John E.Oblong' and Gayle K.Lamppa2. Department of Molecular Genetics and Cell Biology, The University of. Chicago, 920 E. 58th Street, Chicago, IL 60637, ...
The EMBO Journal vol. 1 1 no. 12 pp.4401 - 4409, 1992
Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast John E.Oblong' and Gayle K.Lamppa2 Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 E. 58th Street, Chicago, IL 60637, USA 'Present address: Arizona Cancer Center, The University of Arizona, 1515 N. Campbell Avenue, Tucson, AZ 85724, USA 2Corresponding author Communicated by R.Herrmann
Two proteins of 145 and 143 kDa were identified in pea which co-purify with a chloroplast processing activity that cleaves the precursor for the major light-harvesting chlorophyll binding protein (preLHCP). Antiserum generated against the 145/143 kDa doublet recognizes only these two polypeptides in a chloroplast soluble extract. In immunodepletion experiments the antiserum removed the doublet, and there was a concomitant loss of cleavage of preLHCP as well as of precursors for the small subunit of Rubisco and the acyl carrier protein. The 145 and 143 kDa proteins co-eluted in parallel with the peak of processing activity during all fractionation procedures, but they were not detectable as a homo- or heterodimeric complex. The 145 and 143 kDa proteins were used separately to afinity purify immunoglobulins; each preparation recognized both polypeptides, indicating that they are antigenicaily related. Wheat chloroplasts contain a soluble species similar in size to the 145/143 kDa doublet. Key words: chloroplast/precursor/proteolytic processing enzyme/substrate specificity
Introduction Processing enzymes are essential for the maturation of a multitude of proteins that are synthesized as precursors in the cytoplasm and destined for different cellular compartments and functional complexes. Proteins targeted to the chloroplast are usually proteolytically processed by the removal of an N-terminal extension, the transit peptide, which selectively directs proteins to the chloroplast. Although basic and rich in serine and threonine, transit peptides differ considerably in length and sequence even for the same protein between plant species (for review see Keegstra et al., 1989). The transit peptide appears to mediate precursor recognition by a receptor component of the envelope (Pain et al., 1988; Waegemann and Soll, 1991), which may be part of a shared import apparatus (Perry et al., 1991; Schnell et al., 1991; Oblong and Lamppa, 1992). Mutational analyses indicate that the transit peptide also contains determinants for cleavage, which most probably influence overall precursor conformation (Wasman et al., 1988; Clark et al., 1989; Smeekens et al., 1989; Clark and Lamppa, 1991). The temporal relationship between precursor import and processing has not been established, but precursors can Oxford University Press
be imported without maturation due to mutation at the transit peptide-mature protein junction (Clark et al., 1989) or the absence of an active processing enzyme (Chitnis et al., 1986). We have previously shown (Lamppa and Abad, 1987; Abad et al., 1989) that the chloroplast contains a soluble processing enzyme that cleaves the precursor for the major light-harvesting chlorophyll binding protein (LHCP), one of the most abundant proteins of the thylakoid membranes. Upon in vitro import into chloroplasts, the LHCP precursor (preLHCP) is cleaved at two sites, termed the primary and secondary sites, giving rise to two peptides of 26 and 25 kDa, respectively. However, the processing enzyme only utilizes the secondary site in an organelle-free reaction (Lamppa and Abad, 1987; Clark et al., 1989; Clark and Lamppa, 1992). It is possible that without the translocation step of import, preLHCP remains in a folded conformation that exposes one site for preferential cleavage. Nevertheless, the properties of the enzyme that produces the 25 kDa peptide in the organelle-free assay were found (Abad et al., 1989) to be very similar to those described for a stromal enzyme that cleaves preSSU and prePC (Robinson and Ellis, 1984): it was inhibited by divalent cation chelators, insensitive to PMSF, had a basic pH optimum, and was estimated to be 200 kDa. Furthermore, partial purification (7000-fold) of the enzyme yielded an enriched activity that cleaved not only preLHCP, preSSU and prePC but also preACP, preRA and preHsp2l (Abad et al., 1991). Taken together, these earlier results strongly suggest that the chloroplast contains a general processing enzyme that recognizes a large diversity of imported proteins. However, the identity of the chloroplast processing enzyme has been elusive because of its lability. Mitochondria possess a general processing enzyme for removal of the matrix-targeting signal, and this has now been purified from Neurospora (Hawlitschek et al., 1988), yeast (Yang et al., 1988) and rat tissues (Ou et al., 1989). The enzyme is made up of two subunits (50-57 kDa, depending on the organism), which are encoded in yeast by the essential MASI and MAS2 genes (Witte et al., 1988; Yang et al., 1988). Temperature sensitive mutants accumulate precursors outside the envelope inner membrane (Yaffe and Schatz, 1984), whereas depletion of the MAS subunits results in uncleaved precursors within the mitochondria (Geli et al., 1990). The two subunits are both required for efficient processing and share considerable sequence homology, especially in Neurospora (Pollock et al., 1988). In this study we have identified two proteins, of 145 and 143 kDa, that co-purify with the chloroplast processing activity from pea. Recombinant preLHCP synthesized in Escherichia coli was employed both as a substrate to monitor recovery of the processing activity and as an affinity ligand to rapidly enrich for the enzyme. Using antiserum directed against the 145/143 kDa doublet in immunodepletion experiments, we found a concomitant loss of processing activity with the immunoprecipitation of the two poly-
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peptides. Not only was preLHCP no longer cleaved, but there was a 75 % reduction in the cleavage of the precursors for the small subunit (SSU) of Rubisco and the acyl carrier protein (ACP), both found in the chloroplast stroma. Examination of the size of the holoenzyme suggests that the 145 and 143 kDa species do not form stable hetero- or homodimeric complexes. In addition, our results indicate that the 145 and 143 kDa proteins are antigenically related. A polypeptide of similar size was detected in the monocot wheat.
Results Co-purifcation of 145 and 143 kDa proteins with a chloroplast processing enzyme that cleaves preLHCP We have overexpressed recombinant preLHCP in E. coli and have demonstrated that it is cleavable in an organelle-free reaction by a soluble processing enzyme. Cleavage occurs between residues Lys4O and Ala41, producing the -25 kDa form of LHCP, which was also observed when using preLHCP made in a reticulocyte lysate translation reaction (Abad et al., 1991; Oblong and Lamppa, 1992). Recombinant preLHCP was used to monitor elution of the chloroplast processing enzyme during all chromatographic procedures. A soluble extract was prepared from isolated chloroplasts, ammonium sulfate precipitated and chromatographed twice on a Q Fast-Flow (QFF) anion exchange resin. A major enrichment for the enzyme occurred when an active sample collected from the second QFF chromatography step was applied to a Sepharose 4B column with recombinant preLHCP covalently attached as an affinity ligand. Typically, 4402
-60% of the activity bound at 4°C and was eluted from the affinity matrix with 250 mM NaCl (Figure IA). Earlier experiments had shown that preLHCP processing was inhibited at 40C (Abad et al., 1989). There was no detectable binding of enzymatic activity to a Sepharose 4B column without preLHCP as a ligand (data not shown). The active sample from the preLHCP affinity matrix was then analyzed by anion exchange (Mono Q) and gel filtration (Superose 6) chromatography. Fractions collected from the gel filtration column containing the peak of processing activity (Figure IB, lanes 8 and 9) were analyzed on a final anion exchange column. Eight proteins were identified by silver-stained gels (Figure 1B, lanes 15 and 16). Only two of these eight proteins (see the double-headed arrow in Figure IB) eluted with the identical fractions containing the peak of processing activity (Figure IC) during both Superose 6 and Mono Q chromatography. The Mr of the two proteins were estimated to be 145 and 143 kDa. -
Antiserum raised against the 145/143 kDa doublet recognizes only these proteins in total chloroplast soluble extracts The 145 and 143 kDa proteins were purified by preparative SDS-PAGE and used to prepare polyclonal antiserum. The antiserum recognized both the 145 and 143 kDa proteins on immunoblots in an apparent 1:1 ratio (Figure 2A). LSU, the most abundant protein of the chloroplast stromal phase, was also sometimes detected. In order to remove this crossreactivity with LSU, total soluble chloroplast proteins ranging in size from 30 to 100 kDa were isolated and covalently attached to Sepharose 4B. The chloroplast
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Fig. 2. Antiserum raised against the 145 and 143 kDa proteins recognizes only these two proteins in total soluble chloroplast extracts. The 145 and 143 kDa proteins, purified by preparative gel electrophoresis, were used to generate anti-145/143 kDa serum. (A) Samples from a QFF column were analyzed on a silver-stained gel (lane 1) and on an immunoblot with anti-145/143 kDa serum (lane 2). (B) Anti-145/143 kDa serum (30 Al) was pre-incubated with either 500 A1 of 1 x TBS or with 500 Al of a 50% slurry of 30-100 kDa chloroplast protein-Sepharose 4B (see Materials and methods). The soluble phases from both samples were used for immunoblot analysis of total soluble chloroplast extract (lanes 1 and 2, respectively).
protein - Sepharose 4B matrix was used to pre-clear the anti-145/143 kDa serum, and as a result LSU was no longer detected nor were any other polypeptides besides the 145 and 143 kDa proteins (Figure 2B).
Immunodepletion of the 145/143 kDa doublet results a loss of processing activity Total soluble chloroplast extract was incubated with IgGs from either anti-145/143 kDa or pre-immune antiserum in immunodepletion experiments. Preimmune IgGs covalently attached to a protein A - agarose matrix did not clear the 145 and 143 kDa protein bands upon centrifugation (Figure 3A, 'pre-Imm' bracket) and did not inhibit or remove
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also covalently attached to protein A - agarose. When used in immunodepletion reactions the matrix removed the 145 and 143 kDa proteins in a concentration-dependent manner (Figure 3A, 'anti-145/143 kDa IgG' bracket). Attempts to elute the immunoprecipitated 145 and 143 kDa proteins from protein A-agarose using 100 mM glycine (pH 2.6) did not release any proteins that could be detected on immunoblots. However, immunoblots analyzing a soluble chloroplast extract treated with 100 mM glycine (pH 2.6) and neutralized with 1 M Tris-HCl (pH 8.0) showed a complete loss of both the 145 and 143 kDa proteins due to either degradation, aggregation, or loss of antigenic structural epitopes (data not
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Fig. 3. Immunodepletion of the 145 and 143 kDa proteins and concomitant loss of processing activity. (A) Anti-145/143 kDa IgGs were covalently attached to protein A-agarose and the indicated amounts of IgG conjugated protein A-agarose (50% slurry) were incubated with soluble chloroplast extract. The differences in final volumes (600 Al) were adjusted with 1 x TBS. 'pre-Imm' designates pre-immune IgG conjugated to protein A-agarose. After incubation overnight at 4°C, the matrices were removed by centrifugation and 100 IL of the supematant was analyzed on an immunoblot with anti-145/143 kDa serum. Rabbit IgG binding capacity is 15 mg/ml of protein A-agarose. (B) 100 Al of the supernatant was incubated with preLHCP in organelle-free reactions and the products were analyzed on an immunoblot with anti-LHCP serum. As controls, crude extract (+) or extract diluted to 600 Al with 1 x TBS ('CON') was incubated in organelle-free reactions. (C) 100 I1 of the supernatant was incubated in organelle-free reactions with radiolabeled recombinant preACP and radiolabeled preSSU synthesized in a reticulocyte lysate. The products were analyzed by SDS-PAGE followed by autoradiography. The vertical axis indicates the percent of mature ACP or SSU produced relative to the control reaction using extract incubated with pre-immune IgGs.
Most importantly, as well as immunoprecipitating the 145 and 143 kDa proteins, the IgGs from the anti-145/143 kDa serum conjugated to protein A - agarose also depleted the soluble processing activity that cleaves preLHCP (Figure 3B, 'anti-145/143 kDa IgG' bracket). From this we conclude that the anti-145/143 kDa serum was able to specifically remove the processing enzyme and that this was due to selective recognition of the 145 and 143 kDa proteins in a complex chloroplast protein mixture. In order to determine if the processing activity was either specific for preLHCP or recognizes other precursors, aliquots of the supernatants from the immunodepletion reactions were incubated in organelle-free processing reactions with radiolabeled preACP synthesized in E. coli or radiolabeled preSSU synthesized in a reticulocyte lysate. The products of the processing reactions were analyzed on SDS -polyacrylamide gels followed by quantification of the
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30 kDa) remained besides the 145/143 kDa doublet in the most active fractions, but all exhibited very different patterns of elution from those of the 145 and 143 kDa proteins in the last two chromatographic steps. Hence, none were apparently tightly associated with the 145 and 143 kDa proteins, nor does sedimentation of the holoenzyme on sucrose gradients-the least severe of the procedures employed to estimate its molecular mass-suggest that another factor is involved. Therefore, it seems unlikely that in the immunodepletion experiments a third factor would have co-precipitated with either the 145 or 143 kDa protein as part of a larger complex. Taken together, these results indicate that the 145 and/or 143 kDa proteins contain the catalytic domain of the processing enzyme. Nevertheless, cross-linking studies using preLHCP and soluble extracts from radiolabeled chloroplasts may yet identify a protein present in less than stoichiometric amounts that participates in the cleavage reaction or assists in stabilizing any interaction between the 145 and 143 kDa proteins. We first identified the soluble enzyme that cleaves preLHCP in the organelle-free assay not only in pea, a dicot, but also in the monocot wheat (Lamppa and Abad, 1987). Therefore, we asked in the present study whether the antiserum raised against the 145 and 143 kDa proteins from pea would recognize related polypeptides in wheat. We have found that a soluble chloroplast extract from wheat contains a species that co-migrates during SDS -PAGE with the doublet from pea. However, two more abundant lower molecular weight proteins were detected even in the presence of an array of protease inhibitors. While the species that migrates with the 145/143 kDa doublet is a strong candidate for a component of the processing enzyme in wheat, further experiments are necessary to establish the origin of the lower molecular weight proteins that are recognized by the anti- 145/143 kDa serum. PreLHCP contains two processing sites which are cleaved independently during import (Clark et al., 1989, 1991). It has been puzzling why only the secondary site is utilized in the organelle-free assay (see Introduction), producing the 25 kDa form of LHCP. Site-directed mutagenesis has shown that the sequence motif AKA in wheat preLHCP at the
secondary cleavage site itself (underlined) is essential for efficient processing both in the organelle-free assay and during import into chloroplasts (Clark and Lamppa, 1991). However, the AKA motif is not sufficient; when moved downstream four amino acids by an insertion in this domain, processing is abolished. Thus, this motif must be located within the correct structural context of preLHCP in order to be recognized (Clark et al., 1989). Although the AKA motif is not found in other precursors, alanine is commonly found at position + 1 relative to the processing site. However, preSSU has a methionine in this position. The majority (80%) of transit peptides also have an arginine or lysine near the C-terminus, between residues -1 and -7 (von Heijne et al., 1989). We have shown that preLHCP requires a basic residue at position -4 for efficient cleavage at its primary site, but substitution of uncharged amino acids for the basic residues in the region of interest in preSSU and preRA had no affect on processing (Clark and Lamppa, 1991). It has recently been speculated that a transition from ,B-strand to a-helix near the transit peptide-mature protein junction is important for processing (Gavel and von Heijne, 1990), but this remains to be established. The overall conformation of the precursor polypeptide undoubtedly plays an important role in transit peptide removal. In conclusion, the identification of the 145 and 143 kDa proteins, which function either separately or together as components of the chloroplast processing enzyme now makes it possible to determine the enzyme's primary sequence, and explore its biosynthetic pathway. In addition, the mechanism of precursor recognition and cleavage can be defined more clearly. Future studies should also lead to an understanding of how the expression of the processing enzyme is regulated during chloroplast biogenesis in leaves and plastid differentiation in other organs.
Materials and methods Precursor synthesis A wheat genomic clone (Lamppa et al., 1985) was employed to synthesize preLHCP in E.coli as described earlier (Abad et al., 1991). The precursors for SSU, encoded by a pea construct (see Abad et al., 1989), and ACP, encoded by a spinach gene (Scherer and Knauf, 1987), were synthesized by in vitro transcription (TM polymerase reaction, United States Biochemical Corp.) and translation (with [35S]methionine, 1150 Ci/mmol, in a reticulocyte lysate, Bethesda Research Labs). Generation of preLHCP- Sepharose 4B affinity matrix PreLHCP expressed in E.coli was isolated from inclusion-like bodies in 6 M urea (Abad et al., 1991). The solubilized material was desalted using Sephadex G-25 spin columns first into 20 mM Tris (pH 8.3) (solution A) and then into 100 mM NaHCO3, 500 mM NaCl (pH 8.3). CNBr-activated Sepharose 4B resin (2 g) was swollen in 1 mM HCl for 15 min at room temperature and washed twice for 5 min in 1 mM HCl. The resin was neutralized in 50 ml of 100 mM NaHCO3, 500 mM NaCl (pH 8.3), mixed with 7 ml of recombinant preLHCP (0.57 mg/ml) in 100 mM NaHCO3, 500 mM NaCl (pH 8.3) and incubated overnight at 40C according to the manufacturer's instruction (Pharmacia LKB). The matrix was centrifuged at 2000 g for 2 min and unreacted side chains were neutralized by washing in solution A for 2 h at room temperature. The efficiency of cross-linking was - 95 % (2 mg protein/ml resin). A 1 cm x 10 cm glass column was prepared with the matrix. Enrichment for the processing enzyme Pea chloroplasts were isolated (Bartlett et al., 1982) from 2.5 kg of 9 dayold pea seedlings (Pisum sativum, Laxton's Progress # 9) grown on a 16 h light/8 h dark cycle. Wheat chloroplasts (Triticum aestivum, Era) were isolated from 12 day-old plants; grinding buffer contained 20 mM EDTA and 5 ItM E-64, 50 lsM leupeptin, 1 AM pepstatin and 1 mM PMSF.
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J.E.Oblong and G.K.Lamppa
Chloroplasts were hypotonically lysed in solution A, 0.5 mM ,B-mercaptoethanol for 30 min on ice. For wheat chloroplast lysis, solution A also contained 5 ItM E-64, 50,uM leupeptin, 1 itM pepstatin and 1 mM PMSF. All subsequent handling of protein samples was performed at 4°C. Total membranes were removed by centrifugation at 23 000 g for 20 min. The supematant was precipitated with ammonium sulfate and the 40-70% pellet was resuspended in solution A and desalted twice on Sephadex G-25 spin columns into solution A. The desalted sample was loaded onto a QFF (Pharmacia LKB) anion exchange resin (2.5 x 20 cm, equal to - 85 ml). Activity was eluted by a linear gradient from 150 to 250 mM NaCl in a background buffer of solution A. Fractions containing activity were pooled, concentrated (Centriprep concentrators, 30 kDa size cut-off, Amicon), and desalted on Sephadex G-25 spin columns into solution A. The sample was rechromatographed on QFF and collected fractions were concentrated and desalted as above. Material collected from the second QFF column was then applied to a preLHCP-Sepharose 4B affinity column at a flow rate of 0.1 mIl/min. After the OD280 returned to baseline, the processing activity was eluted with 250 mM NaCl, de-salted as above and loaded onto a Mono Q HR 5/5 anion exchange column. Activity was eluted by a 0-250 mM linear gradient. Active fractions were concentrated to 400 Al and loaded onto a Superose 6 HR 10/30 gel filtration column (Pharmacia LKB). The activity eluted in a volume of 14-15 ml. This sample was then loaded onto the Mono Q column and activity was eluted with the same salt gradient as outlined above. The Superose 6 column was calibrated with Blue Dextran, thyroglobulin (669 kDa), ,B-amylase (200 kDa), alcohol dehydrogenase (150 kDa) and bovine serum albumin (66 kDa) to estimate the apparent molecular size of the processing enzyme by plotting the M, versus Velution/Vvoid (Stellwagen, 1990). Anti- 145/143 kDa serum preparation Soluble chloroplast extract was chromatographed on a QFF anion exchange column and fractions collected, as described above. The fractions ( - 240 mM NaCl) containing processing activity were concentrated and desalted to 4 ml (10 mg/ml), mixed with 2 ml of 3 x SDS-PAGE reducing buffer (6% SDS, 30% glycerol, 15% (3-mercaptoethanol, 187.5 mM Tris-HCl, pH 6.8, 0.5 mg/ml bromophenol blue), and boiled for 3 min. The denatured sample was layered onto a 90 ml 7% polyacrylamide resolving gel with a 15 ml 4% polyacrylamide stacker in a Bio-Rad Model 461 Preparative Cell System. Electrophoresis was carried out at 40 mA for 32 h and 8 ml fractions were collected in 25 mM Tris-HCI, 190 mM glycine and 0.1 % SDS. Aliquots (25 jl) were analyzed on silver-stained gels (Stratagene) for the presence of the 145 and 143 kDa proteins. Fractions containing the 145 and 143 kDa proteins were concentrated and stored at -20°C until further use. Using standard protocols, rabbits were immunized and antiserum was prepared.
Immunoblot analysis
organelle-free processing reactions were performed as described (Abad et al., 1991). In brief, a 5-20 I1 aliquot of soluble chloroplast extract containing the processing enzyme was incubated with 2 t1 of E.coli expressed preLHCP (1 yg/jtl) at 26°C for 1-2 h. Reactions were quenched by the addition of 3 x SDS-PAGE reducing buffer, boiled and run on a 10 or 12% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose in 25 mM Tris and 200 mM glycine (Towbin et al., 1970). Blots were blocked in 1 x TBST [20 mM Tris, 0.5 M NaCl (pH 7.5), 0.1 % Tween-20], 3 % BSA for at least 30 min. Immunoblots analyzing reaction products were In vitro
incubated with a 1:1000 dilution of rabbit antiserum directed against the 25-27 kDa LHCP proteins (Abad et al., 1991). Immunoblots analyzing the presence of the 145 and 143 kDa proteins were incubated with 1:2500 dilution of antiserum which had been first pre-cleared by incubating 25 1l of the anti-145/143 kDa serum with 500 1l of a Sepharose 4B matrix crosslinked with 30-100 kDa chloroplast proteins (see below). Immunoblots were washed with 1 x TBST, incubated with goat anti-rabbit IgGs conjugated with alkaline phosphatase (1:3000 dilution), and developed with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Harlow and Lane, 1988).
Immunodepletion of the 145 and 143 kDa proteins Proteins of - 30-100 kDa were isolated from the soluble chloroplast extract using the Bio-Rad Model 461 Preparative Cell System. The isolated proteins were concentrated and cross-linked to CNBr-activated Sepharose 4B under the same conditions as those used for preLHCP-Sepharose 4B linkage. The anti-145/143 kDa serum was centrifuged to remove coagulants and precleared with Sepharose 4B matrix conjugated with the 30-100 kDa chloroplast proteins. Anti-145/143 kDa serum (1 ml) was mixed with a 50% slurry (3 ml) of the 30-100 kDa chloroplast protein-Sepharose 4B matrix in 1 x TBS, 3% BSA, and incubated at room temperature for 60 min. The matrix was centrifuged at 2000 g and the cleared supernatant containing anti-145/143 kDa IgGs was coupled to protein A-agarose using dimethyl
4408
suberimidate (Affinica Antibody Kit, Schleicher and Schuell). After quenching, the matrix was washed with 1 vol of 100 mM glycine (pH 2.6) to remove unlinked IgGs. Soluble extract was prepared from chloroplasts and 10 Al was mixed with 50-500 41 of a 50% slurry of either preimmune or anti-145/143 kDa IgG protein A-agarose. The final immunoprecipitation reaction volumes were adjusted to 600 Al with 1 x TBS. The reactions were incubated overnight at 4°C and centrifuged at 1000 g for 1 min. 100 1l aliquots of the supernatant were used in either organelle-free processing reactions or analyzed on immunoblots probed with pre-cleared anti-145/143 kDa serum. Processing of preACP or preSSU was analyzed either by densitometry of autoradiograms or liquid scintillation counting of excised gel bands, which were solubilized with TS-1 (Research Products International). Affinity purification of anti- 145/143 kDa IgGs IgGs directed against the 145 and 143 kDa proteins were isolated from nitrocellulose with modifications of Olmsted (1981) and Harlow and Lane (1988). Soluble chloroplast extract was chromatographed on QFF anion exchange resin and fractions containing the 145 and 143 kDa proteins were concentrated and desalted. Aliquots (75 Al) collected from the QFF column (- 10 jig/ml) were loaded onto a 10% polyacrylamide gel and electrophoresed along with pre-stained high molecular weight markers (BioRad). Proteins were transferred to nitrocellulose and stained with Amido black (0.1%, in 20% methanol and 5% acetic acid). Nitrocellulose strips containing the immobilized 145 and 143 kDa proteins were excised and destained with 10% methanol. One group of strips (1-2 cm2) contained the immobilized 145 kDa protein and a second contained the 143 kDa protein. A third group was made up of nitrocellulose pieces from the blot containing no detectable protein. All three groups were blocked with 3% BSA in 1 x TBST for 30 min after which a 1:1000 dilution of anti-145/143 kDa serum was added, and gently rocked for 3 days at 4°C. The soluble phases of the cleared sera were saved and the nitrocellulose strips washed with 5 ml of 1 x TBST. The strips were then washed for 3 min with 2 ml of 100 mM glycine (pH 2.6) and the supernatants were quenched with 1 ml of 1 M Tris-HCl (pH 8.0). A separate blot of soluble chloroplast extract run on a 10% SDS-acrylamide gel was prepared and individual gel lanes containing proteins ranging in size from 60 to 200 kDa were incubated with either the cleared sera or the eluted IgGs, followed by immunoblot analysis. Pore exclusion limit gel electrophoresis A 4-18% linear gradient polyacrylamide gel (3-15 % glycerol) was poured in 0.5 x TB (45 mM Tris-HCI and 45 mM boric acid) with no stacker as described by Westwood et al. (1991). Protein samples were diluted with 3 x native gel loading buffer (45% glycerol, 135 mM Tris-HCI, 135 mM boric acid and 0.1% xylene cyanol FF) and loaded directly into the gels. The gels were electrophoresed in 0.5 x TB at 320 V (16 V/cm gel) until the xylene cyanol FF dye had migrated from the bottom of the gel. Gels were equilibrated in 25 mM Tris-HCI, 200 mM glycine and 0.25% SDS for 10 min at 70°C and proteins were transferred to nitrocellulose at 80 mA overnight. Immunoblots were probed with anti-145/143 kDa serum. Gels were calibrated with thyroglobulin (669 kDa), catalase (240 kDa), /3-amylase (200 kDa), alcohol dehydrogenase (150 kDa) and bovine serum albumin (66 kDa).
Sucrose density gradient centrifugation Sucrose density gradient centrifugation was performed at 4°C as described in Kahana and Edelman (1982). Soluble chloroplast extract (3-4 ml) was mixed with 25 Ag of catalase (240 kDa, 11.3S), (3-amylase (200 kDa, 8.9S), alcohol dehydrogenase (150 kDa, 7.4S) and bovine serum albumin (66 kDa, 4.3S) and layered onto a 17 ml 5-25% sucrose linear gradient in 20 mM Tris-HCI, 10 mM NaCl (pH 8.0) either with or without 1 mM PMSF, 5 IAM E-64 and 30 Ag/ml trypsin inhibitor. Gradients were centrifuged at 95 000 g for 36 h at 4°C. Fractions (500 AI) were collected from the bottom and aliquots (50 Al) were assayed with preLHCP in organelle-free reactions, followed by immunoblot analysis with anti-LHCP serum. Aliquots (50 ,.l) were also analyzed on immunoblots with anti-145/143 kDa serum. The positions of molecular weight markers were identified by Coomassie blue staining of gels. The presence of alcohol dehydrogenase was detected by monitoring the reduction of 3-nicotine adenine dinucleotide (Worthington Enzymes Manual, Worthington Biochemical Corp., 1977).
Chromatofocusing Soluble chloroplast extract (3 mg/ml) was prepared from 5 ml of isolated chloroplasts, precipitated with ammonium sulfate (40-70%) and desalted into 50 mM bis-Tris-HCI (pH 6.3) (buffer C). The sample was applied chromatofocusing column (Pharmacia LKB) preequilibrated in buffer C. Bound protein was eluted from the column in a pH gradient with Polybuffer 74 (pH 4.0) diluted 1:10 as recommended by
to a Mono P HR 5/20
Proteolytic processing of chloroplast precursors the supplier. Aliquots (500 jil) were buffered with 100 yi of 1 M Tris-HCI (pH 8.0) and used in organelle-free reactions with preLHCP and radiolabeled preSSU synthesized in a reticulocyte lysate.
Acknowledgements We thank Li-Ming Yang for generously providing recombinant preACP. This research was supported by National Institutes of Health Grant GM36419 (to G.K.L.) and by Sigma Xi Grants-in-Aid of Research (to J.E.O.).
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Received on July 31, 1992
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