Single-cell imaging of Wnt palmitoylation by the acyltransferase porcupine

Wnts are secreted palmitoylated glycoproteins that are important in embryonic development and human cancers. Here we report a method for imaging the palmitoylated form of Wnt proteins with subcellular resolution using clickable bioorthogonal fatty acids and in situ proximity ligation. Palmitoylated Wnt3a is visualized throughout the secretory pathway and trafficks to multivesicular bodies that act as export sites in secretory cells. We establish that glycosylation is not required for Wnt3a palmitoylation, which is necessary but not sufficient for Wnt3a secretion. Wnt3a is palmitoylated by fatty acids 13-16 carbons in length at Ser209 but not at Cys77, consistent with a slow turnover rate. We find that porcupine (PORCN) itself is palmitoylated, demonstrating what is to our knowledge the first example of palmitoylation of an MBOAT protein, and this modification partially regulates Wnt palmitoylation and signaling. Our data reveal the role of O-palmitoylation in Wnt signaling and suggest another layer of cellular control over PORCN function and Wnt secretion.