Functional dissection of a mercuric ion transporter, MerC, from Acidithiobacillus ferrooxidans.
Topological analysis with a phoA gene fusion suggested that Acidithiobacillus ferrooxidans MerC, a mercury transporter, has two periplasmic loops and four transmembrane domains. Cys-23 and Cys-26 of the protein were involved in Hg(2+)-recognition/uptake, but Cys-132 and Cys-137 were not. Escherichia coli cells producing the MerC were hypersensitive to CdCl(2). In this case, mutation of His72 rendered the host cells less CdCl(2) sensitive, whereas none of the Cys residues affected it. E. coli cells expressing the gene encoding a mercuric ion transporter (merC)-deletion mutant, in which the coding-sequence of the carboxy-terminal cytoplasmic region was removed, retained Hg(2+) hypersensitivity and showed about 55% HgCl(2) uptake ability compared to that of the one expressing the intact merC, indicating that the region is not essential for Hg(2+) uptake. Coexpression of A. ferrooxidans the gene encoding mercuric reductase (merA) and the merC deletion mutation conferred HgCl(2) tolerance to E. coli host cells. Under this condition, the merC deletion gene product was exclusively present as a monomer.
Sasaki
Yoshito
Y
Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba, Sendai, Miyagi 980-8577, Japan.
Minakawa
Takahiro
T
Miyazaki
Atushi
A
Silver
Simon
S
Kusano
Tomonobu
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D008628:Mercury / Q000378:metabolism* / Q000493:pharmacokinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D011994:Recombinant Proteins; C072715:merC protein, Acidithiobacillus ferrooxidans; D006639:Histidine; D010088:Oxidoreductases; C021551:mercuric reductase; D008628:Mercury; D003545:Cysteine