Allelic isoforms of the H+/nucleoside co-transporter (CaCNT) from Candida albicans reveal separate high- and low-affinity transport systems for nucleosides.
Contigs 19-10196 and 19-20196 of the Stanford Candida albicans genome sequence databank encode two putative allelic isoforms of C. albicans CaCNT, a recently characterized 608 amino acid residue H+-coupled fungal member of the CNT family of concentrative nucleoside transport proteins. The single Ser/Gly difference between CaCNT/19-20196 and CaCNT occurs at position 328 in putative TM 7, and corresponds to a Ser/Gly substitution previously shown to contribute to the contrasting pyrimidine and purine nucleoside selectivities of human (h) and rat (r) Na+-dependent CNT1 and CNT2. CaCNT/19-10196 differs from CaCNT by four amino acids, but has Gly at position 328. These new proteins were recreated by site-directed mutagenesis of CaCNT and characterized functionally by heterologous expression in Xenopus laevis oocytes. In marked contrast to h/rCNT1/2, both CaCNT/19-10196 and CaCNT/19-20196 exhibited permeant selectivities for purine nucleosides (adenosine, guanosine and inosine) and uridine similar to that of CaCNT. However, although H+-coupled, CaCNT/19-20196 exhibited a approximately 10-fold higher apparent Km for uridine than either CaCNT or CaCNT/19-10196. CaCNT/19-20196 also exhibited a low apparent affinity for inosine. We conclude that the three proteins correspond to high-affinity (CaCNT, CaCNT/19-10196) and low-affinity (CaCNT/19-20196) allelic isoforms of the C. albicans CNT nucleoside transporter. This is the first example of a single amino acid residue substitution altering a CNT protein's overall apparent affinity for nucleosides.
Slugoski
Melissa D
MD
Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Loewen
Shaun K
SK
Ng
Amy M L
AM
Baldwin
Stephen A
SA
Cass
Carol E
CE
Young
James D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002176:Candida albicans / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D033703:Nucleoside Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009705:Nucleosides / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014981:Xenopus / Q000235:genetics / Q000502:physiology
D005656:Fungal Proteins; D033703:Nucleoside Transport Proteins; D009705:Nucleosides; D020033:Protein Isoforms; D011994:Recombinant Proteins