Temporal Profiling Establishes a Dynamic S-Palmitoylation Cycle
Year: 2018
Type: article
Abstract: 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.
Source: ACS Chemical Biology
Authors: Sang Joon Won, Brent R. Martin
Institution: University of Michigan
Language: English
Subfield: Organic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W2802751954