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One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl ions.

The ATP-supported =NA' uptake by plasma membrane vesicles from the marine microalga, PZatymonas viridis, was studied. At pH 7 in the medium, Na' uptake did not occur in the presence of ATP although dpH across the plasma membrane was generated, The ATP-dependent Na* uptake was induced by adding the protonophore, CICCP. At pH 8, Na' uptake took place when ATP was added even without ClCcP. The ApH generated across the plasma membrane was negligible under these conditions. The Na+ uptake at pH 8 was not affected by ClCCP and amiloride, an inhibitor of the Na+/H' antiporter. It is concluded that the ATP-supported Na+ uptake by PI. viridis vesicles is catalyzed by Na+-ATPase.

Coding sequences of the CLC family genes SaCLCd, SaCLCf, and SaCLCg, the putative orthologs of Arabidopsis thaliana AtCLCd, AtCLCf, and AtCLCg genes, were cloned from the euhalophyte Suaeda altissima (L.) Pall. The key conserved motifs and glutamates inherent in proteins of the CLC family were identified in SaCLCd, SaCLCf, and SaCLCg amino acid sequences. SaCLCd and SaCLCg were characterized by higher homology to eukaryotic (human) CLCs, while SaCLCf was closer to prokaryotic CLCs. Ion specificities of the SaCLC proteins were studied in complementation assays by heterologous expression of the SaCLC genes in the Saccharomyces cerevisiae GEF1 disrupted strain Δgef1. GEF1 encoded the only CLC family protein, the Cl− transporter Gef1p, in undisrupted strains of this organism. Expression of SaCLCd in Δgef1 cells restored their ability to grow on selective media. The complementation test and the presence of both the “gating” and “proton” conservative glutamates in SaCLCd amino acid sequence and serine specific for Cl− in its selectivity filter suggest that this protein operates as a Cl−/H+ antiporter. By contrast, expression of SaCLCf and SaCLCg did not complement the growth defect phenotype of Δgef1 cells. The selectivity filters of SaCLCf and SaCLCg also contained serine. However, SaCLCf included only the “gating” glutamate, while SaCLCg contained the “proton” glutamate, suggesting that SaCLCf and SaCLCg proteins act as Cl− channels. The SaCLCd, SaCLCf, and SaCLCg genes were shown to be expressed in the roots and leaves of S. altissima. In response to addition of NaCl to the growth medium, the relative transcript abundances of all three genes of S. altissima increased in the leaves but did not change significantly in the roots. The increase in expression of SaCLCd, SaCLCf, and SaCLCg in the leaves in response to increasing salinity was in line with Cl− accumulation in the leaf cells, indicating the possible participation of SaCLCd, SaCLCf, and SaCLCg proteins in Cl− sequestration in cell organelles. Generally, these results suggest the involvement of SaCLC proteins in the response of S. altissima plants to increasing salinity and possible participation in mechanisms underlying salt tolerance.

The hypothesis that the primary Na + -pump, Na + -ATPase, functions in the plasma membrane (PM) of halotolerant microalga Dunaliella maritima was tested using membrane preparations from this organism enriched with the PM vesicles. The pH profile of ATP hydrolysis catalyzed by the PM fractions exhibited a broad optimum between pH 6 and 9. Hydrolysis in the alkaline range was specifically stimulated by Na + ions. Maximal sodium dependent ATP hydrolysis was observed at pH 7.5-8.0. On the assumption that the ATP-hydrolysis at alkaline pH values is related to a Na + -ATPase activity, we investigated two ATP-dependent processes, sodium uptake by the PM vesicles and generation of electric potential difference (Dw) across the vesicle membrane. PM vesicles from D. maritima were found to be able to accumulate 22 Na + upon ATP addition, with an optimum at pH 7.5-8.0. The ATP-dependent Na + accumulation was stimulated by the permeant NO À 3 anion and the protonophore CCCP, and inhibited by orthovanadate. The sodium accumulation was accompanied by pronounced Dw generation across the vesicle membrane. The data obtained indicate that a primary Na + pump, an electrogenic Na + -ATPase of the P-type, functions in the PM of marine microalga D. maritima.

Sodium accumulation by the Na + -ATPase in the plasma membrane (PM) vesicles isolated from the marine alga Tetraselmis (Platymonas) viridis was shown to be accompanied by v v8 8 generation across the vesicle membrane (positive inside) and H + efflux from the vesicle lumen. Na + accumulation was assayed with 22 Na + ; v v8 8 generation was detected by recording absorption changes of oxonol VI; H + efflux was monitored as an increase in fluorescence intensity of the pH indicator pyranine loaded into the vesicles. Both ATP-dependent Na + uptake and H + ejection were increased by the H + ionophore carbonyl cyanide m-chlorophenylhydrazone (ClCCP) while v v8 8 was collapsed. The lipophilic anion tetraphenylboron ion (TPB 3 ) inhibited H + ejection from the vesicles and abolished v v8 8. Based on the effects of ClCCP and TPB 3 on H + ejection and v v8 8 generation, the conclusion was drawn that H + countertransport observed during Na + -ATPase operation is a secondary event energized by the electric potential which is generated in the course of Na + translocation across the vesicle membrane. Increasing Na + concentrations stimulated H + efflux and caused the decrease in the v v8 8 observed, thus indicating that Na + is likely a factor controlling H + permeability of the vesicle membrane.z 1999 Federation of European Biochemical Societies.

Proton transport by the vanadate.~nsitive ATPatm in plasma membrane (PM) ~igl~ from the marine uni~llular mi~roalp Ptatynmnas vlrldls was investiltat¢d, The ATPMe~ndent generation of.apH acrof,.s the membran~ of PM vesicl~ was followed by the changml in the abmrbgn~ of the aminoacridine probe. Acridine orange, Nit" caused the d~ay ofdpH Ilenemted by the ATPas¢. the at0.= of the decay b¢iall del~ndent on