Transmembrane topology of the Acr3 family arsenite transporter from Bacillus subtilis.
The transmembrane topology of the Acr3 family arsenite transporter Acr3 from Bacillus subtilis was analysed experimentally using translational fusions with alkaline phosphatase and green fluorescent protein and in silico by topology modelling. Initial topology prediction resulted in two models with 9 and 10 TM helices respectively. 32 fusion constructs were made between truncated forms of acr3 and the reporter genes at 17 different sites throughout the acr3 sequence to discriminate between these models. Nine strong reporter protein signals provided information about the majority of the locations of the cytoplasmic and extracellular loops of Acr3 and showed that both the N- and the C-termini are located in the cytoplasm. Two ambiguous data points indicated the possibility of an alternative 8 helix topology. This possibility was investigated using another 10 fusion variants, but no experimental support for the 8 TM topology was obtained. We therefore conclude that Acr3 has 10 transmembrane helices. Overall, the loops which connect the membrane spanning segments are short, with cytoplasmic loops being somewhat longer than the extracellular loops. The study provides the first ever experimentally derived structural information on a protein of the Acr3 family which constitutes one of the largest classes of arsenite transporters.
Aaltonen
Emil K J
EK
Department of Cell and Organism Biology, Lund University, Sölvegatan 35, S-223 62 Lund, Sweden.
Silow
Maria
M
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry; D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase