Yeast Yeast 2004; 21: 1269–1277. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1178 Research Article Allelic isoforms of the H + /nucleoside co-transporter (CaCNT) from Candida albicans reveal separate high- and low-affinity transport systems for nucleosides Melissa D. Slugoski, 1 Shaun K. Loewen, 1 Amy M. L. Ng, 1 Stephen A. Baldwin, 2 Carol E. Cass 3 and James D. Young 1 * 1 Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 2 School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK 3 Department of Oncology, University of Alberta, and Cross Cancer Institute, Edmonton, Alberta, Canada T6G 2H7 *Correspondence to: James D. Young, Department of Physiology, 7-55 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. E-mail: james.young@ualberta.ca Received: 21 May 2004 Accepted: 25 August 2004 Abstract 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 ∼10-fold higher apparent K m 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. Copyright  2004 John Wiley & Sons, Ltd. Keywords: concentrative nucleoside transporter; Candida albicans; CaCNT; purine nucleoside-selective; site-directed mutagenesis; Xenopus laevis oocyte Introduction Transport of nucleosides and nucleoside analogue drugs across cell membranes in mammalian cells is mediated by two structurally unrelated nucleo- side transporter (NT) protein families: ENT (equi- librative, Na + -independent) and CNT (concentra- tive, Na + -dependent) (Griffith and Jarvis, 1996; Young et al., 2000; Damaraju et al., 2003). In human (h) cells and tissues, there are five identified ENT family members (hENT1-4, CLN3), each encoded by different genes with separate chromo- somal localizations (Griffiths et al., 1997a, 1997b; Crawford et al., 1998; Hyde et al., 2001; Aci- movic and Coe, 2002; Baldwin et al., 2003). Sim- ilarly, there are three non-allelic CNT isoforms (hCNT1-3) (Ritzel et al., 1997, 1998, 2001; Wang et al., 1997). These multiple hENT and hCNT proteins differ from each other in nucleoside selec- tivity, ability to transport nucleobases, inhibitor Copyright  2004 John Wiley & Sons, Ltd.