The Thylakoid lumen of Chloroplast: Isolation, Characterization and Protein Targeting across Thylakoid lumen.

Meghana Rastogi, Amity Institute of Biotechnology, Amity University, Uttar Pradesh, Lucknow Campus, Gomti Nagar Lucknow - 226010, India. Email: rastogi.m0@gmail.com. ABSTRACT The chloroplast is enclosed by the thylakoid membrane, and the lumen, is not properly characterized. Thus it was important to design a stepwise procedure for the isolation of the thylakoid lumen which eventually help in characterization of lumenal proteins and to show the targeting of lumenal protein across the thylakoid membrane .In the procedure the thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, followed by Yeda press fragmentation and the lumenal content was regained in the supernatant by performing centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins which were specific for the different chloroplast compartments. Quantitative immunoblot analyses had also shown that the recovery of soluble lumenal proteins was 6065% (as judged by the presence of plastocyanin), while the contamination with stromal enzymes was less than 1% (ribulose- bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were regained in the luminal fraction, of which several were novel polypeptides. Enzymatic measurements and/or amino-terminal sequencing had shown the presence of proteolytic activities, peroxidase, violaxanthin de-epoxidase, polyphenol oxidase, as well as a novel prolyl cis/trans-isomerase. The membrane system of chloroplast contains a large number of proteins, some of which are synthesized within the organelle, while many others are imported from the cytosol. It has been seen that the targeting of imported proteins into and across the thylakoid membrane is a complex process, with four different targeting pathways. Two of these are used to target membrane proteins: a signal recognition particle (SRP)-dependent pathway and a highly unusual pathway that appears to require none of the known targeting apparatus. Two further pathways are used to translocate lumenal proteins across the thylakoid membrane from the stroma and, again, the two pathways differ efficaciously from each other. One is a Sec-type pathway, in which ATP hydrolysis by SecA drives the transport of the substrate protein through the membrane in an unfolded conformation. The other is the twin-arginine translocation (Tat) pathway, where substrate proteins are transported in a folded state using a unique mechanism that uses the proton motive force across the thylakoid membrane. This article shows the studies done on the isolation and characterization of thylakoid lumen from chloroplast and targeting of lumenal proteins, with reference to the mechanisms involved, their evolution from endosymbiotic progenitors of the chloroplast, and possible elements of regulation. Key words: Isolation, Characterization, Protein Targeting, Sec pathway and Tat pathway. ______________________________________________________________________________
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INTRODUCTION The chloroplasts are found in green algae and higher plants, they are the photosynthetic organelles. They were considered to be originated from photosynthetic bacteria and they had a complex structure. The chloroplast comprises of an envelope membrane, which encloses the soluble stroma and a very highly specialized thylakoid membrane. The stromal compartment contains mainly the components of the C3 cycle, which are required for the fixation of carbon dioxide. The thylakoid, on the other hand, carry out the light reactions of photosynthesis leading to the production of NADPH and ATP. The inner surface of the thylakoid membrane encloses a narrow, continuous compartment, called as lumen (1, 2). Electron microscopy studies of spinach thylakoid had shown that the lumen is a densely packed space (3). Series of steps were designed for isolation of thylakoid lumen; the characterization of the membrane surface of the luminal side became possible (4). This work lead 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). Recent studies had shown that these subunits of photosystem II occur also as soluble lumenal proteins (9). This pool of unassembled subunits PsbO, PsbQ, and PsbP was found to be resistant to the proteolytic degradation and was capable of assembling into photosystem II (10). Further, it was found that during photo-inhibitory conditions the extrinsic proteins were released out 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 analysis had shown that polyphenol oxidases (16, 17) and violaxanthin de-epoxidase were 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 certain chaperones may be located in the lumen (22).So far many of the lumenal proteins had been found to be nuclear encoded and synthesized as a precursors in the cytoplasm. These precursor proteins had characteristic aminoterminal bipartite transit peptides, which help in directing the peptides into the chloroplast stroma and across the thylakoid membrane into the lumen (2325). Although the chloroplast possess its own genome, and majority of chloroplast proteins is encoded by the nuclear genome and posttranslationally transported into chloroplasts by a common import apparatus, named as the TOC/ TIC (translocon at the outer/inner envelope membrane of chloroplasts) complex (26-29). This system helps in importing proteins in an unfolded state at so-called contact sites between the two membranes (30), after which the substrates refold in the stroma , a very large number of proteins are further targeted into the thylakoid systemeither into the membrane, or across the membrane into the lumenal space. In recent years, it has proved that protein trafficking is highly organized, with at least four separate pathways operating for the targeting of thylakoid proteins. This paper had developed a procedure by which a lumenal fraction can be isolated in a highly pure form from spinach thylakoids and systematic characterization of this compartment had been
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done, and showed that the thylakoid lumen contains a high concentration of proteins, among which at least 25 distinct polypeptides can be identified. Several of which had been characterized in terms of enzymatic activity or amino-terminal sequence. To update it, it seems that inner membrane proteins are inserted into thylakoids by two very different pathways. A subset of proteins, namely the abundant light-harvesting chlorophyll-binding proteins (LHCPs) are inserted by a pathway that requires the action of a chloroplast signal recognition particle (cpSRP) and FtsY in the stroma, together with a protein termed Alb3 in the thylakoid membrane. The endosymbiotic origin of chloroplasts, yet the biogenesis of thylakoid membrane proteins differs in one key respect because most of the remaining thylakoid membrane proteins are inserted by a completely different pathway that does not involve SRP or Alb3, or indeed any known targeting apparatus (34).Two further pathways are used for the targeting of soluble proteins into the lumenal space. These substrates are synthesized in the cytosol with bipartite N-terminal pre sequences which contain two targeting signals in tandem. Once they are in the stroma, the first envelope transit domain of the pre sequence is removed by stromal processing peptidase (SPP). This leads to the exposure of a thylakoid signal peptide present on the polypeptide, quickly forming a stromal intermediate which is transported across the thylakoid membrane and processing is done by the thylakoid processing peptidase (TPP). A subset of lumenal proteins is transported in an unfolded state by a Sec-type pathway (cpSec), whereas others are transported by the Tat pathway (cpTat) that appears to function primarily in the transport of fully-folded proteins. Tat systems had now been characterized in a wide variety of bacterial species. Interestingly, unlike the Sec pathway, Tat system transports proteins by a mechanism that does not require nucleoside triphosphatases (NTPs), but which relies totally on the DpH across the thylakoid membrane (32-33).

EXPERIMENTAL PROCEDURES Plant MaterialSpinach (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 It involves three major steps: (i) preparation of chloroplasts, (ii) purification of carefully washed thylakoids, and (iii) rupture of thylakoids by a Yeda press fermentation and isolation of the luminal content. Twice Spinach leaves (200 g) were blended for 510 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. With the help of four layer nylon mesh it was filtered (20 mm), and the filtrate was centrifuged for 1 min at 1000 g. The pellets were resuspended in 330 mM sorbitol, 50 mM Hepes-KOH (pH 7.8), 10 mM KCl, and again centrifuged for 1 min at 1000 g, and resuspended in 1218 ml of the same buffer. The yield was 6070% intact chloroplasts containing 5060 mg of chlorophyll in them. The chloroplasts were diluted with 10 mM sodium pyrophosphate buffer (pH 7.8) to a final chlorophyll concentration of 0.2 mg ml-1 and resuspended in a glass homogenizer. The thylakoids were collected by centrifugation for 5 min at 7500 g and sequentially they were washed in the same way twice with each of the following buffers: I, 10 mM sodium pyrophosphate (pH 7.8) to remove the soluble stromal proteins; II, 2 mM Tricine (pH 7.8), 300 mM sucrose to partially remove ATP synthase, the membrane-attached fraction of Rubisco,1 and unidentified extrinsic thylakoid membrane proteins; III, 30mM sodium phosphate (pH 7.8), 50 mM NaCl, 5 mM MgCl2, 100 mM sucrose (fragmentation buffer) to equilibrate the thylakoids for Yeda press fragmentation. The thylakoid pellets were suspended in a small volume of fragmentation buffer to a concentration of 3.54.5 mg of chlorophyll ml-1 (total yield: 2530 mg of chlorophyll). The washed thylakoids were then passed once through a Yeda press at a nitrogen pressure of 10 megapascals and centrifuged for 1 h at 200,000 g and 2 C. The supernatant was taken second time separately and centrifuged under the same conditions to settle down the residual membrane particles. The entire isolation procedure was performed on ice, and the chloroplasts and thylakoid membranes were purified under green light. The lumenal fraction was stored in liquid nitrogen. Thermolysin Treatment of ThylakoidsThe washed thylakoids (2 mg of chlorophyll ml-1) were incubated with the 10 mM 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 reaction 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 extrinsic 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.
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Measurements of Chlorophyll, Protein, and Oxygen EvolutionChlorophyll concentrations were measured (107).Soluble proteins and membrane proteins were detrmined (108-109). The standard which was 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 (110). Electrophoretic and Immunoblot AnalysisSDS-PAGE was performed in slab gels containing 18% (w/v) polyacrylamide and 2 M urea. Determination of molecular masses was performed by using the low molecular weight electrophoresis calibration kit obtains from Pharmacia Biotech Inc. For immunoblotting analyses proteins were transferred onto a polyvinylidene difluoride membrane in a semi dry Millipore. The antisera used were raised in rabbits against the following spinach proteins: phosphoribulokinase (K.-H. Su ss, Institute for Plant Genetics and Crop Plant Research, Gatersleben); violaxanthin de-poxidase (H.-E. kerlund,University of Lund); plastocyanin (P.-. Albertsson, University of Lund); PsbO, PsbP, PsbQ, the lumen fraction, and Rubisco (our own production). The immunoreactivity was detected using goat anti-rabbit IgGconjugated horseradish peroxidase in combination with enhanced chemiluminescence detection. Quantification was done by using a Fast Scan Personal Densitometer and the Image Quant software from Molecular Dynamics. To compare the results directly 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 AssaysMitochondrial cytochrome c oxidase was assayed 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 (39). Phosphoribulokinase was assayed (40). 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 (41).Peroxidase activity was measured using the substrate 2,29-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 b-subunit of mitochondrialATP synthase of Nicotiana plumbaginifolia (42) as artificial substrates. The activity of violaxanthin de-epoxidase was determined (43). 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 ActivityATP was detected using the firefly luciferase system (44) and the ATP monitoring reagent from BioOrbit. ATPase activity was also tested (45). Amino-terminal Protein Sequence AnalysisProteins were sequenced from polyvinylidene difluoride membrane following resolution by SDS-PAGE essentially as described (46). The amino-terminal sequence analyses were performed by P. I. Ohlsson (University of Ume) 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 (47). Protein Targeting: Sec Pathway: Sec systems pathways are evolutionarily conserved protein translocation machineries that are present in the eukaryotic endoplasmic reticulum (48), the archaeal plasma membrane (49), the eubacterial plasma membrane (50-52), and the thylakoid membranes of plant and algal chloroplasts. A common mechanistic feature of all Sec systems is that the substrates are transported in an unfolded conformation through a protein-conducting channel. In chloroplasts, homologues to SecA (cpSecA), SecY (cpSecY), and SecE (cpSecE) have been identified (53-55) but several components of the bacterial Sec pathway (SecB, SecG, and SecD/F) have not been identified yet. Experiments shows that thylakoid cpSecA/cpSecYE works similarly to the E. coli system, for example, the translocation step across thylakoid membranes is dependent on ATP and it is inhibited by the SecA inhibitor azide (54, 56-57). Moreover antibodies against cpSecY prevent cpSecA-dependent protein translocation, suggesting that, as the bacterial counterparts, cpSecY and cpSecA work in concert (58).It had been shown that cpSecA ATPase activity was stimulated by cpSec-dependent thylakoid signal peptides, but not by E. coli signal peptides, and that stimulation of cpSecA ATPase activity exhibits specific lipid requirements that differ from those of E. coli SecA (59). Recently, it has been shown that cpSecA is very important for photosynthetic development in Arabidopsis, because its absence may lead to severe defects in chloroplast sub-organelle structure and function (60). The inability of the cpSec translocon to transport dihydrofolate reductase (DHFR), in a methotrexate-stabilized folded conformation, demonstrates that the cpSec pathway, similar to bacteria, protein substrates has to be in unfolded state for transport (61-62). In chloroplasts, there is no homologue of the bacterial cytosolic chaperone SecB, which is dedicated to preventing folding of Sec substrates, thus the stromal factors necessary to maintain cpSec pre-proteins in an unfolded state remain unknown. TAT Pathway: The chloroplast Tat translocon consists of three different membrane proteins termed Tha4, Hcf106, and cpTatC, which are homologues of the bacterial TatA, TatB, and TatC proteins, respectively. The first cpTat component was identified only after the isolation of a maize hcf106 (high chlorophyll fluorescence 106) mutant, which was defective in a cpSec-independent protein transport pathway (63-64). After this Tat components of E.coli was identified (65-67). Hcf106
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and Tha4 are single-spanning membrane proteins containing an N-terminal transmembrane domain followed by a short amphipathic -helix and an unstructured C-terminal domain. cpTatC was predicted to contain six transmembrane domains and that both N- and C- termini are located in the stroma. Thylakoid Tat Complexes: Chloroplast Tat components are organized into two distinct complexes. The first, Hcf106cpTatC, is a large; 700 kDa complex which has been characterized by performing BN-PAGE analysis and co-immunoprecipitation of digitonin-solubilized thylakoid membranes (68). Tha4, on the other hand, has not been detected in the Hcf106-cpTatC complex at resting state, but was found to present as a separate homo-oligomer (69-70) proposed that cpTatC might integrate through one of the thylakoid translocation pathways which is still unknown. Import time-course and chase assays confirmed that cpTatC targets to thylakoids via a stromal intermediate. In addition to tis study, the analysis of membranes isolated from Arabidopsis thaliana mutants lacking cpSecY or Alb3 showed that neither is necessary for cpTatC membrane integration nor assembly into the cpTat receptor complex. These data suggested that there is an existence of another translocase, possibly one dedicated to the integration of chloroplast translocases. (71) With the help of photo bleaching studies, it was found that the Hcf106-GFP fusion protein is either immobile within the thylakoid membrane, or its diffusion is tightly restricted within distinct regions. This provides direct evidence for a strict compartmentalization of membrane types within the continuous thylakoid membrane layer. Mechanistic studies strongly suggested that substrates bind first to the Hcf106-cpTatC complex; substrates can be cross linked to this complex under conditions that prevent full translocation (72-75). According to site-directed cross-linking studies on the bacterial system (76), the RR proximal region of the signal peptide is in close contact with TatC, whereas the hydrophobic domain and few parts of the adjacent mature domain are in close contact with TatB (75-76). It seems likely that thylakoid signal peptides interact with the Tat apparatus in a similar manner, although this has not yet been proved with any data yet. Tha4 does cross-link to the cpTatC-Hcf106 complex in the presence of a precursor protein and a thylakoid DpH (74), suggests that the formation of the full translocon occurs under these conditions. Armed with the evidence that the Hcf106-cpTatC complex acts as the substrate-binding unit (with the analogous TatBC complex carrying out a similar role in bacteria) it seems likely that the separate Tha4 complex (TatA in bacteria) could form the translocation pore, or at least contribute to it. Investigation of E. coli TatA using single particle electron microscopy shows that TatA forms ring-shaped structures of different diameter (77), supporting a model in which Tha4/TatA forms a pore-like channel and, also raising the possibility that Tha4/TatA oligomerization and recruitment might be tailored to the size of the protein to be transported. Thus, one of the primary current models for the overall translocation process involves three main phases which are as follow: (i) binding of precursor protein to the Hcf106-cpTatC complex (TatBC complex in bacteria), (ii) coalescence of this complex with the

Tha4 complex (TatA complex in bacteria), and (iii) translocation of the folded substrate protein through the membrane. However, it should be noted that the later stages of translocation are not understood and studied in any real detail, and there is very little information on the nature of the translocation pore. Cross-linking studies had shown that Tha4 undergoes conformational rearrangements during active protein transport, with the amphipathic helix and C-terminal tail interacting only in response to conditions leading to protein transport through the pore (69). Binding of the precursor can occur in the absence of a DpH (72) but the thylakoid proton motive force/ gradient induces a tighter interaction between the signal peptide and cpTatC and Hcf106 such that, during transport, the signal peptide is bound deep within the Tat receptor complex (75). Recently, it has been reported that multiple precursor proteins can bind to sites in a single translocase, and can be transported across membrane together by Tat system in isolated pea chloroplasts. Moreover, oligomers as large as tetramers are collectively transported to the lumen with efficiency nearly as great as that of monomers (78) Substrate specificity of the chloroplast Tat pathway Approximately 50% of thylakoid lumen proteins are translocated either by the cpSec or the cpTat pathway, and the characteristics of their signal peptides have been thoroughly studied in several reports (79-81). All thylakoid cpTat and cpSec targeting signals are very much similar in certain respects, they resembles to the classical bacterial signal peptides in overall structure (81-82). They are characterized by an N-terminal non-polar region, a hydrophobic central core and a polar C-terminal region ending in an Ala-X-Ala terminal site (83). A twin arginine (RR) motif situated in the N-domain of signal peptides is a specific for targeting the signal for the Tat pathway (84), and this explains the pathways nomenclature (twin-arginine translocation).The twin-arginine motif is not the only feature distinguishing the two types of signal peptide, and it is located within a wider consensus motif. In E. coli other determinants within the consensus motif have also been discovered which are assume to be important for translocation. One example is the hydrophilic-1 residue, relative to the twin-arginine motif (typically serine, threonine, aspartate or asparagine). When this serine residue is mutated to alanine in the signal peptides of E. coli TorA and DmsA, it causes a sudden reduction in translocation activity. Other determinants have also been identified. The residue 2 places after the RR pair is usually either a phenylalanine or a leucine. In the signal peptide of TorA no effect on translocation, had been reported if phenylalanine is replaced by serine or alanine, whilst substitution by aspartic acid caused a complete blockage in the transport of protein. The signal peptide of DmsA contains leucine at this position, and substitution by either alanine or aspartic acid caused a complete block in transport. Thus it proves the importance of the residue at this +2 position varies between substrates but an acidic residue at this position cannot be tolerated (85). Plant Tat signal peptides have a shorter consensus sequence (RRXX/) if compared with the extended bacterial one ((S/T) RRXFLK); where / is typically leucine, phenylalanine, valine or methionine, and X can be any amino acid residue (86). While the RR motif is essentially invariant there is no tendency for Phe
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to occur at the +2 position and the presence of a +4 Lys is very rare. Hence plant Tat signals is noticeably different. Tat signals also differ from Sec signal peptides in other respects, including a tendency to contain basic residues in the Tat signal peptide C-domain (87) and they contain a less hydrophobic H-domain when they were compared with Sec signals

Fig 1: Current Model of Tat Translocation process Fig 1 shows a range of Sec and Tat signal peptides. Thylakoid Tat substrates range in size from 4 kDa to over 60 kDa, and it is assumed that these proteins are probably transported in a folded form, although the folding status of nearly all of these substrates remains unknown at present (79). In chloroplasts, however, only a very few cpTat substrates contain redox factors (polyphenol oxidase, for example), and thus it may be possible that the remaining substrates may simply fold too rapidly, or tightly, for the cpSec system to handle them effectively. The reporter system is based on the Streptomyces coelicolor agarase protein, which is secreted into the growth medium by the Tat pathway and whose extracellular activity can be assayed colorimetrically. By replacing the native agarase signal peptide with an uncharacterized one is possible to identify probable Tat substrates from all kingdoms of life. Regulation of Chloroplast Tat system The thylakoid Tat translocation mechanism has been widely studied in vitro using wellestablished translocation assays. These assays have involved the incubation of intact chloroplasts or isolated thylakoids with in vitro-synthesized substrates, and the combined data have shown translocation by the thylakoid Tat system to be efficient, reliant on a DpH, and invariably unidirectional. The maturation of Tat substrates does not depend upon the Dph, this has been shown by in- vivo pulse labeling analysis in Chlamydomonas reinhardtii. The involvement of additional and critical intracellular factors was suggested (88-89) investigated the targeting of an authentic Tat substrate (OE23) and several GFP constructs in transfected tobacco protoplasts. They showed that the constructs are targeted to the chloroplasts and were processed to the mature size. However unlike the in vitro assays, where the cpTat substrates are exclusively found in the lumen, in this in vivo study shows majority of mature GFP and much of the mature OE23
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was found in the stroma in large quantity. Moreover, it was also shown thylakoidal processing peptidase (TPP) carried out the maturation, the active site of which is located on the lumenal face of the thylakoid membrane. RESULTS Preparation and Characterization of the Thylakoid Lumen The main aim 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 chloroplasts obtained were normally 6070% intact and had an oxygen evolving activity of 140 185 mmol of O2 mg Chl-1 h-1. Osmotic shock was given to rupture the membrane of chloroplast, and the thylakoids were collected and purified by several washing steps. Soluble stromal proteins were removed by using 10 mM sodium pyrophosphate (pH 7.8), and 2 mM Tricine (pH 7.8) which contain 300 mM sucrose for the removal of ATP synthase, membrane-bound Rubisco, and other unidentified extrinsic thylakoid proteins. Finally, the thylakoids were equilibrated in fragmentation buffer; these thylakoids retained an oxygen evolution rate of 90165 mmol of O2 mg Chl-1 h-1. The washed thylakoids were then fragmented by a Yeda press fragmentation, and the lumenal content which was released was then separated from the membrane fragments by two ultracentrifugation steps. The final fraction was then eventually free from chlorophyll and had a protein concentration of 0.30.5 mg ml-1, giving a net yield of 23 mg of protein from 200 g of spinach leaves. The separation was done by electrophoresis and analysis of the polypeptide pattern of the starting chloroplasts, the stromal fraction, the washed thylakoids, and the final lumenal preparation (Fig. 2).

Fig 2. SDS-PAGE stained with Coomassie Brilliant Blue for polypeptide analyses of the isolated lumenal fraction and comparative analyses with other chloroplast subfractions. Lane 1, chloroplasts (10 mg of Chl); lane 2, stromal fraction (40 mg of protein); lane 3, thylakoids (10 mg of Chl); lane 4, lumenal fraction (40 mg of protein); lane 5, lumenal fraction (40 mg of protein) after thermolysin treatment.
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The bands of plastocyanin, the extrinsic subunits of photosystem II, LHCII, and Rubisco were identified by immunoblotting and/or amino-terminal sequencing (data not shown). Table1: Polypeptides of isolated thylakoid lumen Appar ent molec ular mass kDa 100 64.5 57.5 54.0 40.0 38.0 and 37.5 32.5 29.0 and 28.5 28.0 27.5 25.0 24.0 23.0 21.0 20.0 18.5 18.0 17.6 17.4 17.2 16.5 15.5 Identity Method identification of

? Polyphenol oxidase ? Large subunit of RUBISCO and ? New protein Ferredoxin NADP+ reductase PsbO Protein New protein

Protein sequencing ? Immunoblotting Protein sequencing Protein sequencing

Immunoblotting, Protein sequencing Protein sequencing

? ? PsbP protein ? ? ? ? ? PsbQ protein ? New protein ? New protin ?

Immunoblotting, Protein sequencing

Immunoblotting, Protein sequencing Protein sequencing Protein sequencing

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15.0 14.0 6-14

Plastocyanin ? Five potein bands

Immunoblotting, Protein sequencing Tricine/SDS-PAGE

The dominant bands seen in the isolated chloroplasts (lane 1) are the larger and smaller subunits of Rubisco and the LHCII. The two subunits of Rubisco and other soluble proteins were also found in the stromal fraction (lane 2). The membrane-bound LHCII and other intrinsic membrane proteins were regained in the thylakoid fraction (lane 3). Finally, lane 4 shows the soluble lumenal proteins which was released after the disruption of the washed thylakoid. Table I summarizes the results, there were more than 25 different polypeptides in the purified lumenal fraction which was recovered. It can be seen clearly that the polypeptide pattern is very much different from the other chloroplast sub- pattern (Fig. 2, lane 4). These polypeptides range from 14 to 100 kDa. There are 5 dominant, 10 to 12 medium abundance, and 8 to 10 low abundance polypeptides. Silver staining of the polypeptides led to the identification of five additional bands in the molecular mass range of 2045kDa. Further, an investigation of the low molecular mass polypeptides of the lumenal fraction was performed by Tricine/SDSPAGE (90) showed five additional low abundance polypeptides in the range of 614 kDa (not shown). Several different approaches were used (summarized in Table I) to identify the individual polypeptides. Initially three of the four major polypeptides (Fig. 2, lane 4) at different positions 32.5, 25, and 18 kDa were immunologically identified as the periferal proteins PsbO, PsbP, and PsbQ of photosystem II. This is consistent with the observation that a soluble unassembled pool of these proteins occurs in the thylakoid lumen (9). The fourth major protein of the luminal fraction, with the apparent molecular mass of 15 kDa, was identified by immunoblotting as plastocyanin. The proteins of the lumenal fraction were systematically studied by amino-terminal protein sequencing. This confirmed the identification of plastocyanin, PsbO, PsbP And PsbQ. Polyphenol oxidase was also identified as a polypeptide with an apparent molecular mass of 64 kDa, and four novel polypeptides were discovered at 40, 29, 17.4, and 16.5 kDa (see Tables I and IV). Finally, ferredoxin NADP1 reductase (FNR) was found to migrate at apparent molecular masses of 38 and 37.5 kDa. The detection of FNR, which is located on the stromal side of the thylakoid surface, showed a more detailed analysis of contamination in the isolated lumenal fraction. Therefore, the washed thylakoids were treated by limited proteolysis with thermolysin before treating it with the Yeda press fragmentation. The conditions of proteolysis were optimized to ensure degradation of most of the extrinsic protein on the stromal side of the thylakoid membrane and to minimize membrane rupture. More extensive proteolysis could cause disruption of the thylakoid membrane, as was previously reported (91). The intensity of the polypeptide bands of the lumenal fraction following SDS-PAGE before (Fig. 2, lane 4) and after (Fig. 2, lane 5) treatment with thermolysin was compared by densitometric analysis of the
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Coomassie Blue-stained SDS gels using the PsbO protein as an internal standard protein. The data obtained from three different preparations demonstrated that most of the polypeptides of the lumenal fraction were not degraded even by the treatment with proteolytic enzymes, arguing that these proteins were located inside the thylakoid lumen. Only in two cases was poly Peptides significantly degraded (Fig. 2). A 40-kDa protein decreased in abundance by approximately 60%, whereas the 38- kDa FNR (Fig. 2) was degraded to 8085%, which is consistent with its location at the stromal surface of the thylakoid membranes (1, 2). The FNR may have been sheared off the outer thylakoid surface during the Yeda press treatment, thereby ending up with the lumenal proteins in the supernatant following centrifugation. Other test for purity of an immunoblot analysis was performed to quantify marker proteins associated with specific chloroplast compartments: plastocyanin as a soluble luminal protein; PsbO as a lumenal extrinsic thylakoid-bound protein; the D1 reaction center protein of photosystem II as an integral membrane protein; the FNR for the outer thylakoid surface; Rubisco as the major stromal protein, and phosphoribulokinase as typical soluble stromal enzyme. As presented in Table II, 6065% of the total amount of plastocyanin and 10% that of the extrinsic PsbO protein were recovered in the lumenal fraction. Table 2: Immunological analysis of chloroplast sub-compartment marker enzymes

Chl oro plas t

Washe d Thyla koid %

Lume nal Fracti on

Resid ual Thyla koid frame nts

Thylakoid lumen Plastocyani n PsbP protein Thylakoid membrane D1 protein

100 100

60-75 80-90

60-65 2-10 10 70-80

100

Ferredoxin- 100 NADP+ reductase Chloroplast stroma RUBISCO 100

100110 40-60

100105 10-40 5-30

8-20

<1

ND
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Phosphorib ulosekinase

100

Under detecti on limit

ND

Furthermore, the lumenal fraction was free of D1 protein demonstrating the absence of contaminating thylakoid membranes. Less than 1% of the total amount of the large subunit of Rubisco was found in the lumenal fraction. The major contamination was FNR. Between 10 and 40% of this enzyme was found in the lumen; this represents approximately 10% of the total protein content of the lumenal fraction. As an additional indication of stromal contaminations as well as to find possible extra chloroplast contaminants, assay for a number of different marker enzymes were done. The marker activities included phosphoribulokinase from the chloroplast stroma, catalase from the cytoplasm, peroxisomes, and vacuole, and the mitochondrial enzymes cytochrome c oxidase (inner membrane) and malate dehydrogenase (matrix). Likewise as shown in Table III, the contamination of the isolated luminal fraction by phosphoribulokinase was only 0.3% of the total activity of the isolated chloroplasts and that of catalase was even lower. The activity of cytochrome c oxidase in the chloroplasts was 0.007 mmol of oxidized cytochrome c min-1 mg protein-1 and that of spinach mitochondria was 8 mmol of oxidized cytochrome c min-1 mg protein-1. Furthermore, the activity of malate dehydrogenase was 0.5 mmol of oxidized NADH min-1 mg protein-1 and that of the Table 3: Activities of marker enzyme Chl oro plas t Washe d Thylak oid % Lume nal Fracti on Residu al Thylak oid framen ts

Thylakoid lumen Polypheno l oxidase

0.0 1

Volaxanth ND in Deepoxidase Chloroplas t stroma Phosphori 7+-

0.09 +0.05 ND

0.25+ -0.05 18+1.8

0.02+0.005 ND

0.07+-

0.024

ND
14

bulosekina se Mitochond ria and cytoplasm Cytochro me c d oxidase

0.001

+0.002 ND

0.0 6+0.0 05 Cytochro 0.0 me c 07+ e oxidase 0.0 006 Malate 0.1 dehydroge 8+nasef 0.0 2 Malate ND dehydroge naseg Catalase 341 +240

0.02+0.007

ND

0.003+ 0 -0.001

ND

0.14+0.002

0.05+ -0.04

ND

ND

0.5+0.3 0.7+0.2

ND

14+-7

ND

Spinach mitochondria was 330 mmol of oxidized NADH min-1 mg protein-1 .Based on these values the contamination of the lumenal fraction by soluble mitochondrial proteins was estimated to be lower than 0.2%. The activity of cytochrome c oxidase was not detectable in the lumenal fraction. Enzyme Activities of the Thylakoid Lumenearlier work had suggested that polyphenol oxidase (17) and violaxanthin de-epoxidase (18) were located inside the lumen of the spinach thylakoids. Hence, we detect 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 luminal 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 luminal fraction was 18 mmol of violaxanthin/g of protein-1 min-1 (Table III), which corresponds to 25% of the value
15

reported for the purified spinach enzyme (92). In addition, the immunoblot shown in Fig. 3 shows that violaxanthin de-epoxidase was highly enriched in the lumenal fraction as compared with the original thylakoid preparation.

Fig 3 Immunoblot analysis of the thylakoids and the lumenal fraction from a lumenal preparation for violaxanthin de-epoxidase. Lane 1, thylakoids (20 mg of Chl), and lane 2, lumenal fraction (20 mg of protein). In searching for other lumenal enzymes, photosystem II can produce hydrogen peroxide (93), and therefore it was tested for the lumenal fraction for peroxidase activity. Surprisingly, it was found to be 60% of the total peroxidase activity of the thylakoids after the recovery of the lumenal fraction, which contrasts with earlier work (94) which suggested that peroxidases were not present in this chloroplast compartment. The lumen-associated peroxidase activity was not affected by limited proteolysis of the thylakoids. The turnover of the D1 protein of photosystem II during photoinhibition involves the proteolytic cleavage of both stromal and lumenal loops (95).Processing of imported precursor proteins and the carboxyl-terminal extension of the D1 protein must occur in the lumen (25, 20). Protease activities were tested in lumenal preparations. It was found that the overall proteolytic specific activity in the chloroplast stroma and that of thylakoids was approximately equal, while the proteolytic activity in the lumenal fraction was only found to be 10% of thylakoids. The optimum pH for the lumenal proteolytic activity was found to be between pH 7 and pH 8. However, in contrast to the thylakoids this activity was still detectable in the pH range of 46. Metalloprotease inhibitors were also found to inhibit the protease activity by 30%. Moreover, the addition of ATP did not stimulate the proteolytic activity. These results corroborate the presence of lumen-located proteases. The lumenal fraction was also tested for the presence of ATP and ATPase activity. The ATP concentration was found to be lower than 100 nM, and no significant ATPase activity could be detected. Identification of Unknown Lumenal Proteins via Amino-terminal Sequencing SDS-PAGE was done to found the novel lumenal proteins, followed by amino-terminal sequencing of polypeptides from the lumenal. This approach led to the identification of four novel polypeptides of the apparent molecular masses of 40.0, 29.0, 17.4, and 16.5 kDa (Table IV).

16

Table 4: Amino terminal sequence of four unknown proteins from the thylakoid luminal fraction kDa 1 40.0 VLISGPXIKD PEALLRYALP IDNKAIREVQ 29.0 ADLIQRRQRS EFQSDIKGIL YTVIKKNPDL 17.4 ANQRLPPLSN DPKRKE.............. 16.5 APLEDEDDLE LLEKVKRDRK KRLERQGAI When the 29-kDa protein was cross checked with the data bases like SwissProt and EMBL, no homology was found. However, the amino-terminal sequence of the 16.5-kDa protein was found to be homologous to partial protein sequences encoded by Arabidopsis thaliana clones 250E4T7 (AC W43350) and 77E7T7 (AC T45153). The cDNAs of both clones overlapped partially and were very similar the alignment of the polypeptide with the translation product (g1327818) of clone 250E4T7. The sequence from A. thaliana contains parts of the pre sequence and the amino terminus. Furthermore, the pre sequence reveals bipartite transit peptide with a hydrophobic core and the motif AXA at the putative processing site (24). The amino-terminal sequence of the 17.4kDa protein was 68% homologous to a protein encoded by a cDNA fragment from A. thaliana (clone 104N18T7, AC T21992). The functions of this sequence is still unknown, it contains a part of a bipartite transit peptide. The motif VIA present at the putative processing site is analogous to the corresponding motif VLA in the precursor of the PsbQ protein from spinach (AC P12301). Furthermore, this motif is flanked by a hydrophobic region that is typical of thylakoid transfer domains present on transit peptides of lumenal proteins. The amino-terminal sequence of the 40.0-kDa protein was homologous found to the putative amino terminus of a hypothetical protein g1001111 from the cyanobacterium Synechocystis sp. PCC6803 (AC D64001). Similar to the Arabidopsis sequences, this protein also contains a transit peptide with a hydrophobic core region and AXA motif at the putative processing site. Interestingly, the carboxyl-terminal 191 residues of hypothetical protein g1001111 are highly homologous to 184 residues of the carboxyl terminus of peptidylprolyl cis/trans-isomerase B (AC D90900, g1651784) from this cyanobacterium (not shown). This suggested that the 40-kDa protein from spinach and the Synechocystis protein g1001111 were functionally related to peptidyl cis/transisomerase. Recently, the spinach 40-kDa protein was isolated, and its corresponding cDNA was cloned and sequenced (AC Y12071) 4. The deduced gene product is a unique high molecular mass immunophilin protein that is located in the thylakoid lumen. The prediction was biochemically confirmed through direct enzymatic analysis of the cis/trans protein isomerization (rotamase) activity of the purified spinach 40-kDa protein4 that is typical for immunophilins (96). In addition, the lumenal fraction from our work was found to have a significant rotamase
17

activity.5.We have not been able to successfully sequence the N termini of the low abundance lumenal proteins. DISCUSSION The thylakoid lumen represents a continuous cellular space that is not properly characterized compared to the other chloroplast compartments. Interest generated in the lumenal side of the thylakoid membrane because of the electron transport events which were associated with the inner membrane surface. Indirectly, it has been considered that this is essential for the generation of proton motiv force which helps in ATP synthesis across the thylakoid membrane (97-98). With the help of advanced and deep understanding of biosynthesis, regulation pathways, and stress protection of the photosynthetic apparatus, the requirement for auxiliary enzymes (99) in the lumenal space has become more apparent. The present isolation procedure of a thylakoid lumenal fraction gives high yield and very low degree of 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 ml per mg of chlorophyll (100) and the yield of 75120 mg 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 ml-1. Thus, soluble proteins in the lumen were at micromolar to millimolar concentrations, which was very much similar to that of the chloroplast stroma. Hence, it can be concluded that the thylakoid lumen is comprised of soluble densely packed components (3). The number of polypeptides in the isolated luminal 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 luminal 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 (101), PsbO (102), and PsbS (103).The PsbO, PsbP, and PsbQ proteins and plastocyanin were found to be the major proteins present in the isolated luminal 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 (1618). Furthermore, the presence of a peroxidase in the lumen may be important for detoxification of hydrogen peroxide produced by photosystem II upon illumination (93). The function of lumenal polyphenol oxidase is not understood. Since chloroplasts from peas lack this enzyme (16), it is not likely to play a
18

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 peripheral proteins of photosystem II brings up the question of proteolytic activities in the thylakoid lumen. The present analyses as well as previous studies (104-105) clearly show the presence of proteases in this compartment. However, the protease activity of the luminal fraction was relatively low, only 10% of that found in the thylakoid reaction, suggesting that most of luminal protease activity is bound to the inner thylakoid surface. The 16.5-kDa luminal 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 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 luminal chaperones (22) as well as experimental indications for phosphorylation of lumenal proteins (106) has 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. The targeting of lumenal proteins has now been studied in some detail and a great deal is known about the overall pathway steps and the components involved. In the case of the cpSec pathway, some elements have yet to be analyzed in much detail; currently very little is known about the fates of cpSec substrates in the stroma, and especially whether they are prevented from folding by specific chaperone molecules. It remains possible that the cpSec translocon is able actively to unfold these proteins (at least to an extent). In general, the cpSec system appears to resemble the well characterized bacterial Sec systems, but it should be emphasized that this is only an assumption based on a relatively limited number of data from the experiments. The more recently-discovered cpTat system shows similarity to Gram negative bacteria. In particular, the roles of the three core cpTat components appear to be similar, and these components form two distinct complexes of broadly similar characteristics. However, in neither case do we understand the translocation mechanism in genuine detail and this has to be the focus of future research. The available data shows that two distinct complexes come together to form a supercomplex after binding of substrate to one of them and there are tantalizing hints of substantial pores in EM images of bacterial TatA complexes. However, at the moment, we have no idea how these complexes form a relatively huge, transient pore that is capable of allowing passage of folded proteins. In fact, there is not even solid evidence for the existence of such a pore at the stage of translocation. Much needs to be done if this remarkable translocase is to be understood properly.

19

ACKNOWLEDGEMENT I would like to express my heartfelt gratitude to Dr. Mohd. I. Ansari, Programme Co-ordinator, M.Tech Biotechnology, Amity University, Lucknow. I also convey my gratitude to all my teachers for their kind support and sound advice. I would like to thank our parents and friends for the moral support they have given me. Finally, I would like to thank the almighty God for his unconditional love and support. References 1. Andersson, B., and Barber, J. (1994) Adv. Mol. Cell Biol. 10, 153 2. Hall, D. O., and Rao, K. K. (1994) Photosynthesis, 5th Ed., Cambridge University Press, Cambridge, UK 3. Weibull, C., and Albertsson, P.-. (1988) J. Ultrastruct. Mol. Struct. Res. 100,5559 4. Andersson, B., and kerlund, H.-E. (1978) Biochim. Biophys. Acta 503,462472 5. kerlund, H.-E., and Jansson, C. (1981) FEBS Lett. 124, 229232 6. kerlund, H.-E., Jansson, C., and Andersson, B. (1982) Biochim. Biophys. Acta 681, 110 7. Murata, N., and Miyao, M. (1985) Trends Biochem. Sci. 10, 122124 8. Vermaas, W. F. J., Styring, S., Schroder, W. P., and Andersson, B . (1993) Photosynth. Res. 38, 249263 9. Ettinger, W. F., and Theg, S. M. (1991) J. Cell Biol. 115, 321328 10. Hashimoto, A., Yamamoto, Y., and Theg, S. M. (1996) FEBS Lett. 391, 2934 11. Hundal, T., Virgin, I., Styring, S., and Andersson, B. (1990) Biochim. Biophys. Acta 1017, 235241 12. Eisenberg-Domovich, Y., Oelmu ller, R., Herrmann, R. G., and Ohad, I. (1995) J. Biol. Chem. 270, 3018130186 13. Haehnel, W., Berzborn, R. J., and Andersson, B. (1981) Biochim. Biophys. Acta 637, 389 399 14. Haehnel, W. (1984) Annu. Rev. Plant. Physiol. 35, 659693 15. He, W.-Z., and Malkin, R. (1992) FEBS Lett. 308, 298300 16. Sommer, A., Neeman, E., Steffens, J. C., Mayer, A. M., and Harel, E. (1994) Plant Physiol. 105, 13011311 17. Sokolenko, A., Fulgosi, H., Gal, A., Altschmied, L., Ohad, I., and Herrmann, R. G. (1995) FEBS Lett. 371, 176180 18. Hager, H., and Holocher, K. (1994) Planta 192, 581589 19. Inagaki, N., Mori, H., Fujita, S., Yamamoto, Y., and Satoh, K. (1995) in Photosynthesis: From Light to Biosphere (Mathis, P., ed) Vol. 3, pp.783786, Kluwer Academic Publishers Group, Drodrecht, Netherlands 20. Oelmu ller, R., Herrmann, R. G., and Pakrasi, H. B. (1996) J. Biol. Chem. 271, 21848 21852

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