title: Proteomic profiling of S-acylated macrophage proteins identifies a role for palmitoylation in mitochondrial targeting of phospholipid scramblase 3.
abstract: S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have been hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine versus mock treatment conditions. Of 1183 proteins identified in four independent experiments, 80 proteins were significant for S-acylation at false discovery rate = 0.05, and 101 significant at false discovery rate = 0.10. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and three presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. Among the candidate novel S-acylproteins was phospholipid scramblase 3 (Plscr3), a protein that regulates apoptosis through remodeling the mitochondrial membrane. Palmitoylation of Plscr3 was confirmed through (3)H-palmitate labeling. Moreover, site-directed mutagenesis of a cluster of five cysteines (Cys159-161-163-164-166) abolished palmitoylation, caused Plscr3 mislocalization from mitochondrion to nucleus, and reduced macrophage apoptosis in response to etoposide, together suggesting a role for palmitoylation at this site for mitochondrial targeting and pro-apoptotic function of Plscr3. Taken together, we propose that manipulation of protein palmitoylation carries great potential for intervention in macrophage biology via reprogramming of protein localization.
authors.affiliation: Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA.
authors.forename: B Alex
authors.initials: BA
authors.lastname: Merrick
authors.forename: Suraj
authors.initials: S
authors.lastname: Dhungana
authors.forename: Jason G
authors.initials: JG
authors.lastname: Williams
authors.forename: Jim J
authors.initials: JJ
authors.lastname: Aloor
authors.forename: Shyamal
authors.initials: S
authors.lastname: Peddada
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Tomer
authors.forename: Michael B
authors.initials: MB
authors.lastname: Fessler
mesh_terms: D000818:Animals; D017209:Apoptosis; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D005047:Etoposide / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D054878:Lipoylation; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010168:Palmitates / Q000737:chemistry / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: C493424:PLSCR3 protein, human; D010168:Palmitates; D049294:Phospholipid Transfer Proteins; C493428:Plscr3 protein, mouse; D005047:Etoposide; D003545:Cysteine