Efficient in vitro import of a cytosolic heat shock protein into pea chloroplasts

In order to further our understanding of the targeting of nuclear-encoded proteins into intracellular organelles, we have investigated the import of chimeric precursor proteins into pea chloroplasts. Two different chimeric precursor proteins were produced by in vitro expression of chimeric genes. One chimeric precursor contained the transit peptide of the small subunit of soybean ribulose 1,5-bisphosphate carboxylase and the mature peptide of the same protein from pea. The second contained the same transit peptide plus 13 amino acids of the pea mature peptide fused to a cytosolic heat shock protein. The extent of import and binding of the two chimeric proteins was examined by using quantitative assays and was compared to the import of pea small subunit precursor. Both precursor proteins imported well into pea chloroplasts, although the extent of import observed with the chimeric small-subunit-heat shock precursor was less than that observed with the soybean-pea small subunit precursor. The heat shock protein alone did not import into nor bind to chloroplasts. The binding of both the chimeric small-subunit-heat shock protein and the soybean-pea small subunit precursor to chloroplasts was physiologically significant, as shown by the fact that when chloroplasts with bound precursors were isolated, these bound precursors could subsequently be imported.     

Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase

Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids. We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy. The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus subtilis hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase. One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues. Both deduced protein sequences share 27.2% identical amino acid residues. The first in vitro translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction. The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steady-state RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth. The mature plastidal and the mitochondrial isoenzyme were overexpressed in E. coli. Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide. The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria.     

Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase

A precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylyase (dimerizing), EC 4.1.1.39] has been identified among the products of cell-free translation of polyadenylated RNA from spinach and pea. In both cases, the precursor is larger than the mature protein by 4000-5000 daltons. Upon incubation of post-ribosomal supernatants of the in vitro protein synthesis mixtures with purified intact chloroplasts, the pea and spinach precursors are transported interchangeably into the chloroplasts and processed to the mature size and charge. Moreover, the newly transported small subunits are found to assemble with endogenous large subunits to form the holoenzyme. In contrast, a precursor to the Chlamydomonas reinhardtii small subunit is not taken up by higher plant chloroplasts, indicating the specificity of the transport events. Together, these results demonstrate that the in vitro reconstruction of the post-translational transport of the higher plant precursors is physiologically significant.     

Cloned DNA sequences complementary to mRNAs encoding precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and a chlorophyll a/b binding polypeptide

Double-stranded cDNA was synthesized from pea poly(A)-containing mRNA and inserted into the Pst I site of the bacterial plasmid pBR322 by the addition of synthetic oligonucleotide linkers. Bacterial colonies containing recombinant plasmids were detected by hybridization to partially purified mRNAs and further characterized by cell-free translation of hybridization-selected mRNAs. To confirm the identity of cDNA clones encoding chloroplast polypeptides, we incubated translation products derived from complementary mRNAs with intact chloroplasts in vitro. After uptake, precursor polypeptides were converted to their mature size and identified by fractionation of the chloroplast stroma and thylakoid membranes. By using these procedures, we have isolated and characterized cDNA clones encoding the two major cytoplasmically synthesized chloroplast proteins: the small subunit of ribulose-1,5-bisphosphate carboxylase and a constituent polypeptide (polypeptide 15) of the light-harvesting chlorophyll a/b-protein complex. Similarly, a third cDNA clone was isolated and shown to encode a 22,000-dalton thylakoid membrane polypeptide.     

Primary structure of a cerulenin-binding beta-ketoacyl-[acyl carrier protein] synthase from barley chloroplasts.

The radioactively labeled beta-ketoacyl thioester synthase inhibitor [3H] cerulenin was used to tag three dimeric barely chloroplast proteins (alpha alpha, alpha beta, and beta beta) from the stromal fraction. Oligonucleotides corresponding to amino acid sequences obtained from the purified proteins were used to generate with the polymerase chain reaction a probe for cDNAs encoding the beta subunit. cDNA sequencing revealed an open reading frame for 462 residues comprising the mature protein and a 35-amino acid transit peptide. The deduced amino acid sequence of the mature protein is homologous to the beta-ketoacyl-[acyl carrier protein] (ACP) synthase I [3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3.1.41] of Escherichia coli. Under analogous experimental conditions [3H]cerulenin tagged a single dimeric protein from spinach chloroplasts.     

A poly(A) binding protein functions in the chloroplast as a message-specific translation factor

High-affinity binding of a set of proteins with specificity for the 5′ untranslated region (UTR) of the Chlamydomonas reinhardtii chloroplast psbA mRNA correlates with light-regulated translational activation of this message. We have isolated a cDNA encoding the main psbA RNA binding protein, RB47, and identified this protein as a member of the poly(A) binding protein family. Poly(A) binding proteins are a family of eukaryotic, cytoplasmic proteins thought to bind poly(A) tails of mRNAs and play a role in translational regulation. In vitro translation of RNA transcribed from the RB47 cDNA produces a precursor protein that is efficiently transported into the chloroplast and processed to the mature 47-kDa protein. RB47 expressed and purified from Escherichia coli binds to the psbA 5′ UTR with similar specificity and affinity as RB47 isolated from C. reinhardtii chloroplasts. The identification of a normally cytoplasmic translation factor in the chloroplast suggests that the prokaryotic-like chloroplast translation machinery utilizes a eukaryotic-like initiation factor to regulate the translation of a key chloroplast mRNA. These data also suggest that poly(A) binding proteins may play a wider role in translation regulation than previously appreciated.     

Characterization of the thyroid Na+/I− symporter with an anti-COOH terminus antibody

The Na⁺/I⁻ symporter (NIS) is the plasma membrane protein that catalyzes active I⁻ transport in the thyroid, the first step in thyroid hormone biogenesis. The cDNA encoding NIS was recently cloned in our laboratory and a secondary structure model proposed, suggesting that NIS is an intrinsic membrane protein (618 amino acids; ≈65.2 kDa predicted molecular mass) with 12 putative transmembrane domains. Here we report the generation of a site-directed polyclonal anti-COOH terminus NIS antibody (Ab) that immunoreacts with a ≈87 kDa-polypeptide present in membrane fractions from a rat thyroid cell line (FRTL-5). The model-predicted cytosolic-side location of the COOH terminus was confirmed by indirect immunofluorescence experiments using anti-COOH terminus NIS Ab in permeabilized FRTL-5 cells. Immunoreactivity was competitively blocked by the presence of excess synthetic peptide. Treatment of membrane fractions from FRTL-5 cells, Xenopus laevis oocytes, and COS cells expressing NIS with peptidyl N-glycanase F converted the ≈87 kDa-polypeptide into a ≈50 kDa-species, the same relative molecular weight exhibited by NIS expressed in E. coli. Anti-NIS Ab immunoprecipitated both the NIS precursor molecule (≈56 kDa) and the mature ≈87 kDa form. Furthermore, a direct correlation between circulating levels of thyroid-stimulating hormone and NIS expression in vivo was demonstrated.     

A chloroplast processing enzyme functions as the general stromal processing peptidase

A highly specific stromal processing activity is thought to cleave a large diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide. The identity of this key component of the import machinery has not been unequivocally established. We have previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII). Here we report the overexpression of active CPE in Escherichia coli. Examination of the recombinant enzyme in vitro revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined for different compartments of the organelle. CPE also processes its own precursor in trans. Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleavage site demonstrating that both forms of the enzyme are sensitive to the same structural modification of the substrate. The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well. These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase. Significantly, recombinant CPE cleaves in the absence of other chloroplast proteins, and this activity depends on metal cations, such as zinc.     

Cloning and structure determination of cDNA for cutinase, an enzyme involved in fungal penetration of plants

The primary structure of cutinase, an extracellular fungal enzyme involved in the penetration of plants by pathogenic fungi, has been determined from the nucleotide sequence of cloned cDNA. Clones containing cDNA made from poly(A)⁺ RNA isolated from fungal cultures induced to synthesize cutinase were screened for their ability to hybridize with the [³²P]cDNA for mRNA unique to the induced culture. The 75 cDNA clones thus identified were screened for the cutinase genetic code by hybrid-selected translation and examination of products with anti-cutinase IgG. This method yielded 15 clones containing cDNA for cutinase, and Southern blots showed that the size of the cDNA inserts ranged from 279 to 950 nucleotides. Blot analysis showed that cutinase mRNA contained 1050 nucleotides, indicating that the clone containing 950 nucleotides represented nearly the entire mRNA. This near-full-length cDNA and the restriction fragments subcloned from it were sequenced by a combination of the Maxam-Gilbert and the phage M13-dideoxy techniques. cDNAs from two other clones, containing the bulk of the coding region for cutinase, were also completely sequenced, and the results confirmed the sequence obtained with the first clone. A peptide isolated from a trypsin digest of cutinase was sequenced and the amino acid sequence as well as the initiation and termination codons were used to identify the coding region of the cDNA. The primary structure of the enzyme so far determined by amino acid sequencing (≈40% of the total) agreed completely with the nucleotide sequencing results. Thus, the complete primary structure of the mature enzyme and that of the signal peptide region were ascertained.     

Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, we started by isolating poly(A)⁺ RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor because: (i) it is rich in proline and poor in leucine, and (ii) the message appears to be more abundant when carrot tissue is wounded. From a cDNA library constructed with poly(A)⁺ RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A)⁺ RNA encoding this 33-kDa peptide. We isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pDC5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clone pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization and DNA sequence analysis indicate that pDC5 is a hybrid clone corresponding to two RNA species. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.     

Primary structure of a key enzyme in plant tetrapyrrole synthesis: glutamate 1-semialdehyde aminotransferase.

The formation of delta-aminolevulinate from glutamate 1-semialdehyde (GSA) is catalyzed by glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8). The active form of the barley enzyme appears to be a dimer of identical subunits with a molecular mass of 46 kDa. From the purified enzyme, amino acid sequences of the N-terminal ends of the mature protein as well as an internal peptide were determined. DNA primers deduced from these peptide sequences were used to amplify with the polymerase chain reaction a cDNA sequence encoding part of the enzyme. Screening a cDNA library with this DNA fragment identified a full-length clone encoding the 49,540-Da precursor of the GSA aminotransferase. The transit peptide for chloroplast import consists of 34 amino acids. GSA aminotransferase and a precursor form were expressed on a multicopy plasmid in Escherichia coli. Both recombinant gene products reacted with an antibody against the barley GSA aminotransferase. Active barley GSA aminotransferase expressed in E. coli was shown to be active in assays of bacterial cell extracts. As a gene symbol for barley GSA aminotransferase, Gsa is proposed.     

Cloning and nitrate induction of nitrate reductase mRNA

Nitrate is the major source of nitrogen taken from the soil by higher plants but requires reduction to ammonia prior to incorporation into amino acids. The first enzyme in the reducing pathway is a nitrate-inducible enzyme, nitrate reductase (EC 1.6.6.1). A specific polyclonal antiserum raised against purified barley nitrate reductase has been used to immunoprecipitate in vivo labeled protein and in vitro translation products, demonstrating that nitrate induction increases nitrate reductase protein and translatable mRNA. A partial cDNA clone for barley nitrate reductase has been isolated and identified by hybrid-selected translation. RNA blot-hybridization analysis shows that nitrate induction also causes a marked increase in the steady-state level of nitrate reductase mRNA.     

A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl–tRNA synthetase in Arabidopsis thaliana

One-fifth of the tRNAs used in plant mitochondrial translation is coded for by chloroplast-derived tRNA genes. To understand how aminoacyl–tRNA synthetases have adapted to the presence of these tRNAs in mitochondria, we have cloned an Arabidopsis thaliana cDNA coding for a methionyl–tRNA synthetase. This enzyme was chosen because chloroplast-like elongator tRNA^Met genes have been described in several plant species, including A. thaliana. We demonstrate here that the isolated cDNA codes for both the chloroplastic and the mitochondrial methionyl–tRNA synthetase (MetRS). The protein is transported into isolated chloroplasts and mitochondria and is processed to its mature form in both organelles. Transient expression assays using the green fluorescent protein demonstrated that the N-terminal region of the MetRS is sufficient to address the protein to both chloroplasts and mitochondria. Moreover, characterization of MetRS activities from mitochondria and chloroplasts of pea showed that only one MetRS activity exists in each organelle and that both are indistinguishable by their behavior on ion exchange and hydrophobic chromatographies. The high degree of sequence similarity between A. thaliana and Synechocystis MetRS strongly suggests that the A. thaliana MetRS gene described here is of chloroplast origin.     

Nucleotide sequence of the gene from the cyanobacterium Anacystis nidulans R2 encoding the Mn-stabilizing protein involved in photosystem II water oxidation.

The gene for the Mn-stabilizing protein (MSP; the so-called extrinsic 33-kDa protein) that is involved in photosystem II water oxidation was cloned and sequenced from the genome of the cyanobacterium Anacystis nidulans R2. The gene (here designated woxA) was shown to be present in a single copy. The deduced amino acid sequence indicated that the translation product consisted of 277 amino acid residues with a Mr of 29,306. The comparison of the sequence with that of mature MSP from spinach chloroplasts suggested that the translation product is a precursor whose amino-terminal 28 amino acid residues represent the signal peptide for the protein to cross the thylakoid membrane into the lumen. The length of the putative signal peptide was less than half that of the transit peptide for thylakoid-lumenal proteins of higher plants, whereas the structural profile of the putative signal peptide was similar to that of the carboxyl-terminal portion of the higher plant transit peptides. The amino acid sequence of the mature A. nidulans R2 MSP showed rather low homology (48-49%) to higher plant MSPs, but the conserved amino acid residues appeared to be clustered. Five clusters were tentatively assigned, in which the homology values were in a range of 66-70%. Domains essential for the functioning of MSP are expected to be situated in these clusters. It is of note that the two cysteine residues in MSP were conserved, and the disulfide linkage between them may play an important role in maintaining the tertiary structure of MSP.     

The general mitochondrial matrix processing protease from rat liver: structural characterization of the catalytic subunit.

A critical step in the import of nuclear-encoded precursor proteins into mitochondria involves proteolytic cleavage of their amino-terminal leader peptides by processing proteases found in the mitochondrial matrix. We report here the characterization of the general matrix processing protease from rat liver mitochondria. The final enzyme preparation consisted of two polypeptides, a catalytically active 55-kDa subunit and a 52-kDa one. To deduce the complete primary structure of the 55-kDa subunit, we first sequenced its mature amino terminus and several tryptic peptides derived from the pure protein. Next, using mixed oligonucleotide primers that had sequences based on two of these peptides, we synthesized a partial cDNA probe by selective amplification of liver RNA with the polymerase chain reaction. The amplified probe was then used to obtain a nearly full-length clone from a rat liver cDNA library. This cDNA codes for 508 amino acid residues, including 16 residues of an amino-terminal leader peptide, the cleavage site of which is located two polypeptide bonds downstream from an arginine residue. The mature portion has a predicted molecular mass of 55.2 kDa; it shows 36% identity with the mitochondrial processing peptidases of Saccharomyces cerevisiae and Neurospora crassa. A conserved structural feature is a putative, negatively charged alpha-helix, located in the amino-terminal half of the subunit; this element might be important for the recognition of positively charged leader peptides characteristic of mitochondrial precursor proteins.     

Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide

A nuclear gene AB80 has been isolated from a phage λ Charon 4 library of pea DNA. The sequence of the gene has been determined and it has been shown to contain an uninterrupted reading frame of 269 amino acids, corresponding to a precursor to a constituent polypeptide of the light-harvesting chlorophyll a/b-protein complex. Primer extension and S1 nuclease studies defined a cap site for AB80. The first methionine codon 3′ from this site is 69 nucleotides away and is the initiating codon of the open reading frame. A “TATA” sequence occurs 31 nucleotides 5′ from the cap site. A second TATA sequence is found 7 nucleotides on the 5′ side of the initiating methionine codon and the sequences surrounding this TATA sequence are strikingly similar to those surrounding the first TATA sequence. The mature polypeptide encoded by AB80 differs by 5 amino acids from the polypeptide corresponding to a previously characterized cDNA sequence pAB96. This result is indicative of heterogeneity within the constituent polypeptides of the light-harvesting chlorophyll a/b-protein complex. The sequence Arg-Lys-Ser-Ala-Thr-Thr-Lys-Lys occurs at, or near, the NH₂-terminus of the mature polypeptide encoded by AB80. This basic peptide is of interest because of its apparent involvement in changes in excitation-energy distribution in chloroplast membranes. Some general similarities, but no extensive sequence homology, is found on comparing the transit sequence for the precursor to the chlorophyll a/b-binding polypeptide with the transit sequences previously determined for the precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase.     

Regulation of a plant pathogenesis-related enzyme: Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin

Two endochitinases (EC 3.2.1.14) of M_r values of ≈34,000 and ≈32,000 have been purified from cultured tissues of Nicotiana tabacum cv. Havana 425. The chitinase content of cloned tobacco pith tissues subcultured on hormone-free medium increases by ≈5-fold to 8% of the soluble protein over a 7-day period. This induction is inhibited >90% by addition of combinations of the plant hormones auxin and cytokinin to the culture medium. Chitinase is also developmentally regulated in the intact plant. Not detectable in leaves near the top of the plant, it is 1-4% of the soluble protein in roots and lower leaves. A cDNA clone of tobacco chitinase was isolated containing a single, large open reading frame of 310 amino acids that includes the complete amino acid sequence of the mature enzyme. Chitinase and chitinase mRNA measured by RNA blot analysis show similar patterns of regulation indicating that chitinase accumulation is controlled, at least in part, at the mRNA level. The patterns were also similar to those obtained with glucan endo-1,3-β-glucosidase (EC 3.2.1.39) suggesting that the two enzymes are coordinately regulated.     

Identification, characterization, and DNA sequence of a functional "double" groES-like chaperonin from chloroplasts of higher plants.

Chloroplasts of higher plants contain a nuclear-encoded protein that is a functional homolog of the Escherichia coli chaperonin 10 (cpn10; also known as groES). In pea (Pisum sativum), chloroplast cpn10 was identified by its ability to (i) assist bacterial chaperonin 60 (cpn60; also known as groEL) in the ATP-dependent refolding of chemically denatured ribulose-1,5-bisphosphate carboxylase and (ii) form a stable complex with bacterial cpn60 in the presence of Mg.ATP. The subunit size of the pea protein is approximately 24 kDa--about twice the size of bacterial cpn10. A cDNA encoding a spinach (Spinacea oleracea) chloroplast cpn10 was isolated, sequenced, and expressed in vitro. The spinach protein is synthesized as a higher molecular mass precursor and has a typical chloroplast transit peptide. Surprisingly, however, attached to the transit peptide is a single protein, comprised of two distinct cpn10 molecules in tandem. Moreover, both halves of this "double" cpn10 are highly conserved at a number of residues that are present in all cpn10s that have been examined. Upon import into chloroplasts the spinach cpn10 precursor is processed to its mature form of approximately 24 kDa. N-terminal amino acid sequence analysis reveals that the mature pea and spinach cpn10 are identical at 13 of 21 residues.     

Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail

Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft. Glyphosate appears to occupy the binding site of the second substrate of EPSP synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme.substrates complex. The elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.