Interactions between charged amino acid residues within transmembrane helices in the sulfate transporter SHST1.

The aim of this study was to identify charged amino acid residues important for activity of the sulfate transporter SHST1. We mutated 10 charged amino acids in or near proposed transmembrane helices and expressed the resulting mutants in a sulfate transport-deficient yeast strain. Mutations affecting four residues resulted in a complete loss of sulfate transport; these residues were D107 and D122 in helix 1 and R354 and E366 in helix 8. All other mutants showed some reduction in transport activity. The E366Q mutant was unusual in that expression of the mutant protein was toxic to yeast cells. The R354Q mutant showed reduced trafficking to the plasma membrane, indicating that the protein was misfolded. However, transporter function (to a low level) and wild-type trafficking could be recovered by combining the R354Q mutation with either the E175Q or E270Q mutations. This suggested that R354 interacts with both E175 and E270. The triple mutant E175Q/E270Q/R354Q retained only marginal sulfate transport activity but was trafficked at wild-type levels, suggesting that a charge network between these three residues may be involved in the transport pathway, rather than in folding. D107 was also found to be essential for the ion transport pathway and may form a charge pair with R154, both of which are highly conserved. The information obtained on interactions between charged residues provides the first evidence for the possible spatial arrangement of transmembrane helices within any member of this transporter family. This information is used to develop a model for SHST1 tertiary structure.