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 tightly 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 proline residues are important for function. Four proline residues were replaced by both alanine and leucine. Only one of these proline residues, Pro-144, was essential for sulphate transport. However, mutation of either Pro-133 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 that all three proline residues are important for transport, and that the conformation 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.
Shelden
M C
MC
Division of Biochemistry and Molecular Biology, The Faculties, Australian National University, Canberra, ACT 0200, Australia.
Loughlin
P
P
Tierney
M L
ML
Howitt
S M
SM
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D007887:Fabaceae / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010946:Plants, Medicinal; D011392:Proline / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000076988:Sulfate Transporters
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C085609:SLC26A4 protein, human; D000076988:Sulfate Transporters; D011392:Proline