title: Dual N- and C-terminal helices are required for endoplasmic reticulum and lipid droplet association of alcohol acetyltransferases in Saccharomyces cerevisiae.
abstract: In the yeast Saccharomyces cerevisiae two alcohol acetyltransferases (AATases), Atf1 and Atf2, condense short chain alcohols with acetyl-CoA to produce volatile acetate esters. Such esters are, in large part, responsible for the distinctive flavors and aromas of fermented beverages including beer, wine, and sake. Atf1 and Atf2 localize to the endoplasmic reticulum (ER) and Atf1 is known to localize to lipid droplets (LDs). The mechanism and function of these localizations are unknown. Here, we investigate potential mechanisms of Atf1 and Atf2 membrane association. Segments of the N- and C-terminal domains of Atf1 (residues 24-41 and 508-525, respectively) are predicted to be amphipathic helices. Truncations of these helices revealed that the terminal domains are essential for ER and LD association. Moreover, mutations of the basic or hydrophobic residues in the N-terminal helix and hydrophobic residues in the C-terminal helix disrupted ER association and subsequent sorting from the ER to LDs. Similar amphipathic helices are found at both ends of Atf2, enabling ER and LD association. As was the case with Atf1, mutations to the N- and C-terminal helices of Atf2 prevented membrane association. Sequence comparison of the AATases from Saccharomyces, non-Saccharomyces yeast (K. lactis and P. anomala) and fruits species (C. melo and S. lycopersicum) showed that only AATases from Saccharomyces evolved terminal amphipathic helices. Heterologous expression of these orthologs in S. cerevisiae revealed that the absence of terminal amphipathic helices eliminates LD association. Combined, the results of this study suggest a common mechanism of membrane association for AATases via dual N- and C-terminal amphipathic helices.
authors.affiliation: Department of Chemical and Environmental Engineering, University of California Riverside, Riverside, California, United States of America.
authors.forename: Jyun-Liang
authors.initials: JL
authors.lastname: Lin
authors.affiliation: Department of Chemical and Environmental Engineering, University of California Riverside, Riverside, California, United States of America.
authors.forename: Ian
authors.initials: I
authors.lastname: Wheeldon
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D066292:Lipid Droplets / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D000123:Acetyltransferases; C116217:ATF2 protein, S cerevisiae; C079238:alcohol O-acetyltransferase
references.citation: Microb Biotechnol. 2010 Mar;3(2):165-77
references.pmid: 21255318
references.citation: FEBS J. 2013 Mar;280(5):1344-57
references.pmid: 23311868
references.citation: Chem Biol. 2014 Jan 16;21(1):86-96
references.pmid: 24239006
references.citation: Yeast. 2004 Mar;21(4):367-77
references.pmid: 15042596
references.citation: Trends Biochem Sci. 2000 Mar;25(3):147-50
references.pmid: 10694887
references.citation: Mol Biol Evol. 2011 Oct;28(10):2731-9
references.pmid: 21546353
references.citation: Plant Cell. 1997 Aug;9(8):1481-93
references.pmid: 9286116
references.citation: Eur J Biochem. 1999 Apr;261(1):317-24
references.pmid: 10103065
references.citation: J Cell Sci. 2005 Jun 15;118(Pt 12):2601-11
references.pmid: 15914536
references.citation: Appl Environ Microbiol. 2000 Feb;66(2):744-53
references.pmid: 10653746
references.citation: J Bacteriol. 1999 Oct;181(20):6441-8
references.pmid: 10515935
references.citation: Plant Cell. 2000 May;12(5):647-62
references.pmid: 10810141
references.citation: Nat Chem Biol. 2014 Apr;10(4):259-65
references.pmid: 24609358
references.citation: Bioinformatics. 2008 Sep 15;24(18):2101-2
references.pmid: 18662927
references.citation: Nature. 2003 Oct 16;425(6959):686-91
references.pmid: 14562095
references.citation: FEBS Lett. 2010 May 3;584(9):1840-7
references.pmid: 19837069
references.citation: Bioprocess Biosyst Eng. 2008 Jun;31(4):299-306
references.pmid: 17891501
references.citation: J Biol Chem. 2008 Oct 17;283(42):28005-9
references.pmid: 18611863
references.citation: Appl Environ Microbiol. 1997 Mar;63(3):910-5
references.pmid: 9055409
references.citation: J Cell Sci. 2013 Oct 15;126(Pt 20):4794-805
references.pmid: 23943872
references.citation: Appl Environ Microbiol. 2003 Sep;69(9):5228-37
references.pmid: 12957907
references.citation: Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20452-7
references.pmid: 19920176
references.citation: Yeast. 2004 Aug;21(11):947-62
references.pmid: 15334558
references.citation: Nucleic Acids Res. 2002 May 15;30(10):e43
references.pmid: 12000848
references.citation: Dev Cell. 2013 Feb 25;24(4):384-99
references.pmid: 23415954
references.citation: Appl Microbiol Biotechnol. 2008 Apr;78(5):783-92
references.pmid: 18309479
references.citation: Proc Int Conf Intell Syst Mol Biol. 1998;6:175-82
references.pmid: 9783223
references.citation: Yeast. 1999 Sep 15;15(12):1183-97
references.pmid: 10487921
references.citation: Yeast. 2006 Jul 15;23(9):641-59
references.pmid: 16845703
references.citation: J Cell Biol. 1995 Feb;128(4):509-23
references.pmid: 7860627
references.citation: Yeast. 2000 Oct;16(14):1287-98
references.pmid: 11015726
references.citation: J Biol Chem. 2006 Aug 4;281(31):22236-22247
references.pmid: 16704979
references.citation: Appl Environ Microbiol. 1994 Aug;60(8):2786-92
references.pmid: 8085822
references.citation: Methods Mol Biol. 2009;579:359-74
references.pmid: 19763485
references.citation: J Biol Chem. 2008 Jun 20;283(25):17065-74
references.pmid: 18430725
references.citation: EMBO J. 2007 Dec 12;26(24):5109-19
references.pmid: 18034159
references.citation: Biochim Biophys Acta. 2011 Dec;1811(12):1165-76
references.pmid: 21820081
references.citation: Annu Rev Biochem. 2012;81:687-714
references.pmid: 22524315
references.citation: J Ind Microbiol Biotechnol. 2003 Jul;30(7):427-32
references.pmid: 12937998
references.citation: J Biol Chem. 2011 Aug 12;286(32):28235-46
references.pmid: 21680734
references.citation: Eur J Biochem. 1992 Dec 15;210(3):1015-22
references.pmid: 1483449
references.citation: J Mol Biol. 2001 Jan 19;305(3):567-80
references.pmid: 11152613
references.citation: Biosci Biotechnol Biochem. 1998 Oct;62(10):1852-7
references.pmid: 9836419
references.citation: J Cell Biol. 2006 Jun 5;173(5):719-31
references.pmid: 16735577
references.citation: Yeast. 1994 Nov;10(11):1421-8
references.pmid: 7871881
references.citation: Cell Metab. 2011 Oct 5;14(4):504-15
references.pmid: 21982710
references.citation: J Biol Chem. 2005 Dec 23;280(51):42325-35
references.pmid: 16207721
references.citation: Mol Cell Biol. 2005 Mar;25(5):1655-68
references.pmid: 15713625
references.citation: J Lipid Res. 2012 Feb;53(2):215-26
references.pmid: 22045929
references.citation: Yeast. 1995 May;11(6):493-536
references.pmid: 7645343
references.citation: Appl Environ Microbiol. 2008 Jan;74(2):454-61
references.pmid: 17993562
references.citation: Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
references.pmid: 7984417
references.citation: Metab Eng. 2004 Oct;6(4):294-9
references.pmid: 15491859