Molecular Cloning and Characterization of a Soluble lnorganic

Plant Physioi. (1 995) 109: 853-860

Molecular Cloning and Characterization of a Soluble lnorganic Pyrophosphatase in Potato’

Patrick du Jardin*, Jorge Rojas-Beltran, Christiane Cebhardt, and Robert Brasseur

Department of Plant Biology (P.d.J., J.R.43.) and Center of Numerical Molecular Biophysics (R.B.), Faculty of Agricultura1 Sciences of Gembloux, B-5030 Gembloux, Belgium; and Max-Planck-lnstitute for Plant Breeding

Research, W-50829 Koln, Germany (C.G.)

A cDNA clone encoding a soluble inorganic pyrophosphatase (EC 3.6.1 .l) of potato (Solanum tuberosum 1.) was isolated by screening a developing tuber library with a heterologous probe. The central domain of the encoded polypeptide i s nearly identical at the sequence leve1 with i t s Arabidopsis homolog (J.J. Kieber and E.R. Signer [1991] Plant MOI Biol 16: 345-348). Computer-assisted analysis of the potato, Arabidopsis, and Escherichia col i soluble pyrophosphatases indicated a remarkably conserved organization of the hydrophobic protein domains. The enzymatic function of the potato protein could be deduced from the presence of amino acid residues highly conserved in soluble pyrophosphatases and was confirmed by i ts capacity to complement a thermosensitive pyrophosphatase mutation in E. coli. The potato polypeptide was purified from complemented bacterial cells and i ts pyrophosphatase activity was shown to be strictly dependent on Mg2+ and strongly inhibited by Ca2+. The subcellular location of the potato pyrophosphatase is unknown. Structure analysis of the N-terminal protein domain failed to recognize typical transit peptides and the calculated molecular m a s of the polypeptide (24 kD) i s significantly inferior to the values reported for the plastidic (alkaline) or mitochondrial pyrophosphatases in plants (28-42 kD). Two unlinked loci could be mapped by restriction fragment length polymorphism analysis in the potato genome using the full-length cDNA as probe.

Inorganic pyrophosphatases (EC 3.6.1.1) are ubiquitous enzymes catalyzing the hydrolysis of PPi into two Pi’s. PPi hydrolysis is highly exergonic (AG” = -33.5 kJ mol-l) and provides a thermodynamic driving force to a range of anabolic processes. Most notably, the synthesis of biologi- cal polymers produces PPi either during the activation of monomers (e.g. ADP-Glc synthesis prior to starch elongation, amino acid activation into amino acyl-tRNA prior to polypeptide elongation) or during the elongation of the polymers (eg. DNA and RNA synthesis, polyisoprene [rubber] synthesis). According to the Kornberg (1962) model, the energy “wasted“ by PPi hydrolysis is needed for making these processes thermodynamically irrevers- ible. Hence, the first and most ubiquitous function of inorganic pyrophosphatases is presumably to drive anabolism.

* This work was supported by the Communauté française de Belgique (Action de Recherche Concertée contract No. 90/ 94-143) and by the European Community (Biotech Project of Technological Priority B102 CT930400, contract No. PL920150).

* Corresponding author; e-mail dujardin8fsagx.ac.be; fax 32- 81-600727.

853

In plants, inorganic pyrophosphatases also participate in the assimilation of mineral nutrients. In particular, PPi hydrolysis is coupled to sulfate activation into adenosine phosphosulfate, hence essential for sulfur metabolism (Schmidt and Jager, 1992).

Remova1 of PPi can also be achieved by conserving the free energy of the phosphoanhydride bond of PPi either in phosphorylated metabolites or as a transmembrane proton gradient. The occurrence of a tonoplastic proton-translocating inorganic pyrophosphatase is remarkable in that re- spect (Rea and Poole, 1993). In the cytosol, two soluble enzymes (UDP-Glc pyrophosphorylase and the PPi-dependent phosphofructokinase) use PPi as a substrate in energy-conserving reactions. Plant tissues are remarkably rich in PPi (Edwards et al., 1984; Smyth and Black, 1984; Weiner et al., 1987; Dancer and ap Rees, 1989; Takeshige and Tazawa, 1989), which is essentially limited to the cytosolic compartment (Weiner et al., 1987; Takeshige and Tazawa, 1989). The physiological importance of the cytosolic PPi pool is substantiated by the observation that expression of a soluble inorganic pyrophosphatase of bacterial origin in the cytosol of transgenic tobacco and potato (Solanum tuberosum L.) plants leads to significant alterations in metabolism, growth, and development (Jelitto et al., 1992; Son- newald, 1992; Lerchl et al., 1995).

Thus far, little information is available concerning the control of PPi levels in the different plant cell compart- ments and most notably the diversity and regulation of soluble inorganic pyrophosphatases. Soluble alkaline pyrophosphatases have been characterized in plant extracts and some of them have been purified (Naganna et al., 1954; Gould and Winget, 1973; Klemme and Jacobi, 1974; Popli and Singh, 1977; Kumar and Singh, 1983; Mukherjee and Pal, 1983; Ho and Khoo, 1985; Ananda Krishnan and Gna- nam, 1988; Gama Branda0 and Aoyama, 1992; Mortain- Bertrand et al., 1992). Based on the subcellular fractionation studies carried out by Gross and ap Rees (1986) and by Weiner et al. (1987), it is likely that most of these soluble alkaline activities correspond to plastidic isoforms. One notable exception is the soluble pyrophosphatase purified from the latex of Hevea brasiliensis (Jacob et al., 1989), a

Abbreviations: GST, glutathione S-transferase; HCA, hydrophobic cluster analysis; IPTG, isopropylthio-/3-galactoside; LB, Luria broth; RFLP, restriction fragment length polymorphism.

854 du Jardin et al. Plant Physiol. Vol. 109, 1995

cytosolic protein participating in rubber synthesis in this very particular cellular environment.

At the gene level, a cDNA and a partia1 genomic sequence in Arabidopsis thaliana have been briefly described (Kieber and Signer, 1991). The pyrophosphatase function of the cloned cDNA was inferred from sequence analysis, and no information was provided regarding the subcellular location of the encoded polypeptide. In the present paper, we describe the cDNA cloning and sequence analysis of a potato soluble pyrophosphatase homologous to the Arabi- dopsis gene product, provide experimental evidence of its pyrophosphatase function, and map two corresponding loci in the potato genome.

MATERIALS A N D METHODS

Plant Material

The potato (Solanum tuberosum L.) cv Désirée was used throughout this work. Plants were grown in a greenhouse under natural light supplemented with fluorescent light.

D N A Techniques and RFLP Mapping

Screening of the developing tuber cDNA library (du Jardin and Berhin, 1991) and dideoxynucleotide sequenc- ing (with T7 DNA polymerase) were performed using standard procedures (Sambrook et al., 1989). Potato DNA ex- traction, restriction digestion, electrophoresis, blotting, and hybridization with 3ZP-labeled probes were performed as previously described (du Jardin, 1990, for the standard Southern blot analysis; Gebhardt et al., 1989, for the RFLP- mapping work).

Expression in bcherichia coli

For expression in E. coli, the potato cDNA was inserted as a BamHI-EcoRI fragment (nucleotides 147-948) into the pGEX3X vector (Pharmacia), resulting in the IPTG-driven expression of a fusion polypeptide comprising residues 21 to 211 of the cDNA product and the vector-encoded GST. For the genetic complementation assay, the pGEX plasmid derivative (named pGEX::ppase) was introduced by standard CaCI, transformation into the E. coli strain K37EKTR(pE), encoding a temperature-sensitive cytoplasmic pyrophosphatase (Chen et al., 1990). Maintenance of the thermosensitive replicon harboring the E. coli pyrophosphatase gene and a chloramphenicol resistance gene is achieved by cultivation of the strain at 30°C on LB plus chloramphenicol (25 mg L-') medium. Loss of the endogenous pyrophosphatase activity was achieved by cultivat- ing the cells of a single colony overnight at 37°C in 2 mL of LB medium. Plating an aliquot of the overnight culture on LB medium failed to develop any colony, indicating the loss of the pyrophosphatase activity. For fusion protein induction, 10 pL of 0.1 M IPTG were added to the agar plates 30 min before inoculation. In liquid cultures, IPTG concentrations between 20 and 100 p~ were used.

Purification of the fusion protein from sonicated bacte- ria1 extracts was achieved by affinity with glutathione- agarose beads (Pharmacia) according to the batch pro-

cedure recommended by the supplier. The fusion polypeptide was cleaved on the agarose beads by the Xa factor (Boehringer) in 18 h at 4°C under the recommended conditions.

Assay of the Mg*+-Dependent Soluble lnorganic Pyrophosphatase Activity

For assaying the pyrophosphatase activity ol' the polypeptide purified from E. coli, the protein fraction released from the glutathione-agarose beads by Xa factor digestion was passed through a Sephadex G-25 column (Pharmacia) and eluted with 50 mM 1,3-bis[tris(hydroxy- methy1)-methylaminolpropane. Enzyme activity (100 ng of protein in 50 pL) was assayed at room temperature for 15 min in 200 p L of the reaction buffer (50 mM 1,3-bis- [tris(hydroxymethyl)-methylaminolpropane, pH 7.0, 1 mM Na,H,P,07, O or 2.5 mM MgCl,). pH 7.0 corresponds to the optimal value for the potato polypeptide. The reactiori was stopped with 750 pL of staining buffer (3.4 mM ammonium molybdate in 0.5 M sulfuric acid, 0.5 M SDS, 0.6 M ascorbic acid, 6:2:1, v / v / v ) as described by Burchell et al. (1988). After 20 min, the A,,, was measured. The standard curve was constructed using known quantities of Pi from a KH,PO, solution replacing the protein extract.

Computer Analysis of the Sequence Data

Primary sequence alignment was performed by the PILEUP program of the Genetics Computer Group soft- ware package (version 8.0; Madison, WI) using the default parameters (GapWeight, 3.000; GapLengthWeight, 0.100), available through the BEN (Belgian EMBL Node) network. Hydrophobic cluster analysis (HCA) was performed using the programs of the Center of Numerical Molecular Bio- physics of the University of Gembloux. A review of the method was presented by Lemesle-Varloot et al. (1990).

RESULTS

cDNA Cloning and Sequence Analysis of a Soluble lnorganic Pyrophosphatase in Potato

A hgtll cDNA library constructed from developing tuber poly(A)' RNA (du Jardin and Berhin, 1991) was screened with a cloned probe corresponding to the pro tein- coding region of the published sequence in A. thdiana (Kieber and Signer, 1991). Two positive phage plaques were isolated out of 200,000. The longest cDNA (0.95 kb) was subcloned in pBluescript and sequenced (Fig. 1). The correspondence between the size of the cDNA and that of the corresponding mRNA (between 1.0 and 1.1 kb, as estimated by northern blot hybridization) indicates that the cDNA is full length or nearly full length, considering that only a few residues of the poly(A) tail are present in the cDNA. The environment of the first ATG (position 79) satisfies the rules for the upstream residues of the ATG translation start (A residues at positions -1, -3, -4, -5), but the downstream triplet AGC (at +4, +5, +6) is less common (Joshi, 1987). The presence of 24 contiguous A residues at the 5' extremity of the cDNA is worth mention-

Soluble lnorganic Pyrophosphatase in Potato 855

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1 M S N E N D D L S P 61 ttaccagaaattgtaaaa ATG AGC AAT GAA AAT GAT GAT TTG TCT CCA

11 109

26 154

41 199

56 244

71 289

06 334

Q R R A P R L N E R I L S S I CAA AGA CGT GCC CCT CGT TTG AAT GAG AGG ATC CTA TCA TCT ATA

S R R S V A A H P W H D L E I TCC AGG AGG TCT GTT GCT GCT CAT CCT TGG CAC GAC CTT GAG ATA

G P E A P S V F N V V I E I S GGA CCT GAA GCT CCA AGT GTT TTC AAT GTT GTC ATT GAG ATT TCA

K G S K V K Y E L D K K T G L AAA GGA AGC AAA GTC AAA TAT GAG CTG GAC AAG AAA ACC GGT CTT

I K V D R I L Y S S V V Y P Q ATT AAG GTT GAT CGC ATC CTA TAC TCT TCA GTG GTT TAC CCT CAA

N Y G F I P R T L C E D N D P AAC TAT GGC TTC ATT CCC CGA ACA CTC TGT GAA GAT AAT GAC CCA

101 M D V L V L M Q E P V L P G C 379 ATG GAT GTA TTA GTC CTC ATG CAG GAA CCT GTC CTT CCA GGT TGT

116 F L R A R A I G L M P M I D Q 424 TTC CTT CGA GCT AGG GCA ATA GGT CTG ATG CCT ATG ATT GAT CAG

131 G E K D D K I I A V C A D D P 469 GGA GAG AAA GAT GAC AAG ATC ATA GCA GTG TGT GCT GAT GAT CCA

146 E Y R H Y T D I K Q L P P H R 514 GAA TAT CGC CAC TAC ACT GAT ATA AAG CAG CTC CCC CCT CAC CGC

161 L A E I R R F F E D Y K K N E 559 CTG GCT GAA ATT CGC CGC TTT TTT GAA GAC TAC AAG AAG AAT GAA

176 N K D V A V D D F L P P N S A 604 AAC AAA GAC GTT GCT GTT GAC GAT TTC CTG CCT CCA AAT TCT GCT

191 V N A I Q Y S M D L Y A E Y I 649 GTC AAT GCC ATT CAG TAC TCC ATG GAT CTG TAT GCT GAA TAC ATA

206 L H S L R K 694 TTA CAC AGC TTG AGG AAG taaggtacaatgggacacatataaaacagaatat=ta

748 taggtacaatgggaaatgaagactgtcacaagtc~c~=gtt~tctt~=tg~tttg~~at=~tatttt

008 t t t t t g t a c t g t t a t t t t c t c t t t g t t t g a a t t t t t g t g t t ~ t a t g a t g t t a t g a c t t a g

868 C a a g g g t t t a t c c c t t g C t g c t c t a a a t t t g t a c t g t t t t t a a t t a t a t t g ~ c ~ t t c a c t a

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Figure 1. Nucleotide and deduced amino acid sequences of the potato cDNA isolated from a developing tuber library.

ing, a feature also found in the leader sequence of the A. thaliana gene (with a few A to G substitutions, Kieber and Signer, 1991). This unusual conserved motif could have some regulatory significance. The polypeptide deduced from the potato cDNA (211 amino acid residues) has a calculated molecular mass of 24.26 kD. The sequence identity between the Arabidopsis 1 potato pyrophosphatases and the other published pyrophosphatases (from both pro- karyotes and eukaryotes) is below 40%, and the plant enzymes are more similar to the E. coli cytoplasmic pyrophosphatase than to any other homolog (for review and sequence alignments, see Cooperman et al., 1992).

The plant tonoplast proton-pumping pyrophosphatases form a very different group of enzymes (Rea et al., 1992). Figure 2A aligns the sequences of the potato, Arabidopsis, and E . coli pyrophosphatases. A central domain (between residues 40 and 201 of the potato polypeptide) shows a high sequence identity with the Arabidopsis polypeptide and a significant sequence similarity with the E. coli pyrophosphatase when conservative substitutions are included. The two plant proteins possess both N-terminal and C- terminal extensions compared with the bacterial protein, with little (N-terminal extremity) or no (C-terminal extremity) sequence similarity with one another. Alignment of eight eukaryotic and prokaryotic soluble pyrophosphatases has identified 24 conserved residues, 15 of which are part of a group of 17 putative active site residues, according to x-ray crystallographic studies in yeast (Cooperman et al., 1992). These residues, thus constituting some kind of

"fingerprint" of soluble inorganic pyrophosphatases, are scattered over the conserved domain of the plant proteins (in potato, the first conserved residue occupies position 53 and the last one occupies position 177).

Further comparison of the three proteins was achieved with the HCA algorithm, which examines the distribution of the globular protein domains along a peptide sequence. This algorithm combines homology detection with second- ary structure analysis (see Lemesle-Varloot et al., 1990, for a detailed description) and is an efficient way for extracting structural and functional information from primary sequences and for the identification of conserved structural elements between distantly related proteins. Figure 2B indicates a common organization of the hydrophobic domains in the potato, Arabidopsis, and E . coli pyrophosphatases. Identical anchor points (homologous hydrophobic clusters and 1 or identical motifs) are visualized and might indicate a common folding pattern for the core of the three proteins. For example, interna1 p-strands sta- tistically occur as short and vertical clusters of hydrophobic residues (circled residues on the HCA plot) and such a motif is found around positions 105, 162, and 72 in the potato, Arabidopsis, and E . coli proteins, respectively. This cluster is probably part of the hydrophobic core of the proteins. Three contiguous short a-helices are also evident, close to the C-terminal extremity of the proteins, visualized as sloping hydrophobic segments in the HCA plots. Based on this algorithm, no particular two-dimensional structure can be assigned to the N- and C-terminal extensions of the plant proteins. Overall, the regular distribution of hydrophobic clusters separated by short hydrophilic areas is characteristic of a classical globular fold. The HCA plot also fails to identify any membrane-spanning helix, typically denoted by long (approximately 20 residues) and horizon- tal hydrophobic clusters. Based on the sequence homology with soluble pyrophosphatases and on the two-dimensional information, the two plant proteins can be reason- ably considered as soluble pyrophosphatases, well apart from the proton-pumping pyrophosphatases inserted in the vacuolar membrane.

Compared with E. coli, the potato and Arabidopsis proteins contain N-terminal extensions of 33 and 40 residues, respectively, with a limited sequence similarity in their C-terminal domains and, as already mentioned, with no remarkable two-dimensional structure. A possible function of N-terminal sequences is the targeting of the polypep- tides to organelles, a question of special significance in the present case, since it is assumed that plant soluble pyrophosphatases are essentially limited to the plastidic compartment. This question can be discussed on the basis of the structural features of the chloroplast transit peptides defined by von Heijne et al. (1989). In the potato sequence, assuming that the mature protein starts at residue 33, because that is where the homology with E. coli begins, a fairly good cleavage site can be found: the segment VAAH corresponds well with the loosely defined consensus V / I- X-A/C i A, and an R residue occupies position -6. More- over, the N-terminal extension is enriched in Ser residues compared with the putative mature protein (19 versus


Figure 2. Comparison of the one- and two-dimensional structures of the soluble inorganic pyrophosphatases from potato (ppast, this work), A. thaliana (ppara, Kieber and Signer, 1991 ), and E. coli (ppacoli, Lahti et al., 1988). A, Align- ment of the three amino acid sequences using the PILEUP program from Genetics Computer Group. The conserved residues, including conservative substitutions, are boxed. Dots indicate t h e 24 amino acid residues highly conserved between all soluble pyrophosphatases. Filled dots correspond to putative active site residues, and open dots correspond to residues with no proposed function (Cooperman et al., 1992). B, HCA plot of the three pyrophosphatases. The basics of the representation are the following (Lemesle-Varloot et al., 1990): the sequences are written on a classical a-helix (3.6 amino acids per t u r n ) smoothed on a cylinder. The cylinder is then cut parallel to its axis and un- folded. Because some adjacent amino acids are separated by the unfolding, the representation is duplicated to restore full connectivity of each residue. Such a two-dimensional plot makes short- and medium-distance interactions readily detectable, since, for a given residue, the first and second neighbors are located in a 17-residue seament. Hvdrophobic residues are circled,

A .IPPdrel M A E I K D E G S A K G Y A F P 1 R N P N V T L N E R N F A A F T H R S A A A H P W H D L E I G P E

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1 7 5

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IPParai ipposti - . - - - - - . IppBcOlll . . - - . - - - - .

K I S I G A F N F V M L I R K H C

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c$$R$sp. A%! allowing the identification of hydrophobic clusters possibly involved in the three-dimensional ppast 0.e-R

fold of the protein. Two-dimensional structure @p$; N;.:a elements tend to have characteristic signatures on such plots. Vertical lines delimit structurally homologous domains, and some typical p- strands (p) and a-helices (a) are positioned under the three plots. Pro/Gly residues are indicated by symbols */#, respectively, considering their specific conformational characteristics.

ppacoli

4.5%) and the 9 N-terminal residues lack both positively charged and Gly/Pro residues. However, despite a net positive charge, the content in acidic residues is not lowered in the putative presequence compared with the mature protein and the last 10 residues of the presequence have no potential to form an amphiphilic p-strand, according to the HCA plot. Based on these observations, it is thus difficult to draw firm conclusions regarding the capacity of the N-terminal extension to target the protein to plastids and, after all, von Heijne's rules are not without exceptions (Dreses-Werringloer et al., 1991; Willey et al., 1991).

Targeting the protein to the mitochondrion has also to be envisaged, since mitochondrial pyrophosphatases have been described in mammalians, in yeasts (for refs., see Cooperman et al., 1992), and more recently in plants (Vi- anello et al., 1991; Zancani et al., 1995). The net positive charge of the potato N-terminal extension and the enrich- ment in Ser residues are properties shared by mitochondrial transit peptides (von Heijne et al., 1989), but the N-terminal domain of the putative presequence does not

a a a (L P

show amphiphilic a-helical properties and the content in acidic residues is not lowered compared with the mature protein, in opposition to the rules proposed by von Heijne et al. (1989). Again, an increasing number of exceptions to these rules have been described (Braun and Schmitz, 1995, and refs. therein) and no firm conclusion can be inferred from this one- and two-dimensional sequence analysis.

Cenetic Complementation of an E. coli Pyrophosphatase- Deficient Mutant

Further evidence that the potato cDNA codes for an inorganic pyrophosphatase was obtained by the genetic complementation of an E. coli strain with a thermosensitive cytoplasmic pyrophosphatase. The mutant mmed K37EKTR(pE) is disrupted in its chromosomal ppa gene encoding the cytoplasmic inorganic pyrophosphatase and contains a wild-type copy of the gene on a thermosensitive replicon (Chen et al., 1990). At 37"C, replication od the plasmid is inhibited, the active ppa gene is lost, and growth

Soluble Inorganic Pyrophosphatase in Potato 857

stops. The potato cDNA was expressed in the mutant fromthe expression vector pGEX-3X (Pharmacia), after fusingthe cDNA in frame with the GST-coding sequence. Expres-sion is controlled by the IPTG-inducible tac promoter. Boththe parental pGEX vector and the recombinant plasmid(pGEX::ppase) were introduced separately in the mutantstrain. Transformed cells were plated either at 30°C on LBplus ampicillin plus IPTG (permissive conditions) or on thesame medium at 37°C after overnight incubation at thesame temperature for eliminating the thermosensitive rep-licon (restrictive conditions). Figure 3A shows that plasmid

B.5

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1

.5

0

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-i

f

i-

- ——

10 2 10 50 100

IPTG (pM)

DK37

• K37EKTR(pE)

• K37EKTR(pE,pGEX::ppase)

Figure 3. Genetic complementation of the thermosensitive E. col!strain K37EKTR(pE), deficient in inorganic pyrophosphatase, by thepotato cDNA. A, The strain was transformed either by the parentalvector (pGEX) or by the recombinant plasmid (pGEX::PPase), andtransformed cells were incubated either in permissive (30°C) or inrestrictive (37°C) conditions. B, IPTG dependence of the comple-mented phenotype. The wild-type strain (K37), the untransformedmutant strain |KE7EKTR(pE)l, and the mutant strain transformed bypGEX::ppase [KE7EKTR(pE, pGEX::ppase)] were cultivated in restric-tive conditions with different IPTG concentrations. A's of the liquidcultures were measured when K37 reached saturation.

Table I. Inorganic pyrophosphatase activity of the cDNA productpurified from E. coli

The assay was performed at room temperature and at pH 7.0(optimum pH of the pyrophosphatase as expressed in E. coli). Meansare of replicate samples.

Assay Conditions Inorganic PyrophosphataseActivity

0 mM Mg2 +

2.5 mM Mg2 +

2.5 mM Mg2+ + 0.05 mM Ca24

2.5 mM Mg2+ + 0.5 mM Ca2 +

nmol PPi hydrolyzedmg~ ' protein 75 min~ '

n.d."683320n.d.°

•' n.d., No detectable activity.

pGEX::ppase allowed the transformed cells to grow inrestrictive conditions, while the pGEX vector was ineffi-cient in complementing the mutation. Complementationwas controlled by IPTG, with an optimal concentrationbetween 2 and 10 /XM in liquid culture (Fig. 3B), and wascorrelated with the expression of a polypeptide with anelectrophoretic mobility expected for the chimeric pyro-phosphatase (not shown).

As a conclusion, the potato cDNA provides the mutantwith the capacity to remove PPi at 37°C, which is essentialfor growth. However, this complemented phenotype is notnecessarily due to an inorganic pyrophosphatase but couldbe conferred by any PPi-consuming enzyme encoded bythe cDNA. It was thus decided to purify the potatopolypeptide from complemented bacteria and to assay itsinorganic pyrophosphatase activity. The fusion proteinwas purified from sonicated bacterial extracts by affinitywith glutathione-agarose beads. The potato polypeptidewas then separated from the carrier protein (GST) by Xafactor digestion. Finally, the potato polypeptide is deletedfrom its 20 N-terminal residues and contains an N-terminalGly residue from GST. The inorganic pyrophosphatase ac-tivity was assayed by measuring the Pi released from PPi,in either the presence or the absence of Mg2+. The datapresented in Table I indicate that the potato cDNA actuallycodes for an inorganic pyrophosphatase, with a strict re-quirement for Mg2+, and that the enzyme is strongly in-hibited by Ca2+, which are typical features of soluble in-organic pyrophosphatases. Incidentally, since the proteinlacks the 20 N-terminal residues, this experiment also in-dicates that this nonconserved domain is dispensable forthe catalytic activity.

At Least Two Separate Genes Code for SolublePyrophosphatases in Potato

Nuclear DNA was isolated from the tetraploid cv Desireeand analyzed by Southern blot with either the full-lengthcDNA or a 3' probe (NcoI-EcoRI fragment corresponding tonucleotides 669-948). This 3' probe covers the 3' untrans-lated region of the cDNA and the last 15 codons of the openreading frame, corresponding to the nonconserved C-ter-minal domain. Both probes identified multiple bandsabove 4 kb (Fig. 4) that might indicate the existence of


B

(kb)

23.13 -

9.42 .

6.56 -

4.36 -

H H

2.322.03

1.35 -

Figure 4. Southern blot experiment using either the full-length cDNA(A) or a 3'-specific probe (B) hybridized with genomic DMA from 5.tuberosum cv Desiree. H, H/ndlll; R, fcoRI.

several copies of the cloned gene or close relatives inpotato. Considering the alternative possibility of multiplealleles for a single locus in the tetraploid background of cvDesiree, a complementary RFLP-mapping analysis wasperformed as previously described (Gebhardt et al., 1989),using the full-length cDNA as probe. The segregating RFLPfragments identified two loci on chromosomes VIII and XII(Fig. 5). Based on the observation of additional homomor-phic fragments, the existence of a third locus cannot beruled out. We propose the terms Ppal(a) and Ppal(b) for thetwo mapped loci, in accordance with the nomenclature ofthe published potato map (Gebhardt et al., 1989) and withthe recommendations of the Commission on Plant GeneNomenclature (1994). The existence of at least two genesper chromosome set in potato contrasts with the conclu-sions of Kieber and Signer (1991) pointing to a single-genecopy in Arabidopsis.

DISCUSSION

In this work, a soluble inorganic pyrophosphatase ex-pressed in developing potato tubers was cloned and char-acterized at the sequence level. Experimental evidence thatthe encoded polypeptide is an Mg2+-dependent inorganicpyrophosphatase was obtained after expression in E. coli.The potato protein is closely related to its Arabidopsishomolog at the sequence level, and comparison of theorganization of the globular protein domains in the potato,Arabidopsis, and E. coli pyrophosphatases (by the HCAalgorithm) revealed a remarkably conserved pattern, pre-sumably indicative of a common three-dimensional fold ofthe core of the proteins. The potato enzyme clearly belongsto the group of soluble pyrophosphatases, including cyto-

plasmic enzymes from prokaryotic and eukaryotic sourcescomposed of a single type of polypeptide (Cooperman etal., 1992). The catalytic subunits of the mitochondrial py-rophosphatases from yeast (Lundin et al., 1991), animals(Volk et al., 1983; Volk and Baykov, 1984), and plants(Vianello et al., 1991; Zancani et al., 1995) can probably beassigned to the same category, based on the publishedsequence of the yeast protein. These mitochondrial pyro-phosphatases are loosely bound to the inner membrane, asparts of multisubunit complexes, and can also be found inthe soluble matrix phase. Finally, the plant vacuolar H+-pumping pyrophosphatases and the pyrophosphatase (re-versible H+-PPi synthase) from Rhodospirillum rubrumchromatophores form a separate group of enzymes, withno significant sequence similarity with any other pyro-phosphatases (Rea et al., 1992).

Since the potato pyrophosphatase is presumably a solu-ble enzyme, its correspondence with the plastidic alkalinepyrophosphatase accounting for most of the soluble activ-ity in plant cells has to be considered. Overall, plant alka-line pyrophosphatases have molecular masses between 28

vnrwx _

GP173 _

GP68 -

CPS3 -

GP217 _

GP36(a) '

GP8S(b)

GP84(a) _

GP189 -

GP170 _

TG45 ~

PAT(a) -

— Ppa1(b)- CP14

- GP40(a)

-GP74(c)

CP158(a) -

GP122 _

TG28 —

_CP74(b) GP168 _Ppa1(a)-^9— GP130 CP20(b)-

-TG16

-GP126

-CP16

— GP171

_GP178(f)

4CL(c) >CP118 XGP178 -Gr\as yGP197 .

GP167(c) '

CP48(b) -

GP34 -

CP103(b) -

CP60 -

-CP134(a)

— GP229

^GP200

GP204-CP112(b)

-GP21S(a)

— GP104(f)-GP96<b)^GP91(c)VGP195

— TG68

_CP114

— ShM

-CP66

Figure 5. Positions of the loci PpaKa) and PpaKb) on the potatomap. Chromosomes VIII and XII were adapted from Gebhardt et al.(1991).

Soluble lnorganic Pyrophosphatase in Potato 859

and 42 kD. Thus, the estimated molecular mass of the potato pyrophosphatase is significantly lower, especially when cleavage of the putative presequence is accounted for (putative mature protein of 20.6 kD). One- and two-dimensional analysis of the N-terminal part of the potato pyrophosphatase failed to recognize a typical chloroplast transit peptide, although some of the rules of von Heijne et al. (1989) are satisfied. The mitochondrial targeting of the potato pyrophosphatase is also worth considering. Again, the molecular mass of the catalytic subunits of mitochondrial pyrophosphatases (28-35 kD) disagrees with that of the potato protein. Some of the structural characteristics of mitochondrial transit peptides are observed in the putative presequence of the potato protein, whereas others are not.

Other subcellular locations cannot be ruled out. Al- though it is generally assumed that the plant cell cytosol lacks a soluble pyrophosphatase, the evidence of a cytosolic pyrophosphatase in the latex of H. brasiliensis (Jacob et al., 1989) indicates that exceptions can be found, probably in relation to specific metabolic pathways. The fact that constitutive (Sonnewald, 1992; Jelitto et al., 1992) or phloem-specific (Lerchl et al., 1995) expression of a bacte- ria1 pyrophosphatase in the cytosol of transgenic tobacco and potato plants has detrimental effects on plant growth and metabolism does not exclude the existence of some endogenous cytosolic pyrophosphatase in specific cellular and physiological contexts. Cytosolic PPi levels might be controlled by distinct sets of enzymes according to the plant and the tissue. Finally, it can be mentioned that a soluble pyrophosphatase (apparently distinct from other phosphohydrolases) has been reported in vacuoles of Sac- charomyces carlsbergensis (Lichko and Okorokov, 1991), but no plant homolog has ever been described.

Clearly, much work is still to be done to gain a good knowledge of plant soluble pyrophosphatases. We think that limiting their existence to the sole plastidic compartment could prove to be a simplistic view.

ACKNOWLEDCMENTS

We are indebted to P. Plateau for providing the E. coli strain K37EKTR(pE). We also thank students for participating in some of the experiments, R. Viola for stimulating discussions at the begin- ning of this work, and A. Locicero and J.C. Martiat for excellent technical assistance.

Received March 17, 1995; accepted August 3, 1995. Copyright Clearance Center: 0032-0889/95/ 109/0853/08. The EMBL/ GenBank accession number for the sequence reported

in this article is 236894.

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Molecular Cloning and Characterization of a Soluble lnorganic