Abstract
Wnt gradients elicit distinct cellular responses, such as proliferation, specification, differentiation and survival in a dose-dependent manner. Porcupine (PORCN), a membrane-bound O-acyl transferase (MBOAT) that resides in the endoplasmic reticulum, catalyses the addition of monounsaturated palmitate to Wnt proteins and is required for Wnt gradient formation and signalling. In humans, PORCN mutations are causal for focal dermal hypoplasia (FDH), an X-linked dominant syndrome characterized by defects in mesodermal and endodermal tissues. PORCN is also an emerging target for cancer therapeutics. Despite the importance of this enzyme, its structure remains poorly understood. Recently, the crystal structure of DltB, an MBOAT family member from bacteria, was solved. In this report, we use experimental data along with homology modelling to DltB to determine the membrane topology of PORCN. Our studies reveal that PORCN has 11 membrane domains, comprising nine transmembrane spanning domains and two reentrant domains. The N-terminus is oriented towards the lumen while the C-terminus is oriented towards the cytosol. Like DltB, PORCN has a funnel-like structure that is encapsulated by multiple membrane-spanning helices. This new model for PORCN topology allows us to map residues that are important for biological activity (and implicated in FDH) onto its three-dimensional structure.
Highlights
Wnt gradients are essential for proper embryonic development and adult homeostasis in both invertebrate and vertebrate organisms
The formation of Wnt gradients requires the activity of Porcupine (PORCN), an O-acyl transferase that primarily resides in the endoplasmic reticulum (ER) [1,2]
We introduced multiple epitope tags throughout the protein in positions that were suggested to be located in the loops between the membrane domains
Summary
Wnt gradients are essential for proper embryonic development and adult homeostasis in both invertebrate and vertebrate organisms. Biochemical studies have confirmed that PORCN catalyses the addition of a monounsaturated fatty acid, palmitoleate (C16:1), to a highly conserved serine residue corresponding to S224 in WNT1 [5,7,10,12,23,24]. Substitution of this conserved serine with an alanine or a cysteine abolishes Wnt palmit(e)oylation, secretion and signalling [5,6,7,10,25].
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