title: Identification of a probable pore-forming domain in the multimeric vacuolar anion channel AtALMT9.
abstract: Aluminum-activated malate transporters (ALMTs) form an important family of anion channels involved in fundamental physiological processes in plants. Because of their importance, the role of ALMTs in plant physiology is studied extensively. In contrast, the structural basis of their functional properties is largely unknown. This lack of information limits the understanding of the functional and physiological differences between ALMTs and their impact on anion transport in plants. This study aimed at investigating the structural organization of the transmembrane domain of the Arabidopsis (Arabidopsis thaliana) vacuolar channel AtALMT9. For that purpose, we performed a large-scale mutagenesis analysis and found two residues that form a salt bridge between the first and second putative transmembrane α-helices (TMα1 and TMα2). Furthermore, using a combination of pharmacological and mutagenesis approaches, we identified citrate as an "open channel blocker" of AtALMT9 and used this tool to examine the inhibition sensitivity of different point mutants of highly conserved amino acid residues. By this means, we found a stretch within the cytosolic moiety of the TMα5 that is a probable pore-forming domain. Moreover, using a citrate-insensitive AtALMT9 mutant and biochemical approaches, we could demonstrate that AtALMT9 forms a multimeric complex that is supposedly composed of four subunits. In summary, our data provide, to our knowledge, the first evidence about the structural organization of an ion channel of the ALMT family. We suggest that AtALMT9 is a tetramer and that the TMα5 domains of the subunits contribute to form the pore of this anion channel.
authors.affiliation: Institute of Plant Biology, University of Zürich, CH-8008 Zurich, Switzerland.
authors.forename: Jingbo
authors.initials: J
authors.lastname: Zhang
authors.forename: Ulrike
authors.initials: U
authors.lastname: Baetz
authors.forename: Undine
authors.initials: U
authors.lastname: Krügel
authors.forename: Enrico
authors.initials: E
authors.lastname: Martinoia
authors.forename: Alexis
authors.initials: A
authors.lastname: De Angeli
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000838:Anions; D017360:Arabidopsis / Q000187:drug effects / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry* / Q000378:metabolism*; D019343:Citric Acid / Q000494:pharmacology; D017124:Conserved Sequence / Q000235:genetics; D015640:Ion Channel Gating / Q000187:drug effects; D008293:Malates / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014026:Nicotiana / Q000187:drug effects / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism*
chemical_list: C584418:ALMT9 protein, Arabidopsis; D000596:Amino Acids; D000838:Anions; D029681:Arabidopsis Proteins; D018118:Chloride Channels; D008293:Malates; D050505:Mutant Proteins; D021122:Protein Subunits; D019343:Citric Acid
references.citation: Science. 1978 Sep 29;201(4362):1187-91
references.pmid: 694508
references.citation: Science. 1992 Nov 6;258(5084):873-4
references.pmid: 1439795
references.citation: Front Plant Sci. 2012 Nov 29;3:263
references.pmid: 23226151
references.citation: Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9738-43
references.pmid: 16740662
references.citation: J Biol Chem. 2005 Mar 11;280(10):8945-50
references.pmid: 15634668
references.citation: Nature. 2002 Jan 17;415(6869):287-94
references.pmid: 11796999
references.citation: J Biol Chem. 1998 Jan 9;273(2):681-4
references.pmid: 9422716
references.citation: Plant Cell Physiol. 2005 May;46(5):812-6
references.pmid: 15769806
references.citation: Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21425-30
references.pmid: 19955405
references.citation: Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):8123-8
references.pmid: 8755614
references.citation: Plant Physiol. 2006 Nov;142(3):1294-303
references.pmid: 17028155
references.citation: Nature. 2011 Jun 2;474(7349):54-60
references.pmid: 21572436
references.citation: Plant J. 2000 Feb;21(3):259-67
references.pmid: 10758477
references.citation: Planta. 1991 Jul;184(4):532-7
references.pmid: 24194244
references.citation: Eur J Biochem. 1988 Jul 1;174(4):671-8
references.pmid: 3134198
references.citation: Nature. 2006 Aug 24;442(7105):939-42
references.pmid: 16878138
references.citation: J Gen Physiol. 1973 Jun;61(6):687-708
references.pmid: 4541078
references.citation: New Phytol. 2005 Sep;167(3):665-91
references.pmid: 16101906
references.citation: Nature. 2008 Mar 27;452(7186):483-6
references.pmid: 18305482
references.citation: Plant J. 2009 Nov;60(3):411-23
references.pmid: 19563436
references.citation: Science. 1991 Feb 22;251(4996):939-42
references.pmid: 2000494
references.citation: J Biol Chem. 1996 Dec 27;271(52):33632-8
references.pmid: 8969232
references.citation: Science. 1990 Oct 12;250(4978):276-9
references.pmid: 2218530
references.citation: J Biol Chem. 1998 May 29;273(22):13469-74
references.pmid: 9593680
references.citation: Pharmacol Rev. 2010 Sep;62(3):405-96
references.pmid: 20716669
references.citation: Crit Rev Biochem Mol Biol. 2008 Jan-Feb;43(1):3-36
references.pmid: 18307107
references.citation: Nat Commun. 2013;4:1804
references.pmid: 23653216
references.citation: Nat Rev Neurosci. 2002 Feb;3(2):115-21
references.pmid: 11836519
references.citation: Plant Physiol. 2008 Aug;147(4):2131-46
references.pmid: 18550686
references.citation: Proc Natl Acad Sci U S A. 2010 Apr 27;107(17):8023-8
references.pmid: 20385816
references.citation: Plant J. 2010 Sep;63(6):1054-62
references.pmid: 20626656
references.citation: Mol Plant. 2013 Sep;6(5):1550-63
references.pmid: 23314055
references.citation: Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2741-4
references.pmid: 8610111
references.citation: Nature. 2010 Oct 28;467(7319):1074-80
references.pmid: 20981093
references.citation: Plant Signal Behav. 2007 Nov;2(6):467-72
references.pmid: 19517008
references.citation: Nature. 2009 Jan 1;457(7225):115-8
references.pmid: 18987630
references.citation: Annu Rev Plant Biol. 2012;63:183-213
references.pmid: 22404463
references.citation: Proc Natl Acad Sci U S A. 2012 Jun 26;109(26):10593-8
references.pmid: 22689970
references.citation: Bioinformatics. 2009 May 1;25(9):1189-91
references.pmid: 19151095
references.citation: Methods Mol Biol. 2006;337:53-63
references.pmid: 16929938
references.citation: Neuron. 2010 Sep 23;67(6):915-28
references.pmid: 20869590
references.citation: Nature. 2008 Mar 27;452(7186):487-91
references.pmid: 18305484
references.citation: Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):741-6
references.pmid: 11209069
references.citation: Annu Rev Physiol. 2009;71:59-82
references.pmid: 18842100
references.citation: J Biol Chem. 1999 Feb 26;274(9):5429-35
references.pmid: 10026154
references.citation: J Membr Biol. 1995 Sep;147(1):23-34
references.pmid: 8531197
references.citation: EMBO J. 1997 Jun 16;16(12):3455-63
references.pmid: 9218788
references.citation: Science. 1998 Jun 5;280(5369):1596-9
references.pmid: 9616121
references.citation: Plant J. 2007 Dec;52(6):1169-80
references.pmid: 18005230
references.citation: Plant J. 2010 Oct;64(1):47-55
references.pmid: 20663086
references.citation: Proteomics. 2008 Oct;8(19):3974-90
references.pmid: 18763698
references.citation: Annu Rev Plant Biol. 2011;62:25-51
references.pmid: 21275645
references.citation: Methods Enzymol. 1992;207:123-31
references.pmid: 1528115
references.citation: Plant J. 2004 Mar;37(5):645-53
references.pmid: 14871306
references.citation: Plant J. 2011 Jul;67(2):247-57
references.pmid: 21443686
references.citation: Physiol Rev. 2012 Oct;92(4):1777-811
references.pmid: 23073631
references.citation: J Biol Chem. 2013 Jul 12;288(28):20758-67
references.pmid: 23709221
references.citation: Science. 1998 Apr 3;280(5360):69-77
references.pmid: 9525859
references.citation: Annu Rev Plant Biol. 2010;61:561-91
references.pmid: 20192751
references.citation: J Neurosci. 2001 Aug 1;21(15):5574-86
references.pmid: 11466429
references.citation: Nature. 1991 Mar 21;350(6315):232-5
references.pmid: 1706481