SummaryThe fluorouridine insensitive 1 (fur1) locus in Arabidopsis thaliana (L.) Heynh. has previously been identified in a screen for growth resistance towards the toxic compound fluorouridine. Mutation of this locus by ethylmethane sulfonate (EMS) allows mutants to grow on this uridine analogue. We identified that the A. thaliana equilibrative nucleoside transporter (AtENT3) was encoded by the fur1 locus. T-DNA insertional mutant plants for AtENT3 resemble the fur1 mutant phenotype: i.e. they grow on fluorouridine, and seedlings as well as leaf discs exhibit a markedly reduced uptake capacity for uridine and cytidine, but a less pronounced reduced uptake for adenosine and guanosine. These results indicate that AtENT3 is an important pyrimidine nucleoside transporter in Arabidopsis. In addition, we identified the mutation in fur1 as a single base-pair exchange, guanine fi adenine, leading to an amino acid exchange G fi R at position 281. Furthermore, we showed that this mutation is indeed responsible for the observed alterations in nucleoside transport in the fur1-1 line, because the introduction of this mutation in AtENT3 promoted fluorouridine resistance in yeast cells expressing this mutated protein. The biochemical characterization of AtENT3 expressed in Xenopus oocytes identified a proton-coupled concentrative mode of nucleoside transport, although this carrier possesses structural features characteristic for equilibrative nucleoside carriers.
The transport activity of the glutamine/neutral amino acid transporter SNAT3 (former SN1, SLC38A3), expressed in oocytes of the frog Xenopus laevis is associated with a non-stoichiometrical membrane conductance selective for Na+ and/or H+ (Schneider, H.P., S. Bröer, A. Bröer, and J.W. Deitmer. 2007. J. Biol. Chem. 282:3788–3798). When we expressed SNAT3 in frog oocytes, the glutamine-induced membrane conductance was suppressed, when carbonic anhydrase isoform II (CAII) had been injected into the oocytes. Transport of substrate, however, was not affected by CAII. The reduction of the membrane conductance by CAII was dependent on the presence of CO2/HCO3
−, and could be reversed by blocking the catalytic activity of CAII by ethoxyzolamide (10 μM). Coexpression of wild-type CAII or a N-terminal CAII mutant with SNAT3 also reduced the SNAT3- associated membrane conductance. The catalytically inactive CAII mutant V143Y coexpressed in oocytes did not affect SNAT3-associated membrane conductance. Our results reveal a new type of interaction between CAII and a transporter-associated cation conductance, and support the hypothesis that the transport of substrate and the non-stoichiometrical ion conductance are independent of each other. This study also emphasizes the importance of carbonic anhydrase activity and the presence of CO2-bicarbonate buffers for membrane transport processes.
The glutamine transporter SNAT3 (SLC38A3), which also transports asparagine and histidine, exchanges sodium for protons, and displays a non-stoichiometrical conductance, which is suppressed by the catalytic activity of carbonic anhydrase II (CAII). In this study, we show that this conductance of rat SNAT3, expressed in Xenopus oocytes, is also suppressed following co-expression with CAI, CAIII, CAIV, and CAII-H64A (mutant with impaired intramolecular H+ shuttling). All CA isoforms and the CAII mutant displayed catalytic activity in intact oocytes, although in vitro studies had reported only very low catalytic activity of CAIII and CAII-H64A. The CA-mediated suppression of conductance was only observed, however, when glutamine, but not when asparagine, was the substrate. We hypothesized that this substrate specificity of the CA action might be due to the different ion selectivity induced by the different amino acid substrates, which induce currents carried by sodium and/or protons. The ion selectivity and conductance was dependent on both pH and extracellular sodium concentration for glutamine and asparagine; however the sodium dependence of the conductance, when asparagine was the substrate, was significantly greater at higher sodium concentrations, which might explain the difference in the sensitivity of the conductance to CAs. Given the presence of CAs in most cells, substrate sensing of SNAT3 would be indicated by different membrane potential changes.
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