©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Genetic Analysis of the Fluorescein Isothiocyanate Binding Site of the Yeast Plasma Membrane H-ATPase (*)

Ana M. Maldonado (§) , Francisco Portillo (¶)

From the (1) Departamento de Bioqumica, Facultad de Medicina, Universidad Autónoma de Madrid and Instituto de Investigaciones Biomédicas del Consejo Superior de Investigaciones Cientficas, Arturo Duperier, 4, 28029 Madrid, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The highly conserved motif of Saccharomyces cerevisiae H-ATPase KGAP has been proposed to participate in the formation of the phosphorylated intermediate during the catalytic cycle (Portillo, F., and Serrano, R. (1988) EMBO J. 7, 1793-1798). In addition, Lys-474 is the FITC binding site of the yeast enzyme (Portillo, F. and Serrano, R. (1989) Eur. J. Biochem. 186, 501-507). We have performed an intragenic suppressor analysis of the K474R mutation to identify the interacting regions involved in these functions. Random in vitro mutagenesis of the K474R allele resulted in seven suppressor (second-site) mutations. One mutation (V396I), located 18 residues away from the Asp-378 residue, which is phosphorylated during catalysis, is allele-specific. This provides genetic evidence of a direct interaction between the KGAP motif and the phosphorylation domain during the catalytic cycle. Three mutations (V484I, V484I/E485K, and E485K/E486K) are located near Lys-474 and may compense the structural alteration introduced by the K474R mutation. Two substitutions at the end of the predicted transmembrane stretch 2 (A165V and V169I/D170N) and another in the predicted ATP binding domain (P536L) may act as allele-nonspecific suppressors, as they are also able to suppress a mutation at the enzyme's carboxyl terminus.


INTRODUCTION

The yeast plasma membrane H-ATPase belongs to the family of ion-translocating ATPases characterized by the formation of a phosphorylated intermediate during the catalytic cycle (Pedersen and Carafoli, 1987). The proton gradient generated by this ATPase plays an essential role in active nutrient uptake and intracellular pH regulation (reviewed by Goffeau and Slayman, 1981 and Serrano, 1985). Comparison of the amino acid sequence of yeast ATPase with other cation-transporting ATPases showed that they share several regions of homology and a common topology (Serrano, 1989; Goffeau and Green, 1990; Wach et al., 1992). DNA sequence analysis and either random or directed mutagenesis have been combined to probe structure-function relationships of this enzyme. These studies have identified several residues essential for catalysis and regulation of the enzyme (reviewed by Gaber (1992)) and have led to the proposal of a hypothetical model relating structure and function in yeast ATPase (Serrano, 1991).

The functionally important Lys-474 is located within the sequence motif KGAP, which is fully conserved among all eukaryotic cation-transporting ATPases. Lys-474 corresponds to the amino acid modified in animal ATPases by the active site inhibitor fluorescein isothiocyanate (FITC)() (Farley et al., 1984; Farley and Faller, 1985; Filoteo et al., 1987; Mitchinson and Green, 1982; Pardo and Slayman, 1988). Substitution of Arg by Lys at position 474 of the yeast ATPase greatly reduces catalytic activity and FITC binding (Portillo and Serrano, 1989). Furthermore, mutagenesis of Lys-474 in this ATPase greatly decreases the steady-state level of the phosphorylated intermediate of the enzyme, but no change in nucleotide specificity is observed (Portillo and Serrano, 1988; 1989), indicating that this residue is not directly involved in ATP binding. Nevertheless, Lys-474 must lie close to the ATP binding site, since FITC labeling of this residue inhibits ATP binding.

To gain insight into the role of Lys-474 in ATPase function and identify interacting domains during the catalytic cycle, we have performed an intragenic suppression analysis of the K474R mutation. Location of an allele-specific suppressor mutation suggests direct interaction of the KGAP motif with the phosphorylation domain. In addition, this reversion analysis led to the identification of three allele-nonspecific suppressors.


MATERIALS AND METHODS

Yeast Strain and Cell Cultures

Mutant ATPases were expressed from centromeric plasmids in Saccharomyces cerevisiae strain RS72 (Cid et al., 1987), a derivative of BWG1-7A (Guarente et al., 1982) in which the chromosomal ATPase gene promoter (PMA1) has been replaced by the galactokinase gene promoter (GAL1). Transformants were grown in medium with 2% glucose or 2% galactose, 0.7% Yeast Nitrogen Base without amino acids (Difco), 40 µg/ml adenine and 30 µg/ml histidine. When indicated, the glucose medium was buffered with 50 m M succinic acid, adjusted to pH 3.0 with Tris (acid glucose medium). Solid media contained 2% agar (Difco).

To express the mutant ATPases, transformants were grown in galactose medium to a final absorbance of 3 at 660 nm, diluted 100-fold and grown for 24 h at 30 °C in glucose medium.

Mutagenesis and Selection of Revertants

Plasmid pSB32 (Rose and Broach, 1991) carrying a 5-kb HindIII fragment containing the yeast ATPase K474R mutant gene (Portillo and Serrano, 1989) was mutagenized with hydroxylamine as described (Serrano et al., 1986b). Yeast cells were transformed with the hydroxylamine-treated plasmid using the lithium acetate procedure (Ito et al., 1983). From 50 µg of mutagenized plasmid, about 16,000 transformants were obtained on galactose plates. Revertants were selected by replica plating onto acid glucose medium. For each revertant, plasmid DNA was rescued, amplified into Escherichia coli (Rose et al., 1990) and used to test the revertant phenotype as above.

Mapping and Sequencing of Revertant Mutants

The K474R mutation destroys an MseI restriction endonuclease recognition site. To determine if the reversion occurred at the site of the original mutation, a 0.49-kb DNA fragment was amplified by the polymerase chain reaction using plasmid DNA rescued from revertants as template and subjected to restriction analysis with MseI. The DNA fragments digested by MseI would indicate that, in the plasmid DNA rescued from the revertants, the original Lys-474 has been restored, while a DNA fragment not digested by MseI would result from a rescued plasmid carrying a second mutation in the ATPase gene, able to suppress the K474R mutation.

The second-site mutations were located within specific restriction fragments of the ATPase gene by constructing chimeric genes containing portions of both the K474R and revertant genes and by testing whether these constructs supported growth on acid glucose medium. The chimeric genes were constructed as described (Eraso and Portillo, 1994).

The DNA fragments conferring on the K474R mutant the ability to grow on acid glucose medium were sequenced completely by the dideoxy method (Sanger et al., 1977) using 17-mer oligonucleotide primers starting at different positions on the fragment.

Construction of Mutant ATPase Genes Containing Only the Second-site Mutation in the Absence of the K474R Mutation

These genes were constructed in two ways. (i) The fragments containing the second-site mutation were exchanged with the corresponding fragment in the wild-type gene. This was the case for A165V, V169I/D170N, V396I (located on a 0.8-kb EcoRI fragment), and P536L (located on a 2.2-kb XbaI fragment) mutations. (ii) The second-site mutant genes carrying V484I, V484I/E485K and E485K/E486K mutations were constructed by site-directed mutagenesis.

Construction of pma1 Alleles Used for Suppression Analysis

The pma1 alleles in were constructed as follows. ( a) Mutant ATPase genes containing the K474R second-site reversions and K474H or K474Q mutations were generated in two ways: (i) by exchange of the fragments containing the second-site reversions with the corresponding fragment in the K474H and K474Q genes, which was the case for A165V, V169I/D170N, V396I, and P536L; (ii) in the case of V484I, V484I/E485K, and E485K/E486K, construction of the pma1 alleles by site-directed mutagenesis. ( b) Mutant ATPase genes containing the K474R mutation and S911A/T912A revertant mutations were created by exchange of either a 2.2-kb XbaI fragment (A565T, G587N, G648S, P669L, and G670S) or a 0.8-kb EcoRI fragment (A350T and A351T) with the corresponding fragment in the K474R mutant gene. ( c) Mutant ATPase genes containing the S911A/T912A mutation and second-site reversions of the K474R mutation were obtained by exchange of a 2.1-kb KpnI fragment containing only the second-site mutation with the corresponding fragment in the S911A/T912A gene.

All the mutant constructions were verified by sequencing.

Site-directed Mutagenesis

A 5.0-kb HindIII fragment containing either the wild type or K474H and K474Q ATPase gene were inserted into pSB32 and after XbaI digestion were religated, resulting in the deletion of the 2.2-kb XbaI fragment. The excised fragments were subcloned into M13mp19 and subjected to mutagenesis by the method of Eckstein (Taylor et al., 1985) with the Mutagenesis System of Amersham (Amersham Corp.). After mutagenesis, the entire XbaI fragments were sequenced to confirm that only the sequence changes introduced by the mutagenic oligonucleotides were obtained. The 2.2-kb XbaI fragments containing the mutation were liberated from the replicative form of M13mp19 and inserted into the deleted ATPase genes described above.

Biochemical Methods

Yeast plasma membranes were purified from glucose-metabolizing cells by differential and sucrose gradient centrifugation (Serrano, 1983). ATPase activity was assayed as described (Serrano, 1988). Immunoquantification of the ATPase in plasma membrane was performed as described (Eraso and Serrano, 1987). Rabbit polyclonal antibodies against yeast ATPase (Serrano et al., 1986a) were affinity purified (Monk et al., 1991). Formation of the phosphorylated intermediate was performed as described (Vara and Serrano, 1983) and electrophoresis of phosphoenzyme was as in Niggli et al. (1979). Protein concentration was measured by the Bradford method (1976) using the Bio-Rad protein assay reagent and bovine IgG as standard.


RESULTS

Isolation and Sequence Analysis of K474R Mutant Revertants

Revertants of the K474R mutant were obtained using essentially the methodology described (Eraso and Portillo, 1994). Briefly, the yeast strain RS72 has the genomic copy of the ATPase gene (PMA1) under the control of a galactose-dependent promoter (Cid et al., 1987). This strain can grow in glucose only if it is transformed with a plasmid carrying PMA1. The ATPase with the K474R mutation supports growth in glucose only if the medium is buffered at neutral pH, while growth in acid glucose medium is abolished because of the low activity level of the mutant enzyme (Portillo and Serrano, 1989). The plasmid carrying the K474R mutant allele was mutagenized with hydroxylamine and introduced into the RS72 strain. Transformants were grown on galactose medium and tested for growth on acid glucose medium. Those transformants able to grow in this medium will be full revertants or have a second mutation in the ATPase gene able to suppress the K474R mutation. Of 16,000 transformants obtained, 99 were able to grow on acid glucose medium.

To ascertain whether ability to grow on acid glucose medium was due to full reversion or to second-site mutations, we took advantage of the destruction by the K474R mutation of an MseI restriction endonuclease site present in the wild-type allele. Plasmid DNA isolated from revertants and analyzed showed that ten of 99 reversions were due to a second-site mutation in the K474R mutant allele.

DNA sequence analysis of 20 full revertants showed that, in 50% of these cases lysine restoration at position 474 occurred by a transition AGA to AAA specific of hydroxylamine (Freese, 1971), and in the remainder, Lys-474 restoration resulted from two simultaneous nucleotide changes which restored the wild-type codon AAG. A possible explanation for this alteration is the replacement of the AGA codon by ectopic gene conversion between PMA1 and PMA2 (Harris et al., 1993).

The position of the second-site mutations was determined by constructing chimeric genes (see ``Materials and Methods''). After localizing each mutation to a single restriction fragment, the changed base pair was determined by DNA sequence analysis of the entire fragment. The positions of the second-site mutations are shown in Fig. 1. Two second-site mutations (A165V and V169I/D170N) were localized at the end of the predicted membrane-spanning helix 2 in the stalk region (MacLennann et al., 1985; Serrano, 1989; Wach et al., 1992), one substitution (V396I) was found near the Asp-378 residue, which is phosphorylated during catalysis (Serrano, 1991), three mutations (V484I, V484I/E485K and E485K/E486K) were located 10 residues away from the Lys-474 residue and another (P536L) was situated within the proposed ATP binding domain (Portillo and Serrano, 1988; Taylor and Green, 1989; Clarke et al., 1990; Capieaux et al., 1993).

Genetic and Phenotypic Analysis of Revertants

The A165V, V169I/D170N, and P536L mutations were previously isolated as suppressors of a mutation (S911A/T912A) which affects activation of the enzyme by glucose (Eraso and Portillo, 1994). This prompted us to determine the suppression spectrum of both S911A/T912A and K474R revertants. We also tested whether the K474R revertants isolated were specific for the K474R mutation or could suppress other amino acids substitutions at this position. For this, we created novel combinations of primary and second-site mutations in cis (see ``Materials and Methods''). The pattern of intragenic suppression obtained is shown in Table I. Only A165V, V169I/D170N and P536L reversions could suppress both the S911A/T912A and K474R mutations. The second-site mutation V396I specifically suppresses the K474R mutation. Three second-site changes (V484I, V484I/E485K and E485K/E486K) suppress the substitution of positive-charged amino acids for the Lys-474 residue. Only second-site mutation P536L was able to suppress any of the amino acid substitutions introduced at position 474.

Before analyzing the effect of the new mutations on the kinetic properties of the ATPase, ATPase activity levels and FITC sensitivity of the K474R revertants, were determined. Table II summarizes these characteristics of the enzyme from mutant cells. The K474R mutant ATPase showed a low level of ATP hydrolysis and high resistance to FITC inhibition as described (Portillo and Serrano, 1989). Although second-site mutations did not restore wild-type levels of activity, they increased the ATPase activity relative to the K474R mutant enzyme. Since K474R substitution affects neither the enzyme's stability nor its targeting to the plasma membrane (Portillo and Serrano, 1989), it seems reasonable to think that second-site mutations improve the efficiency of the mutant enzymes. In addition, all revertant enzymes exhibited an increased sensitivity to FITC inhibition, suggesting alteration of the FITC binding site.

Generation and Biochemical Characterization of Mutant ATPases Containing Only the Second-site Mutations

To further characterize the role of the second-site mutations in enzyme function, we created new pma1 alleles containing only the second-site mutation in the absence of the K474R substitution (see ``Material and Methods''). The effect of the second-site mutation on the ATPase function was evaluated by analyzing the kinetic parameters of the mutant enzymes. Table III and Fig. 2 summarize the behavior of these mutants.

I shows that the amount of mutant enzyme quantified by immunoassay in purified plasma membranes was similar to that of the wild type, except for A165V, V169I/D170N and P536L mutants, which exhibited increased plasma membrane enzyme.

Mutant ATPase containing the K474R mutation showed a very low Vand high resistance to FITC inhibition. Other kinetic properties assayed appeared to be essentially wild type.

All mutant enzymes containing only the second-site mutations were found to increase the Vrelative to the K474R enzyme. In three cases (A165V, V169I/D170N, and P536L), the Vwas even higher than that of the wild type as described (Eraso and Portillo, 1994). Three other mutant enzymes (V484I, V484I/E485K, and E485K/E486K) exhibited an altered Kfor ATP. The nucleotide binding specificity of all mutant enzymes was essentially as in the wild type (data not shown).

The FITC sensitivity of four of the mutant enzymes was modified; the V484I, V484I/E485K, E485K/E486K, and P536L mutation produced an enzyme more resistant than the wild type. This suggests that amino acids Val-484, Glu-485, Glu-486, and Pro-536 could form part of the FITC binding pocket or are able to alter this domain.

Vanadate is a P-ATPase inhibitor of which seems to mimic the transition state for aspartyl-phosphate hydrolysis (Josephson and Cantley, 1977). Mutations V484I, V484I/E485K, and E485K/E486K (together with the previously described A165V and V169I/D170N) rendered an ATPase with increased resistance to vanadate suggesting that amino acids Ala-165, Val-169, Asp-170, Val-484, Glu-485, and Glu-486 contribute to or alter the vanadate (P) binding site.

Erythrosine B inhibits the ATP hydrolysis catalyzed by the yeast H-ATPase by binding to the enzyme's ATP binding site (Wach and Gräber, 1991). Enzymes bearing V484I, V484I/E485K, E485K/E486K, and P536L mutations were more resistant to erythrosine B inhibition than the wild-type, suggesting that these mutations disturb the ATP binding domain structure. It is interesting to note that these mutant enzymes were also resistant to inhibition by FITC.

Mutations at the Lys-474 residue greatly decrease the steady-state level of the phosphorylated intermediate formed during the catalytic cycle of the enzyme (Portillo and Serrano, 1988, 1989). We measured the level of phosphorylated intermediate under steady-state conditions to determine whether or not it was affected by the second-site mutations. All mutant enzymes containing the second-site mutation showed phosphoenzyme levels similar to those of the wild-type enzyme (data not shown).

The pH dependence of the mutant enzymes is shown in Fig. 2. Two types of pH profile were observed. Mutations A165V and P536L rendered an ATPase with a pH optimum displaced to the acid range (Eraso and Portillo, 1993), while mutant enzymes bearing V484I, V484I/E485K, and E485K/E486K mutations exhibited a pH profile with a pH optimum displaced to the neutral range. The pH dependence of V169I/D170N and V396I was as in the wild type.


Figure 2: Effect of pH on the ATPase activity of wild-type and mutant enzymes. ATPase activity was assayed in purified plasma membrane from glucose-metabolizing yeast cells with 2 m M ATP at the indicated pH. ATPase activity is expressed as a percentage of the activity at pH 6.5. Upper panel, - - -, wild type; , A165V; , K474R; , P536L. Lower panel, - - -, wild type; , V484I; , V484I/E485K; , E485K/E486K.




DISCUSSION

In this study, revertant analysis of K474R mutation of the yeast plasma membrane H-ATPase has been used to identify interacting domains within the protein. The Lys-474 residue is located within the KGAP sequence which is fully conserved among all the eukaryotic P-ATPases. Mutation at this amino acid was chosen for revertant analysis as previous studies with the yeast ATPase suggested that it was involved in the phosphorylated intermediate formation (Portillo and Serrano, 1988, 1989). Using this genetic approach, we have isolated revertants located within three functional domains: (i) the stalk region, (ii) the phosphorylation domain, and (iii) the ATP binding domain. This suggests the functional coupling between the KGAP motif and these regions; nevertheless, if there are direct interactions between these regions, then the suppressor mutations might be expected to be allele-specific. The fact that only the V396I revertant specifically suppresses the K474R mutation suggests that only Val-396 and Lys-474 residues may interact directly. The location of V484I, V484I/E485K, and E485K/E486K near Lys-474 and their pattern of intragenic suppression support the idea that the suppressor effect of these mutations is probably due to compensatory structural alterations. The A165V, V169I/D170N, and P536L mutations were isolated previously as suppressors of a mutation (S911A/T912A) located in the regulatory domain. The fact that no other S911A/T912A revertants were able to suppress the K474R mutation suggests suppression specificity. Although no conclusions can yet be drawn about the mechanism of this allele-nonspecific suppression, it can be argued that interaction between Lys-474 and these regions, if it occurs, is probably indirect and mediated by the conformational coupling between the stalk sector and either the ATP binding or the phosphorylation domains described (Harris et al., 1991; Na et al., 1993; Perlin et al., 1992). An alternative possibility is that the suppression patterns of A165V, V169I/D170N, and P536L result from an effect of the mutations on protein stability. Global intragenic suppressors which appear to affect protein stability have been isolated in other systems (Shortle and Lin, 1985; Thomas et al., 1991). Consistent with this idea, A165V, V169I/D170N, and P536L mutants exhibited significantly increased amounts of plasma membrane ATPase.

The role of the second-site mutations on enzyme function was evaluated by creating new pma1 alleles containing only the second-site mutations and analyzing the kinetic properties of the resultant mutant enzymes.

The kinetic properties of the V396I enzyme were not significantly different from those of the wild-type enzyme; thus, the Val-396 residue does not appear to be essential.

Mutant enzymes bearing A165V, V169I/D170N, and P536L mutations exhibited kinetic parameters similar to those described (Eraso and Portillo, 1994). All of these mutant enzymes showed a Vhigher than that of the wild-type enzyme. The higher Vcorrelated with an increased amount of plasma membrane enzyme; the reason for this increase remains to be elucidated. In addition, the P536L mutation led to an enzyme resistant to FITC and erythrosine B inhibition, suggesting that this mutation directly or indirectly affects the binding sites of both inhibitors.

The V484I, V484I/E485K, and E485K/E486K mutations resulted in an ATPase with a complex kinetic behavior. They exhibited a very low Vrelative to wild-type enzyme, as would be expected if this mutation caused a structural alteration in the KGAP motif (see above). In addition, these mutant enzymes show a Kfor ATP lower than that of the wild type. The fact that the sensitivity of the mutant enzymes to ATP binding inhibitors was also altered reinforces the notion that these mutations affect ATP binding. Interestingly, V484I, V484I/E485K, and E485K/E486K second-site mutations render an ATPase with a pH optimum displaced to the neutral range, suggesting that residues implicated in these amino acid substitutions may play an essential role, direct or indirect, in the pH dependence of the ATP binding and/or hydrolysis rates. It has been shown that the equilibrium between the Eand Econformations of the cation-transporting ATPases is influenced by the pH (Pick and Karlish, 1982; Blanpain et al., 1992). Alkaline pH stabilizes the Econformation, which is the high ATP affinity and low vanadate affinity conformation (Goffeau and Slayman, 1981; Serrano, 1985). Considering these findings, it is reasonable to think that V484I, V484I/E485K, and E485K/E486K mutations could mimic the pH effect and displace the conformational equilibrium toward the Econformation, thus accounting for the higher ATP affinity observed (I). Future research involving intrinsic fluorescence studies should confirm the alteration of the steady-state distribution of Eand Ecatalytic intermediates in the mutant enzymes.

  
Table: Suppression spectrum of K474R and S911A/T912A revertants

Suppression is defined as the ability to support growth on acid glucose media of the RS72 yeast strain. +, suppression; -, no suppression; ND, combination of mutations was not done.


  
Table: ATP hydrolysis activity and FITC sensitivity of enzymes from intragenic suppressors of the K474R mutant


  
Table: Kinetic properties of mutant ATPases containing only the second-site mutations



FOOTNOTES

*
This work was supported in part by Spanish Grant DGICYT-PB91-0063. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a predoctoral fellowship from the Gobierno Vasco.

To whom correspondence should be addressed: Instituto de Investigaciones Biomédicas del CSIC C/Arturo Duperier, 4, 28029 Madrid, Spain. Tel.: 34-1-5854616; Fax: 34-1-5854587; E-mail: fportillo@biomed.iib.uam.es.

The abbreviations used are: FITC, fluorescein isothiocyanate; kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Drs. H. Celis, P. Eraso, M. J. Mazón, and R. Serrano for critical reading of the manuscript.


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