Measuring S-Depalmitoylation Activity In Vitro and In Live Cells with Fluorescent Probes

S-palmitoylation is a reversible lipid posttranslational modification (PTM) that can mediate protein localization, trafficking, interaction with membranes, and a host of other biophysical characteristics. Over the past decade, a suite of chemoproteomic strategies have uncovered the breadth of S-palmitoylation, revealing widespread susceptibility to modification by this PTM throughout the human proteome. A focal point of research toward understanding the role of S-palmitoylation in varied cellular processes has focused on understanding how "writer" and "eraser" proteins function together to control the levels of S-palmitoylation of target proteins. The spatial and temporal regulation of S-palmitoylation by its "erasers"-acyl protein thioesterases (APTs)-is not fully understood. Tools which enable monitoring of the activity levels of the APTs in real-time in live cells illuminate how spatial control of these enzymes redecorate the lipidation state of the local proteome. To this end, we have developed fluorescence-based depalmitoylation probes (DPPs), which report S-depalmitoylase activity in live cells. Using DPPs, we have demonstrated that S-depalmitoylase activity changes in response to growth factor stimulation, unveiling potential regulation of cell growth and metabolism by APTs. Additionally, we recently discovered APTs in mitochondria using targeted DPPs, indicating new roles for S-depalmitoylation in this critical cellular compartment. Here, we present detailed protocols on how to carry out in vitro S-depalmitoylase activity assays and live cell fluorescence imaging employing the growing DPP toolbox.