Subcellular distribution and membrane topology of the mammalian concentrative Na+-nucleoside cotransporter rCNT1

The rat transporter rCNT1 is the archetype of a family of concentrative nuc leoside transporters (CNTs) found both in eukaryotes and in prokaryotes. In the present study we have used antibodies to investigate the subcellular d istribution and membrane topology of this protein. rCNT1 was found to be ex pressed predominantly in the brush-border membranes of the polarized epithe lial cells of rat jejunum and renal cortical tubules and in the bile canali cular membranes of liver parenchymal cells, consistent with roles in the ab sorption of dietary nucleosides, of nucleosides in the glomerular filtrate, or of nucleosides arising from the action of extracellular nucleotidases, respectively. The effect of endoglycosidase F treatment on wild-type and mu tant rCNT1 expressed in Xenopus oocytes revealed that the recombinant trans porter could be glycosylated at either or both of Asn(605) and Asn(643), in dicating that its C terminus is extracellular. In contrast, potential N-gly cosylation sites introduced near the N terminus, or between putative transm embrane (TM) helices 4 and 5, were not glycosylated. The deduced orientatio n of the N terminus in the cytoplasm was confirmed by immunocytochemistry o n intact and saponin-permeabilized Chinese hamster ovary cells expressing r ecombinant rCNT1. These results, in conjunction with extensive analyses of CNT family protein sequences using predictive algorithms, lead us to propos e a revised topological model, in which rCNT1 possesses 13 TM helices with the hydrophilic N-terminal and C-terminal domains on the cytoplasmic and ex tracellular sides of the membrane, respectively. Furthermore, we show that the first three TM helices, which are absent from prokaryote CNTs, are not essential for transporter function; truncated proteins lacking these helice s, derived either from rCNT1 or from its human homolog hCNT1, were found to retain significant sodium-dependent uridine transport activity when expres sed in oocytes.