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

Remco N P Rodenburg, Joost Snijder, Michiel van de Waterbeemd, Arie Schouten, Joke Granneman, Albert J R Heck, Piet Gros

Research output: Contribution to journalArticleAcademicpeer-review


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.

Original languageEnglish
Article number1280
JournalNature Communications
Issue number1
Publication statusPublished - 3 Nov 2017


  • Mass spectrometry
  • Membrane proteins
  • Post-translational modifications
  • Structural biology


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