School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UNITED KINGDOM.
Active, sodium-dependent nucleoside transport systems have been kinetically described in a number of different mammalian tissues. The recent cloning of several active, mammalian nucleoside transporters belonging to a novel gene family has provided an opportunity to examine both the structure/function relationships and the tissue distributions of these proteins in detail, and thus to elucidate their physiological roles. The first of these transporters to be cloned was rCNT1, a protein from rat jejunal epithelium which exhibits pyrimidine-selective, cit-type transport activity. The present study involved a bi-directional approach to the investigation of this protein by the analysis of its distribution in rat tissues at both the cellular and subcellular levels, in addition to structural investigation of rCNT1 by protein chemistry and gene manipulation techniques.
A model originally proposed for the topology of this protein, based largely upon hydropathy analysis of its sequence, suggested that the N- and C-terminal hydrophilic regions of this protein were cytoplasmic. However the present study has shown, from the effect of endoglycosidase F treatment of the protein expressed in xenopus oocytes, that the C-terminal hydrophilic domain of the protein is N-glycosylated and so must in fact be extracellular. Furthermore, mutagenesis of rCNT1 cDNA allowed for the production of an aglyco-rCNT1 mutant into which novel glycosylation sites were introduced as a means of probing topology. Evidence from this mutagenesis has indicated that the N-terminus of rCNT1 is cytoplasmic, therefore suggesting that rCNTI exists in the lipid bilayer with thirteen putative transmembrane domains and not fourteen as originally proposed. Analysis of the protein sequence using predictive algorithms suggests that the originally-predicted transmembrane helix 6 is in fact extramembranous.
Analysis of the distribution of rCNT1 in rat tissues by immunoblotting has revealed that this active transporter is more widespread than originally suggested by the results of Northern blotting experiments. Moreover, it has been possible by immunocytochemical analysis not only to identify the cell types in which rCNT1 is expressed but also to show that its distribution is restricted to certain domains of the plasma membrane. rCNT1 in the jejunum and kidney has been localized at the intestinal and renal brush-borders respectively. While in the liver it is abundant in the bile canalicular membranes. This location is consistent with roles for the concentrative transporter in the absorption of nucleosides from the diet or their salvage from the extracellular breakdown of nucleotides. Unexpectedly, rCNT1 also appears to be abundant in the hippocampus, and in the actively dividing seminiferous epithelium of the testis. Furthermore, its presence has also been suggested in cardiac muscle, skeletal muscle and the spleen, although these observations await further confirmation.