In vitro reconstitution in membrane environment reveals critical lipid dependence of substrate S-acylation by protein palmitoyltransferase enzymes.

Protein S-acylation, also known as protein palmitoylation, is a common form of post-translational modification and the most abundant form of lipid modification of proteins with several thousand substrates. Protein S-acylation is critically important in a host of physiological processes, including protein trafficking, stability, and synaptic organization. Twenty-three transmembrane enzymes belonging to the zDHHC family catalyze this modification in humans. Yet, how the membrane environment modulates zDHHC activity and their interactions with substrates are poorly understood. Here, we report a direct fluorescence polarization-based assay in completely reconstituted membrane environments using two zDHHC members, zDHHC15 and zDHHC20 and their substrates, postsynaptic density protein-95 and epidermal growth factor receptor, respectively. Our assay enables investigation of the role of lipid composition on the activity of zDHHC enzymes. We show, using this assay, that cholesterol modulates the activity of zDHHC enzymes. We use the assay reconstituted in giant unilamellar vesicles to directly visualize the localization of S-acylated proteins to cholesterol-rich membranes, a phenomenon known for many decades through cell-based experiments. Our work demonstrates the role of lipid composition in zDHHC activity and opens up avenues to dissect the role of different lipids in the activity of 23 zDHHC enzymes. Our nanodisc assay will facilitate high-throughput screening of small-molecule probes and the discovery of novel therapeutic leads for human diseases by targeting protein S-acylation.