Subcellular Distribution and Membrane Topology of the Mammalian Concentrative Na+-Nucleoside Cotransporter rCNT1
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".