Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6873-6877, September 1986 Cell Biology Translocation of the precursor of 5-enolpyruvylshikimate-3- phosphate synthase into chloroplasts of higher plants in vitro (shikimate pathway/transit peptide/glyphosate) GUY DELLA-CIOPPA*, S. CHRISTOPHER BAUER, BARBARA K. KLEIN, DILIP M. SHAH, ROBERT T. FRALEY, AND GANESH M. KISHORE Monsanto Company, Plant Molecular Biology Group, Division of Biological Sciences, 700 Chesterfield Village Parkway, St. Louis, MO 63198 Communicated by Esmond E. Snell, June 16, 1986 ABSTRACT 5-enolPyruvylshikimate-3-phosphate syn- transferase; EC 2.5.1.19) catalyzes the transfer of the thase (EPSP synthase; 3-phosphoshikimate 1-carboxyvinyl- carboxyvinyl moiety of phosphoenolpyruvate (P-ePrv) to transferase; EC 2.5.1.19) is a chloroplast-localized enzyme of shikimate-3-phosphate yielding EPSP and inorganic phos- the shikimate pathway in plants. This enzyme is the target for phate. EPSP synthase is an intermediate in the shikimate the nonselective herbicide glyphosate (N-phosphonomethyl- pathway that gives rise to the aromatic amino acids L- glycine). We have previously isolated a full-length cDNA clone phenylalanine, L-tyrosine, and L-tryptophan (8). In addition ofEPSP synthase from Petunia hybrida. DNA sequence analysis to the biosynthesis of these amino acids, the shikimate suggested that the enzyme is synthesized as a cytosolic precur- pathway is utilized for the production of p-amino- and sor (pre-EPSP synthase) with an amino-terminal transit, pep- p-hydroxybenzoic acids, and a variety of other natural plant tide. Based on the known amino terminus of the mature products (8). The biosynthesis of aromatic amino acids has enzyme, and the 5' open reading frame ofthe cDNA, the transit been shown to occur in isolated chloroplasts in vitro (9), and peptide of pre-EPSP synthase would be maximally 12 amino shikimate-pathway enzymes (including EPSP synthase) have acids long. To confirm this prediction and to assay directly for been identified in the chloroplast compartment (10). EPSP translocation of pre-EPSP synthase into chloroplasts in vitro, synthase is of considerable agronomic importance because it we cloned the full-length cDNA into an SP6 transcription is known to be the target ofthe broad-spectrum, nonselective system to produce large amounts of mRNA for in vitro herbicide glyphosate (N-phosphonomethylglycine) (11). The translation. The translation products, when analyzed by primary mode of action of glyphosate is to competitively NaDodSO4/PAGE autoradiography, indicate a relative molec- inhibit the binding of the substrate phosphoenolpyruvate to ular mass for pre-EPSP synthase of -55 kDa. Uptake studies the active site of the enzyme. Glyphosate is thus thought to with intact chloroplasts, in vitro, indicate that pre-EPSP inhibit plant growth by blocking the synthesis of'aromatic synthase was rapidly taken up into chloroplasts'and proteo- amino acids (12). lytically cleaved to the mature -48-kDa enzyme. The transit We have described the isolation of a full-length cDNA peptide was shown to be essential' for import of the precursor clone for EPSP synthase from a Petunia hybrida cell line that enzyme into the chloroplast. To our knowledge, post-transla- overproduces the enzyme (13). Based on the known amino tional import into chloroplasts of a precursor enzyme involved terminus of the mature enzyme, the DNA sequence analysis in amino acid biosynthesis has not been reported previously. suggested that the enzyme is synthesized as a precursor Furthermore, enzymatic analysis of translation products indi- polypeptide with an amino-terminal extension that would be cates that pre-EPSP synthase is catalytically active and has a maximally 72 amino acids long. For EPSP synthase, we were similar sensitivity to the herbicide glyphosate as the mature interested in characterizing the precursor protein and deter- enzyme. To our knowledge, pre-EPSP synthase represents the mining whether it could be translocated into chloroplasts in only example of a catalytically competent chloroplast-precur- vitro and subsequently processed. In this report, we show sor enzyme. that EPSP synthase is synthesized as a precursor (designated pre-EPSP synthase, -55 kDa) that is rapidly translocated In addition to the reactions of photosynthesis, chloroplasts into chloroplasts in vitro and processed to its mature form are known to play a central role in many important aspects of (-48 kDa). The amino-terminal transit peptide was found to plant cellular metabolism. The biochemical pathways for be essential for uptake of the precursor enzyme into the biosynthesis of fatty acids (1), terpenoids (2), porphyrins (3), chloroplast. Furthermore, we report that pre-EPSP synthase and several amino acids (4) are compartmentalized princi- is a catalytically active enzyme that is inhibited in vitro by the pally within the chloroplast. In recent years it has become herbicide glyphosate. clear that the majority of chloroplast-localized proteins are METHODS encoded by nuclear genes and are synthesized as precursor on free Materials. Analytical grade glyphosate and shikimate-3- proteins cytoplasmic ribosomes (5). The import of phosphate (a gift from R. D. Sammons) were provided by these precursors into chloroplasts has been shown to be Monsanto Agricultural Products. The following products mediated by amino-terminal sequences (termed transit pep- were obtained from Promega Biotec (Madison, WI): cloning tides) which post-translationally direct the uptake process vectors pQEM-1 and pGEM-2, SP6 polymerase, T7 poly- (6). After uptake into the chloroplast, the transit peptide merase, RNasin, and rabbit reticulocyte lysate in vitro sequence is removed by a sequence-specific metalloprotease translation systems. Restriction enzymes were obtained from (7). New England Biolabs and used according to the manufac- The enzyme 5-enolpyruvylshikimate-3-phosphate syn- turer's directions. [35S]Methionine (1000-1100 Ci/mmol; 1 Ci thase (EPSP synthase; 3-phosphoshikimate 1-carboxyvinyl- Abbreviations: EPSP, 5-enolpyruvylshikimate-3-phosphate; The publication costs of this article were defrayed in part by page charge glyphosate, N-phosphonomethylglycine; kb, kilobase(s); P-ePrv, payment. This article must therefore be hereby marked "advertisement" phosphoenolpyruvate; 2D, two dimensional. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6873 Downloaded by guest on September 28, 2021 6874 Cell Biology: della-Cioppa et al. Proc. Natl. Acad. Sci. USA 83 = 37 GBq) and phosphoenol["4C]pyruvate (['4C]P-ePrv) (11 sterile 330 mM sorbitol in 50 mM Hepes KOH}(OI t7)], mCi/mmol) were obtained from Amersham. A SynChropak assayed for chlorophyll (16), and adjusted to a findchloro- AX-100 HPLC column was from P. J. Cobert Associates (St. phyll concentration of 4 mg/ml (using sorbitol/HeMes)':kThe Louis, MO). A radioactive flow detector for HPLC yield of intact chloroplasts from a single head of letme~was (Radiomatic FLO-ONE/Beta IC) was from Radioanalytic 3-6 mg of chlorophyll. These chloroplasts wermedeemed (Tampa, FL). High-temperature silicone oil was from homogeneous based on phase contrast and transmission Aldrich. All other chemicals were of the highest grade electron microscopy. available. A typical 300-pl uptake experiment (modified from ref. 15) EPSP Synthase cDNA Clones. A full-length cDNA clone of contained 5 mM ATP, 8.3 mM unlabeled methionine, 322 mM EPSP synthase from P. hybrida was obtained as described sorbitol, 58.3 mM Hepes-KOH (pH 8.0), 50 ,ul of reticulocyte (13). This cDNA clone contains 27 nucleotides of the 5'- lysate translation products, and intact chloroplasts from L. untranslated leader, 1.5 kilobases (kb) that codes for the 72 sativa (200 ,/g ofchlorophyll). The uptake mixture was gently amino acid transit peptide and 444 amino acids of the mature rocked at room temperature (in 10 x 75 mm glass tubes) enzyme, and 0.4 kb of the entire 3'-flanking sequence. The directly in front of a fiber optic illuminator set at maximum full-length EPSP synthase cDNA was cloned as a 2.1-kb Bgl light intensity (150-W bulb). Aliquots of the uptake mix II/Sal I fragment into the BamHI/Sal I sites of the plasmid (50-125 dul) were removed at various times and fractionated pGEM. The plasmids pMON6140 and pMON6145 containing over 100-,ul silicone-oil gradients (in 250-pul polyethylene the EPSP synthase coding region are transcribed 5' to 3' from tubes) by centrifugation at 11,000 x g for 30 sec. Under these the T7- and SP6-promoters, respectively. conditions, the intact chloroplasts form a pellet under the In Vitro Transcription/Translation of Plasmid DNA with silicone-oil layer and the incubation medium (containing the SP6 and T7 RNA Polymerases. Plasmid DNA (pMON6140 and reticulocyte lysate) floats on the surface. After centrifuga- pMON6145) containing the full-length EPSP synthase cDNA tion, the silicone-oil gradients were immediately frozen in dry was linearized at the only Pvu I site located in the 3'- ice. The chloroplast pellet was then resuspended in 50-100 Pl1 untranslated region. The linearized plasmid DNA was tran- of lysis buffer (10 mM Hepes-KOH, pH 7.5/1 mM scribed in vitro (uncapped) with SP6 or T7 polymerase phenylmethylsulfonyl fluoride/1 mM benzamidine/5 mM essentially as described (14). The standard reaction buffer e-aminocaproic acid/aprotinin at 30 ,ug/ml) and centrifuged at contained 40 mM Tris HCl (pH 7.9), 6 mM MgCl2, 10 mM 15,000 x g for 20 min to pellet the thylakoid membranes. The dithiothreitol, 2 mM spermidine, 80 units of RNasin ribonu- clear supernatant (stromal proteins) and an aliquot of the clease inhibitor, 0.5 mM each of ATP, GTP, CTP, and UTP, reticulocyte-lysate incubation medium from each uptake in a final reaction volume of 100 ,ul. The final RNA pellet was experiment were mixed with an equal volume of 2 x resuspended in 20 Al of sterile water and stored at -80°C. A NaDodSO4/PAGE sample buffer for electrophoresis (see standard translation reaction contained 200 pl of nuclease- below).