The proline permease of Aspergillus nidulans: functional replacement of the native cysteine residues and properties of a cysteine-less transporter.
The major proline transporter (PrnB) of Aspergillus nidulans belongs to the Amino acid Polyamine Organocation (APC) transporter superfamily. Members of this family have not been subjected to systematic structure-function relationship studies. In this report, we examine the functional replacement of the three native Cys residues (Cys54, Cys352 and Cys530) of PrnB and the properties of an engineered Cys-less PrnB protein, as background for employing a Cys-scanning mutagenesis approach. We show that simultaneous replacement of Cys54 with Ala, Cys352 with Ala and Cys530 with Ser results in a functional Cys-less PrnB transporter. We also introduce the use of a biotin-acceptor domain tag to quantitate protein levels of the engineered PrnB mutants by Western blot analysis. Finally, by using the background of the Cys-less PrnB transporter, we evaluate the functional importance of amino acids Q219, K245 and F248 of PrnB, which our previous data had suggested to be involved in the mechanism of PrnB-mediated proline uptake. In the current study, we show that K245 and F248 but not Q219 are critical for PrnB-mediated proline uptake.
Kafasla
Panagiota
P
Institute of Biology, National Center for Scientific Research Demokritos, Aghia Paraskevi, 153 10 Athens, Greece.
Bouzarelou
Demetra
D
Frillingos
Stathis
S
Sophianopoulou
Vicky
V
D019943:Amino Acid Substitution; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry / Q000502:physiology*; D000596:Amino Acids / Q000502:physiology; D001233:Aspergillus nidulans / Q000201:enzymology*; D001692:Biological Transport; D003545:Cysteine / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D017354:Point Mutation; D011392:Proline / Q000378:metabolism; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; C033082:proline transporter; D011392:Proline; D003545:Cysteine