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Temporal Profiling Establishes a Dynamic S-Palmitoylation Cycle.

ACS chemical biology (2018-05-08)
Sang Joon Won, Brent R Martin
RESUMEN

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.

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Sigma-Aldrich
Anti-α-tubulina monoclonal antibody produced in mouse, clone B-5-1-2, purified from hybridoma cell culture
Sigma-Aldrich
Tetraethylammonium borohydride, technical, ≥95% (T)