Prenylome profiling reveals S-farnesylation is crucial for membrane targeting and antiviral activity of ZAP long-isoform.

S-prenylation is an important lipid modification that targets proteins to membranes for cell signaling and vesicle trafficking in eukaryotes. As S-prenylated proteins are often key effectors for oncogenesis, congenital disorders, and microbial pathogenesis, robust proteomic methods are still needed to biochemically characterize these lipidated proteins in specific cell types and disease states. Here, we report that bioorthogonal proteomics of macrophages with an improved alkyne-isoprenoid chemical reporter enables large-scale profiling of prenylated proteins, as well as the discovery of unannotated lipidated proteins such as isoform-specific S-farnesylation of zinc-finger antiviral protein (ZAP). Notably, S-farnesylation was crucial for targeting the long-isoform of ZAP (ZAPL/PARP-13.1/zc3hav1) to endolysosomes and enhancing the antiviral activity of this immune effector. These studies demonstrate the utility of isoprenoid chemical reporters for proteomic analysis of prenylated proteins and reveal a role for protein prenylation in host defense against viral infections.