title: Evidence for a molecular diode-based mechanism in a multispecific ATP-binding cassette (ABC) exporter: SER-1368 as a gatekeeping residue in the yeast multidrug transporter Pdr5.
abstract: ATP-binding cassette multidrug efflux pumps transport a wide range of substrates. Current models suggest that a drug binds relatively tightly to a transport site in the transmembrane domains when the protein is in the closed inward facing conformation. Upon binding of ATP, the transporter can switch to an outward facing (drug off or drug releasing) structure of lower affinity. ATP hydrolysis is critically important for remodeling the drug-binding site to facilitate drug release and to reset the transporter for a new transport cycle. We characterized the novel phenotype of an S1368A mutant that lies in the putative drug-binding pocket of the yeast multidrug transporter Pdr5. This substitution created broad, severe drug hypersensitivity, although drug binding, ATP hydrolysis, and intradomain signaling were indistinguishable from the wild-type control. Several different rhodamine 6G efflux and accumulation assays yielded evidence consistent with the possibility that Ser-1368 prevents reentry of the excluded drug.
authors.affiliation: Department of Biology, Catholic University of America, Washington, D. C. 20064.
authors.forename: Jitender
authors.initials: J
authors.lastname: Mehla
authors.affiliation: Institute of Biochemistry, Biocenter of the Goethe University, Frankfurt, Germany 60438, and.
authors.forename: Robert
authors.initials: R
authors.lastname: Ernst
authors.affiliation: Department of Biology, Catholic University of America, Washington, D. C. 20064.
authors.forename: Rachel
authors.initials: R
authors.lastname: Moore
authors.affiliation: Department of Biology, Catholic University of America, Washington, D. C. 20064.
authors.forename: Adina
authors.initials: A
authors.lastname: Wakschlag
authors.affiliation: Department of Biology, Catholic University of America, Washington, D. C. 20064.
authors.forename: Mary Kate
authors.initials: MK
authors.lastname: Marquis
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Ambudkar
authors.affiliation: Department of Biology, Catholic University of America, Washington, D. C. 20064,. Electronic address: golin@cua.edu.
authors.forename: John
authors.initials: J
authors.lastname: Golin
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics; D000255:Adenosine Triphosphate / Q000737:chemistry; D000494:Allosteric Regulation; D001693:Biological Transport, Active; D020134:Catalytic Domain; D025141:Drug Resistance, Fungal; D005456:Fluorescent Dyes / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D012235:Rhodamines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D012694:Serine / Q000235:genetics
chemical_list: D018528:ATP-Binding Cassette Transporters; D005456:Fluorescent Dyes; C092438:PDR5 protein, S cerevisiae; D012235:Rhodamines; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases
keywords: ABC Transporter; ATPase; Drug Transport; Membrane Protein; Multidrug Transporter
references.citation: Biochemistry. 2005 Jul 19;44(28):9703-13
references.pmid: 16008355
references.citation: Lancet. 2002 Mar 30;359(9312):1135-44
references.pmid: 11943280
references.citation: Nature. 2006 Sep 14;443(7108):180-5
references.pmid: 16943773
references.citation: Mol Biol Cell. 1998 Feb;9(2):523-43
references.pmid: 9450972
references.citation: J Struct Biol. 2011 Feb;173(2):333-44
references.pmid: 21034832
references.citation: Nat Rev Cancer. 2002 Jan;2(1):48-58
references.pmid: 11902585
references.citation: Proc Natl Acad Sci U S A. 2008 Apr 1;105(13):5069-74
references.pmid: 18356296
references.citation: Biochemistry. 2010 Jun 1;49(21):4440-9
references.pmid: 20426485
references.citation: Science. 2008 Jul 18;321(5887):367-9
references.pmid: 18635793
references.citation: J Biol Chem. 2008 Dec 12;283(50):35010-22
references.pmid: 18842589
references.citation: Nature. 2012 Oct 25;490(7421):566-9
references.pmid: 23000902
references.citation: Biochim Biophys Acta. 1997 Aug 22;1361(2):159-68
references.pmid: 9300797
references.citation: Nature. 2007 Nov 22;450(7169):515-21
references.pmid: 18033289
references.citation: Biochemistry. 2013 Apr 2;52(13):2184-95
references.pmid: 23464591
references.citation: J Biol Chem. 2013 Oct 18;288(42):30420-30431
references.pmid: 24019526
references.citation: Biol Chem. 2011 Jan;392(1-2):53-60
references.pmid: 21194365
references.citation: Biochem Biophys Res Commun. 2007 Apr 27;356(1):1-5
references.pmid: 17316560
references.citation: Science. 2011 Jun 3;332(6034):1202-5
references.pmid: 21566157
references.citation: Proc Natl Acad Sci U S A. 2000 Mar 14;97(6):2515-20
references.pmid: 10716986
references.citation: J Biol Chem. 2014 May 30;289(22):15272-9
references.pmid: 24733388
references.citation: J Biol Chem. 2003 Feb 21;278(8):5963-9
references.pmid: 12496287
references.citation: Appl Environ Microbiol. 2011 Feb;77(3):786-93
references.pmid: 21115699
references.citation: Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15152-6
references.pmid: 21825153
references.citation: Antimicrob Agents Chemother. 2013 Feb;57(2):1025-34
references.pmid: 23254431
references.citation: Biochim Biophys Acta. 1997 Aug 22;1361(2):169-76
references.pmid: 9300798
references.citation: Biochemistry. 2007 Nov 13;46(45):13109-19
references.pmid: 17956128