Proteome scale characterization of human S-acylated proteins in lipid raft-enriched and non-raft membranes

Mol Cell Proteomics. 2010 Jan;9(1):54-70. doi: 10.1074/mcp.M800448-MCP200. Epub 2009 Oct 2.


Protein S-acylation (palmitoylation), a reversible post-translational modification, is critically involved in regulating protein subcellular localization, activity, stability, and multimeric complex assembly. However, proteome scale characterization of S-acylation has lagged far behind that of phosphorylation, and global analysis of the localization of S-acylated proteins within different membrane domains has not been reported. Here we describe a novel proteomics approach, designated palmitoyl protein identification and site characterization (PalmPISC), for proteome scale enrichment and characterization of S-acylated proteins extracted from lipid raft-enriched and non-raft membranes. In combination with label-free spectral counting quantitation, PalmPISC led to the identification of 67 known and 331 novel candidate S-acylated proteins as well as the localization of 25 known and 143 novel candidate S-acylation sites. Palmitoyl acyltransferases DHHC5, DHHC6, and DHHC8 appear to be S-acylated on three cysteine residues within a novel CCX(7-13)C(S/T) motif downstream of a conserved Asp-His-His-Cys cysteine-rich domain, which may be a potential mechanism for regulating acyltransferase specificity and/or activity. S-Acylation may tether cytoplasmic acyl-protein thioesterase-1 to membranes, thus facilitating its interaction with and deacylation of membrane-associated S-acylated proteins. Our findings also suggest that certain ribosomal proteins may be targeted to lipid rafts via S-acylation, possibly to facilitate regulation of ribosomal protein activity and/or dynamic synthesis of lipid raft proteins in situ. In addition, bioinformatics analysis suggested that S-acylated proteins are highly enriched within core complexes of caveolae and tetraspanin-enriched microdomains, both cholesterol-rich membrane structures. The PalmPISC approach and the large scale human S-acylated protein data set are expected to provide powerful tools to facilitate our understanding of the functions and mechanisms of protein S-acylation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acyltransferases / metabolism
  • Binding Sites
  • Cell Line, Tumor
  • Cell Membrane / metabolism*
  • Humans
  • Immunoblotting
  • Lipoylation
  • Mass Spectrometry
  • Membrane Microdomains / metabolism*
  • Membrane Proteins / analysis*
  • Membrane Proteins / classification
  • Membrane Proteins / metabolism
  • Microscopy, Fluorescence
  • Palmitic Acid / metabolism
  • Proteome / analysis*
  • Proteome / metabolism
  • Proteomics / methods
  • Ribosomal Protein L10
  • Ribosomal Proteins / analysis
  • Ribosomal Proteins / metabolism
  • Zinc Fingers


  • Membrane Proteins
  • Proteome
  • RPL10 protein, human
  • Ribosomal Proteins
  • Palmitic Acid
  • Acyltransferases