Abstract
Palmitoylation affects membrane partitioning, trafficking and activities of membrane proteins. However, how specificity of palmitoylation and multiple palmitoylations in membrane proteins are determined is not well understood. Here, we profile palmitoylation states of three human claudins, human CD20 and cysteine-engineered prokaryotic KcsA and bacteriorhodopsin by native mass spectrometry. Cysteine scanning of claudin-3, KcsA, and bacteriorhodopsin shows that palmitoylation is independent of a sequence motif. Palmitoylations are observed for cysteines exposed on the protein surface and situated up to 8 Å into the inner leaflet of the membrane. Palmitoylation on multiple sites in claudin-3 and CD20 occurs stochastically, giving rise to a distribution of palmitoylated membrane-protein isoforms. Non-native sites in claudin-3 indicate that membrane-protein function imposed evolutionary restraints on native palmitoylation sites. These results suggest a generic, stochastic membrane-protein palmitoylation process that is determined by the accessibility of palmitoyl-acyl transferases to cysteines on membrane-embedded proteins, and not by a preferred substrate-sequence motif.
Highlights
Palmitoylation affects membrane partitioning, trafficking and activities of membrane proteins
Palmitoylation is mediated by membraneembedded palmitoyl-acyl transferases (PAT) that contain a palmitoylated cysteine-rich domain with a conserved Asp-His-HisCys (DHHC) motif, which is required for the palmitoylation activity[14–18]
Protein palmitoylation remains generally elusive in shotgun proteomics experiments, since palmitoylated peptides do typically not make it to mass spectrometric detection, due to their hydrophobicity, general insolubility, poor fragmentation behavior and hydrolysis susceptibility of the thioester-linked palmitate[15, 31]
Summary
Palmitoylation affects membrane partitioning, trafficking and activities of membrane proteins. Nonnative sites in claudin-3 indicate that membrane-protein function imposed evolutionary restraints on native palmitoylation sites These results suggest a generic, stochastic membrane-protein palmitoylation process that is determined by the accessibility of palmitoyl-acyl transferases to cysteines on membrane-embedded proteins, and not by a preferred substrate-sequence motif. We use native mass spectrometry (MS) to quantify the palmitoylation states of three members of the four-helical tightjunction protein family claudins (Cld), Cld[3], Cld[4], and Cld[6], the four-helical B-lymphocyte antigen CD20 and cysteine-engineered constructs of the light-activated proton-pump bacteriorhodopsin and the potassium-channel KcsA. Based on our data we propose a model in which palmitoylation of cysteines is not dependent on a local sequence motif In these cases, modification of cysteines is rather determined by membrane-protein structure, in that it occurs generally on accessible residues within a depth of 8 Å of the lipid bilayer. These results provide insights into a membrane-protein palmitoylation process that is independent of a substratesequence motif
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