The role of cysteine residues in the sulphate transporter, SHST1: Construction of a functional cysteine-less transporter

We investigated the role of cysteine residues in the sulphate transporter, SHST1, with the aim of generating a functional cysteine-less variant. SHST1 contains five cysteine residues and none was essential for function. However, replacement of C421 resulted in a reduction in transport activity. Sulp...

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Bibliographic Details
Published in:Biochimica et biophysica acta 2005-05, Vol.1669 (2), p.95-100
Main Author: Howitt, Susan M.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Blotting, Western
Carrier Proteins - genetics
Carrier Proteins - metabolism
Congenital chloride diarrhoea
Cysteine
Cysteine - genetics
Cysteine - metabolism
Diastrophic dysplasia
Fabaceae - genetics
Fabaceae - metabolism
Molecular Sequence Data
Mutation
Pendred syndrome
Protein Engineering
Protein Structure, Secondary
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Sequence Alignment
Site-directed mutagenesis
Sulphate transporter
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Summary:We investigated the role of cysteine residues in the sulphate transporter, SHST1, with the aim of generating a functional cysteine-less variant. SHST1 contains five cysteine residues and none was essential for function. However, replacement of C421 resulted in a reduction in transport activity. Sulphate transport by C205 mutants was dependent on the size of the residue at this position. Alanine at position 205 resulted in a complete loss of function whereas leucine resulted in a 3-fold increase in sulphate transport relative to wild type SHST1. C205 is located in a putative intracellular loop and our results suggest that this loop may be important for sulphate transport. By replacing C205 with leucine and the other four cysteine residues with alanine, we constructed a cysteine-less variant of SHST1 that has transport characteristics indistinguishable from wild type. This construct will be useful for further structure and function studies of SHST1.
ISSN:0005-2736
0006-3002
1879-2642
DOI:10.1016/j.bbamem.2005.01.002