citation: J Immunol. 2004 Aug 15;173(4):2594-606
references.pmid: 15294976
references.citation: J Immunol. 2013 Aug 15;191(4):1765-74
references.pmid: 23842753
references.citation: J Biol Chem. 2003 Aug 1;278(31):29336-43
references.pmid: 12732635
references.citation: Nat Rev Mol Cell Biol. 2012 Jan 11;13(2):75-88
references.pmid: 22233676
references.citation: Protein Eng Des Sel. 2008 Nov;21(11):639-44
references.pmid: 18753194
references.citation: PLoS Pathog. 2009 Sep;5(9):e1000577
references.pmid: 19750224
references.citation: Infect Immun. 2005 Jun;73(6):3278-86
references.pmid: 15908352
references.citation: Curr Opin Microbiol. 2011 Aug;14(4):414-21
references.pmid: 21783405
references.citation: J Exp Med. 2006 Sep 4;203(9):2063-71
references.pmid: 16940170
references.citation: Genome Biol. 2005;6(11):R92
references.pmid: 16277747
references.citation: J Exp Med. 2001 Jul 16;194(2):181-8
references.pmid: 11457893
references.citation: Science. 2003 Oct 24;302(5645):654-9
references.pmid: 14576437
references.citation: J Biol Chem. 2000 Jun 16;275(24):18520-6
references.pmid: 10764749
references.citation: Biochem Soc Trans. 2013 Feb 1;41(1):84-8
references.pmid: 23356263
references.citation: Nat Cell Biol. 2010 Dec;12(12):1154-65
references.pmid: 21102437
references.citation: PLoS One. 2011;6(12):e28323
references.pmid: 22163006
references.citation: J Biol Chem. 2011 Sep 2;286(35):30471-30480
references.pmid: 21757726
references.citation: PLoS Pathog. 2009 Feb;5(2):e1000288
references.pmid: 19197351
references.citation: J Biol Chem. 2000 Jan 7;275(1):261-70
references.pmid: 10617614
references.citation: Prog Lipid Res. 2009 May-Jul;48(3-4):117-27
references.pmid: 19233228
references.citation: PLoS One. 2010 Jan 13;5(1):e8648
references.pmid: 20072621
references.citation: Blood. 2011 Aug 11;118(6):1525-33
references.pmid: 21633090
references.citation: Proc Natl Acad Sci U S A. 2000 Jan 18;97(2):751-5
references.pmid: 10639151
references.citation: Biochimie. 2011 Jan;93(1):18-31
references.pmid: 21056615
references.citation: Cell. 2004 Dec 17;119(6):753-66
references.pmid: 15607973
references.citation: Nat Immunol. 2009 Aug;10(8):907-17
references.pmid: 19620982
references.citation: Mol Biol Cell. 2011 Jun 1;22(11):1930-42
references.pmid: 21471001
references.citation: Biochim Biophys Acta. 2011 Dec;1808(12):2981-94
references.pmid: 21819967
references.citation: PLoS One. 2011;6(6):e20568
references.pmid: 21698150
references.citation: J Leukoc Biol. 2010 Feb;87(2):333-43
references.pmid: 19920210
references.citation: Cell Microbiol. 2007 May;9(5):1099-107
references.pmid: 17359233
references.citation: Nature. 2007 Jun 7;447(7145):661-78
references.pmid: 17554300
references.citation: Am J Physiol Gastrointest Liver Physiol. 2013 Oct 15;305(8):G573-84
references.pmid: 23989005
references.citation: PLoS Pathog. 2005 Nov;1(3):e24
references.pmid: 16304607
references.citation: Nat Genet. 2007 Jul;39(7):830-2
references.pmid: 17554261
references.citation: Cell Host Microbe. 2012 Oct 18;12(4):432-44
references.pmid: 23084913
references.citation: EMBO J. 2008 Oct 8;27(19):2495-509
references.pmid: 18772884
references.citation: Mol Cell Proteomics. 2011 Oct;10(10):M110.006007
references.pmid: 21785166
references.citation: PLoS Pathog. 2013;9(6):e1003414
references.pmid: 23785284
references.citation: J Cell Biol. 2010 Dec 27;191(7):1229-38
references.pmid: 21187327
references.citation: Nat Immunol. 2000 Aug;1(2):113-8
references.pmid: 11248802