Proteome scale characterization of human S-acylated proteins in lipid raft-enriched and non-raft membranes.
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.
Urological Diseases Research Center, Department of Urology, Children's Hospital Boston, Boston, Massachusetts 02115, USA.
Wei
W
Yang
Dolores
D
Di Vizio
Marc
M
Kirchner
Hanno
H
Steen
Michael R
MR
Freeman
D000217:Acyltransferases / Q000378:metabolism; D001665:Binding Sites; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D054878:Lipoylation; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis* / Q000145:classification / Q000378:metabolism; D008856:Microscopy, Fluorescence; D019308:Palmitic Acid / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000032:analysis / Q000378:metabolism; D016335:Zinc Fingers
D008565:Membrane Proteins; D020543:Proteome; C000630686:RPL10 protein, human; D012269:Ribosomal Proteins; D019308:Palmitic Acid; D000217:Acyltransferases