Proteomic profiling of S-acylated macrophage proteins identifies a role for palmitoylation in mitochondrial targeting of phospholipid scramblase 3

Mol Cell Proteomics. 2011 Oct;10(10):M110.006007. doi: 10.1074/mcp.M110.006007. Epub 2011 Jul 23.


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.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Apoptosis
  • Cell Membrane / enzymology
  • Cell Membrane / metabolism
  • Cell Nucleus / metabolism
  • Cysteine / chemistry
  • Cysteine / metabolism*
  • Etoposide / pharmacology
  • HEK293 Cells
  • Humans
  • Lipoylation*
  • Macrophages / enzymology*
  • Mice
  • Mitochondria / enzymology*
  • Mutagenesis, Site-Directed
  • Palmitates / chemistry
  • Palmitates / metabolism
  • Phospholipid Transfer Proteins / genetics
  • Phospholipid Transfer Proteins / metabolism*
  • Protein Processing, Post-Translational
  • Proteomics
  • Tandem Mass Spectrometry


  • PLSCR3 protein, human
  • Palmitates
  • Phospholipid Transfer Proteins
  • Plscr3 protein, mouse
  • Etoposide
  • Cysteine