Proc. Nati. Acad. Sci. USA Vol. 84, pp. 3288-3292, May 1987 Cell Biology Protein import into chloroplasts requires a chloroplast ATPase (cell-free translation/precursor of small subunit of pea ribulose-1,5-bisphosphate carboxylase/pea chloroplasts/ nonhydrolyzable ATP analogs/ionophores) DEBKUMAR PAIN AND GUNTER BLOBEL Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021 Contributed by Gunter Blobel, January 29, 1987 ABSTRACT We have transcribed mRNA from a cDNA the rbcS-E9 gene (9) and contains the entire mature polypep- clone coding for pea ribulose-1,5-bisphosphate carboxylase, tide (S) sequence, including two introns. A portion of translated the mRNA in a wheat germ cell-free system, and pUC3AE9 containing the two introns but not the transit studied the energy requirement for posttranslational import of sequence was excised with Sph I and Kpn I and an identical the [35S]methionine-labeled protein into the stroma of pea Sph I-Kpn I fragment from SS15, which lacked the two chloroplasts. We found that import depends on ATP hydrolysis introns, was substituted. The resulting clone contained the within the stroma. Import is not inhibited when HI, K+, Na+, complete uninterrupted coding region of pS. or divalent cation gradients across the chloroplast membranes The pS coding DNA was excised from the pUC vector with are dissipated by ionophores, as long as exogenously added Xba I and Cla I and ligated to pT7-1 cut with Xba I and Acc ATP is also present during the import reaction. Our data I. The resulting plasmid (pT7-pS) was purified on a sucrose suggest that protein import into the chloroplast stroma requires gradient and linearized downstream from the gene with Pst I a chloroplast ATPase that does not function to generate a before transcription. In vitro transcription using phage T7 membrane potential for driving the import reaction but that RNA polymerase (United States Biochemicals, Cleveland, exerts its effect in another, yet-to-be-determined, mode. We OH) was done essentially according to supplier's assay have carried out a preliminary characterization of this ATPase conditions except that the nucleoside triphosphates and the regarding its nucleotide specificity and the effects of various cap analog diguanosine triphosphate [G(5')ppp(5')G] were ATPase inhibitors. used at 0.25 mM each. After transcription, the solution was extracted with phenol/chloroform and the mRNA was pre- The in vitro import of cytoplasmically synthesized proteins cipitated with LiCl. About 2 ,ug oftranscript was obtained per into chloroplasts has been previously demonstrated. Trans- ,ug of DNA template (as judged by A260). lation of mRNA for a cytoplasmically synthesized polypep- Cell-Free Translation. The previously described wheat tide of the chloroplast stroma, the small subunit (S) of germ cell-free translation system (10) was supplemented with ribulose-1,5-bisphosphate carboxylase, yielded a large pre- 10 ,uM S-adenosyl-L-methionine and contained 200 ng of cursor (pS) (1), and posttranslational incubation with isolated mRNA and 2.8 ,ul of wheat germ extract per 10-,ul translation chloroplasts resulted in import of pS into the chloroplast mixture. The final concentrations ofthe other components of stroma accompanied by conversion of pS into S (2, 3) by the translation mixture were adjusted to 43 mM Hepes-KOH means of cleavage of an NH2-terminal transit sequence (4). at pH 7.5, 112 mM KOAc, 2.1 mM Mg(OAc)2, 0.25 mM Import is an energy-dependent process (5). When the spermidine, 3 mM dithiothreitol, 0.5 mM ATP, 20 ,uM GTP, import reaction was carried out in the dark, addition of ATP 8 mM creatine phosphate, creatine kinase at 65 ,ug/ml, each stimulated uptake into the chloroplast. Likewise, illumina- of the 20 amino acids except for methionine at 26 ,uM, and tion of the import reaction stimulated uptake, and this [35S]methionine at 0.9-1.2 mCi/ml (1 Ci = 37 GBq). After stimulation was abolished when uncouplers were present, 1-hr incubation at 25°C, the translation mixture was centri- again suggesting that ATP is required for import (5). fuged for 22 min at 4°C in a Beckman Airfuge at 30 psi Recently, Cline et al. (6) succeeded in experimentally (140,000 x g), yielding a postribosomal supernatant (PRS) separating the import reaction into two steps. In the first that contained the newly synthesized pS. reaction precursors were bound to the surface of ATP- Posttranslational Import Assay. Intact chloroplasts were depleted chloroplasts. In a second reaction much of the isolated from the leaves of 2- to 3-wk-old pea (Pisum sativum, bound precursor was imported into the chloroplast, depend- Progress no. 9) seedlings and purified on a Percoll step ing on exogenously added ATP. gradient as described (11). Isolated chloroplasts were resus- In the present paper we have addressed the questions of pended in 50 mM Hepes-KOH, pH 7.7/0.33 M sorbitol at a how and where ATP is used to drive the import ofpS into the concentration of 2-4 mg of chlorophyll per ml. To deplete chloroplast stroma. chloroplasts of their endogenous ATP, we incubated them in the dark for 15 min at room temperature prior to use in the import assay. The import reaction was carried out essentially MATERIALS AND METHODS as in ref. 5 with some modifications. The basic import assay Subcloning and Phage T7 Transcription. A clone represent- mixture (300 ,u) contained 0.5 ul of PRS (see above), ing the complete coding sequence ofpS was constructed from preincubated chloroplasts equivalent to 100 ,g of chloro- phyll, bovine serum albumin at 100 ,ug/ml, and 50 mM a partial cDNA clone, SS15, that lacks part of the NH2- Hepes-KOH, pH 7.7/0.33 M sorbitol/40 mM KOAc/2 mM terminal transit sequence (7) and a genomic clone, pUC- Mg(OAc)2/1.5 mM dithiothreitol/10 mM methionine. In most 3AE9. The plasmid pUC3AE9 contains two S gene segments; instances, the import assay mixture was further modified as one is derived from the rbcS-3A gene (8) and contains the specified in the figure legends. The import reaction was complete transit peptide of pS, and the other is derived from Abbreviations: S, small subunit of ribulose-1,5-bisphosphate car- The publication costs of this article were defrayed in part by page charge boxylase; pS, precursor of S; PRS, postribosomal supernatant; CF1, payment. This article must therefore be hereby marked "advertisement" coupling factor; pmf, protonmotive force; CCCP, carbonylcyanide in accordance with 18 U.S.C. §1734 solely to indicate this fact. m-chlorophenylhydrazone. 3288 Downloaded by guest on September 26, 2021 Cell Biology: Pain and Blobel Proc. Natl. Acad. Sci. USA 84 (1987) 3289 carried out at room temperature for 30 min with occasional -<---- Dark - <- Room Light -O gentle mixing, either in the dark or in the room light as specified. Chloroplasts - + Quantitative Analysis of Import. After incubation, each ATP - _ I - import mixture was chilled on ice, diluted with 5 ml of cold Proteases - - - - - 50 mM Hepes-KOH, pH 7.7/0.33 M sorbitol and centrifuged X-100 + for 1 min at 4000 x g. The pelleted chloroplasts were lysed Triton - - - -- - in 0.8 ml of2 mM EDTA, pH 7.5, by vigorous mixing. Sodium chloride was added to a final concentration of 0.24 M and the lysate was centrifuged at 12,000 x g for 15 min. To the resulting supernatant, representing the stroma fraction, ice- cold trichloroacetic acid was added to a final concentration of 10%o. The precipitate was'analyzed by electrophoresis on 12% polyacrylamide gels in NaDodSO4 (12). The gels were fixed in 10% acetic acid/35% methanol (vol/vol), treated with Enlightning (New England Nuclear), dried, and exposed to S- Fuji'x-ray film for 6-24 hr at -80'C. An AMBIS ,8-scanner was used to quantitate the radioactivity in pS or S in dried gels. Protease Protection. The procedure for protease protection 1 2 3 4 5 6 7 was identical to that above for the given quantitative analysis FIG. 1. Protein import into the chloroplast stroma depends on of import, except that prior to lysis the pelleted chloroplasts ATP. Lane 1 shows the 3"S-labeled products present in a 0.5-,ui were gently resuspended in a final volume of 300 ,ul of aliquot of a PRS after translation. Lanes 2-7 show the products of a ice-cold 50 mM Hepes-KOH, pH 7.7/0.33 M sorbitol. Either 0.5-1.l aliquot of PRS imported into the chloroplast stroma fraction. 250 ,tg/ml each of trypsin and chymotrypsin or the same Where indicated, ATP was present at a final concentration of 1 mM. amount of proteases plus Triton X-100 to a final concentra- The data are from a fluorograph of a dried Na1DodSO4/polyacryl- tion of 0.3% was added. After incubation for 30 min on ice, amide gel. the reaction mixture was diluted with 0.8 ml of cold 2 mM EDTA, pH 7.5 containing protease inhibitors (2.5 mM p- ground" import (e.g., import in the dark without exogenously aminobenzamidine/10 mM e-amino-n-caproic acid/i mM added ATP) that was observed in the earlier studies (5), for phenylmethylsulfonyl fluoride), and mixed vigorously. which the ATP concentration contributed to the import reaction by the translation mixture was calculated to be 166 RESULTS AM instead of 0.8 AM in the present study. ATP Acts Within the Stroma. Chloroplasts are capable of ATP Is Required for Import. mRNA for S was obtained by synthesizing ATP in the dark when supplied with dihydroxy- in vitro transcription of cloned DNA (see Materials and acetone phosphate/oxalacetic acid/phosphate buffer (13).