title: Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase.
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. The MBOAT family 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.
authors.affiliation: From the Department of Pharmacology and Molecular Sciences,; the High Throughput Biology Center and Department of Molecular Biology and Genetics.
authors.forename: Martin S
authors.initials: MS
authors.lastname: Taylor
authors.affiliation: the Department of Cell Biology.
authors.forename: Travis R
authors.initials: TR
authors.lastname: Ruch
authors.affiliation: From the Department of Pharmacology and Molecular Sciences.
authors.forename: Po-Yuan
authors.initials: PY
authors.lastname: Hsiao
authors.affiliation: From the Department of Pharmacology and Molecular Sciences.
authors.forename: Yousang
authors.initials: Y
authors.lastname: Hwang
authors.affiliation: the Program in Gene Expression and Regulation, The Wistar Institute, Philadelphia, Pennsylvania 19104.
authors.forename: Pingfeng
authors.initials: P
authors.lastname: Zhang
authors.affiliation: the High Throughput Biology Center and Department of Molecular Biology and Genetics.
authors.forename: Lixin
authors.initials: L
authors.lastname: Dai
authors.affiliation: the High Throughput Biology Center and Department of Molecular Biology and Genetics,; the McKusick-Nathans Institute of Genetic Medicine.
authors.forename: Cheng Ran Lisa
authors.initials: CRL
authors.lastname: Huang
authors.affiliation: the Howard Hughes Medical Institute and Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Berndsen
authors.affiliation: the McKusick-Nathans Institute of Genetic Medicine.
authors.forename: Min-Sik
authors.initials: MS
authors.lastname: Kim
authors.affiliation: the McKusick-Nathans Institute of Genetic Medicine.
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Pandey
authors.affiliation: the Howard Hughes Medical Institute and Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Wolberger
authors.affiliation: the Program in Gene Expression and Regulation, The Wistar Institute, Philadelphia, Pennsylvania 19104; the Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Ronen
authors.initials: R
authors.lastname: Marmorstein
authors.affiliation: the Department of Cell Biology,. Electronic address: machamer@jhmi.edu.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Machamer
authors.affiliation: the High Throughput Biology Center and Department of Molecular Biology and Genetics,. Electronic address: machamer@jhmi.edu.
authors.forename: Jef D
authors.initials: JD
authors.lastname: Boeke
authors.affiliation: From the Department of Pharmacology and Molecular Sciences,. Electronic address: pcole@jhmi.edu.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Cole
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002645:Chickens; D019295:Computational Biology; D004285:Dogs; D054439:Ghrelin / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D054439:Ghrelin; D000217:Acyltransferases; C542776:MBOAT4 protein, human
keywords: Endoplasmic Reticulum (ER); Enzyme; Lipids; Membrane Proteins; Metabolism; Peptide Hormones
references.citation: PLoS One. 2012;7(2):e32100
references.pmid: 22363801
references.citation: PLoS One. 2009;4(5):e5426
references.pmid: 19412546
references.citation: Methods Enzymol. 2012;514:205-28
references.pmid: 22975055
references.citation: Nucleic Acids Res. 1989 Jun 26;17(12):4895
references.pmid: 2748349
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10750-5
references.pmid: 18669668
references.citation: Nat Chem Biol. 2013 Apr;9(4):247-9
references.pmid: 23416332
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D348-52
references.pmid: 23197659
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 25;103(30):11142-7
references.pmid: 16847258
references.citation: J Biol Chem. 2007 Nov 23;282(47):34288-98
references.pmid: 17890783
references.citation: J Cell Biol. 2002 Dec 23;159(6):1029-37
references.pmid: 12486113
references.citation: Neurogastroenterol Motil. 2013 Feb;25(2):e140-50
references.pmid: 23279217
references.citation: J Biol Chem. 2008 Aug 8;283(32):22076-88
references.pmid: 18534984
references.citation: PLoS One. 2008 Jun 11;3(6):e2399
references.pmid: 18545655
references.citation: Proc Natl Acad Sci U S A. 2008 Apr 29;105(17):6320-5
references.pmid: 18443287
references.citation: Genes Dev. 2004 Mar 15;18(6):641-59
references.pmid: 15075292
references.citation: J Biol Chem. 2012 May 25;287(22):17942-50
references.pmid: 22474325
references.citation: Biophys J. 2000 Mar;78(3):1606-19
references.pmid: 10692345
references.citation: Biochem Biophys Res Commun. 2002 Dec 20;299(5):739-43
references.pmid: 12470640
references.citation: J Cardiovasc Pharmacol. 2002 Jun;39(6):779-83
references.pmid: 12021570
references.citation: Am J Physiol Gastrointest Liver Physiol. 2002 Jun;282(6):G948-52
references.pmid: 12016119
references.citation: Nature. 1999 Dec 9;402(6762):656-60
references.pmid: 10604470
references.citation: Methods Enzymol. 2000;327:175-90
references.pmid: 11044982
references.citation: Nature. 2009 Mar 26;458(7237):453-60
references.pmid: 19325625
references.citation: J Biol Chem. 2003 Mar 21;278(12):10208-13
references.pmid: 12524434
references.citation: Nat Med. 2009 Jul;15(7):741-5
references.pmid: 19503064
references.citation: Science. 2010 Dec 17;330(6011):1689-92
references.pmid: 21097901
references.citation: Comput Appl Biosci. 1994 Dec;10(6):685-6
references.pmid: 7704669
references.citation: Mol Microbiol. 2012 Dec;86(5):1156-66
references.pmid: 23016825
references.citation: Nucleic Acids Res. 2009 Jul;37(Web Server issue):W465-8
references.pmid: 19429891
references.citation: J Biol Chem. 2012 Dec 14;287(51):42881-9
references.pmid: 23112049
references.citation: Bioinformatics. 2001 Sep;17(9):849-50
references.pmid: 11590105
references.citation: Endocrinology. 2008 Oct;149(10):5172-6
references.pmid: 18599547
references.citation: Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1760-5
references.pmid: 19181854
references.citation: Nat Methods. 2011 Sep 29;8(10):785-6
references.pmid: 21959131
references.citation: Trends Biochem Sci. 2000 Mar;25(3):111-2
references.pmid: 10694878
references.citation: PLoS One. 2010 Jun 23;5(6):e11195
references.pmid: 20585641
references.citation: Anal Biochem. 2000 Mar 15;279(2):151-63
references.pmid: 10706784
references.citation: Am J Physiol Endocrinol Metab. 2009 Jul;297(1):E1-9
references.pmid: 19141679
references.citation: J Biol Chem. 2005 Nov 11;280(45):37814-26
references.pmid: 16154994
references.citation: J Biol Chem. 2011 Oct 21;286(42):36438-47
references.pmid: 21849510
references.citation: J Biol Chem. 2010 Nov 26;285(48):37377-87
references.pmid: 20876538
references.citation: J Biol Chem. 2006 Feb 24;281(8):4646-53
references.pmid: 16373356
references.citation: Addict Biol. 2010 Jul;15(3):304-11
references.pmid: 20477752
references.citation: Mol Biol Cell. 2006 Jun;17(6):2636-45
references.pmid: 16597698
references.citation: Biochem Biophys Res Commun. 2009 Jun 5;383(3):320-5
references.pmid: 19361486
references.citation: BMC Bioinformatics. 2012 Jul 17;13:169
references.pmid: 22805427
references.citation: Mol Biol Cell. 2003 Jun;14(6):2447-60
references.pmid: 12808042
references.citation: Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W390-3
references.pmid: 15215417
references.citation: Dev Cell. 2006 Dec;11(6):791-801
references.pmid: 17141155
references.citation: Endocrinology. 2005 May;146(5):2255-64
references.pmid: 15677766
references.citation: Cell Metab. 2008 May;7(5):389-99
references.pmid: 18460330
references.citation: ACS Synth Biol. 2013 Aug 16;2(8):473-7
references.pmid: 23654272
references.citation: Trends Mol Med. 2012 Apr;18(4):206-14
references.pmid: 22342806
references.citation: Cold Spring Harb Symp Quant Biol. 2011;76:121-7
references.pmid: 21785007
references.citation: J Cell Biol. 1991 Oct;115(1):19-30
references.pmid: 1655802
references.citation: J Mol Biol. 2001 Jan 19;305(3):567-80
references.pmid: 11152613
references.citation: Nucleic Acids Res. 2007 Jul;35(Web Server issue):W429-32
references.pmid: 17483518
references.citation: J Cell Biol. 1987 Aug;105(2):633-45
references.pmid: 3305520
references.citation: Mol Biol Cell. 2008 Mar;19(3):1174-84
references.pmid: 18094042
references.citation: Bioinformatics. 1998;14(4):378-9
references.pmid: 9632836
references.citation: J Biol Chem. 1993 Mar 15;268(8):5798-801
references.pmid: 8449946
references.citation: Nat Neurosci. 2006 Mar;9(3):381-8
references.pmid: 16491079
references.citation: Cell. 2008 Feb 8;132(3):387-96
references.pmid: 18267071
references.citation: Nucleic Acids Res. 2004 Mar 19;32(5):1792-7
references.pmid: 15034147
references.citation: J Biol Chem. 1999 Dec 17;274(51):36139-45
references.pmid: 10593897
references.citation: Bioinformatics. 2007 Mar 1;23(5):538-44
references.pmid: 17237066