The chloride dependence of the human organic anion transporter 1 (hOAT1) is blunted by mutation of a single amino acid

Organic anion transporter 1 (OAT1) is key for the secretion of organic anions in renal proximal tubules. These organic anions comprise endogenous as well as exogenous compounds including frequently used drugs of various chemical structures. The molecular basis for the polyspecificity of OAT1 is not known. Here we mutated a conserved positively charged arginine residue (Arg(466)) in the 11(th) transmembrane helix of human OAT1. The replacement by the positively charged lysine (R466K) did not impair expression of hOAT1 at the plasma membrane of Xenopus laevis oocytes but decreased the transport of p-aminohippurate (PAH) considerably. Extracellular glutarate inhibited and intracellular glutarate trans-stimulated wild type and mutated OAT1, suggesting for the mutant R466K an unimpaired interaction with dicarboxylates. However, when Arg(466) was replaced by the negatively charged aspartate (R466D), glutarate no longer interacted with the mutant. PAH uptake by wild type hOAT1 was stimulated in the presence of chloride, whereas the R466K mutant was chloride-insensitive. Likewise, the uptake of labeled glutarate or ochratoxin A was chloride-dependent in the wild type but not in R466K. Kinetic experiments revealed that chloride did not alter the apparent K(m) for PAH but influenced V(max) in wild type OAT1-expressing oocytes. In R466K mutants the apparent K(m) for PAH was similar to that of the wild type, but V(max) was not changed by chloride removal. We conclude that Arg(466) influences the binding of glutarate, but not interaction with PAH, and interacts with chloride, which is a major determinant in substrate translocation.     

Organic anion transporters involved in the excretion of bestatin in the kidney

Bestatin, a dipeptide, a low molecular weight aminopeptidase inhibitor, has been demonstrated to be an immunomodulator with an antitumor activity. However, the transporter-mediated renal excretion of bestatin is not fully understood. The purpose of this study was to elucidate the transporter-mediated renal excretion mechanism for bestatin. The plasma concentration of bestatin was increased markedly and both the accumulative renal excretion and renal clearance of bestatin were decreased significantly after intravenous administration of bestatin in combination with probenecid. p-Aminohippuric acid (PAH), a substrate of organic anion transporter (OAT) 1, benzylpenicillin (PCG), a substrate of OAT3 and JBP485, a substrate of OAT1 and OAT3, reduced the uptake of bestatin in rat kidney slices and in hOAT1- or hOAT3-HEK 293 cells. The accumulation of bestatin in hOAT1-HEK and hOAT3-HEK 293 cells was significantly greater than that in vector-HEK, and the K(m) and V(max) were 0.679 ± 0.007 mM and 0.807 ± 0.006 nmol/mg protein/30s for OAT1, 0.632 ± 0.014 mM and 1.303 ± 0.015 nmol/mg protein/30s for OAT3 respectively. PAH and JBP485 inhibited significantly the uptake of bestatin in hOAT1-HEK with the K(i) values of 92 ± 9 μM and 197 ± 21 μM; and PCG, JBP485 inhibited significantly the uptake of bestatin in hOAT3-HEK 293 cells with the K(i) values of 88 ± 12 μM and 160 ± 16 μM. Our results are novel in demonstrating for the first time that OAT1 and OAT3 are involved in the renal excretion of bestatin.     

Functional expression of pig renal organic anion transporter 3 (pOAT3)

With the cloning of pig renal organic anion transporter 1 (pOAT1) (Biochimie 84 (2002) 1219) we set up a model system for comparative studies of cloned and natively isolated membrane located transport proteins. Meanwhile, another transport protein involved in p-aminohippurate (PAH) uptake on the basolateral side of the proximal tubule cells was identified, designated organic anion transporter 3 (OAT3). To explore the contribution of pOAT1 to the PAH clearance in comparison to OAT3, it was the aim of this study to extend our model by cloning of the pig ortholog of OAT3. Sequence comparisons of human organic anion transporter 3 (hOAT3) with the expressed sequence tag (EST) database revealed a clone and partial sequence of the pig renal organic anion transporter 3 (pOAT3) ortholog. Sequencing of the entire open reading frame resulted in a protein of 543 amino acid residues encoded by 1632 base pairs (EMBL Acc. No. AJ587003). It showed high homologies of 81%, 80%, 76%, and 77% to the human, rabbit, rat, and mouse OAT3, respectively. A functional characterization of pOAT3 in Xenopus laevis oocytes yielded an apparent Km (Kt) for [3H]estrone sulfate of 7.8 +/- 1.3 microM. Moreover, pOAT3 mediated [3H]estrone sulfate uptake was almost abolished by 0.5 mM of glutarate, dehydroepiandosterone sulfate, or probenecid consistent with the hallmarks of OAT3 function.     

Ubiquitin-specific peptidase 8 regulates the trafficking and stability of the human organic anion transporter 1

BackgroundOrganic anion transporter 1 (OAT1) plays a vital role in avoiding the potential toxicity of various anionic drugs through the involvement of kidney elimination. We previously demonstrated that ubiquitin conjugation to OAT1 led to OAT1 internalization from cell surface, followed by degradation. Ubiquitination is a dynamic process, where deubiquitination is catalyzed by a class of ubiquitin-specific peptidases.MethodsThe role of ubiquitin-specific peptidase 8 (USP8) in hOAT1 function, expression and ubiquitination was assessed by conducting transporter uptake assay, biotinylation assay and ubiquitination assay.ResultsWe demonstrated that USP8 overexpression in hOAT1-expressing cells led to an increased hOAT1 transporter activity and expression, which correlated well with a reduced hOAT1 ubiquitination. Such phenomenon was not observed in inactive USP8 mutant-transfected cells. In addition, the knockdown of endogenous USP8 by USP8-specific siRNA resulted in an increased hOAT1 ubiquitination, which correlated well with a decrease in hOAT1 expression and transport activity. Biotinylation experiments demonstrated that USP8-induced increase in hOAT1 expression and transport activity occurred through a deceleration of the rates of hOAT1 internalization and degradation.ConclusionsThese results indicated the regulatory role of USP8 in OAT1 function, expression, trafficking, and stability.General significanceUSP8 could be a new target for modulating OAT1-mediated drug transport.     

Structurally conserved aromaticity of Tyr249 and Phe264 in helix 7 is important for toxicity of the Bacillus thuringiensis Cry4Ba toxin

Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry delta- endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, Trp(243), Phe(246), Tyr(249) and Phe(264), in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at Tyr(249) and Phe(264) still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/ Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of Tyr(249) and Phe(264) within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin.     

Role of Phe286 in the recognition mechanism of cyclomaltooligosaccharides (cyclodextrins) by Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII). X-ray structures of the mutant TVAIIs, F286A and F286Y, and kinetic analyses of the Phe286-replaced mutant TVAIIs

Phe286 located in the center of the active site of alpha-amylase 2 from Thermoactinomyces vulgaris R-47 (TVAII) plays an important role in the substrate recognition for cyclomaltooligosaccharides (cyclodextrins). The X-ray structures of mutant TVAIIs with the replacement of Phe286 by Ala (F286A) and Tyr (F286Y) were determined at 3.2 A resolution. Their structures have no significant differences from that of the wild-type enzyme. The kinetic analyses of Phe286-replaced variants showed that the variants with non-aromatic residues, Ala (F286A) and Leu (F286L), have lower enzymatic activities than those with aromatic residues, Tyr (F286Y) and Trp (F286W), and the replacement of Phe286 affects enzymatic activities for CDs more than those for starch.     

Conserved pore residues in the AAA protease FtsH are important for proteolysis and its coupling to ATP hydrolysis

Like other AAA proteins, Escherichia coli FtsH, a membrane-bound AAA protease, contains highly conserved aromatic and glycine residues (Phe228 and Gly230) that are predicted to lie in the central pore region of the hexamer. The functions of Phe228 and Gly230 were probed by site-directed mutagenesis. The results of both in vivo and in vitro assays indicate that these conserved pore residues are important for FtsH function and that bulkier, uncharged/apolar residues are essential at position 228. None of the point mutants, F228A, F228E, F228K, or G230A, was able to degrade sigma32, a physiological substrate. The F228A mutant was able to degrade casein, an unfolded substrate, although the other three mutants were not. Mutation of these two pore residues also affected the ATPase activity of FtsH. The F228K and G230A mutations markedly reduced ATPase activity, whereas the F228A mutation caused a more modest decrease in this activity. The F228E mutant was actually more active ATPase. The substrates, sigma32 and casein, stimulated the ATPase activity of wild type FtsH. The ATPase activity of the mutants was no longer stimulated by casein, whereas that of the three Phe228 mutants, but not the G230A mutant, remained sigma32-stimulatable. These results suggest that Phe228 and Gly230 in the predicted pore region of the FtsH hexamer have important roles in proteolysis and its coupling to ATP hydrolysis.     

Autoreduction of ferryl myoglobin: discrimination among the three tyrosine and two tryptophan residues as electron donors

Ferric myoglobin undergoes a two-electron oxidation in its reaction with H(2)O(2). One oxidation equivalent is used to oxidize Fe(III) to the Fe(IV) ferryl species, while the second is associated with a protein radical but is rapidly dissipated. The ferryl species is then slowly reduced back to the ferric state by unknown mechanisms. To clarify this process, the formation and stability of the ferryl forms of the Tyr --> Phe and Trp --> Phe mutants of recombinant sperm whale myoglobin (SwMb) were investigated. Kinetic studies showed that all the mutants react normally with H(2)O(2) to give the ferryl species. However, the rapid phase of ferryl autoreduction typical of wild-type SwMb was absent in the triple Tyr --> Phe mutant and considerably reduced in the Y103F and Y151F mutants, strongly implicating these two residues as intramolecular electron donors. Replacement of Tyr146, Trp7, or Trp14 did not significantly alter the autoreduction, indicating that these residues do not contribute to ferryl reduction despite the fact that Tyr146 is closer to the iron than Tyr151 or Tyr103. Furthermore, analysis of the fast phase of autoreduction in the dimer versus recovered monomer of the Tyr --> Phe mutant K102Q/Y103F/Y146F indicates that the Tyr151-Tyr151 cross-link is a particularly effective electron donor. The presence of an additional, slow phase of reduction in the triple Tyr --> Phe mutant indicates that alternative but normally minor electron-transfer pathways exist in SwMb. These results demonstrate that internal electron transfer is governed as much by the tyrosine pK(a) and oxidation potential as by its distance from the electron accepting iron atom.     

Liver X receptor activation downregulates organic anion transporter 1 (OAT1) in the renal proximal tubule

Liver X receptors (LXRs) play an important role in the regulation of cholesterol by regulating several transporters. In this study, we investigated the role of LXRs in the regulation of human organic anion transporter 1 (hOAT1), a major transporter localized in the basolateral membrane of the renal proximal tubule. Exposure of renal S2 cells expressing hOAT1 to LXR agonists (TO901317 and GW3965) and their endogenous ligand [22(R)-hydroxycholesterol] led to the inhibition of hOAT1-mediated [(14)C]PAH uptake. This inhibition was abolished by coincubation of the above agonists with 22(S)-hydroxycholesterol, an LXR antagonist. Moreover, it was found that the effect of LXR agonists was not mediated by changes in intracellular cholesterol levels. Interestingly, the inhibitory effect of LXRs was enhanced in the presence of 9-cis retinoic acid, a retinoic X receptor agonist. Kinetic analysis revealed that LXR activation decreased the maximum rate of PAH transport (J(max)) but had no effect on the affinity of the transporter (K(t)). This result correlated well with data from Western blot analysis, which showed the decrease in hOAT1 expression following LXR activation. Similarly, TO901317 inhibited [(14)C]PAH uptake by the renal cortical slices as well as decreasing mOAT1 protein expression in mouse kidney. Our findings indicated for the first time that hOAT1 was downregulated by LXR activation in the renal proximal tubule.     

Characterization of ochratoxin A transport by human organic anion transporters.

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporters (hOAT1 and hOAT3, respectively) using the second segment of proximal tubule (S2) cells from mice stably expressing hOAT1 and hOAT3 (S2 hOAT1 and S2 hOAT3). S2 hOAT1 and S2 hOAT3 exhibited a time- and dose-dependent, and a saturable increase in uptake of [3H]-OTA, with apparent Km values of 0.42 microM (hOAT1) and 0.75 microM (hOAT3). These OTA uptakes were inhibited by several substrates for the OATs. Para-aminohippuric acid (PAH), probenecid, piroxicam, octanoate and citrinin inhibited [3H]-OTA uptake by hOAT1 and hOAT3 in a competitive manner (Ki = 4.29-3080 microM), with the following order of potency: probenecid > octanoate > PAH > piroxicam > citrinin for hOAT1; probenecid > piroxicam > octanoate> citrinin > PAH for hOAT3. These results indicate that hOAT1, as well as hOAT3, mediates a high-affinity transport of OTA on the basolateral side of the proximal tubule, but hOAT1- and hOAT3-mediated OTA transport are differently influenced by the substrates for the OATs. These pharmacological characteristics of hOAT1 and hOAT3 may be significantly related with the events in the development of OTA-induced nephrotoxicity in the human kidney.     

Determination of the external loops and the cellular orientation of the N- and the C-termini of the human organic anion transporter hOAT1

The OAT (organic anion transporter) family mediates the absorption, distribution and excretion of a diverse array of environmental toxins and clinically important drugs. OAT dysfunction significantly contributes to renal, hepatic, neurological and fetal toxicity and disease. As a first step to establish the topological model of hOAT1 (human OAT1), we investigated the external loops and the cellular orientation of the N- and the C-termini of this transporter. Combined approaches of immunofluorescence studies and site-directed chemical labelling were used for such purpose. Immunofluorescence microscopy of Myc-tagged hOAT1 expressed in cultured cells identified that both the N- and the C-termini of the transporter were located in the cytoplasm. Replacement of Lys59 in the predicted extracellular loop I with arginine resulted in a mutant (K59R), which was largely inaccessible for labelling by membrane-impermeable NHS (N-hydroxysuccinimido)-SS (dithio)-biotin present in the extracellular medium. This result suggests that loop I faces outside of the cell membrane. A single lysine residue introduced into putative extracellular loops III, V and VI of mutant K59R, which is devoid of extracellular lysine, reacted readily with membrane-impermeable NHS-SS-biotin, suggesting that these putative extracellular loops are in the extracellular domains of the protein. These studies provided the first experimental evidence on the extracellular loops and the cellular orientation of the N- and the C-termini of hOAT1.     

Contributions of aromatic pairs to the folding and stability of long-lived human γD-crystallin

Human γD-crystallin (HγD-Crys) is a highly stable protein that remains folded in the eye lens for the majority of an individual's lifetime. HγD-Crys exhibits two homologous crystallin domains, each containing two Greek key motifs with eight β-strands. Six aromatic pairs (four Tyr/Tyr, one Tyr/Phe and one Phe/Phe) are present in the β-hairpin sequences of the Greek keys. Ultraviolet damage to the aromatic residues in lens crystallins may contribute to the genesis of cataract. Mutant proteins with these aromatic residues substituted with alanines were constructed and expressed in E. coli. All mutant proteins except F115A and F117A had lower thermal stability than the WT protein. In equilibrium experiments in guanidine hydrochloride (GuHCl), all mutant proteins had lower thermodynamic stability than the WT protein. N-terminal domain (N-td) substitutions shifted the N-td transition to lower GuHCl concentration, but the C-terminal domain (C-td) transition remained unaffected. C-td substitutions led to a more cooperative unfolding/refolding process, with both the N-td and C-td transitions shifted to lower GuHCl concentration. The aromatic pairs conserved for each Greek key motif (Greek key pairs) had larger contributions to both thermal stability and thermodynamic stability than the other pairs. Aromatic-aromatic interaction was estimated as 1.5-2.0 kcal/mol. In kinetic experiments, N-td substitutions accelerated the early phase of unfolding, while C-td substitutions accelerated the late phase, suggesting independent domain unfolding. Only substitutions of the second Greek key pair of each crystallin domain slowed refolding. The second Greek keys may provide nucleation sites during the folding of the double-Greek-key crystallin domains.     

Pressure-dependent ionization of Tyr 9 in glutathione S-transferase A1-1: contribution of the C-terminal helix to a "soft" active site.

The glutathione S-transferase (GST) isozyme A1-1 contains at its active site a catalytic tyrosine, Tyr9, which hydrogen bonds to, and stabilizes, the thiolate form of glutathione, GS-. In the substrate-free GST A1-1, the Tyr 9 has an unusually low pKa, approximately 8.2, for which the ionization to tyrosinate is monitored conveniently by UV and fluorescence spectroscopy in the tryptophan-free mutant, W21F. In addition, a short alpha-helix, residues 208-222, provides part of the GSH and hydrophobic ligand binding sites, and the helix becomes "disordered" in the absence of ligands. Here, hydrostatic pressure has been used to probe the conformational dynamics of the C-terminal helix, which are apparently linked to Tyr 9 ionization. The extent of ionization of Tyr 9 at pH 7.6 is increased dramatically at low pressures (p1/2 = 0.52 kbar), based on fluorescence titration of Tyr 9. The mutant protein W21F:Y9F exhibits no changes in tyrosine fluorescence up to 1.2 kbar; pressure specifically ionizes Tyr 9. The volume change, delta V, for the pressure-dependent ionization of Tyr 9 at pH 7.6, 19 degrees C, was -33 +/- 3 mL/mol. In contrast, N-acetyl tyrosine exhibits a delta V for deprotonation of -11 +/- 1 mL/mol, beginning from the same extent of initial ionization, pH 9.5. The pressure-dependent ionization is completely reversible for both Tyr 9 and N-acetyl tyrosine. Addition of S-methyl GSH converted the "soft" active site to a noncompressible site that exhibited negligible pressure-dependent ionization of Tyr 9 below 0.8 kbar. In addition, Phe 220 forms part of an "aromatic cluster" with Tyr 9 and Phe 10, and interactions among these residues were hypothesized to control the order of the C-terminal helix. The amino acid substitutions F220Y, F2201, and F220L afford proteins that undergo pressure-dependent ionization of Tyr 9 with delta V values of 31 +/- 2 mL/mol, 43 +/- 3 mL/mol, and 29 +/- 2 mL/mol, respectively. The p1/2 values for Tyr 9 ionization were 0.61 kbar, 0.41 kbar, and 0.46 kbar for F220Y, F220I, and F220L, respectively. Together, the results suggest that the C-terminal helix is conformationally heterogeneous in the absence of ligands. The conformations differ little in free energy, but they are significantly different in volume, and mutations at Phe 220 control the conformational distribution.     

HUMAN RENAL ORGANIC ANION TRANSPORTER 1 (hOAT1) AND ITS ROLE IN THE NEPHROTOXICITY OF ANTIVIRAL NUCLEOTIDE ANALOGS

hOAT1 is a renal membrane protein able to efficiently transport acyclic nucleoside phosphonates (ANPs). When expressed in CHO cells, hOAT1 mediates the uptake and cytotoxicity of ANPs suggesting that it plays an active role in the nephrotoxicity associated with cidofovir CMV therapy and high-dose adefovir HIV therapy. Although efficiently transported by hOAT1, tenofovir did not show any significant cytotoxicity in isolated human proximal tubular cells, which correlates with the lack of nephrotoxicity observed in HIV-infected patients on prolonged tenofovir therapy.     

Human renal organic anion transporter 1 (hOAT1) and its role in the nephrotoxicity of antiviral nucleotide analogs

hOAT1 is a renal membrane protein able to efficiently transport acyclic nucleoside phosphonates (ANPs). When expressed in CHO cells, hOAT1 mediates the uptake and cytotoxicity of ANPs suggesting that it plays an active role in the nephrotoxicity associated with cidofovir CMV therapy and high-dose adefovir HIV therapy. Although efficiently transported by hOAT1, tenofovir did not show any significant cytotoxicity in isolated human proximal tubular cells, which correlates with the lack of nephrotoxicity observed in HIV-infected patients on prolonged tenofovir therapy.     

Aromatic residues at the extracellular ends of transmembrane domains 5 and 6 promote ligand activation of the G protein-coupled alpha-factor receptor

The alpha-factor receptor (STE2) stimulates a G protein signaling pathway that promotes mating of the yeast Saccharomyces cerevisiae. Previous random mutagenesis studies implicated residues in the regions near the extracellular ends of the transmembrane domains in ligand activation. In this study, systematic Cys scanning mutagenesis across the ends of transmembrane domains 5 and 6 identified two residues, Phe(204) and Tyr(266), that were important for receptor signaling. These residues play a specific role in responding to alpha-factor since the F204C and Y266C substituted receptors responded to an alternative agonist (novobiocin). To better define the structure of this region, the Cys-substituted mutant receptors were assayed for reactivity with a thiol-specific probe that does not react with membrane-imbedded residues. A drop in reactivity coincided with residues likely to be buried in the membrane. Interestingly, both Phe(204) and Tyr(266) are located very near the interface region. However, these assays predict that Phe(204) is accessible at the surface of the receptor, consistent with the strong defect in binding alpha-factor caused by mutating this residue. In contrast, Tyr(266) was not accessible. This correlates with the ability of Y266C mutant receptors to bind alpha-factor and suggests that this residue is involved in the subsequent triggering of receptor activation. These results highlight the role of aromatic residues near the ends of the transmembrane segments in the alpha-factor receptor, and suggest that similar aromatic residues may play an important role in other G protein-coupled receptors.     

Mutational analysis of stress-responsive peanut dual specificity protein kinase. Identification of tyrosine residues involved in regulation of protein kinase activity

We recently reported that Arachis hypogaea serine/threonine/tyrosine (STY) protein kinase is developmentally regulated and is induced by abiotic stresses (Rudrabhatla, P., and Rajasekharan, R. (2002) Plant Physiol. 130, 380-390). Other than MAPKs, the site of tyrosine phosphorylation has not been documented for any plant kinases. To study the role of tyrosines in the phosphorylation of STY protein kinase, four conserved tyrosine residues were sequentially substituted with phenylalanine and expressed as histidine fusion proteins. Mass spectrometry experiments showed that STY protein kinase autophosphorylated within the predicted kinase ATP-binding motif, activation loop, and an additional site in the C terminus. The protein kinase activity was abolished by substitution of Tyr(297) with Phe in the activation loop between subdomains VII and VIII. In addition, replacing Tyr(148) in the ATP-binding motif and Tyr(317) in the C-terminal domain with Phe not only obliterated the ability of the STY protein kinase protein to be phosphorylated, but also inhibited histone phosphorylation, suggesting that STY protein kinase is phosphorylated at multiple sites. Replacing Tyr(213) in the Thr-Glu-Tyr sequence motif with Phe resulted in a 4-fold increase in autophosphorylation and 2.8-fold increase in substrate phosphorylation activities. Mutants Y148F, Y297F, and Y317F displayed dramatically lower phosphorylation efficiency (k(cat)/K(m)) with ATP and histone, whereas mutant Y213F showed increased phosphorylation. Our results suggest that autophosphorylation of Tyr(148), Tyr(213), Tyr(297), and Tyr(317) is important for the regulation of STY protein kinase activity. Our study reveals the first example of Thr-Glu-Tyr domain-mediated autoinhibition of kinases.     

Molecular cloning and characterization of two novel human renal organic anion transporters (hOAT1 and hOAT3)

The cloned organic anion transporters from rat, mouse, and winter flounder (rOAT1, mOAT1, fROAT) mediate the coupled exchange of alpha-ketoglutarate with multiple organic anions, including p-aminohippurate (PAH). We have isolated two novel gene products from human kidney which bear significant homology to the known OATs and belong to the amphiphilic solute facilitator (ASF) family. The cDNAs, hOAT1 and hOAT3, encode for 550- and 568-amino-acid residue proteins, respectively. hOAT1 and hOAT3 mRNAs are expressed strongly in kidney and weakly in brain. Both genes map to chromosome 11 region q11.7. PAH uptake by Xenopus laevis oocytes injected with hOAT1 mRNA is increased 100-fold compared to water-injected oocytes. PAH uptake is chloride dependent and is not further increased by preincubation of oocytes in 5 mM glutarate. Uptake of PAH is inhibited by probenicid, alpha-ketoglutarate, bumetanide, furosemide, and losartan, but not by salicylate, urate, choline, amilioride, and hydrochlorothiazide.     

Role of glycosylation in the organic anion transporter OAT1

Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We reported previously (Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519-1524) that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting an important role of glycosylation in mOAT1 function. In the present study, we investigated the effect of disrupting putative glycosylation sites in mOAT1 as well as its human counterpart, hOAT1, by mutating asparagine to glutamine and assessing mutant transporters in HeLa cells. We showed that the putative glycosylation site Asp-39 in mOAT1 was not glycosylated but the corresponding site (Asp-39) in hOAT1 was glycosylated. Disrupting Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 without affecting their cell surface expression, suggesting that the loss of function is not because of deglycosylation of Asp-39 per se but rather is likely because of the change of this important amino acid critically involved in the substrate binding. Single replacement of asparagines at other sites had no effect on transport activity indicating that glycosylation at individual sites is not essential for OAT function. In contrast, a simultaneous replacement of all asparagines in both mOAT1 and hOAT1 impaired the trafficking of the transporters to the plasma membrane. In summary, we provided the evidence that 1) Asp-39 is crucially involved in substrate recognition of OAT1, 2) glycosylation at individual sites is not required for OAT1 function, and 3) glycosylation plays an important role in the targeting of OAT1 onto the plasma membrane. This study is the first molecular identification and characterization of glycosylation of OAT1 and may provide important insights into the structure-function relationships of the organic anion transporter family.     

Site-directed mutagenesis of conserved C-terminal tyrosine and tryptophan residues of PsbO, the photosystem II manganese-stabilizing protein, alters its activity and fluorescence properties

The extrinsic photosystem II PsbO subunit (manganese-stabilizing protein) contains near-UV CD signals from its complement of aromatic amino acid residues (one Trp, eight Tyr, and 13 Phe residues). Acidification, N-bromosuccinimide modification of Trp, reduction or elimination of a disulfide bond, or deletion of C-terminal amino acids abolishes these signals. Site-directed mutations that substitute Phe for Trp241 and Tyr242, near the C-terminus of PsbO, were used to examine the contribution of these residues to the activity and spectral properties of the protein. Although this substitution is, in theory, conservative, neither mutant binds efficiently to PSII, even though these proteins appear to retain wild-type solution structures. Removal of six residues from the N-terminus of the W241F mutant restores activity to near-wild-type levels. The near-UV CD spectra of the mutants are modified; well-defined Tyr and Trp peaks are lost. Characterizations of the fluorescence spectra of the full-length WF and YF mutants indicate that Y242 contributes significantly to PsbO's Tyr fluorescence emission and that an excited-state tyrosinate could be present in PsbO. Deletion of W241 shows that this residue is a major contributor to PsbO's fluorescence emission. Loss of function is consistent with the proposal that a native C-terminal domain is required for PsbO binding and activity, and restoration of activity by deletion of N-terminal amino acids may provide some insights into the evolution of this important photosynthetic protein.