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J. Biol. Chem., Vol. 276, Issue 30, 27981-27988, July 27, 2001
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From the The rat
transporter rCNT1 is the archetype of a family of concentrative
nucleoside transporters (CNTs) found both in eukaryotes and in
prokaryotes. In the present study we have used antibodies to
investigate the subcellular distribution and membrane topology of this
protein. rCNT1 was found to be expressed predominantly in the
brush-border membranes of the polarized epithelial cells of rat jejunum
and renal cortical tubules and in the bile canalicular membranes of
liver parenchymal cells, consistent with roles in the absorption 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 mutant rCNT1 expressed in Xenopus oocytes revealed that
the recombinant transporter could be glycosylated at either or both of
Asn605 and Asn643, indicating that its C
terminus is extracellular. In contrast, potential
N-glycosylation sites introduced near the N terminus, or
between putative transmembrane (TM) helices 4 and 5, were not glycosylated. The deduced orientation of the N terminus in the cytoplasm was confirmed by immunocytochemistry on intact and
saponin-permeabilized Chinese hamster ovary cells expressing
recombinant rCNT1. These results, in conjunction with extensive
analyses of CNT family protein sequences using predictive algorithms,
lead us to propose a revised topological model, in which rCNT1
possesses 13 TM helices with the hydrophilic N-terminal and C-terminal
domains on the cytoplasmic and extracellular 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 helices, derived
either from rCNT1 or from its human homolog hCNT1, were found to retain significant sodium-dependent uridine transport activity
when expressed in oocytes.
Subcellular Distribution and Membrane Topology of the Mammalian
Concentrative Na+-Nucleoside Cotransporter rCNT1*
§,,,
,,, and§§
School of Biochemistry and Molecular
Biology, University of Leeds, Leeds LS2 9JT, the United Kingdom,
Institute of Cell and Molecular Biology, University of
Edinburgh, Edinburgh EH9 3JR, United Kingdom, and the Membrane
Transport Research Group, Departments of ¶ Physiology and
** Oncology (Cross Cancer Institute), University of Alberta, Edmonton,
Alberta T6G 2H7, Canada
*
This work was supported by the Medical Research Council and
the Biotechnology and Biological Sciences Research Council of the
United Kingdom, the National Cancer Institute of Canada, with funds
from the Canadian Cancer Society, the Alberta Cancer Board, and the
Natural Sciences and Engineering Research Council of Canada.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Heritage Scientist of the Alberta Heritage Foundation for
Medical Research.
§§
To whom correspondence should be addressed. Tel.:
44-113-233-3173; Fax: 44-113-233-3167; E-mail:
s.a.baldwin@leeds.ac.uk.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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