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Determination of the Membrane Topology of the PORCN O‐Acyl Transferase
Author(s) -
Fraley J Gabriel,
Galli Lisa,
Burrus Laura,
Sanchez Luis
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb53
Subject(s) - membrane topology , topology (electrical circuits) , cytosol , wnt signaling pathway , palmitoylation , microbiology and biotechnology , biology , membrane protein , signal transduction , membrane , genetics , biochemistry , mathematics , combinatorics , cysteine , enzyme
WNT signaling is critical for proper embryonic development and adult tissue homeostasis. WNT proteins require palmitoylation for optimal secretion and receptor binding. Porcupine (PORCN), a membrane bound O‐acyltransferase (MBOAT) family member is the protein that palmitoylates WNT. PORCN is localized to the Endoplasmic Reticulum (ER). Though PORCN is required for proper tissue patterning in developing embryos, its membrane topology is poorly understood. Determining the topology of PORCN may have useful applications in areas of embryonic development studies. Although numerous bioinformatics algorithms that predict membrane topology are available, no clear consensus for the topology of PORCN has emerged. Therefore, our goal is to experimentally determine the topology of PORCN. To determine topology, we developed two strategies. The first strategy utilized differential solubilization and immunofluorescent imaging of single, and dual tagged PORCN variants. Results from single Myc‐tagged N‐and C‐terminus PORCN variants in transfected COS‐7 cells orient the N‐terminus in the ER lumen, and the C‐terminus in the cytosol. To further determine topology, we developed twelve dual tagged PORCN variants with Myc epitopes within predicted loops, and a C‐terminal 3X FLAG tag. The construction of dual tagged variants provided us with qualitative information, as well as quantitative values using Mander's coefficients of colocalization. Our studies show that five of the PORCN variant loops exhibit cytosolic orientation while one loop was oriented toward the lumen. As a control, we showed that the N‐ and C‐terminal tags remained lumenal and cytosolic, respectively. However, the remaining constructs provided conflicting results. Additionally, a PORCN antibody was generated against an immunogenic region that includes residues 282–303. Immunofluorescent imaging and colocalization quantitation orient this epitope towards the cytosol. As a second strategy, we developed ten mutants which resulted in the introduction of ectopic consensus sites for the addition of N‐linked carbohydrates (NxT/NxS). These PORCN mutants were transfected into HEK‐293T cells and subjected to Western blot analysis to identify glycosylated variants. This analysis identified two glycosylated residues at A134N and D283N, which indicates localization of these two residues to the lumen of the ER. Intriguingly, these results do not agree with those from our differential solubilization studies. We are now carrying out additional controls to help us distinguish between these two results. Support or Funding Information NIH‐MARC T34‐GM008574