Temporal Profiling Establishes a Dynamic S-Palmitoylation Cycle

ACS Chem Biol. 2018 Jun 15;13(6):1560-1568. doi: 10.1021/acschembio.8b00157. Epub 2018 May 23.


S-palmitoylation is required for membrane anchoring, proper trafficking, and the normal function of hundreds of integral and peripheral membrane proteins. Previous bioorthogonal pulse-chase proteomics analyses identified Ras family GTPases, polarity proteins, and G proteins as rapidly cycling S-palmitoylated proteins sensitive to depalmitoylase inhibition, yet the breadth of enzyme regulated dynamic S-palmitoylation largely remains a mystery. Here, we present a pulsed bioorthogonal S-palmitoylation assay for temporal analysis of S-palmitoylation dynamics. Low concentration hexadecylfluorophosphonate (HDFP) inactivates the APT and ABHD17 families of depalmitoylases, which dramatically increases alkynyl-fatty acid labeling and stratifies S-palmitoylated proteins into kinetically distinct subgroups. Most surprisingly, HDFP treatment does not affect steady-state S-palmitoylation levels, despite inhibiting all validated depalmitoylating enzymes. S-palmitoylation profiling of APT1-/-/APT2-/- mouse brains similarly show no change in S-palmitoylation levels. In comparison with hydroxylamine-switch methods, bioorthogonal alkynyl fatty acids are only incorporated into a small fraction of dynamic S-palmitoylated proteins, raising the possibility that S-palmitoylation is more stable than generally characterized. Overall, disrupting depalmitoylase activity enhances alkynyl fatty acid incorporation, but does not greatly affect steady state S-palmitoylation across the proteome.

Publication types

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

MeSH terms

  • Enzyme Inhibitors / pharmacology
  • Fatty Acids, Unsaturated / chemistry
  • HEK293 Cells
  • Humans
  • Indicators and Reagents / chemistry
  • Kinetics
  • Lipoylation
  • Mass Spectrometry / methods
  • Organofluorophosphonates / pharmacology
  • Protein Processing, Post-Translational
  • Proteome / metabolism*
  • Proteomics / methods
  • Thiolester Hydrolases / antagonists & inhibitors


  • Enzyme Inhibitors
  • Fatty Acids, Unsaturated
  • Indicators and Reagents
  • Organofluorophosphonates
  • Proteome
  • 17-octadecynoic acid
  • Thiolester Hydrolases