EXPERIMENTAL PARASITOLOGY 90, 181–188 (1998) ARTICLE NO. PR984320 Crithidia luciliae: Functional Expression of Nucleoside and Nucleobase Transporters in Xenopus laevis Oocytes Simone T. Hall,* ,1 Jeffrey I. Penny,² Annette M. Gero,* and Sanjeev Krishna² *School of Biochemistry and Molecular Genetics, The University of NSW, Sydney, NSW, 2052, Australia; and ² Division of Infectious Diseases, St. George’s Hospital Medical School, London, SW17 ORE, U.K. Hall, S. T., Penny, J. I., Gero, A. M., and Krishna, S. 1998. Crithidia INTRODUCTION luciliae: Functional expression of nucleoside and nucleobase transport- ers in Xenopus laevis oocytes. Experimental Parasitology 90, 181–188. The expression of purine-specific nucleoside and base transporters of Crithidia luciliae has been demonstrated in Xenopus laevis oocytes. Parasitic protozoa lack a de novo purine biosynthetic path- Poly(A) + -mRNA from C. luciliae, cultured in either purine-replete or way and hence depend on the salvage of purines for nucleic purine-starved conditions, was microinjected into X. laevis oocytes. acid synthesis by specific transport proteins. The trypanoso- For “purine-replete” mRNA, expression of adenosine and hypoxanthine matid Crithidia luciliae is closely related to the pathogenic uptake in microinjected X. laevis oocytes was increased on average trypanosomatids Leishmania spp. and Trypanosoma spp. 9- and 3-fold above water-injected controls, respectively. Expression of adenosine and hypoxanthine uptake in oocytes microinjected with Biochemical evidence indicates that C. luciliae possess at “purine-starved” mRNA was 8 and 3-fold above water-injected con- least three distinct purine transport systems. Two of these trols, respectively. Substrate competition indicated an adenosine/deoxy- mediate uptake of purine nucleosides: one for adenosine adenosine transporter and a separate base transporter specific for hypo- and its analogues and one for guanosine, its analogues, and xanthine. In contrast to C. luciliae in vivo, where the level of activity inosine (Hall et al. 1993). The third represents a separate of adenosine and hypoxanthine transport was regulated by the level of purines in the medium, the heterologous expression of these transporters base transporter with high affinity for hypoxanthine and (from both purine replete and deplete cultures) in X. laevis oocytes adenine; although adenosine is also transported by this sys- was independent of the extracellular purine concentration. These results tem (Day and Gero 1997). Purine transporters with similar may suggest that the presence of specific transporter message is inde- substrate specificities have been characterised in L. bra- pendent of the extracellular purine content, indicating that the regulation ziliensis (Hansen et al. 1982), L. donovani promastigotes of activation and expression of these transporters in C. luciliae may not be under transcriptional control.  1998 Academic Press (Aronow et al. 1987), L. major promastigotes (Baer et al. Index Descriptors and Abbreviations: Crithidia luciliae; Xenopus 1992), T. brucei brucei procyclic and bloodstream forms laevis; functional expression; adenosine transporter; hypoxanthine (deKoning and Jarvis 1997), and C. fasciculata (Kidder et transporter; 3'-nucleotidase/nuclease, 3'-NTase (EC 3.1.3.6); DEPC, al. 1978). diethylpyrocarbonate; MBM, modified Barths’ medium; 2'DOG, C. luciliae exhibits an unusual phenomenon in which the 2'-deoxy-D-glucose; IQR, interquartile range. level of activity of transporters which mediate uptake of purine nucleosides and nucleobases is dependent upon the concentration of available purines in the culture medium. It has been observed that a dramatic increase in the level of adenosine, guanosine, and hypoxanthine transport occurs in C. luciliae grown in purine-deficient medium compared to 1 To whom correspondence should be addressed. Fax: (612) 9385- cells grown in purine-replete medium (Alleman and Gottlieb 1483. E-mail: S.Hall@unsw.edu.au. 1996; Hall et al. 1996; Day and Gero 1997; Gero et al. 181 0014-4894/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.