Transmembrane topology of the arsenite permease Acr3 from Saccharomyces cerevisiae.
Acr3 is a plasma membrane transporter, a member of the bile/arsenite/riboflavin transporter (BART) superfamily, which confers high-level resistance to arsenicals in the yeast Saccharomyces cerevisiae. We have previously shown that the yeast Acr3 acts as a low affinity As(III)/H+and Sb(III)/H+antiporter. We have also identified several amino acid residues that are localized in putative transmembrane helices (TM) and appeared to be critical for the Acr3 activity. In the present study, the topology of Acr3 was investigated by insertion of glycosylation and factor Xa protease cleavage sites at predicted hydrophilic regions. The analysis of the glycosylation pattern and factor Xa cleavage products of resulting Acr3 fusion constructs provide evidence supporting a topological model of Acr3 with 10 TM segments and cytoplasmically oriented N- and C-terminal domains. Next, we investigated the role of the hydrophilic loop connecting TM8 and TM9, the large size of which is unique to members of the yeast Acr3 family of metalloid transporters. We found that a 28 amino acid deletion in this region does not affect Acr3 folding, trafficking substrate binding, or transport activity. Finally, we constructed a homology-based structural model of Acr3 using the crystal structure of the Yersinia frederiksenii homologue of the human bile acid sodium symporter ASBT.
Institute of Experimental Biology, University of Wroclaw, 50-328 Wroclaw, Poland.
Donata
D
Wawrzycka
Institute of Experimental Biology, University of Wroclaw, 50-328 Wroclaw, Poland.
Katarzyna
K
Markowska
Institute of Experimental Biology, University of Wroclaw, 50-328 Wroclaw, Poland.
Ewa
E
Maciaszczyk-Dziubinska
Institute of Experimental Biology, University of Wroclaw, 50-328 Wroclaw, Poland.
Magdalena
M
Migocka
Institute of Experimental Biology, University of Wroclaw, 50-328 Wroclaw, Poland. Electronic address: robert.wysocki@uwr.edu.pl.
Robert
R
Wysocki
D000595:Amino Acid Sequence; D018053:Arsenites / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D015870:Gene Expression; D006031:Glycosylation; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C109281:ACR3 protein, S cerevisiae; D018053:Arsenites; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C468437:SUC2 protein, S cerevisiae; D043324:beta-Fructofuranosidase
Antiporter; Arsenite; Plasma membrane; Transmembrane topology; Yeast