Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase

Martin S. Taylor, Travis R. Ruch, Po Yuan Hsiao, Yousang Hwang, Pingfeng Zhang, Lixin Dai, Cheng Ran Lisa Huang, Christopher E. Berndsen, Min Sik Kim, Akhilesh Pandey, Cynthia Wolberger, Ronen Marmorstein, Carolyn Machamer, Jef D. Boeke, Philip A. Cole

    Research output: Contribution to journalArticle

    Abstract

    Ghrelin O-acyltransferase (GOAT) is a polytopic integral membrane protein required for activation of ghrelin, a secreted metabolism-regulating peptide hormone. Although GOAT is a potential therapeutic target for the treatment of obesity and diabetes and plays a key role in other physiologic processes, little is known about its structure or mechanism. GOAT is a member of the membrane-bound O-acyltransferase (MBOAT) family, a group of polytopic integral membrane proteins involved in lipid- biosynthetic and lipid-signaling reactions from prokaryotes to humans. Here we use phylogeny and a variety of bioinformatic tools to predict the topology of GOAT. Using selective permeabilization indirect immunofluorescence microscopy in combination with glycosylation shift immunoblotting, we demonstrate that GOAT contains 11 transmembrane helices and one reentrant loop. Development of the V5Glyc tag, a novel, small, and sensitive dual topology reporter, facilitated these experiments. TheMBOATfamily invariant residue His-338 is in the ER lumen, consistent with other family members, but conserved Asn-307 is cytosolic, making it unlikely that both are involved in catalysis. Photocross-linking of synthetic ghrelin analogs and inhibitors demonstrates binding to the C-terminal region of GOAT, consistent with a role of His-338 in the active site. This knowledge of GOAT architecture is important for a deeper understanding of the mechanism of GOAT and other MBOATs and could ultimately advance the discovery of selective inhibitors for these enzymes.

    Original languageEnglish (US)
    Pages (from-to)32211-32228
    Number of pages18
    JournalJournal of Biological Chemistry
    Volume288
    Issue number45
    DOIs
    StatePublished - Nov 8 2013

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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  • Cite this

    Taylor, M. S., Ruch, T. R., Hsiao, P. Y., Hwang, Y., Zhang, P., Dai, L., Huang, C. R. L., Berndsen, C. E., Kim, M. S., Pandey, A., Wolberger, C., Marmorstein, R., Machamer, C., Boeke, J. D., & Cole, P. A. (2013). Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase. Journal of Biological Chemistry, 288(45), 32211-32228. https://doi.org/10.1074/jbc.M113.510313