Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase

Martin S. Taylor, Travis R. Ruch, Po Yuan Hsiao, You Sang Hwang, Pingfeng Zhang, Lixin Dai, Cheng Ran Lisa Huang, Christopher E. Berndsen, Min Sik Kim, Akhilesh Pandey, Cynthia Wolberger, Ronen Marmorstein, Carolyn E 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

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Acyltransferases
Ghrelin
Organizations
Enzymes
Membrane Proteins
Topology
Glycosylation
Lipids
Peptide Hormones
Enzyme Inhibitors
Phylogeny
Bioinformatics
Indirect Fluorescent Antibody Technique
Medical problems
Computational Biology
Catalysis
Fluorescence Microscopy
Immunoblotting
Metabolism
Catalytic Domain

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase. / Taylor, Martin S.; Ruch, Travis R.; Hsiao, Po Yuan; Hwang, You Sang; Zhang, Pingfeng; Dai, Lixin; Huang, Cheng Ran Lisa; Berndsen, Christopher E.; Kim, Min Sik; Pandey, Akhilesh; Wolberger, Cynthia; Marmorstein, Ronen; Machamer, Carolyn E; Boeke, Jef D.; Cole, Philip A.

In: Journal of Biological Chemistry, Vol. 288, No. 45, 08.11.2013, p. 32211-32228.

Research output: Contribution to journalArticle

Taylor, MS, Ruch, TR, Hsiao, PY, Hwang, YS, Zhang, P, Dai, L, Huang, CRL, Berndsen, CE, Kim, MS, Pandey, A, Wolberger, C, Marmorstein, R, Machamer, CE, Boeke, JD & Cole, PA 2013, 'Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase', Journal of Biological Chemistry, vol. 288, no. 45, pp. 32211-32228. https://doi.org/10.1074/jbc.M113.510313
Taylor, Martin S. ; Ruch, Travis R. ; Hsiao, Po Yuan ; Hwang, You Sang ; Zhang, Pingfeng ; Dai, Lixin ; Huang, Cheng Ran Lisa ; Berndsen, Christopher E. ; Kim, Min Sik ; Pandey, Akhilesh ; Wolberger, Cynthia ; Marmorstein, Ronen ; Machamer, Carolyn E ; Boeke, Jef D. ; Cole, Philip A. / Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase. In: Journal of Biological Chemistry. 2013 ; Vol. 288, No. 45. pp. 32211-32228.
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AU - Hwang, You Sang

AU - Zhang, Pingfeng

AU - Dai, Lixin

AU - Huang, Cheng Ran Lisa

AU - Berndsen, Christopher E.

AU - Kim, Min Sik

AU - Pandey, Akhilesh

AU - Wolberger, Cynthia

AU - Marmorstein, Ronen

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AU - Boeke, Jef D.

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