(Received for publication, April 10, 1997, and in revised form, June 5, 1997)
From the Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018
On the basis of a revised topological model of
the vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1)
derived from the analysis of four published sequences using two
structure-predicting programs, TopPred II and MEMSAT, eight acidic
amino acid residues located near or within transmembrane -helices
were identified. The codons specifying these amino acids in the
cDNA encoding the V-PPase from Arabidopsis thaliana
were singly mutated to examine their involvement in pyrophosphate
(PPi) hydrolysis and PPi-dependent H+ translocation and the functional significance of the
similarities between the sequences encompassing Glu229
(227-245) of the V-PPase and the
N,N
-dicyclohexylcarbodiimide (DCCD)-binding transmembrane
-helix of the c-subunits of F-ATPases (Nyren, P., Sakai-Nore, Y.,
and Strid, A. (1993) Plant Cell Physiol. 34, 375-378).
Three functional classes were identified after heterologous expression
of mutated enzyme in Saccharomyces cerevisiae. Class I
(E119Q, E229Q, D573N, E667Q, and E751Q) mutants exhibited
PPi hydrolytic and H+ translocation activities
and DCCD sensitivities similar to wild type. The one class II mutant
obtained (E427Q) was preferentially impaired for H+
translocation over PPi hydrolysis but retained sensitivity
to DCCD. Class III (E305Q and D504N) mutants exhibited a near complete abolition of both PPi hydrolysis and H+
translocation and residual activities with decreased DCCD sensitivity. In none of the mutants was diminished insertion of the V-PPase into the
membrane or an increase in the background conductance of the membrane
to H+ evident. The decoupled character of E427Q mutants and
the enhancement of H+ pumping in E427D mutants by
comparison with wild type, in conjunction with the retention of DCCD
inhibitability in both E427Q and E427D mutants, implicate a role for
Glu427 in DCCD-insensitive H+ translocation by
the V-PPase. The proportionate diminution of PPi hydrolytic
and H+ translocation activity and conservation of wild type
DCCD sensitivity in E229Q mutants refute the notion that
Glu229 is the residue whose covalent modification by DCCD
is responsible for the abolition of
PPi-dependent H+ translocation.
Instead, the diminished sensitivity of the residual activities of E305Q
and D504N mutants, but not E305D or D504E mutants, to inhibition by
DCCD is consistent with the involvement of acidic residues at these
positions in inhibitory DCCD binding. The results are discussed with
regard to the possible involvement of Glu427 in coupling
PPi hydrolysis with transmembrane H+
translocation and earlier interpretations of the susceptibility of the
V-PPase to inhibition by carbodiimides.
The membranes constituting the vacuolysosomal complex of plant cells are unusual in possessing an H+ translocating inorganic pyrophosphatase (V-PPase1; EC 3.6.1.1) (2). The V-PPase bears no systematic resemblance to soluble PPases at the sequence level (3, 4) and is considered to belong to a fourth class of H+-phosphohydrolase distinct from the F-, P- and V-ATPases (4). Moreover, unlike the V-ATPase, which is ubiquitous in the membranes bounding the acidic intracellular compartments of all eukaryotic cells, the V-PPase appears to be restricted to plants and a few species of phototrophic bacteria (2, 5). Notwithstanding the intrinsic evolutionary interest of this phenomenon, it poses a problem: the lack of sequence-divergent homologs from phylogenically remote organisms. Because all published V-PPase sequences are from the same group of organisms, vascular plants, and exhibit greater than 85% sequence identity at the amino acid level (6), most attempts to identify conserved amino acid residues of potential mechanistic significance by sequence alignment procedures have been unproductive. Crucial, therefore, has been the development of methods for the expression of functional pump in the yeast, Saccharomyces cerevisiae (7, 8). When constructs of the yeast-Escherichia coli shuttle vector pYES2, containing the entire open reading frame of the cDNA (AVP; Ref. 9) encoding the Mr 66,000 substrate-binding subunit2 of the V-PPase from Arabidopsis thaliana are employed to transform S. cerevisiae, endomembrane-associated enzyme active in PPi-dependent H+ translocation is generated (7). 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 S. cerevisiae, 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 the V-PPase.
By the parallel application of mutational and protein chemical methods, we have demonstrated a specific requirement for a cytosolically oriented Cys residue at position 634 for inhibition of the V-PPase by maleimides and the dispensability of all conserved Cys residues, including Cys634, for catalysis (8, 10). Our current studies of the V-PPase are directed at elucidating the involvement of acidic (Asp, Glu) residues located near or within hydrophobic spans in substrate turnover and/or H+ translocation.
Two factors prompted investigation of these acidic residues. The first
was the need to gain insight into the identity and location of acidic
residues with the potential for undergoing cycles of protonation and
deprotonation within the hydrophobic core of the membrane. On the basis
of analyses of other H+ pumps and H+-coupled
transporters, acidic residues associated with transmembrane spans might
be expected to directly participate in H+ uptake,
translocation, and release by the V-PPase. The second factor was the
observations of Nyren et al. (1), who noted that the
sequences encompassed by positions 227-245 of the V-PPase from
Arabidopsis bear a resemblance to the C-terminal regions of
the c-subunits of F-ATPases. The C-terminal sequence flanking Glu229 in AVP is 71, 65, and 67% similar (35, 47, and 39%
identical) to Rhodospirillum rubrum c-subunit (positions
58-74), Pisum sativum chloroplast subunit III (positions
61-77), and P. sativum mitochondrial subunit 9 (positions
55-72) (Fig. 1). Since the c-peptide,
the most highly conserved subunit of the H+-conductive
Fo sector of F-ATPases, binds the hydrophobic carboxyl reagent, N,N-dicyclohexylcarbodiimide (DCCD) at an acidic
residue located in the middle of the second of the two transmembrane
helices of this polypeptide, to abolish H+
translocation, Nyren et al. (1) have proposed that the
sequence flanking Glu229 of the V-PPase may assume an
analogous function. Specifically, in view of the sensitivity of the
V-PPase to inhibition by DCCD (11), it has been suggested that
Glu229 is the residue whose covalent modification by this
carbodiimide is responsible for the inhibition of
PPi-dependent H+ translocation.
Here we present (i) a revised topological model of the V-PPase, which, unlike those reported previously (9, 12), is derived from the concerted application of multiple computer-based structural criteria to the deduced sequences of the polypeptides specified by the cDNAs from several independent sources; and (ii) the single substitution of most of the conserved Asp and Glu residues inferred to be located near or within transmembrane spans on the basis of this topological model. The results of modeling are consistent with a structure for the V-PPase incorporating 15 transmembrane spans, while the results of mutagenesis demonstrate that Glu229 is unlikely to play a role in H+ translocation or inhibition of the V-PPase by DCCD. Instead, the characteristics of the mutants, combined with the inferred topology of the V-PPase, are better accommodated by a scheme in which membrane-embedded residues Glu305 and Asp504 contribute to DCCD binding, whereas Glu427, which is located at the interface between a transmembrane span and its adjoining cytosolic loop, is required for coupling PPi hydrolysis with H+ translocation.
The
cDNA encoding the V-PPase from A. thaliana
(AVP; Ref. 9) was heterologously expressed in vacuolar
protease-deficient S. cerevisiae haploid strain BJ5459
(MATa, ura3-52, trp1, lys2-801, leu21, his3-
200,
pep4::HIS3, prb
1.6R, can1, GAL) (8, 13). Transformation of BJ5459 with yeast-E. coli shuttle vector
pYES2, containing the entire open reading frame of AVP inserted between the GAL1 promoter and CYC1 termination sequences
(pYES2-AVP; Ref. 7), isolation of the Ura+ transformants
and growth of the cells for the preparation of membranes were performed
as described (8). E. coli DH5
and CJ236
(dut
ung
) were
employed for the amplification of pYES2-AVP and the generation of
single-stranded, uracilated template for site-directed mutagenesis, respectively.
Mutagenesis was performed directly on pYES2-AVP vector (8). In all
cases the mutagenic oligonucleotides were designed to singly substitute
each conserved Asp or Glu codon with an Asn or Gln codon on the basis
of the cDNA sequence of AVP (9). The sequences of the eight
oligonucleotides (positions of conserved Asp or Glu codons shown
in bold type and positions of degeneracy shown in brackets) were:
Glu119 Gln,
CGGCTCTGTT[C]AGGGATTCAGCAC; Glu229
Gln, TCTTTTT[C]AGGCTATTACTGG;
Glu305
Gln,
GGATCATATGCT[C]AAGCATCATGCGC;
Glu427
Gln,
GTTTCGTCA-CT[C]AGTACTACACTAG;
Asp504
Asn,
GGCAATT[A]ATGCTTATGGTCCC; Asp573
Asn, CCACACCGTA[A]ATGTTTTGACC;
Glu667
Gln,
CTTTGGAGTT[C]AGACCCTC-TCTGG; Glu751
Gln, CATGGCTGTT[C]AGTCTCTTGTC. At four
positions (Glu229, Glu305, Glu427,
and Asp504), mutants in which the Asp codons were replaced
by Glu codons or vice versa were also generated. The
sequences of the four oligonucleotides used for this purpose were:
Glu229
Asp,
TCTTTTTGA[C]GCTATTACTGG; Glu305
Asp, GGATCATATGC-TGA[T/C]GCATCATGCGC;
Glu427
Asp,
GTTTCGTCACTGA[C]TACTACACTAG; Asp504
Glu, GGCAATTGA[G]GCTTATGGTCCC.
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 mutant oligonucleotides (14, 15). In all cases, mutagenesis was confirmed by sequencing the target region before yeast transformation. In selected cases, when a pronounced alteration of V-PPase function was observed, the sequence of the target region of the AVP insert of pYES2-AVP was determined after extraction of the vector from the yeast transformants.
Preparation of Vacuolar Membrane-enriched VesiclesYeast vacuolar membrane-enriched vesicles were prepared as described (8).
Reaction of V-PPase with N,NThe standard mixture for reaction with DCCD contained 30 mM Tris-Mes buffer (pH 8.0), the indicated concentrations of ligands (Mg2+ as MgSO4, K+ as KCl, PPi as Tris-PPi) and membrane protein (9.7-10.7 µg/ml). Reaction was initiated by the addition of DCCD (0-500 µM dissolved in ethanol), and the samples were incubated at 37 °C for the times indicated. After terminating the reaction by the addition of Mg2+ (1.3 mM), the samples were cooled on ice before assaying aliquots for V-PPase activity. Control samples were treated in an identical manner after the addition of equal volumes of ethanol. All stock DCCD solutions were prepared fresh daily.
Measurement of V-PPase Activity and ProteinPPi hydrolytic activity was measured as the rate of liberation of Pi from PPi at 37 °C in reaction media containing 0.3 mM Tris-PPi, 1.3 mM MgSO4, 100 mM KCl, 1 mM NaF, 5 µM gramicidin-D, 1 mM Tris-EGTA, and 30 mM Tris-Mes (pH 8.0) (8). Since yeast-soluble PPase, unlike the V-PPase, is exquisitely sensitive to inhibition by fluoride (Kiapp (soluble PPase) = 20 µM; Kiapp (V-PPase) = 3.4 mM) (16), inclusion of 1 mM NaF in the assay media effectively abolishes the contribution of the former to total hydrolysis (8).
PPi- and ATP-dependent H+
translocation was assayed fluorimetrically using acridine orange (2.5 µM) as transmembrane pH difference indicator in assay
media containing vacuolar membrane-enriched vesicles (200 µg), 100 mM KCl, 0.4 M glycerol, 1 mM
Tris-EGTA, and 5 mM Tris-HCl (pH 8.0). Reaction was
initiated by the addition of Tris-PPi (1.0 mM)
to media containing MgSO4 (1.3 mM) in the case
of V-PPase-mediated H+ translocation or by the addition of
MgSO4 (3 mM) to media containing Tris-ATP (3 mM) in the case of V-ATPase-mediated H+
translocation. The decrease in fluorescence was measured at excitation and emission wavelengths of 495 and 540 nm, respectively (8). The
initial rate of H+ translocation and steady state pH
gradient were estimated as F%/mg/min (at time zero) and
F%/mg (after 5-10 min), where
F% = percentage decrease in fluorescence as described (17). Coupling ratio
(the ratio of the rate of H+ pumping to the rate of
PPi hydrolysis) was estimated as
(
F%/min)/(µmol of PPi hydrolyzed/min).
Protein was estimated by a modification of the method of Peterson (18).
For Western analyses of the heterologously
expressed V-PPase, membrane samples were delipidated by extraction with
acetone:ethanol (1:1; 20 °C) (19), dissolved in denaturation
buffer, and subjected to one-dimensional SDS-polyacrylamide gel
electrophoresis on 11% (w/v) slab gels in a Bio-Rad minigel apparatus
(7). The electrophoresed samples were electrotransferred to 0.45-µm
nitrocellulose filters in standard Towbin buffer (20), containing 10%
(v/v) methanol for 30 min at a current density of 2.5 mA/cm2 in a Millipore semi-dry blotting apparatus. After
reversible staining of the transferred protein bands with Ponceau-S,
the filters were processed for reaction with antibody
(PABHK1) raised against synthetic peptide with the sequence
HKAAVIGDTIGDPLK, corresponding to positions 720-734 of AVP (9).
Immunoreactive bands were visualized by successive incubations of the
membrane filters with horseradish peroxidase-conjugated sheep
anti-rabbit immunoglobulin G and buffer containing 0.03% (w/v)
H2O2, 0.5 mg/ml diaminobenzidine, and 0.03%
(w/v) NiCl2 (21).
Data were fitted by nonlinear least squares analysis (22) using the Ultrafit nonlinear curve-fitting package from BioSoft (Ferguson, MO).
Computer Programs for Modeling V-PPase TopologyTwo programs were employed to model the overall topology of the V-PPase: TopPred II and MEMSAT (membrane structure and topology). The TopPred II program, developed by Manuel G. Claros and Gunnar von Heijne (Karolinska Institute, Stockholm, Sweden) for Macintosh computers is a public domain software package for predicting the topology of both prokaryotic and eukaryotic membrane proteins by the concerted application of hydropathy analyses, the "positive-inside" rule and "charge-difference" rule (23). The MEMSAT program, developed by Jones et al. (24) for IBM PCs, is based on expectation maximization. From the distributions of amino acids compiled from membrane proteins, or portions thereof, of defined topology, the log-likelihood ratios (si) for domain classes are calculated for each of the 20 amino acids according to the expression si = ln (qi/pi) where pi is the relative frequency of occurrence of amino acid i in all the sequences in the data set and qi is the relative frequency of occurrence of the same amino acid in a particular domain. These si values or propensities are then used to equate a given sequence with a given topology.
The deduced amino acid sequences of the V-PPases encoded by the cDNAs isolated from A. thaliana (AVP, GenBankTM accession no. M81892) (9), Beta vulgaris (BVP1, L32792; BVP2, L32791) (25), and Hordeum vulgare (HVP, D13472) (12) were processed in parallel using both programs.
A revised topological model of the
V-PPase was derived from the deduced sequences of the polypeptides
encoded by four cDNAs: AVP from A. thaliana
(9), BVP1 and BVP2 from B. vulgaris
(25), and HVP from H. vulgare (12). The model
shown in Fig. 2 was the only one of the
three predicted by the TopPred II and MEMSAT programs of Claros and von
Heijne and Jones et al. (24) capable of accommodating a
cytosolic orientation for both the C terminus and the hydrophilic loop
containing the N-ethylmaleimide (NEM)-reactive cysteine,
Cys634, inferred from the characteristics of apoaequorin
fusions (26) and the results of peptide mapping and Cys mutagenesis,
respectively (8, 10).
Examination of the structure of the Mr 66,000 subunit of the V-PPase by TopPred II consisted of three main stages. (i) The first stage was the construction of hydrophobicity profiles using a trapezoid sliding window (27). Depending on the height and width of the hydrophobicity maxima and the preset "upper cutoff" and "lower cutoff" values for the computed hydrophobicity indices, spans were categorized as either "certain" or "putative." (ii) The second stage was enumeration of the difference in representation of positively charged amino acid residues between the two sides of the membrane and tests of the adherence of any given model to the positive-inside rule, with the bias in favor of Arg and Lys residues in hydrophilic loops with a cytosolic disposition in most polytopic membrane proteins (28). (iii) The third stage was application of the charge-difference rule (29), wherein the net charge difference between the 15 N-terminal and the 15 C-terminal residues flanking the most N-terminal transmembrane span is computed. Transmembrane orientation is correlated with the disposition of charged residues in the immediate vicinity of the first membrane span. The segment C-terminal to the first span is generally positively charged with respect to the N-terminal flanking regions in membrane proteins possessing a luminally oriented N terminus (29).
Deployment of the MEMSAT program entailed analysis of segments of the sequence of the V-PPase in terms of their likelihood of being located within a particular topological element. Based on statistical analysis of the distribution of amino acids in membrane proteins, the MEMSAT program ranks amino acids according to their propensities for being associated with each of five types of topological element: two classes of hydrophilic loop, designated cytoplasmic (inside) loop (Li) and luminal (outside) loop (Lo), and three classes of transmembrane helix domain, designated helix inside (Hi), helix middle (Hm), and helix outside (Ho) (24).
The consensus structure consistent with the predictions from both programs, the disposition of Cys634, and the C-terminal apoaequorin fusion data was a 15-span model containing a luminally localized N terminus and cytoplasmically localized C terminus (Fig. 2). While MEMSAT ranked a 16-span model highest, with the additional span encompassing residues 743-761, two models containing 14 and 15 spans ranked just below this model. In the 14-span model, the two lowest scoring transmembrane spans in the 16-span model (V and VI) were excluded, thus preserving the orientation of the N and C termini and the remaining C-terminal spans. In the 15-span model, the last transmembrane span in the 16-span model (XVI) was excluded, thus transferring the C terminus from the luminal to the cytosolic face of the membrane while preserving the orientation of all of the other spans.
All three models were consistent with a cytosolic disposition for Cys634 (8, 10), but only one, the 15-span model, was compatible with the finding that fusion of apoaequorin with the C terminus of AVP generates a vacuolar membrane-associated polypeptide capable of sensing cytosolic Ca2+ in transgenic A. thaliana plants (26). Assuming that the fusion of apoaequorin with the C terminus does not, itself, change the topology of the V-PPase, these data constrain the C terminus to the cytosolic face of the membrane and exclude the 16- and 14-span models.
Notable is the basic equivalence between the predictions deriving from TopPred II and MEMSAT. Whereas TopPred II constrains the length of transmembrane spans at a specific value (21 amino acid residues in this study) and is based on the assumption that all spans are perpendicular to the phospholipid bilayer, MEMSAT selects the best fit within a user-defined range of minimum and maximum lengths (17-25 amino acids residues in this study), thereby diminishing bias in favor of any one angle of intersection. Nevertheless, the margins of 11 of the 15 spans predicted by the two programs differed by no more than 4 amino acid residues and the average length of the spans (21.5 by MEMSAT versus a fixed value of 21 for TopPred II) were virtually identical. Of the spans predicted by MEMSAT, only two, spans IX (408-425) and XV (671-687), were shorter than the 20 amino acids required to traverse the entire bilayer, but in both cases the counterpart helices predicted by TopPred II included all 17 of these residues. Accordingly, when the MEMSAT settings were altered to increase the minimum span length from 17 to 19 residues, the overall topology of the V-PPase was unchanged; spans IX and XV were simply lengthened.
The three transmembrane spans (V (230-252), VI (292-316), X (452-472)) not identified in the original 13-span model (9) were neglected because of the proximity of adjacent maxima in the hydrophobicity profiles (span X) and the use of window sizes so broad as to obscure hydrophobic segments adjacent to regions of extreme hydrophilicity (spans V and VI). TopPred II, by contrast, permitted better resolution of the neighboring transmembrane spans by the application of a narrower sliding window (11 amino acid residues).
Two of the three transmembrane spans overlooked in the original 13-span model, helices V and VI, were the least likely in terms of their hydrophobicity and expectation maximization scores. Their MEMSAT scores (339 and 449, respectively) were significantly higher than the default cutoff value of 100 but markedly lower than the average score of 2713 for the other transmembrane spans. However, because the low scores of these helices were largely attributable to Arg246 (in helix V) and Asp298 (in helix VI), each residue of which was predicted to be displaced by seven positions from the cytosolic face of the membrane according to MEMSAT, and therefore appropriately positioned for mutual electrostatic screening, both spans were retained in the model.
The orientation of the N terminus was deduced from the
charge-difference rule (29). Examination of the N-terminal residues immediately adjacent to the first transmembrane span (positions 14-34)
revealed no positively charged residues and two negatively charged
residues (Glu9, Glu13), giving a net charge of
2. The corresponding regions of BVP1, BVP2 (25), and HVP (12) had the
same charge, attributable to Glu13 in all three sequences,
plus Glu9 in HVP and Asp9 in BVP1 and BVP2. Of
the 15 amino acid residues on the C-terminal side of the first span of
AVP, two (Arg36, Lys38) were positively charged
and one (Asp42) was negatively charged, giving a net charge
of +1. BVP1 and BVP2 had three positive charges and one negative charge
(net charge +2), and HVP had six positive charges and no negative
charges (net charge +6) in this region. A net charge difference of at least 3 (
2 for the N-terminal 15 residues before the first span versus +1 for the C-terminal 15 residues after the span for
AVP, 4 for BVP1 and BVP2, 8 for HVP) in all four cases was consistent with a luminal orientation for the N terminus.
The cytosolically disposed loops of the 15-span model for AVP contain a significantly greater number of Arg and Lys residues than the luminally oriented loops (83% versus 17%, respectively). Further, the majority of the residues located in hydrophilic loops are cytosolically oriented (79.7%, 45.6% of total), in accord with the inside-positive rule (28) and with the expectation that the overall distribution of hydrophilic loops would be biased toward the side of the membrane responsible for catalysis and ligand binding.
Three Classes of MutantAccording to the 15-span model (Fig.
2), a total of eight conserved acidic amino acid residues
(Glu119, Glu229, Glu305,
Glu427, Asp504, Asp573,
Glu667, and Glu751) were tentatively identified
as being near or within putative transmembrane spans. To examine their
involvement in PPi hydrolysis, H+
translocation, and DCCD inhibition, these residues were singly substituted. In all cases acidic residues were replaced with their corresponding amides; in those cases where acid amide substitutions had an influence on V-PPase activity, enzyme containing structurally conservative Asp
Glu or Glu
Asp substitutions was also
generated.
Three classes of V-PPase mutant were distinguishable on the basis of their hydrolytic and pumping activities after heterologous expression in S. cerevisiae strain BJ5459: (i) those exhibiting rates of PPi hydrolysis and PPi-dependent H+ translocation similar to wild type, (ii) those exhibiting selective impairment of H+ translocation, and (iii) those exhibiting gross impairment of both PPi hydrolysis and PPi-dependent H+ translocation (Table I and Fig. 3).
|
In all class I mutants except one (D573N), PPi hydrolytic
activity and the rate and extent of
PPi-dependent H+ translocation were
diminished proportionately. Glu Gln substitutions at positions 119, 229, 667, and 751 generated enzyme with at least 15% of wild type
PPi hydrolytic and PPi-dependent
H+ pumping activity and coupling ratios, enumerated as
F%/µmol of PPi hydrolyzed, similar to wild
type (Type I). In the case of position 573, an Asp
Asn substitution
resulted in an approximately 1.8-fold increase in coupling ratio,
which, since the rate of PPi hydrolysis was unaffected, was
almost exclusively attributable to an increase in the rate of
PPi-dependent H+ translocation
(Table I and Fig. 3). When Glu229 was replaced with Asp,
rather than Gln, PPi hydrolytic activity and
PPi-dependent H+ translocation were
increased coordinately to values 1.4- and 1.7-fold greater,
respectively, than wild type (Table I and Fig. 3).
The one class II mutant obtained, E427Q, retained 50% of wild
type hydrolytic activity and less than 5% pumping activity and exhibited a coupling ratio of 12% of wild type (Table I). A
conservative Glu Asp substitution at this position restored wild
type PPi hydrolytic activity and generated enzyme with a
pump capacity 1.4-fold greater than wild type (Table I and Fig. 3).
The remaining two acid amide mutants, E305Q and D504N, fell into
class III. Both were completely deficient in
PPi-dependent H+ translocation and
mediated PPi hydrolysis at less than 10% of the wild type
rate (Table I and Fig. 3). In contrast to what was found with class I
and class II mutants, Glu
Asp and Asp
Glu substitutions at
positions 305 and 504, respectively, did not restore
PPi-dependent H+ translocation
(Table I and Fig. 3). While replacement of the E305Q substitution with
an E305D substitution increased PPi hydrolytic activity
from 5% to 26% of wild type, the equivalent substitution, to Glu
instead of Asn, at position 504 had the converse effect, decreasing
activity from 10% to less than 1% of wild type (Table I).
In none of the mutants was there an increase in
membrane H+ conductance of sufficient magnitude to account
for the effects of these substitutions. Vacuolar membrane-enriched
vesicles harboring any of the V-PPase mutants achieved similar rates
and extents of MgATP-dependent H+ translocation
by the endogenous, chromosomally coded V-ATPase associated with this
fraction as membranes containing wild type V-PPase (Fig.
4A), indicating that the
background conductance of the membrane to H+ was unaltered
by mutagenesis of the V-PPase. Moreover, the possibility of a substrate
(Mg2PPi) elicited increase in H+
conductance associated with the decoupled mutant, E427Q, was excluded
by the finding that the rate of MgATP-dependent
intravesicular acidification of membranes containing this form of the
enzyme was the same regardless of whether or not PPi was
added to the V-ATPase assay medium (Fig. 4B). Similarly, the
decreased PPi hydrolytic and/or H+ pumping
activities of some of the mutants were not explicable in terms of a
decrease in the amounts of intact membrane-associated V-PPase through
premature maturation, changes in expression level, or expression of
polypeptide with decreased stability. Vacuolar membrane-enriched
vesicles from cells expressing either wild type or mutated V-PPase
contained similar levels of PABHK1-reactive, Mr 66,000 (AVP-specific) polypeptide,
irrespective of the type or position of the substitution, and in none
of the membrane samples was there an indication of a change in the
electrophoretic mobility of the PABHK1-reactive band (Fig.
5).
Kinetics of Inhibition by DCCD
If DCCD inhibits the V-PPase
through its interaction with a carboxyl group located in a
transmembrane span, introduction of acid amide substitutions at
these positions in the heterologously expressed enzyme would be
predicted to confer decreased sensitivity to this reagent.
As a prelude to optimizing the conditions for reaction with DCCD, the
ligand requirements for inhibition were investigated. Of the ligands
tested, Mg2+ was the only one that influenced the
susceptibility of the wild type heterologously expressed enzyme to
inhibition by DCCD (Fig. 6). In contrast
to the requirements for protection of the V-PPase from inhibition by
maleimides (8, 10), free PPi and K+ did not
influence the inhibitory action of DCCD, and substrate, Mg2+ + PPi, did not confer any greater
protection than Mg2+ alone. The I50
values for inhibition by DCCD were 150, 130, 120, and greater than 500 µM for membranes incubated in the absence of ligands and
those incubated in the presence of 0.3 mM PPi, 50 mM K+, and 1.3 mM
Mg2+, respectively (Fig. 6).
The kinetics of inhibition of wild type V-PPase by DCCD were consistent
with a scheme in which the modification of two reactive sites on the
enzyme is necessary for inactivation and Mg2+ confers
protection by binding to a high affinity site. The time dependence of
inhibition by DCCD was described by the integrated second order rate
equation 1/A = 1/Ao + kt
such that a plot of the reciprocal of V-PPase activity (A)
at time t approximated a straight line of slope k
and ordinate intercept 1/Ao, where
Ao is activity at time zero and k is the
rate constant (Fig. 7A).
Mg2+ decreased the second order rate constant for
inactivation by DCCD as a hyperbolic function of Mg2+
concentration to yield an apparent affinity constant of 10-15 µM (Fig. 7B).
A screen of all eight acid amide mutants for
Mg2+-protectable inhibition by DCCD revealed that only two
(E305Q and D504N) were markedly less sensitive to DCCD (Fig.
8 and Table
II). While acid
amide substitutions
at positions 119, 229, 427, 573, 667, and 751 had little or no effect
on DCCD inhibitability versus wild type, the corresponding
substitutions at positions 305 and 504 diminished the inhibitability of
the residual activity by more than 3- and 4-fold, respectively (Fig.
8). In none of the mutants, with the exception of E427Q, which showed
an approximately 2-fold decrease, was Mg2+ protectability
affected. In agreement with a requirement for acidic residues at
positions 305 and 504 for inhibition by DCCD, replacement of the E305Q
and D504N substitutions with E305D and D504E substitutions,
respectively, restored wild type DCCD inhibitability (Fig. 8 and Table
II).
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Both of the predictive methods applied to the substrate-binding subunit of the V-PPase, TopPred II and MEMSAT, yielded a tentative model containing two transmembrane spans in addition to the 13 proposed previously (9) and an opposed orientation for the N and C termini (luminal N terminus; cytosolic C terminus). Although tests of the validity of the new model will depend eventually on the results of direct, independent, and complementary structural studies, we consider it a significant improvement over those proposed previously. The outcomes from both programs were remarkably similar for all four of the sequences analyzed, with only minor differences in the lengths and locations of a few of the transmembrane spans. The results of both were compatible with the available, albeit limited, biochemical data on the localization of Cys634 (8, 10) and the characteristics of AVP-apoaequorin fusions (26). By contrast, the 12-span model of the V-PPase proposed by Tanaka et al. (12), on the basis of hydropathy analyses of the deduced sequence of HVP alone, places Cys634 on the wrong side (on the luminal face) of the membrane and contains a highly improbable transmembrane span (span VI in their model) incorporating 30-40 amino acid residues.
It was by inspection of the revised topological model of the V-PPase that the eight acidic residues located within or near transmembrane spans were identified as targets for substitution. Although six other acidic residues with a similar disposition were evident from the model (Fig. 2), these were either not conserved in all four of the sequences analyzed (Glu13, Glu298, Asp324) or located in relatively hydrophilic environments (Glu225, Asp351, Glu645) and considered less likely to participate in transmembrane H+ translocation and/or DCCD binding. Three main conclusions, discussed below, derive from the results of substituting these acidic residues.
Glu229 Is Not Essential for PPi Hydrolysis or H+ TranslocationE229Q mutated V-PPase shows some impairment of PPi hydrolysis and H+ translocation but, since both processes are diminished in parallel, the diminution of coupling ratio is small. Moreover, in direct opposition to the imputed role of Glu229 in inhibition by DCCD, E229Q-substituted V-PPase is no less sensitive to inhibition by DCCD than wild type or E229D mutated enzyme, indicating that an acidic residue at this position is not required for inhibition by this reagent. On this basis, and in contrast to the speculations in Ref. 1, it is unlikely that the alignments between the V-PPase sequences C-terminal to Glu229 and the second DCCD-reactive transmembrane span of the c-peptides of F-ATPases (Fig. 1) signifies a functional equivalence. Similarly, Asp573 and Glu residues 119, 667, and 751 do not appear to be critical for PPi hydrolysis, PPi-dependent H+ translocation, or inhibition by DCCD. Substitution of these residues by their corresponding amides exerts little or no effect on PPi hydrolytic activity, H+ pumping, coupling ratio, or DCCD inhibitability.
Glu305 and Asp504 Are Critical for Catalysis and Contribute to DCCD BindingE305Q and D505N mutants
exhibit less than 10% wild type PPi hydrolytic activity,
no detectable PPi-dependent H+
translocation, and residual activities markedly less sensitive to
inhibition by DCCD than that of wild type enzyme. These
characteristics, together with the recovery of DCCD inhibitability
shown by E305D and D504E mutants, are consistent with the involvement
of acidic residues at these positions in inhibition by DCCD. It is
unlikely, however, that Glu305 and Asp504 are
the sole residues involved in inactivation of the V-PPase by this
carbodiimide. Doubly mutated enzyme containing acid amide
substitutions at both of these positions is no less sensitive than
either single mutant to DCCD, implying the participation of residues
other than Glu305 and Asp504. The finding that
structurally conservative Asp
Glu or Glu
Asp substitutions
cause a decrease in hydrolytic activity in the case of
Glu504 and only a minor increase in activity in the case of
Asp305 suggests that the steric constraints for catalysis
are more stringent than for DCCD binding. Evidently, a difference of
one methyl group in the carboxyl side chain is sufficient to severely
impair catalytic function while leaving DCCD inhibitability
unaffected.
Mg2+ appears to protect the V-PPase from inhibition by DCCD by interacting with a high affinity binding site. In agreement with the results from earlier studies, the kinetics of protection against DCCD inhibition are consistent with an affinity constant for Mg2+ of 10-15 µM. The steady state kinetics of substrate hydrolysis by the V-PPase approximate a scheme in which the binding of two Mg2+ ions, in addition to those associated with the substrate, dimagnesium pyrophosphate (Mg2PPi), is required for activity: a high affinity binding site with a binding constant of about 25 µM and a lower affinity site with a binding constant of 0.25-0.46 mM (30, 31). Protection of the V-PPase from inhibition by the water-soluble carboxyl-selective reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), exhibits an Mg2+ concentration dependence consistent with a Km for free Mg2+ of 20-23 µM (32). However, despite their kinetic equivalence, it is improbable that these results denote a direct interaction between Mg2+ and the residues or domains that undergo covalent modification in all cases. Although the notion of direct protection is consistent with the finding that high affinity Mg2+ protection of the V-PPase not only applies to the carboxyl reagents, DCCD and EDAC, but also NEM, a sulfhydryl reagent, and phenylglyoxal, a reagent that preferentially reacts with guanidino side chains (31, 32) and the fact that one cytosolic loop, that between transmembrane spans XIII and XIV, containing the Cys residue (Cys634) responsible for substrate-protectable inhibition of the V-PPase by NEM (8, 10), also contains Arg, Asp, and Glu residues, it does not reconcile two critical findings. It fails to explain why inhibition by DCCD is second order while EDAC, NEM, and phenylglyoxal inhibit the enzyme with first order kinetics (32)3; it cannot account for the diminution of Mg2+-protectable DCCD inhibitability consequent on substituting Glu305 and Asp504, neither of which are located in this hydrophilic loop, with their corresponding amides. We therefore propose that Mg2+ exerts its high affinity effects indirectly through conformational coupling rather than through direct screening of the residues that would otherwise be covalently modified. By implication, Glu305 and Asp504, although contributing to DCCD binding, may not themselves bind Mg2+.
Another explanation compatible with the effects of mutagenesis on
Mg2+-protectable inhibition of the V-PPase by DCCD is that
neither Glu305 nor Asp504 directly participate
in DCCD binding or that either Glu305 or Asp504
does but not the other. For instance, by neutralizing the - or
-carboxyl groups on the side chains of these residues, acid
amide substitutions at one or both of these positions may simulate the
electrostatic screening action of Mg2+ binding and thereby
diminish the sensitivity of the V-PPase to inhibition by DCCD in the
absence of Mg2+, while at the same time impairing overall
catalytic activity.
E427Q
mutants, although still active in PPi hydrolysis, mediate
H+ translocation at less than 6% of the wild type rate to
yield an 8-9-fold diminished coupling ratio. While it may be premature to conclude that these results demonstrate a direct role for
Glu427 in H+ transfer, since its substitution
by Gln might cause a structural change that indirectly effects enzyme
function, the large recovery of wild type H+ pumping
versus the modest increase in PPi hydrolytic
activity by a Glu Asp substitution nonetheless implies an important
role for an acidic residue at this position for H+
translocation per se. Whereas the capacity for
PPi hydrolysis is increased by only 2-fold when the E427Q
substitution is replaced with an E427D substitution, the rate of
H+ translocation is increased by more than 20-fold.
If Glu427 does indeed directly participate in H+ transfer, two corollaries follow. First, since E427Q- or E427D-mutated V-PPase is as sensitive to DCCD as wild type enzyme, inhibition by DCCD does not have a direct bearing on H+ translocation. By analogy with the results of protein chemical studies of F- and V-ATPases, the DCCD inhibitability of the V-PPase has been interpreted in terms of the participation of a DCCD-reactive residue in transmembrane H+-conduction (11). From the results presented here and the known reaction specificity of DCCD, such a conclusion is not warranted. Given that stable incorporation of dicyclohexylisourea and irreversible modification of a protein is contingent on the exclusion of water or other nucleophiles from the site of reaction (33), inhibition by DCCD simply indicates that catalytic activity is directly or indirectly dependent on carboxyl functions sequestered from bulk phase water; it does not automatically imply that the residue modified participates in H+ translocation. By the same token, lack of interaction of an acidic residue with DCCD, as is the case for Glu427, does not exempt it from involvement in H+ translocation.
Second, according to the revised topological model of the V-PPase,
Glu427 has a cytosolic disposition (Fig. 2). If this
residue is involved in H+ translocation, it seems likely
that it forms part of an input channel responsible for the entry of
H+ at the cytosolic face of the membrane. It is therefore
conceivable that the E427Q mutation acts to neutralize the -carboxyl
group that would otherwise be present at this position, thereby
blocking protonation of the other H+-carrying residues of
the pump. Providing that the other H+-carrying residues in
the transmembrane relay can switch into their output configuration at
sufficient velocity, possibly as a result of a local increase in
H+ activity due to a block in H+ transfer from
this site in E427Q mutants, the pump will mediate futile cycling
(become decoupled) and the coupling ratio will fall.
Similar decoupled mutants have been reported for bacteriorhodopsin (34), E. coli cytochrome-bo ubiquinol oxidase (35) and Rhodobacter sphaeroides cytochrome-c oxidase (36). In bacteriorhodopsin, four membrane-embedded Asp residues are essential for H+ translocation (37) and substitution of one of these has been shown to abolish H+ translocation while leaving the photocycle unaffected (34). In subunit I of E. coli cytochrome-bo ubiquinol oxidase, mutation of an Asp residue residing in a hydrophilic domain decouples H+ translocation from electron transfer (35). In R. sphaeroides cytochrome-c oxidase, mutation of an Asp residue at a position equivalent to that of cytochrome bo has a similar decoupling action (36).
The results reported here provide an indication of the identity of one potential starting point for PPi-driven H+ movement across the phospholipid bilayer by the V-PPase and dispel previous misconceptions of the mode of action of DCCD in this system, but many basic questions remain. It has yet to be determined if Glu305 and/or Asp504, because of their deeper insertion into the membrane, act at a point in the H+ relay downstream of Glu427 such that pump mutated at these positions, unlike pump mutated at position 427, is arrested in its output configuration, so stalling both H+ translocation and PPi hydrolysis. It is not known if H+-dissociable side chains other than those on acidic residues also participate in H+ translocation.
We thank Dr. Elizabeth Jones (Carnegie Mellon University, Pittsburgh, PA) for the kind gift of S. cerevisiae strain BJ5459 and Dr. Yolanda Drozdowicz for critically reading this manuscript.