Structure-function analysis of the highly conserved charged residues of the membrane protein FT1, the main folic acid transporter of the protozoan parasite Leishmania.
The main plasma membrane folate transporter FT1 of Leishmania belongs to the novel FBT family which is part of the major facilitator superfamily. We have investigated the role of the 10 most conserved charged amino acids of FBTs by site directed mutagenesis. The functions of the mutated proteins were tested for their capacity to transport FA, to sensitize methotrexate resistant cells to methotrexate, for protein production, and for protein localisation. Of the 10 conserved charged amino acids that were mutated to neutral amino acids, all had effects on FT1 transport activities. Only four of the 10 initial mutants (K116L, K133L, R497L, and D529V) retained between 15% and 50% of FT1 activity. The R497 residue was shown to be involved in substrate binding. When the charged conserved residues at position 124, 134, 179, 514, 537 and 565 were changed to neutral amino acids, this led to inactive proteins but the generation of new mutants D124E, R134K, D514E and D537E regained between 20% and 50% of wild-type FT1 activity suggesting that the charge is important for protein function. The mutated protein D179E had, under our standard experimental conditions, no activity, while E565D was completely inactive. The differential activity of the mutated proteins was due either to changes in the apparent K(m) or V(max). Mutagenesis experiments have revealed that charged amino acids were essential for FT1 stability or activity and led to a plausible model for the transport of folic acid through FT1.
Dridi
Larbi
L
Centre de Recherche en Infectiologie du CHUL, Université Laval, 2705 Boul, Laurier, Québec, Québec G1V4G2, Québec, Canada.
Haimeur
Anass
A
Ouellette
Marc
M
D000595:Amino Acid Sequence; D000818:Animals; D027321:Anion Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D001224:Aspartic Acid / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D005492:Folic Acid / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D007891:Leishmania / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*
D027321:Anion Transport Proteins; D026901:Membrane Transport Proteins; D015800:Protozoan Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D005492:Folic Acid