A Novel Calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal Autoinhibitory Domain*

Jeffrey F. HarperDagger §, Bimei HongDagger , Ildoo Hwang, Hong Qing GuoDagger , Robyn StoddardDagger , Jing Feng HuangDagger , Michael G. Palmgrenpar , and Heven Sze

From the Dagger  Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037,  Department of Plant Biology, University of Maryland, College Park, Maryland 20742-5815, and par  Institute of Molecular Biology, Copenhagen University, Oster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta  residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Calcium (Ca2+) appears to function as an important second messenger in all eukaryotes (1, 2). Ca2+ also plays an important role in regulating the processing of proteins in the secretory pathway (3, 4). Thus, cells require transport systems to carefully regulate Ca2+ concentrations in different cellular compartments. In plants, high affinity Ca2+-translocating ATPases are thought to function in the endoplasmic reticulum, Golgi apparatus, tonoplast, plastid inner membrane, and plasma membrane (5-8). In addition, plants have low affinity Ca2+/H+ antiporters (9). However, little is known at the molecular level about the genes which encode different Ca2+ transporters, or the specific functions of different Ca2+-transport pathways.

Ca2+ pumps are members of a large superfamily of P-type ATPases and have been classified as endoplasmic reticulum (ER)1 or plasma membrane (PM)-type Ca2+-ATPases (type IIA and IIB, respectively), based on enzymes first identified in animal systems (10). In plants, ECA1/ACA3 was the first cloned ER-type pump to be shown to have a localization and enzymatic activity analogous to an animal homolog (11). At present only two plant genes encoding homologs of "PM-type" Ca2+-ATPases have been identified, ACA1 from Arabidopsis thaliana (12) and BCA1 from Brassica oleracea (13). Interestingly, these plant pumps differ from animal homologs not only in subcellular localization, but also in their structural arrangement. ACA1p is thought to be targeted to a plastid inner membrane (12), while BCA1p appears to be localized to the tonoplast (13). The enzyme activity of ACA1p has not been investigated. However, BCA1 appears to encode a calmodulin-stimulated Ca2+ pump, based on correspondence between the peptide sequence obtained from a Ca2+-ATPase preparation and the predicted sequence of BCA1p (13, 14). A notable feature of both ACA1p and BCA1p is the absence of a long C-terminal regulatory domain, which is required for calmodulin activation in a typical PM-type Ca2+-ATPase (15, 16). The structural divergence of these pumps raises two important questions: 1) does calmodulin still regulate members of this subfamily, and 2) how does calmodulin activation occur without a C-terminal calmodulin binding regulatory domain?

Here we report the predicted primary structure for ACA2p, a Ca2+-ATPase most similar to ACA1p and BCA1p. We provide direct evidence that ACA2-like pumps constitute a novel subfamily of calmodulin-regulated Ca2+-ATPases, distinguished by a unique N-terminal regulatory domain and their presence in non-plasma membrane locations. ACA2p is the first homolog of this family to be functionally expressed in yeast. We show that a truncation of the N-terminal domain results in a constitutively active pump that can complement a yeast strain harboring a disruption of its endogenous Ca2+ pumps. These biochemical and genetic studies are significant for two reasons. First, they show that P-type ATPases can be regulated by autoinhibitors at either the N- or C-terminal end. Second, they establish a yeast expression system to genetically dissect the autoinhibitory mechanism of a calmodulin-regulated P-type ATPase.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

A. thaliana Columbia was used for plant material. DNA cloning was done in Escherichia coli strain XL1-Blue (Stratagene) or DH10alpha (a derivative of DH5alpha , Stratagene). Unless otherwise noted, standard molecular techniques were performed according to Sambrook et al. (17).

ACA2 cDNA Cloning-- A lambda -Zap (Stratagene)-based cDNA library constructed from A. thaliana 3-day-old seedlings (18) was screened under moderate stringency (19) using a genomic fragment of ACA2 as a hybridization probe. This genomic fragment was generated by PCR using degenerate DNA oligomers (see "Results"). The "PEGL" 20-mer primer had the sequence dGC(G/C/T)GT(G//C/T)CC(G/C/T)GAGGG(G/C/T)(C/T)T(G/C/T)CC. One full-length cDNA was identified after screening approximately 100,000 phage. The insert was subcloned into the vector pBlueScript II SK(-) and named pACA2.

Constructs-- Standard PCR reactions and subcloning procedures were used to modify ACA2 sequences in clones described below. PCR was performed using Ampli-Taq (Perkin-Elmer) or PanVera X-Taq (PanVera Corporation) for 15 cycles as follows: 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. All PCR-derived sequences were sequenced to ensure the absence of PCR mistakes.

pACA2-wt encodes a full-length ACA2p and was derived from pACA2 by deletion of the 5'-untranslated region. The resulting sequence at the 5' end is GTC GAC ATG (SalI underlined, followed by the codon for M1). Two restriction sites were also introduced as silent mutations in the coding region: CTT CG AAA CC TAT GAA GCC GCG GCG (BstBI and SacII underlined) corresponding to the peptide sequence LSKRYEAAA-52.

pACA2-Delta P80 encodes an N-terminally truncated ACA2p and was derived from pACA2 by a deletion of DNA sequence corresponding to residues 2-80. The resulting sequence at the 5' end is GTC GAC ATG AGT (SalI underlined, followed by codons for M1 and "S81").

pYX112-TEV was derived from pYX112 (Novagen) by the insertion into the EcoRI/BamHI site of a 5'-untranslated sequence from tobacco etch virus (TEV) (20). The sequence of the polylinker is shown in Fig. 1.


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Fig. 1.   Sequence of a polylinker engineered into pYX-112 (TEV) and used to aid construction of ACA2 yeast expression clones.

pYX-ACA2-1 encodes a full-length ACA2p and was subcloned from pACA2-wt as a SalI/XbaI fragment into the XhoI/NheI site of pYX112-TEV.

pYX-ACA2-2 encodes an N-terminal deletion of ACA2 and was subcloned from pACA2-Delta P80 as a SalI/XbaI fragment into the XhoI/NheI of pYX112-TEV.

pMC2(1-72) encodes the first 72 residues of ACA2p as a C-terminal fusion to maltose binding protein. It was constructed by the insertion of a PCR fragment into the SalI/PstI site of the vector pMAL-CR1 (New England Biolabs).

pGC2(1-58) encodes the first 58 residues of ACA2p as a fusion protein with glutathione S-transferase at the N-terminal end and a green fluorescence protein (GFP)/6 × histidine tag at the C-terminal end. It was constructed in the vector pGEX-KG (21) as a SalI/HindIII insert. The sequence at the fusion site between ACA2 and GFP is CAG GCC GCA GAT CTA AAG ATG (first codon = Gln58 from ACA2, BglII site underlined, last codon = M1 from GFP). The sequence at the 3' end of the GFP reads ATG GAT GAA CTA GAC CCG GGA ATG CAT CAC CAT CAC CAT CAC GGA TCC TGA coding for mdelDPGMHHHHHHGS* (GFP sequence shown in italic lowercase letters). All deletion clones, pGC2(1-9) to pGC2(1-58) were made by an ExoIII nuclease deletion using the parent clone pGFP-ACA2-NL-1 (not described).

pGL10 encodes residues Val119-Phe161 of ACA2p as a fusion with an N-terminal maltose binding protein and a C-terminal end 6 × histidine tag (Phe161-PGMHHHHHHGS* encoded by the DNA sequence TTC CCG GGA ATG CAT CAC CAT CAC CAT CAC GGA TCC TGA). It was constructed from a PCR-amplified fragment cloned into a modified version of vector pMAL-CR1. The insert can be subcloned as a SalI/PstI fragment.

pACA2-Ns encodes the same sequence (Val119-Phe161) encoded by pGL10, except that it has an N-terminal glutathione S-transferase (GST) affinity tag instead of maltose-binding protein (MBP). It was derived by subcloning the SalI/PstI fragment from pGL10 into the XhoI/PstI site of pGEX-KG (ampicillin resistance gene was reformed).

DNA Sequencing-- DNA sequencing was done in the Scripps Biochemistry Core Facility using an automated ABI Prism373XL sequencer. All cDNA sequence was confirmed by sequencing both strands.

Fusion Protein Purification-- Fusion proteins were expressed in E. coli DH10alpha . Fusion proteins constructed as an affinity sandwich with an N-terminal GST or MBP tag and a C-terminal 6 × histidine tag were purified by sequential affinity purification for the 6 × histidine tag then GST or MBP (22). Purifications based on single affinity tag selections were done with minor modifications of standard procedures (New England Biolabs and Invitrogen).

Antibodies-- Anti-ACA2 (number 1471) rabbit polyclonal antiserum was produced at the Scripps Animal Resource Facility. Anti-ACA2 was raised against a fusion protein, encoded by the plasmid pACA2-Ns, containing residues Val119-Phe161. Purified fusion protein samples were injected into New Zealand White rabbits with RIBI adjuvant as recommended by the manufacturer (RIBI ImmunoChem Research).

In some cases affinity-purified antibodies were used. Serum was first precipitated with 50% ammonium sulfate and redissolved in phosphate-buffered saline. Anti-GST antibodies were removed using a column with GST-coupled protein. Anti-ACA2 antibodies were then allowed to bind to a column containing the fusion protein encoded by pACA2-Ns. Columns were made using cyanogen bromide-activated Sepharose 4B according to manufacturer (Pharmacia Biotech Inc.). Anti-ACA2 antibodies were eluted with 0.1 M glycine, pH 2.7, and immediately neutralized with 0.1 volume of 1 M Tris-HCl, pH 8.0, and dialyzed against phosphate-buffered saline. Control preimmune serum was purified over a protein A-Sepharose CL-4B column (Sigma).

Total Protein Extracts-- Leaves, flowers, and siliques were obtained from soil-grown plants grown in a growth room at 22 °C. Roots were obtained from 3-week-old plants grown in liquid culture consisting of Gamborg's B5 medium (Life Technologies, Inc.) supplemented with 2% sucrose and 0.5% (w/v) MES, pH 5.7. Cultures were grown on a shaker at approximately 125 rpm under constant illumination at 22 °C. Tissue samples were pulverized in liquid nitrogen with a mortar and pestle, and total protein was extracted in homogenization buffer (0.3 M sucrose, 10 mM EDTA, 2 mM EGTA, 100 mM Tris-HCl, pH 7.8, 35 mM beta -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin). The protein extract was clarified by a 5-min centrifugation in an Eppendorf microcentrifuge.

Aqueous Two-phase Partitioning-- Plant microsomal membranes were prepared by modifications of a previously described procedure (23). All manipulations were conducted on ice or in a cold room, with prechilled buffers. Membranes were prepared from roots of plants grown in liquid culture. Roots were frozen in liquid nitrogen and pulverized with mortar and pestle or ground with dry ice in a coffee grinder. Pulverized tissues were mixed with an equal weight of extraction buffer (290 mM sucrose, 25 mM EDTA, 250 mM Tris-HCl, pH 8.5, 2 mM phenylmethylsulfonyl fluoride, and 76 mM beta -mercaptoethanol). Homogenates were filtered through cheesecloth to remove large debris and then centrifuged at 5,000 × g to remove intact organelles and cell walls. Supernatants were spun at greater than 40,000 × g for at least 40 min to pellet microsomal membranes. The resulting supernatants were used as the soluble protein fraction. Membrane pellets were resuspended in resuspension buffer (0.33 M sucrose, 4 mM KCl, 5 mM KHPO4, pH 7.8). Plasma membranes were partially purified by aqueous two-phase partitioning (24). Each fractionation was conducted with 5-10 g of fresh weight tissue (liquid-grown plants) with yields around 75 µg of upper phase membrane protein.

Western Blots-- Protein concentrations were determined by Bradford (25) assays using bovine serum albumin as a standard. For SDS-PAGE, samples were mixed with 3 × loading buffer (100 mM Tris, pH 6.8, 3.7% (w/v) SDS, 5% (w/v) dithiothreitol, 20% (w/v) sucrose or glycerol, 0.3% (w/v) bromphenol blue) and incubated for 15 min at 37 °C. After electrophoresis, proteins were transferred to nitrocellulose (Schleicher & Schuell), using a Bio-Rad transfer apparatus. Transfer buffer consisted of 192 mM glycine, 25 mM Tris-HCl, pH 8.3, 20% (v/v) methanol, and 0.02% (w/v) SDS.

Blots were blocked for at least 2 h in 20 mM Tris, pH 7.6, 137 mM NaCl, 0.5% (w/v) Tween-20 (TBS-T) with 5% (w/v) non-fat dry milk. Primary antisera were normally diluted in blocking buffer at the following concentrations: anti-ACA2 at 1:1000 and anti-CTF2 (26) at 1:5000. The secondary antibody used for immunodetection was a donkey anti-rabbit IgG conjugated with horseradish peroxidase (Amersham Corp.), and was diluted at 1:5000 in blocking buffer. Primary and secondary antibody incubations were for 1 h at room temperature, and were followed by four 15-min washes in TBS-T. Secondary antibodies were detected by enhanced chemiluminescence (ECL, Amersham Corp.) and exposure to x-ray film.

Yeast Transformations-- Saccharomyces cerevisiae strains used for complementation studies were W303-1A (MATa, leu2, his3, ade2, ura3) and K616 (MATa pmr1::HIS3 pmc1::TRP1 cnb1::LEU2, ura3) (27). For protein expression and ATPase assays we used strain G19 (MATalpha ura3, his3, leu2, trp1, ade2, ena1Delta ::His3::ena4Delta ) (28) (generously provided by A. Rodriguez-Navarro and J. Schroeder).

For transformations W303-1A and K616 were grown in standard YPD media supplemented with 10 mM CaCl2 for K616 cells; G19 cells were grown in synthetic medium minus histidine. Yeast were transformed with either pYX112 vector alone, pYX-ACA2-1, or pYX-ACA2-2 by the LiOAc/PEG methods (29, 30) and selected for uracil prototrophy by plating on synthetic medium minus uracil (SC-URA): 6.7 g/liter yeast nitrogen base without amino acids, 2 g/liter of drop-out mix without uracil, 2% glucose or sucrose as a carbon source, and 1.5% agar (31). The Ura+ colonies were picked and grown for 2-3 days on SC-URA agar plates. For complementation studies, colonies were streaked again on SC-URA agar plates containing 10 mM EGTA, pH 6.0, and incubated for 2-3 days at 30 °C as described previously (11).

Isolation of Yeast Membranes-- Yeast cells were homogenized and fractionated on a continuous gradient of 20-60% sucrose with modifications of Villalba et al. (32). A 500-ml culture of yeast was grown to late log phase with shaking at 30 °C, harvested, washed with cold H2O, and processed for a single sucrose gradient. Twelve 1-ml fractions were collected from the top, frozen in liquid N2, and stored at -70 °C until use.

ATPase Assays-- ATPase assays were conducted with modifications of Baginsky et al. (33). Samples (10-15 µl) were assayed at 22 °C in a buffer of 20 mM MOPS, 8 mM MgSO4, 50 mM KNO3, 0.25 mM K2MoO4, 5 mM NaN3, and 1 mM EGTA (to chelate free Ca2+). Ca2+ stimulation was tested by the addition of 1.1 mM CaCl2. Vanadate was added to some reactions at 0.1 mM. Calmodulin (bovine brain or spinach) was obtained from Sigma.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Isolation of ACA2 cDNA-- ACA2 was identified as a full-length cDNA in a lambda -Zap-based cDNA library (18) and subcloned as a plasmid pACA2 (Fig. 2). To identify this cDNA we used a partial genomic fragment of ACA2 as a hybridization probe. This genomic fragment was obtained by two sequential rounds of PCR amplification (i.e. nested PCR) using degenerate primers. In both reactions the 3' primer corresponded to a sequence conserved in all P-type ATPases (MTGDGVNDA) (primer number 1025) (34). In the first reaction the 5' primer (PEGL) corresponded to a sequence conserved in Na+ and Ca2+ pumps (A(I/V)PEGLP). In the second reaction we used a nested 5' primer corresponding to a more generally conserved sequence (CSDKTGTLT) (34). A critical feature of our strategy was the PEGL primer which biased reactions into amplifying putative Ca2+ pumps. Without such a bias, the primary reaction products were fragments of proton pump genes, perhaps reflecting the large size of this gene family in Arabidopsis (34).


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Fig. 2.   Diagram of clone pACA2-wt. Diagnostic and useful restriction enzyme sites within the cDNA sequence are shown above. Restriction sites from the vector polylinker are shown below. During the deletion of the 5'-untranslated sequence, *BstBI and *SacII sites were introduced in the coding region as silent mutations. S81 marks the penultimate residue in the N-terminal deletion mutant encoded by pACA2-2. The cross-hatched region corresponds to the position of the peptide sequence used to generate ACA2 antiserum.

Primary Structure Shows Unique N-terminal Domain-- Fig. 3 shows the predicted 110-kDa protein encoded by ACA2. The cDNA sequence contained a single long open reading frame with an in-frame stop codon upstream of the predicted start codon. ACA2p shows greatest identity to a subfamily of PM-type Ca2+-ATPases, including ACA1p/PEA1p from Arabidopsis (12) (78% identity), BCA1p from Brassica (13) (62% identity) (shown aligned in Fig. 3). ACA2p shows approximately 44% identity to a mammalian PM-type Ca2+-ATPase, but less than 32% identity to ER-type Ca2+-ATPases, including the rabbit SERCA1p (35) or Arabidopsis ACA3p/ECA1p (11).


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Fig. 3.   Primary structure of ACA2p aligned to ACA1p (12) (GenBankTM accession number L08469) and BCA1p (13) (EMBL accession number X99972). The black background indicates identity between two or more isoforms. Dashes indicate the absence of a residue. Predicted transmembrane domains are indicated above as M1 to M10. A double line indicates the possible position of a calmodulin-binding sequence. A dashed line indicates the sequence used for generating an ACA2 antiserum. The alignment was done by the method of Jotun-Hein using a PAM250-weighted comparison and DNAstar software.

Ten transmembrane domains are predicted for ACA2p based on programs predicting hydropathy and topology (Predict Protein and PSORT programs) (36, 37). The N- and C-terminal ends of ACA2p are predicted on the cytoplasmic side of the membrane. However, the N-terminal domain contains an additional 19-residue hydrophobic segment from Ile63 to Ser82, which may provide an 11th transmembrane domain. The C-terminal end does not show a putative ER retention motif (KXKXX) (38, 39) as seen for the plant ER-type Ca2+-ATPases.

In comparison to animal homologs the most distinct feature of ACA2p is the absence of a long C-terminal regulatory domain. Instead, there is a unique, relatively long N-terminal domain of 160 residues, compared with 94 residues for a typical mammalian pump (e.g. hPMCA2p) (40). It is possible to align ACA2p with hPMCA2p starting around position Glu87, but the identity up to the first transmembrane remains poor (22%).

ACA2p Is Most Abundant in Roots and Flowers-- To determine the relative tissue distribution of ACA2p, we produced a polyclonal antibody (anti-ACA2) against a 43-residue sequence in the N-terminal domain (Val119-Phe161). This region shows high variability, with only 56% identity to ACA1p. This variable region was used in hopes of generating an isoform specific antibody.

Fig. 4 shows a Western blot analysis of total proteins extracted from different tissues. Anti-ACA2 specifically detected a 110-kDa protein, most abundant in roots and flowers. Cross-reaction of anti-ACA2 with other Arabidopsis isoforms (e.g. ACA1p) has not been tested. However, no cross-reaction was observed with proteins extracted from tobacco or onions (data not shown), suggesting that this antiserum is highly specific.


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Fig. 4.   Western blot analysis showing tissue-specific expression for anti-ACA2 cross-reacting proteins. Total protein extracts (10-µg samples) from roots (R), leaves (L), flowers (F), and siliques (S) were subjected to SDS-PAGE (8% gel), transferred to nitrocellulose, and probed with preimmune (0.25 µg/ml protein A-purified) or anti-ACA2 (0.25 µg/ml affinity-purified) serum. Immunoreactive bands were detected by ECL and exposure to x-ray film. The arrow marks the expected position of ACAp (110 kDa).

ACA2p Is an Endomembrane Pump-- To test the hypothesis that ACA2p is a plasma membrane pump, we fractionated microsomal membranes by an aqueous two-phase partitioning procedure (Fig. 5). Plasma membranes partitioned to the upper phase, as shown by immunodetection of a plasma membrane H+-ATPase marker. In contrast, ACA2p partitioned to the lower phase, indicating an endomembrane localization. In controls, an ER-type Ca2+-ATPase (ACA3p/ECA1p) also partitioned to the lower phase (not shown). Endomembrane localization was confirmed by showing immunodecoration of subcellular structures using confocal microscopy on root tip cells (not shown).


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Fig. 5.   Western blot analysis showing ACA2p fractionation with endomembranes in aqueous two-phase partitioning. Root microsomal membranes were fractionated into a soluble fraction (S), total microsomal membranes (M), upper phase (U), and lower phase (L). Parallel Western blots were processed with protein samples (5 µg) subjected to SDS-PAGE (8% gel), transferred to nitrocellulose, and probed with affinity-purified anti-ACA2 serum (0.25 µg/ml) or an anti-H+-ATPase serum (anti-CTF-2). Immunoreactive bands were detected by ECL and exposure to x-ray film.

ACA2-2p Complements a Disruption of Yeast Ca2+ Pumps-- To genetically test for Ca2+ pump activity, ACA2p was expressed in a mutant yeast strain, K616. K616 (pmr1 pmc1 cnb1) has a disruption of three genes involved in Ca2+ homeostasis, PMR1, PMC1, and CNB1. PMR1 and PMC1 encode Ca2+-ATPases in the yeast vacuole and Golgi apparatus, respectively (41, 42), and CNB1 encodes a Ca2+-dependent phosphatase (calcineurin), which regulates a H+/Ca2+ exchanger (43). Due to the disruption of the yeast Ca2+ pumps, this strain displays poor growth on Ca2+-depleted medium (+10 mM EGTA, pH 6.0). ACA2p was expressed as a full-length protein, ACA2-1p (construct pYX-ACA2-1), and an N-terminally truncated mutant, ACA2-2p (construct pYX-ACA2-2). Both ACA2-1 and -2 were cloned in a yeast vector (pYX112) to provide a moderate level of constitutive expression from a triose phosphate isomerase promoter (Novagen).

In controls, wild type yeast transformed with vector alone grew on Ca2+-depleted medium, while the K616 mutant transformed with the vector alone did not (Fig. 6). When the K616 host was transformed with pYX-ACA2-1 or -2, only the truncation mutant was able to restore growth. The expression levels for both ACA2-1p and ACA2-2p were comparable, as indicated by immunodetection of a 110- and 102-kDa polypeptide, respectively, using anti-ACA2 antiserum (not shown).


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Fig. 6.   Complementation of yeast mutant K616 through expression of ACA2-2p. Yeast were grown on medium supplemented with 10 mM EGTA, pH 6.0. Diagram indicates the strain genotypes. The vector used was pYX112. Delta N and full-length correspond to ACA2-2p and -1p, respectively.

ACA2-1p and ACA2-2p Have Ca2+-ATPase Activity-- To biochemically determine the activity of ACA2p, we used sucrose gradients to fractionate yeast microsomal membranes containing either ACA2-1p or ACA2-2p. As a control we fractionated membranes from yeast transformed with a vector only. Sucrose gradients were analyzed for ATPase activities in four separate experiments with equivalent results. Approximately equal amounts of protein were loaded on each gradient.

Results from a typical set of fractionations are shown in Fig. 7. All gradients showed a peak of vanadate-sensitive ATPase activity (Ca2+-independent) at approximately 48% sucrose, consistent with the expected position of the plasma membrane H+-ATPase (23). In the vector-only control, there was very little Ca2+- or Ca2+/calmodulin-dependent ATPase, consistent with the reported low levels of endogenous Ca2+ pump activity in yeast (41).


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Fig. 7.   ATPase activity of ACA2-1p and ACA2-2p in yeast membranes. Membranes were fractionated from yeast transformed with a vector only control, pYX-ACA2-1 (full-length) or pYX-ACA2-2 (N-termDelta ). The top panel shows the percent sucrose measured in each gradients. Total ATPase activity is shown for fractions assayed without Ca2+ (-C), plus 100 µM Ca2+ (+C), plus 100 nM Ca2+/calmodulin (+C/CaM) or inhibited with 0.1 mM vanadate (-C/vanadate). The bottom panel shows Ca2+-dependent (+C) and Ca2+/calmodulin-dependent (+C/CaM) activities calculated from total activities by subtraction of the Ca2+-independent activity in each fraction. A dotted line provides a guide for analyzing the peak fractions of activity for ACA2-1p and ACA2-2p.

In contrast to the vector-only control, gradients containing ACA2-1p and -2p always showed a broad peak of Ca2+-stimulated activity between 30 and 45% sucrose (fractions 4-7). The Ca2+-stimulated activity was consistently higher (3-7-fold) in gradients containing the truncation mutant ACA2-2p, compared with the full-length ACA2-1p. However, only the full-length protein displayed significant calmodulin stimulation (around 5-fold).

To confirm that ACA2-1p could be activated by Ca2+/calmodulin, samples from fraction 5 were assayed with increasing concentrations of calmodulin (Fig. 8). Calmodulin activated ACA2-1p in a dose-dependent fashion, with a half-maximal activation around 30 nM. Calmodulin activation was Ca2+-dependent and vanadate-sensitive, as shown by controls assayed in the presence of 100 nM calmodulin with 1 mM EGTA or 0.1 mM vanadate, respectively (not shown). In parallel, the addition of up to 100 nM Ca2+/calmodulin failed to stimulate activity from the truncation mutant or vector only control.


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Fig. 8.   Calmodulin activation of ATPase activity for ACA2-1p. Samples from fraction 5 were analyzed in detail for calmodulin-stimulated activity. Relative amounts of ACA2-1p and ACA2-2p in each assay are shown in Fig. 9A, as detected on a Western blot by anti-ACA2 serum. ATPase assays included 100 µM free Ca2+. Calmodulin was varied from 0 to 2000 nM. A dotted line indicates the estimated 50% activation point. Assays were done with 1-7 µg of membrane protein with no significant difference observed for the estimated 50% activation point. 50% activation occurred between 20 and 50 nM calmodulin with three different enzyme preparations tested. We estimated ACA2-1p in our assays to be significantly less than 2 nM (based on the observation that ACA2-1p appears to represent less than 1% of the membrane protein, as indicated by the failure to detect ACA2-1p in a Coomassie-stained SDS-PAGE gel).

In the detailed analysis shown in Fig. 8, the truncation mutant showed a higher specific activity than the full-length protein, in the presence or absence of calmodulin. In this assay there was less truncation mutant protein than full-length enzyme, as determined by immunodetection on three independent Western blot analyses (e.g. see Fig. 9A). Therefore, the truncation mutant appears more active than the full-length protein on a per mole of enzyme basis. This higher relative activity was observed in all four sucrose gradient fractionations.


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Fig. 9.   A, a Western blot analysis shows the relative levels of ACA2-1p and ACA2-2p assayed in fraction 5, and a mixed sample of ACA2-1p and ACA2-2p shows that the two enzymes can be resolved as separate bands by SDS-PAGE. Protein samples (approximately 5 µg) were taken from fraction 5 and precipitated with 11% trichloroacetic acid. The 1 + 2 mix is a sample containing a mixture of ACA2-1p and -2p cofractionated on the same gradient (see B). Samples were redissolved in loading buffer and subjected to SDS-PAGE (8% gel), transferred to nitrocellulose, and probed with anti-ACA2 serum. Immunoreactive bands were detected by ECL and exposure to x-ray film. Arrows mark the positions of ACA2-1p and -2p, respectively. B, sucrose gradient fractionation of ACA2-1p and ACA2-2p shows colocalization in yeast endomembranes. Samples (100 µl) from each gradient fraction were precipitated with 11% trichloroacetic acid and subjected to a Western blot analysis as described above. The peak fraction for both ACA2-1p and -2p was around 39% sucrose.

To confirm that a deletion of the N-terminal end in ACA2-2p did not dramatically alter its subcellular localization in yeast, a mixture of microsomal membranes containing ACA2-1p and ACA2-2p were fractionated on the same gradient, and a Western blot analysis was used to analyze their fractionation profile. A control shown in Fig. 9A demonstrates that ACA2-1p and ACA2-2p can be mixed and still resolved as separate bands by SDS-PAGE. Fig. 9B shows that the two proteins cofractionate when run as a mixture on the same gradient. Cofractionation was tested in two separate experiments and is consistent with results obtained from each enzyme fractionated separately. The peak fraction for both ACA2-1p and ACA2-2p was between 31 and 45% sucrose, consistent with the broad peak determined by ATPase activity measurements.

The N-terminal Domain Contains a Calmodulin-binding Sequence-- To determine if the N-terminal domain contained a calmodulin-binding sequence, we performed binding assays on a series of fusion proteins (e.g. MC2(1-72)p) (Fig. 10). In Fig. 11, a calmodulin gel overlay analysis shows that calmodulin binds in a Ca2+-dependent fashion to all fusion proteins containing the first 36 residues of the N-terminal domain. Calmodulin did not bind to a control MBP or GST, nor to a fusion protein containing residues 1-19. This suggests that the calmodulin binding sequence lies within the first 36 residues, and probably downstream of residue 19. 


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Fig. 10.   N-terminal domain fusion proteins used to show a calmodulin-binding sequence within the first 36 residues. Fusion proteins diagrammed here were tested for calmodulin binding as shown in Fig. 11. A portion of the protein sequence is shown for two fusions, MC2(1-72)p and GC2(1-58)p. MC2(1-72)p stands for Maltose-binding protein fusion with Ca2+-pump ACA2 residues 1-72. In GC2(1-58)p the G denotes a glutathione S-transferase fusion protein. Markings on top of the sequences (left) indicate the positions of factor X and thrombin protease sites, respectively. A series of deletions were made to delineate the minimal sequence required for calmodulin binding. The truncation points in GC2-deletion constructs are indicated by a number under GC2(1-58)p. These fusions have an ACA2p sequence sandwiched between an N-terminal GST and a C-terminal GFP. They were made by an ExoIII deletion strategy which resulted in slightly different linkers between the ACA2p sequence and GFP, as shown at the right (GFP sequence shown in italic lowercase letters). Black filling, fusion proteins displayed strong Ca2+-dependent calmodulin-binding; no filling, no calmodulin binding. The shortest construct to bind calmodulin was GC2(1-36). A putative binding site is marked by a bracket on top of the MC2(1-72)p sequence.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Two lines of evidence indicate that ACA2 encodes a Ca2+-ATPase most similar to a PM-type Ca2+ pump (type IIB). First, ACA2p has 44% sequence identity with PM-type Ca2+-ATPases and only 32% identity with an ER-type Ca2+-ATPase. Second, ACA2p displayed calmodulin-stimulated activity, a biochemical hallmark of PM-type Ca2+-ATPases. Nevertheless, we show here that ACA2p is fundamentally distinct in two respects. First, ACA2p is localized to an endomembrane system (i.e. not the plasma membrane), as shown by aqueous two-phase partitioning of microsomal membranes (Fig. 5). Second, ACA2p is regulated by an autoinhibitor located in the N-terminal instead of C-terminal domain, as shown by (i) genetic complementation studies in yeast (Fig. 6), (ii) ATPase activity assays on full-length and truncated pumps (Fig. 8), and (iii) identification of a calmodulin-binding sequence in the N-terminal instead of C-terminal domain (Figs. 10 and 11). Thus, our data provide strong support for the hypothesis (13) that ACA2-like pumps constitute a unique subfamily of calmodulin-regulated Ca2+-ATPases, distinguished by their novel structural arrangement and their presence in non-plasma membrane locations.


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Fig. 11.   Calmodulin gel overlays show calmodulin binding to a sequence in the N-terminal domain of ACA2p. Fusion proteins were purified and subjected to SDS-PAGE (10% gel), transferred to nitrocellulose, and probed with 60 nM biotinylated calmodulin (52). Bound calmodulin was detected by ECL and exposure to x-ray film. The top panel shows a Coomassie-stained gel of the fusion proteins. Calmodulin-probed blots are shown below. No binding was observed in the absence of Ca2+. A description for each fusion is provided in Fig. 10. Although the MC2(1-72)p fusion is smaller than the GC2 fusions, it actually contains the longest N-terminal sequence.

Yeast Complementation-- The expression of ACA2 in yeast (mutant K616) provides genetic evidence that ACA2 encodes a Ca2+ pump. Expression of ACA2-2p (N-terminal truncation mutant) allowed the K616 strain to grow on Ca2+-depleted medium. K616 (pmc1 pmr1 cnb1) contains a disruption of the endogenous Ca2+ pumps, PMC1 and PMR1, as well as calcineurin (CNB1), a Ca2+-stimulated phosphatase (43). K616 cells fail to grow on Ca2+-depleted medium, presumably because they cannot scavenge sufficient Ca2+ into an endomembrane compartment. Thus, the ability of ACA2-2p to restore growth on Ca2+-depleted medium is consistent with the hypothesis that it can function as a high affinity endomembrane Ca2+ pump.

Interestingly, complementation of the K616 conditional growth phenotype was observed only with the truncation mutant, and not the full-length enzyme. We offer two explanations. First, the truncated pump may provide more activity in vivo than the full-length pump. This hypothesis is consistent with in vitro ATPase assays showing that the truncated pump is comparatively more active, even when the full-length pump is fully stimulated by calmodulin. In addition, it is likely that the full-length enzyme is normally autoinhibited in vivo, since its activation by Ca2+/calmodulin would require continuously high levels of cytosolic Ca2+, a condition not expected for K616 cells grown on Ca2+-depleted medium. However, even if high concentrations of cytosolic Ca2+ occur, the full-length pump may still remain unactivated if (i) levels of the endogenous yeast calmodulin are too low, or (ii) the yeast calmodulin cannot functionally interact with the plant pump. Our second alternative explanation is that only the truncated pump may be targeted to the proper functional location to permit complementation. Although a targeting problem remains a formal possibility, both truncated and full-length pumps co-fractionated to similar densities on the same sucrose gradient, suggesting a similar subcellular localization. Nevertheless, the sensitivity of this fractionation assay may not have detected a small, but functionally important difference in their subcellular localization.

Ca2+-ATPase Activity-- Biochemical ATPase assays on full-length ACA2-1p demonstrate that ACA2 encodes a calmodulin-regulated Ca2+-ATPase. Using yeast membranes fractionated by a sucrose gradient, the activity associated with the full-length ACA2-1p was shown to be Ca2+/calmodulin-dependent, displaying half-maximal activation around 30 nM calmodulin. This indicates that the pump has a relatively high affinity for calmodulin, similar to a typical PM-type Ca2+-pump (e.g. hPMCA4p) (44).

ATPase assays on the truncated ACA2-2p provide evidence that the N-terminal domain functions as a calmodulin-regulated autoinhibitor. In comparison to ACA2-1p, the truncation mutant displayed a 7-fold higher basal activity and was calmodulin-independent. This deregulated activity is similar to that observed after limited trypsin proteolysis of several purified calmodulin-dependent Ca2+-ATPases from plants (45, 46). Together these data indicate that the N-terminal domain contains a calmodulin-regulated autoinhibitor and its removal results in a constitutively active enzyme. We speculate that the deregulated activity associated with ACA2-2p allowed the K616 yeast to grow on Ca2+-depleted medium.

Activation of ACA2p by an N-terminal truncation mutation appears to be functionally analogous to a C-terminal truncation of hPMCA4p (47), a typical PM-type Ca2+ pump. Both truncations produced highly active pumps which were calmodulin-independent. The development of the ACA2-2 truncation mutant provides the potential to constitutively activate a specific Ca2+-transport pathway in transgenic plants. An interesting question is whether such a dominant mutant would alter Ca2+-signal transduction, or disrupt the function of a specific organelle.

N-terminal Calmodulin Binding Autoinhibitor-- Calmodulin binding assays demonstrate that the N-terminal domain of ACA2p contains a calmodulin-binding sequence. Calmodulin binding to a fusion protein containing only the first 36 residues of ACA2p was Ca2+-dependent. Within this region there is one sequence (Val20-Val31) with a reasonable potential to form an amphipathic basic helix, typical of many calmodulin-binding sequences (48). However, a complete binding sequence may include additional downstream residues. In the N-terminal domain of BCA1p, an analogous binding site has been proposed. However, the BCA1p sequence displayed a Ca2+-independent interaction with calmodulin, as detected using a 25-residue peptide spotted on nitrocellulose (13). Although such Ca2+-independent binding may be an artifact of using a short peptide, it may also reflect a functional difference between the putative binding sequences in BCA1p and ACA2p. In plants, evidence for pumps with both low and high affinity calmodulin-binding sequences have been reported (14, 46, 49).

Our findings demonstrate that P-type ATPases can be regulated by autoinhibitors located at either N or C termini. In general, autoinhibitors may function by: 1) directly binding to an active site on the enzyme and thereby sterically blocking access of a substrate (intrasteric inhibition), or 2) binding or altering the structure of a region removed from the active site and locking the enzyme in an inactive conformation. In a typical PM-type Ca2+-ATPase the C-terminal autoinhibitor is thought to interact with multiple regions, including the central cytoplasmic loop (15). However, this need not be the case for the ACA2 N-terminal autoinhibitor. If a portion of the N-terminal domain is also part of the core enzyme structure, then an N-terminal autoinhibitor may directly regulate that component. For example, the N-terminal domain of many ATPases is thought to control access of ions to the channel domain (50). Thus, it might be possible to regulate the enzyme activity by converting this N-terminal structure into a regulated ion gate. This raises the question of whether ACA2p employs a novel strategy of autoinhibition, made possible by its unique structural arrangement.

In a comparison of primary structures, we failed to identify any significant identities between the N-terminal domain of ACA2p and the C-terminal regulatory domain of a true PM-type Ca2+-ATPase. Thus, it is unclear if the two regulatory domains evolved independently or diverged after a domain swapping event in a common ancestor. With ACA2 as a possible exception, there has been no other evidence for "domain shuffling" in P-type ATPases (51). Regardless of origin, since both domains function as calmodulin-regulated autoinhibitors, a comparative investigation into their structure and function should provide valuable insights into the regulation of calmodulin-dependent Ca2+ pumps.

Function-- The biological functions of ACA2p are not known. Since ACA2p is activated by calmodulin, it may provide a Ca2+-regulated feedback system to control Ca2+ levels in the cytoplasm. It may also regulate Ca2+ levels within a specific organelle, such as a plastid, Golgi apparatus, or vacuole. In contrast to animal cells, plant cells appear to have calmodulin-regulated Ca2+-ATPases in multiple subcellular locations, in addition to the plasma membrane (7). Whether the plant plasma membrane-localized pumps are also members of this structurally unique subfamily is not known. However, they are reported to be larger than ACA2p (110 kDa) and are closer to the 127-136-kDa size typical of an animal PM-type Ca2+ pump (5, 46, 49). Thus, it remains an open question whether plant cells function with two distinct types of calmodulin regulated Ca2+ pumps, or operate exclusively with variants of the subfamily described here.

    ACKNOWLEDGEMENTS

We thank Lone Baunsgaard, Anja Fuglsang, and Thomas Jahn for assistance in ATPase assays.

    FOOTNOTES

* This research was supported in part by donations from Mrs. Frederick Garry and Mr. and Mrs. Kenneth E. Hill for the operation of the laboratory at The Scripps Research Institute, and by United States Department of Agriculture Grant 92-37304-7889), Department of Energy Grant DE-FG03-94ER20152, and Triagency-DOE/NSF/USDA Plant Biology Program 92-37105-7675, and a joint grant from the National Aeronautics and Space Administration and National Science Foundation (IBN-9416038) (to J. F. H.), and by Department of Energy Grant DE-FG02-95ER20200 (to H. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF025842.

§ To whom correspondence should be addressed: Dept. of Cell Biology, BCC283, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-2862; Fax: 619-784-9840; E-mail: Harper{at}Scripps.edu.

1 The abbreviations used are: ER, endoplasmic reticulum; PM, plasma membrane; PCR, polymerase chain reaction; TEV, tobacco etch virus; GFP, green fluorescence protein; MBP, maltose-binding protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; SC-URA, synthetic medium minus uracil; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid.

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Abstract
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Discussion
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