Transposon-Disruption of a Maize Nuclear Gene, Thal, Encoding a Chloroplast Seca Homologue: Zn Vivo Role of Cp-Sed in Thylakoid Protein Targeting

Transposon-Disruption of a Maize Nuclear Gene, thal, Encoding a Chloroplast SecA Homologue: Zn Vivo Role of cp-Sed in Thylakoid Protein Targeting

Rodger Voelker, Janet Mendel-Hartvig,’and Alice Barkan

Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403 Manuscript received August 16, 1996 Accepted for publication October 14, 1996

ABSTRACT A nuclear mutant of maize, thal, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the thal locus facilitated the cloning of portions of the thal gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that thal encodes a Sed homologue (cp-SecA). The thal-ref allele is either null or nearly so, in that thal mRNA is undetectable in mutant leaves and cpSecA accumulation is reduced 240-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-Sed function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome J and in proteintargeting in the etioplast, a nonphotosyntheticplastid type. Our finding that theaccumulation of properly targeted plastocyanin and cytochrome fin thal-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.

ITOCHONDRIA and chloroplasts are highly com- chloroplasts and thylakoid membranes demonstrated M partmentalized organelles believed to be evolved that sodium azide, a potent inhibitor of the bacterial from ancient endosymbiotic bacteria. Of the proteins secA gene product, inhibits the translocation of a subset localized to these organelles, only a minority are synthe- of proteins across pea thylakoid membranes (HENRY sized on organellar ribosomes. The majority are synthe- et al. 1994; KNOTT and ROBINSON1994). In addition, sized in the cytosol and subsequently targeted to the homologues of bacterial secA and secY have been found organelle. Regardless oftheir origin,all organellar pro- on the plastid genomes of several algae (SCARAMUZZIet teins must ultimately be sorted to the correct intra- al. 1992; FLACHMANNet al. 1993; REITH and MUNHOL organellar compartment. LAND 1993; VALENTIN1993), and the nuclear genomes A “conservative sorting” hypothesis has been pro- of higher plants encode chloroplast-localized SecA (cp posed whereby nuclear-encoded proteins are initially SecA) and SecY homologues (NAKAIet al. 1994; BERG targeted to the mitochondrial matrix or chloroplast HOEFER et al. 1995; LAIDLER et al. 1995; NOWet al. stroma (derived from the cytoplasmof the ancestral 1995). Finally, cp-Sed can function in uitro to facilitate endosymbiont) and then “secreted” across internal or- the translocation of several proteins across the thyla- ganellar membranes via mechanisms evolved from the koid membrane (YUAN et al. 1994; Nomet al. 1995). endosymbiont’s secretory system (WTLet al. 198’7; Intrachloroplast protein sortinghas been studied pri- SMEEKENSet al. 1990). This notion was first suggested marily with in uitro targeting experiments in which ra- when structural and functional similarities were noted diolabeled protein precursors are incubated with iso- between bacterial signal peptides and the domains that lated chloroplasts or isolated thylakoid membranes. target proteins to the thylakoid lumen or mitochondrial These experiments have provided evidence €or at least intermembrane space. Although a conservative sorting three pathways by which proteins can translocate into mechanism for targeting to the mitochondrial inter- or across the thylakoid membrane. Nuclear-encoded membrane space remains controversial (GLICKet al. proteins destined for the thylakoid lumen are synthe- 1992), there is increasing evidence that such a mecha- sized with cleavable lumenal targeting sequences that nism is involved in the sortingof certain proteins to the resemble bacterial signal peptides (reviewed by ROE thylakoid lumen. In uitro experiments involving isolated INSON and KLOESGEN 1994; CLINEand HENRY1996). A subset of these engages a “set"-like pathway, which Corresponding authorc Alice Barkan, Institute of Molecular Biology, requires ATP and is facilitated by cp-SecA in uitro. Oth- Klamath Hall, University of Oregon, Eugene, OR 97403. E-mail: [email protected] ers are translocated across the membrane by a mecha- ‘fiesent address: Oregon Health Sciences University, Portland, OR nism that does not requireATP nor any soluble factors 97201. but that is strictly dependent upon a trans-thylakoidal

Grnrtirs 145 467-478 (Fehruary, 1997) 468 R. Voelker, J. Mendel-Hartvig and A. Barkan

ApH. A third pathway is engaged by the nuclear-en- In each case, we observed the characteristic "fingerprint" of coded protein LHCP, whose integration into thethyla- the thal-refmutation [Le., the loss of the core subunits of the electron transport complexes and the over-accumulation of koid requires GTP butdoes not involve a cleavable tar- the stromal precursor to plastocyanin (VOELKERand BARKAN geting sequence. LHCP integrationis facilitated in vitro 1995)], which confirmed that these were homozygous thal- by 54CP, a homologue of the signal recognition particle refseedlings. protein SW54 (HOFFMANand FRANKLIN 1994; LI et al. Etiolated thal-refleaves were obtained by germinating and 1995). growing the F, progeny of a thal-reJ/+ plant in the absence of light. After 9 days of growth at 26", plants were numbered These in vitro experiments have revealed the mecha- under a green safelight and individual etiolated leaf tips were nistic complexityof thylakoid membrane assembly. harvested and immediately frozen. The plants were then trans- However, to fully appreciate the nature of these tar- ferred to continuous light for 24 hr and thal-refplants were geting pathways and their interrelationships in vivo, it identified by virtue of their reduced pigmentation and in- is essential to study the consequences of genetic disrup-creased chlorophyll fluorescence when viewed with a hand- held ultraviolet light (MILES 1982). Leaftips of several tion of thylakoid membrane targeting. We described greened thal-refand wild-type seedlings were harvested. two maize mutants, hcfl06 and thal, with defects in the The thal locus was mapped at the University of Missouri- localization of different sets of proteins to the thylakoidColumbia Maize RFLP Laboratory to chromosorne 3 in Bin lumen (VOELKERand BARKAN1995). The thal refer- 3.04. enceallele described previously (thal-ref)interferes Isolation and analysis of plant DNA: DNAwas extracted from the above-ground portion of single 2-wk-old seedlings. with targeting of nuclear-encoded proteins thought to Plants were ground in liquid nitrogen to a fine powder and engage the see-like pathway and also with the targeting thawed in a lysis buffer consisting of 7 M urea, 0.25 M NaCl, 50 of the chloroplast-encoded protein cytochrome $ In mM Tris-HCl (pH 8.0), 1% N-lauryl-sarcosine,20 mM EDTA, contrast, the hc-106 mutation disrupts the targeting of 0.25% P-mercoptoethanol. After a 5-min incubation at 37", proteins that engage the ApH-dependent pathway in samples were extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with an equal volume of vitro. Thesephenotypes evidencethat the provided isopropanol. Samples were resuspended in TE [IO mM Tris- ApH-dependent and sec-like pathways are genetically HCl (pH 7.5), 1 mM EDTA] containing RNAse A (20 pg/ separable in vivo. ml), extracted once with phenol/chloroform/isoamyl alcohol Somatic instability of the hcfl06 and thal-ref muta- and once with chloroform/isoamyl alcohol. After the addition tions indicated that both were the result of transposableof an equal volume of a solution containing 1.6 M NaCl, 13% polyethylene glycol 8000, samples were incubated at room element insertions. The hcfl06 gene was cloned pre- temperature for 30 min and the DNA pelleted by centrifuga- viously by virtue of its association with a Mutator (Mu) tion at 12,000 X g for 15 min. Pellets were rinsed with 70% transposon (MARTIENSSEN et al. 1989). Here, we de- ethanol and the DNA was resuspended in TE. scribe the hlu-facilitated cloning of the thal gene and For Southern analysis, 4 pg of DNA was digested overnight report that the thal gene encodesa homologue of the with 15 units of restriction enzyme. DNAwas fractionated by electrophoresis (1.5 V/cm, 20 hr) in 0.8% agarose gels bacterial and plantSecA proteins. The accumulation of prepared with 1X TAE [40 mM Tris-Acetate, 1 mM EDTA (pH cp-Sed is exceedingly low in leaves of thal-refplants. S)] and 0.2 pg/ml ethidium bromide. DNA in the gel was These results, in conjunctionwith the specific targeting depurinated, denatured, neutralized, and transferred to Mag- defects associated with the thal-ref mutation, confirm nacharge nylon membrane (MSI) as described (SAMBROOKet and extend the roles for cpSecA that were postulated al. 1989). DNAwas fixed to the membrane by exposure to light (1200 pJ/cm') in a Fisher UV Crosslinker. based upon the results of in vitro experiments.It is W Membranes were prehybridized in hybridization buffer [SX interesting, however, that even those lumenal proteins SSC (SAMBROOKet al. 1989), 0.1% Nlaurylsarcosine, 0.02% whose translocation is affected in thal-ref mutants do SDS, 1% casein, 200 pg/ml salmon DNA] for 1-2 hr. DNA accumulate within the thylakoid lumen to substantial probes, prepared by polymerase chain reaction amplification levels. This observation suggests thepossibility that cp- of plasmid inserts in the presence of digoxigenin-labeled SecA, while afacilitator of translocation across thethyla- dUTP (dUTP:dTTP = 1:20), were denatured by incubation in boiling water for 5 min and added to the hybridization koid, is not absolutely required for the translocation of solution. Hybridization proceeded for 16-24 hr. Filters were any protein yet examined. washed six times for 15 min each in 0.5X SSC, 0.1% SDS. Standard prehybridization, hybridization, and washes were at 65" (Figure 1). Low stringency hybridization and washes were MATERIALSAND METHODS at 54" (Figure 5). Bound probe was detected with the GENIUS Plant material: The thal-ref mutation was recovered from chemiluminescence system (Boehringer Mannheim). a Mu-active maize line propagated in the laboratories of S. Genomic cloning: DNA (30 pg) from a homozygous Ihal- HAKE(USDA Plant Gene Expression Center, Albany, CA)and ref seedling was digested with EcoRI and fractionated in an M. FREELINC(University of California, Berkeley). Numerous agarose gel, as described above. A gel slice containing DNA thal-ref/ + plants were propagated in parallel for several gen- fragments of 2000-3000 bp was excised. DNA was extracted erations by crossing with inbred lines. Heterozygous plants from the gel with QJAEX beads, according to the manufactur- were then self-pollinatedto recover homozygous mutant seed- er's instructions (Qiagen). The DNAwas ligated into Blue lings. Mutant seedlings used for DNA and RNA extraction Script SK+ plasmid (Stratagene) that had been digested with were identified initially by virtue of their pale green pigmenta- EcoRI, and electroporated into XL1-Blue MRF' cells (Stra- tion. The protein composition of a leaf tip harvested from tagene). Colony lifts (SAMBROOKet al. 1989) were probed by each potential thal-refseedlingwas analyzed on Western blots. hybridization with a radiolabeled Mu1 probe. R ole ofIn Vivo Role Chloroplast469 SecA

Reverse transcription-PCR amplification of a partial thal to probe for actin mRNA was obtained from WIMVAN HEECK- cDNA: A degenerate primer, 5'-(G/A/C) GC(G/C) GT(G/ EREN (University of Oregon). C)CC(G/C) GTCAT (G/A/T/C)CG3', was designed against Isolation and analysis of protein: Leaf or chloroplast pro- the highly conserved SecA peptide sequence gly-met-thr-gly- teins were extracted and analyzed on Western blots as de- thr-ala and was used to prime reverse transcription from poly scribed previously (VOELKERand BARKAN 1995). Antisera A+ leaf RNA isolated from B73 inbred maize seedlings. Each raised against pea cpSecA and LHCP were generously pro- reaction (20 pl) contained 1 ng of poly A+ RNA, 100 pmol vided by T. ENDO (NagoyaUniversity) and BILLTAYLOR of primer, 20 units RNAsin ribonuclease inhibitor, 15 units (CSIRO, Canberra),respectively. Antisera specificfor OEC23, AMV reverse transcriptase, 1 mM each dNTP, 10 mM Tris-HC1 cytochrome f; and plastocyaninwere described previously (pH 8.8), 50 mM KCl, 5 mM MgC12, 0.1% Triton X-100, 50 (VOELKERand BARKAN1995). ng/pl actinomycin-D. Reactions were incubated at 21" for 10 min and thenfor 1 hr at 42". The template RNA was degraded RESULTS by the addition of 4 pl 1 M NaOH/ 0.1 mM EDTA and incubation at 60" for 1.5 hr. Nucleic acids were precipitated by add- Molecular cloning of the thal mutation: The thal-ref ing 2.5p1 3 M NaAcetate, pH 5.2, and 75p1 ethanol, and recovered by centrifugation. The resulting pellets were rinsed mutation arose in a maize line with active Mu transpo- with 70% ethanol, dried, and resuspended in 20 p1 water. sons. The frequent appearanceof small phenotypically PCR amplification using the cDNA as a template was per- normal sectors on the pale green mutant leaves sup- formed by using a thul gene specific primer, 5"AGCCCGAGG ported the notion that the mutation resulted from a TCT CCGCGG3' (see Figure 3), and a degenerate primer, 5'- Mu insertion. To allow the segregation of thal from (G/C) CC(G/C)GT(G/A) AA(T/C) TC(G/A)TC(G/A/C) AC(G/T/A) AT-3', designed according to the highlycon- unlinked Mu insertions, numerous heterozygous plants served SecA motif ile-Val-aspglu-phe-thr-gly.Based upon the were outcrossed to inbredlines. Resulting heterozygous sequence of pea cpSecA, we expected that this primer pair plants were again outcrossed. After several rounds of would amplify a cDNA fragment of -900 bp. Each 100 pl this crossing scheme, DNA was prepared from individ- reaction contained 10 p1 of the reverse transcription product, ual mutant plants representing distantly related 100 pmol of each primer, 0.2 mM each dNTP, 50 mM KC1, 10 mM TrisHCl (pH 8.3), 1.5 mM MgClp, 0.01% gelatin, 0.05% branches of the pedigree. DNA was also prepared from Tween-20. The reactions were incubated at 95" for 5 min after homozygous wild-type plants closely related to the mu- which 5 units Taq DNA polymerase were added and allowed tant plants used in this analysis. to amplify for five cycles of 95"/30 sec, 37"/30 sec, and 72"/ Mu insertions were visualized by Southern hybridiza- 90 sec and 25 cycles of 95"/30 sec, 45"/30 sec, and 72"/90 tion with probes corresponding to members of the Mu sec. A second round of amplification was performed using 5 p1 of the initial PCR products and the same buffer conditions family (CHANDLERand HARDEMAN1992). Probes for used above. However, the reactions contained only 10 pmol Mu3, Mug, and MuDRfailed to detect any insertions in of each primer and the following "touchdown" temperature common to all mutant individuals. However, a Mu1 profile was used for amplification: 20 cyclesof 94"/30 sec, probe revealed a 2.4kb EcoN fragment in all mutant 56"/30sec (-0.5"/cycle), and 72"/60sec and 20 cyclesof individuals that was absent in all homozygous wild-type 94"/30 sec, 45"/30sec, and 72"/60sec. The 906bp DNA fragment was gel-purified and subcloned into a Bluescript individuals (representative results are shown in Figure SK+ plasmid. 1A). Genomic DNA comigrating with this fragment was Multiple sequence alignments Sed homologues from a gel-purified and ligated into a plasmid, and bacterial variety of organisms (GenBank accession numbers: Pisum sati- transformants were screened by hybridization with a vum (common pea), X82404, Synechococcus PCC7942, X74592, Mu1 probe. The cloned 2.4kb fragment contained the Escherichia coli, M20791, Spinacia okacea (spinach) 249124) 1.4kb Mu1 element flanked by 333 bp and 719 bp of were compared with the thal protein sequence deduced from the partial genomic and cDNA clones (GenBank accession genomic DNA (Figure 1B).The genomic Southern numbers U71124 and U71123, respectively).Alignments were blots used for the linkage analysis were reprobed with calculated using the GCG program package (Wisconsin Ge- each of these flanking DNA segments (Figure 1C and netics Computer Group). Default alignment parameters were data not shown). Both flanking probes detected just a chosen. Homologies were determined using the GES index single band in all thal samples, of 2.4 kb. This band scale for 85% homology. was absent in all homozygous wild-type samples. The PCR amplification of genomic DNA: Amplification reactions (100 pl) were performed containing20-50 ng of maize genomic wild-type plants were polymorphic with regard to the DNA, 20 pmol of each primer (primer-A: 5'-ACCGTGGC GCG cloned locus (compare lane 1 with lanes 6 and 12), the TATC AAG3', primer-B CTGCAATC TGATACGAGAGT-3', Mu- size of the hybridizing fragments correlating with the primer: 5'GGCCTCCATTT CGTCGAATCG3'), 0.2mM of each line from which the wild-type allele arose. These results dNTP, 50 mM KCl, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgCl2, 0.01% gelatin, 0.05% Tween-20. After a 5-min incubation at indicate that the cloned fragment is genetically linked 95", 5 units of Taq DNA polymerase were added and allowed to the thal locus. to amplify for 30cycles as follows: 94"/30 sec, 56"/30 sec, If the cloned genomic fragment contains the Mu in- and 72"/45 sec. 10 pl of each reaction was analyzed by gel sertion responsible for the thal-ref phenotype,one electrophoresis and DNA was visualized with ethidium bro- would expect that excision of the Mu1 element from mide. Isolation and analysis of mRNA Total and poly A+ leaf these sequences would result in phenotypic reversion. RNA were purified and analyzed in Northern and RNAse- TOtest this prediction, thestructures of the correspond- protection experiments as described previously (MARTIENSSEN ing genomic sequences in sectors of revertant tissue et al. 1989; BARKAN 1993; BARKANet al. 1994). The clone used were analyzed. DNA was extracted from revertant sec- a thcrl s thal 3khc- ""'1""

- 4.0

- 3.0 b + + + - - - + + + prirnerA "- + + + + + + k\rrimerB - 2.0 + + + + + + - - - Mu primer

1 2 3 4 5 6 7 8 91011 12 b EcoRI MuI EC"Hl Y/&

C C i -..c' -A:':,: QTAC~TACCCAMTATATM~CQ~CACTCQT~CCTCACTACAQ -b .:.G.:.'ZGGG CATQCATOOQTTTATATATTQCCCQTQAQCATAGQAGTCTGTC. 2.0

1 .s In Vivo Role of Chloroplast SecA 471 in DNA fragments whosesizes were consistent with openreading frame in thethal genomic cloneand those predicted (198 and 270 bp, respectively). No am- sequences encoding pea and spinach cp-SecA ended 19 plification was observed from the homozygous thal-ref bp 5’ to the Mu1 insertion and did not continue on template when the gene-specific primers A and B were the other side of the Mu element, suggesting that Mu1 paired. This was expected since the polymerase chain had inserted near the 5‘end of an intron (data not reaction fails to amplify across intact Mu elements shown; GenBank accession No. U71124). (A. B. and M. WALKER,unpublished observations). To determine definitively whether the cloned frag- DNA extracted from a revertant sector that flanked ment derives from a secA homologue, reverse transcrip- the mutant tissue used for the thal control reactions tion followed by PCR amplification was used to obtain was analyzed in the same way. Primers A and B paired downstream mRNA sequence. Published sequences of with the Mu primer amplified the two fragments pre- secA homologues in other organisms were used to de- dicted for the mutant allele (Figure 2B, lanes 3 and 6), sign two degenerate primers that were expected to an- as expected ifjust one of the two alleles had reverted. neal with highly conserved mRNA regions -1000 and Primer A paired with primer Bamplified a fragmentof 900 nucleotides downstream of those encoded by the -340 bp (Figure 2B, lane 9). This amplification pro- cloned genomic sequences. Themore downstream vided evidence that Mu1 had excised from one of the primer was used to prime a reverse transcription reac- mutant alleles in the cell that gave rise to the revertant tion on a template of wild-type leaf mRNA. The nested sector. The revertant allele, however, was -40 bp primer was then used in a polymerase chain reaction, smaller than the progenitor wild-type allele in the re- in conjunction with a “thal-specific” primer comple- gion between primers A and B. These results suggest mentary to the genomic clone in a region that is less that imprecise excision of Mu1 caused a small deletion conserved in known secA genes (see horizontal arrow of flanking genomic sequences and that this excision Figure 3). This yielded a 906-bp cDNA fragment (data nonetheless resulted in reversion to a wild-type pheno- not shown). type. Nucleotide sequence analysis indicated that this A second revertant sector, excised from a different cDNA contained a continuous open readingframe with plant, was analyzed in the same way and with similar the capacity to encode a proteinwith extensive similar- results (data not shown). Onceagain, the Mu1 insertion ity to Sedhomologues in many organisms (Figure 3). was homozygous in the phenotypically mutant tissue The nucleotide sequence of the cDNA clone in the and was heterozygous in the revertant tissue. Excision region adjacent to the “thal-specific” primer was iden- of Mu1 was again accompanied by a small deletion of tical for 126 bases to that of the genomic clone, diverg- flanking sequences, although the size of this deletion ing in sequence 19 bp upstream of the Mu1 insertion differed slightly between the two revertant samples. The (data notshown; GenBank accession No. U71123). This amplification products derived from both excision site of sequence divergence between the genomic and events were cloned and their DNA sequences were de- cDNA clones corresponds precisely with a 5’-splice site termined. In both cases, the deletions (27 and 38 bp) consensus sequence (shown in Figure 2C) (LUEHRSEN extended into what proved to be intron sequences (Fig- et al. 1994) and with the point of divergence between ure 2C and results below) anddid not disrupt se- pea cp-SecA and the deduced productof the thalgeno- quences essential for splicing at the 5’ splice junction mic clone (as described above). We concluded, there- (LUEHRSENet al. 1994). The thal gene would likely be fore, that the amplified cDNA does correspond to the expressed fairly normally in these derivative allelessince genomic clone, that Mu1 had inserted within an intron it is unlikely that these small deletions of intronic se- 19 bp from the 5’-splicejunction, and that thal encodes quences would interfere with splicing. Taken together, a SecA homologue. these results indicate that excision of Mu1 from geno- Both the pea and spinach cpSecA proteins are pre- mic sequences corresponding to the clone correlates dicted to be synthesized with a 60-80 amino acid N- with reversion to a wild-type phenotype, providing terminal extension when compared to bacterial SecA strong evidence that the cloned sequences represent homologues (BERGHOEFERet al. 1995; NOHARAet al. the thal gene. 1995) (Figure 3). These N-terminal sequences facilitate The thl gene encodes a Sedhomologue: DNA se- protein targeting across the chloroplast outer envelope. quence analysis revealed a 627-bp open reading frame We did not directly determine the start codon for the in the 719-bp cloned genomic region flanking the Mu1 thal gene product. However, translation initiated at an insertion (Figure 1B). This region has the potential to AUG 420 nt upstream of the Mu1 insertion site would encode the aminoacids shown upstream of thevertical result in an N-terminal extension comparable in length arrow in Figure 3. The deduced amino acid sequence to those predicted for thepea and spinach cpSecA pre- shows similarity to the amino-terminus of the pea (P. proteins (see * in Figure 3). Thepredicted sequence of sativum) and spinach (S. ohacea) chloroplast-localized the N-terminus of a proteinstarting at this site exhibits SecA homologue, cp-SecA (Figure 3) (NAKAIet al. 1994; properties characteristic of stromal targeting sequences BERGHOEFERet al. 1995). The similarity between the (reviewed by CLINEand HENRY1996): it is rich in hy- 472 R. Voelker, 1. Mendel-Hartvig and A. Barkan

Ihn I 1 MPGSAWCGODTFGLKLNMGSVVGPPKFLIRRLLRLHPHSTATPQSLPTTOTRGGLS 56 P-salhrm 1 ...... 0 s.olpraose 1 ...... M 1 E&I 1 ...... 0 Synech. sp 1 ...... * 0 Iha I 57 PCAPPPPPHEAPEKDIKRTPMAMTPUASAATVAVTPALRFPHTLSATGLSSPPLAG 112 P-snlhrum 1 ... . MATSS LCSSFTSOTCNPHSRPHRKT LT LPGSVF LCROFHLNSPS...... 44 s*rsraoea 2 ESCARSASOMSSSSCCR-CSSFN~KLKOGGIGGGSLPVSFSCVMIGGGGGRRLIDO~~ Ed, ...... 0 Swh. sp 1 ...... 0

that 113 GCGGCRVRFRPSORGRGTQGRUGG HVSRVG LLGTV 0 GRDDGEA P-sathrm 45 ...... VSKTRRIRTROSGVASLG LLGGIFCG --DTGEA RKO AI1 86 SQB~51 ERGKVRGRERKIGE-MOVRASACG LLNLGULLCU-FK@ [D-.~PA~S~~~~~ Ed! 1 ...... h' 0IKL!TIVFG~RNJ;-LRP'.'~~V 24:l: Synech. sp 1 ...... u:

lhdl 169 P.snIhrm 81 Sabmc3.4 109 Ed 25 Synech. Sp 21

lhnl 211 P-safhrm 135 EASKRVLGLRPFDVOLIGGMV LHKGE I AEMRTGEGKTLVAl LPAY LNA KGVHV 190 soblaawl 157 ~EASKRVLGLRPFDVOLlGGMVLHKGElAEMRTGEGKTLVAI LPAYLNA~~~KIGIVHVl212 E&! 73 FASKRVFG'"~R~FDVnLLGGMVLNER; I AEKDTGEGKT 1 -AT I PA" I NA I TGCGVHV 128 Symh. SD 71 EASKRVLGLnHFDVnMIGGMI LHDGOIAEMKTGEGKTLVATLPSVLNALSGKGAHV 132

lhal 273 TVNDYLARRDCEWVGOVPFLGLO GLIOONMTDE P-SBfhrWn 191 VTVNDY LARRDCEWVGOVPQF LGMK GLI OONMTSEQKKENY LCD1 TYVTNSELGF248 S.olsmc3.4 213 VTVNDYLARRDCEWVGOVAQFLGLK GLVOONMTSEVRRFNYLCOITYVTNSELGF 268 VTVNDY LAORDAF:IN~Ptr~=Is17v~l >!I -';UDAC,~KRFAVAADIR~S TY~TN~JF'.'GC 328184 €Coli 129 ryE~R YVI Synech. SD 133 VTVNDY LA9RDAEWMGQVHRF LGLSVGLI OOGMSPEERRRNYNCDI TYATNSELGF 188

(ha1 329 VlDEVDSl LlDEARTPLl ISGL P-selhrm 247 T~~LUL~B [lmrDF LRDNLA-S EEL I QGFNVlDEVDSl LlDEARTPLl ISG SObremn 269 DFLRDNLATSVlDEVDSlLPGDEL LlDFARTPLl 1SGoEKP E& 185 ~YLRDN\'AFS"F€~VUFKIHYALVDFVD~lLIDEARTPL! ISGPIFDSSEb'VI3'J:240 Synech sp I 89 DYLPDN~AAVIEEVVQP~'NYAVlDEVDS1LlDEARTPLl ISGOVDRPSEKVV9AS 244

lh81 385 ...... P-selhrun 303 ..... SO- 325 Ed1 241 Swh.Sp 245 EVAALLORSTNTDSEE.EPD~DYFV~EK~~RNVLLTDOGFlNAEQLL...... GVS 292

lhal 423 467 P.salhrm 341 385 Sobfnma 363 407 Edf 297 ~.!VS~ANIL'.M~HVTA4LRAUA~FTP~VDVlVU:,:~FV~IVDFPTGR .... 342 Synech. sp 293 DLFDSND-PWAHYIFNAIKAKELFIK~VNYIVRGGEIVIVDEFTGR.... 331 FIGURE3.-Comparison of the deduced /ha1 gene product with other SecA homologrm. The amino acid sequence shown for the lhal gene product was deduced from the combined sequence analysis of the genomic and cDNA clones. The genomic and cDNA sequence data haw beendeposited in GenRank/EMRI,/DDF%J under accession numbers Ui1124 and Uil123, respectively. The N-terminal methionine shown for thn1 is encoded in genomic DNA but may not represent the authentic Stilrt codon; the putative /hnl start codon (as described in RISLXTS) is designated ("). The region corresponding to the primer used for the amplification of the /hnl cDNA is indicated with a horizontal arrow. An intron in the /hlgrne maps between the codons for amino acids 209 and 210 (1).Amino acid identity displayed lmwccn the /hIgene product and thr pea (1'. srhncnr) and spinach (S. olmmn) chloroplast cpSecA homologs is indicated by hoxrs. Amino acid similarity rlisplayccl between these proteins and the E. rofi and Synechococcus SecA proteins is indicated by shading. Alignment and homology were determined as indicated in MATERIAIS AND METIIODS. The sequences shown correspond to approximately one third of the full-length proteins. droxylated amino acids (20%), devoid of acidic resi- digested with HindIII, two genomic fragments were de- dues, and contains an alanine at the second position. tected. M'e expected that Hind111 would cut the geno- Together,these observations support the conclusion mic sequence corresponding to the probe intotwo frag- that the thnl gene encodesa chloroplast-localized SecA ments since the probewas known to contain an internal homologue. HindIII site (data not shown) andsince the probe and The fhal gene is unique in the maize genome: The the genomic DNA on the blot were derived from the genomic clone encoding thefirst exon detect.? a single same inbred line. Together, these results support the band on genomic Southern blots (Figure 1C and data notion that there is only a single SecA homologue en- not shown), indicating that the 5"portion of the /ha1 coded in the maize genome. gene has no close homologues in the maize genome. Accumulation of cp-Sed in thal mutants is very However, sequences that aremost highly conserved below: To use the /ha1 mutant productively in studies of tween all SPCAgenes occur in the second exon and fur- cpSecA function,it is important toknow to what extent tlw cIownsLrcan1. T1lc:sc: liighly conserved regions 01' lllc Mu insertion decreases cpSecA protein accumula- the thnl cDNA (encoding aminoacids 175-467 in Fig- tion. This was addressed by quantieing both the thnl ure 3) were used to probe genomic Southern blots at mRNA and cpSecA proteinin mutant leaves. Northern reduced stringency(Figure 4). \%en DNA from the hybridizations were used to visualize thal mRNA in /hnl- inbredline B73 was cut with the restrictionenzyme rffor wild-type seedling leaves. A probe consisting of SstI, a single genomic fragmentwas detected, andwhen the 906bp /hnl cDNA revealed a mKNA of -3.5 kb in In Vivo Role of Chloroplast SecA 45.7

e e a thal b actin WT thal WT thal "" T.7

aff

thal - - + + + + C UctiI?+ + - - ++

-8 -7 -6 710- * -5 489- -,-4 404- 325- -3

-2 242- -1.5 123 4 5 67 8910 0 -1 FIGURE.i.-Abundance of /lrnl mRNA in /hnl leaf tissue. (A) Northern blot containing 3 pg of polyA+ cnriched RNA isolated from hrl and wild-type seedling leaves. The blot was probed with the F)O(i-hp thnl cDNA. (R) The same blot was reprobed with a maize actin cDNA fragment. (C) RNAse- protection analysis. The probe for/Ira1 mRNA was transcribed 12 from a clone of the 719-bp /hnl genomic fragment flanking the MuZ insertion (see Figure 1). The probe for actin mRNA FI(;URE4.-Southern blot probed at redwed stringency was transcribed from a 232-bp partial actin genomic clone. with a highly conserved region of the lhal gene. DNA oh The actin probe was synthesized such that its specific-activit). tained from the inbred line B73 was digested with the indi- was one-tenth that of the /ha1 probe, in order that the final cated restriction enzyme. Hybridization was performed in 5X signal intensities be comparable. Totalleaf RNA (30 pg) from SSC at .54" and washes were with 0.5X SSC at 54". This blot wild-type or tAnl seedlings was hybridized with the /lrnI probe, was probed with a DNA probe made from the entire 906bp the actin probe, or both, as indicated. Hybrids were digrsted cDNA fragment drrived from the /ha1 locus in R73. with RNAse TI andfractionated in a denaturing gel. Controls included a lane in which tRNA was substituted ['or maize RNA wild-type polvA+ enriched leaf RNA (Figure 5A). This (lane S), undigested actin probe (lane2) and undigested /hcrl mRNA is sufficient in size to encode a protein of "1 10 probe (lane 4). Lane 1 contains DNA size standards. kD, the size of the previously described cpSecA proteins (NAUI P/ al. 1994; YUAN r/ al. 1994; RERGHOEFER were assayed. These results indicate that theIMUI inser- rt al. 1995). Even after long exposure, this mRNA was tion in thal-rffcauses a dramatic decrease in the accu- notdetected in the /Anl-rPf sample.When an actin mulation of the tho1 mRNA. The precise level of resid- cDNA was used to reprobe the same blot, it became ual mRNA was impossible to determine since it was apparent that the fhnl-r$ sample contained consider- below the limit of detection in both experiments. ably more mRNA thanthe wild-type sample(Figure The amount of cpSecA protein in /Iltzl-rrj chlorc- 5R), making the absence of thal mRNA still more sig- plasts relative to wild-type chloroplasts was quantified nificant. on a Westernblot probed with anantiserum raised The abundance of tlml mRNA was also assayed in against a highly conserved portion of pea cpSecA (NA- RNAase-protection experiments (Figure 5C). Totalleaf KAI Pt al. 1994). This antiserum should recognize not RNA from wild-type seedlings protected a probe frag- just the thnl gene product hutalso the products of any ment of -700 nucleotides (based upon the mobility of nonallelic genes encoding cpSecA. A prominent band DNA markers). This corresponds to the first protein- of the expected size (110 kD) was detected in extracts coding exon of the mRNA. An equivalent amount of of wild-type chloroplasts but was nearly undetectable in total RNA from /hal-rpfleaves did not protect the probe extracts of /hal-r~fchloroplas~(Figure 6). Comparison to detectable levels. A probe complementary to actin with a dilution series of the wild-type sample demon- mRNA was protected to a similar extent in the hvo strated that the abundanceof cpSecA in /k1-rffchloro- samples, demonstrating that equal amounts of mRNA plasts is no more thana few percent of that in wild-type 474 R. Voelker, J. Mendel.-Hartvig and A. Barkan

a %WT a etiolated green thal 100 50 20 10 5 s* "112 - " m s M s &jbb>-N--bbx 123456 033s8828332 b E'- - -112 kD -u- . "m

79- - - -53 kD 123456 FIGURE6.-Western blot showing loss of cp-SecA in thal chloroplasts. Total proteins (7 pg)obtained from thal or wild- type chloroplasts were loaded in lanes 1 and 2, respectively. Dilutions of the wild-type sample were analyzed in lanes 3- 6. (A) The blot was probed with an antibody raised against ".- OEC23 a highly conserved fragment of pea cpSecA (NM et al. 1994). (B) The filter shown in A was stained with Ponceau S iPC before antibody probing. This demonstrates that equal PC amounts of mutant and wild-type chloroplast proteins were I I analyzed.

bv"0- "" cytf chloroplasts. These results demonstrate that the thal gene is the only significant source ofcpSecA in young c 01x23 maize leaves and that the Mul insertion causes nearly 1) a complete loss of cpSecA in thal-refchloroplasts. d Cp-SecA functionsin etioplasts: The role of cpSecA -1 LHcp 1 I has not been studied in plastid typesother than chloroplasts. To assess its role in the biogenesis of etioplasts, FIGURE7.-Etiolated and green thal leaves exhibit similar we examined the phenotype of etiolated thal-refleaves protein deficiencies. Protein was extracted from leaf tips excised from two etiolated thal seedlings and two etiolated wild- (Figure 7). Protein was extracted from the leaf tips of type siblings.The plants were then exposed to light for 24 hr two etiolated thal-ref plants and from two normal and protein was extracted from leaves of the same plants. siblings.Protein was then extracted fromleaves of Total leaf proteins (5 pg) or the indicated dilutions of a wild- the same plants after they had been exposed to light type sample, were fractionated by SDSPAGE and transferred for 24 hr. to a nitrocellulose membrane. (A) Proteins bound to the filter werevisualized by staining with Ponceau S. (B) The filter To illustrate that the etiolated leaves had developed shown in panel (A) was probed with antibodies specific for in the absence of light, we took advantage of the fact plastocyanin and OEC23. (C) The filter shown in B was re- that the major light harvesting chlorophylla/b binding probed with antibody specific for cytochromef:(D) The filter protein (LHCP) failsto accumulate in etioplasts and shown in C was stripped and reprobed with an antibody spe- accumulates rapidlyupon exposure to light (NELSONet cific for LHCP, the major light harvesting chlorophyll a/6/ binding protein. al. 1984). A strong signal was obtained with just 2.5% of the LHCP in the greened seedlings, but no signal was detected in the etiolated tissue (Figure 7D). There- 7B). We have observed that the accumulation of i-PC fore, the etiolated leaves had not been exposed to sig- is variable, even among light-grown mutant seedlings nificant light. (unpublished observations). The unusually high ratio The protein deficiencies in etiolated and greened of i-PC to mature PC observed in the greened samples thal-refleaves were similar: a fivefold reduction in ma- shown here mayhave resultedfrom their unusual ture plastocyanin and cytochrome f was accompanied growth regime:the shift of etiolated leaves from dark to by an increase in the accumulation of the plastocyanin light may have overwhelmed the already compromised stromal intermediate (i-PC) (Figure7, B and C).These targeting machinery with newly synthesized substrates results indicatethat cpSecA functions in the biogenesis for transport. of etioplasts and that the thal gene (rather than a non- The results of this experiment alsosuggest that allelic gene encoding cp-SecA)provides cp-SecA to OEC23 and OEC16 are efficiently translocated across both chloroplasts and etioplasts. internal etioplast membranes: these proteins accumu- The ratio of i-PC to mature PCwas higher in the lated to similar levels in etioplastsand chloroplasts and greened than in the etiolated mutant samples (Figure their stromal intermediates did not accumulate de- In Vivo Role of Chloroplast SecA 475

FIGURE8.-Ultrastructure of thul chloroplasts. (A) Sec- tion of awild-type leaf. A mesophyll cell is shown on the bottom and two bundle sheath cells on the top. (B) Section of a thul leaf. A mesophyll cell is shown on the bottom and a bundle sheath cell on the top. Bars, 1 pm.

tectably in etioplasts (Figure 7, B and C, and data not chloroplasts (data not shown). In contrast, the bundle shown). Thisis intriguing in that these two proteins are sheath thylakoids in hcfl06 are clearly aberrant in their strictly dependent upon a trans-thylakoidal delta pH organization (MARTIENSSEN et al. 1987, 1989). for their translocation in vitro (reviewed by CLINEand HENRY1996). These results suggest that either a suffi- DISCUSSION cient pH gradient is generated in the absence of light and chlorophyll or that these proteins are targeted via Mutations in the nuclear genes thal and hcfl06 block an alternative mechanism in etioplasts. the translocation of distinct sets of proteins across the Chloroplast ultrastructure in thal mutants Thyla- thylakoid membrane (VOELKERand BARKAN1995). The koid membrane organization varies with growth envi- proteins affected by hcjl06 require a ApH to cross the ronment and cell type. Mesophyll chloroplasts in maize thylakoid in vitro, while the proteins affected by thal-ref contain clusters of stacked thylakoid membranes require ATP. The possibility that the pathway blocked (grana) that are interconnectedby unstacked lamellae in thal mutants and the secA/Y/E pathway for protein (LAETSCH 1974). Chloroplasts in maize bundle sheath secretion in Eschm'chia coli are closely related was first cells contain primarily unstacked thylakoid membranes suggested by the fact that they are both ATPdependent thatare distributed throughout the stroma and ar- and sensitive to azide. Recently, this possibility gained ranged in parallel to the long axis of the chloroplast. further supportby the discovery that cp-SecA, a chloro- The cp-SecA deficiency in thal-ref mutants disrupts plast-localized homologue of the bacterial secA gene thestructure of thylakoid membranes in mesophyll product, facilitates the translocation of plastocyanin chloroplasts (Figure 8). Mesophyllthylakoid mem- and OEC33 across thylakoid membranes in vitro (NAKAI branes are arranged in giant grana that often extend et al. 1994; YUANet al. 1994). Our finding that thal the entire lengthof the chloroplast and that exhibit an encodes a chloroplast-localized SecA homologue dem- abnormally high electron density. These structures are onstrates that cpSecA functions in targeting OEC33, reminiscent of those observed in the mesophyll chlore plastocyanin and PSI-F in vivo. These results also dem- plasts of hcj106 mutants (MARTIENSSEN et al. 1987, onstrate that our mutant screen and phenotypic analy- 1989). However, they are also quite similar to structures ses led us to an authentic component of the targeting observed in several nonallelic barley mutants that lack machinery and give confidence that we can use a ge- photosystem I1 core particles specifically (SIMPSONand netic approach to probemechanisms by which proteins VON WEITSTEIN1980; SIMPSONet al. 1989). This struc- are targeted to the thylakoid membrane. ture may be a consequence of lesions that lead to the Our results indicate that the Mul insertion in the loss of photosystem I1 core particles but notof the light thal-refallele causes a dramatic decreasein thal expres- harvesting complexes. Bundle sheath thylakoid mem- sion. Thal mRNA is not detectable in mutant tissue and branes are organized similarly in wildtype and thal immunoreactive cp-SecA protein accumulates to no (Figure 8), despite the fact that the protein targeting more than afew percent of normal levels. The residual defect is equally severe in bundle sheath and mesophyll cp-SecA in thal-ref chloroplasts may result from the 476 R. Voelker, J. Mendel-Hartvigand A. Barkan translation of residual mRNA that is synthesized despite coded proteins: Most studies of thylakoid protein tar- the Mu1 insertion. Alternatively, the thal-refallele may geting have focused on nuclear-encoded proteins. Little be null, theresidual “cp-Sed’ signal arising from non- information is available concerning the integration of specificity of the antiserum, from a nonallelic gene in the numerous thylakoid membrane proteins encoded the maize genome (which seems unlikely considering by chloroplast genes. Of these, cytochrome fis the only the results of the genomic analysis) or from the pres- one known to be synthesized with a typical cleavable ence of small revertant sectors. To be certain that we signal sequence. Cytochrome f is an integral membrane will be dealing with a null allele in future experiments, protein with a single membrane-spanning domain, its we are now seeking a derivative allele in which Mu exci- N-terminus lyingwithin the thylakoid lumen (GRAY sion was accompanied by a flanking deletion of essential 1992). Mutations in sequences encodingthe cyto- thal sequences. chrome fsignal sequencein Chlamydomonas (Chlamy- Role of cp-SecA in the targeting of nuclearencoded domonas reinhardtii) interfered with the insertion of the proteins to the thylakoid lumen:Previously, it was estab- protein into the membrane, establishing a functional lished that cp-SecA can facilitate the translocation of role for the signal sequence in vivo (SMITHand KOHORN plastocyanin and OEC33 acrossisolated thylakoid mem- 1994). Our results indicate that cytochrome frequires branes in vitro (NAKAIet al., 1994; YUAN et al., 1994). A cp-SecA for efficient insertion into the membrane.This role for cp-SecA in the targeting of PSI-F was also de- is consistent with the observation that pea cytochrome duced, based upon the fact that PSI-F translocation is f; when expressed in E. coli, inserts into the E. coli cyto- inhibited by azide (KARNAUCHOV et al. 1994; MANT et plasmic membrane in a secA-dependent manner al. 1994).The thal mutation causes a defect in the (ROTHSTEINet al. 1985). translocation of all three of these proteins (VOELKER SMITHand KOHORN (1994) identified mutations in and BARKAN1995), demonstrating thatcp-SecA is capa- nuclear genes that suppressed the targeting defects ble not only of promoting their translocation in vitro, caused by alteration of the cytochrome f signalse- but is actually required for normalrates of translocation quence. In the context of our results, it would not be in vivo. surprising if some of the nuclear suppressors alter the Azide does not significantly inhibit the translocation cpSecA gene. The reduced accumulation of LHCP, a of the nuclear-encoded proteins OEC16 and OEC23 nuclearencoded integral thylakoid protein, caused by across isolated thylakoids(reviewed by CLINEand onemutation in the cytochrome f signal sequence HENRY1996) nor is their translocation facilitated by cp- (SMITHand KOHORN1994) suggested that cytochromef SecA in vitro (NAKAIet al. 1994; YUAN et al. 1994). The and LHCP might engage a common component during rates of translocation of OEC16 and OEC23 in vivo are their integration. This common component is unlikely not detectably reduced in thal-ref (VOELKERand BAR- to be cp-SecA since the thal mutant has no apparent KAN 1995) despite its near absence of cp-SecA. Thus, defect in the accumulation or integration ofLHCP these two nuclear-encoded proteins depend little, if at (VOELKERand BARKAN1995) and purified cp-SecA does all, upon cpSecA to cross the thylakoid in vivo. not facilitate the integration of LHCP into isolated thy- Although cp-SecA clearly facilitatesthe translocation lakoid membranes (YUAN et al. 1994). Perhaps cyto- of several proteins in vivo,our results suggestthat none chrome fintegration is facilitated by both cp-SecA and of the proteins yet examined absolutely require cp- SecA. Mature, lumenal OEC33, plastocyanin, cyto- by 54CP, a homologue of SRP54that functions in LHCP integration in vitro (FRANKLINand HOFFMAN1993; LI et chrome f; and PSI-F accumulate in thal mutants to 30- 50% of their normal levels, despite their reduced rates al. 1995). of translocation (VOELKERand BARKAN1995). There cp-SecA functions in a variety of plastid types: The are several possible explanations for this observation. plastids comprise a group of related organelles that Our preferred hypothesis is that a redundant transport contain chloroplast DNA and develop from a common mechanism for these proteins is exploited in the ab- progenitor. Plastid types other than chloroplasts con- sence of cp-SecA. A likely candidate for an alternative tain internal membranes, although these are less abun- pathwayis the OEC16/OEC23 pathway, since this is dant than the chloroplast thylakoid membrane. The engaged by signal sequences that closely resemble those localization of OEC33 within the lumen of an internal that target proteins to the Sed pathway. The analysis membrane system inetioplasts (RYRIEet al. 1984;HASHI- of double mutantswith defects in both pathways should MOTO et al. 1993) suggested the presence of a targeting allow us to evaluate this possibility. It is also possible, machinery internal to etioplasts. Our results indicate however, that residual cpSecA in mutant chloroplasts that cp-SecA is a component of this machinery, since permits very slow translocation of these proteins, the the thal mutation affects etioplast and chloroplast pro- translocated forms then accumulating to disproportion- tein accumulation in similar ways. Ectopic expression ately high levels due to a decrease in their degradation of plastocyanin in transgenic tomato plants resulted in rate relative to that in wild-type chloroplasts. the accumulation of mature plastocyanin in root and Role of cp-SecA in the targeting of chloroplast-en- petal plastids (BOERet al. 1988), suggesting that the In Vivo Role of Chloroplast SecA 477 targeting pathway mediated by cp-SecA isalso function- GTP requirement for the integration of a chlorophyll &binding polypeptide into thylakoid membranes. Plant Physiol. 105: ing in these plastid types. 295-304. Mutants with defects in thylakoid protein targeting KARNAYCHOV, I., D. CAI, I. SCHMIDT,R. G. HERRMANNand R.B. are useful not only for identifylng components of the KI.ORSC;EN, 1994 The thylakoid translocation of subunit 3 of photosystem I, the psali gene product, depends on a bipartite targeting machinery but also for establishing the roles transit peptide and proceedsalong an azide-sensitive pathway. J. of these components in vivo. Further studyof thal Biol. Chem. 269: 32871-32878. should elucidate roles of cp-SecA that are difficult to KNOTT,T. G., and C. ROBINSON,I994 The Sed inhibitor, azide, reversibly blocks the translocation of a subset of proteins across address in vitro. Loss-of-function mutants like thal as the chloroplast thylakoid membrane. J. Biol. Chem. 269: 7843- well as mutants identified by their ability to suppress 7846. signal sequence mutations (SMITHand KOHORN 1994) LAETSCH, W. M., 1974 The C4 syndrome: a structural analysis. Annu. Rev. Plant Physiol. 25: 27-52, will be essential tools for understanding mechanisms LAIILER,V., A. M. CHADDOCK,T. G. KNOTT, D. WALKERand C. ROB- involved in thylakoid protein targeting. INSOK, 1995 A SecY homolog in Arabidopsis thaliana. J. Biol. Chem. 270: 17664-17667. We are grateful to LAURA ROYand WCIEWAI.KER for experttechni- LI, X., R. HYNKY,J. Yum, K. CLINEand N. E. HOFFMAN,1995 A cal assistance and to Smrf HAKE.,MICHAEI. FREEI.ING and coworkers chloroplast homologue of the signal recgiion particle subunit for access to their Mutatorstocks. We also appreciate gifts of plasmids SRP54 is involved in the posttranslational integration of a protein encoding Mutator elements and actin from VICN CHANDLER.We are into thylakoid membranes. Proc. Natl. Acad. Sci. USA 92: 3789- especially grateful to TOSHNAENDO for the antibody to pea cp-SecA. 3793. LUEHKSt,N, K. R., S. Tmand V. 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