(Received for publication, October 24, 1994)
From the
A characteristic feature of the vacuolar
H-translocating inorganic pyrophosphatase (V-PPase) of
plant cells is its high sensitivity to irreversible inhibition by N-ethylmaleimide (NEM) and other sulfhydryl reagents. Previous
investigations in this laboratory have demonstrated that the primary
site for substrate-protectable covalent modification of the V-PPase by
C-labeled NEM maps to a single M
14,000 V8 protease fragment (V8
) (Zhen, R.-G., Kim,
E. J., and Rea, P. A.(1994) J. Biol. Chem. 269,
23342-23350). Here, we describe site-directed mutagenesis of the
cDNA encoding the V-PPase from Arabidopsis thaliana, its
heterologous expression in Saccharomyces cerevisiae and single
substitution of all 9 conserved Cys residues to either Ser or Ala. In
all cases, except one, Cys mutagenesis exerts little or no effect on
either the catalytic activity or susceptibility of the enzyme to
inhibition by NEM. By contrast, and in complete agreement with the
results of peptide mapping experiments, substitution of
Cys
, the sole conserved cysteine residue encompassed by
V8
, with Ser or Ala generates enzyme that is insensitive
to NEM but active in both PP
hydrolysis and
PP
-dependent H
translocation. The specific
requirement for Cys
for inhibition by NEM and the
dispensability of all of the conserved Cys residues, including
Cys
, for V-PPase function indicate that the inhibitory
action of maleimides reflects steric constraints imposed by the
addition of a substituted alkyl group to the side chain of Cys
rather than direct participation of this amino acid residue in
catalysis.
Two primary electrogenic H pumps reside on the
vacuolar membrane of plant cells: a V-type H
-ATPase
(V-ATPase) (
)(Sze et al., 1992) and a
H
-translocating inorganic pyrophosphatase (V-PPase)
(Rea and Poole, 1993). The V-ATPase is common to the membranes bounding
the acidic intracellular compartments of all eukaryotic cells, but the
V-PPase appears to be restricted to the vacuolysosomal membranes of
plants and possibly the chromatophores of a few species of
photosynthetic bacteria (Rea and Poole, 1993). Elucidation of the role
played by pyrophosphate (PP
) as an alternate energy source
for a number of critical metabolic reactions, specifically sucrose
mobilization via sucrose synthase (Huber and Azakawa 1986; Black et
al., 1987) and glycolysis via PP
:fructose-6-phosphate
1-phosphotransferase (Black et al., 1987; Dennis and Greyson,
1987), has given rise to the notion that, rather than simply
dissipatively hydrolyzing PP
, plants have retained or
developed the capacity to scavenge a significant fraction of the energy
contained in this phosphoanhydride. By extension, recognition of the
capacity of the V-PPase for vacuolar energization (Rea and Sanders,
1987) and the results of enzyme localization studies indicating that
the V-PPase may be the sole enzyme responsible for the disposal of
cytosolic PP
(Quick et al., 1989) have implicated
this enzyme not only as a scavenger of the free energy of PP
but also as an important element in the regulation of steady
state cytosolic PP
concentrations (Rea and Poole, 1993).
To understand the relationship between its cellular function and
molecular organization, it is necessary to define the
structure-function relations of the V-PPase. To date, however, two
inherent characteristics of the enzyme have impeded such analyses: its
novelty and pronounced sequence conservation. There are no known
homologs of the V-PPase; it bears no systematic resemblance to soluble
PPases at the polypeptide level (Cooperman et al., 1992; Rea et al., 1992b) and is considered to belong to a fourth
category of primary ion translocase, distinct from the previously
defined F-, P- and V-ATPases (Pedersen and Carafoli, 1987; Rea et
al., 1992b). Those V-PPase sequences that are known show unusually
high levels of sequence similarity; the four published deduced amino
acid sequences for the major M 66,000
substrate-binding subunit (
)of the V-PPase are more than 85%
identical (Kim et al., 1994a). The identification of amino
acid residues that might be critical for catalysis by the application
of sequence alignment procedures has consequently been largely
unproductive. As a result, there has had to be a near exclusive
reliance on protein chemical methods for the initial delineation of
catalytically important amino acid residues. One such approach has been
the use of maleimides to modify and localize residues that might be
involved in V-PPase function.
A well characterized property of the
V-PPase is its sensitivity to inhibition by the sulfhydryl reagent, N-ethylmaleimide (NEM). NEM irreversibly inhibits the enzyme
in a substrate (``Mg +
PP
'') protectable manner with single site kinetics. On
the basis of this finding and the fact that NEM and the
membrane-impermeant cysteine reagent,
3-(N-maleimidylpropionyl) biocytin, inhibit the V-PPase with
similar kinetics and compete for a common binding site on the M
66,000 substrate-binding subunit, a single
residue located in a cytosolically disposed extramembranous domain is
inferred to undergo covalent modification in both cases (Zhen et
al., 1994). Peptide mapping of this residue through selective
labeling of the V-PPase with
C-labeled NEM, purification
of the M
66,000 subunit and its digestion with V8
protease generates a single
C-labeled band
(V8
) migrating at M
14,000 on
Tris-tricine gels that aligns with the carboxyl-terminal segment of the
substrate-binding subunit. Because V8
encompasses only 1
cysteine residue at position 634 that is conserved between the V-PPases
from Arabidopsis thaliana (Sarafian et al., 1992), Beta vulgaris (Kim et al., 1994a), and Hordeum
vulgare (Tanaka et al., 1993), this residue, located in
putative hydrophilic loop X, is concluded to contain the cytosolically
oriented sulfhydryl group whose alkylation by maleimides is responsible
for inactivation of the enzyme (Zhen et al., 1994).
Although instructive, the results of these protein chemical
investigations are subject to two limitations. (i) They provide insight
into the topology of the V-PPase but are incapable of resolving the
direct participation of Cys in catalysis and/or substrate
binding. The fact that reaction of an enzyme with a group-specific
reagent causes irreversible inhibition does not necessarily imply that
the functional group is in the active site. Covalent modification of a
nonessential residue could, for instance, result in a conformational
change that inactivates the enzyme. By the same token, protection
against inhibition, by substrate, does not mean that the susceptible
group is in the active site. It is equally likely that conformational
changes accompanying substrate binding result in the occlusion of
reactive residues that are otherwise remote from the active site. (ii)
Technical constraints have prohibited direct identification of the
modified residue (Zhen et al., 1994). There is a possibility,
albeit small, that the
C-labeled residue is not a cysteine
residue or that nonsequenceable amino-terminally blocked peptides, for
example, other than V8
are responsible for the signal
seen on fluorograms. Thus, whenever practicable, it is important to
test the necessity of specific amino acid residues for catalysis and/or
substrate binding by approaches other than through the use of
group-specific reagents so as to circumvent some of the interpretative
difficulties associated with these compounds.
Recent experiments
have demonstrated that when constructs of the yeast-Escherichia
coli shuttle vector pYES2 containing the entire open reading frame
of the cDNA (AVP) encoding the substrate-binding subunit of the V-PPase
from Arabidopsis are employed to transform Saccharomyces
cerevisiae, vacuolarly localized functional enzyme active in
PPdependent H
translocation is generated
(Kim et al., 1994b). Since the heterologously expressed pump
is indistinguishable from the native plant enzyme, thereby establishing
the sufficiency of AVP for the elaboration of active V-PPase in Saccharomyces, approaches based on site-directed mutagenesis,
epitope tagging, and expression of fusion proteins are now applicable
to investigations of the membrane organization and catalytic mechanism
of this pump. In the specific context of the significance of
Cys
, site-directed mutagenesis has the potential of not
only providing independent criteria of identity for the Cys residue, or
residues, required for inactivation of the enzyme by maleimides but
also a means of determining whether one or more of these residues is
essential for catalysis.
In this communication, we report the single
substitution of all of the conserved Cys residues of the V-PPase from Arabidopsis. Analyses of the patterns of inhibition of
heterologously expressed wild type and mutant enzyme by NEM demonstrate
the necessity of Cys for inhibition of the V-PPase by
maleimides but dispensability of this, and all other conserved Cys
residues, for catalytic activity.
Potassium fluoride was included in the NEM
reaction and PPase assay media to diminish PP hydrolysis by
contaminating yeast-soluble PPase. Yeast-soluble PPase is exquisitely
sensitive to inhibition by potassium fluoride
(K
= 20 µM)
whereas the V-PPase is relatively insensitive
(K
= 3.4 mM) (Baykov et al., 1993b; Kim et al., 1994b). Inclusion of
100-500 µM potassium fluoride thereby largely
abolishes soluble PPase-catalyzed consumption of protective substrate
during incubation with NEM and minimizes the contribution of soluble
PPase to total PP
hydrolysis during the activity assays.
Figure 1:
Effects of
Mg + PP
(MgPP
) and Tris-PP
alone (free PP
) on inhibition of heterologously
expressed wild type (A) and Cys
Ser- or
Cys
Ala-substituted A. thaliana V-PPase (B) by NEM. Membrane vesicles purified from
pYES2-AVP-transformed S. cerevisiae BJ5459 expressing wild
type or C634S or C634A mutant AVP were treated with NEM for 5 min at 0
°C in reaction medium containing MgPP
(0.3 mM Tris-PP
+ 1.3 mM MgSO
) or
free PP
(0.3 mM Tris-PP
). Reaction
with NEM was terminated by the addition of 0.5 mM dithiothreitol, and aliquots of the mixture were assayed for
PP
hydrolysis as described under ``Materials and
Methods.''
These
findings are in complete agreement with the results of previous
investigations of plant vacuolar membrane vesicles showing that
Mg alone confers negligible protection from
inhibition by NEM by comparison with Mg
+
PP
, in combination, while free PP
has a
potentiating action versus membranes treated with NEM in the
absence of both Mg
and PP
(Britten et
al., 1989). A specific requirement for the simultaneous presence
of Mg
and PP
for quantitative protection
from NEM is consistent with the results of steady state reaction
kinetic modeling analyses, which demonstrate that magnesium, probably
dimagnesium (Mg
PP
), pyrophosphate is the active
substrate species (Baykov et al., 1993a; Leigh et
al., 1992).
Figure 2: Tentative topological model of V-PPase from A. thaliana showing positions of conserved Cys residues. The putative transmembrane spans (boxedsequences) were predicted from the HELIXMEM program of PC/GENE. The conserved Cys residues, common to the deduced sequences from A. thaliana (Sarafian et al., 1992), B. vulgaris (isoforms 1 and 2) (Kim et al., 1994a), and H. vulgare (Tanaka et al., 1993), are shown in white against a blackbackground.
Uracilated
single-stranded template DNA was isolated from pYES2-AVP-transformed E. coli CJ236, and site-directed mutations were introduced by
second strand synthesis from the template using mutagenic
oligonucleotides designed to substitute each conserved Cys codon with a
Ser or Ala codon. After amplification of the mutated plasmid in E.
coli DH and confirmation of the mutations by DNA sequencing, S. cerevisiae BJ5459 (Ura
) was transformed
with the mutated plasmid, and the resulting Ura
transformants were grown in liquid culture under inducing
conditions. Partially purified microsomes were prepared from the
transformants and V-PPase activity was characterized. The results are
summarized in Fig. 1, Fig. 3, and Fig. 4.
Figure 3:
NEM sensitivity of V-PPase singly mutated
from Cys to Ser at positions 19, 78, 128, 136, 308, 343, 411, and 444.
Membranes purified from S. cerevisiae BJ5459 expressing
mutated V-PPase were tested for inhibition by NEM (0-20
µM) in media containing MgPP (
) or free
PP
(
) as described in Fig. 1.
Figure 4:
PP-dependent
H
translocation by membrane vesicles prepared from
pYES2-AVP-transformed S. cerevisiae BJ5459 expressing either
wild type or mutant (C634S or C634A) V-PPase. The membranes were
incubated with 50 µM NEM in reaction medium containing
MgPP
(0.3 mM Tris-PP
+ 1.3 mM MgSO
) or free PP
(0.3 mM Tris-PP
) for 5 min at 0 °C. Reaction with NEM was
terminated by the addition of 0.5 mM dithiothreitol, and the
membranes were washed free of NEM and ligands. Aliquots (50 µg) of
the washed membranes were assayed for H
translocation
(intravesicular acidification) with the fluorescent transmembrane pH
difference indicator acridine orange in a total reaction volume of 1 ml
after the addition of 1.3 mM MgSO
to medium
containing 1 mM Tris-PP
.
All
of the substitutions, except for C634S and C634A, exert little or no
effect on the activity of the V-PPase or its sensitivity to inhibition
by NEM (Fig. 3). Cys Ser substitutions at positions 19,
78, 308, 343, and 411 yield enzyme exhibiting a pattern of
(Mg
+ PP
)-protectable, free
PP
-potentiated inhibition by NEM similar to wild type
V-PPase ( Fig. 1and Fig. 3). Likewise, although C128S,
C136S, and C444S mutants are slightly less sensitive to inhibition by
NEM in the presence of free PP
than wild type enzyme, they
are nevertheless quantitatively protected by Mg
+ PP
(Fig. 3). Furthermore, the specific
activity of the V-PPase approximates wild type in all cases. Hence,
wild type enzyme and the C19S, C78S, C128S, C136S, C308S, C343S, C411S,
and C444S mutants exhibit activities of 30, 36, 19, 16, 22, 27, 25, 37,
and 20 µmol/mg/h, respectively. In striking contrast, substitution
of Cys
generates enzyme that is insensitive to NEM
irrespective of whether Mg
+ PP
or
free PP
are included in the NEM reaction medium (Fig. 1). Replacement of Cys
with either Ser or
Ala yields heterologously expressed enzyme that is active in both
PP
hydrolysis (Fig. 1B) and
PP
-dependent H
translocation (Fig. 4) but insensitive to NEM concentrations in excess of 50
µM. The PP
hydrolytic activities of C634S and
C634A, 28 and 26 µmol/mg/h, respectively, approximate wild type
enzyme as do both the rates and extents of PP
-dependent
intravesicular acidification. It therefore appears that although
Cys
is required for inhibition of the V-PPase by NEM, it
is not required for PP
hydrolysis or
PP
-dependent H
translocation. The finding
that the C634S and C634A mutants are indistinguishable with respect to
both NEM insensitivity and catalytic activity (Fig. 1B)
implies that both functional attributes are independent of whether the
amino acid at position 634 possesses a polar side chain containing an
electronegative center (-CH
-SH or
-CH
-OH) or nonpolar (-CH
)
substituent.
The mutational analyses described demonstrate that only one
conserved Cys residue, Cys, is required for inhibition of
the V-PPase by NEM. Together with the results from previous protein
chemical investigations of the mode and site of action of maleimides
(Zhen et al, 1994), these studies identify the sulfhydryl
group of Cys
as the reactive moiety. Two inferences
therefore follow. (i) A cytosolic orientation must be assigned to the
putative hydrophilic loop (loop X) encompassing Cys
(residues 594-646, Fig. 2). The studies showing that
NEM, a permeant maleimide, and 3-(N-maleimidylpropionyl)
biocytin, an impermeant maleimide, inhibit the enzyme with similar
kinetics in a substrate-protectable manner and compete for a common
binding site (Zhen et al., 1994) in conjunction with the
demonstrated necessity for a Cys residue at position 634 by
mutagenesis, unequivocally establish a cytosolic disposition for this
residue and those immediately flanking it. (ii) Earlier speculations
concerning the location of the NEM-reactive site, or sites, are
refuted. It has been noted that Cys
and
Cys
, located in putative hydrophilic loop II, are flanked
by sequences possessing a spacing and alternation of acidic and basic
amino acid residues equivalent to those regions of the soluble PPase
from Saccharomyces known, from site-directed mutagenesis and
x-ray crystallography, to be critical for catalysis (Rea et
al., 1992b; Cooperman et al., 1992). Hence, while the
V-PPase and soluble PPases appear to be remote evolutionarily, it has
been proposed that they may share convergent motifs related to the need
for both classes of enzyme to interact with the same substrates,
inhibitors, and activators (MgPP
,
Mg
PP
, Mg
,
Ca
). Further, in view of the proximity of Cys
and Cys
to these motifs, the sensitivity of the
V-PPase to covalent modification and inhibition by maleimides has been
attributed to alkylation of one or both of these Cys residues (Rea et al., 1992b). Although the direct participation of loop II
residues other than Cys in substrate binding and/or turnover is not
addressed here, the finding that C128S and C136S mutants are not only
active in catalysis but also retain sensitivity to (Mg
+ PP
)-protectable, free
PP
-potentiated inhibition by NEM clearly contradicts their
proposed involvement in inhibition by maleimides.
An unexpected but
simplifying finding and an insight that could not have been gained
other than through mutagenesis is that C634S and C634A mutants retain
catalytic activity. The situation with the V-PPase is therefore
reminiscent of the A subunit of yeast V-ATPase (Taiz et al.,
1994) and E. colilac permease (Kaback, 1992). In the
case of the V-ATPase, C261S mutants acquire insensitivity to NEM while
retaining wild type hydrolytic activity (Taiz et al., 1994).
In the case of lac permease, only Cys appears to
be important for transport, but even this residue is not essential
(Kaback, 1992). When Cys
is replaced with Val and each of
the other Cys residues is replaced with Ser, about 30% of the initial
rate of transport and about 60% of the steady state level of
accumulation of lactose is achieved by the ``C-less''
permease versus wild type, although the former is rendered
insensitive to NEM (van Iwaarden et al., 1991). By analogy
with these two transporters and the dispensability of all of the
conserved Cys residues of the V-PPase, the inhibitory action of
maleimides on wild type enzyme is specifically attributed to structural
deformation imposed through the introduction of a bulky substituted
alkyl group on Cys
. Thus, while the substrate
protectability of enzyme inhibition and covalent modification of
Cys
by maleimides indicates that this residue is close to
or conformationally coupled with the substrate-binding site, direct
participation of this or any other Cys residue in the catalytic cycle
of the V-PPase is improbable.