From the Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden
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ABSTRACT |
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The chloroplast compartment enclosed by the thylakoid membrane, the "lumen," is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60-65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase.
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INTRODUCTION |
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The chloroplast is the photosynthetic organelle of green algae and higher plants. The chloroplast architecture comprises an envelope membrane, which encloses the soluble stroma as well as the highly specialized thylakoid membrane. The stromal compartment contains mainly the components of the Calvin cycle, which are required for the fixation of carbon dioxide. The thylakoids, on the other hand, carry out the light reactions of photosynthesis leading to the production of NADPH and ATP. The thylakoid membrane has a characteristic flat shape and is differentiated into appressed grana stacks and single non-appressed stroma-exposed lamellae. The inner surface of the thylakoid membrane encloses a narrow, continuous compartment, the lumen (1, 2). Electron microscopy studies of spinach thylakoids have suggested that the lumen is a densely packed space (3). No isolation method has so far been available for obtaining a high yield of pure thylakoid lumen. Thus, the present knowledge of the lumen from a compositional and functional point of view is fragmentary and is gathered from several independent approaches, addressing only single aspects of this compartment.
By developing a technique for obtaining inside-out thylakoids, the investigation of the membrane surface of the lumenal side became possible (4). This work contributed to the discovery of the extrinsic proteins PsbO, PsbP, and PsbQ (5, 6) that bind to the lumenal side of photosystem II and are thought to stabilize the water oxidizing complex (7, 8). More recent studies have shown that these subunits of photosystem II occur also as soluble lumenal proteins (9). This pool of unassembled PsbO, PsbQ, and PsbP was resistant to proteolytic degradation and was capable of assembling into photosystem II (10). Furthermore, it was found that during photoinhibitory conditions the extrinsic proteins were released from the membrane into the lumen (11, 12). Other important components of the thylakoid lumen are plastocyanin, the primary electron donor of photosystem I (13, 14), and PsaN, a photosystem I subunit that is extrinsically bound to the lumenal side of the thylakoid membrane (15).
Recent investigations have revealed that polyphenol oxidases (16, 17) and violaxanthin de-epoxidase are also present in the thylakoid lumen (18). Furthermore, the carboxyl-terminal processing protease for the D1 protein (19, 20) and a processing protease for plastocyanin (21) were found on the lumenal surface of the thylakoids, whereas chaperones may be located in the lumen (22).
So far all lumenal proteins have been found to be nuclear-encoded and synthesized as precursors in the cytoplasm. These precursor proteins have characteristic amino-terminal bipartite transit peptides, which direct their import into the chloroplast stroma and across the thylakoid membrane into the lumen (23-25). On the basis of this property, bipartite transit peptides have become typical markers for lumenal proteins. However, not all chloroplast proteins encoded with such presequences are routed into the lumenal space. The PsbW protein and CFoII, for instance, are synthesized with bipartite transit peptides but have been shown to be integral proteins of the thylakoid membrane (26-28).
In this study we have developed a procedure by which a lumenal fraction can be isolated in a highly pure form from spinach thylakoids. We have carried out the first systematic characterization of this compartment, and we show that the thylakoid lumen contains a high concentration of proteins, among which at least 25 distinct polypeptides can be identified. Several have been characterized in terms of enzymatic activity or amino-terminal sequence.
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EXPERIMENTAL PROCEDURES |
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Plant Material-- Spinach (Spinacia oleracea) was grown hydroponically for 6 weeks with alternating periods of 10 h light and 14 h darkness.
Isolation of the Thylakoid Lumenal Content-- The general approach applied consists of three principal steps as follows: (i), preparation of chloroplasts, (ii) purification of carefully washed thylakoids, and (iii) rupture of thylakoids by a Yeda press and isolation of the lumenal content (Fig. 1).
Spinach leaves (200 g) were blended twice for 5-10 s in 330 mM sorbitol, 50 mM Hepes-KOH (pH 7.8), 10 mM KCl, 1 mM EDTA, 0.15% (w/v) bovine serum albumin, 4 mM sodium ascorbate, and 7 mM cysteine. The resulting slurry was filtered through four layers of nylon mesh (20 µm), and the filtrate was centrifuged 1 min at 1000 × g. The pellets were resuspended in 330 mM sorbitol, 50 mM Hepes-KOH (pH 7.8), 10 mM KCl, centrifuged for 1 min at 1000 × g, and resuspended in 12-18 ml of the same buffer. The yield was 60-70% intact chloroplasts containing 50-60 mg of chlorophyll. The chloroplasts were diluted with 10 mM sodium pyrophosphate buffer (pH 7.8) to a final chlorophyll concentration of 0.2 mg mlThermolysin Treatment of Thylakoids--
The washed thylakoids
(2 mg of chlorophyll ml1) were incubated in the presence
of 10 µM thermolysin (0.4 mg ml
1) for 2 min
on ice in 100 mM sucrose, 30 mM Hepes (pH 7.8),
50 mM NaCl, 5 mM MgCl2, and 2 mM CaCl2. The digestion was stopped by adding
EDTA to final concentration of 50 mM, and the thylakoids were washed twice with 100 mM sucrose, 30 mM
Hepes (pH 7.8), 50 mM NaCl, and 50 mM EDTA.
These conditions were balanced to degrade the major part of peripheral
proteins on the stromal side of the thylakoid membrane, without
degrading too much of the integral membrane proteins. Harsher
treatments were found to lead to leaky membranes followed by a loss of
especially the lumenal part of the exposed stromal lamellae.
Measurements of Chlorophyll, Protein, and Oxygen Evolution-- Chlorophyll concentrations were measured as described (29). Determination of soluble proteins was carried out according to Ref. 30 and that of membrane proteins was performed essentially as described (31). The standard used was bovine serum albumin. Oxygen evolution activities and intactness of the chloroplasts were measured with a Clarke-type electrode at 20 °C using potassium hexacyanoferrate(III) as the electron acceptor (32).
Electrophoretic and Immunoblot Analysis-- SDS-PAGE was performed by the method described in Ref. 33 in slab gels containing 18% (w/v) polyacrylamide and 2 M urea. Determination of molecular masses were performed by using the low molecular weight electrophoresis calibration kit from Pharmacia Biotech Inc. For immunoblotting analyses proteins were transferred onto a polyvinylidene difluoride membrane in a semidry electroblotter system (Millipore). The antisera used were raised in rabbits against the following spinach proteins: phosphoribulokinase (K.-H. Süss, Institute for Plant Genetics and Crop Plant Research, Gatersleben); violaxanthin de-epoxidase (H.-E. Åkerlund, University of Lund); plastocyanin (P.-Å. Albertsson, University of Lund); PsbO, PsbP, PsbQ, the lumen fraction, and Rubisco (our own production). The immunoreactivity was detected using goat anti-rabbit IgG-conjugated horseradish peroxidase in combination with an enhanced chemiluminescence detection. Quantification was performed using a Fast Scan Personal Densitometer and the ImageQuant software from Molecular Dynamics. To be able to compare directly the results for the chlorophyll-free lumenal fractions with those of the chloroplast and thylakoids, a chlorophyll equivalent was used for the lumenal fraction. It was calculated as follows: [Lumeneq] = [Chl]thyl × Vthyl/Vlumen. However, the amount of plastocyanin and ferredoxin-NADP reductase (FNR) in the lumenal fraction was determined from the difference between the content in the washed thylakoids and their residual membrane fragments.
Enzymatic Assays--
Mitochondrial cytochrome c
oxidase was assayed according to Ref. 34 and malate dehydrogenase
according to the Worthington manual.2 As controls
mitochondria from potato and spinach were used (kindly provided by P. Pavlov and X. Zhang, Stockholm University). Catalase activity was
determined by oxygen evolution in the presence of 60 mM
hydrogen peroxide (32). Phosphoribulokinase was assayed according to
Ref. 35. To prevent oxidative inactivation of this enzyme during the
lumenal isolation, all buffers contained 5 mM dithiothreitol. Diphenolase activity of polyphenol oxidase was determined at pH 4.6 using the substrate 3,4-dihydroxyphenylpropionic acid (36). Peroxidase activity was measured using the substrate 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) as described by
Sigma.3 Protease activity was
tested using a dye-linked peptide (PepTag, Promega) and the in
vitro translated, [35S]methionine-labeled
-subunit of mitochondrial ATP synthase of Nicotiana
plumbaginifolia (37) as artificial substrates. The activity of
violaxanthin de-epoxidase was determined as in Ref. 38. All enzymatic
activities were measured on freshly prepared samples (except those for
violaxanthin de-epoxidase) at 25 °C in the presence of saturating
substrate concentrations.
Assay for ATP and ATPase Activity-- ATP was detected using the firefly luciferase system (39) and the ATP monitoring reagent from BioOrbit. ATPase activity was tested according to Ref. 40.
Amino-terminal Protein Sequence Analysis-- Proteins were sequenced from polyvinylidene difluoride membrane following resolution by SDS-PAGE essentially as described (41). The amino-terminal sequence analyses were performed by P. I. Ohlsson (University of Umeå) using an Applied Biosystems pulsed liquid phase sequenator (ABS 477A). Searches in the data bases of EMBL and SwissProt as well as sequence alignments were carried out using the Wisconsin GCG software (42).
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RESULTS |
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Preparation and Characterization of the Thylakoid Lumen--
The
purpose of the present study was to obtain a preparation of thylakoid
lumen in a yield and purity sufficiently high to make it generally
useful for characterizing this chloroplast compartment. The developed
method, as outlined in the scheme of Fig.
1, starts with the isolation of intact
spinach chloroplasts. The chloroplasts obtained were normally 60-70%
intact and had an oxygen evolving activity of 140-185 µmol of
O2 mg Chl1 h
1. In the next step
the chloroplasts were disrupted by osmotic shock, and the thylakoids
were collected and purified by several washing steps. Soluble stromal
proteins were effectively removed by 10 mM sodium
pyrophosphate (pH 7.8), and 2 mM Tricine (pH 7.8) containing 300 mM sucrose was used to remove ATP synthase,
membrane-attached Rubisco, and other unidentified peripheral thylakoid
proteins. Finally, the thylakoids were equilibrated in fragmentation
buffer; these thylakoids retained an oxygen evolution rate of 90-165
µmol of O2 mg Chl
1 h
1. The
washed thylakoids were then fragmented by a Yeda press, and the
released lumenal content was separated from the membrane fragments by
two ultracentrifugation steps. The final fraction was free of
chlorophyll and had a protein concentration of 0.3-0.5 mg
ml
1, giving a total yield of 2-3 mg of protein from
200 g of spinach leaves.
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Enzyme Activities of the Thylakoid Lumen-- Earlier work had suggested that polyphenol oxidase (17) and violaxanthin de-epoxidase (18) are located in the lumen of the spinach thylakoids. Therefore, we analyzed the isolated lumenal fraction for the presence of these two enzymes. The specific activity of polyphenol oxidase increased from the chloroplasts to the washed thylakoids and attained a maximum in the lumenal fraction (Table III). The fraction of the polyphenol oxidase activity that remained with the thylakoid fragments was 8% of that present in the lumenal fraction. The presence of polyphenol oxidase in the lumenal fraction was also confirmed by amino-terminal protein sequencing of the polypeptide with an apparent molecular mass of 64.5 kDa (Table I).
The specific activity of violaxanthin de-epoxidase in the lumenal fraction was 18 µmol of violaxanthin/g of protein
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Identification of Unknown Lumenal Proteins via Amino-terminal Sequencing-- To find new lumenal proteins, amino-terminal sequencing of polypeptides from the lumenal fraction was performed after their separation by SDS-PAGE. This approach led to the identification of four polypeptides of the apparent molecular masses of 40.0, 29.0, 17.4, and 16.5 kDa (Table IV).
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DISCUSSION |
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The thylakoid lumen represents a continuous exoplasmic cellular space that is poorly characterized compared with the other chloroplast compartments. Interest in the lumenal side of the thylakoid membrane has mainly focused on electron transport events associated with the inner membrane surface. Indirectly, the thylakoid lumen has also been considered as essential for the generation of the trans-thylakoid proton gradient that drives ATP synthesis and for the anion and cation currents established by ion channels in the thylakoid membranes (49, 50). More recently, with an increased understanding of biosynthesis, regulation, and stress protection of the photosynthetic apparatus, the requirement for auxiliary enzymes (51) in the lumenal space has become more apparent.
The present isolation of a thylakoid lumenal fraction gives high yield
and low degree contamination, thereby providing a new tool for
biochemical analysis of this chloroplast compartment. Based upon a
volume to chlorophyll ratio for thylakoids of 3.3 µl per mg of
chlorophyll (52) and the yield of 75-120 µg of lumenal protein per
mg of chlorophyll in the starting thylakoid material, the protein
concentration in the lumenal space is estimated to be higher than 20 mg
ml1. Thus, soluble proteins in the lumen are at
micromolar to millimolar concentrations, which is similar to that of
the chloroplast stroma. Hence, the thylakoid lumen is comprised of a
densely packed core of soluble components, as suggested from electron
microscopy studies (3).
The number of resolved polypeptides in the isolated lumenal fraction was approximately 25, of which 15 remain to be functionally identified. In contrast to what is observed for the thylakoid proteins only a few of these are of low molecular mass.
The purity of the isolated lumenal fraction was very high as judged by its low contamination of stromal and thylakoid integral membrane proteins. The one notable exception was FNR, which is functionally associated with the outer thylakoid surface and which could not be removed by various pre-washes. Moreover, FNR is a notoriously "sticky" protein that contaminates various protein preparations including those of cytochrome b/f (53), PsbO (54), and PsbS (55).
The PsbO, PsbP, and PsbQ proteins and plastocyanin were found to be the major proteins present in the isolated lumenal fraction. The presence of extrinsic PsbO, PsbP, and PsbQ polypeptides is consistent with the previous observation of an unassembled, stable, lumenal pool of these polypeptides (9, 10), particularly under photoinhibitory conditions (11, 12). The amount of soluble PsbO in the isolated lumenal fraction was 10% relative to the total content of chloroplasts. This agrees with the finding that 8% of the PsbO of spinach occurs in an unassembled state in the thylakoid lumen (9).
The identification of violaxanthin de-expoxidase and polyphenol oxidase as soluble polypeptides of the lumen corroborates previous work suggesting the occurrence of these enzymes in this compartment (16-18). Furthermore, the presence of a peroxidase in the lumen may be important for detoxification of hydrogen peroxide produced by photosystem II upon illumination (45). The function of lumenal polyphenol oxidase is not understood. Since chloroplasts from peas lack this enzyme (16), it is not likely to play a role crucial for photosynthesis. The polyphenol oxidase might be transported into the lumen where it would be stored safely separated from its substrate in the vacuole.
The stability of the three unassembled extrinsic proteins of photosystem II brings up the question of proteolytic activities in the thylakoid lumen. The present analyses as well as previous studies (56, 57) clearly reveal the presence of proteases in this compartment. However, the protease activity of the lumenal fraction was relatively low, only 10% of that found in the thylakoid fraction, suggesting that most of lumenal protease activity is bound to the inner thylakoid surface.
The 16.5-kDa lumenal protein is highly homologous to the deduced sequence of the A. thaliana clone 250E4T7 (g1327818), which shows a typical bipartite transit peptide. However, the three arginine residues close to the putative processing site (Fig. 4) represent an unknown motif for previously determined bipartite transit peptides of chloroplast proteins. It would be of interest to determine the specific import pathway by which this protein is transported from the cytoplasm into the thylakoid lumen.
The possibility of lumenal chaperones (22) as well as experimental indications for phosphorylation of lumenal proteins (58) have been reported. This work does not support this view since we could not show the presence of ATP or demonstrate ATPase activity in the lumen. The presence of nucleotides in the thylakoid lumen will require more detailed studies.
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ACKNOWLEDGEMENTS |
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We thank Drs. P.-Å. Albertsson and H.-E. Åkerlund (University of Lund) and Dr. K.-H. Süss (Institute for Plant Genetics and Crop Plant Research, Gatersleben) for the generous supply of antibodies. We greatly appreciate the excellent technical assistance of Ann-Christine Holmström.
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FOOTNOTES |
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* This work was supported in part by grants from the Swedish Forestry and Agriculture Research Council, the Swedish Natural Science Research Council, and the Carl Trygger Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a guest research grant by The Swedish Institute.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden. Tel.: 46-8-164392; Fax: 46-8-153679; E-mail: Wolfgang{at}biokemi.su.se.
1 The abbreviations used are: Rubisco, ribulose-bisphosphate carboxylase; AC, accession number; Chl, chlorophyll; FNR, ferredoxin-NADP+ reductase; LHCII, light harvesting complex II; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine.
2 Available on-line at the following address: http//:www.worthington-biochem.com/manual/manIndex.html
3 Available on-line at the following address: http://www.sigma.sial.com.
4 Fugolsi, H., Vener, A. V., Altschied, L., Herrmann, R. G., and Andersson, B. (1998) EMBO J., in press.
5 A. V. Vener, unpublished data.
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REFERENCES |
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