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.
- SourceAvailable from: Francoise Gisou van der Goot
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- "Four profiling studies (Martin and Cravatt, 2009; Yang et al, 2010; Yount et al, 2010; Merrick et al, 2011) identified, with high confidence, calnexin as an S-acylated protein in mammalian cells. By immunoprecipitation of either the endogenous or tagged calnexin from 3 H-palmitate-labelled cells, we validated these profiling studies: the protein indeed incorporated radiolabelled palmitate, which could be removed by hydroxylamine hydrochloride treatment, indicating the involvement of a thioester bond (Figure 1A). "
ABSTRACT: A third of the human genome encodes N-glycosylated proteins. These are co-translationally translocated into the lumen/membrane of the endoplasmic reticulum (ER) where they fold and assemble before they are transported to their final destination. Here, we show that calnexin, a major ER chaperone involved in glycoprotein folding is palmitoylated and that this modification is mediated by the ER palmitoyltransferase DHHC6. This modification leads to the preferential localization of calnexin to the perinuclear rough ER, at the expense of ER tubules. Moreover, palmitoylation mediates the association of calnexin with the ribosome-translocon complex (RTC) leading to the formation of a supercomplex that recruits the actin cytoskeleton, leading to further stabilization of the assembly. When formation of the calnexin-RTC supercomplex was affected by DHHC6 silencing, mutation of calnexin palmitoylation sites or actin depolymerization, folding of glycoproteins was impaired. Our findings thus show that calnexin is a stable component of the RTC in a manner that is exquisitely dependent on its palmitoylation status. This association is essential for the chaperone to capture its client proteins as they emerge from the translocon, acquire their N-linked glycans and initiate folding.The EMBO Journal 02/2012; 31(7):1823-35. DOI:10.1038/emboj.2012.15 · 10.75 Impact Factor
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ABSTRACT: Protein lipidation is the covalent attachment of a lipid group to protein. Lipids modify large numbers of eukaryotic proteins and regulate protein function and localization. The realization that S-prenylation is essential for proper protein function has stimulated the development of small molecule S-prenyltransferase inhibitors to block the activity of oncogenic Ras and other S-prenylated proteins that malfunction in various disease states. Protein N-myristoylation refers to the irreversible addition of myristic acid to eukaryotic and viral proteins through an amide linkage to an N-terminal glycine residue. The process of depalmitoylation is less well characterized. The lysosomal enzyme responsible for degradation of S-palmitoylated proteins may be protein palmitoylthioesterase 1 (PPT1). Short amino acid sequences that encode the recognition motifs for modification are sufficient for lipidation to occur in cells. For example, the last 10 amino acids of H-Ras are sufficient for CaaX processing and S-palmitoylation in cells when transplanted onto a soluble protein.Chemical Reviews 09/2011; 111(10):6341-58. DOI:10.1021/cr2001977 · 45.66 Impact Factor
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ABSTRACT: Protein palmitoylation is a widespread lipid modification in which one or more cysteine thiols on a substrate protein are modified to form a thioester with a palmitoyl group. This lipid modification is readily reversible; a feature of protein palmitoylation that allows for rapid regulation of the function of many cellular proteins. Mutations in palmitoyltransferases (PATs), the enzymes that catalyze the formation of this modification, are associated with a number of neurological diseases and cancer progression. This review summarizes the crucial role of palmitoylation in biological systems, the discovery of the DHHC protein family that catalyzes protein palmitoylation, and the development of methods for investigating the catalytic mechanism of PATs.Science China-Chemistry 12/2011; 54(12). DOI:10.1007/s11426-011-4428-2 · 1.52 Impact Factor