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INTRODUCTION |
An essential protein of the plant plasma membrane is the
H+-ATPase. It is a major protein at this membrane, where it
controls cytoplasmic and apoplastic pH by generating the transmembrane electrochemical proton gradient that serves as a driving force for
nutrient uptake (1, 2). The plasma membrane H+-ATPase
belongs to the family of P-type ATPases, which include the plasma
membrane-bound Ca2+- and
Na+/K+-ATPases, and has several other members
found throughout prokaryotic and eukaryotic cells (3, 4). Members of
the P-type ATPase family transport a variety of cations including
H+, Na+, K+, Cu2+,
Ca2+, and Mg2+.
Structures of P-type ATPases reveal the presence of 10 transmembrane
helices including large cytoplasmic extensions (5-7). The catalytic
mechanism of P-type ATPase transport involves at least four different
enzyme conformational states named E1,
E1P, E2P, and
E2 (the P designating phosphorylated
intermediates), with the
E1P-E2P transition
accompanying the transfer of ion(s) across the membrane (8). High
resolution three-dimensional crystal structures of the
E1 and the E2
conformations, respectively, have been solved for the animal SR
Ca2+-ATPase (7, 9). These structures indicate substantial
domain movements of P-type ATPases in both cytoplasmic and
transmembrane parts during catalysis. Significant progress has been
made to map the transport pathways in representative P-type ATPases
such as Ca2+- and Na+/K+-ATPases
(7, 9-16). These studies have established the presence of
cation-binding pockets, each of which are formed by several coordinating groups situated in transmembrane helices M4, M5, M6, and
M8. However, the molecular determinants of cation specificity have not
been determined for any P-type ATPase and the mechanism of proton
transport by P-type ATPases is not well characterized.
Protons in solution (as H3O+) form solvation
structures characteristic of cationic solvation. This suggests that the
plant H+-ATPase possibly could mediate hydronium ion
transport (17). However, in a number of different proton pumps that
have been described in molecular detail, H+ seems to be the
transported species, and H+ is in these proton pumps
believed to be transported along a proton transduction chain (a proton
wire) comprising alternating groups of proton donors and proton
acceptors. Whether P-type proton pumps also could mediate
H+ transport along a proton transduction chain is unknown.
In this study, we investigated whether two charged residues,
Arg655AHA2 and
Asp684AHA2, situated in transmembrane segments
M5 and M6, respectively, might play a role in the proton pumping mechanism of plasma membrane H+-ATPase. Transmembrane
segment M5 and M6 are connected by a very short stretch of
extracellularly located amino acid residues and are therefore expected
to form a hairpin loop. During proteolysis of the related
Na+/K+- and
H+/K+-ATPases, inclusion of potassium ions
stabilizes the M5M6 hairpin loop, and it has been suggested that this
moiety moves in and out of the membrane during catalysis (18, 19). It
is evident from the crystal structures of SERCA1
Ca2+-ATPase in two conformations (7, 9) that the M5M6
segment is a mobile structure, somehow coupling ATP hydrolysis to
cation transport. The selected residues
Arg655AHA2 and
Asp684AHA2 are conserved in all plant plasma
membrane H+-ATPases and are close to the predicted
proton-binding site in atomic models of plant (20) and fungal (21)
P-type H+-ATPases. Asp684AHA2 is
strictly conserved in H+-, Na+/K+-
(Asp808), H+/K+-
(Asp804), and Ca2+-ATPases
(Asp800). In the SERCA1 Ca2+-ATPase it
coordinates both of the two Ca2+ ions bound (7). In a
homology model of AHA2 (20), Arg655AHA2 seems
to be equivalent to Glu771SERCA, a residue
involved in direct coordination of Ca2+ in SERCA1. One of
the residues, the negatively charged
Asp684AHA2, has previously been implicated in
enzyme conformational changes in the plant H+-ATPase (22).
The other, the positively charged
Arg655AHA2, has not been assigned a
role so far.
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EXPERIMENTAL PROCEDURES |
Expression in Yeast--
The Saccharomyces cerevisiae
strain RS-72 (23) was transformed and cultured essentially as described
previously (24). In RS-72 (MATa ade1-100 his4-519
leu2-3, 112), the natural constitutive promoter of the endogenous
yeast plasma membrane H+-ATPase PMA1 is replaced
by the galactose-dependent promoter of GAL1.
Since PMA1 is essential for yeast growth, RS-72 grows only on galactose medium. Using this strain, plasmid-borne plant
H+-ATPases brought under control of the constitutive
PMA1 promoter can be tested for their ability to rescue
pma1 mutants on glucose medium.
pma1 Complementation Test in Yeast--
Yeast was grown for 3 days at 30 °C in liquid medium containing 2% galactose.
Approximately 103 cells in 10 µl were spotted onto solid
synthetic minimal medium containing either 2% galactose at pH 5.5 or
2% glucose at pH values of 6.5, 5.5, and 4.5, respectively. Growth was
recorded after incubation for 3 days at 30 °C.
Site-directed Mutagenesis--
As a starting point for
mutagenesis experiments, a modified cDNA of the Arabidopsis
thaliana plasma membrane H+-ATPase isoform
AHA2 under control of the PMA1 promoter subcloned into the yeast multicopy vector YEp-351 (25) was used. The modified cDNA (pMP-1280; Ref. 26) has eight unique novel restriction enzyme
sites created by silent mutagenesis for facilitation of cassette
mutagenesis and furthermore encodes the plant H+-ATPase
AHA2 with (i) a COOH-terminal deletion of 73 amino acid residues and
(ii) a COOH-terminal Met-Arg-Gly-Ser-His6
(MRGSH6) tag. The COOH-terminal deletion transforms the
enzyme into a constitutively activated state, which simplifies
functional studies. Addition of the His6 tag allows
purification of the recombinant enzyme by affinity chromatography,
whereas the additional residues (Met-Arg-Gly-Ser) provide the
H+-ATPase with a convenient antibody epitope.
Site-directed mutagenesis was performed by overlap extension polymerase
chain reaction (27) with mutagenic primers designed to replace the
codons for Arg655 and Asp684. The codon for
Arg655 (CGT) was replaced with codons encoding Lys (AAA),
Ala (GCC), and Asp (GAC), respectively. The codon for
Asp684 (GAC) was replaced with codons for Glu (GAA), Asp
(AAC), Ala (GCC), Val (GTC), and Arg (CGC), respectively. Products of
the polymerase chain reaction were used to replace the corresponding fragments in pMP-1280. DNA sequencing of the cassettes was used to
verify the presence of the desired mutations and the absence of
unwanted base changes. Arg655 and Asp684 double
mutants were constructed by replacing respective fragments in
Asp684 mutant backgrounds with Arg655 mutations.
Purification of ATPase by Ni2+ Affinity
Chromatography--
Growth of yeast for protein purification was the
same as described in Ref. 22. Microsomal yeast membrane vesicles were
isolated as described previously (24). Microsomal membranes were
solubilized by the n-dodecyl-
-D-maltoside at
a detergent to protein ratio of 3:1, after which heterologously
expressed plant H+-ATPases were purified by
Ni2+-nitrilotriacetic acid affinity chromatography
as described (22), except that the elution buffer contained 0.5 mg/ml
of L-phosphatidylcholine. Samples of pure protein were
frozen in liquid nitrogen and stored at 80 °C. All experiments were
repeated with at least three independent membrane preparations.
ATPase Assay--
ATPase activity was determined as described
previously (24). The assay was carried out at 30 °C in a buffer (300 µl) containing 20 mM
MOPS,1 4 mM
MgSO4, and 3 mM ATP. The pH of the buffer
was adjusted to pH 6.5 with
N-methyl-D-glucamine. The reaction was initiated
by the addition of purified membrane protein in reactivation buffer (20% (v/v) glycerol, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothretiol, 10 µg/µl
L-phosphatidylcholine, 0.7% (w/v)
n-dodecyl-D-maltoside).
Reconstitution of ATPase into Artificial Liposomes and
Measurement of Proton Transport--
Protein was reconstituted at a
lipid to protein ratio of 200:1 as described previously (28).
Reconstitution was performed in 10 mM Mes-KOH, pH 6.5, 50 mM K2SO4, and 20% (v/v) glycerol. Proton transport was measured as described (29) by monitoring fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA), a
dye that upon protonation accumulates in an impermeant form inside
vesicles. The reaction medium contained 10 mM Mes-KOH, pH
6.5, 1 mM ATP, 1 µM ACMA, and 50 mM K2SO4. Proton pumping reactions were started by addition of MgSO4 to a final concentration
of 2 mM.
Gel Electrophoresis and Western Blotting--
Membrane fractions
or purified proteins were separated by SDS-PAGE on 10% acrylamide
using the system of Fling and Gregerson (30). After electrotransfer of
the proteins to an Immobilon-P membrane (Millipore), protein blots were
probed with an antibody against the MRGSH6 epitope present
at the COOH terminus (Qiagen, Chatsworth, CA).
Phosphorylation and Dephosphorylation of the Phosphorylated
Intermediate--
Phosphorylation by [
-32P]ATP and
dephosphorylation initiated with 10 mM EDTA or 10 mM EDTA supplemented 2 mM ADP were performed as
described previously (22).
Protein Determination--
Protein concentrations were
determined by the method of Bradford (31) with the Bio-Rad protein
assay reagent and employing bovine serum albumin as a standard.
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RESULTS |
Selection of Residues to Be Studied--
Charged residues in the
hairpin loop formed by transmembrane segments M5 and M6 have been shown
to contribute to ion coordination in a number of related
P2-type ATPases (7, 32). Fig.
1 illustrates this region of the plant
plasma membrane H+-ATPase AHA2 aligned with the
corresponding segment of SERCA1 Ca2+-ATPase, the only
P-type ATPase for which high resolution structural data are available
(7, 9). In transmembrane segments M5 and M6 of this
Ca2+-ATPase, the charged residues Glu771 and
Asp800, together with polar side chains, contribute to
coordination of bound Ca2+. To explore the functional role
of the charged residues Arg655 and Asp684
situated in the corresponding region of the AHA2 plant
H+-ATPase, site-directed mutants of these two positions
were constructed and expressed in yeast.

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Fig. 1.
Amino acid sequence alignment of
transmembrane spanning helices M5 and M6 of the SERCA1
Ca2+-ATPase (Swiss-Prot number P04191) and the plant
H+-ATPase AHA2 (Swiss-Prot number P19456). The
alignment was performed using ClustalW
(www.ebi.ac.uk/clustalw/) employing default parameters. The
transmembrane segments M5 and M6 are highlighted in rectangles.
Conserved residues are indicated by a light gray underlay,
and the residues from M5 and M6 involved in ion coordination in SERCA1
are marked with a black underlay. Residues
Arb655AHA2 and
Asp684AHA2 subjected to analysis in this study
are underlined.
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Replacement of PMA1 in Yeast with Mutant Plant
H+-ATPase--
In the yeast S. cerevisiae, PMA1
is an essential gene, encoding a P-type plasma membrane
H+-ATPase. To test whether any of the plant plasma membrane
H+-ATPase AHA2 substitutions were able to complement a
pma1 mutation, each mutant was expressed in the yeast strain
RS-72, in which the constitutive promoter of the PMA1 gene
has been replaced by the galactose-dependent
GAL1 promoter at the chromosomal level (23). Thus, this
strain is only able to grow on galactose medium and fails to grow on
other carbon sources unless the pma1 deficiency has been
complemented. The plant plasma membrane H+-ATPase employed
(aha2
73; termed wild type (WT) in this study for clarity) carries a
truncation of 219 base pairs in the 3' end of the cDNA. As
nucleotides encoding the COOH-terminal auto-inhibitory domain of AHA2
in this way are removed, this construct readily complements
pma1 (22). Among the Arg655 substitutions
tested, only the R655K substitution was able to fully support yeast
growth, whereas the R655A and R655D mutants supported yeast growth at a
significantly reduced rate (Fig. 2). Of
the Asp684-substituted proteins, only D684E was able to
support yeast growth to some extent. All other substitutions of
Asp684 (D684N, D684A, D684V, and D684R) failed to replace
yeast Pma1p. Altogether, the complementation test pointed to the
importance of both Arg655 and Asp684 for the
proper functioning of plant plasma membrane H+-ATPase.

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Fig. 2.
Complementation test for the growth of a
pma1 yeast mutant expressing wild-type and mutant
Arg655- or Asp684-substituted plant plasma
membrane H+-ATPases. Yeast transformed with empty
vector served as a control. Approximately 103 of
transformed yeast cells in 10 µl of water were spotted on plates
containing either 2% galactose (GAL) or 2% glucose
(GLU) at pH 6.5, 5.5, or 4.5. Growth was recorded after 3 days at 30 °C. Yeast PMA1 ATPase was only expressed on
galactose, whereas plasmid-borne plant ATPases were expressed on both
glucose and galactose medium.
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Expression and Purification of Mutant
H+-ATPases--
To test whether mutant proteins were
successfully produced in the heterologous host, isolated yeast
microsomal membrane vesicles were examined by Coomassie Blue staining
following separation of membrane polypeptides by SDS-polyacrylamide gel
electrophoresis (Fig. 3A). In
all cases a new band corresponding to a polypeptide with an apparent
molecular weight of 92 kDa was observed. The identity of the
polypeptides as plant H+-ATPases was confirmed following
immunoblotting using an antibody directed against the
MRGSH6 epitope fused to the COOH-terminal end of
recombinant plant H+-ATPase (Fig. 3B).
Interestingly, mutant ATPase protein typically reached levels above
those of the wild-type enzyme (Fig. 3B). Following
heterologous expression in transgenic yeast, all of the plant
H+-ATPase mutants described in this work could be purified
by Ni2+-nitrilotriacetic acid affinity chromatography to
near homogeneity (Fig. 3C). This allowed for a detailed
kinetic characterization of the enzymatic properties of the mutant
enzymes.

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Fig. 3.
Affinity purification of wild-type and
Arg655- and Asp684-substituted mutant plant
H+-ATPases after expression in yeast. A,
Coomassie Blue-stained microsomal membrane protein (15 µg) from yeast
transformed with empty vector or yeast expressing wild-type and mutant
H+-ATPases following separation on SDS-PAGE. The purified
wild-type plant H+-ATPase protein runs approximately as a
92-kDa protein on SDS-PAGE gels; B, Western blotting of an
equivalent gel. After blotting, the blot was probed with monoclonal
antiserum raised against the MRGSH6 epitope; C,
Coomassie Blue-stained SDS-PAGE gel preloaded with 2-µg samples of
affinity-purified wild-type and mutant plant
H+-ATPases.
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ATP Hydrolysis and Kinetic Properties--
A way to screen for
qualitative changes in mutant H+-ATPases is to measure
several standard kinetic properties including
Vmax, the Km for ATP,
IC50 for orthovanadate, and pH dependence. Notably, the
apparent ATP affinity of all Arg655 and Asp684
substitutions remained essentially unaltered (Table
I). This is evidence that the
nucleotide-binding pocket was unaffected by the substitutions, strongly
suggesting that the enzymes were correctly folded. However, when other
kinetic parameters were determined for the eight mutants listed above,
major differences were observed from the wild-type control.
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Table I
Biochemical characteristics of affinity purified WT and Arg655
and Asp684 single point-substituted H+-ATPases
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Among the Arg655 substitutions, all led to enzymes with
significantly reduced ATP hydrolytic activities (Table I). Furthermore, the non-conservative substitutions R655A and R655D led to mutant proteins that were almost insensitive toward the inhibitor vanadate, which specifically binds to the E2 conformation
of P-type ATPases. Thus, importantly, removal of the positive charge at
the position of Arg655 seems to cause an appreciable shift
in the equilibrium between the E1 and
E2 conformational states of the enzyme, since
such a shift would be expected to result in a change of
Ki for vanadate (2). The conservative substitution
R655K did not change the vanadate sensitivity of the enzyme.
The pH dependence of P-type H+-ATPases has often been taken
as an indicative for their proton affinity, since the activating effect
of protons on ATP hydrolytic activity resembles that of the transported
cations in other pumps and with comparable affinities. Notably, as was
observed in all cases (Fig. 4A), pH
profiles of the Arg655 substitutions were comparable with
that of wild-type H+-ATPase.
Substitutions at position Asp684 similarly led to reduced
ATP hydrolytic activities (Table I). In addition, most substitutions had dramatic effects on other kinetic properties of the enzyme. The
Asp684 substitutions were all more or less insensitive
toward vanadate. In the D684E substitution, Ki for
vanadate was radically shifted, whereas the D684N, D684A, D684V, and
D684R substitutions were completely vanadate-insensitive. This
indicates that albeit all the tested substitutions are able to
hydrolyze ATP, they all have difficulties proceeding from the
E1 to the E2 conformation.
The conservative substitutions D684N and D684E resulted in a minor
shift in the pH dependence profile toward more acidic values (Fig.
4B). The slight shift in the pH dependence of the
H+-ATPase (Fig. 4B) was more pronounced with
non-conservative substitutions such as D684A and D684V. Furthermore, a
D684R substitution, which replaces a negative charge with a positive
charge, produced a dramatic shift in pH dependence (Fig.
4B).
Proton Pumping--
To ask whether any of the charged residues are
essential for proton pumping, the eight enzymatically active mutants
were further assayed for their ability to pump protons in an
ATP-dependent manner (Fig.
5). Indeed, all the tested
Arg655 substitutions, including R655D, were able to carry
out ATP dependent proton transport, albeit at significantly reduced
rates (Fig. 5A). For all Arg655 substitutions
tested, proton transport seemed efficiently coupled to ATP hydrolysis
(Table I).

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Fig. 5.
Proton pumping in reconstituted
proteoliposome vesicles. Proton pumping was initiated by addition
of MgSO4 (2 mM) to reconstituted
proteoliposomes containing either: WT, R655K, R655A, or R655D
(A) or D684E, D684N, D684A, D684V, or D684R (B)
plant H+-ATPase. In both A and B
proton pumping was measured in 10 mM Mes-KOH, pH 6.5, 50 mM K2SO4, 1 mM ATP, 1 µM ACMA, and 0.15 µM valinomycin. The
decrease in ACMA fluorescence reflects proton accumulation into the
lumen of the vesicles. Rates of proton pumping are presented in Table
I.
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However, among the Asp684 substitutions, only D684E pumped
protons at reduced rates (Fig. 5B). All other attempts to
substitute Asp684 resulted in mutant enzymes that were
completely deficient in proton pumping (Fig. 5B). It is
notable that mutants having non-conservative substitutions at
Asp684 possessed basal ATP hydrolytic activities (Table I).
Thus, in all these mutants ATP hydrolysis appeared to be completely
uncoupled from proton translocation. The proton pumping activity of the D684E mutant seemed efficiently coupled to ATP hydrolysis (Table I).
The E1P-phosphorylated Intermediate Accumulates in
R655A, R655D, and D684N Mutant H+-ATPases--
The amount
of phosphorylated intermediate was measured under steady-state
conditions for the R655K, R655A, R655D, D684E, D684N, D684A, D684V, and
D684R mutants (Fig. 6A). Both
the R655A and R655D H+-ATPases accumulated in the form of
the phosphorylated intermediate (EP) at levels significantly
higher (approximately four times higher) than the WT ATPase (Fig.
6A). In contrast to the E2P
conformational state of P-type ATPases, the E1P
conformational state is able to transfer the covalently bound phosphate
back to ADP. By initiating dephosphorylation of R655A and R655D
proteins with or without ADP, the EP of R655A and R655D was
demonstrated to be sensitive to the application of ADP (Fig.
6B). Thus, these proteins accumulate in the
E1P conformational state, and the low level of
proton transport by these proteins can be ascribed to a reduced rate of
the E1P-E2P conformational transition.

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Fig. 6.
Phosphorylation and dephosphorylation of
purified plasma membrane H+-ATPases. A,
measurement of steady-state phosphorylation levels of WT, R655K, R655A,
R655D, D684E, D684N, D684A, D684V, and D684R ATPases. Phosphorylation
was initiated by addition of 1 µM
[ -32P]ATP and was terminated after 20 s by acid
quenching. Values of the amount of phosphorylated intermediate
(EP) are the mean ± S.E. Levels of steady-state
EP are given relative to wild-type steady-state
EP level; B, dephosphorylation and ADP
sensitivity of the phosphorylated intermediate in WT, R655A, R655D, and
D684N proteins. Proteins were phosphorylated for 20 s with 1 µM [ -32P]ATP, after which
phosphorylation via [ -32P]ATP was stopped by the
addition of EDTA in the absence ( ) or in the presence (+) of ADP. The
dephosphorylation reaction was quenched by acid at t = 0 (before initiation of dephosphorylation, black bars),
after 1 s of dephosphorylation (white bars) or after
3 s of dephosphorylation (gray bars), and the amount of
phosphorylated intermediate determined. The amount of phosphorylated
intermediate present at t = 0 was set to 100%. All
values are the mean ± S.E.
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Whereas the conservative D684E substitution resulted in an enzyme with
EP half of those of the WT enzyme (Fig. 6A), the
level of EP was found to be approximately five times higher
in the D684N mutant as compared with the unmodified enzyme (Fig.
6A). The phosphorylated intermediate was sensitive toward
application of ADP (Fig. 6B). This corresponds to previous
observations that the D684N substitution is blocked in the
E1P-E2P transition (22).
If protons, like cations in other P-type ATPases, are occluded
following phosphorylation and ADP dissociation, these results might
imply that the E1P (H+) occluded
state is greatly stabilized in the D684N mutant. However, whether
protons get occluded within the pump molecule during catalysis is unknown.
With the non-conservative substitutions, accumulation of
EP was much less evident. Thus, D684A and D684V failed to
accumulate EP to the same degree as the D684N mutant and,
with the D684R substitution, only negligible amounts of EP
intermediate could be detected (Fig. 6A). These results
indicate that the catalytic machinery in the D684A, D684V, and D684R
mutants not only is blocked in the
E1P-E2P conformational
transition accompanying proton transport, but also is disturbed in a
step before phosphorylation, namely the
E1-E1P transition.
Genetic Analysis of Arg655 and Asp684
Double Mutants--
In a three-dimensional structural model of the
AHA2 H+-ATPase (20), the side chains of Arg655
and Asp684 are located in close vicinity to each other.
From an energetic point of view, arginine residues within the membrane
spanning segments are expected to be either strongly hydrogen-bonded or members of ions pairs (33). Therefore, it seems possible that an
electrostatic interaction between Arg655 and
Asp684 could take place. In the yeast plasma membrane
H+-ATPase Pma1p, Arg695Pma1p and
Asp730Pma1p in M5 and M6, respectively, were
found to be involved in the formation of a structural important
salt-bridge (34). Sequence alignments point to
Asp684AHA2 as being analogous to
Asp730Pma1p, whereas the residue corresponding
to Arg695Pma1p is equivalent to an
Ala649AHA2 in the plant H+-ATPase.
Arg655AHA2 aligns in sequence comparisons with
His701Pma1p. Single substitutions of
Arg695Pma1p,
His701Pma1p, or
Asp730Pma1p have been difficult to characterize
due to protein mis-folding (34), but both swapping of
Arg69Pma1p and
Asp730Pma1p and insertions of alanines at these
positions in the yeast H+-ATPase resulted in active ATPases
capable of ATP hydrolysis and proton transport at near normal rates
(34).
To address the effect of complementary charge swapping and charge
elimination in the plant H+-ATPase, all the single point
substituted enzymes analyzed in this work (R655K, R655A, R655D) and
(D684E, D684N, D684A, D684V, D684R) were combined. Among the 15 double
mutants, R655K,D684E could replace Pma1p to some extent (Fig.
7). All other double mutants failed to
support growth of the pma1 mutant yeast strain (Fig. 7),
indicating that neither simple charge neither swapping nor charge
elimination do result in a functional proton pump.

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Fig. 7.
Complementation test for the growth of a
pma1 yeast mutant expressing plant plasma membrane
H+-ATPases carrying amino acid substitutions at both
positions Arg655 and Asp684. Transformed
yeast cells (~103 cells) spotted onto synthetic
medium, pH 5.5, containing either: 2% glucose, pH 6.5 (A), or 2% galactose (B). Yeast transformed with
the wild-type plant H+-ATPase and yeast transformed with
empty vector served as positive and negative control, respectively, for
functional complementation of the yeast endogenous plasma membrane
H+-ATPase. Spotting of yeast cells in A and
B was similar.
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Biochemical Characterization of Asp684 and
Arg655 Double Mutants--
Two mutant proteins, namely
R655D,D684R (charge swapping) and R655A,D684A (charge elimination) were
affinity purified to apparent homogeneity (data not shown). When
reconstituted in lipid vesicles, no proton transport could be
demonstrated for any of the double mutated species (Fig.
8B), although both exhibited
ATP hydrolytic activity (R655D,D684R: 0.1 ± 0.02 µmol of
Pi produced per min/mg of protein and R655A,D684A: 1.9 ± 0.1 µmol of Pi produced per min/mg of protein).
Because the apparent affinities for ATP were comparable with that
of the wild-type enzyme (R655D,D684R: 152 µM ± 32 µM and R655A,D684A: 103 ± 18 µM),
the nucleotide-binding site of both substitutions appeared to be
intact. Since both double mutants seem to be folded correctly, the two
residues in the plant protein are not likely to be engaged in an
essential structural salt bridge.

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Fig. 8.
pH dependence and proton pumping capability
of double substituted R655A,D655A and R655D,D684R membrane
proteins. A, pH dependence of ATP hydrolysis;
B, proton pumping of purified, recombinant enzymes measured
following reconstitution of protein in proteoliposomes.
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R655D,D684R and R655A,D684A were completely vanadate-insensitive (data
not shown). Interestingly, the pH dependence profiles of the double
mutants could be super imposed upon the corresponding pH profiles of
the respective Asp684 single point substituted ATPases
(D684R and D684A; Figs. 4 and 8A). In fact, for all of the
kinetic parameters tested, the double mutants behaved similarly to the
respective Asp684 single point mutants. This would suggest
that the rate-limiting step in the catalytic cycle of the
Arg655 and Asp684 double mutants is
determined by the Asp684 substitution.
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DISCUSSION |
The goal of the present study was to determine the role in proton
transport of conserved residues Arg655 and
Asp684 situated in the middle of transmembrane segments M5
and M6, respectively, of the plant plasma membrane
H+-ATPase. Our results provide new insight into the
mechanism by which P-type proton pumps transport protons across the membrane.
The Positive Charge at Arg655 Is Involved in the
E1P-E2P Transition--
All tested
Arg655 substitutions (R655K, R655A, and R655D) were capable
of catalyzing ATP-coupled proton transport albeit with different
efficiencies. Thus, whereas the R655K substituted enzyme was almost as
efficient as the wild-type plant H+-ATPase in carrying out
ATP hydrolysis and proton transport, the two non-conservative
substitutions (R655A and R655D) resulted in enzymes that were severely
inhibited in both ATP hydrolysis and proton transport.
R655A and R655D mutant ATPases were almost insensitive toward vanadate,
an inhibitor of the E2 conformation, but, as
judged from their slight ability to carry out ATP dependent proton
transport, both mutants were able to carry out a complete catalytic
cycle. The phosphorylated intermediate accumulated in the R655A and the R655D proteins to levels corresponding to 3-4 times the value in the
unmodified protein. It was demonstrated that the high level of
EP in these proteins could be ascribed the
E1P phosphoform. Based on the unaltered pH
dependence and this high level of E1P accumulating in the R655A and R655D H+-ATPases, this study
do not suggest a role for Arg655 in the initial
proton-binding event allowing E1P formation to proceed. Rather, in R655A and R655D, the
E1P-E2P conformational transition occurs at a significantly reduced rate. Arginine is the most
basic amino acid (pKa ~12.5),
suggesting that Arg655 is likely to be found in the
protonated form. We conclude that the positive charge of
Arg655 must be ascribed an important, although not
essential, role in the
E1P-E2P conformational
transition. We did not find evidence for a structural salt bridge
between Arg655 and Asp684.
The Amino Acid Side Chain at Position 684 Influences Proton
Affinity and the E1-E1P Conformational
Transition--
In catalytic models of P-type ATPases, the species to
be transported must be bound before any ATP hydrolysis can take place and before the E1-E1P
conformational change. All Asp684 substitutions of the
plant H+-ATPase tested showed capabilities of both
formation of the E1P phosphorylated intermediate
and ATP hydrolysis. Thus, the biochemical characteristics of the amino
acid side chain at position 684 appear not to be essential for the
specific proton-binding event in E1 that allows
the formation of E1P. Alternatively, removal of
the negative charge at position Asp684 mimics the
protonated state of this residue.
The Carboxylate of Asp684 as a Molecular Determinant
for Proton Transport--
By insertion of a single CH2
group into the aspartate at position 684 (D684E) in the plant
H+-ATPase, ATP hydrolytic activity was hampered (~15% of
wild-type). Reconstitution experiments demonstrated that ATP hydrolysis
was coupled to proton transport. Therefore, a precise location of the
carboxylate at position 684 must be important for efficient ATP
hydrolysis and proton transport. However, although the precise location
of this carboxylate is not essential for ATP dependent proton
transport, it seems that the presence of a carboxylate at position 684 is absolutely necessary for any coupling between ATP hydrolysis and
proton transport to take place. Thus, all Asp684
substitutions in which the proton acceptor capabilities of
Asp684 have been removed (D684N, D684A, D684V, and D684R)
showed a complete lack of coupling between ATPase activity and proton
translocation. Still the mutant proteins show relatively high levels of
ATP hydrolysis and steady-state phosphoenzyme.
Among the substitutions of Asp684 tested, it appeared that
D684N, D684A, D684V, and D684R exhibited a complete block in normal P-type enzymatic E1P-E2P transition. Thus, in
addition to the fact that no proton pumping accompanied ATP hydrolysis,
these mutant proteins were completely insensitive toward vanadate. In particular, following replacement of the Asp684 carboxylate
by carbamide (D684N), the mutated H+-ATPase accumulated in
the E1P conformational state, and the rate-limiting step
regarding ATP hydrolysis in this mutant is most likely determined by
the rate of spontaneous E1P dephosphorylation.
Taken together, the absence of a carboxylate at position 684 in the
H+-ATPase completely abolishes any enzymatic transition
from E1P to E2P. Our
results therefore demonstrates that the carboxylate group of
Asp684 is absolutely essential for the transfer of protons
during the E1P-E2P
conformational transition, which is hypothesized to accompany transport
of the bound species across the membrane. This strongly suggests that
the carboxylate of Asp684 function as a molecular
determinant for proton translocation across the membrane in plant
P-type H+-ATPases.
Mechanism of Proton Transport by P-type Proton Pumps--
This
study provides some clues into the biochemistry of the initial
proton-binding event allowing E1P formation (the
E1 proton-binding site). The nature of the amino
acid side chain at position 684 influences binding of the proton to
this E1 proton-binding site. Several groups
including Asp684, none of which need to be essential, may
contribute to initial coordination of the transported species but
further conformational transitions involve Asp684 as an
essential residue. The E1 proton-binding site
could include a proton acceptor group or a group of atoms coordinating
a hydrated proton (H3O+). A
H3O+ coordination center could include the
Asp684 carbonyl side chain oxygen as one of the
coordinating groups. In such a scenario, the proton could arrive to an
already bound water molecule.
The function of Asp684AHA2 could resemble the
functions of Asp85 and Asp61 in the non-related
proton pumps bacteriorhodopsin (BR; 35, 36) and
F1F0-ATPase (37, 38), respectively. In an
essential protonation event, the carboxylates of these residues
receives the proton during catalysis. Among the amino acid residues
involved in the proton transfer reaction of BR, Asp85 is
the only indispensable. A substitution to other than glutamate of
Asp85 results in mutant proteins completely
devoid of proton transporting activity (39, 40). As has been suggested
for the Neurospora PMA1 H+-ATPase (21), the presence of a
conserved arginine
(Arg655AHA2/Arg82BR)
close to a conserved aspartate working as the essential proton acceptor
group during catalysis
(Asp684AHA2/Asp85BR)
could resemble the proton-binding site in bacteriorhodopsin. The
pKa of Asp85BR is influenced
by the orientation of Arg82BR, and by
modulating pKa values of other groups also,
Arg82BR modulates the proton transfer trough
the protein (40, 41). Our finding that non-conservative substitutions of Arg655 in the plant H+-ATPase significantly
slows down the E1P-E2P
transition would be in accordance with an equivalent role of
Arg655 in the plant H+-ATPase.
The major finding of this communication is that the carboxyl group of
Asp684 contributes to the proton-binding site of the AHA2
P-type H+-ATPase. A negative charged oxygen of the
Asp684 carboxyl side chain group could function as an
essential proton acceptor/donor during transport of protons across the
membrane. This suggests that the mechanism of proton transport by
P-type H+-ATPases basically could be similar to the
mechanism utilized by proton pumps such as BR and
F1F0 ATP synthase, in which proton transfer
utilizes protonable side chain groups as proton donors and proton
acceptors in a timely fashion.