Temporal Profiling Establishes a Dynamic <i>S</i>‑Palmitoylation Cycle

<i>S</i>-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 <i>S</i>-palmitoylated proteins sensitive to depalmitoylase inhibition, yet the breadth of enzyme regulated dynamic <i>S</i>-palmitoylation largely remains a mystery. Here, we present a pulsed bioorthogonal <i>S</i>-palmitoylation assay for temporal analysis of <i>S</i>-palmitoylation dynamics. Low concentration hexadecylfluorophosphonate (HDFP) inactivates the APT and ABHD17 families of depalmitoylases, which dramatically increases alkynyl-fatty acid labeling and stratifies <i>S</i>-palmitoylated proteins into kinetically distinct subgroups. Most surprisingly, HDFP treatment does not affect steady-state <i>S</i>-palmitoylation levels, despite inhibiting all validated depalmitoylating enzymes. <i>S</i>-palmitoylation profiling of APT1^–/–/APT2^–/– mouse brains similarly show no change in <i>S</i>-palmitoylation levels. In comparison with hydroxylamine-switch methods, bioorthogonal alkynyl fatty acids are only incorporated into a small fraction of dynamic <i>S</i>-palmitoylated proteins, raising the possibility that <i>S</i>-palmitoylation is more stable than generally characterized. Overall, disrupting depalmitoylase activity enhances alkynyl fatty acid incorporation, but does not greatly affect steady state <i>S</i>-palmitoylation across the proteome