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

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

B Alex Merrick  1 , Suraj Dhungana, Jason G Williams, Jim J Aloor, Shyamal Peddada, Kenneth B Tomer, Michael B Fessler
Affiliations

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

B Alex Merrick et al. Mol Cell Proteomics. 2011 Oct.

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.

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Figures

Fig. 1.
Fig. 1.
Enrichment of candidate S-acylproteins in the macrophage through acyl- biotinyl exchange. A, RAW 264.7 membranes were subjected to acyl-biotin exchange under hydroxylamine (HA+) or mock (HA−) treatment conditions. Proteins precipitated by avidin-Sepharose were then resolved by 10% SDS-PAGE and detected by Sypro ruby stain. B, Mean spectral counts under HA+ and HA− treatment conditions was plotted for n = 297 proteins evaluated in the statistical analysis. Proteins identified as candidate S-acylproteins at FDR = 0.05 (see Table I) are shown in figure as open dots. Inset shows greater detail.
Fig. 2.
Fig. 2.
Principal component analysis of candidate S-acylproteins. PCA plots of the data were performed using (A) the full set of 297 proteins that were entered into the statistical analysis of S-acylation, or (B) just the 80 S-acylprotein candidates that were significant at FDR = 0.05 (Table I). These unsupervised plots, accounting for 77.5% (A) and 93.6% (B) of the variability in the data, respectively, reveal discrete clustering of proteins on the basis of HA treatment. This serves to confirm the selectivity of the ABE-statistical procedure, verifying that the 80 S-acylprotein candidates identified clearly discriminate between the HA+ and HA− conditions.
Fig. 3.
Fig. 3.
Comparative MS analysis of proteins enriched and not enriched by acyl-biotinyl exchange procedure. A, MS/MS of ion m/z 714.4, corresponding to a tryptic peptide of M2-pyruvate kinase, a protein not enriched by acyl-biotinyl exchange procedure (see text). Analysis is consistent with NEM modification of the cysteine residue. B, Extracted ion chromatogram (EIC) of ion m/z 714.4 from HA+ -treated condition (dashed line) and HA− -treated condition (solid line). C, MS/MS of ion m/z 861.6, corresponding to tryptic peptide 12 of stomatin, a known palmitoylprotein that was enriched by acyl-biotinyl exchange (see text). Analysis indicates that the cysteine residue is unmodified. D, EIC of ion m/z 885.5, corresponding to peptide in C with cysteine oxidized to cysteic acid, from HA+ -treated condition (dashed line) and HA− -treated condition (solid lines).
Fig. 4.
Fig. 4.
Identification of palmitoylated residues in Plscr3. HEK293 cells were transiently transfected with empty vector (EV), wild type (WT) Plscr3, or with cysteine-to-alanine mutant Plscr3 in which all 5 cysteines (5A), the 3 C-terminal cysteines (3A), or the 2 N-terminal cysteines (2A) of the 159CGCSCCPC166 sequence were mutagenized. Transfected cells were exposed to 0.1mCi/ml 3H-palmitate (4h), Plscr3 was immunoprecipitated, and precipitates were then probed for Plscr3 by immunoblot and imaged by autoradiography. Samples in right figure represent biological duplicates. Data are representative of three independent experiments.
Fig. 5.
Fig. 5.
Palmitoylation of Plscr3 regulates its subcellular localization. A, HEK293 cells were transiently transfected with empty vector (EV), WT Plscr3, or with 5A, 3A, or 2A cysteine-to-alanine Plscr3 mutants as described in Fig. 4. Cells were then fractionated into mitochondrial and nuclear fractions, and both whole cell lysate (WCL) and fractions immunoblotted (IB) for Plscr3. Data are representative of three independent experiments. B, HEK293 cells stably transfected with EV, wt Plscr3, or 5A mutant Plscr3 were imaged (63×/1.4 NA oil objective) after immunostaining with anti-Plscr3 antibody (primary) and Alexa Fluor 488-conjugated donkey anti-goat secondary antibody, MitoTracker Red to detect mitochondria, and DAPI to detect nuclei. Two examples of 5A Plscr3-transfected fields are shown to illustrate extranuclear (middle panels) and nuclear (bottom panels) staining patterns. C, Mitochondrial localization of WT and 5A mutant Plscr3 in transfected HEK293 cells was quantified by determining the weighted colocalization coefficient to reveal intensity-independent colocalization of Plscr3 with Mitotracker Red-stained mitochondria. Data are mean ± S.E. (n = 9 fields [1–4 cells/fields] per condition; *, p < 0.0001).
Fig. 6.
Fig. 6.
Palmitoylation-deficient mutant Plscr3 confers reduced apoptotic function. A, RAW 264.7 macrophages stably expressing EV, wt Plscr3, or 5A mutant Plscr3 were treated with vehicle (DMSO) or different concentrations of etoposide (Etop) for 24 h. Cells were then treated with a cell-permeant caspase activity probe (FAM-FLIVO™, Immunochemistry) and 7-aminoactinomycin D (AAD) to probe membrane integrity. Apoptosis was quantified by flow cytometry as the sum of FLIVO+ 7-AAD− (lower right quadrants in B) and FLIVO+ 7-AAD+ (upper right quadrants in B) cells by flow cytometry. Data are mean ± S.E., and are representative of two independent experiments performed in duplicate. B, Representative flow cytometry plots for data in A. C, Cells as in A were treated with vehicle or 8 nm etoposide for 8 h, and then apoptosis was quantified as in A. Data are mean ± S.E., and are representative of two independent experiments performed in triplicate.
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