Proteomic Profiling of S-acylated Macrophage Proteins Identifies a Role for Palmitoylation in Mitochondrial Targeting of Phospholipid Scramblase 3

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

doi:10.1074/mcp.m110.006007

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.

Highlights

EXPERIMENTAL PROCEDURESReagents—Sequencing grade porcine trypsin was purchased from Promega Corp. (Madison, WI)
S-Palmitoylation is the post-translational modification of specific cysteine residues in proteins with the 16-carbon fatty acid palmitate, a process that promotes tethering of proteins to cellular membranes and thereby contributes to specifying protein subcellular localization
We propose that proteomic analysis may offer important new insights on a global scale into subcellular targeting of proteins in the macrophage, and we speculate that manipulation of protein palmitoylation may represent a fruitful opportunity for intervention in macrophage biology

Summary

EXPERIMENTAL PROCEDURES

Reagents—Sequencing grade porcine trypsin was purchased from Promega Corp. (Madison, WI). Each portion was dissolved in 4% SDS buffer followed by addition of 4 volumes of low HPDP-biotin binding buffer (150 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 0.2 mM HPDP-biotin, 0.2% Triton X-100) and another 1 h incubation with rotation and three sequential chloroform-methanol extractions to remove excess crosslinker. Media was removed and cells were washed with phosphate-buffered saline (PBS) prior to lysis (1% Nonidet P-40, 50 mM Tris HCl pH 8, 1 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 300 mM NaCl, protease inhibitors [Roche #1836153], 5 mM sodium fluoride, 1 mM sodium-o-vanadate and 1 mM PMSF). Nuclei were isolated (18) by solubilizing cell membranes from the 700 ϫ g pellet with 500 ␮l of low salt lysis buffer (20 mM Hepes, pH 7.4, 5 mM NaCl, 5 mM MgCl2, 0.5% Nonidet P-40, 0.1% sodium deoxycholate, protease inhibitors) for 10 min on ice, followed by centrifugation (1000 ϫ g, 7 min, 4 °C).

RESULTS

Membrane Trafficking

Mtdh b

DISCUSSION