PDZ domain-dependent regulation of NHE3 protein by both internal Class II and C-terminal Class I PDZ-binding motifs.

NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to the NHE3 C-terminal four amino acids. The internal NHERF-binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminal PBM mutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determines NHE3 stability in the PM, and the latter determines total expression and percent PM expression.

Analysis of substrate-binding elements in OxlT, the oxalate:formate antiporter of Oxalobacter formigenes.

An OxlT homology model suggests R272 and K355 in transmembrane helices 8 and 11, respectively, are critical to OxlT-mediated transport. We offer positive evidence supporting this idea by studying OxlT function after cysteine residues were separately introduced at these positions. Without further treatment, both mutant proteins had a null phenotype when they were reconstituted into proteoliposomes. By contrast, significant recovery of function occurred when proteoliposomes were treated with MTSEA (methanethiosulfonate ethylamine), a thiol-specific reagent that implants a positively charged amino group. In each case, there was a 2-fold increase in the Michaelis constant (K(M)) for oxalate self-exchange (from 80 to 160 microM), along with a 5-fold (K355C) or 100-fold (R272C) reduction in V(max) compared to that of the cysteine-less parental protein. Analysis by MALDI-TOF confirmed that MTSEA introduced the desired modification. We also examined substrate selectivity for the treated derivatives. While oxalate remained the preferred substrate, there was a shift in preference among other substrates so that the normal rank order (oxalate > malonate > formate) was altered to favor smaller substrates (oxalate > formate > malonate). This shift is consistent with the idea that the substrate-binding site is reduced in size via introduction of the SCH(2)CH(2)NH(3)(+) adduct, which generates a side chain that is approximately 1.85 A longer than that of lysine or arginine. These findings lead us to conclude that R272 and K355 are essential components of the OxlT substrate-binding site.

Plasmid-encoded asp operon confers a proton motive metabolic cycle catalyzed by an aspartate-alanine exchange reaction.

Tetragenococcus halophila D10 catalyzes the decarboxylation of L-aspartate with nearly stoichiometric release of L-alanine and CO(2). This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an L-aspartate-beta-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter --> aspD --> aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known L-aspartate-beta-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of L-aspartate-beta-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.