Bipartite transite peptide. PDZ-domain. N. C. Peptidase. GMDVSGKS. CSILQQS. Figure 6. Schematic drawing showing the domain organisation of the carboxy-.
The chloroplast lumen proteome of Arabidopsis thaliana
Maria Schubert
Department of Biosciences and Nutrition, Karolinska Institute and School of Life Sciences, Södertörns högskola Stockholm 2006
Doctoral thesis © Maria Schubert, 2006 Printed by: Intellecta DocuSys, Göteborg ISBN: 91-7140-654-9
To my family
Abstract In plants, the chloroplast organelles host the photosynthetic machinery, which catalyzes the conversion of light energy to chemical energy used for synthesis of carbohydrates. Inside the chloroplast, the lumen compartment forms an integral part of the thylakoid network that performs the light reactions of photosynthesis. Despite intensive research within the field of photosynthesis, the lumen located proteins were relatively unexplored. To get insight into the lumen proteins and their roles in photosynthesis this thesis aimed at characterising the chloroplast lumen proteome. A 2-dimensional protein map of the lumen proteome of Arabidopsis thaliana revealed a high protein content within this chloroplast compartment. Thirty-eight proteins were experimentally identified demonstrating that the chloroplast lumen contains its own specific proteome. Comparison of the Arabidopsis chloroplast lumen proteome with the spinach lumen proteome showed good correlation and demonstrates that Arabidopsis can serve as a model for characterising the lumen proteins. An in silico determination of the chloroplast lumen proteome from the Arabidopsis genome sequence data showed that the experimentally identified proteins are good representatives of the proteome. Combining the in silico proteome with the experimental proteome, the chloroplast lumen estimates to contain at least 80 different proteins. The putative ascorbate peroxidase TL29 detected in the thylakoid lumen was biochemically characterised. The protein associated to the PSII-enriched grana membrane fraction by electrostatic forces and accumulated upon high illumination. Functional analysis showed that the TL29 protein is not a peroxidase but was able to bind ascorbate and may be involved in regulating the ascorbate levels in the chloroplast lumen. The dynamics of the lumen proteome were studied during the cold acclimation process. The lumen proteome was relatively insensitive to cold stress but important changes to the proteome were observed in the long-term developmental response to cold. These included changes in abundance of the different isoforms of the extrinsic PSII subunits, the PSII assembly factor Hcf136 and immunophilins. In comparison, the stroma proteome responded at an earlier stage in the acclimation process. Changes to the stroma proteome involved proteins related to photosynthesis, other plastid metabolism, hormone biosynthesis and stress & signal transduction.
List of publications This thesis is based on the following publications, which will be referred to by their Roman numerals in the text. Paper I and Paper II were reproduced with permission from the publishers.
I.
Kieselbach, T., Bystedt, M., Hynds, P., Robinson, C. and Schröder, W.P. (2000) A peroxidase homologue and novel plastocyanin located by proteomics to the Arabidopsis chloroplast thylakoid lumen. FEBS lett. 480, 271-276.
II.
Schubert, M., Petersson, U.A., Haas, B.J., Funk, C., Schröder, W.P. and Kieselbach, T. (2002) Proteome map of the chloroplast lumen of Arabidopsis thaliana. J. Biol. Chem. 277, 8354-8365. Erratum in J. Biol Chem. 278, 13590.
III.
Schubert, M. and Schröder, W.P. (2006) The TL29 protein a proposed ascorbate regulator in the thylakoid lumen of Arabidopsis thaliana. Manuscript.
IV.
Goulas, E.*, Schubert, M.*, Kieselbach, T., Kleczkowski, L.A., Gardeström, P., Schröder, W.P. and Hurry, V. (2006) Cold acclimation: a proteomic study of the chloroplast lumen and stroma of Arabidopsis thaliana by fluorescence two-dimensional difference gel electrophoresis and mass spectrometry. Submitted. *These authors contributed equally.
Other publication Schubert, M., Wirén, M., Kieselbach, T. and Schröder, W.P. (2001) Effect of low temperature on the protein content of the thylakoid lumen of Arabidopsis thaliana. Proceedings of the 12th International Congress in Photosynthesis, Brisbane, Australia. S41-019.
Table of contents Abbreviations________________________________ 8 Introduction _________________________________ 9 Photosynthesis ______________________________________________ 9 The chloroplast _____________________________________________ 10 The photosynthetic reactions__________________________________ 10 The light reactions_________________________________________ 12 Carbon fixation reactions ___________________________________ 13 Responding to the environment _______________________________ 14 This study _________________________________________________ 16
The chloroplast lumen _______________________ 17 From an unexplored compartment to new perspectives for thylakoid function ___________________________________________________ 17 Import of proteins __________________________________________ 18 The bipartite transit peptide _________________________________ 19 Experimental determination of the chloroplast lumen proteome ____ 19 The in silico chloroplast lumen proteome _______________________ 22 Functions of the lumenal proteins______________________________ 23 Photosystem II subunits_____________________________________ 23 Assembly factors and proteases_______________________________ 26 Plastocyanins and cytochrome c6A ____________________________ 27 Immunophilins____________________________________________ 28 Protective enzymes ________________________________________ 30 Proteins with unknown functions______________________________ 32 Nucleotides in the lumen ____________________________________ 34 Cold acclimation____________________________________________ 35 Cold induced changes to the lumen proteome____________________ 35 Cold induced changes to the stroma proteome ___________________ 37
Summary __________________________________ 38 Perspectives________________________________ 39 Acknowledgements __________________________ 41 References _________________________________ 44
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Abbreviations A0 primary electron acceptor of photosystem I A1 secondary electron acceptor of photosystem I APX ascorbate peroxidase ATPase ATP synthase CF0/CF1 subunits of the chloroplastic ATP synthase complex CYP cyclophilin (cyclosporine A-binding immunophilin) Cyt b6f cytochrome b6 f complex D1 32-kDa protein of the photosystem II reaction centre D1_pP D1 processing protease, tail specific peptidase DegP serine-type ATP-independent protease Fd ferredoxin FNR ferredoxin-NADP+ reductase FKBP FK506-binding immunophilin FX, FA, FB iron-sulphur centres LHC light harvesting chlorophyll protein complex MS mass spectrometry NPQ non-photochemical quenching P680 primary electron donor of photosystem II P700 primary electron donor of photosystem I PC plastocyanin PDZ structural protein domain involved in protein-protein interactions Pheo pheophytin, primary electron acceptor of photosystem II PQH2 plastoquinol, reduced and protonated form of plastoquinone (PQ) PrxQ Peroxiredoxin Q PSI photosystem I PSII photosystem II Psa photosystem I subunit Psb photosystem II subunit PPI peptidyl prolyl cis/trans isomerase QA, QB primary and secondary plastoquinone in photosystem II RC reaction centre ROS reactive oxygen species Rubisco ribulose bisphosphate carboxylase/oxygenase SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis Tat twin arginine translocation TPR tetratrico repeat involved in mediating protein-protein interaction 2-D two-dimensional VDE violaxathin de-epoxidase WOC water-oxidising complex YZ tyrosine 161 in the D1 protein P
B
B
8
Introduction Photosynthesis The sun is the ultimate source of energy, sustaining all life on Earth through the process of photosynthesis. It provides us with the food we eat and the oxygen we breathe. The fossil fuels that we use today are photosynthetic products some millions years of age. Most species, humans and animals alike, cannot utilize the energy from the sun directly. First, plants, algae and cyanobacteria must convert the light energy into chemical energy that subsequently is used to produce carbohydrates from the simple inorganic molecules carbon dioxide and water (Figure 1). These delicate life-sustaining mechanisms have fascinated scientists for a long time. Besides leading to great contributions within plant biology in general, of which some was awarded the Nobel Prize, there are also practical and economical aspects to photosynthetic research. Key areas are to increase crop productivity and attempt to do artificial photosynthesis for energy conversion [1]. It is therefore important to understand how the photosynthetic mechanisms are regulated and how stationary plants cope with the changes in the environment, such as light, temperature and nutrient availability.
Light
CO2 + H2O
(CH2O)n + O2
Figure 1. Photograph of an Arabidopsis thaliana plant with the overall equation of photosynthesis inserted.
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The chloroplast In eukaryotes, like plants and algae, the chloroplast organelle hosts the photosynthetic reactions. All green tissue of plants holds chloroplasts and a mature leaf mesophyll cell of Arabidopsis thaliana contains approximately 120 chloroplasts. Chloroplasts are semiautonomous and carry their own genome and protein translation machinery. Although best known for its role in photosynthesis, the chloroplast also synthesize many different compounds such as plant hormones, amino acids, fatty acids and lipids, vitamins, starch, purine and pyrimindine nucleotides, tetrapyrroles and isoprenoids. Several of the metabolic pathways link to each other with intermediates from one pathway utilised in other pathways. The chloroplast contains six different compartments, three soluble fractions and three membranes. The chloroplast envelope, consisting of a double membrane and the soluble intermembrane space, separates the organelle from the surrounding cytosol. Within the chloroplast, there is an internal membrane system, the thylakoids, which gives the chloroplast its characteristic structure. This attributes to the folding of the thylakoid membrane into stacked grana regions connecting to each other via the non-stacked membrane lamellae, which are in close contact with the surrounding soluble stroma. The thylakoid membrane in turn encloses the third narrow soluble compartment, the chloroplast lumen (Figure 2). To form a continuous membrane system with an intact lumen space, a refined 3-D model describes how the non-stacked stroma lamellae thylakoid forms right-handed helices wound around the appressed grana cylinders [2].
The photosynthetic reactions The photosynthetic process divides into two different parts, 1) the light reactions involving the capture of light energy and conversion into chemical energy, and 2) the carbon fixation reactions, which utilise the energy produced in the light reactions to fix carbon dioxide and produce carbohydrates. The former occurs within the thylakoid membrane and involves four different multisubunit protein complexes, the two photosystems I and II (PSI and PSII); cytochrome b6 f (Cyt b6 f) and ATP-synthase (ATPase). These complexes are heterogeneously distributed in the membrane with the majority of PSII found in the grana thylakoids, PSI and ATPase located in the stroma lamellae thylakoids and Cyt b6 f evenly distributed in the different regions [3]. Meanwhile, all enzymes necessary for the carbon fixation reactions are present in the surrounding stroma
10
compartment where the reaction takes place through a series of enzymatic steps also known as the Calvin cycle. The proteins in the photosynthetic protein complexes are encoded both by the nuclear genome and by the chloroplast genome.
Chloroplast 5-10 μm
Lumen
25 nm
5-10 nm Stroma
Chloroplast envelope
Stroma thylakoids
Grana thylakoids
Figure 2. (A) Schematic drawing of the chloroplast organelle showing the different compartments. The internal thylakoid membrane folds into stacked grana thylakoids that interconnect via the stroma thylakoids. The thylakoid membrane encloses the narrow lumen compartment. (B) Electron micrograph of an Arabidopsis chloroplast (Courtesy: Ellinor Thidholm, Södertörns University College).
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The light reactions The first phase of photosynthesis involves the light-dependent water oxidation, NADP reduction and production of ATP. Several biophysical, genomic and biochemical, including structural studies have contributed to our understanding of the complex architecture of oxygenic photosynthesis [4, 5]. The non-cyclic electron transport, first defined by Hill and Bendall in 1960 [6] describes how electrons moves from water on the donor lumenal side of PSII to reduce NADP+ on the stromal acceptor side of PSI (Figure 3). The photosystems capture the light energy through large pigment antenna complexes and transfer it, in case of PSII, to the primary electron donor P680 that becomes excited and passes an electron to the primary acceptor pheophytin (Pheo). The electron translocates further to a tightly bound quinone QA that passes it on to the mobile quinone QB. A nearby tyrosine residue (YZ) reduces the oxidised P680+ with electrons withdrawn from a four manganese cluster and subsequently water. This is referred to as the water splitting reactions and deposits protons in the lumen and releases oxygen to the atmosphere. After yet another round, the dually reduced QB2- takes up protons in the stroma forming PQH2 and moves through the lipid bilayer to transfer the electrons to the Cyt b6 f releasing the protons in the lumen. At the same time, an oxidised QB from the membrane quinone pool replaces the PQH2. Plastocyanin (PC), a small copper containing protein located within the lumen compartment mediates the transfer of electrons between Cyt b6 f and PSI. Light activates photosystem I in the same way as photosystem II. The special chlorophyll pair P700 becomes excited and passes an electron to a chlorophyll a molecule (A0), which works as the primary electron acceptor. Subsequently, the electron transfers via a phylloquinone (A1) and several iron-sulphur clusters to ferredoxin (Fd). Finally, NADP+ is reduced to NADPH catalysed by ferredoxin NADP+ reductase (FNR). The docking of plastocyanin to PSI on the lumenal side facilitates transfer of electrons from PC to the primary electron donor P700, making the photosystem ready for another round of electron transfer. B
B
P
During the electron transport, protons translocates from the stroma side of the membrane to the lumen compartment generating a proton gradient. The splitting of water by the water-oxidising complex (WOC) on the lumenal side of PSII also adds to the proton gradient. The accumulated protons in the lumen move down their concentration gradient through the ATP-synthase, thus couple the electron transport and proton movement with ATP synthesis. In this way, the non-cyclic electron transport generates NADPH and ATP in fixed relationships [7]. In addition, cyclic electron transport occurs around PSI producing no NADPH but
12
generates a proton gradient allowing the production of ATP. The chloroplast lumen plays an important role in upholding the proton gradient and counterbalances the flow of protons with uptake of Cl- and Ca2+, and efflux of Mg2+ [8].
Stroma
Light
H
PSII QA LHCII
QB
H+ + NADP+
NADPH Light FNR
+
P680
A1
RFeS
CF1 LHCI
CF0
A0
ADP + Pi H+
Cyt f
YZ
P700
PC
WOC Mn
H+
2H+ H2O
FB FX
Pheo PQH2
Fd FA
Cyt b6f
PQ
ATP
½ O2 + 2H+
Lumen
Figure 3. Schematic presentation of the linear electron transport and proton translocation in the chloroplast thylakoid membrane. For abbreviations, see text or abbreviation list page 8.
Carbon fixation reactions The second part of photosynthesis, the fixation of carbon dioxide, uses the ATP and NADPH produced during the light reactions. In most plants, the fixation produces a 3-carbon compound through a process named the Calvin cycle after its elucidator Melvin Calvin who described the pathway together with Andrew Benson and James A. Bassham in the 1950’s [9]. Sometimes, the fixation of carbon dioxide (CO2) is referred to as the dark reactions of photosynthesis. This is misleading because in most plants, the carbon fixation takes place during light and several of the enzymes involved in the Calvin cycle are light regulated [10, 11]. The pathway proceeds in the stroma through 13 steps in three different phases:
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carboxylation, reduction and regeneration. In the first phase consisting of only one step, the enzyme ribulose bisphosphate carboxylase/oxygenase (Rubisco) catalyses the reaction of CO2 and ribulose- 1,5 bisphosphate (RuBP) to produce two molecules of 3-phosphoglycerate. Rubisco is the most abundant soluble protein in plants constituting some 30 % of total protein in many leaves [12] and possibly the most abundant enzyme on earth [13]. Two steps accounts for the second reduction phase in the Calvin pathway forming glyceraldehydes 3phosphate (GAP) through 1,3-bisphosphoglycerate. The second phase, catalysed by phosphoglycerate kinase in the first step and glyceraldehydes-3-phosphate dehydrogenase in the second step, consumes ATP and the reducing agent NADPH. Regeneration of ribulose 1, 5-bisphosphate from five of the six GAPs occurs in the last phase through ten different steps using additional ATP. Three enzymes are unique to this pathway meanwhile the other 10 are common with the degradation pathway for carbohydrates. To ensure optimal activity it is necessary to regulate the mechanisms in specific ways. The increases in pH and magnesium ions in the stroma because of the light driven electron transport regulate several Calvin enzymes. These regulatory steps provide coupling between the light reactions and the carbon fixation reactions. In the case of Rubisco, the enzyme shows a complex pattern of regulation at multiple levels. These include posttranslational modifications such as carbamylation and conformational changes upon ligand binding [14, 15]. Light activation of Rubisco requires the presence of the Rubisco activase that sense the ADP/ATP ratio and redox state of the chloroplast [14, 16].
Responding to the environment Plants are sessile and need to be able to adjust to their changing environment on molecular level. During the course of the day or season, light, temperature, water and available nutrients varies. In addition, herbivore attacks on plants add to the sometime stressful situation. Plants respond to stress through a variety of mechanisms. If the plant cannot compensate for the encountered stress, it will lead to death meanwhile resistance mechanisms allow the plant to survive. Resistance can be grouped into stress avoidance or stress tolerance. These mechanisms follow two major lines. The genotypic response, where alternations in the genome are stable and remain in the population over generations is termed adaptation. These features are present whether or not the plants are exposed to stress. On the other hand, acclimation is an induced response to the environmental cue in the individual plant and causes a phenotypic not genotypic change. The ability to 14
acclimate may of course be inherited, which can determine the difference between stress tolerance and stress susceptible plants. The photosynthetic reactions are essential for photoautotrophic growth and therefore the changes in the environment have a specific impact on the photosynthetic apparatus, especially since it is a major site of damage under stress conditions. Photosynthesis inevitably leads to production of reactive oxygen species (ROS), such as singlet oxygen, superoxide radicals, hydroxyl radicals and hydrogen peroxide. ROS are produced at different locations in the chloroplast with three major sites being the light harvesting complex of PSII, the PSII reaction centre and the acceptor side of PSI [17-20]. These reactive species cause oxidative damage to the photosynthetic apparatus and may lead to loss of photosynthetic efficiency or photoinhibition by, for example PSII damage [21] or PSI damage [22]. Changes in environmental conditions such as light intensity and cold influence the energy balance between the absorb energy by photochemistry and the energy utilised for plant metabolism causing increased damage to the photosynthetic apparatus. Plants have evolved several strategies to adjust to the incoming light and minimise loss of photosynthetic capacity. At the chloroplast level, these include; 1) Regulating the light harvesting by short-term state transition [23, 24] or long-term regulation by synthesis [25] and degradation [26, 27]. 2) Non-photochemical quenching (NPQ), in which the rapid reversible ΔpHdependent dissipation of excess energy as heat dominates under most circumstances [28, 29]. 3) Changes in enzyme activities, gene expression and protein abundance of components involved in assimilatory electron transport [14, 25, 30-33] 5) Oxygen-dependent electron transport, either through photorespiration [34] or through photoreduction of oxygen by PSI in the so-called water-water cycle [35, 36]. 6) Scavenging of reactive oxygen species by nonenzymatic antioxidants such as carotenoids, ascorbate, tocopherols, glutathione and the scavenging enzymes superoxide dismutase (SOD), ascorbate peroxidases (APX) and gluthatione peroxidases (GPX) [19, 37-39]. 7) Damage and repair of PSII, which involves selective degradation and synthesis of proteins, mainly the D1 reaction centre protein [40]. These mechanisms are influenced not only by light but also by a variety in environmental factors. In this way, photosynthesis functions as a sensor in which the redox state of the photosynthetic electron transport components and redoxactive molecules act as regulating parameters [41-45]. Depending on plant species, developmental stage and environmental factors, the responses may vary greatly in detail and extent. However, to execute the adjustments numerous additional proteins participate to allow all processes with protein modifications, gene 15
expression, synthesis and degradation, assembly/disassembly of protein complexes, for example. Thus, knowledge of what proteins localises to the chloroplast and if their amounts change under different conditions may provide important information into these mechanisms progression and regulation.
This study Within the chloroplast, the lumen compartment forms an integral part of the thylakoid network that performs the light reactions of photosynthesis. Despite intensive research within the field of photosynthesis, not much was known about the lumen located proteins. The discovery of a lumenal proteome [46] was an important step towards a deeper understanding of the functions of the chloroplast lumen. The studies that were carried out in the course of this thesis continued that work and aimed to get detailed insights of the lumenal protein families and their roles in photosynthesis. To meet this task a proteomic approach was designed that enabled a detailed analysis of the lumenal chloroplast proteome. In the first phase of the study, the original method for the isolation of chloroplast lumen was modified to fit for the model plant Arabidopsis thaliana, whose genome was being sequenced by that time. In addition, a suitable 2-D electrophoresis system was set up to separate and display the lumenal chloroplast proteins for a general image analysis and a further identification by mass spectrometry and microsequencing. (Paper I). In the second phase, a comprehensive map of the prevalent proteins of the chloroplast lumen was acquired that provided an overview of the potential functions of lumenal chloroplast proteins and their import pathways. The map of the lumenal chloroplast proteome was also compared to an in silico produced protein map based on the finalised genome sequence data from Arabidopsis thaliana (Paper II). This was followed by more traditional biochemical functional studies of one of the newly identified lumen proteins, the TL29 protein (Paper III). Lastly, to gain information about the dynamics of the proteome during different physiological conditions, the lumen proteome was studied during the cold acclimation process by using 2-D difference gel electrophoresis (2-D DIGE) (Paper IV).
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The chloroplast lumen From an unexplored compartment to new perspectives for thylakoid function During years of extensive photosynthetic research, some proteins with lumen location have been characterised. They include proteins mostly involved in the photosynthetic reactions. One of the most studied is plastocyanin (PC). This small copper binding protein was first found in algae but later also in higher plants [47]. Its role in photosynthesis was early established although the localisation of the protein was first debated [48]. Now the protein sequence of plastocyanin has been described in more than 35 organism of cyanobacteria, algae and plants and its lumen location is since long widely accepted where it transfer electrons from the cytochrome b6 f complex to photosystem I. To process the PC protein a protease situated on the lumenal surface of the thylakoid membrane with its active site in the lumen has been described [49]. In the late 1970´s, the development of inside-out thylakoids from spinach [50] led to the identification of the photosystem II extrinsic proteins PsbO, PsbP and PsbQ [51-53]. Later on it was shown that these proteins also exist as soluble lumen proteins [54], they are long-lived and assembly-competent [55]. In addition, PsbT, a nuclear encoded photosystem II protein with unknown function [56] and an extrinsic photosystem I subunit, PsaN [57], reside in the lumen. The function of the latter is not established but the electron transport in PSI was not affected by its removal. In addition to the proteins involved in the light reactions of photosynthesis, the xantophyll cycle protein violaxanthin de-epoxidase [58], a D1 processing protease in spinach and barley [59], a DegP1 protease in pea [60] and a photosystem II assembly/stability factor Hcf136 [61] localises to the chloroplast lumen. Polyphenol oxidase has been identified within the lumen in tomato [62] and spinach [63]. When the procedure to yield a highly pure lumen fraction was developed for spinach (Spinacia oleracea), it became clear that the lumen protein concentration was high. It was estimated that the lumen fraction had a similar protein concentration as the stroma with at least 20 mg mL-1 giving protein abundance in the micro- to milli-molar range. This first systematic study of the lumen proteome in spinach revealed the presence of at least 25 lumenal proteins [46]. In the same study four of the proteins, with molecular weights of 40 kDa, 29 kDa, 17.4 kDa 17
and 16.5 kDa were identified as unknown proteins by amino-terminal sequencing. The subsequent cDNA characterisation of the 17.4 kDa protein showed that the protein was related to a novel pentapeptide repeat family [64] and the 16.5 kDa protein [65] was one of the early polypeptides shown to utilise the Tattranslocation pathway (see next section). The 40-kDa protein was independently identified as a new lumenal immunophilin-like protein, TLP40, in spinach [66].
Import of proteins According to the endosymbiont theory, the chloroplasts originate from engulfed ancient cyanobacteria by protoeukaryotic cells. The chloroplast of today still contains its own genome, encoding around 100 proteins depending of plant species, and has its own protein translation machinery. In Arabidopsis thaliana, the chloroplast genome contains 87 putative protein-coding genes, including 8 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes [67]. The number of proteins found in chloroplasts far outnumbers the few encoded by the organelle. The “endosymbiotic gene transfer” [68] explains this, as during the evolution most of the genes have been transferred to the nucleus and the proteins are post-translationally imported into the chloroplast. This applies to all lumenal proteins found to date. In order to reach their lumen location proteins are synthesised as precursors in the cytoplasm with an Nterminal bipartite transit peptide, which consist of a chloroplast transit peptide and a lumenal transit peptide. The import of lumen proteins proceeds through two steps where the precursor protein translocates across the chloroplast envelope and once in the stroma the chloroplast transit peptide is cleaved off by a stromal processing peptidase generating an intermediate form. The intermediate is then transported into the lumen via one of two distinct pathways [69] and processed to mature form by the thylakoid processing protease [49, 70, 71]. The two known transport pathways routing proteins into the lumen are the Sec pathway and the twin arginine translocation (Tat) pathway [69, 72-74]. The Sec pathway is dependent on ATP and transports proteins in an unfolded state, meanwhile the Tat pathway shows no requirement for ATP and is capable of transporting fully folded proteins [75-78]. The Tat-pathway is also known as the ΔpH-dependent pathway because it requires a transmembrane pH gradient [79, 80]. However, the need for a proton gradient in vivo was questioned when it was shown in Chlamydomonas reinhardtii that different Tat substrates was transported by the Tat system in vivo independent on the ΔpH [81]. This data was later
18
revisited with proposal of the transmembrane difference in electric field (Δψ) also would be able to fuel the Tat translocase [82]. Recently, Di Cola et al. addressed this issue and showed that the thylakoid ΔpH/Δψ was not required for the initial stages of Tat-dependent transport of proteins in tobacco protoplasts and considerable differences between the common in vitro assay and the in vivo system were observed [83]. From this, it is clear that several features of transport using the Tat-system remain to be clarified.
The bipartite transit peptide The bipartite transit peptide found in lumenal precursor proteins consists of a hydrophilic serine and threonine rich region for the transit across the envelope [73]. The lumenal transit peptide consists of a charge N-terminal domain followed by a hydrophobic core close to the processing site [84]. There is no consensus sequence for the hydrophobic core but there is a clear preference for AxA at the -1 and -3 position prior to the cleavage site of the thylakoid transit peptide [85]. In addition, proteins routed with the Tat pathway have a distinct twin ariginine motif, hence the name, in the beginning of the hydrophobic core. On the other hand, proteins transported via the Sec pathway have a single lysine close to the start of the hydrophobic core (figure 2 in Paper II). The import analysis of the putative ascorbate peroxidase TL29, carried out in our first analysis of the lumen proteins from Arabidopsis demonstrated that TL29 was only the seventh protein shown to utilise the Tat system (Paper I). The following analysis of the lumen transit peptides in the identified lumen proteins showed that more than half of the proteins seem to be transported by the Tat-pathway based on the presence of the twin arginine motif (Paper II). This illustrates the impact of this translocation pathway for protein traffic into the chloroplast lumen.
Experimental determination of the chloroplast lumen proteome When characterising the chloroplast lumen proteome, we used Arabidopsis thaliana to take advantage of the accumulating knowledge of the genome sequence. We adapted the isolation method from spinach and employed 2-D gel electrophoresis. With this approach, we clearly demonstrated and confirmed the high protein content in the chloroplast lumen in plants (Paper I). Using a broad pH range from 3 to 10 revealed the presence of approximately 300 protein spots in the lumen 2-D protein map of Arabidopsis (Paper I, Paper II). Most of these proteins
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were present in the acidic area and for detail analysis we created a protein map in the pH range from 4 to 7 detecting around 200 protein spots (Paper II, figure 4). The pattern of the protein map from Arabidopsis was very similar to the protein map made for the spinach chloroplast lumen where approximately 250 proteins spots were detected (Figure 1B in Paper II).
Figure 4. 2-D protein map of the Arabidopsis chloroplast lumen proteome (Paper II).
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So far, 47 of the lumen proteins from Arabidopsis have been experimentally identified (figure 5). Apart from the 36 proteins identified in paper II, 31 proteins were identified in the study of Peltier and colleagues of which all except 5 overlapped with the work in paper II [85]. The additional six proteins are the cytochrome c6 protein (Atc6) [86], two PsbQ-like proteins [87], a lumen located peroxiredoxin Q-like protein (Petersson U.A., Kieselbach, T., Funk, C. and Schröder, W.P., manuscript in preparation), the PsbP2 protein and a putative FKBP-type isomerase, AtFKBP16-1 (paper IV). The identified proteins evidently show that the chloroplast lumen contains its own specific proteome. Interestingly, many of the proteins located to the Arabidopsis chloroplast lumen belong to rather small protein families of which a relatively large portion within each family is part of the lumen proteome [88]. The large group of FKBP-type peptidyl-prolyl cistrans isomerases gives a good example. Twelve of the 23 putative Arabidopsis FKBP-type isomerases are predicted to be chloroplast located. Of these, 11 seem to be lumen located and eight have so far been experimentally identified in the lumen proteome (Paper II; Paper IV; [85, 88-90]). The novel family of PsbPdomain proteins of which all nine Arabidopsis sequences encode experimentally identified lumen located proteins (Paper II; Paper IV) demonstrates the same. Several proteases belong to the lumen proteome and we identified two C-terminal D1 processing proteases and three DegP-proteases, showing a lumen preference for ATP-independent proteases. A putative ascorbate peroxidase (Paper I-III), a set of proteins of unknown function, such as two pentapeptide proteins, and proteins containing no known motifs or functional domains are found in the chloroplast lumen of Arabidopsis (Paper II; [85]). The 2-D protein map shows several proteins appear in multiple spots both with different molecular weights and with different isoelectric points (Paper II, figure 4). Alternative splicing variants are suggested for a few lumen located proteins, such as the pentapeptide proteins, the DegP8 protein, the C-terminal D1 processing proteases and the PsbP domain protein T215. However, aligning the alternative forms revealed that for some of the proteins the difference is found only within the precursor protein (e.g pentapeptide protein TL17) and not in the mature form. Still these proteins and numerous other lumen proteins without reported splicing variants appear in multiple spots suggesting they are posttranslationally modified. Comparing the Arabidopsis lumen proteins with the spinach lumen proteins we observed a good correlation between the proteomes (Paper II), demonstrating the Arabidopsis lumen proteome can serve well as a model for other plants (Paper II).
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This holds true when comparing the 15 experimentally identified proteins in pea and the putative lumen proteins encoded by the rice genome [88, 91].
Cyt b6f
PSII VDE TL29 PrxQ
PSII subunits O1 O2 P1
P2
Q1
Q2
At1g03600
PsbP-domain 8 proteins
PC
ATPase
PSI
PC Cyt c6A
N
Proteases DegP1 D1-pP DegP5 D1-pPDegP8 like
PsbQ-domain 2 proteins
Immunophilins 11 proteins Pentapeptide proteins TL17 TL15
Unknown function 5 proteins
Figure 5. Schematic overview of the experimental chloroplast lumen proteome. The four major protein complexes in the thylakoid membrane are also shown.
The in silico chloroplast lumen proteome To complement our experimental data, the Arabidopsis genome sequence data [92] was analysed to identify putative lumen located proteins (paper II). For this purpose, the characteristic transit peptide of lumen proteins can be used for theoretical prediction of the lumen proteome (Paper II) [85, 88, 93]. Target P is a neural network based tool for large-scale sub-cellular location prediction [94]. The program predicts nuclear encoded proteins to the chloroplast, mitochondria, secretory pathway or other cell location based on the N-terminal signal peptide. TargetP predicted 3972 chloroplast proteins from 27288 Arabidopsis sequences [88] or including different gene models, 4255 gene models (4013 genes) from 28581 gene models (27170 genes) were predicted to produce chloroplast located proteins [93]. These predictions show a chloroplast proteome size of 14.6-14.9% of the total Arabidopsis proteome and agree well with our prediction of 14.6 %,
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based on an earlier gene annotation version (Paper II). To assess the lumen located proteins from the chloroplast predicted ones, would call for careful analysis. The programme SignalP [95, 96] provides a good tool to predict the bipartite transit peptide found in lumenal proteins. However, some thylakoid membrane proteins, such as PsbW, PsbX and PsaF, carry the lumenal transit peptide and to exclude membrane proteins from the predicted lumen proteome, screening for putative transmembrane helices need to be included. A fully automated prediction of the lumen proteome using TargetP and SignalP, estimated the lumen to contain 201 proteins of which 109 contained a Tat-motif. A closer examination of these 109 proteins resulted in a drop to 71 predicted lumenal proteins with a Tat-motif [85]. In our approach, which included a manual analysis for each positive SignalP sequence to exclude false positives and keep the number of missed lumenal proteins to a minimum, we predicted 55 lumenal proteins (Paper II). Combining the experimentally proteome with the predicted proteome, we estimated the chloroplast lumen to contain 80 proteins. The predictor program LumenP [97], trained specifically to predict thylakoid lumen proteins was released in 2003. A direct approach using LumenP in combination with TargetP, predicts 1.5 %-3% of all proteins encoded by the genomes of Arabidopsis and rice (Oryza sativa) to be located in the chloroplast lumen [97]. Applying additional filters, such as signal peptide length and screening for transmembrane helices, the number of predicted proteins was lowered to 1 % of the total Arabidopsis proteome using TargetP in combination with either LumenP (291 proteins) or SignalP (285 proteins) [93]. It is clear that prediction of proteins at this subcellular level is still very difficult. Nevertheless, our prediction of lumenal proteins in paper II correlated very well with the experimental proteome and demonstrated the experimentally identified proteins are good representatives of the chloroplast lumen proteome.
Functions of the lumenal proteins Photosystem II subunits The recently discovered lumenal proteins raise several questions about the physiological functions not only for the new proteins but expand to include the previously described. The PsbO, PsbP and PsbQ proteins were initially believed to be essential for the water oxidation by photosystem II (PSII) within the lumen. Later studies modified the view; the functions of the extrinsic proteins seem to be to form a structure together with the PSII intrinsic proteins that provides a stable environment for Mn ligation and the retention of both Ca2+ plus Cl-. In this
23
structure, PsbO is thought to stabilise the manganese ligation, therefore also named manganese stabilising protein [98, 99]. The roles for PsbP and PsbQ in stabilising the calcium and chloride environment are less clear [99] but recently it was shown in tobacco (Nicotiana tabacum) that PsbP, but not PsbQ, was indispensable for normal PSII function in vivo [100]. We discovered early on in the analysis of the lumen proteome of Arabidopsis, the presence of two forms of PsbO and PsbQ, at approximately equal amounts (Paper I). The stochiometric amounts of extrinsic proteins bound to PSII are not clearly resolved [99] but structural studies suggest one copy of the PsbO protein [101]. The presence of isoforms raises questions as to whether different forms participate in the water oxidising complex. Is there a heterogeneous mix of supercomplexes based on the extrinsic proteins? The feature of two genes encoding PsbO proteins has so far only been observed in Arabidopsis although predictions suggest the presence of duplicate PsbO genes in Brassica rapa and Brassica napus [102] as well as suggestions for multigene families encoding PsbO in tobacco [103] and pea [104]. The Arabidopsis isoforms have similar primary structure and it was shown in a mutant missing the PsbO1 protein [105] that PsbO2 can functionally replace the former although with less PSII photochemical efficiency [102]. The lower efficiency may be related to the decrease in total amount of PsbO [106]. In tobacco, a multigene family of four isogenes [107] encodes the PsbP protein. Recently, these PsbP members were demonstrated to have equivalent functions in vivo based on the observation that the PSII activity linearly correlated with the total amount of PsbP, independent of which PsbP protein accumulated [108]. However, in virus-infected tobacco different forms of the PsbP protein were shown to decrease in abundance differently in response to the encountered stress [103]. It seems therefore, that optimal PSII activity is dependent on the full expression of the different forms of PsbP and PsbO under controlled circumstances and differential accumulation may be one way to adjust PSII activity during different environmental conditions. For the PsbO protein there may well be that it has a dual role within the wateroxidising complex. Recently it was shown that the PsbO protein in pea possessed carbonic anhydrase activity [109], which catalyses the interconversion of carbon dioxide and bicarbonate. Bicarbonate stimulates PSII activity and one plausible hypothesis for this function of PsbO could be to provide PSII with bicarbonate although the role of bicarbonate in donor side PSII chemistry is not well understood [110]. A lumen located alpha carbonic anhydrase (Cah3) has previously been shown in the green algae Chlamydomonas reinhardtii [111] and the protein was shown to be associated with PSII [112]. Later studies with the
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Cia3-mutant lacking the Cah3 protein suggested the carbonic anhydrase to be required for both stability and function of the water-oxidising complex in C. reinhardtii [113]. In higher plants, an extrinsic carbonic anhydrase (CAext) associated with PSII was reported in the mesophyll cells of maize [114]. The CAext protein co-migrated on a SDS-gel with the PsbO protein but was at that time believed not be the same protein as PsbO since it was immunodetected with antibodies directed against the C. reinhardii cah3 protein. However, the cross reactivity of the pea PsbO protein with the C. reinhardii cah3 antibodies and the inhibited carbonic anhydrase activity after incubation with either PsbO antibodies or the algae αCA-antibodies [109], may suggest that the putative CAext identified in maize is the PsbO protein. The physiological relevance of this activity for PsbO is still to be clarified but another explanation besides providing PSII with bicarbonate could be to have a system where protons fast can be withdrawn to minimize local acidification that may cause damage to the water-oxidising complex during high illumination [113]. The CO2-byproduct could then diffuse to the stroma and be used for carbon assimilation. The CO2 hydration and the bicarbonate dehydration could be of importance during different stages of the PSII complex, one that provides bicarbonate during the assembly of the water oxidising complex and the other with removal of protons that protects the complex under conditions that cause increased local acidification, such as high light. In addition to the extrinsic PSII subunits PsbO, PsbP and PsbQ, the Arabidopsis lumen proteome include several proteins with PsbP-domains and two proteins with PsbQ-domains (Paper I; Paper II). In cyanobacteria, the common view is that PsbU & PsbV replace the PsbP & PsbQ, respectively as extrinsic PSII proteins. However, genomes of different cyanobacteria contain genes encoding proteins with homologues regions to both PsbP and PsbQ. In Synechocystis sp. PCC 6803, the PsbP-like protein (Sll1418) is homologous to three of the Arabidopsis lumenal PsbP domain proteins [88, 115] and the PsbQ-like protein (Sll1638) is a homologue to the Arabidopsis PsbQ1 protein [116]. Both these proteins have been shown to associate with PSII in Synechocystis [115-118]. Thornton et al. reported reduced photoautotrophic growth for both PsbP-knockout and PsbQ-knockout mutants grown under limited Ca2+ or Cl- conditions. Purified PSII from these mutants showed reduced oxygen evolution in comparison to wild type PSII. Based on their findings they suggested that PsbQ is present with one copy per PSII in cyanobacteria, PsbP is present in 3 % of all PSII complexes and that these proteins are involved in regulation of the PSII activity [117]. In contrast, two later studies of the Synechocystis PsbP-knockout mutants showed no reduced photoautotrophic growth in either normal or nutrient limited media [115, 118]. However, these
25
results would still fit with the PsbP protein having a regulatory role as proposed by Thornton et al. and being present only in 3% of PSII complexes in Synechocystis. Moreover, the effect of deleting the PsbQ-like protein was studied under various conditions both in strains with wild-type background and in strains with inactivated PSII extrinsic proteins in Synechocystic sp. PCC 6803 [116]. In that study, the authors concluded that the cyanobacterial PsbQ protein is important for the stability of PSII and not solely associated with the CaCl2 requirement, and that the PsbQ protein is required for photoautotrophic growth in the bacteria’s natural environment. There are no functional studies described for the PsbP-domain and PsbQ-domain proteins in higher plants. No reports describe the association of these proteins to PSII. Nevertheless, the findings in cyanobacteria may point to regulatory roles for these proteins in plants and the association with the PSII supercomplex will probably be discovered for at least some of them. Mutant plants lacking these proteins studied in plants natural habitat and/or under different stress-conditions could help deciphering their functions.
Assembly factors and proteases The accurate assembly of PSII is a prerequisite for proper function of the complex within the plant and requires the lumenal HCF136 protein [61]. HCF136 localises to the stroma thylakoids [61] and Hcf 136 mutant plants were shown not to form any PSII reaction centres [119]. Using blue native-PAGE and affinity chromatography, Plucken et al. could show that the HCF136 protein interacted with a pre-reaction centre (pRC) complex encompassing D2 and cytochrome b559. Based on these results the authors proposed a model where HCF136 act on the lumenal side of the membrane as an assembly or folding catalysts interacting with D2 and cytochrome b559 that subsequently assemble with D1. In the absence of HCF136, the D1 protein is synthesised but assembly into the pRC is blocked and D1 degraded [119]. On the other hand, the D1 protein has been shown to cotranslational assemble with D2 and the prePSII complex without detection of free D1 protein after terminated translation [120]. It is clear the assembly/disassembly process of PSII is a complex process with tight regulation at multiple levels [121, 122] and it will be interesting to see how the soluble lumen protein HCF136 mechanistically is involved in this process. In addition, the PSII assembly process involves another lumenal protein, the D1 C-terminal processing protease [59, 123125]. The D1 precursor protein includes a C-terminal extension that needs to be cleaved off for proper assembly of the water-oxidising complex [123, 126]. The C-terminal processing protease responsible for the processing shows strict
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substrate specificity for the D1 precursor protein [123] and the cleavage site recognition relies on the formed structure between the protease and the substrate rather than the primary amino acid sequence [127]. Two of the three C-terminal processing proteases predicted to reside within the lumen compartment have been experimentally identified (Paper II) but no functional analyses have been reported for the Arabidopsis proteins. It may be possible that the proteins have different physiological purposes within the lumen compartment as will be further discussed (see section Proteins with unknown function). Proteases function not only in processing proteins but fulfil important roles in highly specific and regulated degradation of proteins [128-130]. The DegP1 protein has been identified in pea [60] where it was shown to associate to the lumenal surface of the thylakoid membrane and its abundance was stimulated by increased temperature. Deg proteases belong to a family of serine-type ATPindependent proteases of which the E. coli DegP (or HtrA) is the best characterised [131]. This E. coli heat shock protein forms a hexamer and combines chaperone and protease activities with the chaperone activity dominating at low temperatures [132]. In Arabidopsis, DegP1, DegP5 and DegP8, form part of the lumen proteome (Paper II). In vitro studies have shown that the DegP1 protein is capable of degrading lumenal proteins with increased protease activity at elevated temperatures [133]. The lumenal Deg proteases have been named DegP because they all contain the same catalytic triad His-Asp-Ser as the E. coli DegP enzyme [134]. However, in contrast to the bacterial DegP that contains two C-terminal PDZ-domains, the DegP1 and DegP8 proteins have one PDZ-domain meanwhile the DegP5 lacks an obvious PDZ-domain. The PDZ-domains are implicated in regulating protein-protein interaction and are believed to function as gatekeepers by recognition of substrates in the DegP hexamer in E. coli [135] as well as a stress sensor in the E. coli DegS protein that contains only one copy of the PDZdomain [136]. The high sequence similarity of the lumenal Deg P proteases to the bacterial enzyme suggests that the DegP proteins might form complexes [129], which are sustained by the observation of oligomerisation in vitro of the DegP1 protein [133]. Further analyses will reveal if these complexes are homo- and/or hetero-oligomers.
Plastocyanins and cytochrome c6A Two proteins comprise the plastocyanin pool in Arabidopsis (Paper I) but no functional dissection between the two proteins has been reported. The model of plastocyanin as the only protein mediating electron transfer between the
27
cytochrome b6 f complex and photosystem I in higher plants was significantly challenged with the discovery of a cytochrome c6-like protein (Atc6) in Arabidopsis [86]. In cyanobacteria and eukaryotic algae, it has long been known that both plastocyanin and cytochrome c6 may function as electron carriers whilst plants lack cytochrome c6. Gupta et al. reported that the Atc6 protein could functionally replace plastocyanin and restore photosynthetic electron transport in a reconstitution assay with inside out thylakoids from Arabidopsis. To address the physiological function, the plastocyanin genes were silenced both in a wild type plant and in an Atc6-knockout plant. The depletion of plastocyanin in plants with wild type background showed retarded growth on soil and the Atc6-knockout mutant with depleted plastocyanin did not survive when grown photoautotrophically. With this, the authors reported against current dogma that neither plastocyanin nor Atc6 was essential for Arabidopsis growth and development [86]. In contrast, Weigel et al. found that a double mutant that lacked both plastocyanins but expressed a functional Atc6 protein could not grow photoautotrophically because of the complete blockage in light-driven electron transport [137]. Instead, they discussed a possible regulatory or accessory role for the Atc6 protein [138]. Another recent review addressed the functional role of the Atc6 protein, now renamed to cytochrome c6A where the author did not favour the cytochrome c6A protein as a substitute for plastocyanin. Howe proposed a regulatory role for cytochrome c6A involving redox signalling mediated by the heme, two conserved cysteins and immunophilin-binding [139]. The interaction with immunophilins was inferred from the original data where the cytochrome c6A was initially identified as an interaction partner of an immunophilin [86, 140].
Immunophilins Among the identified lumenal proteins, immunophilins constitute the largest group so far. Originally identified as targets of immunosuppressive drugs, immunophilins group into two families: FK506-binding proteins or FKBPs and cyclosporin A-binding proteins or cyclophilins [141]. The ubiquitous immunophilins share similar enzymatic activity of peptidyl-prolyl cis-trans isomerization (PPI) of polypeptides and organism proteomes ranging from bacteria to humans and plants encompass these proteins [90]. The functions of the individual immunophilins are far from clear. In plants, the multidomain AtCYP40 immunophilin functions in the vegetative development and is hypothesised to regulate the activity of specific signalling pathways within Arabidopsis leaf development [142]. In contrast, mutations in the gene (PASTICCINO1) coding for
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the AtFKBP72 immunophilin, give a highly pleitropic phenotype, suggesting a critical function for the protein for many aspects of plant growth and development [143]. Both AtCYP40 and AtFKBP72 contain three tetratricopeptide repeats (TPRs) in their C-terminal parts. The repeats form a scaffold known to mediate protein-protein interactions [144], which is not seen in any of the lumenal immunophilins. The largest cyclophilin found in the lumen is AtCYP38, which is a multidomain protein consisting of an N-terminal leucine zipper domain, a central acidic region and a C-terminal CYP domain [145]. The spinach homologue, TLP40, was the first lumen cyclophilin characterised in detail [66]. The protein co-purified with a PP2A-like protein phospatase situated in the thylakoid membrane with its active site in the stroma [66, 146]. Through the reversible binding TLP40 was suggested to regulate the phosphatase activity, involved in dephophorylation of PSII reaction centre proteins [146]. Upon an abrupt elevated temperature, TLP40 was demonstrated to dissociate from the membrane followed by an increase in phosphatase activity [147], suggesting a regulatory role for TLP40 in PSII protein turnover as response to heat shock. The single domain AtCYP20-2 immunophilin is a light regulated cyclophilin associated with the grana membrane fraction [148]. The function of the protein is not established but its spinach homologue, TLP20, was suggested to be the general folding catalysts within the lumen compartment [149]. In the similar theme, the FKBP-type immunophilin AtFKPB13 regulates the accumulation of the Rieske-subunit, an essential subunit of the electron transport chain within the Cyt b6 f complex [140]. Using a two-hybrid screen, the Rieske protein interacted with the FKBP13 precursor protein and silencing the fkbp13 gene lead to accumulation of the Rieske protein. Interestingly, only the precursor form of FKBP13 and not the processed mature protein interacted with the Rieske subunit. Gupta et al. proposed a model where AtFKBP13 functions as an anchor chaperone, preventing the targeting of Rieske to the thylakoids and coordinating the Cyt b6 f complex subunits to assure efficient assembly. Detailed structural studies of the AtFKBP13 protein revealed the presence of two redox-active disulfide bonds [150]. Upon reducing the protein with thioredoxin, inactivated the peptidyl-prolyl isomerase activity, demonstrating the redox regulation of this lumenal protein. One key finding was that oxidation rather than reduction, as seen for many biosynthetic enzymes within the stroma, activated the AtFKBP13 protein, suggesting a dependence on the redox milieu of the host compartment. Recently, three functional distinct forms of AtFKBP13 were proposed [90]. When the oxidised precursor form enters the chloroplast, AtFKBP13 precursor may keep the Rieske protein in an inactive soluble form by acting as a chaperone. Following
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conditions up-regulating cytochrome b6 f synthesis, thioredoxin might reduce AtFKBP13 and the immunophilin-Rieske complex may subsequently be recruited to the membrane. After release of the Rieske subunit, the AtCYP13 might be processed to mature form and released into the lumen compartment. Within the lumen, the immunophilin PPI-activity may be activated by oxidation and AtFKBP13 function in folding other lumenal or thylakoid membrane proteins.
Protective enzymes Violaxathin-de epoxidase (VDE) operates within the xantophyll cycle where it converts violaxanthin to zeaxanthin through the intermediate antheraxanthin [151]. The xanthophyll cycle form part of the protective mechanism against excess light energy known as non-photochemical quenching (NPQ) [28, 152]. Consequently, Arabidopsis mutant plants (npq1) defective in the gene encoding VDE showed greatly reduced NPQ [153]. Analysis of plant fitness using the npq1 mutant showed that this protective mechanism was important for plants encountering fluctuation in light in a controlled environment but did not affect plant fitness under constant light conditions [154]. From these results, the authors suggested that this advantage was due to increase in plant tolerance to variation in light rather than tolerance to high-intensity light itself. VDE uses ascorbic acid as cofactor [155] and deficient ascorbate levels was shown to limit VDE activity in vivo [156]. This need for ascorbate implies that ascorbate is present within the lumen compartment although this has not been shown. The specific ascorbate levels in chloroplasts are difficult to measure but based on leaf ascorbate content the chloroplast concentrations estimate to range between 10-50 mM, with increased levels upon high light treatment [38, 157]. Assuming an ascorbate concentration within the chloroplast stroma of 50 mM at pH 8 and that the ascorbic acid diffuses across the membrane, Eskling et al. calculated that the ascorbic acid concentration within the lumen would be 8 µM. To meet the need of ascorbate for VDE with a determined Km of 100 µM for ascorbic acid [155] a carrier was proposed to transport ascorbate in to the lumen and dehydroascorbate out [157]. Besides donating electrons to VDE, ascorbate has been shown to function as an alternative electron donor to PSII when the wateroxidising complex was impaired [158, 159]. Ascorbate serves a central function as substrate for the two ascorbate peroxidases, tAPX and sAPX, which operates on the stromal side of the thylakoid membrane to scavenge hydrogen peroxide [19, 160]. In the lumen, we identified
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the TL29 protein as a putative ascorbate peroxidase in tomato and Arabidopsis (paper 1). The TL29 protein is an abundant protein in the chloroplast lumen and BLAST searches in the expressed sequence tag databases suggest the TL29 protein to be a general feature of all higher plants. TL29 was found to associate with the grana region of the thylakoids and co-purified with PSII enriched membrane fractions, i.e. BBY-particles (Paper III). In the same study, we demonstrated that the levels of TL29 protein was induced by light and further increased upon high illumination. The sequence similarity to ascorbate peroxidase implicates the TL29 protein to form part of the plant defence system against reactive oxygen species and TL29 has therefore been given an additional name APX4 in Arabidopsis [160-163]. To investigate the function for TL29 as a putative ascorbate peroxidase within the lumen we used three approaches. First: A “native” manner was applied by isolation of thylakoid lumen in the presence of ascorbate and analyses of ascorbate dependent peroxidase activity. Second: we overexpressed recombinant TL29 protein followed by reconstitution assays with hemin and third: we measured and compared the peroxidase activity in isolated lumen fractions from TL29-knockout plants and wild type plants. All lines of investigation failed to show any peroxidase activity associated with the TL29 protein, strongly suggesting that TL29 is not an ascorbate peroxidase (Paper III). This finding is supported by the previous observation of no detectable ascorbate dependent peroxidase activity within the lumen compartment of spinach [164]. The result from our functional analysis high-lightens the need to confirm with experimental data the putative function inferred from sequence similarity to other proteins. The bioinformatics data provide an excellent tool for finding structural motifs or functional domains that offer a first clue to a putative function of the protein. However, caution must be taken so that the putative function ones assigned based on a sequence similarity is not lost in genome wide and proteomic wide analysis that will perhaps interfere with the interpretation of the data. We were not able to detect any ascorbate dependent peroxidase activity connected to the TL29 protein. Instead, we were able to show that the TL29 protein can bind ascorbate and that the protein is found close to PSII (Paper III). This opens for an interesting proposal that the TL29 protein provides PSII with ascorbate that when needed can be utilized for photochemical quenching. In this way, TL29 could regulate the ascorbate concentration within the lumen by locally increasing the concentration of ascorbate around the PSII complex. This will enable PSII to use ascorbate as electron donor for photochemical quenching when the water oxidising system is impaired despite that ascorbate is at the same time needed for non-photochemical quenching by the action of VDE. The increased
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amounts of the TL29 protein in response to high light would also be explained. The TL29 protein is routed across the membrane by the Tat-pathway (Paper I), which allows transport of folded proteins. One might speculate that part of the increased amounts of TL29 comes from newly imported proteins that bind ascorbate in the stroma and transport ascorbate across the thylakoid membrane. However, more probable is that TL29 bind ascorbate within the lumen, thus “tunnelling” the ascorbate to the PSII complex thereby lowering the concentration of free ascorbate, which in turn facilitates the transfer of ascorbate from the stroma into the lumen. No reports describe how ascorbate enters the lumen, thus this working hypothesis needs to be further tested.
Proteins with unknown functions Numerous additional proteins reside in the lumen compartment without known or putative functions. For some of the proteins, structural motifs group them together, such as the pentapeptide proteins TL17 and TL15. The pentapeptide repeat family compose of more than 500 members found in both prokaryotes and eukaryotes [165]. Few of the members are functionally defined and most certainly this family include functionally very divergent proteins. Interestingly, in a search for thioredoxin targets the TL17 protein was identified as a putative target using the cytosolic TRX h3 from Arabidopsis [166]. Thioredoxin reduces disulphide groups and play an important role in enzyme regulation. The link between electron transport and numerous metabolic enzymes in the stroma via the ferrodoxin/thioredoxin system is well documented [11, 167, 168]. Within the lumen compartment, the area of redox regulation is of growing interest [169], exemplified by the AtFKBP13 immunophilin (see section Immunophilins). Whether or not the redox signal is mediated by a yet unidentified thioredoxin, the proposed action of cytochrome c6A or the thioredoxin-like Hcf164 situated on the lumenal surface of the thylakoid membrane still awaits clarification. The in silico lumen proteome includes five tetratrico peptide repeat (TPR) proteins (gene loci At2g37400; At3g09490; At5g02590; At4g39470; At3g53560) (Paper II, this study), of which one was experimentally identified [87]. They appear to have structural motifs consisting of repeats of 34 amino acids that fold into helices [144]. The TPR-domain contains three repeats, which forms a scaffold that mediates protein-protein interaction and assembly of multiprotein complexes. Like the pentapeptide repeat domain, the TPR-domain is found in various proteins such as the PSI-specific chaperone Ycf3 that assists the assembly of the complex with suggested interactions between the TPRs and individual proteins of PSI
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[170]. Another example is the Tic 40 protein, a co-chaperone facilitating protein translocation across the chloroplast envelope [171]. The predicted lumen located TPR proteins may be candidates for mediating protein-protein interactions between themselves and targets and might form complexes with other lumenal proteins such as the immunophilins or proteases to ensure the correct folding and transport needed for proper functioning of the chloroplast thylakoids. More intriguingly is that two of the predicted lumen proteins (At5g02590; At4g39470) also group within the structural family of prenyl transferases by Interpro (SSF 48439; IPR008940). Prenyl transferases catalyse the posttranslational lipid modification of proteins by attaching 15- (farnesyl) or 20(geranylgeranyl) carbon isoprenes via a thioether link to C-terminal cysteines of proteins [175]. Chloroplast proteins have been shown to be prenylated and the majority of these proteins were discovered to be modified by attachment of a phytol within the organelle [172-174]. The prenylation of proteins within the chloroplast was proposed to occur through a yet unidentified binding mechanism and not via the common thioether link [172]. The observed prenyl transferase activity in spinach chloroplasts was almost exclusively confined to the thylakoids with no detected activity in the stroma [173]. Given that prenyl transferases are normally soluble and that they act as heterodimers, a highly speculative idea is that the predicted lumenal proteins may be the source of the prenyl transferase activity detected in chloroplasts. Considering the interplay between the lumen proteins and the thylakoid membrane, proteins modified with lipids might be present within the lumen compartment. With a lipid modification, the proteins could be anchored to the membrane or the lipid may mediate protein-protein interaction. The proteins may be modified by the hypothesised internal system through unknown mechanisms or by the external systems via the conventional thioether link. For the latter system, this would require a C-terminal cystein within a structural motif. Indeed, this motif is found within the carboxy-terminal proteinase D1-like protein (At5g46390.2). Two alternative forms are reported and one of them contains the C-terminal CSILdomain (figure 6) typically used as prenylation site by the geranylgeranyl transferase type 1 (GGTase-I) [175]. Interestingly, the GGTase-I directs the localisation of regulatory proteins and target proteins are found within the Rop family of small GTPases, for example [175]. Relevant is that the carboxy-terminal proteinase D1-like protein has an ATP/GTP binding site (P-loop) within its PDZdomain and may bind a nucleotide. These observations may suggest a regulatory role for the protein and support an idea that the D1-processing proteases have different physiological functions within the lumen.
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Bipartite transite peptide
PDZ-domain
Peptidase C
N GMDVSGKS
CSILQQS
Figure 6. Schematic drawing showing the domain organisation of the carboxyterminal proteinase D1-like protein (At5g46390.2). The putative ATP/GTPbinding site within the PDZ-domain and the putative prenylation site at the Cterminus are enlarged.
Nucleotides in the lumen The putative binding of ATP/GTP for the carboxy-terminal proteinase D1-like protein speaks in favour of nucleotides within the chloroplast lumen. This controversial issue has been addressed and Schröder et al. investigated the presence of ATPase activity within the spinach lumen but no significant activity could be detected [176]. Conversely, Spetea et al. demonstrated nucleoside diphosphate kinase (NDPK) activity in the lumen of spinach and Arabidopsis [177]. The detected activity was subscribed to the NDPKIII protein (At4g11010) and import analysis showed that this protein was imported into chloroplast. The NDPKIII showed a preference for GDP as substrate and it was suggested that the PsbO protein was a potential GTP binding protein. Fitting the missing transporter, they suggested a 36.5 kDa protein was involved in transporting nucleotides into the lumen compartment. The data presented by Spetea et al. points to the presence of nucleotides in the lumen. However, it should be noted that the NDPKIII has been reported to localise to the intermembrane space of the mitochondria. There the protein was present in sufficient amounts to be detected with silver staining when only small amounts (50 μg) of total mitochondria proteins were separated with 2-D PAGE [178]. Thus, the location in mitochondria is unlikely to have been a chloroplast cross-contamination. It may be that NDPKIII localises to both mitochondria and the chloroplast as this would then explain the different reported locations. The question of ATP or GTP within the lumen compartment remains open. Investigation into the ATP/GTP-binding capacity and ATP/GTP-
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dependence of the carboxy-terminal proteinase D1-like protein may provide insight into this area.
Cold acclimation Many plant species, like tomato, potato and maize have limited capacity to cope with low temperature. On the other hand, cold tolerant species, including Arabidopsis and crops such as winter cereals grow at low temperature and survive freezing temperatures [179, 180]. These plants increase their freezing tolerance upon exposure to low but non-freezing temperatures during a period ranging from days to weeks, an adaptive process known as cold acclimation [181]. The constraints low temperature place on growth as well as distribution of many important crops and horticultural plants has resulted in much effort trying to understand the phenomena of cold acclimation and the attained freezing tolerance. Cold acclimation is a complex process, which will induce changes in many different cellular processes, for example membrane modifications, osmotic regulation with increases in soluble sugars, multiple changes in gene expression and changes in energy balance [182]. From the observed modifications in metabolism and gene expression, the relative importance of each factor in increasing freezing tolerance is not all clear, nor is the exact signalling cascades established. However, studies combining transcriptome and metabolome data show that the alternations in metabolism associated with cold acclimation are energetically costly [183-185] and that these changes in the metabolome remain dynamic even during long-term acclimation [186]. The energetic cost of cold acclimation emphasizes the importance of the acclimation of plastid metabolism in plants exposed to low growth temperatures.
Cold induced changes to the lumen proteome To increase our knowledge of the lumen proteins and to understand how the chloroplast lumen proteome adapt to low temperature, we investigated the proteome dynamics during short-term cold stress and long-term cold acclimation (Paper IV). In the lumen proteome, we identified eight proteins that displayed at least a 2-fold change in abundance in response to low temperature (table 4 in paper IV). Most of these changes to the lumen proteome were observed in leaves that had developed at 5ºC following 40 days in the cold. Four of the proteins were extrinsic photosystem II proteins. Interestingly, the PsbO2 protein increased in abundance meanwhile the PsbO1 protein decreased. In addition, a complex pattern
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of appearance for the PsbP1 protein was detected with both increased and decreased levels of the protein migrating to different positions in the 2-D gel suggesting various forms of the protein accumulating differently. The gene encoding the PsbP2 protein, At2g30790 has been shown to be repressed by cold treatment [187] and our analysis showed a decrease in abundance for the PsbP2 protein as well. No functional data has been presented for the PsbP2 protein but the observed alternations in abundance for the various forms of extrinsic PSII proteins implicate important changes in stability of the water-oxidising complex of PSII during cold acclimation. These alternations of the extrinsic proteins in cold acclimated leaves may adjust the PSII efficiency, and possibly form part of the increased resistance to photoinhibition reported in cold acclimated Arabidopsis [188]. We detected decreased levels of the photosystem II assembly factor HCF136 in the lumen fraction from cold acclimated leaves. The Hcf136 protein has been shown to interact with the preRC of PSII [119] to facilitate the assembly of PSII. The reason for the decreased abundance observed in paper IV, is not clear but one hypothesis might be that the interaction with preRC during the assembly process tightens the association of Hcf136 to the membrane. If there is an increased D1 repair in the cold acclimated leaves, this may lead to a larger set of Hcf136 proteins being more tightly associated to the membrane. In turn, this may decrease the pool of Hcf136 in the soluble lumen fraction that was analysed. Remarkably, we observed a strong increase in abundance of the cyclophilin AtCYP20-2 protein. Previously we had only detected the AtCYP20-2 protein in one single protein spot in the 2-D protein map (Paper II). In paper IV, the protein was identified in multiple protein spots, all with increased abundance and was one out of only two proteins displaying a change in abundance at an early stage of acclimation as well as in cold acclimated leaves. In addition, two FKBP-type immunophilins, AtFKBP16-1 and AtFKBP18, decreased in abundance in cold developed leaves. This verified the predicted lumen location for the AtFKBP16-1 [88] that had not been experimentally identified in the chloroplast lumen before. The two FKBP-type immunophilins have not been characterised and their function is not clear. Nevertheless, the current understanding of the role of immunophilins as part of the signalling system in plants [146] (see section Immunophilins) suggests that these proteins may have such roles in the lumen during acclimation to abiotic stresses such as low temperature. Two additional proteins were found to increase in abundance in cold acclimated leaves. However, even though these proteins increased in abundance 30-fold and
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8-fold, respectively the amounts were too low for successful detection. Still this illustrates the need to investigate the lumen proteome during different physiological conditions both as a tool to discover additional lumen located proteins and to explore their functions. In conclusion, the lumen proteome was relatively insensitive to cold stress and only a small set of eight proteins of the estimated 100 proteins (8%) changed during the cold acclimation process. This may reflect that a major part of the lumen proteins are important for maintenance of functional thylakoids and need to be present in stable amounts even during changing environmental conditions.
Cold induced changes to the stroma proteome The stroma compartment is much better defined in terms of metabolic functions than the lumen compartment. In the stroma proteome, we identified 35 proteins with changed abundance involved in photosynthesis, other plastid metabolic functions, hormone biosynthesis, and stress & signalling transduction (Paper IV). Similar to the lumen proteome, no substantive changes were seen among the proteins in response to cold shock. In contrast to the lumen proteome, short-term acclimation resulted in changes of several stroma proteins that either increased or decreased in abundance, which was the case in cold acclimated leaves too. In Arabidopsis, the activities of several Calvin cycle enzymes have been shown to increase during cold acclimation [189, 190]. In paper IV, we observed a decrease in abundance for many Calvin cycle enzymes, which was analysed with respect to the stroma protein pool. This demonstrates the significance of assessing changes in protein abundance in the relevant cellular compartment, and supports the data showing that the enzyme activities per unit chlorophyll increased because of a general increase in leaf protein during cold acclimation [190]. We detected increased levels of proteins known to increase in abundance in response to oxidative stress and our results suggest that this response is maintained even during long-term acclimation. Proteins related to hormone biosynthesis were identified and suggests the involvement of jasmonic acid in response to low temperature, and the observed strong accumulation of a novel protein related to PII uridylyl-transferases may indicate this protein to be involved in sensing the metabolic status of the chloroplast in response to cold stress and acclimation.
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Summary •
A 2-dimensional protein map showed that the lumen proteome of Arabidopsis resolves into more than 300 protein spots revealing a high protein content within this chloroplast compartment. Thirty-eight proteins were experimentally identified demonstrating that the chloroplast lumen contains it own specific proteome, with a large group of immunophilins as a special feature. Comparison of the Arabidopsis chloroplast lumen proteome with the spinach lumen proteome showed good correlation and demonstrates that Arabidopsis can serve as a model for characterising the lumen proteins.
•
An in silico determination of the chloroplast lumen proteome from the Arabidopsis genome sequence data showed that the experimentally identified proteins are good representatives of the proteome. Combining the in silico proteome with the experimental proteome, the chloroplast lumen estimates to contain at least 80 different proteins.
•
The chloroplast lumen localised TL29 protein was shown to use the Tatpathway for routing across the thylakoid membrane. The protein accumulated upon high illumination, distributed mainly to the grana region of the thylakoids where it associated to the PSII-enriched membrane fraction with electrostatic forces. Functional analysis revealed that this putative ascorbate peroxidase is not a peroxidase but was able to bind ascorbate and may be involved in regulating the ascorbate concentration in the lumen compartment.
•
The lumen proteome was relatively insensitive to cold stress but important changes to the proteome were observed in the long-term developmental response to cold (cold acclimation). These included changes in abundance of the different isoforms of the extrinsic PSII subunits, the PSII assembly factor Hcf136 and immunophilins. In comparison, the stroma proteome responded at an earlier stage in the acclimation process. Changes to the stroma proteome involved proteins related to photosynthesis, other plastid metabolism, hormone biosynthesis, and stress & signal transduction.
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Perspectives The discovery of the lumen proteome over the past few years has broadened the horizon of the impact these proteins have in maintaining a functional chloroplast. This has undoubtedly raised numerous questions of the function of the individual proteins and their interaction partners. To understand the functionality of the lumen compartment a few points need clarification. Some are: •
Several of the lumen proteins contain structural domains known to be involved in protein-protein interactions. Studies of the oligomeric state of the lumen would be of interest. There are unfortunately technical problems because of dilution effects when isolating the lumen fraction that may interfere. This might not allow a straight forward analysis but valuable information may be given by combining different approaches such as: 1) separation of lumen fraction using native PAGE in combination with SDSPAGE, 2) use of antibodies either in immuno-affinity chromatography or immuno-precipitations to analyse co-purified or co-precipitated products, 3) use of recombinant lumen proteins to screen for interaction partners.
•
Several lumenal proteins are thioredoxin targets in vitro and may be so in vivo as well. Studies of the potential thioredoxin-signalling system in the lumen would be of interest to understand its role and possible cross-talk with the light regulated thioredoxin-signalling system in the stroma compartment.
•
Many proteins remain to be experimentally identified within the lumen. Applying different stress conditions as a tool may help to explore the lumen proteins further and identify putative regulatory proteins that can be further characterised.
•
The TL29 protein appears in various forms with different molecular weight and pI. There may be that different forms accumulate differently which can be assessed in the stress analysis (see above). The reason for this behaviour is not clarified. Detailed mass spectrometry analysis might help identifying possible post- translational modifications that are present in the various forms. The TL29 may be involved in regulating the ascorbate levels within the lumen. The TL29-knockout mutant does not show any visible phenotype when grown under standard growth room conditions. Studies of the mutant under different stress conditions may give information into the physiological relevance of this TL29 feature.
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•
The carboxy-terminal proteinase D1-like protein (At5g46390.2) contains some structural interesting motifs, which include a putative prenylation site and a putative ATP/GTP-binding site. This may point to an important regulatory role of the protein and it may be involved in intracellular signalling. Biochemical analysis of the protein as well as analysis at the plant level with knockout or anti-sense plants will help deciphering the function and importance of this interesting protein.
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Acknowledgements I spot land and my goal of this journey is getting close. It has been fascinating and many people have contributed to making this trip fun, exciting, easy but also sometimes endurable. With your help, my fellow travellers, the completion of this thesis was possible. I would like to THANK YOU all, and especially: Professor Wolfgang Schröder, my supervisor, for always believing in me, for encouragement and support, and for utmost freedom to carry out my projects. I thank you also for your sincere understanding that combining work and family is not always easy. Many thanks to you and your family for letting me stay in your home during my visits to Umeå. Dr Thomas Kieselbach, for introducing me to the thylakoid lumen, teaching me about MS, for providing valuable comments to the thesis manuscript and for always taking the time you never had to answer all my questions. Thank you also for all cakes during our coffee breaks and for popping in at SH with small Easter and Christmas treats when the group was spread out at different places. It really counts. The WSC-group and roommates at SH, Ulrika and Ellinor. I greatly thank you for being good friends, for your support with both scientific discussions and overall pep talks during the time in Moas båge when we were on our own. Thanks Ulrika for being my link to SH after I moved and for your encouragement during the completion of the thesis. Thanks to roommate Sofia for being friendly, supportive and providing a link to Gävle. Good luck to all of you! To passed and present members of the WSC-group. Erik thanks for discussions concerning 2-DE & MS, and for all the fun when we shared lab. José thanks for being fun, friendly and supportive within the TL29 project. LanXin, Iréne and Ulrika thanks for friendliness. Dmitri thanks for all help with fluorescence measurements. Marianna, so dear, it was a pleasure supervising you, thanks for nice teamwork, good luck with your graduate studies in your new group. Professor Lisbeth Jonsson, thank you for letting me to be part of your group, for taking interest into my project, for being a source of inspiration and for social gettogethers in your home. To all in the LJO-group, past and present: Jeanette, for being a great friend at work and private ever since we studied molecular biology together, your determination is outstanding. Gaby, thanks for sharing your great knowledge in molecular biology and for friendly chats. Thanks Kristina, Andrea, Terese, and all others that passed view in this group for making the lab a nice
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place to work in. Thanks to Karolina for taking interest into my work and the ASL-group, Annika, Alessia, and Lars for all being friendly and helpful lab-mates. A BIG thanks to all co-authors for fruitful collaboration, a special thanks to Dr Vaughan Hurry, for critically questioning my analysis of the data, I learned a lot writing paper IV with you. Thanks to my co-teachers for making teaching fun: Sissi, we worked hard to get the practical biochemistry course working, it was great and created a special bond between us. Ida, for interesting discussion on enzyme kinetics, fine collaboration and being a good friend. Anna, for friendliness and nice teamwork. You are all so dear. Thanks to Drs Gabriela Delp and Magnus Johansson for evaluating and discussing my project at my pre-dissertation seminar, Prof. Lisbeth Jonson, Prof. Anthony Wright and Dr Lotta Wikström for evaluating and discussing my project at the halftime control. Friends at School of Life Sciences, too numerous to mention them all, thanks for creating a nice environment, for friendly chats, for sharing equipment, protocols & scientific discussions and for organising pub-evenings and social lunches. Thanks to all administrative personnel, MarieG for all help with semesterdagar & föräldradagar, MarieBo for always trying to solve teaching schedules for the best, LottaG for all help with ordering and stuff, Elisabeth, Eilert, Lotta, Helena, Sam, Johan and everybody at the course-lab for making things function and School of LifeSciences a nice place to work in. Thanks to people at MedNut, Professor Jan-Åke Gustavsson for accepting me as a PhD-student, Lena, Marie & Monica for help and friendliness, Drs Johanna Zilliacus & Lotta Wickström for their time spent on guiding graduate students and Johanna for the examination. Mamma, som utan att blinka tagit tåget till Göteborg ett flertal gånger för att sköta markservicen när Niclas var bortrest och jag behövde få lite tid för arbete. Utan din hjälp så hade det inte gått att få avhandlingen klar i tid. TACK! Fortsätt gärna komma ofta nu när jag har tid att umgås också. Thanks to my sister and brothers and their families, for taking interest in my studies and for being great friends. I feel privileged in your company. A special thanks to Anette for trying hard to proofread the thesis manuscript. Thanks to my in-laws for caring and trying to understand my project.
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Thanks to all my friends outside work, no names, my thoughts are with you all.
”Månen bor i rymden. Den lyser upp mörkret så natten inte glömmer att det snart blir en ny dag.”* Niclas, tack för att du är min måne. Tack för allt. Johanna & Alice, mina solstrålar, tack för att ni ger mig perspektiv. Psst Johanna, sommaren är snart här - nu städar vi ur ditt och mitt arbetsrum och inreder något nytt. Lastly, many thanks to Södertörns högskola for financially supporting my studies, The Royal academy of Science for travelling stipend and The Swedish Research Council (VR) for financially supporting the project.
*Ann-Sofie, 8 år, Ur ”Gud som haver barnen kär har du någon ull” av Mark Levengood och Unni Lindell.
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