Stochastic palmitoylation of accessible cysteines in membrane proteins revealed by native mass spectrometry

Nat Commun. 2017 Nov 3;8(1):1280. doi: 10.1038/s41467-017-01461-z.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD20 / chemistry
  • Bacterial Proteins / chemistry
  • Bacteriorhodopsins / chemistry
  • Binding Sites
  • Claudin-3 / chemistry
  • Claudin-4 / chemistry
  • Claudins / chemistry
  • Cysteine / chemistry
  • HEK293 Cells
  • Humans
  • Lipoylation
  • Mass Spectrometry
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Models, Molecular
  • Potassium Channels / chemistry
  • Protein Domains
  • Protein Processing, Post-Translational
  • Stochastic Processes


  • Antigens, CD20
  • Bacterial Proteins
  • CLDN3 protein, human
  • CLDN4 protein, human
  • Claudin-3
  • Claudin-4
  • Claudins
  • Membrane Proteins
  • Potassium Channels
  • prokaryotic potassium channel
  • Bacteriorhodopsins
  • Cysteine
  • claudin 6