| Summary: | Equilibrative nucleoside transporter (ENT) family members are widely distributed in many mammalian cell types. They transport not only the naturally occurring nucleosides but also a number of nucleoside. analogue drugs. The first of the human ENT family r . members to be identified, hENT1, remains the best characterised ofthese transporters and was used as a model for· investigation of the nucleoside transport mechanism. in the present study. In particular, the roles ofseveral residues in putative transmembrarie helix 8 in the structure and function of the protein were investigated by site-directed mutagenesis. A key finding was that a conserved pair of residues, aspartate 341 and arginine 345, plays a critical role both in the transport function of the prQtein and in the binding ofthe inhibitory nucleoside analogue nitrobenzylthioinosine. Investigation ofthe homologous transporter CeENT2 from the nematode Ctienorhabditis elegans, which contains amino acids with uncharged side-chains at the corresponding positions, indicated that these residues are likely to be located in the permeant translocation pathway. Cysteine mutagenesis ofthe conserved hENTI residue asparagine 338 revealed that this residue is also likely to be located in the translocation pathway and appears to be close to the bin~ing site for the coronary vasodilator dilazep. While not essential for - permeant binding, the properties of mutants at this position suggest that asparagine 338 also plays an important role in protein folding into a functional state. Members of the concentrative nucleoside transporter (CNT) family act in concert with ENTs to regulate extracellular and intracellular nucleoside concentrati6ns in mammals and other organisms, u~ing the energy ofcation gradients to drive the concentrative flux of nucleosides' across the cell membrane. To complement work on the mammalian equilibrative transporter hENT1, a prokaryotic member of the CNT family, NupC from Escherichia coli, was also investigated as an experimentally amenable homologue ofthe mammalian CNT proteins. ~y fluorescent labelling of single cysteine mutants, the topo.logy ofNupC was experimentally established for the first time, and shown to differ substantially from published models that had been based primarily on hydropathy analysis. The N- and C-termini ofthe NupC proteinwere both shown to be located on the cytoplasmic side of the membrane, and ten putatively a-helical transmembrane (TM) segments were definitively identified. The topology ofthe region between TM2 and TM3~ previously suggested to contain a single membrane-spanning region, rema~ns unclear but the available evidence suggests that it is located on the periplasmic side ofthe membrane. Site-directed mutagenesis studies on NupC, performed in the light ofthe newly described topology ofthe protein, identified a number of residues critical fo~ its transport function. Ofparticular interest were the conserved residues E149 in putative TM3, E321 in putative TM8 and S355 in putative TM9. Mutants at these positions lacked transport activity but were shown, using solid state NMR, to retain the ability to bind uridine. It i~ hypothesised that these mutants are 'locked' into one or other ofthe alternating conformations through which the protein cycles during permeant translocation. If this is the case, such mutants should be ofgreat use in future studies of the protein by 2-D and 3-D crystallization.
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