Proline residues in two tightly coupled helices of the sulphate transporter, SHST1, are important for sulphate transport

The sulphate transporter SHST1, from Stylosanthes hamata, features three ti ghtly coupled transmembrane helices which include proline residues that are conserved in most related transporters. We used site-directed mutagenesis and expression of the mutant transporters in yeast to test whether these pr oline residues are important for function. Four proline residues were repla ced by both alanine and leucine. Only one of these proline residues, Pro-14 4, was essential for sulphate transport. However, mutation of either Pro-13 3 or Pro-160 resulted in a severe decrease in sulphate transport activity; this was due more to a decrease in transport activity than to a decrease in the amount of mutant SHST1 in the plasma membrane. These results suggest t hat all three proline residues are important for transport, and that the co nformation of the three tightly coupled helices may play a critical role in sulphate transport. We also show that SHST1 undergoes a post-translational modification that is required for trafficking to the plasma membrane.