title: Identification of critical residues for transport activity of Acr3p, the Saccharomyces cerevisiae As(III)/H+ antiporter.
abstract: Acr3p is an As(III)/H(+) antiporter from Saccharomyces cerevisiae belonging to the bile/arsenite/riboflavin transporter superfamily. We have previously found that Cys151 located in the middle of the fourth transmembrane segment (TM4) is critical for antiport activity, suggesting that As(III) might interact with a thiol group during the translocation process. In order to identify functionally important residues involved in As(III)/H(+) exchange, we performed a systematic alanine-replacement analysis of charged/polar and aromatic residues that are conserved in the Acr3 family and located in putative transmembrane segments. Nine residues (Asn117, Trp130, Arg150, Trp158, Asn176, Arg230, Tyr290, Phe345, Asn351) were found to be critical for proper folding and trafficking of Acr3p to the plasma membrane. In addition, we found that replacement of highly conserved Phe266 (TM7), Phe352 (TM9), Glu353 (TM9) and Glu380 (TM10) with Ala abolished transport activity of Acr3p, while mutation of Ser349 (TM9) to Ala significantly reduced the As(III)/H(+) exchange, suggesting an important role of these residues in the transport mechanism. Detailed mutational analysis of Glu353 and Glu380 revealed that the negatively charged residues located in the middle of transmembrane segments TM9 and TM10 are crucial for antiport activity. We also discuss a hypothetical model of the Acr3p transport mechanism.
authors.affiliation: Institute of Experimental Biology, University of Wroclaw, 50-328, Wroclaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Markowska
authors.affiliation: Institute of Experimental Biology, University of Wroclaw, 50-328, Wroclaw, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Maciaszczyk-Dziubinska
authors.affiliation: Institute of Experimental Biology, University of Wroclaw, 50-328, Wroclaw, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Migocka
authors.affiliation: Institute of Experimental Biology, University of Wroclaw, 50-328, Wroclaw, Poland.
authors.forename: Donata
authors.initials: D
authors.lastname: Wawrzycka
authors.affiliation: Institute of Experimental Biology, University of Wroclaw, 50-328, Wroclaw, Poland.
authors.forename: Robert
authors.initials: R
authors.lastname: Wysocki
mesh_terms: D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D017920:Antiporters / Q000378:metabolism*; D001151:Arsenic / Q000737:chemistry / Q000378:metabolism*; D018053:Arsenites / Q000378:metabolism; D001646:Bile / Q000378:metabolism; D001692:Biological Transport; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D021381:Protein Transport; D012256:Riboflavin / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D017920:Antiporters; D018053:Arsenites; D026901:Membrane Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; C015001:arsenite; D001151:Arsenic; D000409:Alanine; D012256:Riboflavin