title: Single liposome analysis of peptide translocation by the ABC transporter TAPL.
abstract: ATP-binding cassette (ABC) transporters use ATP to drive solute transport across biological membranes. Members of this superfamily have crucial roles in cell physiology, and some of the transporters are linked to severe diseases. However, understanding of the transport mechanism, especially of human ABC exporters, is scarce. We reconstituted the human lysosomal polypeptide ABC transporter TAPL, expressed in Pichia pastoris, into lipid vesicles (liposomes) and performed explicit transport measurements. We analyzed solute transport at the single liposome level by monitoring the coincident fluorescence of solutes and proteoliposomes in the focal volume of a confocal microscope. We determined a turnover number of eight peptides per minute, which is two orders of magnitude higher than previously estimated from macroscopic measurements. Moreover, we show that TAPL translocates peptides against a large concentration gradient. Maximal filling is not limited by an electrochemical gradient but by trans-inhibition. Countertransport and reversibility studies demonstrate that peptide translocation is a strictly unidirectional process. Altogether, these data are included in a refined model of solute transport by ABC exporters.
authors: Institute of Biochemistry and.
authors: Tina
authors: T
authors: Zollmann
authors: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, 9747 AG Groningen, The Netherlands.
authors: Gemma
authors: G
authors: Moiset
authors: Institute of Biochemistry and.
authors: Franz
authors: F
authors: Tumulka
authors: Institute of Biochemistry and Cluster of Excellence Frankfurt Macromolecular Complexes, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany; and.
authors: Robert
authors: R
authors: Tampé
authors: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, 9747 AG Groningen, The Netherlands abele@em.uni-frankfurt.de B.Poolman@rug.nl.
authors: Bert
authors: B
authors: Poolman
authors: Institute of Biochemistry and abele@em.uni-frankfurt.de B.Poolman@rug.nl.
authors: Rupert
authors: R
authors: Abele
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008081:Liposomes; D010455:Peptides / Q000378:metabolism*; D010843:Pichia / Q000235:genetics; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D008081:Liposomes; D010455:Peptides; D011994:Recombinant Proteins
keywords: ABCB9; DCFBA; lysosome; trans-inhibition; uphill transport
references: J Mol Biol. 1999 Dec 17;294(5):1203-13
references: 10600378
references: Nat Commun. 2014;5:5419
references: 25377891
references: Biochemistry. 2001 Dec 25;40(51):15733-42
references: 11747450
references: Annu Rev Biochem. 2002;71:537-92
references: 12045106
references: J Biol Chem. 2002 Nov 22;277(47):45688-94
references: 12244102
references: Curr Opin Struct Biol. 2002 Dec;12(6):754-60
references: 12504680
references: Methods. 2003 Jan;29(1):74-85
references: 12543073
references: Anal Chem. 2003 Apr 1;75(7):1664-70
references: 12705600
references: Arch Biochem Biophys. 1973 Jan;154(1):387-99
references: 4632118
references: Arch Biochem Biophys. 1973 May;156(1):244-54
references: 4730472
references: J Biol Chem. 1977 May 10;252(9):2900-7
references: 16007
references: J Biol Chem. 1979 Jul 25;254(14):6691-7
references: 447744
references: Nature. 1991 Sep 26;353(6342):326-9
references: 1922338
references: J Bacteriol. 1997 Nov;179(22):6979-85
references: 9371443
references: J Bacteriol. 1998 Sep;180(17):4718-23
references: 9721316
references: Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1375-8
references: 9990031
references: J Biol Chem. 2005 Jun 24;280(25):23631-6
references: 15863492
references: Naunyn Schmiedebergs Arch Pharmacol. 2006 Mar;372(6):444-50
references: 16525794
references: Nature. 2006 Sep 14;443(7108):180-5
references: 16943773
references: Biophys J. 2007 Feb 15;92(4):1233-40
references: 17142294
references: Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):19005-10
references: 18024585
references: J Biol Chem. 2007 Dec 28;282(52):37836-43
references: 17977821
references: Proc Natl Acad Sci U S A. 2008 Apr 1;105(13):5069-74
references: 18356296
references: J Biol Chem. 2008 Jun 20;283(25):17083-91
references: 18434309
references: Science. 2008 Jul 11;321(5886):246-50
references: 18511655
references: Methods. 2008 Oct;46(2):123-30
references: 18667165
references: Nat Rev Mol Cell Biol. 2009 Mar;10(3):218-27
references: 19234479
references: Biochemistry. 2009 May 26;48(20):4423-30
references: 19334674
references: J Mol Biol. 2009 Oct 30;393(3):574-85
references: 19715704
references: Traffic. 2010 Mar;11(3):383-93
references: 20377823
references: Pharm Res. 2010 May;27(5):796-810
references: 20204471
references: Nat Chem Biol. 2010 Aug;6(8):572-80
references: 20644544
references: Structure. 2011 Mar 9;19(3):430-9
references: 21397193
references: J Exp Bot. 2011 Apr;62(7):2403-10
references: 21282327
references: FEBS Lett. 2011 Nov 16;585(22):3533-7
references: 22020218
references: Protein Sci. 2012 Jan;21(1):84-96
references: 22095702
references: J Cell Sci. 2012 Sep 15;125(Pt 18):4230-40
references: 22641697
references: Biophys Chem. 2013 Jan;171:31-7
references: 23176825
references: J Biol Chem. 2013 Dec 27;288(52):37039-47
references: 24196954
references: Science. 2014 Mar 7;343(6175):1133-6
references: 24604198
references: Elife. 2014;3:e02740
references: 24837547
references: J Biol Chem. 2000 Jul 28;275(30):23287-94
references: 10748049