Báo cáo khoa học: The highly conserved extracellular peptide, DSYG(893–896), is a critical structure for sodium pump function docx

The highly conserved extracellular peptide, DSYG(893–896), is a critical structure for sodium pump function Susanne Becker*, Heike Schneider* and Georgios Scheiner-Bobis Institut fu ¨ r Biochemie und Endokrinologie, Fachbereich Veterina ¨ rmedizin, Justus-Liebig-Universita ¨ t Giessen, Germany The peptide sequence DSYG(893–896) of the s heep sodium pump a1 subunit is highly conserved among all K + -trans- porting P-type ATPases. To obtain information about its function, single mutations were introduced and the mutants were expressed in yeast and analysed for enzymatic activity, ion recognition, and a/b subunit interactions. Mutants of Ser894 or Tyr895 were all active. Conservative phenylalan- ine and tryptophan mutants of Tyr895 displayed properties that were similar to the properties of the wild-type enzyme. Replacement of the same amino acid by cysteine, however, produced heat-sensitive enzymes, indicating that the aromatic group contributes to the stability of the enz yme. Mutants of the neighbouring Ser894 recognized K + with altered apparent affinities. Thus, the Ser894fiAsp mutant displayed a threefold higher apparent affinity for K + (EC 50 ¼ 1.4 ± 0.06 m M ) than the wild-type enzyme (EC 50 ¼ 3.8 ± 0.33 m M ). In contrast, the mutant Ser894fiIle had an almost sixfold lower apparent affinity for K + (EC 50 ¼ 21.95 ± 1.41 m M ). Mutation of Asp893 or Gly896 produced inactive proteins. When an anti-b1 subunit immunoglobulin was used to co-immunoprecipitate the a1 subunit, neither the Gly896fiArg nor the Gly896fiIle mutant could be visualized by subsequent probing with an anti-a1 subunit immunoglobulin. On the other hand, co-immunoprecipitation was obtained with the inactive Asp893fiArg and Asp893fiGlu mutants. Thus, it might be that Asp893 is involved in enzyme conformational transitions required for ATP hydrolysis and/or ion translo- cation. The results obtained h ere demonstrate the import- ance of the highly conserved peptide DSYG(893–896) for the function of a/b heterodimeric P-type ATPases. Keywords:Na + /K + -ATPase; a/b subunit interactions; immunoprecipitation; ouabain binding; thermal stability. The sod ium pump (Na + /K + -ATPase, EC 3.6.3.9) is an a/b-oligomeric enzyme embedded in the plasma membrane of animal cells. The enzyme hydrolyzes ATP to transport three Na + ions out of the cell and two K + ions into the cell. Although ATP binding and ion occlusion seem to be tightly connected to the a subunit, the o verall catalytic activity, defined as ATP-driven ion transport, requires the inter- action of both a and b subunits of the enzyme. The subunits are known to interact with each other at extracellular s ites. On the a subunit, a stretch of 26 amino acids localized within the peptide loop that connects M7 and M8 membrane-spanning domains (hereafter denoted L7/8) was first identified as being important for interactions with the b subunit [1]. In a more detailed study, using a yeast two-hybrid system, the SYGQ(894–897) sequence from the 26-amino acid peptide was identified as an essential component for a/b subunit interactions following replacement with four alanine residues: SYGQ(894– 897)fiAAAA(894–897) [2]. (Note that the peptide number- ing corresponds to the sheep Na + /K + -ATPase a1 subunit.) Using the same system, either Ser894 or Tyr895 were identified as being essential for a/b interactions [3]. Besides being important for interactions with the b subunit, the L7/8 region of the a subunit also seems to be involved in ion translocation or rec ognition. This is supported by the results of various investigations involving either enzymatic analysis of a subunit mutants [4] or metal ion-catalysed oxidative cleavage of a and b subunits, resulting in the loss of Rb + occlusion [5]. Recognition of K + or Na + was also found to depend on the b subunit structure, a finding that was based on subunit- substitution studies involving various chimeras between the sodium pump b subunit and the gastric proton pump b subunit [6,7]. Thus, besides its role in stabilization of the a subunit and its function as a vehicle for bringing the a subunit from the en doplasmic reticulum to the p lasma membrane [8], the b subunit may be directly involved in ion recognition or transport. To better understand a/b interactions and their involve- ment in ion transport, we introduced mutations within the DSYG(893–896) sequence of the L7/8 peptide o f the sheep a1 subunit, which is highly conserved among the hetero- dimeric P-type ATPases. The mutant a1 subunits were expressed in yeast and investigated with respect to their enzymatic properties and their interaction with the coex- pressed b subunit. The results described here demonstrate Correspondence to G. Scheiner-Bobis, Institut fu ¨ r Biochemie und Endokrinologie, Fachbereich Veterina ¨ rmedizin, Justus-Liebig-Uni- versita ¨ t Giessen, Frankfurter Str. 100, D-35392 Giessen, Germany. Fax: +49 641 9938189, Tel.: +49 641 9938180, E-mail: Georgios.Scheiner-Bobis@vetmed.uni-giessen.de Abbreviations:Na + /K + -ATPase, sodium- and potassium-activated adenosine triphosphatase; NaCl/P i -T, phosphate-buffered saline containing 0.1% (v/v)Tween TM 20. Enzymes:Na + /K + -ATPase (EC 3.6.3.9). *Note: Both authors contributed equally to the scientific work presented here. (Received 9 June 2004, revised 21 July 2004, accepted 26 July 2004) Eur. J. Biochem. 271, 3821–3831 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04305.x that single mutations within the highly conserved DSYG(893–896) peptide influence enzyme activity, enzyme stability, interac tions with K + , a nd assembly of the a and b subunits. Experimental procedures Vectors and strains The shuttle vectors YhNa1, GhNb1 and pCGY1406ab, used for the expression of a1andb1 subunits of Na + /K + - ATPase in the yeast Saccharomyces cerevisiae, have been described previously [9,10]. The pBluescriptÒ TM KS II+ (Stratagene, La Jolla, CA, USA) was used for the introduction of mutations and the amplification of DNA in Escherichia c oli strain DH5aF¢ (Life Technologies, Eggen- stein, Germany). Conditions for cell growth and media compositions have been described previously [9]. Introduction of mutations Mutations of Asp893, Ser894 and Gly896 were introduced by inverse PCR [11] using, as a template, the plasmid LPSKH5-7 [4] (a derivative of the pBluescriptÒII KS + )and appropriate amplification primers (Roth, Karlsruhe, Ger- many) shown in Table 1. PCR reaction mixtures o f 100 lL contained 1 l M mutation primer, 1 l M reverse primer, 1 ng of the plasmid LPSKH5-7, 1.5 m M MgCl 2 ,2UTfl DNA polymerase (Promega, Madison, WI, USA), 0.2 m M each dNTP, a nd the appropriate amount of buffer provided by the supplier. After 20 PCR cycles, the amplification product was isolated by agarose gel electrophoresis, treated with T4 DNA polymerase (Promega) to remove the dA overhang produced by the Tfl DNA polymerase, and recircularized by the use of T4 DNA ligase (MBI Fermentas, Vilnius, Lithuania). After amplification in E. coli, the plasmids were tested by restriction analysis with AseI(allrestriction enzymes purchased from MBI Fermentas), and DNA sequencing was carried out according to Sanger et al.[12] using T7 DNA polymerase (Amersham Life Science, Little Chalfont, Bucks., UK) and [ 35 S]dATP (ICN Radiochemi- cals, Irvine, CA, USA). A 589 bp MunI/BglII fragment, fully sequenced to exclude additional unintended mutations, was removed from the LPSKH5-7 plasmid and inserted into the MunI/BglII site of the yeast exp ression vector, pCGY1406a. T he pCGY1406ab vectors now carrying t he desired mutations in the a1 subunit cDNA (Table 1) and the wild-type cDNA for the sodium pump b1 subunit [9] were used to transform yeast [13]. Mutations of Tyr895 were introduced by PCR using the QuikChange TM Site-Directed Mutagenesis Kit (Stratagene Europe, Amsterdam, the Netherlands) and the primers shown in Table 1. The template plasmid was pBluescript TM KS II+ containing, in the multiple cloning site, a 1528 bp BglII/AflII fragment of the a1 subunit cDNA. The protocol of the provider was used for the amplification of the mutant cDNA. After complete automatic sequencing, the 1528 bp BglII/AflII fragments carrying the desired mutations were ligated back into the yeast vector and used for yeast transformations. Isolation of membranes containing native or mutant sodium pumps The methods involved in the isolation of membranes from yeast cells, and for the preparation of SDS-treated microsomes enriched in the sodium pump, have been described previously in great detail [14,15]. The Na + / K + -ATPase activity in the i solated fractions was d eter- mined by a coupled spectrophotometric assay in the presence or absence of 1 m M ouabain [9,16]. The protein concentration of the microsomal preparations was deter- mined by the method of Lowry [17] using BSA as a standard. Table 1. Primers used for mutations. Mutation Oligonucleotide Primers used for mutations by inverse PCR Wild type 5¢-GTGGAGGACAGCTATGGGCAGCAG-3¢ Asp893fiArg 5¢-GTGGAGCGCAGCTATGGGCAGCAG-3¢ Asp893fiGlu 5¢-GTGGAGGAGAGCTATGGGCAGCAG-3¢ Asp893fiAla 5¢-GTGGAGGCCAGCTATGGGCAGCAG-3¢ Ser894fiAsp 5¢-GTGGAGGACGACTATGGGCAGCAG-3¢ Ser894fiIle 5¢-GTGGAGGACATCTATGGGCAGCAG-3 Gly896fiArg 5¢-GTGGAGGACAGCTATAGGCAGCAG-3¢ Gly896fiIle 5¢-GTGGAGGACAGCTATATCCAGCAG-3¢ Second primer for all of the above 5¢-GTCATTAATCCAACGGTCATCCCA-3¢ a Primers used for mutations by PCR and the QuikChange TM Site-Directed Mutagenesis Kit Wild type 5¢-GTGGAGGACAGCTATGGGCAGCAGTGG-3 ¢ Tyr895fiCys 5¢-GTGGAGGACAGCTGTGGGCAGCAGTGG-3¢ Second primer 5¢-CCACTGCTGCCCACAGCTGTCCTCCAC-3¢ Tyr895fiPhe 5¢-CGATGTGGAGGACAGCTTTGGCCAGCAGTGGACCTATG-3¢ Second primer 5¢-CATAGGTCCACTGCTG G CCAAAGCTGTCCTCCACATCG-3¢ b Tyr895fiTrp 5¢-CGATGTGGAGGACAGCTGGGGGCAGCAGTGGACC-3¢ Second primer 5¢-GGTCCACTGCTGCCCCCAGCTGTCCTCCACATCG-3¢ a Silent mutations produce a diagnostic restriction site for AseI. b The silent mutation produces a diagnostic restriction site for EaeI. 3822 S. Becker et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Co-immunoprecipitation of a1 and b1 subunits by an antibody against b1 subunits Unless otherwise specified, all of the following steps were carried out at 4 °C. A total of 1 mg of microsomes enriched in Na + /K + -ATPase [9] was centrifuged for 20 min at 13 000 g and subsequently suspended in 1.5 mL of a buffer consisting of 50 m M Tris/HCl, pH 7 .5, 150 m M NaCl, 1 m M Na 2 EDTA, 0.2% BSA (w/v), 1% Triton X-100 (w/v), 2m M dithiothreitol, 0.2 m M phenylmethanesulfonyl fluor- ide, 0.5 mgÆmL )1 leupeptin and 0.7 mg ÆmL )1 pepstatin. Thereafter, 0.5 lLoftheanti-b1 immunoglobulin (Alexis Corporation, Gru ¨ nberg, Germany) w as added t o the solu- tion and the reaction was allowed to proceed for 5 h under continuous, g entle shaking. Then, 20 lLofproteinG– SepharoseÒ 4B (Sigma, Deisenhofen, Germany) was added and the incubation extended for an additional 16 h. The solution was then c entrifuged at 900 g for5sandthe supernatant removed by aspiration. The pellet w as then subjected to a washing routine involving alternating steps of careful suspension of the complex i n the extraction buffer followed b y centrifugation at 900 g for 5 s a t 4 °C, as described i n t he protocol of Tamkun & Fambrough [18]. The Sepharose beads were first washed three times in 1.5 mL of buffer A [150 m M NaCl, 50 m M Tris/HCl, pH 7 .5, 1 m M Na 2 EDTA, 0.5% Triton X-100 (w/v)], then once with 1.5 m L of a solution consisting of 300 m M NaCl, 50 m M Tris/HCl, pH 7.5, 0.1% SDS (w/v), 0.1% Triton X-100 (w/v), th en once with 1.5 mL of 1 M NaCl, 50 m M Tris/HCl, pH 7 .5, 0.5% Triton X-100 (w /v), twice with 1.5 mL of buffer A, and finally with 1.5 mL of 1% Triton X-100 (w/v). Thereafter, the antigen/antibody/Sepharose bead com- plex was suspended in 20 lL of sample buffer consisting of 250 m M Tris/HCl, pH 6.8, 10% SDS (w/v), 10% 2-mercaptoethanol (v/v), 1 m gÆmL )1 Coomassie Brilliant BlueÒ and 25% glycerol (v/v), and heated for 2 min at 100 °C. The Sepharose beads were pelleted by centrifuga- tion and 20 lL of the supernatant was mixed with 20 lLof 8 M urea and heated for 5 min at 70 °C. Proteins in 20 lL of this mixture were separated by electrophoresis on a polyacrylamide gel containing 10% polyacryla mide and 0.3% N,N¢-methylene-bisacrylamide [19]. T he gel was then equilibrated for 30 min in 0.1% S DS (w/v), 12.5 m M Tris, 96 m M glycine, 20% methanol (v/v), pH 8.4, and blotted onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) for 2 h at 2.5 AÆcm )2 [20]. The nitrocellulose membrane was first blocked overnight in NaCl/P i (PBS) containing 0.1% Tween 20 (v/v) ( NaCl/ P i -T) and 5% (w/v) nonfat dried milk, then washed three times (10 min each wash) with NaCl/P i -T, and subsequently incubated for 2 h with either an anti-a1mAb(diluted 1 : 300 in NaCl/P i -T) o r a n anti-b1 m Ab (diluted 1 : 1000 in NaCl/P i -T), bo th raised in mouse (Alexis Cor poration). The detection of antibody-bound a1orb1 subunits was carried out by incubating the nitrocellulose membrane for 90 m in with a n alkaline phosphatase-conjugated anti- mouse IgG (SeroTec, Oxford, UK; diluted 1 : 2500 in NaCl/P i -T) and subsequently adding the alkaline phos- phatase substrates 5-bromo-4-chloro-3-indoyl-phosphate (Molecular Probes, Eugene, OR, USA) for the detection of a1 subunits, or Nitro Blue tetrazolium (Serva, Heidel- berg, Germany) f or the detection of b1 subunits, according to the corresponding protocols of the providers. The chromogenic reaction was interrupted by adding 20 m M EDTA in NaCl/P i -T. Metabolic labelling and immunoprecipitation of a1 subunits Single yeast colonies were grown in 5 mL of selective minimal medium for 16 h at 30 °C to mid-logarithmic phase [i.e. an attenuance (D), at 600 n m, of 0.5–1]. A total of 2.5 · 10 6 cells, corresponding to a D 600 of 2.5, were then centrifuged at room temperature for 5 min at 1800 g and washed with sterile H 2 O. T his procedure was repeated. Thereafter, cells were suspended i n 1.25 mL of minimal medium and incubated at 3 0 °C for 60 min with continu- ous, gentle shaking. Cells were pelleted again, a s described above, and subsequently suspended in 1.25 m L of the minimal medium now containing 100 lCiÆmL )1 of [ 35 S]methionine. Incubation was allowed to proceed with gentle shaking at 30 °C for a further 30 min. Labelled c ells we re centrifuged, as described above, and washed with 1 mL o f ice-cold water. After a second w ash, the pelleted cells were suspended in 100 lLofanextraction buffer comprising 50 m M Tris/HCl, pH 7.4, 150 m M NaCl, 10 m M MgCl 2 ,1m M EDTA, 10% glycerol (w/v), 0.2% BSA (w/v), 2 m M dithiothreitol, 0.2 m M phenylmethane- sulfonyl fluoride, 0.5 mgÆmL )1 and 0.7 mgÆmL )1 pepstatin (phenylmethanesulfonyl fluoride, leupeptin and pepstatin were obtained from Boehringer Ingelheim, Heidelberg, Germany). Then,  100 lL of glass beads (0.25–0.3 mm in diameter) were added and the cells were broken by 10 bursts of 20 s of vigorous mixing at the highest speed in a vortex mixer, each time followed by a 40 s cooling phase on ice. The supernatant was then transferred to a new vial and the glass beads washed twice with 100 lL of the extraction buffer. The combined supernatants ( 300 lL) were cen- trifuged at 7500 g for 20 min at 4 °Ctoremovedebris. After adjusting the radioactivity in the supernatants with extraction buffer to be the same in a final volume of 750 lL, 0.5 lLofananti-a1 immunoglobulin (Alexis Corporation) was added and the s olution w as incub ated w ith g entle shaking for 16 h at 4 °C. Subsequently, 25 lL of a protein G + agarose suspension ( Sigma) were added and incuba- tion was continued for another 4 h. Thereafter, the antigen/antibody/protein-G + agarose complex was sedimented at 4 °C by centrifugation for 5 s at 900 g, and the supernatant was removed b y aspiration. The pellet was then washed, essentially as described above, for the immunoprecipitation using the anti-b immuno- globulin. After the last wash, the antigen/antibody/protein G + agarose complex was equilibrated with 20 lLofa buffer containing 125 m M Tris/HCl, pH 6.8, 4 M urea, 5% SDS (w/v), 5% 2-mercaptoethanol (v/v), 12.5% glycerol (v/ v), 0.5% of a solution of 0.1% eth anol saturated w ith bromophenol blue solution (v/v), and heated at 70 °Cfor 15 min . Then, the protein G + agarose was removed by centrifugation at 900 g for5sat4°C and solubilized proteins in the supernatant were separated by SDS/PAGE on gels containing 10% polyacrylamide and 0.3% N,N¢- methylene-bisacrylamide, prepared according to Laemmli [19]. After electrophoresis, proteins in t he gel were stained Ó FEBS 2004 DSYG(893–896) in the Na + /K + -ATPase a1 subunit (Eur. J. Biochem. 271) 3823 with Coomassie Brilliant BlueÒ (Serva) and, after drying, exposed for 2 days at )80 °CtoaKodakX-OmatX-ray film. Immunodetection of wild-type and Tyr895 mutant a1 subunits by Western blotting A total of 50 lg of SDS-extrac ted yeast membrane proteins [9], containing either native or mutant Na + /K + -ATPase, was suspended in 10 lL of load ing buffer a nd separated by SDS/PAGE following established protocols [19]. Mem- brane extracts from untransformed yeast served as the negative control. Protein was then transferred onto nitro- cellulose membranes followin g the instructions provided by the commercially available ECL Western blotting system PRN 2180 kit (Amersham Pharmacia Biotech, Freiburg, Germany). Following the same protocol, the a1orb1 subunit of the Na + /K + -ATPase was detected using specific antibodies (Alexis Corporation) raised in mice, each used at a dilution of 1 : 2500. The s econdary antibody was a horseradish peroxidase-coupled anti-mouse IgG provided by the kit. Binding of [ 3 H]ouabain under various conditions To obt ain a relative af finity f or ATP, a tot al o f 2 50 lgof microsomal protein isolated from yeast cells expressing either wild-type or mutant sodium pumps was incubated a t 30 °C for 5 min in a mixture containing 10 m M Tris/HCl, pH 7.4, 50 n M [ 3 H]ouabain, 50 m M NaCl, 5 m M MgCl 2 and various concentrations of ATP (Tris salt). The total volume of each sample was 250 lL. Thereafter, the protein was pelleted by centrifugation at 13 000 g for 2 min, washed twice with H 2 Oat4°C, and dissolved in 200 lL of 1 M NaOH by incubation at 80 °C for 10 min. After the addition of 200 lLof1 M HCl, the samples were mixed with 3.5 m L of scintillation cocktail (Roth) and counted for radioactivity. To obtain a relative affinity of the enzyme and its mutants for Na + , this last experiment was performed using a constant concentration of 100 l M ATP (Tris salt) and varying t he concentration of N a + . B efore the m icrosomes were used, however, they were washed twice in 1 mL of 10 m M Tris/HCl, pH 7.4, to remove any Na + from the microsomes storage buffer, which was composed of 25 m M imidazole/1 m M Na 2 EDTA, pH 7.4 [9,15]. All other con- ditions were unchanged. To obtain a relative affinity for K + , the enzyme or its mutants were incubated in 1 0 m M Tris/HCl, pH 7 .4, for 60minwith50n M [ 3 H]ouabain, 5 m M phosphate (Tris salt), 5 m M MgCl 2 and various concentrations of KCl. The other conditions were as described above. Thermal stability of wild-type Na + /K + -ATPase and mutants This experiment was carried out according to a p reviously published protocol [21]. Briefly, a total of 125 lgof microsomal protein w as incubated on ice to serve as a control. An equivalent amount of protein was heated for 5minat50°C. Then, both samples were incubated for an additional 15 min on ice followed by 30 min at 30 °Cwith 5m M phosphate (Tris salt), 5 m M MgCl 2 ,10m M Tris/HCl, pH 7.4, and 50 n M [ 3 H]ouabain. T he total volume w as 500 lL. Bound radioactivity was determined as described above. Results Na + /K + -ATPase activity Yeast membrane preparations contain endogenous ATP- ases. Unlike the mammalian sodium pump, these are ouabain in sensitive. Therefore, in order to distinguish yeast endogenous ATPases from heterologously expressed Na + / K + -ATPase, ATPase activity was determined in SDS- extracted membrane preparations in the presence or absence of 1 m M ouabain. In Fig. 1A, it can be seen that no significant Na + /K + -ATPase activity was detected in mem- brane preparations from cells expressing any of the Asp893 or Gly896 mutants. The same applied for membranes from nontransformed cells. A significant, ouabain-sensitive ATPase activity was, however, detected in membrane preparations from cells expressing the wild-type enzyme Fig. 1. Na + /K + -ATPase activity in yeast membrane preparations. (A) Inactive mu tants. (B) Active mutants. Na + /K + -ATPase activity was determined, a s de scribed in the Experimental pro cedure s, by a coupled spectrophotometric assay as the o uabain-sensitive fraction of a ll ATPase activity that is present in t he preparations. All resu lts r epresent the mean ± SD of three indep endent experiments. Membran e pre p- arations from cells expressing either Asp893fiArg or Gly896fiIle did not display any Na + /K + -ATPase-specific activity. The unit 1 mU is defined as the enzymatic activity that hydrolyzes 1 nmol of ATP in 1 min at 37 °C. NT, nontransformed y east cells. *Significantly lower (P < 0.05) than the activity obtained with the wild-type preparation. 3824 S. Becker et al. (Eur. J. Biochem. 271) Ó FEBS 2004 and all of the Ser894 or Tyr89 5 mutants (Fig. 1B). While the Ser894 mutants and Tyr895fiCys or Tyr895fiPhe mutants displayed ATPase activities comparable to that of the wild- type enzyme, t he activ ity of the Tyr895fiTrp m utant was significantly reduced. Coimmunoprecipitation of a1 and b1 subunits by an anti-b1 immunoglobulin As the mutations are all localized within a peptide sequence of the a subunit that has been shown to interact with the b subunit [ 1,3], i t was im portant to evaluate whether the observed loss of ATPase activity shown in Fig. 1A was caused by the loss of a/b interactions. Following a well-established protocol for co-immuno- precipitation of a1andb1 b y using an anti-b1 immunoglobulin [18], it was possible to demonstrate co-immunoprecipitation of t he wild-type a1withtheb1 subunit (Fig. 2A). Co-immunoprecipitation was also observed with t he inactive Asp893fiArg and Asp893fiGlu mutants of the a1 subunit (Fig. 2A). In contrast, a1 did not co-immunoprecipitate with b1 when membrane prepara- tions from yeast expressing the inactive forms of the a1 subunit (Gly896fiArg or Gly896fiIle) were used (Fig. 2A). The b1 subunits, however, were found, as expected, in all immunoprecipitates except in membranes from nontrans- formed cells (Fig. 2B), verifying that the absence of Gly896fiArg or Gly896fiIle in Fig. 2A was not c aused by the lack of b1 s ubunit e xpression or by degradation of this protein. Nevertheless, the quantities of the b1 subunits detected varied among the lanes, indicating either different levels of expression or variations in experimental recovery. Hence, in order to obtain a value that relates to the abundance of co-immunoprecipitated a1 subunits to the precipitated b1 subunits, the protein bands in Fig. 2B were analysed by densitometry using the image analysis system of Biostep (Jahnsdorf, Germany). By setting the abundance of b1 subunits in lane 4 (Asp894fiGlu) of Fig. 1B to 100%, the b1 a bundance in lane 1 (wild type), lane 3 (Asp893fiArg), lane 5 (Gly896fiArg) and l ane 6 (Gly896fiIle) were 91%, 40%, 54% and 51%, respectively. Lane 2 (nontransformed cells) w as considered to represent the background signal. In an analogous way, the a1 subunit detected in lane 1 (wild type) of Fig. 2A was set to represent the 100% value. In comparison, the relative abundance of the Asp893fiArg mutant a1 subunit (lane 3) only accounted for 47% of this value. The equivalent value for the Asp894fiGlu (lane 4) mutant wa s 9 0%. L ane 2 was set to indicate the back- ground. No protein bands were detected in lanes 5 and 6, containing the Gly896 mutants, by the auto-detect function of the software program. The values obtained from the densitometric scans were used to define the stoichiometry between co-immunopre- cipitated a1andb subunits by forming a quotient between the relative abundance of these subunits. For all the a1/b heterodimers that co-immunoprecipitated (wild type, Asp894fiArg/b subunit, or Asp894fiGlu /b subunit), the quotient of a1 abundance to b1 abundance was approxi- mately 1, indicating proportional expression and recovery levels. Detection of [ 35 S]methionine-labelled wild-type and mutant a1 subunits expressed in yeast Our inability to co-immunoprecipitate the Gly896fiArg and Gly896fiIle mutants of the a1 subunits (Fig. 2A) might have been caused by a lack of e xpression. Therefore, yeast cells transformed with plasmids coding for Asp893fiArg, Asp893fiGlu, Gly896fiArg or Gly896fiIle mutants of the a1 subunit of the sodium pump were m etabolically labelled with [ 35 S]methionine and subsequently used to isolate membrane fractions. Mutant or wild-type a1 subunits were isolated from this mixture by immunoprecipitation with an anti-a1 immunoglobulin. As shown in Fig. 3, it is apparent Fig. 2. Coimmunoprecipitation of inactive mutant a1 subunits with an antibody a ga inst b1 subunits. (A) As w ith t he wild-t ype a1 subunit (lane 1), the inactive Asp893fiArg (lane 3) and Asp893fiGlu (lane 4) mutants c o-immunoprecipitate w ith the b1 subunits, indicating that a/b assembly is not affected by these two mu tations. In contrast, the Gly896fiArg (lane 5) or Gly896fiIle (lane 6) mutants do not co- immunoprecipitate w ith the b1 subunits. The co-immunoprecipitated subunits were visualized in a Western blot using an antibody against the a1 subunit as a primary antibo dy and a n alkaline phosphatase- conjugated anti-imm unoglobuli n G (IgG) as a secondary a ntibody, with 5-bromo-4-chloro-3-indoyl-ph osphate as a chromogenic alkaline phosphatase substrate. (B) All b1 subunits precipitate as an antigen/ antibody/protein G–Sepharose complex and can be detected in the Western blot using an antibody against b1 subunits as a primary antibody and the same secondary antibody mentioned above. The chromogenic substrate of alkaline phosphatase used he re was N itro Blue tetrazolium. Neither a1norb1 subunits were visualized in membranes from nontransformed yeast cells (lane 2). Fig. 3. Immunoprecipitation of metabolically labelled, inactive mutant a1 subunits. [ 35 S]Methionine-labelled proteins were immunoprecipi- tated by an anti-a1 immunoglobulin, as described in the Experimental procedures. After SDS/PAGE, labelled proteins were detected by autoradiography. Wild-type (lane 1) or mutant a1 subu nits (Asp893fiArg, lane 3; Asp8 93fiGlu, lane 4; Gly896fiArg, lane 5; Gly896fiIle, lane 6) were present in the membrane preparations from transformed cells. A labelled protein of  110 kDa was n ot found in membranes from nontransformed cells (lane 2). Ó FEBS 2004 DSYG(893–896) in the Na + /K + -ATPase a1 subunit (Eur. J. Biochem. 271) 3825 that a labelled protein of 110 kDa was found only in membrane preparations from yeast cells expressing either the wild-type or the mutant a subunits. As the sodium pump a1 subunit displays a relative molecular mass of  110 kDa, and because no similar p rotein was detected i n m embrane preparations from nontransformed cells (lane 2), it is very likely that t he precipitated proteins are the wild-type a1 subunits and its mutants. Furthermore, these results dem- onstrate that the a1 subunits are expressed at approximately the same level. Immunodetection of the Tyr895 mutants by Western blotting The Tyr895 mutants are all active (Fig. 1 ). Nevertheless, the Tyr895fiTrp mutant displayed a significantly de- creased activity when compared to the wild-type enzyme and to the mutants Tyr895fiCysorTyr895fiPhe. In order to evaluate whether this difference was caused by different e xpre ssion levels, SDS-extracted membranes of cells expressing either of these mutants or the wild-type enzyme were probed in a Western blot w ith antibodies against the a1orb subunit of the sodium pump. As s hown in Fig. 4 , t he abundance of the Tyr895fiTrp mutant is considerably reduced when compared to the abundance o f t he wild-type enzyme a nd the other mutants. Comparison of the signals for the wild-type and the Ty r895fiCys and Tyr895fiPhe mutants indicates similar expression of these enzymes, as determined by optical densitometry using the image analysis s ystem of Biostep, described above. Using the same system, the quotient of a1 abundance to b1 abundance was approxi- mately 1 for all the a1/b heterodimers that were detected in this Western blot (wild type/b subunit, Tyr895fiCys/b subunit, T yr895fiPhe/b subunit or Tyr895fiTrp/b sub- unit), indicating proportional expression and recovery levels. Binding of [ 3 H]ouabain as a function of ATP concentration In the presence of ATP, Na + ,Mg 2 + and [ 3 H]ouabain, the sodium pump a1 subunit forms a stable [phosphoen- zymeÆ[ 3 H]ouabain] complex that can be easily measured [4]. Figure 5 shows the binding of [ 3 H]ouabain to yeast membrane preparations as a function of the ATP concen- tration in the presence of Na + and M g 2 + .After5minof incubation, [ 3 H]ouabain binding was detectable with mem- branes containing the wild-type enzyme o r either of the Ser894 mutants. The EC 50 for ATP was 0.77 ± 0.11 l M for the wild-type enzyme and 0.46 ± 0.10 l M or 0.94 ± 0.11 l M for the Ser894fiAsp and Ser894fiIle mutant enzymes, respectively. Similar results were obtained with the Tyr895 mutants (Table 2). These values and the values obtained with the Ser894 mutants a re all in g ood agreement with K D values determined for ATP binding to sodium pumps from mammalian tissues [22]. Binding of [ 3 H]ouabain as a function of Na + concentration When [ 3 H]ouabain binding was measured as a function of the Na + concentration i n the presence of 100 l M ATP, Na + -enhanced [ 3 H]ouabain binding to the w ild-type enzyme, with an EC 50 of 1.26 ± 0.38 m M , was observed (Fig. 6 ). The corresponding values determined with mem- branes containing either the Ser894fiAsp or Ser894fiIle mutants were 1.46 ± 0.58 m M and 1.56 ± 0.58 m M , respectively, and indicate the action of Na + on cytosolic sites [23,24]. No specific binding was seen under these conditions with membranes from nontransformed cells. The Tyr895 mutants displayed s imilar sensitivities towards Na + (Table 2). Fig. 4. Immunodetection of active Tyr895 mutants. The W estern blot experiment demonstrates reduced expression for the Tyr895fiTrp a1 (A) a nd b1 subunits ( B), t hus e xplaining t he reduced Na + /K + -ATPase activity observed with membrane preparations containing this mutant. The wild-type, T yr895fiCysorTyr895fiPhe a1(A)orb1 subun its ( B) were expressed at comparable levels. The multiple protein bands r eflect various glycosylation states of the b1 subunit [9]. Fig. 5. Binding of [ 3 H]ouabain as a function of the ATP concentration. Yeast membranes from cells expressing either the wild-type (h)orthe Ser894fiAsp (s) and Ser894fiIle ( .) mutants were incubated for 5minwith50n M [ 3 H]ouabain, 50 m M NaCl, 5 m M MgCl 2 and var- ious concent rations o f A TP ( Tris sa lt; s ee th e E xperim ental p rocedures for details). ATP promotes [ 3 H]ouabain binding to wild-type and mutant enzymes with similar EC 50 values of  1 l M . 3826 S. Becker et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Binding of [ 3 H]ouabain as a function of K + concentration In order to obtain a value for the relative affinity for K + of the wild-type enzyme and the S er894fiAsp or Ser894fiIle mutants, the binding of [ 3 H]ouabain to these enzymes in the presence of phosphate and Mg 2 + was measured as a function of the K + concentration. Under these conditions, K + caused a reduction in [ 3 H]ouabain binding to the wild- type enzyme, showing an EC 50 of 3.8 ± 0.33 m M (Fig. 7 ). The corresponding value obtained with the Ser894fiAsp mutant was 1.4 ± 0.06 m M . A similar experiment carried out with the Ser894fiIle mutant yielded an EC 50 of 21.95 ± 1.41 m M , and the Tyr895 mutants revealed K + sensitivities comparable to that of the wild-type enzyme (Table 2). Table 2. Properties of the Tyr895 mutants. Mutant EC 50 for ATP (l M ) EC 50 for Na + (m M ) EC 50 for K + (m M ) Detection of a and b subunits in the Western blot Na + /K + -ATPase activity (mUÆmg protein )1 ) ab Tyr895fiCys 0.72 ± 0.3 1.44 ± 0.11 3.74 ± 1.14 + + 13.16 ± 1.95 Tyr895fiPhe 1.07 ± 0.15 1.36 ± 0.71 1.75 ± 0.78 + + 13.30 ± 1.61 Tyr895fiTrp 0.8 ± 0.2 1.87 ± 0.31 2.78 ± 0.59 + + 7.60 ± 1.89 Fig. 6. Binding of [ 3 H]ouabain as a function of Na + concentration. (A) Yeast membranes from cells expressing the wild-type (h), the Ser894fiAsp (s) or the Ser894fiIle (.) mutants were incubated for 5minwith50n M [ 3 H]ouabain , 5 m M MgCl 2 , 100 l M ATP (Tris salt) and various concentrations of Na + . The latter promotes [ 3 H]ouabain binding to wild-type and mutant enzymes. (B) T he double re ciprocal plot of the values obtained above reveals that Na + promotes ouabain binding to the wild-type and mutant enzymes with similar EC 50 values of  1.5 m M . Fig. 7. Inhibition of [ 3 H]ouabain binding by K + . Yeast membranes from cells expressing the wild-type (h), the Ser894fiAsp (s)orthe Ser894fiIle (.) m utants were incubated in 10 m M Tris/HCl, pH 7.4, for 60 min with 50 n M [ 3 H]ouabain, 5 m M phosphate (Tris salt), 5 m M MgCl 2 , and various concentrations of KCl. The other conditions were as described above. In all cases, K + leads t o a reduction of ouabain binding. (B) By plottin g the reciprocal binding of [ 3 H]ouabain against the K + concentration, the EC 50 for K + can be obtained from the intercept of the straight lines with the abscissa. Ó FEBS 2004 DSYG(893–896) in the Na + /K + -ATPase a1 subunit (Eur. J. Biochem. 271) 3827 Thermal stability of the Tyr895 mutants The binding of [ 3 H]ouabain to membrane preparations that have been preheated at 50 °C ca n serve as a measu re for the stability of the a/b heterodimer that forms the catalytically active enzyme [21]. After preheating the wild-type sodium pump at 50 °C f or 5 min, the enzyme was c apable of binding only 62.0 ± 8.0% of the ouabain that was bound by the same control enzyme that had been incubated for the same length of time on ice (Fig. 8). The Tyr895 mutants displayed a similar behaviour. Thus, after preheating, the Tyr895fiPhe mutant bound 67.6 ± 16.0% of the ouabain bound by the control, while the corresponding value w ith the Tyr895fiTrp mutant was 51.4 ± 5.9%. Therefore, these two conservative mutants did not convert into more temperature-sensitive forms. The nonconservative Tyr895fiCys mutant, however, was able to bind only 36.5 ± 6.5% of the ouabain that was bound by the unheated control, indicating a higher thermal sensitivity than the wild-type enzyme or its conservative Tyr895 mutants (Fig. 8). This result, which shows a significantly lower value than that obtained w ith the wild-type enzyme, points towards an involvement of the aromatic group in enzyme stabilization (Fig. 8). Discussion The extracellularly localized peptide of the sodium pump a subunit that connects the M7 and M8 membrane-spanning domains (L7/8) contains 26 amino acids that are important for assembly with the b subunit [1]. Corresponding peptides of oth er K + -transporting ATPases seem to play a compar- able role. Results from st udies of chimeric constructs formed by replacing the 26 amino acids of the r at a3 subunit (Asn886)Ala911) with the corresponding region from either the gastric (Gln905-Val930) or the distal (Asn908-Ala933) colon H + /K + -ATPase of the rat demon- strated interaction of the chimeras with the H + /K-ATPase b subunits and h elped t o i dentify Val904, Tyr898, and Cys908 of the sodium pump a3 subunit as important for assembly with the b subunit [21,25]. In addition, the L 7/8 loop confers sensitivity towards specific inhibitors of P 2 -type ATPases, as shown u sing chimeric constructs between the a subunits of Na + /K + -ATPaseandthegastricH + /K + - ATPase [26]. This segment was also demonstrated to be important for ion conduction, as shown for Asp884 and Asp885 mutants [4], to affect interactions with Na + or K + , as demonstrated with a/b heterohybrids [ 7,27], or to be associated with the loss of Rb + occlusion, as shown by Cu 2+ -catalysed oxidative cleavage near His875 [5]. Finally, the i mportance o f t he L7/8 loop is underlined by the high degree of homology of the 26-amino acid peptide seen in all K + -transporting P 2 -type ATPases (Fig. 9). The sequence DSYG(893–896) is – with one exception seen in Hydra – absolutely conserved in all of the K + -transporting P 2 -type ATPases, indicating that some important role for this peptide was conserved in the course of evolution. Thus, to investigate the function of this tetrapeptide, single mutations were introduced within this area of the a1 subunit. The investigation of these mutants revealed that each of the altered a mino acids had an impact on the enzyme proper- ties, although in somewhat different ways. In general, however, we can distinguish between catalytically inactive and catalytically active mutants. Catalytically inactive mutants All mutants of Asp893 and Gly896 were i nactive (Fig. 1) . Lack of expression might be one plausible reason for the lack of measurable activities. Alternatively, for some of the mutants it could be that a1andb1 subunit interactions were disturbed, as all of the mutations are within the 26-amino acid peptide that w as found to be important for a ssembly [1]. A possible c hange in expression caused b y t he mutations was investigated by applying an immunoprecipitation protocol after metabolic labelling of the wild-type and mutant a1 subunits. The immunoprecipitation method was preferred over a standard Western blot to prevent possible degrada- tion of the mutant a1 subunits, which are known to be a target for proteases unless they form heterodimers with the b subunit [28,29]. This experiment, however, verified that all Asp893 and Gly896 m utants were expressed similarly to that of the wild-type a1 subunit (Fig. 3). B ased on that result, the loss of activity is not caused by the lack of expression. A possible loss of a/b interaction because o f the mutations was investigated by applying a co-immunopre- cipitation protocol, described previously [18], which verified alossina/b interactions when Gly896 is replaced with either Arg or Ile. This may provide an explanation for the lack of any detectable enzymatic activity with these two mutants, as formation of the a/b-heterodimer is a presupposition for Na + /K + -ATPase activity [9]. Although rather unusual, single mutations that entirely change, or e ven obliterate, protein–protein i nteractions are not that uncommon. An arginine residue was found to be absolutely essential for oligomerization o f ribulose-1, 5-bisphosphate carboxylase [30]. A similar experience was Fig. 8. Thermal stability of t he wild-type ATPase and the Tyr895 mutants. When the wild-type sodium pump is heated for 5 min at 50 °C, the enzyme binds only 62.0 ± 8.0% of the [ 3 H]ouabain bound by the unheated control. The Tyr895fiPhe and the Tyr895fiTrp mutants behave similarly. Ouabain binding to the nonconservative Tyr895fiCys mutant, however, is only 36.5 ± 6.5% of that obtained with the unheated control under these conditions. This result is signi- ficantly lower (*P < 0.05) than that obtained with the wild-type enzyme. For all measurements n ¼ 3, error bars represent ± SD. 3828 S. Becker et al. (Eur. J. Biochem. 271) Ó FEBS 2004 also observed with s odium pump/proton pump hybrids where the amino acids Tyr898, Val904, and Cys908 of the a3 subunit of the sodium pump (corresponding to Tyr901, Val907 and Cys911 of the sheep a1 subunit u sed in the current investigation) were found to be important for assembly with the b subunit [ 21]. On the other hand, co-immunoprecipitation was ob- tained with the inactive Asp893fiArg and Asp893fiGlu mutants that w as similar i n relative a mount to t he co- immunoprecipitated wild-type a1 subunit (Fig. 2). Thus, the very strong negative effects of the Asp893 mutations on enzyme activity cannot be explained by a lack of association of a and b subunits. One possibility is that th e mutation of this amino acid results in reduced affinity for ouabain, and therefore binding of [ 3 H]ouabain was n ot detectable under the e xperimental c onditions applied in this investigation. Although this explanation cannot be excluded, two facts speak against it: first, in the coupled spectrophotometric assay, preincubation with 1 m M ouabain did not result in different A TPase a ctivities for the assays performed i n the presence or absence of t he glycoside. Unless ouab ain sensitivity is completely abolished by the mutation, 1 m M ouabain should be sufficient to detect some inhibition. Second, comparison of the primary sequen ces reveals that the aspartic acid investigated is highly conserved in all K + - transporting P-type ATPases, regardless of w hether or not they bind ouabain (Fig. 8). This latter fact suggests that this highly conserved a spartic acid i s unlikely to b e directly involved in ouabain binding. As Asp893 is within an area of the p rotein that interacts w ith the b subunit [1,2], and because the b subunit h as been shown not only to influence enzyme properties [6,26,27] but also to be absolutely essential for catalytic activity [9,31], it m ight be that Asp893 is involved in enzyme conformational transitions required for ATP hydrolysis and/or ion translocation. This assumption is difficult to investigate further, however, because in various experiments that were not shown here it was not possible to detect any partial activities that are typical for the sodium pump [32] with any of the Asp893 mutants. Catalytically active mutants Mutants of S er894 and Tyr895 were all active (Fig. 1 ). This was a rather unexpected result, because, by using the tw o- hybrid system, p revious reports had identified these amino acids as being critical for a/b assembly and for enzyme activity [3]. In addition, interactions of the Tyr895 mutants with cations or ATP were not altered when compared w ith the properties o f the wild-type enzyme (Table 2 ). The fact, however, that in Hydra the amino acid corresponding to Tyr895 is a phenylalanine (Phe910; Fig. 9) indicates that although Tyr895 might not be a critical amino acid, the presence of an aromatic group might be important for enzyme stability. This seems to be the case, as the Tyr895fiCys mutant displayed a significantly higher thermal sensitivity than the wild-type enzyme or the Tyr895fiPhe and Tyr895fiTrp mutants (Fig. 8). The Ser894 mutants were all active and d isplayed, in most cases, properties similar to th ose of the w ild-type enzyme. Their interactions with ATP or Na + were essentially unaffected (Figs 5 and 6). Nevertheless, the interactions of Fig. 9. Comparison of primary structures. The 26-amino acid p eptide of the sodium p um p a1 s u bunit that is known t o be important for th e assembly with b subunits is c ompared with equivalent a reas of other K + -transporting P-type ATPases. The A sp893, Ser894 and Gly896 residues inv estigated here (underlined) correspond to highly conserved aspartic acid, serine and glycine residues present in all K + -transporting P-type ATPases. Similar aminoacidsarenotfoundintheCa 2+ ATPases or in the Na + ATPases. The Tyr895 is replaced with a phenylalanine (Phe910) in Hydra.*The numbering here takes into consid eration the deduction of a 5-amino acid propeptide. Ó FEBS 2004 DSYG(893–896) in the Na + /K + -ATPase a1 subunit (Eur. J. Biochem. 271) 3829 the mutants Ser894fiAsp and Ser894fiIle with K + were clearly different from these of the wild-type enzyme. As showninFig.6,K + reduces [ 3 H]ouabain binding to membranes containing the wild-type enzyme, with an EC 50 of 3.8 ± 0.33 m M . The membranes containing the Ser894fiAsp mutants, however, display an EC 50 of 1.4 ± 0.06 m M , an almost threefold-higher relative affinity towards K + than that of the wild-t ype enzyme (Fig. 6). In thecaseoftheSer894fiIle mutant, the relative affinity for K + was 21.95 ± 1 .41 m M , which is about six t imes lower than the a ffinity of the wild-type e nzyme. How can mutation of Ser894 affect the interactions of the enzyme with K + ? Here, too, one can only assume that this highly conserved s erine stabilizes a s tructure of the p rotein important for enzyme interactions with K + on the extra- cellular surface. The fact that replacement o f the serine hydroxyl group by the aspartic acid carboxyl group, with its higher dipole character, results in an enzyme with higher apparent affinity for K + , whereas replacement by the nonpolar isoleucine results in an enzyme with lower affinity for t he cation, could indicate (but does not necessarily require) a direct involvement of Ser894 in the process involved in the uptake of K + from the extracellular milieu. A different explanation should also be considered: because the b subunit is known to influence the interactions of the enzyme w ith K + and because Ser894 is within a sequence known to interact with the b subunits, it is possible that mutations of this amino acid influence i nteraction between the subunits sufficiently enough to affect K + recognition without being absolutely essential for activity. Based on the present data, it is not possible to discern between these two possibilities. The results obtained h ere demonstrate a variety of functional implications associated with the highly conserved peptide DSYG(893–896). While Tyr895 appears to be rather neutral for the enzyme properties, Ser894 is involved – directly or indirectly – in enzyme interactions with K + . Also, Asp893 and Gly896 were proven to be absolutely essential for activity. Nevertheless, while mutations of Gly896 demonstrate that t his a mino acid is critical for assembly between a and b subunits, the results obtained with the Asp893 mutants demonstrate that loss of a ctivity cannot be entirely explained on t he basis of structural disturbances leading to the loss of a/b subunit interactions. Together with the results of previous investigations, show- ing the involvement of amino acids from within the L7/8 loop in Na + conduction [4] and studies showing the loss of Rb + occlusion after Cu 2+ -catalysed cleavage of the L7/8 peptide [5], the study presented here supports and underlines the importance of the L7/8 peptide for the function of a/b heterodimeric P-type ATPases by addressing the functional role of single amino acids from the highly conserved peptide DSYG(893–896). Acknowledgements The authors thank E. A. Martin son for reading the manuscript and R. A. Farle y for t he generous gift of the vectors YhNa1 and GhNb1. This work was supported b y t he Deut sche Forschungsgemeinschaft, Sche 397/5-1 a nd 397/5-2. S. B. was supported throu gh the Gradu- iertenkolleg ÔMolekulare Biologie und Pharmakolo gieÕ of the Justus- Liebig-Univ ersit y Giessen. References 1. Lemas, M.V., Hamrick, M., Takeyasu, K. & Fambrough, D.M. (1994) 26 amino acids of an extracellular domain of the Na,K-ATPase alpha-subunit are suffi cient f or a ssembly with the Na,K-ATPase b eta-subunit. J. Biol. Chem. 269, 8255–8259. 2. Colonna, T.E., Huynh, L. & F ambrough , D.M. (1997) Su bu nit interactions in the Na,K-ATPase explored with the yeast two- hybrid system. J. Biol. Chem. 272, 12366–12372. 3. Fambrough, D.M., Huynh, L . & Hwang, B. (2000) The Na,K-ATPase a-b subunit a ssembly site. In Na/K-AT Pase and Related ATPases (Taniguchi, K. & Kaya, S., eds), pp. 103 –106. Elsevier Science BV, Tokyo. 4. Schneider, H. & Scheiner-Bobis, G. (1997) Involvement o f the M7/ M8 extracellular loop of the sodium pu mp a subunit in ion transport. Struc tural and functional homology to P-loops of ion channels. J. Biol. Chem. 27 2, 16158–16165. 5. Shimon, M.B., Goldshleger, R. & Karlish, S.J. (1998) Specific Cu 2+ -catalyzed oxidative cleavage of Na,K-ATPase at the extracellular surface. J. Biol. Chem. 273, 34190–34195. 6. Eakle,K.A.,Kabalin,M.A.,Wang,S.G.&Farley,R.A.(1994) The influence of b subunit structure on th e stabilit y of Na + /K + - ATPase complexes and interaction with K + . J. Biol. Chem. 269, 6550–6557. 7. Eakle, K.A., Lyu, R M. & Farley, R.A. (1995) The influence of b sub unit structure o n the i nteractio n of Na + /K + -ATPase complexes with Na + . A chimeric a subunit reduces the Na + dependence of phosphoenzyme formation from ATP. J. Biol. Chem. 270, 13937–13947. 8. Geering, K. (1991) Posttranslational modifications a nd intracel- lular transport of sodium pumps: imp ortance o f subunit assembly. Soc. Gen. Physiol. Ser. 46, 31–43. 9. Horowitz, B., Eakle, K.A., Scheiner-Bobis, G., Randolph, G.R., Chen,C.Y.,Hitzeman,R.A.&Farley,R.A.(1990)Synthesisand assembly of functional Na,K-ATPase in yeast. J. Biol. Chem . 265, 4189–4192. 10. Muller-Ehmsen, J., Juvvadi, P., Thompson, C.B., Tumyan, L., Croyle, M., Lingrel, J.B., Schwinger, R.H., McDonough, A.A. & Farley, R.A. (2001) Ouabain and substrate a ffinities of hum an Na + -K + -ATPase a1/b1, a2/b1, and a3/b1 when expressed sepa- rately in yeast cells. Am. J. Physiol. Cell Physiol. 281, C1355– C1364. 11. Hemsley, A., A rnheim, N ., To ney, M.D., Cortopassi, G. & Galas, D.J. (1989) A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res. 17, 6545–6551. 12. Sanger, F., Nickler, S. & Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl Acad. Sci. USA 74, 5463–5467. 13. Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983) Transfor- mation of intact yeast cells treated with alkali cations. J. Ba ct eriol. 153, 163–168. 14. Horowitz, B., Hensley, C. B., Quintero, M., A zuma, K.K., Putnam, D. & McDonough, A.A. (1990) Differential regulation of Na,K-ATPase alpha 1, alpha 2, and beta subu nit mRNA and protein levels by thyroid hormone. J. Biol. Chem. 265, 14308– 14314. 15. Scheiner-Bobis, G. & Farley, F.A. (1994) Subunit requirements for the expression of functional sodium pumps in yeast cells. Biochim. Biophys. Acta 1193, 226–234. 16. Scheiner-Bobis, G. & Schoner, W. (1985) Demonstration of a Mg 2+ -induced conformational change by photoaffinity labelling of the h igh a ffinity ATP-binding site of (Na + +K + )-ATPase with 8-azido-ATP. Eur. J. Biochem. 152, 739–746. 17. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. & Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. 3830 S. Becker et al. (Eur. J. Biochem. 271) Ó FEBS 2004 [...]... 2608–2615 Jaisser, F., Canessa, C.M., Horisberger, J.D & Rossier, B.C (1992) Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase The beta subunit modulates potassium activation of the Na,K -pump J Biol Chem 267, 16895–16903 Noguchi, S., Higashi, K & Kawamura, M (1990) A possible role of the beta-subunit of (Na,K)-ATPase in facilitating correct assembly of the alpha-subunit... 1437–1445 25 Wang, S.G., Eakle, K .A. , Levenson, R & Farley, R .A (1997) Na+K+-ATPase a- subunit containing Q905–V930 of gastric H+-K+ATPase alpha preferentially assembles with H+-K+-ATPase beta Am J Physiol 272, C923–C930 26 Farley, R .A. , Schreiber, S., Wang, S.G & Scheiner-Bobis, G (2001) A hybrid between Na+,K+-ATPase and H+,K+-ATPase 27 28 29 30 31 32 is sensitive to palytoxin, ouabain, and SCH 28080... ion-stimulated adenosine triphosphatase J Biol Chem 246, 5234–5240 23 Inagaki, C., Lindenmayer, G.E & Schwartz, A (1974) Effects of sodium and potassium on binding of ouabain to the transport adenosine triphosphatase J Biol Chem 249, 5135–5140 24 Post, R.L., Sen, A. S & Rosenthal, A. S (1965) A phosphorylated intermediate in adenosine triphosphate-dependent sodium and potassium transport across kidney membranes... ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit interferes with holoenzyme formation J Biol Chem 267, 10576–10582 Scheiner-Bobis, G., Meyer zu Heringdorf, D., Christ, M & Habermann, E (1994) Palytoxin induces K+ efflux from yeast cells expressing the mammalian sodium pump Mol Pharmacol 45, 1132–1136 Scheiner-Bobis, G (2002) The sodium pump Its molecular properties and mechanics of ion transport... 2004 DSYG(893–896) in the Na+/K+-ATPase a1 subunit (Eur J Biochem 271) 3831 18 Tamkun, M.M & Fambrough, D.M (1986) The (Na+ + K+)ATPase of chick sensory neurons Studies on biosynthesis and intracellular transport J Biol Chem 261, 1009–1019 19 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 20 Towbin, H., Staehelin, T & Gordon,... transfer of proteins from polyacryamide gels to nitrocellulose sheets: procedure and some applications Proc Natl Acad Sci USA 76, 4350–4354 21 Wang, S.G & Farley, R .A (1998) Valine 904, tyrosine 898, and cysteine 908 in Na,K-ATPase a subunits are important for assembly with b subunits J Biol Chem 273, 29400–29405 22 Hegyvary, C & Post, R.L (1971) Binding of adenosine triphosphate to sodium and potassium... into the membrane J Biol Chem 265, 15991–15995 Jaunin, P., Horisberger, J.D., Richter, K., Good, P.J., Rossier, B.C & Geering, K (1992) Processing, intracellular transport, and functional expression of endogenous and exogenous alpha-beta 3 Na,K-ATPase complexes in Xenopus oocytes J Biol Chem 267, 577–585 Flachmann, R & Bohnert, H.J (1992) Replacement of a conserved arginine in the assembly domain of . type 5¢-GTGGAGGACAGCTATGGGCAGCAG-3¢ Asp893fiArg 5¢-GTGGAGCGCAGCTATGGGCAGCAG-3¢ Asp893fiGlu 5¢-GTGGAGGAGAGCTATGGGCAGCAG-3¢ Asp893fiAla 5¢-GTGGAGGCCAGCTATGGGCAGCAG-3¢ Ser894fiAsp 5¢-GTGGAGGACGACTATGGGCAGCAG-3¢ Ser894fiIle 5¢-GTGGAGGACATCTATGGGCAGCAG-3 Gly896fiArg. type 5¢-GTGGAGGACAGCTATGGGCAGCAG-3¢ Asp893fiArg 5¢-GTGGAGCGCAGCTATGGGCAGCAG-3¢ Asp893fiGlu 5¢-GTGGAGGAGAGCTATGGGCAGCAG-3¢ Asp893fiAla 5¢-GTGGAGGCCAGCTATGGGCAGCAG-3¢ Ser894fiAsp 5¢-GTGGAGGACGACTATGGGCAGCAG-3¢ Ser894fiIle 5¢-GTGGAGGACATCTATGGGCAGCAG-3 Gly896fiArg 5¢-GTGGAGGACAGCTATAGGCAGCAG-3¢ Gly896fiIle 5¢-GTGGAGGACAGCTATATCCAGCAG-3¢ Second