Proc. Natl. Acad. Sci. USAVol. 90, pp. 10066-10070, November 1993Plant Biology

Characterization of a gene encoding a Ca2 -ATPase-like protein inthe plastid envelope

(ion pump/P-type ATPase/calcium transport)

LAIQIANG HUANG*t, TOM BERKELMAN*, AMIE E. FRANKLIN*t, AND NEIL E. HOFFMAN*§*Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA 94305; and *Department of Biological Sciences, Stanford University,Stanford, CA 94305

Communicated by Winslow R. Briggs, July 26, 1993

ABSTRACT By screening an Arabidopsis expression li-brary with an antiserum against chloroplast envelope proteins,we have isolated a partial cDNA with an open reading framethat encodes a polypeptide similar to P-type cation-transporting ATPases. The corresponding genomic done wasisolated and the complete coding sequence was deduced afteridentification and mapping of introns. The gene has beendesignated PEAI (plastid envelope ATPase) and the predictedpolypeptide PEAlp. PEAlp has 946 amino acids and a molec-ular mass of 104 kDa. This protein is 40-44% identical tovarious mammalian plasma membrane Ca2+-ATPases butlacks the C-terminal calmodulin binding domain present in the

mmalian polypeptides. When aligned with mammalianplasma membrane Ca2+-ATPases, PEAlp has a 70- to 80-amino acid N-terminal region that extends beyond the Nterminus of these enzymes. This extension has some similarityto the transit peptide of the plastid envelope phosphate trans-locator and may function to target the protein to the plastid.Antibodies raised against a portion ofPEAlp recognize a single90- to 95-kDa polypeptide in chloroplast inner envelope prep-arations. Transcript abundance as determined by RNase pro-tection was found to be 7- to 9-fold higher in roots than inleaves. Possible roles for a plastid envelope calcium pump aresuggested.

Higher plant plastids are bounded by a double-membranesystem termed the plastid envelope. The envelope plays anactive role in maintaining distinct intracellular environmentsin the stroma and the cytoplasm and mediates communicationbetween the plastid and the rest of the cell. The two mem-branes constituting the envelope are termed the outer enve-lope and the inner envelope. The inner envelope is known tocontain several metabolite transporters and various biosyn-thetic enzymes (1), although primary structural informationfor these polypeptides is sparse. To date, only a handful ofgenes encoding inner envelope proteins have been cloned andsequenced. These include genes for the phosphate translo-cator (2, 3), the product of the maize Btl gene (4), and aprotein ofunknown function (5). To extend our knowledge ofthe structure and function of plastid envelope proteins, aproject was initiated in which cDNA clones for potentialchloroplast envelope polypeptides were selected from anexpression library using antisera against size-selected poolsof polypeptides from purified chloroplast envelopes. Thispaper describes anArabidopsis gene cloned by this approach,verification of the subcellular localization of the polypeptideproduct, and a preliminary analysis of its expression indifferent plant tissues.The polypeptide encoded by this gene belongs to the

superfamily of P-type ATPases based on sequence homologyand overall structure. P-type ATPases are cation transporters

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

found in both prokaryotes and eukaryotes. Distinguishingcharacteristics of P-type ATPases include formation of aphosphorylated enzyme intermediate during the reactioncycle, inhibition by vanadate, and a requirement for Mg2+ aswell as the ion(s) transported. P-type ATPases share severalregions of high sequence identity, particularly in regionsinvolved in ATP-binding and phosphorylation (6,7). They areintrinsic membrane proteins with at least eight transmem-brane helices predicted by sequence analysis.Ca2+-ATPases from animals are among the best studied

P-type ATPases. Their function is to establish steep Ca2+gradients across cellular membranes and maintain cytoplas-mic Ca2+ at submicromolar concentrations. This allowstransient increases in cytoplasmic Ca2+ to mediate the trans-duction of various signals. These enzymes fall into twocategories: Ca2+-ATPases located in the plasma membrane(PM Ca2+-ATPases) and Ca2+-ATPases located in the sar-coplasmic/endoplasmic reticulum membranes (SER Ca2+-ATPases). Primary structure similarity between these twotypes of enzyme is -30%, whereas similarity within thesetypes is >50%o (8).We have cloned a P-type ATPase unique in both its

subcellular localization and its structure. It is found in theplant plastid envelope, a membrane not previously known tocontain a P-type ATPase. Its deduced primary structure mostclosely resembles that of mammalian PM Ca2+-ATPases. Itis, however, clearly distinct from the mammalian polypep-tides and appears to represent an unusual type of calciumtransporter.

MATERIALS AND METHODScDNA Cloning. An Arabidopsis thaliana (L. cv. Columbia)

shoot cDNA library in AYES (9) was screened with anantiserum against spinach chloroplast envelope proteins inthe 55- to 75-kDa size range, and positive clones were isolatedand characterized as described (10). This resulted in thecloning and identification ofPEAJa (see Fig. 1). Subsequentisolation ofa longer cDNA, PEA]b (see Fig. 1), was achievedby screening the same library by hybridization at high strin-gency (11) with a random-primed 32P-labeled DNA probemade from a 254-bp EcoRI/Kpn I fragment of PEAJa (uti-lizing an EcoRI site on the polylinker and the 5' Kpn I site).Genomic Cloning. The gene for PEAl (gPEAJ) was cloned

by screening an A. thaliana (L. cv. Columbia) genomiclibrary in AGEM-11 (Promega) using a random-primed 32p-labeled DNA probe made from a 298-bp Xho I/Xmn Ifragment of PEAJb (utilizing an Xho I site on the polylinker

Abbreviations: PM, plasma membrane; SER, sarcoplasmic/endoplasmic reticulum.tPresent address: Department of Biological Sciences, Stanford Uni-versity, Stanford, CA 94305.§To whom reprint requests should be addressed.IThe sequences reported in this paper have been deposited in theGenBank data base (accession nos. L08468 and L08469).

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and the 5' Xmn I site) with hybridization at high stringency[final wash, 0.lx standard saline citrate (SSC) at 65°C] (11).DNA Sequencing. Restriction fragments of the cloned DNA

were subcloned into pBluescript SK vectors (Stratagene).Both strands of the denatured double-stranded plasmid tem-plates were sequenced in entirety by the dideoxynucleotidemethod (12) with Sequenase (United States Biochemical).Products of asymmetric PCR were sequenced by the samemethod.Sequence Analysis. DNA and peptide sequence analysis

employed various programs of the GCG (Genetics ComputerGroup) sequence analysis software package, includingTFASTA for data base homology searches, PEPPLOT for hy-dropathy analysis, GAP for alignment of individual sequences,and PILEUP for multiple sequence alignments.

Nucleic Acid Preparations. Total RNA was purified fromleaves or roots of 6-week-old Arabidopsis plants as described(13) with an extraction buffer consisting of 6% p-aminosalicylic acid (sodium salt), 6% butanol, 2% polyvinylpyr-rolidone, 2% triisopropylnaphthalene sulfonic acid, 50 mMNaCl, 10 mM Tris HCl (pH 8.5), 0.5% 2-mercaptoethanol.Polyadenylylated RNA was prepared with biotinylated oli-go(dT) and magnetic streptavidin beads (Promega) accordingto the manufacturer's instructions. DNA was prepared fromwhole liquid-cultured Arabidopsis as described (14).RNA Analysis. RNase protection analysis using Esche-

richia coli RNase I (Promega) was done essentially as sug-gested by the manufacturer. Total RNA prepared as de-scribed (10 jig), or yeast RNA used as a control, washybridized to 5 x 104 cpm of a 32P-labeled antisense RNAprobe generated by in vitro transcription from pBluescript(Stratagene) containing either an insert subcloned fromgPEAJ or a 160-bp EcoRI/HindlIl fragment of an Arabidop-sis cytoplasmic cyclophilin cDNA. Quantitation of the ra-dioactivity in the protected fragments was done by radioan-alytical imaging of the dried gel with a PhosphorImager(Molecular Dynamics).For RNA-based PCR, polyadenylylated RNA from leaves

of 6-week-old Arabidopsis plants (1 ,ug) was treated withRNase-free DNase and reverse-transcribed with avian my-eloblastosis virus reverse transcriptase (Promega). This ma-terial was subjected to PCR using two gene-specific 21-bpoligonucleotide primers flanking the intron location deter-mined by RNase protection analysis. PCR products to besequenced were amplified asymmetrically with one primer at50 pmol per 100 ,ul of reaction mixture and the other at 1 pmolper 100 ,ul of reaction mixture (15). Unincorporated nucleo-tides and residual primers were removed by centrifugationthrough Centricon 100 membranes.

Preparation of Antibodies. Polyclonal antisera against chlo-roplast envelope proteins and against a fusion protein be-tween theE. coli maltose-binding protein and the polypeptideexpressed from an internal 702-bp EcoRI/Xho I fragment ofthe PEAla cDNA were prepared and affinity purified asdescribed (10).Immunoblot of Plant Subcellular Fractions. Pea chloro-

plasts were isolated and further fractionated as described(10). Envelope membranes were prepared and further sepa-

rated into inner and outer membranes (16). Pea microsomalmembranes were isolated (17). Samples containing 50 ,ug ofprotein were solubilized, electrophoresed, and immunoblot-ted with purified antibodies against the peptide expressedfrom the PEAJa cDNA as described (10).Genomic DNA Hybridization Analysis. Arabidopsis DNA

prepared as described was digested to completion with re-

striction enzymes, electrophoresed through 0.7% agarose,

capillary blotted onto Hybond N+ membrane (Amersham),and probed with a random-primed 32P-labeled DNA probemade from a 1.24-kb EcoRI fragment containing the 5' end ofPEAla. Procedures described in ref. 11 were followed.

RESULTS

Isolation and Analysis of cDNA and Genomic Clones. Thescreen for cDNA clones encoding chloroplast envelope poly-peptides led to the isolation of seven types of non-cross-hybridizing clones (10). One ofthese types was represented bya single isolate, PEAla. Sequence analysis of the 2447-bpinsert (Fig. 1) revealed it to be a partial cDNA clone with an

open reading frame encoding a polypeptide with extensivesequence homology to mammalian PM Ca2+-ATPases.Screening an additional 500,000 plaques with a 5' fragment ofthe PEAJ a cDNA yielded two additional cDNA clones, one ofwhich (PEAlb) was longer (2904 bp) yet still partial (Fig. 1).A genomic clone, designated gPEAI, was isolated as

described. A 4667-bp region of this clone encompassing thecDNA was sequenced. Comparison between the genomicand cDNA sequences revealed the presence of 5 intronsranging in size from 75 to 174 bp. An additional intron of 470bp in the region upstream from the 5' end of the incompletePEAJb cDNA was identified and mapped by RNase protec-tion analysis and sequencing of PCR-amplified cDNA. Thisallowed determination of the entire PEAl open reading frame(Fig. 2). The translation initiation codon (ATG) was identifiedbased on a number of criteria: it is the first ATG in all threereading frames downstream of a probable TATA box; thereare stop codons in all frames between the ATG and theprobable TATA box; and it specifies the only in-framemethionine upstream of the 5' end of the partial cDNAPEA] b. The deduced open reading frame ofPEA] encodes apolypeptide with 946 amino acids and a molecular mass of103.7 kDa. The discrepancy between the size of the peptideindicated by the isolated cDNA and the molecular mass ofthepeptides used for antigen might be attributable to proteolyzedPEAlp in our antigen preparation.

Sequence Analysis. PEAlp shares 45% amino acid identitywith rat brain PM Ca2+-ATPase isoform II (18) and 20-33%amino acid identity with other P-type ATPases. It has thetypical hydrophobicity profile of P-type ATPases (8) with 10hydrophobic stretches long enough to be transmembranehelices and a large extramembrane loop between hydropho-bic regions 4 and 5 and a smaller one between hydrophobicregions 2 and 3. The conserved phosphorylation site (Asp-456) and fluorescein isothiocyanate binding site (Lys-581) arereadily identifiable. An -71-amino acid N-terminal region inthe plant polypeptide is not present in the PM or SERCa2+-ATPases and may be a chloroplast transit peptide (seeDiscussion) (Fig. 2). The PM Ca2+-ATPases contain a hy-drophilic C-terminal domain of 130-210 amino acids, whichis completely absent in the plant polypeptide. This domain isknown to be involved in interaction with calmodulin andprotein kinases (21). The PM Ca2+-ATPases are also larger

gPEA1

PEAlbXmnlP'

PEAla4 P.-- -1

&I-Il' S

1 2 3 4 kb

FIG. 1. Structure of the Arabidopsis PEA] gene. The sequencedportion of the genomic clone gPEAJ is shown with the positions ofselected restriction enzyme sites. Exons are represented as solidboxes. The cDNA clones PEAJa and PEAJb are shown below themap ofgPEAI. Lines above PEAJa and PEAlb indicate probes usedto isolate PEAlb and gPEAI.

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Proc. Natl. Acad. Sci. USA 90 (1993)++ + + + ++ + t + +$ SPEAlp MESYLNENFGDVKPKNSSDEALQRWRKLCWIVKNPKRFRFTANLSKRSEAEAIRRSNQEKFRVAVLVSQAALQFINSLLSSEYTLSEEVRKAGFEICPDELGSIVEGHD.... IKKLKIHGGTEGLTEKLS 129

P ....................................................................... MGDMTNS .DFYSKNQRNESSHGGEFGCSMEELRSLMELRGTEAVVKIKETYGDTESICRRLK 61SER ........................................................................ MENAHTKTVEEVLGHFGVNESTGLSL. 26

++ + + +$ + + S+$+++$ ++ Al S S+ ++ + + S + ++ A++ + ++ $ + +PEAlp TSIASGISTSEDLLSVRKEIYGINQFTESPSRGFWLFVWEALQDTTIM ILAACAFVSLIVG LM........ EGWPIGAHD ......... CLIVASI1LLVrVTATDYRQSLQFKDL.... DAEKKKIV 239

PM TSPVEGLPGTAPDLEKRKQIFGQNFIPPKKPKTFLQLVWEALQDVTLI ILEIAAI ISLGLSFYHPPGESNEGCATAQGGAEDEGEAEAGWIEGAAILLSVICVVLVTAFNDWSKEKQFRGLQSRIEQEQKFTV 194SER EQVKKLKERWGSNELPAEEGKTLLELVIEQFEDLLVRILLLAACISFVLAWF ................. EEGEETITAFVEPFVILLILVANAIVGVWQERNAENAIEALK... EYEPEMGK 128

$ + $ $$ t$$+++ + + + +4+$$$$$ + ++ + 4+$*$ + ++ + $+ +$ + $ + ++ +PEAlp VQVTRDKLRQKISIYDLLPGDVVHLGIGDQIPADGLF. ISGFSVLINESSLTGES ... ........ EPVSVSVE HPFLLSGTKVQDGSCKMLVTTVGMRTQWGKLMATLSEGGDDE.. 343

PM V... RAGQVVQIPVAEIIWGDIAQIKYGDLLPADGLF.. IQGNDLKIDESSLTGES ........... DQVRKSVDKDPMLLSGTHVMEGSGRMVVTAVGVNSQTGI IFTLLGAGGEEEEKKDKKAKQQDGAAA 311SER VYR2DRKSV4RIKAKDIVPGDIVEIAVGDKVPADIRLTSIKSTTLRVDQSILTGESVSVIKHTDPVPDPRAVNQDKKNMLFSGTN5IAAGKAMGVWATGVNTEIGKIRDEMVATEQE 245

t4 4$ + ++4 ++ S+ ++ + + S A+ A+j+**t+Jt .. +4$ ++$+ + +4$ ++$$PEA1p .. . TPLQVKLNCVKL =DTGLANITFQVLVQGLANQKRLDNSHWIWTADE... LMAMLEYFAVAVTIVVAWEGLPIAVTILSLAFAMKKMMNDKALVRNLAACET 446

PM MEMQPLKSAEGGDADDKKKANMHKKEKSVLQGKLTKIAVQIIGKAGLVMSAITVI ILVLYFTVDTFVVNKKPWLTECTPVYVQYFVKFFIIGVTVLWAVPEGLPLAVTISLAYSVKKMMKDNNLVRHLDACET 444SER .RTPLQQKLDEFGEQLSK... VISLICIAVWI INIG........HFNDPVHGGSWIRGAIYYFKIAVALAVAAIPEGLPAVITTCLALGTRRMAKKNAIVRSLPSVET 341

+$ ++ $$$$$$$$ $ $+$+ + ++ + + + ++ + + + + + ++PEAlp MGSATTICSDKTGTLTTNHMTVVKACI ........ CE.QAKEVNGPDAAMKFASGIPESAVK ..... LLLQSIFTNTGGEIIWGKGNKTEIL....GTPTETALLEFGLSLG ...... GDFQEVR ... QASNV 552

MGNATAICSDKTGTLTTNRMTVVQAYV........ GDVHYKEIPDP ....... SSINAKTLE.....LLVNAIAINSAYTTKILPPEKEGALPRQVGNKTECGLLGFVLDLR ......QDYEPVRSQMPEEKL 551SER LGCTSVICSDKTGTLTTN4MSVCRMFILDKVDGDTCSLNEFTITGSTYAPIGEVHKDDKPVKCHQYDGLVELAT7ICALCNDSALDYNEAGVYEKVGEATETALTCLVEKMNVFDTELKGLSKIERANACNSV 474

$ + $++ ++ + ++ 444+4+4+4+ 4+ + 4+ + $$++$+PEAlp VK ......VEPFNSTKKRMGVVIELPERH FRAHCKGASEIVLDSCDKYINKDGEVVPLDEKSTSHL KNIIEEF .. ASEALRTLCLAYFEI.. 634MYK......VYTFNSVRKSMSTVIKMPDES FRMYSKGASEIVIKKCCKILSGAGEPRVFRPRDRDEMVKKVIEPM ..ACDGLRTICVAYRDFPSSPE ... .PDWDNEND LNELTCICWGIEDPVRPE 667

SER IKQLMKKEFTLEFSRDRKSMSVYCTPNKPSRTSMSKMFVKGAPEGVIDRCTHI. RVGSTKVPMTAGVKQKIMSVIREWGSGSDTLRCLAIATHDNPLRREEMHLKDSANFIKYETNLTFVGCVGMLDPPRIE 605

+ + +++ + $$++$ +4+++4 + ++$+$$$$+$$ $+ 444+PEAlp .. GDEFR.... EKSDEELLKLIPK4LQVMARSSPMDKHTLVRLL RTMFQEWAVTGDGTNDAPALHEADIGLAMGISGTE 707PM VPEAIRKCQRAGiTVRMVTGDNiNTARAIAiKCGiiHPGEDFL.. CLEGKEFNRRIRNEKGEIEQERIDKI WPKLRVLARSSPTDKHTLVKGI IDSTHTEQRQVVAVTGDGTNDGPALKKADVGFAMGIAGTD 798

SER VASSVKLCRQAGIRVIMITGDNKGTAVAICRRIGIFGQEEDVTAKAFTGREF ........ DELNPSAQRDACLNMARCFARVEPSHKSKIVEFLQS ..... FDEITAMTGDGVNDAPALKKAEIGIAMG. SGTA 7244$4+ + + 4444$ +44 +t + +$ ++++++$I++ ++ $ $+ + +$ +A $$$$ ++$+ +**+++t4 + + $ ++ 44 + ++t+Jt.A + + + +

PEAlp VAKESADVI ILDDNFSTIVTVAKWGRSVYINIQKF KLTGNWPLIVN L LT EPPQ DLM SPVQDDU4KRSPVGRKGNFISNVMWRNTIIALYQLVII*CLTKKTM 840PM VAKEASDIILTDDNFSSIVKAVMWGRNVYDSISKFLQFQLTVNWAVIVAFTGACITQDSPLKAVQMLWVLIMDTFASLAIATEPPTETLLLRKPYGRNKPLISRTMMKNILGHAWQLTLIFTLLFVGEKM 931

SER VAKTASEMVLADDNFSTIVAAVEEGRAIYNNMKQFI RYALISSNVGEWCIFLTAALGFPEALIPVQLLWVNLVTDGIATALGFNPPDDIMNPPRNPEPLISGWLFFRYLAIGCY..VGAATVGAAAWWF 855

+ $ $+ $ +++ +$ + +$++ ++ ++ ++ + + + + ±..........++ .....+PEAp FGLD.GP ................ DSDL.. TLN. TLIFNIFVFCQVFNEI SSREM.EKIDVFKGILKNYVFVAVLTC... 91TVVFQVIII GTFADTTPLNLGQWLVSIILGFIPVAAAMMI 941

PM FQIDSGR .............. NAPLHSPPSEHY .....TIIFNTFVMMQLFNEINARKIHGERNVFDGIFRNPIFCTIVLG...TFAIQIVIVQFGGKPFSCSPLQLDQWMWCIFIGLGELVWGQVIATI 1039SER IAADGGPRVSFYQLSHFLQCKEDNPDFEGVDCAIFESPYPMTMALSVLVTIEMCNALNS. LSENQSLLRMPPWENIIWLVGSICLSMSLHFLILYVEPLPLIFQITPLNVTQWLMVLKISLPVILMDETLKFV 986

+

PEAlp PVGSH ........................................ 946P4 PTSRLFKAGRLTKEE IPEEELNEDEEIDAERELRRGQILERGLNR IQI RW>FRSS LYEGLEKPESRTS I HNFPEF I E 1172

SER ARNYLEPAILE ............................................................. 997

PM NLTTDTSKSATSSSPGSPIHSLETSL 1198

FIG. 2. Deduced amino acid sequence of PEAlp, aligned with isoform II of the PM Ca2 -ATPase from rat brain (PM) (18) and the SERCa2+-ATPase of rabbit muscle (SER) (19). Amino acids that are identical between PEAlp and the PM Ca2+-ATPase are indicated with a + abovethe position. Amino acids that are identical in all three polypeptides are indicated with a t above the position. Amino acids reported to be involvedin Ca2+-binding by the SER Ca2+-ATPase (20) are indicated with a * below the sequence. The predicted membrane-spanning regions are indicatedwith a line above the sequence. The conserved phosphorylation and fluorescein isothiocyanate binding sites are marked with a o below thesequence. This sequence was deduced from the sequence of the genomic clone. The cDNA clone differed by one nucleotide, causing asubstitution of I for T in position 727 of the amino acid sequence.

than PEAlp in the hydrophilic regions found between theputative transmembrane regions 2 and 3 and the putativetransmembrane regions 4 and 5.

Immunodetection of the Product of PEAI. To confirmlocalization of the product of PEAI to the chloroplast enve-lope and to determine whether the polypeptide is resident inthe inner or outer envelope, an antiserum against the purifiedpeptide expressed from a fragment of the PEAla cDNAcorresponding to amino acids Glu-637-Ser-871 (Fig. 2) wasgenerated, affinity purified with the antigen, and used forimmunoblot analysis of chloroplast and crude microsomalfractions of pea leaves. The crude microsomal fraction con-tained both plasma membrane and endoplasmic reticulum.Results of a representative experiment are shown in Fig. 3.The antiserum recognized the antigen and reacted specifi-cally with a protein of 90-95 kDa only in the inner envelope

Stain Pre-Immune Immune

S~~ F- ^ .F O _F- S X XF- S

9766 -X

45 -

31

21 - _*14

FIG. 3. Immunoblot of membrane fractions from pea with anantiserum against PEAlp. An antiserum raised as described againstan expressed portion of PEAlp was used to probe immunoblots ofproteins from various membrane fractions. Lanes: MW, molecularsize standards; A, fusion protein (1 pg for the Coomassie-stained geland 0.03 ug for the immunoblots); IE, chloroplast inner envelope;OE, chloroplast outer envelope; T, thylakoid membranes; M, mi-crosomes. The sizes of the standards are indicated in kDa.

fraction. The preimmune serum (which was not affinitypurified) did not recognize either the antigen or the innerenvelope protein recognized by the antiserum, although it diddetect a faint band slightly smaller than the 95-kDa protein inthe inner envelope. This was verified by splitting an innerenvelope lane into halves and challenging one with thepreimmune serum and the other with the antiserum (Fig. 3).Genomic DNA Hybridization Analysis. A probe corre-

sponding to part of the PEAJa cDNA hybridized to blots ofArabidopsis DNA cleaved with three different restrictionendonucleases (Fig. 4). In each case, only ont band wasapparent, indicating that PEAI is probably a single copy genein Arabidopsis.Comparison of PEAI Transcript Levels Between Leaves and

Roots. Relative transcript levels were compared betweenleaves and roots by an RNase protection assay. This assaywas performed with 32P-labeled antisense RNA probes forPEA) as well as Arabidopsis cytoplasmic cyclophilin. Re-sults of a representative experiment are displayed in Fig. 5.The cyclophilin probe was used as a control, as the level ofthis transcript is reported to be similar between leaves androots (22). The cyclophilin transcript was found to be 1.5-foldmore abundant in the root RNA than in the leaf RNApreparations. The PEA) transcript was found to be 7- to9-fold more abundant in the root RNA than in the leaf RNApreparations. The presence of relatively large amounts ofundigested PEA) probe after RNase treatment in the pres-ence of Arabidopsis leaf and root RNA may be due to thepresence of unspliced message. The PEA) transcript isthought to be of low abundance in leaf tissue because it ispoorly represented in the cDNA library (-1 clone in 250,000)and was not detected by standard RNA blotting procedures.

DISCUSSIONComparison to other sequences in the GenBank data baseindicates that the product of PEA) is a member of thesuperfamily of P-type ATPases. It is most similar to mam-

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Ft QN

14140-

8454-

5686-

4822-

3675-

2323-

1829-

FIG. 4. Southern blot hybridization of genomic DNA with a

probe generated from PEA]. Each lane was loaded with 5 pg ofArabidopsis DNA digested to completion with the indicated en-zymes. Blots were probed with a 32P-labeled 1.24-kb 5' EcoRIfragment of PEAla and washed at high stringency (O.1x SSC at65°C). The molecular sizes of the standards are indicated in bp.

malian PM Ca2+-ATPases. This similarity is most striking inthe regions between amino acids 405 and 471, which is74-79%o identical to the equivalent region in mammalian PMCa2+-ATPases and amino acids 677-795, which is 68-70%oidentical. The latter region has been shown to be particularlywell conserved among all Ca2+-ATPases (7). Site-directedmutagenesis studies have identified 6 amino acids as probableconstituents of calcium-binding sites on the SER Ca2+-ATPase (20) (Fig. 2). Based on sequence comparisons of thecorresponding amino acids among 30 P-type ATPases,Maguire (23) observed that the composition of the 6 aminoacids was predictive of the ion transported by the ATPase.All six of the corresponding amino acids of PEAlp are

identical to the corresponding residues in the mammalian PMCa2+-ATPases. Therefore, based on the mutagenesis studiesand the trends observed from sequence analysis, PEAlp

400-

undigested

probe _

protentedpro Uct _p,15.f

Iic

cyclophilinprobe

z

;n

I

z

x96

.:::....:.:I...-

PEAl

probe

FIG. 5. Relative transcript levels in roots and leaves determinedby RNase protection. Total RNA from Arabidopsis leaves, Arabi-dopsis roots, or yeast was probed with 32P-labeled antisense RNAprobes generated either from a cDNA for Arabidopsis cytoplasmiccyclophilin as described or from a subcloned 367-bp EcoRI/Sac Ifragment from gPEAI. The size of the undigested cyclophilin probeis 204 nucleotides and the expected size of the protected fragment is160 nucleotides. The size of the undigested PEA] probe is 375nucleotides and the expected size of the protected fragment is 213nucleotides. The molecular sizes of the standards are indicated innucleotides.

should be typed as a Ca2+-ATPase. However, in the absenceof biochemical confimation, we cannot ignore the possibilitythat PEAlp may transport ions other than Ca2+.The immunochemical evidence presented indicates that

the product of PEA) is found in the chloroplast inner enve-lope (Fig. 3). The N-terminal extension on the predictedPEA] gene product, which is not homologous to other P-typeATPases, may therefore function as a chloroplast transitpeptide. The difference in sizes estimated by immunoblottingand by sequence analysis is consistent with this hypothesis.Chloroplast transit peptides for proteins targeted to thestroma or thylakoid have been extensively studied. Commonfeatures include a high proportion of hydroxylated aminoacids and small hydrophobic amino acids, a net positivecharge, and a lack of negatively charged amino acids (24).Few transit peptides for proteins targeted to the inner enve-lope have been characterized; however, it has been notedthat the transit peptide of the phosphate translocator differsfrom typical chloroplast transit peptides in several respects.These include charged residues close to the N terminus, ahigher proportion of acidic residues, lower proportions ofhydroxylated amino acids and small hydrophobic aminoacids, and a high arginine/serine ratio (3). The N-terminaldomain of PEAlp shares these features. In addition, the first3 amino acids (Met-Glu-Ser) are identical between the PEAlpN terminus and the transit peptides of all three sequencedphosphate translocators (refs. 2 and 3; B. Schultz, GenBankaccession no. X67045).There are not, to our knowledge, any reports of calcium-

transporting ATPase activity in the plastid envelope. Ca2+-ATPases have, however, been described in both the plasmamembrane and the endoplasmic reticulum of plant cells (25).We consider it unlikely that PEAlp is one of these for thefollowing reasons. (i) Immunochemical data indicate thatPEAlp is localized in the inner chloroplast envelope (Fig. 3).(ii) PEAlp may have a chloroplast transit peptide. (iii) Thesize of PEAlp is considerably smaller than the 140 kDareported for the plant PM Ca2+-ATPase (25). (iv) PEAlp hasrelatively little sequence identity with the tomato endoplas-mic reticulum Ca2+-ATPase (33%) (26).Attempts made in our laboratory to identify Ca2+-

dependent ATPase activity or ATP-dependent Ca2+ uptake inpreparations of spinach chloroplast envelope have thus farbeen negative (data not shown). This indicates the followingpossibilities. (i) The activity of PEAlp is low and cannot bemeasured against a background of Ca2+-independent ATPaseor passive Ca2+ uptake. (ii) PEAlp was inactive under theconditions used. (iii) The membranes were not ionicallysealed. (iv) The membrane vesicles prepared were of theopposite orientation required to observe ATP-dependentuptake of Ca2+. (v) PEAlp is not a Ca2+-ATPase.

Isolated chloroplasts take up calcium upon illumination(27). Kreimer et al. (28) argue that the Ca2+ influx is notmediated by an ATPase because the Ca2+ uptake is insensi-tive to vanadate and is inhibited by lipophilic ions andruthenium red, an inhibitor of the uniport type carrier ofmammalian mitochondria. The role of the envelope ATPasemight instead be to export Ca2+ from the stroma into thecytosol. Consistent with this idea, cytoplasmic membranesfrom the cyanobacteria Anabaena (29) and Synechococcussp. PCC 7942 (T.B., unpublished results) contain a Ca2+_ATPase that most likely exports Ca2+. Based on the endo-symbiont hypothesis, PEAlp may be related to the cyano-bacterial Ca2+-ATPase. If so, it would be expected to exportCa2+ into the cytoplasm. As all known Ca2+-ATPases pumpCa2+ out of the cytoplasm, this would be an unusual functionfor such an enzyme.

Miller and Sanders (30) have suggested that Ca2+ may playa role in signaling between the plastid and the rest of the cellbased on their observation that cytosolic Ca2+ was influenced

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Proc. Natl. Acad. Sci. USA 90 (1993)

by chloroplast metabolism. If PEAlp exports calcium, itcould play a role in such a communication process. There area number of responses where Ca2+ has been implicated as asignaling molecule and the plastid has been implicated as thestimulus receptor. These include control of stomatal apertureby light and CO2 (31, 32), cytosolic distribution of chloro-plasts in response to light (33), and root gravitropic curvature(34). Conceivably, the relatively high expression ofPEAlp inroots is related to the gravitropic response.A Ca2+-transporting ATPase in the chloroplast envelope

may also help maintain low levels of free Ca2+ in the stroma.These levels have been estimated to be in the micromolarrange (35), whereas total chloroplast Ca2+ concentration isseveral millimolar. Calcium is thought to play a role in theregulation of stromal fructose 1,6-bisphosphatase and se-doheptulose 1,7-bisphosphatase (36), the gating betweenlocalized and delocalized proton gradient coupling in thethylakoid (37), and oxygen evolution by photosystem II (36).By regulating free Ca2+ in the stroma, PEAlp may also playa role in the regulation of chloroplast metabolism.

We would like to thank Dr. Ron Davis and his laboratory forproviding the genomic and cDNA libraries; Dr. Chuck Gasser for thecDNA clone of the Arabidopsis cytoplasmic cyclophilin gene; andDrs. Kirk Apt, David Kehoe, and Fitnat Yildiz for critical evaluationof the manuscript. This work was supported by National Institutesof Health Grant GM42609-02 (to N.E.H.). This is Carnegie Institu-tion of Washington Department of Plant Biology publication no.1166.

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