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Determining The Topology of The Membrane Bound O‐acyltransferase Porcupine
Author(s) -
Sanchez Luis,
Fraley Gabriel,
Bueno Raymund,
Galli Lisa,
Burrus Laura
Publication year - 2015
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.29.1_supplement.574.20
Subject(s) - membrane topology , cytoplasm , topology (electrical circuits) , biology , palmitoylation , microbiology and biotechnology , chemistry , membrane protein , biochemistry , membrane , enzyme , mathematics , combinatorics , cysteine
Porcupine (PORCN), a membrane bound O‐acyltransferase (MBOAT) of the endoplasmic reticulum (ER), is essential for Wnt palmitoylation, transport, secretion, and activity. Because all human Wnts require PORCN for their activity, the inhibition of PORCN has become an attractive therapeutic target for the treatment of various cancers displaying an overexpression of Wnt ligands. In order to design and understand how potential PORCN inhibitors work, the topology of PORCN must be determined. Although various bioinformatics algorithms offer numerous predictions, no clear membrane topology of PORCN has emerged. Therefore, we aimed to experimentally establish the membrane topology of PORCN. Preliminary data orient the N and C terminus toward the ER lumen and cytoplasm, respectively. Additional experiments in which consensus glycosylation sites were introduced into the PORCN sequence suggest that the loop containing A134 is localized to the ER lumen. Therefore, we hypothesized that the A134 containing loop is entirely localized to the ER lumen. To test this hypothesis, we expressed two PORCN constructs with FLAG tags inserted at residues 118 and 145 respectively, and a myc tag at the C‐terminus, in COS‐7 cells. Contrary to our hypothesis, immunostaining of COS‐7 cells selectively permeabilized with Triton X‐100 or digitonin revealed that the FLAG tag on both variants was localized to the cytoplasm. These apparently contradictory data suggest that PORCN may exist in multiple conformations. Together, our findings provide structural information about PORCN that can help in the design and understanding of PORCN inhibitors that could be used for the treatment of Wnt driven cancers This research was made possible by a grant received from the National Institute of Health (NIH).