Detection and Localization of Porcupine: a Conserved Membrane-bound O-acyltransferase Involved in the Post-translational Lipid Modification of Wnts

Wnts are secreted signal molecules that are important for patterning of tissues during embryogenesis. Wnt secretion is dependent on the lipidation of two conserved residues. Lipidation of the conserved serine is facilitated by Porcupine (Porcn), a membrane-bound O-acyltransferase. To better understand the role of Porcn in Wnt secretion, we sought to characterize the subcellular localization of overexpressed and endogenous Porcn. Overexpressed Porcn was previously shown to be localized to the endoplasmic reticulum (ER) and to promote the association of Wnt1 with lipid rafts. Thus, we hypothesize that endogenous Porcn is localized to the ER and lipid rafts. To test this, we developed polyclonal antibodies against Porcn. We first confirmed the localization of transiently overexpressed Porcn to the ER of cultured cells. We next demonstrated the presence of overexpressed Porcn in lipid rafts and on the cell surface. We then tested the localization of endogenous Porcn. In contrast to overexpressed Porcn, the majority of endogenous Porcn was detected in the Golgi. To confirm the presence of endogenous Porcn in lipid rafts, we immunostained cells for Flotillin-1 (a lipid raft marker) and Porcn. Endogenous Porcn in cell lines partially co-localizes with Flotillin-1, further supporting the localization of Porcn to lipid rafts. These findings challenge the current dogma about Porcn localization and suggest novel roles for Porcn.