Sari Sirpiö

Yagut Allahverdiyeva

Marjaana Suorsa

Virpi Paakkarinen

Julia Vainonen

Natalia Battchikova

Eva-Mari Aro
email: evaaro@utu.fi

TLP18.3, A NOVEL THYLAKOID LUMEN PROTEIN REGULATING PHOTOSYSTEM II REPAIR CYCLE

Keywords: Arabidopsis thaliana, D1 protein, photosystem II repair, polysome-nascent chain complex, proteome analysis, thylakoid lumen Abbreviatios: BN, blue native, CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, chl, chlorophyll, cyt, cytochrome, 2-D, two-dimensional, DMBQ, 2, 6-dimethyl-p-benzoquinone, DUF, domain electrospray ionization, FNR, ferredoxin-NADP + reductase

A proteome analysis of Arabidopsis thaliana thylakoid-associated polysome-nascent chain complexes was performed to find novel proteins involved in the biogenesis, maintenance and turnover of thylakoid protein complexes, particularly the photosystem II (PSII) complex exhibiting a high turnover rate. Four unknown proteins were identified, of which TLP18.3 (thylakoid lumen protein of 18.3 kDa) was selected for further analysis. The Arabidopsis mutants (SALK_109618 and GABI-Kat 459D12) lacking the TLP18.3 protein showed higher susceptibility of PSII to photoinhibition. The increased susceptibility of ∆TLP18.3 plants to high light is likely to originate from an inefficient reassembly of PSII monomers into dimers in the grana stacks as well as from an impaired turnover of the D1 protein in stroma exposed thylakoids. Such dual function of the TLP18.3 protein is in accordance with it' s even distribution between the grana and stroma thylakoids. Notably, the lack of the TLP18.3 protein does not lead to a severe collapse of the PSII complexes, suggesting a redundance of proteins assisting these particular repair steps to assure functional PSII. The ∆TLP18.3 plants showed no clear visual phenotype under standard growth conditions but when challenged by fluctuating light during growth, the retarded growth of ∆TLP18.3 plants was evident.

INTRODUCTION

Thylakoid membrane-embedded multi-subunit protein complexes photosystem I (PSI), photosystem II (PSII), cytochrome b 6 f (cyt b 6 f) and ATP-synthase are responsible for light harvesting and solar energy transduction into chemical energy. Although the photosynthetic apparatus and light driven electron transport have been extensively studied, the biosynthesis and maintenance of these complexes have remained largely unknown. PSII performs dangerous chemistry of water oxidation and as a most oxidizing protein complex in nature, it exerts photodamaging effects on its own protein components in a process known as PSII photoinhibition [START_REF] Anderson | Unifying model for the photoinactivation of photosystem II in vivo under steady-state photosynthesis[END_REF][START_REF] Asada | The water-water cycle in chloroplasts: Scavenging of active oxygens and dissipation of excess photons[END_REF][START_REF] Baena-Gonzalez | Biogenesis, assembly and turnover of photosystem II units[END_REF][START_REF] Barber | Too much of a good thing: Light can be bad for photosynthesis[END_REF]. The light-induced photoinhibition of PSII results in an irreversible damage of the D1 protein, one of the heterodimeric polypeptides of the PSII core, and as a consequence the PSII core is subjected to a multistep repair cycle thereby maintaining the photosynthetic light reactions functional [START_REF] Baena-Gonzalez | Biogenesis, assembly and turnover of photosystem II units[END_REF][START_REF] Aro | Photoinhibition of photosystem II. inactivation, protein damage and turnover[END_REF].

Only a few proteins related to the regulatory network of the PSII complex have been characterized so far.

However, the accomplishment of the Arabidopsis genome sequencing project and multiple proteomic studies localizing unknown proteins to different chloroplast compartments [START_REF]Analysis of the genome sequence of the flowering plant arabidopsis thaliana[END_REF][START_REF] Friso | In-depth analysis of the thylakoid membrane proteome of arabidopsis thaliana chloroplasts: New proteins, new functions, and a plastid proteome database[END_REF][START_REF] Peltier | Central functions of the lumenal and peripheral thylakoid proteome of arabidopsis determined by experimentation and genome-wide prediction[END_REF][START_REF] Schubert | Proteome map of the chloroplast lumen of arabidopsis thaliana[END_REF] have established a basis for identification of novel proteins possibly associated with the dynamics of the PSII complex. One such protein, PsbS, has been known for a long time and is involved in energy-dependent quenching of excess light energy thus partially protecting the PSII complex against severe photoinhibition under fluctuating high light conditions [START_REF] Li | Molecular and global time-resolved analysis of a psbS gene dosage effect on pH-and xanthophyll cycle-dependent nonphotochemical quenching in photosystem II[END_REF]. Despite the mechanisms protecting the D1 protein from excess light, the light-induced damage and repair do constantly take place. These processes include (i) a reversible phosphorylation of the PSII core proteins at least partially catalysed by the Stn8 protein kinase [START_REF] Bonardi | Photosystem II core phosphorylation and photosynthetic acclimation require two different protein kinases[END_REF] while the phosphatases involved in the process still remain unknown, (ii) migration of the damaged PSII core from grana stacks to stromaexposed membranes, (iii) degradation of the D1 protein by the FtsH and DegP proteases [START_REF] Bailey | A critical role for the Var2 FtsH homologue of arabidopsis thaliana in the photosystem II repair cycle in vivo[END_REF][START_REF] Haussuhl | A chloroplast DegP2 protease performs the primary cleavage of the photodamaged D1 protein in plant photosystem II[END_REF], (iv) de novo synthesis of D1 protein and its co-translational insertion into PSII core in stroma-exposed membranes [START_REF] Zhang | Co-translational assembly of the D1 protein into photosystem II[END_REF] followed by (v) the re-assembly of functional PSII. Several proteins have been recognized that regulate the stability and translation of the psbA mRNA encoding the D1 protein, as well as the D1 processing protease CtpA [START_REF] Anbudurai | The ctpA gene encodes the C-terminal processing protease for the D1 protein of the photosystem II reaction center complex[END_REF][START_REF] Danon | Light regulated translational activators: Identification of chloroplast gene specific mRNA binding proteins[END_REF][START_REF] Somanchi | A nuclear gene of chlamydomonas reinhardtii, Tba1, encodes a putative oxidoreductase required for translation of the chloroplast psbA mRNA[END_REF][START_REF] Yohn | Translation of the chloroplast psbA mRNA requires the nuclear-encoded poly(A)-binding protein, RB47[END_REF]. On the contrary, only a few auxiliary proteins, like Psb29, Lpa1, HCF136 and Psb27 have been identified to be essential in the PSII assembly process [START_REF] Keren | Psb29, a conserved 22-kD protein, functions in the biogenesis of photosystem II complexes in synechocystis and arabidopsis[END_REF][START_REF] Peng | LOW PSII ACCUMULATION1 is involved in efficient assembly of photosystem II in arabidopsis thaliana[END_REF][START_REF] Plucken | The HCF136 protein is essential for assembly of the photosystem II reaction center in arabidopsis thaliana[END_REF][START_REF] Chen | A Psb27 homologue in arabidopsis thaliana is required for efficient repair of photodamaged photosystem II[END_REF].

To get further insights into the biogenesis, maintenance and turnover of the thylakoid protein complexes, we have taken a proteomic approach focusing on thylakoid-associated polysome-nascent chain complexes, referred hereafter as polysomes. Translation of most proteins encoded by the chloroplast genome takes place on ribosomes attached to the stroma-exposed membranes [START_REF] Falk | Rough thylakoids: Polysomes attached to chloroplast membranes[END_REF]. Of all thylakoid proteins the PSII core protein D1 has the highest turnover rate [START_REF] Mattoo | Regulation of protein metabolism: Coupling of photosynthetic electron transport to in vivo degradation of the rapidly metabolized 32-kilodalton protein of the chloroplast membranes[END_REF], and therefore the proteome analysis of polysomes is a promising approach in attempts to find novel proteins particularly involved in the PSII repair cycle. In this study 40 proteins of Arabidopsis polysomes were determined of which five possibly function as auxiliary proteins in the PSII repair. One of those proteins, At1g54780, enriched in the polysomes, was selected for further analysis. The At1g54780 protein, earlier unambiguously demonstrated to locate on the lumenal side of the thylakoid membrane [START_REF] Peltier | Central functions of the lumenal and peripheral thylakoid proteome of arabidopsis determined by experimentation and genome-wide prediction[END_REF][START_REF] Schubert | Proteome map of the chloroplast lumen of arabidopsis thaliana[END_REF] The Plastid Proteome Database], was named as TLP18.3 (thylakoid lumen protein of 18.3 kDa) and two Arabidopsis TLP18.3 T-DNA insertion mutant lines were subjected to detailed characterization.

EXPERIMENTAL Plant material and growth conditions

Arabidopsis thaliana ecotype Columbia wild type (WT) plants and homozygous TLP18. [START_REF] Baena-Gonzalez | Biogenesis, assembly and turnover of photosystem II units[END_REF] T-DNA insertion mutants on Columbia background (SALK_109618 and GABI-Kat 459D12) were used in the experiments [START_REF] Alonso | Genome-wide insertional mutagenesis of arabidopsis thaliana[END_REF][START_REF] Rosso | An arabidopsis thaliana T-DNA mutagenized population (GABI-kat) for flanking sequence tag-based reverse genetics[END_REF]. Information about insertion mutants was obtained from the Salk Institute Genomic Analysis Laboratory website at http://signal.salk.edu and from the GABI-Kat Arabidopsis thaliana T-DNA mutagenized population website at http://www.gabi-kat.de. Plants were analysed by PCR using primers specific to the flanking sequences either on the left (LP) or on the right (RP) side of At1g54780 gene together with a primer within the T-DNA insert (LB). The following primers were used (5' → 3 ' ) : L P 1 (CCATATTGACCATCATACTCATTGC) for analysis of GABI-kat 459D12 plants. Immunoblot analysis using a peptide antibody raised against the TLP18.3 protein was carried out to confirm that the TLP18.3 protein was lacking from the selected mutant plants.

Plants were grown in a growth chamber under a photon flux density of 150 µmol photons m -2 s -1 in 8 h light / 16 h dark regime at 23ºC (GL). Full-grown rosettes of 5-week old plants were used for experiments. All experiments (with the exception of the pulse labeling and fluorescence induction measurements in the presence of lincomycin) were conducted two hours after the lights were turned on. For specific experiments, intact plants or detached leaves were treated at 900 µmol photons m -2 s -1 of white light (HL) as indicated. In search for the visual phenotype for ∆TLP18.

Statistical analyses

The numerical data was subjected to statistical analysis by Student's T-test with statistical significance at the P-values < 0.001.

Isolation of polysomes

Polysomes were isolated from mature Arabidopsis leaves. The leaves were homogenized in 250 ml of the grinding buffer (50 mM Hepes-KOH pH 7.6, 330 mM sorbitol, 5 mM EDTA, 1 mM MgCl 2 , 5 mM ascorbate, 0.05% (w/v) BSA), filtrated through Miracloth and centrifuged at 1000g, 4°C for 1-2 min. The pellet was resuspended in the HSE buffer (50 mM Hepes-KOH pH 8.0, 330 mM sorbitol, 2 mM EDTA) and loaded on percoll step gradients (70%: 3.5 ml 100% percoll + 1.5 ml HSE, 40%: 6 ml 100% percoll + 9 ml HSE). The percoll step gradients were centrifuged at 4500g, 4°C for 5 min and the lower part of the gradient containing intact chloroplasts was collected and diluted with 2 -3 fold excess of the HSE buffer.

Chloroplasts were pelleted at 1300g, 4°C for 2 min, resuspended in the HSE buffer and after centrifugation at 1000g, 4°C for 1 min again resuspended in the HSE buffer. Intact chloroplasts (equivalent to 250 µg of chlorophyll (Chl)) were lysed by adding 1 ml of lysis buffer containing a mixture of protease inhibitors (50 mM Hepes-KOH pH 8.0, 5 mM MgOAc, 50 mM KOAc, 250 µg/µl chloramphenicol, 0.5 mg/ml heparin, 2 mM dithiothreitol, 100 µg/ml pefabloc, 2 µg/ml antipain, 2 µg/ml leupeptin) and thylakoids were collected at 3500g, 4°C for 1 min. The lysis step was repeated twice. Thylakoids were solubilized with 1% (w/v) dodecyl β-D-maltoside in the lysis buffer (0.5 mg chl/ml), loaded on the top of the 500 µl sucrose cushion (1M sucrose in the lysis buffer) and the polysomes were collected by centrifugation at 270000g, 4°C for 1 h. After centrifugation, the supernatant was removed carefully and the pellet, if not used immediately, was promptly frozen by liquid nitrogen and stored at -80 C.

In vivo pulse labeling of thylakoid proteins

Radioactive methionine was incorporated into detached leaves through petioles overnight (20 µCi ml -1 [ 35 S] Methionine and 0.4% (v/v) Tween 20). Subsequently, the pulse labeling was performed under HL at 23°C and the samples were collected after 15, 30 and 60 min. For chase experiments, the leaves were washed with unlabeled methionine (10 mM L-methionine, 0.4% (v/v) Tween 20) followed by 1 h incorporation of unlabeled methionine under darkness. Chase experiments were performed under HL at 23°C and the samples were collected after 1, 2 and 3 h. The rate constant for D1 degradation and the relative rates for D1 protein synthesis were calculated from the amount of radioactivity incorporated into D1 relative to that which was stably incorporated into CF1 (α-and βsubunits of the ATP-synthase) under HL and remained unchanged during the chase period. The relative synthesis rate of the D1 protein was determined from the shortest 15 min pulse samples since the use of 30 min and 60 min pulses appeared to be hampered by the degradation of the newly labeled D1 protein (rapid D1 turnover at HL).

Isolation and fractionation of thylakoid membranes

Total thylakoids were isolated as described in [START_REF] Rintamaki | Differential D1 dephosphorylation in functional and photodamaged photosystem II centers. dephosphorylation is a prerequisite for degradation of damaged D1[END_REF]. Chl was determined according to [START_REF] Porra | Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophyll a and b with four different solvents: Verification of the concentration of chlorophyll by atomic absorption spectroscopy[END_REF] and Chl per leaf area as described in [START_REF] Inskeep | Extinction coefficients of chlorophyll a and b in N,Ndimethylformamide and 80% acetone[END_REF]. For subfractionation, leaves were collected from GL conditions and after HL treatment of plants for 6 days, thylakoids were isolated, diluted in the suspension buffer (50 mM Hepes-NaOH pH 7.5, 10 mM MgCl 2 , 100 mM sorbitol) to a Chl concentration of 0.2 µg / µl and 0.2 % digitonin was added. The samples were vortexed for 4 s, centrifuged briefly and resuspended in the same buffer. After centrifugation at 1000g, 4°C for 3 min, the supernatant was collected and centrifuged at 29000g, 4°C for 30 min to precipitate the grana membranes. To collect the stroma-exposed membranes the supernatant was centrifuged at 185 000g, 4°C for 90 min.

2-D IEF

Polysomal proteins were separated by isoelectric focusing in the first dimension followed by SDS-PAGE into the second dimension. Protein concentrations were determined by Lowry assay (BioRad). Proteins (100 µg) were solubilized in the RB-buffer (8M urea, 2M thiourea, 4% (w/v) CHAPS, 100 mM dithiothreitol, 0.5% (v/v) biolytes pH 3-10) and then applied during 12 h active rehydration to a ReadyStrip™ IPG Strips pH 4-7 (Bio-Rad Laboratories, Inc.). The proteins were focused using a program: 150 V for 15 min, 300 V for 40 min, 500 V for 1h, 1000 V for 2h, followed 8000 V until 65000 V-h was reached. After focusing the strips were incubated in 2% (w/v) dithiothreitol in the EB buffer (6M urea, 0.375 M tris pH 8.8, 2% (w/v) SDS, 20 % (v/v) glycerol) at room temperature for 15 min followed by incubation in 2.5% (w/v) iodoacetamide in the EB buffer at room temperature for 15 min in darkness. Subsequently the strips were subjected of denaturing electrophoresis in the second dimension (14% SDS-PAGE) [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. After electrophoresis, the proteins were visualized by silver staining [START_REF] O'connell | Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption/ionization mass spectrometry of in situ enzymatic digests[END_REF].

Blue-native gels

Blue-native-PAGE (BN-PAGE) was performed as described in [START_REF] Rokka | Synthesis and assembly of thylakoid protein complexes: Multiple assembly steps of photosystem II[END_REF]. Thylakoid membranes were first resuspended in the 25BTH20G buffer (25 mM BisTris-HCl pH 7.0, 20% (w/v) glycerol and 0.25 mg ml -1 pefabloc) to a final Chl concentration of 0.5 mg ml -1 . An equal volume of 2% (w/v) dodecyl β-D-maltoside was added and the thylakoids were solubilized on ice for 3 min. Traces of unsolubilized material was removed by centrifugation at 18000g, 4°C for 20 min. Supernatant was supplemented with 1/10 volume of 100 mM BisTris-HCl pH 7.0, 0.5 M ε-amino-n-caproic acid, 30% (w/v) sucrose and 50 mg ml -1 Serva Blue G. Proteins were loaded on BN-PAGE (5-12.5% (v/v) acrylamide gradient) in amounts corresponding to 4 µg Chl per well. Electrophoresis (Hoefer Mighty Small, Amersham Pharmacia Biotech, Uppsala, Sweden) was performed at 0°C by gradually increasing the voltages as follows: 50 V for 30 min, 75 V for 30 min, 100

V for 1h, 125 V for 30 min, 150 V for 1h, 175 V for 30 min followed 200 V until the stain reached the end of the gel. After electrophoresis the lanes were excised and solubilized using the Laemmli buffer [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] containing 5% (v/v) -mercaptoethanol for 1 h at room temperature. The strips were run in the second dimension by SDS-PAGE (15% polyacrylamide, 6 M urea).

SDS-PAGE, immunoblotting and protein quantification

The polypeptides were separated with SDS-PAGE (15% polyacrylamide, 6 M urea) [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. After electrophoresis, the proteins were electroblotted to a PVDF membrane (Millipore, Watford, Herts, UK) and western blotting with chemiluminescence detection was performed according to standard procedures using protein specific antibodies or the antibody raised against the PSI complex. The TLP18.3 antibody was produced against synthetic peptides: DGQPDPGGPTVKDSK and RESNFKTKEETDEKR (Eurogentec, Seraing, Belgium). Lhcb1, Lhcb4 and PsbS antibodies were purchased from Agrisera (Vännäs, Sweden) and the antibody raised against the D1 protein was produced against oligopeptides as described earlier [START_REF] Zhang | Co-translational assembly of the D1 protein into photosystem II[END_REF]. The

In gel trypsin digestion and sample preparation for mass spectrometry

In gel trypsin digestion and sample preparation for mass spectrometry were performed as described in [START_REF] Rokka | Synthesis and assembly of thylakoid protein complexes: Multiple assembly steps of photosystem II[END_REF].

MALDI-TOF (matrix-assisted laser-desorption-ionization time-of-flight) analysis was performed in reflector mode on a Voyager-DE PRO mass spectrometer (Applied Biosystems). ESI (electrospray ionization) mass spectrometry was performed on a hybrid mass spectrometer API QSTAR Pulsar i (Applied Biosystems) equipped with a nanoelectrospray ion source (MDS Protana).

Photosynthetic activity measurements

PSII photochemical efficiency was determined as a ratio of variable fluorescence to maximal fluorescence (F V /F M ) measured from intact leaves with Hansatech Plant Efficiency Analyzer (King' s Lynn, UK) after 30min dark incubation. In experiments with inhibition of translation, 1mM lincomycin was incorporated into leaves through petioles overnight.

Non-photochemical quenching was determined using PAM-2000 Fluorometer (Walz, Effeltrich, Germany).

Leaves were adapted to darkness for 30 min before the measurements. Non-photochemical quenching was induced by exposing the leaves to actinic white light of 1000 µmol photons m -2 s -1 and was calculated as (F M -F M ' )/F M ' [START_REF] Bilger | Relationships among violaxanthin deepoxidation, thylakoid membrane conformation, and nonphotochemical chlorophyll fluorescence quenching in leaves of cotton (gossypium hirsutum L.)[END_REF].

Light-saturated rates of steady-state oxygen evolution from thylakoids were recorded using the Hansatech oxygen electrode fitted to the DW1 chamber (Hansatech, King' s Lynn, UK) in the presence of 1 mM DMBQ (2,6-dimethyl-p-benzoquinone) as an artificial electron acceptor.

Flash-induced increase and subsequent relaxation of Chl fluorescence yield were measured by the doublemodulation fluorometer (PSI Instruments, Brno) in the 150 µs to 100 s time range as described before [START_REF] Allahverdiyeva | The function of D1-H332 in photosystem II electron transport studied by thermoluminescence and chlorophyll fluorescence in site-directed mutants of synechocystis 6803[END_REF].

The redox change of P700 was monitored by absorbance at 810 minus 860 nm, using the PAM-Fluorometer PAM-101/102/103 (Walz, Germany) equipped with the ED-P700DW-E emittor-detector unit. P700 was oxidized by far-red light from a photodiode (FR-102, Walz, Germany) for 30 s, and the subsequent rereduction of P700 + in darkness was monitored.

RESULTS

Novel proteins associated with the polysomes

We have set up a proteomic system to search for novel thylakoid-associated regulatory proteins from the polysomes. Forty proteins were identified by mass spectrometry (Fig 1, Table I), most of which were subunits of the photosynthetic complexes or the translation apparatus, as expected. Subunits α, β, γ and ε of ATP synthase as well as NdhH protein of NDH-1 complex, CytF and Rieske FeS proteins of Cyt b 6 f complex, and PsbE, PsbO1 and PsbO2 subunits of PSII were observed. Light harvesting complex (LHC) proteins were also abundant: Lhca1, Lhca3, Lhca4 and Lhca5 proteins of LHCI, and Lhcb1, Lhcb3 and Lhcb5 proteins of LHCII complex were identified. Ferredoxin-NADP + reductase (FNR) and FNR-like protein were detected as two spots having equal molecular mass but different isoelectric point, possibly representing phosphorylated and dephosphorylated forms of the proteins. The detection of soluble FNR and FNR-like proteins corroborate with earlier studies showing that a pool of FNR proteins are associated with Cyt b 6 f [START_REF] Zhang | Ferredoxin:NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b6f complex[END_REF], NDH-1 [START_REF] Guedeney | Evidence for an association of ndh B, ndh J gene products and ferredoxin-NADP-reductase as components of a chloroplastic NAD(P)H dehydrogenase complex[END_REF] and PSI complexes [START_REF] Andersen | The PSI-E subunit of photosystem I binds ferredoxin:NADP+ oxidoreductase[END_REF]. Rubisco large subunit, being the most abundant stromal product of chloroplast protein synthesis, was not present as a contamination but most probably represented proteins under translation process [START_REF] Muhlbauer | The stromal protein large subunit of ribulose-1,5bisphosphate carboxylase is translated by membrane-bound ribosomes[END_REF]. For translation apparatus, S1, S5 and L4 ribosomal proteins as well as PSRP-2, representing a ribosomal regulatory protein, were found. In addition, the FtsH protease Var2 and four potential auxiliary proteins were identified: TLP18.3 and At1g15980 proteins found as one isoform and the PsbQ domain protein At1g14150 and At1g64770 protein, both present in two isoforms. Of these proteins TLP18.3 was selected for further analysis.

The TLP18.3 protein was found enriched in association with the polysomes whereas lower amount of TLP18.3 was found from the total thylakoid preparation (data not shown). To clarify whether the TLP18.3

protein is a tightly bound subunit of some thylakoid protein complex, the thylakoids from WT Arabidopsis were subjected to 2-D BN/SDS-PAGE analysis enabling the separation of the protein complexes according to their size. Immunoblot analysis of the 2D-BN/SDS-PAGE gel using antibodies raised against the D1, CP43 and TLP18.3 proteins showed that the TLP18.3 protein after BN-electrophoresis was not associated with any thylakoid protein complex but instead migrated in the gel as a free protein (Fig 2A). The migration of the protein was highly dependent on the composition of the SDS-PAGE gel: if the separation of the polypeptides was carried out using a traditional SDS-PAGE gel without urea, the TLP18.

Localization of the TLP18.3 protein

In order to localize the lumenal TLP18.3 protein more precisely, WT thylakoids isolated from growth conditions were fractionated into the stroma lamellae and grana membranes by digitonin. The purity of the fractions was evaluated using the PSII core protein CP43, antenna protein Lhcb4 and the PSI core proteins PSIA/B as controls. As expected, the PSI core proteins were observed exclusively in the stroma-exposed

Characterization of ∆TLP18.3 plants from normal growth conditions

Arabidopsis plants lacking the TLP18.3 protein did not reveal any visible phenotype under standard growth conditions (data not shown). Chl a/b ratio, as well as the total amount of Chl per leaf area were similar in WT and mutant plants (Table IIA). Activity of the PSI complex, monitored in vivo as redox changes of P700, the primary electron donor of the PSI complex, did not reveal any significant differences between WT and mutant plants (data not shown). However, the oxygen evolution activity of PSII was slightly higher in the mutant thylakoids compared to WT (Table IIA). To investigate whether the lack of the TLP18. To test whether the distribution of the PSI and PSII complexes between grana and stroma membranes is affected by TLP18.3, an immunoblot analysis was performed from WT and GABI-Kat 459D12 thylakoids isolated from GL and HL, and subfractionated into grana and stroma membranes. As shown in Figure 4B, the amount and distribution of the representative proteins of the PSII and PSI complexes were unaltered under both conditions.

Susceptibility of ∆TLP18.3 plants to short-term high light illumination

We next investigated the impact of the TLP18. IIIA). The apparent synthesis rate for D1 protein was calculated from the relative amount of radioactivity incorporated into the D1 protein during the 15 min pulse experiment. Like D1 degradation, also the relative synthesis rate of the D1 protein was lower in mutant plants compared to WT, although the difference in synthesis was less pronounced than in the degradation (Table IIIB). These results would indicate that the TLP18.3 protein had a direct role in the turnover of the D1 protein on the stroma-exposed thylakoids.

Heterogeneity of the PSII complexes in WT and ∆TLP18.3 plants

To clarify the impact of the lack of the TLP18. IV). Moreover, while WT plants seemed to accelerate the PSII repair cycle under short-term HL treatment by increasing the proportion of the PSII monomers from 30 % under GL to 36% under HL, the relative amount of PSII monomers in the ∆TLP18.3 plants remained at 45 % despite of the increase in light intensity (Table IV). This suggests that the ∆TLP18.3 plants were unable to alter their capacity to repair PSII upon increasing light intensity. Increasing instability of PSII dimers was seen in WT plants after HL shift as evidenced by high standard deviations at HL, and was likely originating from general instability of the PSII complexes under photoinhibitory conditions. Such instability was evident to PSII dimers of the ∆TLP18.3

plants even under GL (Table IV).

Retarded growth of the ∆TLP18.3 plants under fluctuating light

Since the ∆TLP18.3 plants showed no visible phenotype when grown under normal growth conditions (150 µmol photons m -2 s -1 in 8 h light / 16 h dark regime) or at high light intensity (900 µmol photons m -2 s -1 in 8 h light / 16 h dark regime), we decided to challenge the light reactions by using fluctuating light during growth. Relatively low irradiances were given in the beginning and end of the light period whilst during midday the light intensity varied between 15 and 600 µmol photons m -2 s -1 (for exact programming of lamp, see Materials and Methods). Under these conditions the growth of the ∆TLP18.3 plants was compromised as compared to WT (Fig 6).

GABI-Kat 459D12 mutant plants accumulated a truncated form of TLP18.3 protein upon prolonged HL treatment

When studying the GABI-Kat 459D12 mutant thylakoids isolated after prolonged HL treatment of the plants, the antibody raised against the TLP18.3 protein recognized a protein of circa 2 kDa smaller than the I). The method presented is especially promising in search for novel proteins involved in the synthesis of PSII subunits exhibiting a high turnover rate [START_REF] Mattoo | Regulation of protein metabolism: Coupling of photosynthetic electron transport to in vivo degradation of the rapidly metabolized 32-kilodalton protein of the chloroplast membranes[END_REF].

Enrichment in the polysome fraction of auxiliary proteins that generally are present only in minor amounts in the chloroplast, was an additional benefit of using the polysomes for identification of novel regulatory proteins.

Thylakoid solubilization with dodecyl β-D-maltoside releases the ribosome-nascent chain complexes associated with fragments of the stroma thylakoids, and indeed, stroma-exposed thylakoid proteins were strongly represented among identified proteins. Ribosomal proteins, on the other hand, were underrepresented. The majority of ribosomal proteins are highly basic having an isoelectric point in the pH range 9

-10 [40] and therefore they were not separated by IPG strips selected for the search of novel auxiliary proteins in the pH range 4 to 7 (Fig 1). However, four ribosomal proteins were identified as well as one translation regulation protein, PSRP-2 (Table I). PSRP-2 contains two RNA-binding domains, thus suggesting a function in recruiting of stored chloroplast mRNAs for active protein synthesis [START_REF] Yamaguchi | Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit[END_REF].

Interestingly, one known auxiliary protein, FtsH protease Var2, involved in the degradation of PSII D1 protein [START_REF] Bailey | A critical role for the Var2 FtsH homologue of arabidopsis thaliana in the photosystem II repair cycle in vivo[END_REF] was found enriched in polysomes as well as four auxiliary protein candidates: At1g14150 (a PsbQ family protein), At1g64770, At1g15980 and TLP18.3. The TLP18.3 protein, which was in earlier studies shown to be located in the thylakoid lumen of Arabidopsis [START_REF] Peltier | Central functions of the lumenal and peripheral thylakoid proteome of arabidopsis determined by experimentation and genome-wide prediction[END_REF][START_REF] Schubert | Proteome map of the chloroplast lumen of arabidopsis thaliana[END_REF], was selected for further analysis.

Analysis of the sequence of the TLP18.3 protein did not reveal any known functional domains. However, it is interesting to note that the TMH domain appears to play a role in the functional association of TLP18.

TLP18.3 is a novel lumenal protein required for high light tolerance of PSII

Characterisation of the ∆TLP18.3 thylakoids from normal growth conditions revealed only small differences as compared to WT thylakoids. These include a higher amount of PSII reaction centers as evidenced by the higher amplitude of the flash-induced fluorescence yield and by the higher steady-state oxygen evolution rate (Table IIA small differences under growth conditions, they are clear symptoms of disturbances in the light tolerance of the PSII complex [START_REF] Hashimoto | Unassembled subunits of the photosynthetic oxygen-evolving complex present in the thylakoid lumen are long-lived and assembly-competent[END_REF]. Indeed, the physiological role of the TLP18.3 protein in protection against HL was revealed when plants were exposed to moderately high irradiances that enhance the PSII photoinhibitionrepair cycle. Observed higher susceptibility of the ∆TLP18.3 plants to HL can, in principle, originate from two sources; either the PSII complexes are more susceptible to light induced damage or the repair of the damaged PSII complexes is inefficient [START_REF] Adir | Photoinhibition -a historical perspective[END_REF]. To dissect between these two possibilities, the photoinhibition experiments were repeated in the presence of lincomycin to block the repair process. Now the enhanced susceptibility of ∆TLP18.3 plants to photoinhibition disappeared indicating that the TLP18.3 protein facilitates the repair of the PSII centers after photodamage (Fig 5B). The repair of photodamaged PSII complexes is a complex process and occurs in multiple steps including the monomerization and partial disassembly of the dimeric complex in grana stacks [START_REF] Aro | Photoinhibition of photosystem II. inactivation, protein damage and turnover[END_REF][START_REF] Hankamer | Structure and membrane organization of photosystem II in green plants[END_REF], migration of the PSII monomers to the stroma-exposed thylakoids [START_REF] Aro | Dynamics of photosystem II: A proteomic approach to thylakoid protein complexes[END_REF][START_REF] Van Wijk | Kinetic resolution of the incorporation of the D1 protein into photosystem II and localization of assembly intermediates in thylakoid membranes of spinach chloroplasts[END_REF] followed by proteolysis of the damaged D1 protein [START_REF] Aro | Photoinhibition of photosystem II. inactivation, protein damage and turnover[END_REF][START_REF] Haussuhl | A chloroplast DegP2 protease performs the primary cleavage of the photodamaged D1 protein in plant photosystem II[END_REF][START_REF] Lindahl | Identification, characterization, and molecular cloning of a homologue of the bacterial FtsH protease in chloroplasts of higher plants[END_REF]. After cotranslational insertion of the newly synthesized D1 protein into the membrane [START_REF] Zhang | Co-translational assembly of the D1 protein into photosystem II[END_REF], the D1 protein is posttranslationally processed, the PSII monomers are re-assembled [START_REF] Anbudurai | The ctpA gene encodes the C-terminal processing protease for the D1 protein of the photosystem II reaction center complex[END_REF][START_REF] Zhang | Biogenesis of the chloroplastencoded D1 protein: Regulation of translation elongation, insertion, and assembly into photosystem II[END_REF] and migrated back to the grana thylakoids where dimerisation takes place.

Functional role of the TLP18.3 protein in PSII repair

It is demanding to specify the role of the TLP18.3 protein in the repair of PSII due to the fact that the protein does not contain functionally known domains. It is also important to emphasize that the amount of the TLP18.3 protein is not regulated by light, but the content of the TLP18.3 protein remained constant in the thylakoid membrane regardless of the irradiance level (Fig 7 ), suggesting an intimate interaction with the PSII complex. The observed higher levels of PSII monomers in ∆TLP18.3 plants confirm that the monomerization of the PSII complex is not the limiting factor slowing down the repair process (Table IV).

Neither the migration of the PSII complexes from grana to stroma-exposed thylakoids seems to be a problem in the ∆TLP18.3 plants since similar amounts of PSII complexes were recorded in the stroma thylakoids of both the WT and ∆TLP18.3 plants (Fig 4B). This latter conclusion is further supported by the fact that also cyanobacteria, which completely lack the thylakoid heterogeneity, have a homolog of TLP18.3, the Sll1390 protein. Sll1390 was found in substoichiomertic concentrations from active oxygen-evolving PSII complexes purified from Synechocystis sp. PCC 6803 using His-tagged CP47, strongly suggesting a role for TLP18.3 as an auxiliary protein of PSII [START_REF] Kashino | Proteomic analysis of a highly active photosystem II preparation from the cyanobacterium synechocystis sp. PCC 6803 reveals the presence of novel polypeptides[END_REF]. TLP18.3 thus seems to belong to evolutionarily conserved auxiliary proteins facilitating the assembly and repair of PSII, together with the previously characterized proteins Psb27 (slr1645) and Psb29 (Sll1414), both also found in purified Synechocystis PSII preparations [START_REF] Keren | Psb29, a conserved 22-kD protein, functions in the biogenesis of photosystem II complexes in synechocystis and arabidopsis[END_REF][START_REF] Chen | A Psb27 homologue in arabidopsis thaliana is required for efficient repair of photodamaged photosystem II[END_REF][START_REF] Kashino | Proteomic analysis of a highly active photosystem II preparation from the cyanobacterium synechocystis sp. PCC 6803 reveals the presence of novel polypeptides[END_REF][START_REF] Nowaczyk | Psb27, a cyanobacterial lipoprotein, is involved in the repair cycle of photosystem II[END_REF].

Two steps in the repair process of PSII are clearly malfunctioning in the absence of the TLP18.3 protein, namely the turnover of the damaged D1 protein and the dimerization of the PSII complexes (Table III Indeed, it has been shown earlier that the assembly-competent OEC proteins can be stored into the thylakoid lumen for PSII repair [START_REF] Hashimoto | Unassembled subunits of the photosynthetic oxygen-evolving complex present in the thylakoid lumen are long-lived and assembly-competent[END_REF][START_REF] Ettinger | Physiologically active chloroplasts contain pools of unassembled extrinsic proteins of the photosynthetic oxygen-evolving enzyme complex in the thylakoid lumen[END_REF][START_REF] Hashimoto | Assembly of newly imported oxygen-evolving complex subunits in isolated chloroplasts: Sites of assembly and mechanism of binding[END_REF] and that the PsbP and PsbQ proteins are not bound to stromal PSII monomers but instead to the PSII dimers in the grana [START_REF] Rokka | Synthesis and assembly of thylakoid protein complexes: Multiple assembly steps of photosystem II[END_REF][START_REF] Hashimoto | Assembly of newly imported oxygen-evolving complex subunits in isolated chloroplasts: Sites of assembly and mechanism of binding[END_REF].

Taken together, our results provide evidence that the TLP18.3 protein is involved in regulation of both the degradation / synthesis steps of the damaged D1 protein of the PSII core in stroma-exposed membranes and

(directly or indirectly) in the assembly of PSII monomers into dimers in grana stacks. HL illumination of the mutant plants, however, demonstrated that the PSII repair cycle does not completely collapse in the absence of the TLP18.3 protein but rather the TLP18.3 protein represents an auxiliary protein that normally augments specific steps in the complicated processes of PSII repair. The beneficial role of TLP18.3 for optimal growth was particularly evident under fluctuating illumination where the growth phenotype of the ∆TLP18.3 plants was compromised. This presumably resulted from perturbations in the capacity of PSII repair to rapidly adjust to varying rates of PSII damage during variations in illumination intensity. The protein spots were identified with MALDI-TOF (identified proteins are listed in Table 1). It should be noted that there were no statistically significant differences between the two TLP18.3 mutant lines in their susceptibility to photoinhibition (data not shown). A. Short-term HL treatment.

FIGURES AND TABLES

Measurements were carried out using full-grown plants from GL (150 µmol photons m -2 s -1 ) 2h after the lights were turned on. Plants were then transferred to HL (900 µmol photons m -2 s -1 ) and samples were taken Coding system of Arabidopsis protein coding genes, RNA coding genes and pseudogenes in MIPS. 2 Mascot protein score is -10*Log(P), where P is the probability that the observed match is a random event.

Table II. Characteristics of the photosynthetic apparatus in ∆TLP18.3 plants (GABI-Kat 459D12, SALK_109618). Chlorophyll (Chl) a/b -ratio, total Chl/leaf area (µg Chl/cm 2 ) and oxygen evolution capacity (µmol O 2 /mg Chl h -1 ) of WT and ∆TLP18.3 plants. (A) Measurements from steady-state GL conditions (150 µmol photons m -2 s -1 ). (B) after 3h mild HL treatment (900 photons µmol photons m -2 s -1 ). The HL values are presented as percentage value derived from the corresponding GL value. The values are the means ± SD of n = 7 for both Chl measurements and n = 3 -6 for oxygen evolution measurements.

A.

Growth light

WT ∆TLP18.3 3

  CP43 and PsbO antibodies were kindly provided by Dr R. Barbato and the PsbP and PsbQ antibodies were kindly provided by Dr C. Spetea and Dr T. Hundal, respectively. [ 35 S] Methionine labeled proteins were detected on PVDF membrane by autoradiography (X-ray film). Quantification of different proteins was carried out by densitometry analysis of the films with Fluor Chem™ 8000 image analyzer (Alpha Innotech Corporation, San Leandro, CA, USA).

  3 protein migrated as a 18 kDa protein (no 37 in Fig 1) whereas when the peptides were separated by the SDS-PAGE containing 6 M urea, the protein migrated as a 25 kDa protein (Fig 2A).

  thylakoids and the PSII proteins in the grana thylakoids except for a small amount of PSII proteins in the stroma-exposed membrane representing the PSII complexes under repair (Fig 2B). Interestingly, the TLP18.3 protein was found more evenly distributed between the grana stacks and the stroma thylakoids indicating that the protein might have a function in both locations (Fig 2B). Screening of the ∆TLP18.3 T-DNA insertion mutants To address the function of the TLP18.3 protein, the T-DNA insertion mutants were obtained from SALK and GABI-Kat collections (SALK_109618; GABI-Kat 459D12). The SALK_109618 line plants had the T-DNA insert in the first intron of the At1g54780 gene (see Supplementary Fig 1A) and in the GABI-Kat line 459D12 the T-DNA insert was located in the beginning of the last exon of the At1g54780 gene, between predicted domain of unknown function 477 (DUF477) and a transmembrane helix (TMH) (see Supplementary Fig 1A,B). SALK_109618 and GABI-Kat 459D12 plants selected for experiments were homozygous according to PCR analysis (data not shown) and did not contain detectable amounts of the TLP18.3 protein in thylakoids (see Supplementary Fig 1C).

  [START_REF] Baena-Gonzalez | Biogenesis, assembly and turnover of photosystem II units[END_REF] protein has an influence on the acceptor and/or donor side of PSII, flash-induced increase and subsequent relaxation of fluorescence yield in the presence and absence of DCMU was performed. The results showed slightly slower relaxation kinetics in mutant thylakoids compared to WT in the absence of DCMU (Fig 3B), demonstrating modifications on the acceptor side of PSII complex. Interestingly, in the presence of DCMU, the overall fluorescence relaxation kinetics was identical in the WT and ∆TLP18.3 thylakoids. However, the total amplitude of fluorescence in the presence of DCMU, reflecting the amount of active PSII centers, was around 8% higher in ∆TLP18.3 thylakoids (Fig3A). No statistically significant differences were recorded in the induction of non-photochemical quenching between WT and ∆TLP18.3 plants (data not shown).To further analyse the impact of the lack of the 18.3 kDa protein, an immunoblot analysis of thylakoid proteins was performed. Notable differences to WT were observed only in the PSII oxygen evolving complex (OEC) PsbQ protein and to a lesser extent in the PsbP protein, both being up-regulated in the ∆TLP18.3 plants (Fig4A). The amounts of the PSI A/B proteins and the PSII core proteins D1 and CP43 as well as the PSII antenna proteins Lhcb1-6 (Lhcb2-6; data not shown) and the PsbS protein required for energy-dependent quenching seemed to be unaltered in the mutant plants compared to WT plants (Fig4A). It should be noted that the immunoblot analysis did not detect the minor increase in the quantity of the PSII complexes observed in the ∆TLP18.3 thylakoids with biophysical measurements.

  photoinhibition of the PSII complex even under a mild HL illumination.

  3 protein for the organization of the PSII complexes, the relative amounts of the PSII dimers (PSII supercomplexes & PSII core dimers), PSII monomers and PSII monomers without CP43 subunit in the WT and ∆TLP18.3 thylakoids were quantified by 2-D BN/SDS-PAGE combined with the immunoblot analysis of the D1 protein. Interestingly, under GL conditions the mutant plants had less of the PSII dimers and more of the PSII monomers, possibly under repair, than WT plants (Table

TLP18. 3 (

 3 Fig 7). This protein accumulated during HL treatment, in particular during the subsequent recovery at GL while the content of the TLP18.[START_REF] Baena-Gonzalez | Biogenesis, assembly and turnover of photosystem II units[END_REF] protein in WT remained unchanged, irrespective of the light condition (Fig 7). Interestingly, mass spectrometric identification revealed the protein as TLP18.3 (see Supplementary Fig 1D, E). Sequencing of the GABI-Kat 459D12 At1g54780 gene Biochemical Journal Immediate Publication. Published on 19 Jun 2007 as manuscript BJ20070460 (data not shown) confirmed that the T-DNA was inserted into the gene after base pair 1,841 of the unspliced At1g54780 gene corresponding to amino acid 259 of the protein sequence. This led to the translation of a truncated TLP18.3 protein (termed TLP18.3*) where the last 26 amino acids from the C-terminus of the native protein were replaced with residues ITYIS (see Supplementary Fig 1D). Using protein domain prediction programs (TMHMM, SignalP, InterPro) the predicted TLP18.3* protein comprised the chloroplast-targeting signal, the DUF477 domain but not the carboxyl-terminal TMH sequence (see Supplementary Fig 1A,B). The absence of TLP18.3* in thylakoids isolated from the mutant plants grown under normal growth conditions is consistent with the requirement of the TMH sequence for the binding of TLP18.3 (albeit loosely, Fig 2A) to thylakoid membranes. However, after exposure to prolonged HL increasing amounts of TLP18.3* peptides were isolated with the thylakoids from the GABI-Kat 459D12 mutant. The basis for this curious accumulation of TLP18.3* following HL treatment remains to be resolved.DISCUSSIONProteome search for auxiliary PSII proteinsWe have addressed the regulatory network related to PSII function and found potential novel photosynthetic regulatory proteins from thylakoids of Arabidopsis thaliana. During the past few years hundreds of Arabidopsis chloroplast proteins have been localized into the inner envelope membrane, thylakoid membrane and thylakoid lumen with prediction programs and different proteomic approaches giving further insights into the function of unknown proteins[START_REF] Friso | In-depth analysis of the thylakoid membrane proteome of arabidopsis thaliana chloroplasts: New proteins, new functions, and a plastid proteome database[END_REF][START_REF] Peltier | Central functions of the lumenal and peripheral thylakoid proteome of arabidopsis determined by experimentation and genome-wide prediction[END_REF][START_REF] Schubert | Proteome map of the chloroplast lumen of arabidopsis thaliana[END_REF][START_REF] Ferro | Integral membrane proteins of the chloroplast envelope: Identification and subcellular localization of new transporters[END_REF]. In this study, the isoelectric focusing/SDS-PAGE gel system linked to mass spectrometric identification of proteins was used as a method to search unknown auxiliary proteins from polysomes likely to contain proteins involved directly or indirectly in the process of translation and assembly of chloroplast-encoded proteins (Fig 1, Table

3 with

 3 thylakoid membranes. As expected the GABI-Kat 459D12 mutant plants showed no trace of the TLP18.3 protein but did accumulate the C-terminally truncated TLP18.3* variant during prolonged HL treatment and the subsequent recovery period (Fig7). According to our hypothesis the full-length TLP18.3 protein is loosely bound to the thylakoid membrane by the TMH, possibly in interaction with an unknown subunit of the PSII complex (see below) and thus the TLP18.3 protein is protected from degradation. This conclusion is based on two facts, firstly the TLP18.3 protein must be bound to the membrane because it was detected in the polysomal preparation, which does not contain thylakoid lumenal proteins, and secondly the linkage of TLP18.3 to PSII is loose because in the BN analysis the TLP18.3 protein was observed as a free protein (Fig 1, Fig 2A). Unlike the TLP18.3 protein, the truncated TLP18.3* protein produced by the GABI-Kat 459D12 plants is not bound to the membrane and is rapidly degraded under normal growth conditions (see Supplementary Fig 1C), but becomes more stabilized during prolonged HL stress possibly due to the inactivation of the protease (Fig 7).

  , Fig 3A), accompanied by an upregulation of the PsbP and PsbQ proteins (Fig 4A). Despite only

  ) and the enrichment of both the TLP18.3 protein and the D1 degrading protease FtsH in the same fraction further support the co-operating of these two proteins (Fig1). Turnover of the photodamaged D1 protein in stromaexposed thylakoid membranes precedes the dimerization of the PSII complex, and thus it would be logical to assign the function for the TLP18.3 protein solely in degradation and synthesis steps of D1 in stromaexposed membranes. Nevertheless, the TLP18.3 protein was quite evenly distributed in both the grana stacks and stroma lamellae suggesting a function also in the appressed membranes (Fig2B). The increased amount of PSII monomers in TLP18.3 thylakoids as compared to WT thylakoids (TableIV) must reside in grana membranes since the content of PSII centers was similar in grana and stroma thylakoids between WT and TLP18.3 (Fig 4B). Moreover, under growth conditions these complexes were clearly active (Fig 3A)indicating that they do not represent damaged PSII monomers but active ones, apparently just after repair and migration back to the grana. Notably, the ∆TLP18.3 plants also exhibited increased amounts of the OEC proteins PsbP and PsbQ, which reflects a dysfunction of the dimerisation of the PSII complex (Fig4A).

Fig 1 .

 1 Fig 1. Silver-stained gel after 2-D isoelectric focusing/SDS-PAGE separation of proteins from Arabidopsis thylakoid associated polysomes. The polysomes were isolated from WT Arabidopsis plants grown under standard conditions, proteins were separated by 2-D electrophoresis with denaturing isoelectric focusing on immobilized pH gradient between 4 -7 in the first dimension and with SDS-PAGE in the second dimension.

Fig 2 .Fig 3 .

 23 Fig 2. Localization of the TLP18.3 protein in Arabidopsis thylakoids. A. Thylakoid membranes isolated from WT plants were subjected to 2-D BN/SDS-PAGE, proteins were transferred to a PVDF membrane followed by the analysis with antibodies against D1, CP43 and TLP18.3. B. Thylakoid membranes were fractionated into stroma (ST) and grana (GT) thylakoids by digitonin and proteins were separated by SDS-PAGE, followed by transfer to PVDF membrane and analysis with antibodies against TLP18.3, CP43, Lhcb4 and PsaA/B.

Fig 4 .Fig 5 .

 45 Fig 4. Immunoblots of the thylakoid membrane proteins in WT and ∆TLP18.3 (GABI-Kat 459D12) mutant.A. Thylakoid membranes isolated from GL conditions. 0.25 µg of Chl was loaded, except for the detection of PsbS (0.5 µg), PsbR (0.5 µg), PsbP (0.5 µg) and PsbQ (2 µg) in order to maintain the amount of the protein in the linear range for the antibody used. A representative blot from three independent experiments is shown. B. Thylakoid membranes isolated from GL and HL conditions and fractionated into stroma and grana thylakoids by digitonin. 0.25 µg of Chl was loaded in order to maintain the amount of the protein in the linear range for the antibody used.

Fig 6 .

 6 Fig 6. Mutant phenotype. The phenotype of WT, SALK_109618 and GABI-Kat 459D12 plants grown under fluctuating light conditions (see Materials and Methods).

Fig 7 .

 7 Fig 7. Truncated form of the TLP18.3 protein (TLP18.3*) is expressed in GABI-Kat 459D12 plants. WT and ∆TLP18.3 (GABI-Kat 459D12) plants growing under GL (150 µmol photons m -2 s -1 ) were transferred to HL(900 photons µmol photons m -2 s -1 ) for 5h, 1d, 3d and 6d. Thereafter plants were transferred back to GL (Rec) for 5h and 1d. Thylakoids were isolated, polypeptides separated by SDS-PAGE (1 µg of Chl per well) and an immunoblot analysis using the antibody raised against the TLP18.3 protein was carried out.

Fig 1 Biochemical

 1 Fig 1

Fig 2 Biochemical

 2 Fig 2

Fig 3 Biochemical 29 Fig 4 Fig 5 Fig 6

 329456 Fig 3

  3 plants, the plants were grown in 12 hour dark / light rhythm and fluctuating light was supplied from OSRAM Power Star HQIT metal halide lamps. Light intensity was

	adjusted with 1h steps as follows: 15, 250, 350, 600, 250, 350, 600, 15, 600, 350, 250, 15 µmol photons m -2 s -
	1 by switching on and off the lamps.

Biochemical Journal Immediate Publication. Published on 19 Jun 2007 as manuscript BJ20070460

Biochemical Journal Immediate Publication. Published on 19 Jun 2007 as manuscript BJ20070460

Table IV ,

 IV , SupplementaryFig 2). Slower D1 degradation was measured from ∆TLP18.3 plants (TableIIIA

Table I .

 I Arabidopsis proteins identified from polysomes.

	Spot	Protein						AGI 1	MW	Peptides matched /	Cover	Score 2
	no.								(kDa)	total	(%)
	1	ATP synthase alpha subunit (AtpA)			ArthCp007	55,3	17/21	39	302
	2	ATP synthase beta subunit (AtpB)			ArthCp029	54,0	21/26	69	359
	3	ATP synthase gamma chain (AtpC1)			At4g04640 33,5	11/15	38	161
	4	ATP synthase epsilon chain (AtpE)			ArthCp028	14,5	4/6	38	83
	5	ATP	synthase	delta	(OSCP)	chain,	putative	At4g09650 25,6	6/8	29	113
		H+ -transporting ATPase
	6	PS I type III chlorophyll a/b binding protein (lhca3)		At1g61520 29,1	6/10	27	91
	7	PSI LHC protein Lhca5				At1g45474 27,8p 4/7	19	85
	8	PSI LHC protein Lhca4				At3g47470 27,7p 4/5	23	90
	9	PSI LHC protein Lhca1				At3g54890 26,1	6/11	18	78
	10	PSI LHC protein Lhca1				At3g54890 26,1	8/11	25	109
	11	PS I type III chlorophyll a/b-binding protein (Lhca3)		At1g61520 29,1	8/14	28	110
	12	Lhcb1 protein					At2g34430	28,2	8/15	44	107
								At2g34420
	13	Lhcb1 protein					At2g34430	28,2	8/12	44	124
								At2g34420
	14	Lhcb3 protein					At5g54270 28,7	7/9	43	102
	15	Lhcb1 protein					At1g29920	28,2	8/13	63	125
								At1g29910
								At1g29930
	16	Lhcb1 protein					At1g29920	28,2	7/13	47	98
								At1g29910
								At1g29930
	17	CP26 antenna protein of PS II (Lhcb5)			At4g10340 30,2	10/15	41	131
	18	33 kDa oxygen-evolving enhancer protein (PsbO1)		At5g66570 35,3	12/15	43	221
	19	33 kDa oxygen-evolving enhancer protein (PsbO2)		At3g50820 35,2	10/14	44	169
	20	Cytochrome B559 alpha chain (PsbE)			ArthCp039	9,3	5/7	28	85
	21	Cytochrome F (PetA)					ArthCp035	35,5	8/12	38	155
	22	Rieske FeS protein (PetC)				At4g03280 22,8	5/6	29	115
	23	NADH dehydrogenase, subunit 7			ArthCp080	45,6	16/19	55	252
	24	30 S Ribosomal protein S1				At5g29771 45,3	15/18	35	223
	25	30 S ribosomal protein S5				At2g33800 32,7	6/11	18	65
	26	30 S ribosomal protein S5				At2g33800 32,7	10/19	26	89
	27	50 S ribosomal protein L4				At1g07320 30,1	10/18	41	137
	28	RNA recognition motif (RRM)-containing protein	At3g52150 27,7	10/16	18	95
		(PSRP-2)
	29	Rubisco large subunit (RbcL)				ArthCp030	48,0	13/19	31	182
	30	Ferredoxin-NADP+ reductase (FNR)			At5g66190 40,6	10/15	33	119
	31	Ferredoxin-NADP+ reductase (FNR)			At5g66190 40,6	17/22	42	204
	32	Similar to Ferredoxin-NADP+ reductase (FNR-like)		At1g20020 39,1	15/19	37	214
	33	Similar to Ferredoxin-NADP+ reductase (FNR-like)		At1g20020 39,1	13/20	25	137
	34	FtsH protease (Var2)					At2g30950 74,3	14/19	29	169
	35	PsbQ domain protein family				At1g14150 22,2	4/8	21	64
	36	PsbQ domain protein family				At1g14150 22,2	5/7	31	113
	37	Thylakoid lumen 18.3 kDa protein (TLP18.3)		At1g54780 31.1	7/12	17	115
	38	Expressed protein At1g15980				At1g15980 51,3	11/14	29	184
	39	Expressed unknown protein At1g64770			At1g64770 38,3	7/11	24	123
	40	Expressed unknown protein At1g64770			At1g64770 38,3	5/7	16	79
	1

Biochemical Journal Immediate Publication. Published on 19 Jun 2007 as manuscript BJ20070460

	Chl a/b	3.2 ± 0.02	.1 ± 0.09 (GABI-Kat)
			3.1 ± 0.06 (SALK)
	Chl/leaf area	22.3 ± 1.2	21.1 ± 0.7 (GABI-Kat) 22.5 ± 0.7 (SALK)
	O 2 evolution	426 ± 5	474 ± 3 (GABI-Kat) 486 ± 5 (SALK)
	B.	High light
		WT	∆TLP18.3
	O 2 evolution	94% ± 2%	80% ± 2% (GABI-Kat) 69% ± 2% (SALK)

Table III .

 III Comparison of the rate constants (k PI ) for D1 protein degradation and the relative synthesis rates of the D1 protein in the WT and ∆TLP18.3 thylakoids. Detached leaves from WT and ∆TLP18.3 plants were pulse labeled under HL (900 µmol photons m -2 s -1 ) with [ 35 S] methionine for 15, 30 and 60 min followed by 1, 2 and 3 h chase in the presence of unlabeled methionine (see Supplementary Fig 2). A. k PI and half-times (T ½ , min) for the D1 protein degradation were calculated from data fitted to first-order reaction kinetics (r = k[A]). Data shown are means ± SD, n = 3 for WT and GABI-Kat 459D12 mutant and n = 1 for SALK_109618 mutant. B. Relative D1 protein synthesis rates were determined by comparing the amount of radioactivity incorporated into the D1 protein normalized to that incorporated into ATP synthase -/subunits in WT and ∆TLP18.3 thylakoids during radioactive pulse of 15 min at HL. Data shown are means ± SD, n = 3.

	A.	k PI × 10 3 , min -1 T ½ , min
	WT	8.1 ± 1.1	87 ± 13
	GABI-Kat 459D12	4.4 ± 1.1	163 ± 42
	SALK_109618	5.0	138
	B.	Relative synthesis rate
	WT	1.00 ± 0.00
	GABI-Kat 459D12	0.69 ± 0.15
	SALK_109618	0.69 ± 0.05

Table IV .

 IV Quantification of the PSII complexes separated by 2-D BN/SDS-PAGE (WT and GABI-Kat 459D12 thylakoids). Relative amounts of the PSII dimers (PSII supercomplexes & PSII core dimers), PSII monomers and PSII monomers without CP43 subunit were defined using immunoblotting with D1 antibody and results from three independent experiments were analyzed. Thylakoids were isolated from steady-state GL conditions (150 µmol photons m -2 s -1 ) and after 3h mild HL treatment (900 photons µmol photons m -2 s -1 )

	of plants.
	GL	WT	∆TLP18.3
	PSII dimers	70% ± 0.7 54% ± 3.5
	PSII monomers	25% ± 2.1 36% ± 3.5
	PSII monomers w/o CP43 5% ± 2.8 10% ± 0.1
	HL	WT	∆TLP18.3
	PSII dimers	64% ± 7.8 56% ± 7.9
	PSII monomers	26% ± 4.0 30% ± 5.1
	PSII monomers w/o CP43 10% ± 4.4 14% ± 2.9

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 19 Jun 2007 as manuscript BJ20070460 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

This work was financially supported by the Academy of Finland, Biological Interactions Garduate School, Jenny and Antti Wihuri Foundation and Kone Foundation. The Proteomics and Mass Spectrometry Unit in the Turku Center for Biotechnology is thanked for technical advices in protein analysis. We thank the Salk Institute Genomic Analysis Laboratory and GABI-Kat for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. Funding for the SIGnAL indexed insertion mutant collection was provided by the National Science Foundation and GABI-Kat by the German Plant Genomics Program of BMBF.


Ülo Langel

Samir El-Andaloussi

Peter Järver

Henrik J Johansson

Cargo dependent cytotoxicity and delivery efficacy of cellpenetrating peptides-a comparative study

Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study

Introduction

The ability to cross the lipid bilayer of cells and access the cell interior is still one of the major obstacles to overcome in order to progress current drug development. Therefore, various techniques have been developed in order to improve cellular uptake of bioactive agents [START_REF] Patil | DNA-based therapeutics and DNA delivery systems: a comprehensive review[END_REF].

Cell-penetrating peptides (CPPs) have, since their discovery in 1994 [START_REF] Derossi | The third helix of the Antennapedia homeodomain translocates through biological membranes[END_REF], been widely used to deliver a wide range of bioactive compounds across cellular membranes of several cell types both in vivo and in vitro, reviewed in [START_REF] El-Andaloussi | Cell-penetrating peptides: mechanisms and applications[END_REF][START_REF] Järver | The use of cell-penetrating peptides as a tool for gene regulation[END_REF]. Delivery seems to be independent of cell type and can be directed to diverse compartments inside the cell [START_REF] Langel | Cell-penetrating peptides: Processes and Applications[END_REF]. No proper definition of CPPs has been formulated, but most, if not all, carry a net positive charge, are less than 30 amino acids long and have the ability to rapidly translocate large molecules into cells. Still it is unclear what mechanism is responsible for uptake of CPPs and their cargos. Originally it was believed that CPPs translocated cell membranes in a receptor-and energy independent manner. However, recent studies suggest that the uptake for most CPPs is an energy dependent process with initial binding of peptides to proteoglycans on the cell surface and concomitant endocytosis of peptides [6][START_REF] Nakase | Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement[END_REF][START_REF] El-Andaloussi | Induction of splice correction by cell-penetrating peptide nucleic acids[END_REF]. However, there are still reports claiming that membrane translocation is independent of endosome formation and that uptake is, in line with early reports, occurring directly through the outer cellular membrane [START_REF] Simeoni | Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells[END_REF][START_REF] Deshayes | On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids[END_REF].

There is a constantly growing number of CPPs introduced in the literature with different chemical properties and abilities to ferry various cargos across cellular membranes.

However, studies on CPP-mediated cargo delivery performed so far have focused on achieving high delivery yields, and few studies have focused on the toxicity that these peptides might exhibit. Lindsay et al analyzed the toxicity of CPP mediated peptide delivery in 2005 [11], and Saar et al studied the toxicity of free CPP uptake 2005 [START_REF] Saar | Cell-penetrating peptides: a comparative membrane toxicity study[END_REF]. Yet, the results are divergent regarding uptake efficacy and toxicity of single CPPs and different cargo molecules, making comparisons between one study and another very difficult. Further, different cells, cell passages, incubation times, concentrations et cetera, have been used, making comparisons between studies all the more complicated. Since no comprehensive study has been performed to evaluate the relative efficacy of single CPPs to deliver different cargos, and whether the cargo type might influence the toxicity of peptides, a study using the same conditions would offer a more accurate comparison concerning these aspects.

Here we aim to evaluate the delivery efficiency and cytotoxicity of three well characterized CPPs, Tat, transportan 10 (TP10), and penetratin [START_REF] Derossi | The third helix of the Antennapedia homeodomain translocates through biological membranes[END_REF][START_REF] Vivés | A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus[END_REF][START_REF] Soomets | Deletion analogues of transportan[END_REF] (Table 1), using different cargos. These cargo molecules include carboxyfluorescein that is used on a routine basis to assess the cellular uptake of peptides, double stranded DNA (dsDNA) that can serve as model for decoy oligonucleotides or possibly short interfering RNAs (siRNAs), and two model proteins streptavidin and avidin. Proteins have been transported into cells by CPPs and shown to be biologicaly activity [START_REF] El-Andaloussi | Cell-penetrating peptides: mechanisms and applications[END_REF][START_REF] Järver | The use of cell-penetrating peptides as a tool for gene regulation[END_REF], and in this study streptavidin and avidin were utilized as model proteins as they have been widely studied and have very high affinity for biotinylated peptides, making conjugation less cumbersome. In addition, since different proteins have different isoelectric points (pI), choosing these two proteins as models is appropriate as they have significantly different pI (streptavidin 5.5 and avidin 10.5). The study does not consider the biological activity of the delivered cargos (see [START_REF] Snyder | Cell penetrating peptides in drug delivery[END_REF] for a thorough review) but rather the cargo dependent toxicity that the CPP translocation exhibit.

We show here that the cellular uptake of CPPs is cargo dependent, and our results also imply that the cytotoxicity of these peptides is cargo dependent. Furthermore, we show that the cytotoxicity and internalization level of TP10 varies significantly depending on cargo coupling position within the peptide. Taken together, we believe that these results could be useful in future experiments utilizing CPPs to facilitate the choice of peptide depending on cargo and choice of conjugation strategy.

Experimental Procedures

Peptide synthesis and purification

The peptides (Table 1) were synthesized in a stepwise manner in a 0.1 mmol scale on an automated peptide synthesizer (Applied Biosystems, Model 433A) using t-Boc solid-phase peptide synthesis strategy. tert-Butyloxycarbonyl amino acids were coupled as hydroxybenzotriazole (HOBt) esters to a p-methylbenzylhydrylamine resin (amino acids and resin purchased from Neosystem, Strasbourg, France) to obtain C-terminally amidated peptides. Deprotection of the formyl protecting group on tryptophan was carried out in 20% piperidine in DMF for 1 h. Peptides used for uptake studies were labeled N-terminally and TP10 was also labeled orthogonally on Lys 7 with carboxyfluorescein using 5 eq. 5,6carboxyflorescein, 5 eq, 1,3-diisopropylcarbodiimide, 5 eq. HoBt and 20 eq. N,Ndiisopropylethylamine dissolved in dimethyl sulfoxide/dimethylformamide 1:1 over night [START_REF] Fischer | Extending the applicability of carboxyfluorescein in solid-phase synthesis[END_REF]. Biotin (Sigma, Sweden) was coupled as hydroxybenzotiazole ester to the N-terminus of tat and penetratin or to the ε-amino group of lys 7 of TP10.

The peptide was finally cleaved from the resin using liquid HF at 0 °C for 1 h in the presence of p-cresol (1:1). Peptides were purified using reversed phase HPLC on a C18 column, 20-100% Acetonitrile (0.1% TFA) gradient, and the molecular weight was determined by MALDI-TOF mass spectrometry using Perkin Elmer prOTOF™ 2000 MALDI O-TOF Mass Spectrometer (Perkin Elmer, Sweden). The peptide purity was >90% as determined by analytical HPLC.

Cell culture

HeLa cells were grown in Dulbecco's Modified Eagle's Media (DMEM) with glutamax supplemented with 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10 % FBS, 100 U/ml penicillin, and, 100 mg/ml streptomycin. CHO cells were grown in minimal essential media (MEM) with glutamax supplemented with 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10 % FBS, 100 U/ml penicillin, and, 100 mg/ml streptomycin.

Cells were grown at 37 °C in 5 % CO 2 atmosphere. All media and chemicals were purchased from Invitrogen (Sweden).

Quantitative cellular uptake 200.000 HeLa or CHO cells were seeded 24 h prior to experiments in 12-well plates to reach 70 % confluency. In the uptake studies on free peptide, cells were treated with 1 or 5 µM fluoresceinyl labeled peptides in 500 µl serum free media. In transfection studies of dsDNA, peptides in various concentrations were co-incubated for 30 min in 50 µl 0.9 % NaCl solution. Cells were then treated with 0.2 µM fluoresceinyl labeled dsDNA and CPPs in 500 µl serum free media. In the protein transduction experiments, streptavidin or avidin were either complexed with 1 µM biotinylated peptides in serum free media media or co-incubated with free peptides at various molar ratios in 0.9% NaCl solution to a final volume of 50 µl.

Cells were then treated with 0.2 µM protein and CPPs in 500 µl serum free media. One hour after treatment or 90 min after treatment with proteins, cells were washed twice in Hepes Krebb Ringer (HKR) buffer before trypsination. The trypsination step is crucial in order to remove membrane associated peptides and/or cargo [START_REF] Holm | Studying the uptake of cell-penetrating peptides[END_REF]. Cells were centrifuged at 1000g for 5 min at 4 °C and cell pellets were lysed with 300 µl 0.1 M NaOH for 60 min after which 250 µl lysate was transferred to a black 96-well plate. Fluorescence was measured at 494/518 nm on a Spectra Max Gemeni XS fluorometer (Molecular devices, USA) and recalculated to the amount of internalized compound and normalized to the amount of total protein (Lowry BioRad, USA). Fluoresceinyl labeled peptides, dsDNA, and proteins displayed linear correlation between concentration and fluorescence. DNA was purchased from CyberGene AB (Sweden) and proteins (streptavidin and avidin) from Invitrogen (Sweden). Single stranded DNA, fluoresceinyl labeled in the 3´end of the antisense strand, were hybridized to the sense strand in MQ water containing 0.9 % NaCl for 1h at 37 °C to achieve dsDNA with two nucleotides overhang at the 3´ end. The DNA sequences are presented in Table 1.

LDH leakage assay

Membrane integrity was measured using the Promega CytoTox-ONE TM assay (Promega, Sweden). In brief, 10 4 HeLa or CHO were seeded in 96-well plates two days before treatment with 100 µl of the above mentioned compounds at different concentrations in serum free media. After 30 min, 80 µl media was transferred to a black fluorescence plate and incubated for 10 min with 80 µl CytoTox-ONE TM reagent followed by 40 µl stop solution.

Fluorescence was measured at 560/590 nm. Untreated cells were defined as no leakage and 100% leakage was defined as total lactate dehydrogenase (LDH) release by lysating cells in 0.18% triton X100 in HKR buffer.

Wst-1 assay

HeLa or CHO cells were seeded onto 96-well plates, 10.000 cells/well, two days prior treatment. Cells were treated according to the same procedure as in the LDH-leakage assay but for 24 h. Cells were then exposed to wst-1 according to manufacturers protocol (Sigma, Sweden). Absorbance (450-690nm) was measured on absorbance reader Digiscan (Labvision, Sweden). Untreated cells were defined as 100% viable. Although the wst-1 assay measures dehydrogenase activity in mitochondria, it is generally distributed and used as a measure of proliferation [START_REF] Bykov | Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound[END_REF].

Results

TP10 induces membrane leakage and reduces proliferation dose-dependently

To get a general view of the toxic profile displayed by the selected CPPs, the cytotoxic properties of unlabeled peptides was assessed in HeLa and CHO cells. In order to get a comprehensive analysis of the cytotoxicity, both membrane disturbance and mitochondrial activity was studied. A LDH leakage assay was used to measure the acute membrane disturbance caused by the peptides, and a wst-1 assay was employed to study the more long term change in proliferation (i.e. mitochondrial dehydrogenase activity). As seen in Table 2, neither penetratin nor Tat affect proliferation at concentrations up to 50 µM. TP10 conversely displays long-term toxic side effects in HeLa and CHO cells at 20 µM concentration. Overall, CHO cells appear more resistant to peptide treatment than HeLa cells. Penetratin and Tat have no effect on membrane integrity (Fig. 1c, and data not shown) while TP10 induces LDH leakage (approximately 20 %) at 10 µM concentration in HeLa cells (Fig. 2b). The same was observed in CHO cells (data not shown).

Uptake of fluoresceinyl labeled CPPs: cargo attachment changes the cytotoxic properties of CPPs

As fluorescein labeled, the selected peptides have vastly different cell-penetrating properties. From Fig. 1, the following order of uptake yields was observed; TP10 > Penetratin >> Tat, where the uptake of Tat is nearly undetectable (Fig. 1a,b). The yield of internalized peptide is overall slightly higher in CHO cells (Fig. 1a,b). To see whether the fluorescein moiety alters the toxic properties of the peptides, the membrane integrity and proliferation was compared between free or fluoresceinyl labeled peptides. Penetratin shows no signs of toxicity, neither as a free peptide or when flouresceinylated in any of the two assays (data not shown). Tat, in contrast, displays slightly higher membrane leakage as free peptide (Fig. 1c) but, interestingly, proliferation is reduced significantly already at 20 µM concentration with the labeled peptide (Fig. 1d) but not as a free peptide. Since the fluoresceinyl moiety apparently influence the cell viability, it is highly possible that it also alters the uptake levels of some peptides. If this is true, many of the uptake studies performed on CPPs might only show the uptake of the fluorescein labeled peptide, which do not inevitably correlate with the uptake of the unlabeled CPP.

Internalization and cytotoxicity of TP10 is dependent on cargo coupling position within the peptide

On a routine basis TP10 has been orthogonally conjugated with cargo molecules.

Although orthogonally coupled TP10 has been shown to efficiently deliver various cargos [START_REF] El-Andaloussi | TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein[END_REF], we wanted to elucidate whether coupling carboxyfluorescein N-terminally to TP10 would change the uptake pattern and cytotoxic profile of the peptide. Surprisingly, internalization of N-terminally labeled TP10 is decreased four-fold compared to the orthogonally labeled peptide (Fig. 2a), suggesting that cargo coupling position is an important aspect to consider when designing conjugates, at least in case of TP10. This observation also further supports the hypothesis that uptake of free peptides might deviate significantly from that of fluoresceinyl labeled ones. Furthermore, orthogonally labeled TP10 displays significantly lower membrane perturbation and long term toxicity compared to free TP10 or TP10 labeled in the N-terminus (Fig. 2b,c). Additionally, N-terminally labeled peptide exhibit both higher membrane leakage and long term toxicity compared to unlabeled peptide (Fig. 2b,c). As seen in Fig. 3, both unlabeled and N-terminally labeled TP10 slightly alter the morphology and proliferation of HeLa cells treated with 10 µM peptide, while the orthogonally labeled peptide has negligible effect on cell morphology.

CPPs promote internalization of dsDNA in a relatively non-toxic fashion

As seen in Fig. 4a, all three peptides dose-dependently promote cellular internalization of dsDNA with the following efficiency order: TP10 > Penetratin ≥ Tat. This pattern is in agreement with the one observed for fluoresceinyl labeled peptides (Fig. 1a,b) with the exception for Tat, that seems more efficient in translocating dsDNA than when used as a free peptide (Fig 1a,b, Fig. 4a).

The same fluoresceinyl labeled dsDNA was further used in complex with increasing amounts of CPPs to examine the cytotoxicity of peptides in non-covalent complexes with dsDNA. Both penetratin and Tat are together with dsDNA nontoxic at concentrations up to 50 µM (Fig. 4b,c). There is a tendency that Tat in complex with dsDNA decreases the wst-1 activity at concentrations above 20 µM, although this decrease is not significant (Fig. 4c).

Intriguingly, it appears that the cargo decreases the toxic side effects of TP10, as no sign of toxicity was observed at 10 µM TP10 together with dsDNA (Fig. 4b,c). This is compared to free TP10, which displays approximately 20 % LDH leakage (Fig. 2b) and about 11 % decrease in wst-1 activity, at 10µM (Table 2).

Protein uptake varies depending on CPP and delivery strategy used

The selected CPPs were studied in two different protein delivery strategies with two dissimilar proteins (streptavidin and avidin). First, the peptides were co-incubated with the proteins and second, the peptides were conjugated to the proteins through a biotin linker.

When comparing data from co-incubation studies on streptavidin, the translocation efficacy is; Penetratin ≥ TP10 > Tat (Fig. 5a). No significant difference in uptake was observed with avidin with or without peptide co-incubation, most probably since avidin has a cationic nature that prohibit electrostatic interactions with positively charged CPPs (Fig. 5b). Again, penetratin and Tat show no toxicity displayed by LDH leakage (Fig. 5d). Also the toxic side effects of TP10 seem to decrease together with protein compared to free peptide, suggesting that peptide-protein interactions somewhat shield the cellular membrane from perturbation (Fig. 5d). However, when measuring the mitochondrial activity of cells 24 h after streptavidin treatment, we observed a strong reduction of approximately 75 % in cell viability, concluding that streptavidin is toxic if applied for longer periods of time (Fig. 5e).

Interestingly, when co-incubating streptavidin with increasing concentrations of TP10, the long-term toxicity clearly decreases, suggesting that the peptide might form stable complexes with the protein that remain unaltered inside cells and thereby shield the cytotoxic epitopes of the protein (Fig. 5e). This pattern was not observed for Tat or penetratin, indicating that these peptides form less stable complexes with streptavidin (data not shown).

The CPP efficacy pattern changes completely when using biotinylated CPPs that form nearly irreversible interactions with streptavidin. Then the observed uptake is as follows; TP10 ≥ Tat > Penetratin (Fig. 5c). The same pattern was observed with avidin but overall the level of uptake was higher, most probably since avidin is more cationic allowing interactions with negatively charged cell surfaces (Fig. 5c). Strikingly, Tat efficiently internalizes to cells when conjugated via biotin to proteins but is poorly internalized as fluorescein coupled peptide compared to TP10 and penetratin (Fig. 5c and

Discussion

In this study, we compared three of the major CPPs by means of their delivery yield and toxic side effects. We selected the two first discovered CPPs, penetratin and Tat, where the latter has been most extensively used, reviewed in [20]. Both peptides are highly cationic and they have a low amphipathic moment. The third peptide in this study, TP10, is longer, less cationic and more amphipathic than the other two. Common for all the peptides (Table 1) is their ability to promote cellular uptake of various cargos.

The most common way to determine the cell-penetrating efficiency of different CPPs has so far been through labeling with fluorophores, such as carboxyfluorescein. On a routine basis, peptides have been labeled N-terminally, with the exception of transportan and its analog TP10, that have been labeled orthogonally on Lys 13 or Lys 7 , respectively [START_REF] Pooga | Cell penetration by transportan[END_REF]. We wanted to see whether this labeling strategy could influence the cytotoxic properties of different CPPs which thereby also might influence the detected cellular uptake. Non-labeled penetratin showed no different in toxicity compared to fluorescein labeled. Tat on the other hand displays slightly higher membrane perturbation and significantly reduced proliferation The striking differences in uptake and toxicity observed between N-terminally and orthogonally labeled TP10 (Fig. 1) might be assigned to the hydrophobic property of fluorescein. When coupled N-terminally, it increases the already hydrophobic nature of the peptide tail resulting in increased membrane destabilization and elevated cytotoxicity. When coupled orthogonally, fluorescein is attached to the cationic part of the peptide, possibly reducing the amphipathicity of the peptide, and hence, the cytotoxicity. This is just speculation, but it has previously been shown that there might be a correlation between amphipathicity and toxicity of CPPs [START_REF] Saar | Cell-penetrating peptides: a comparative membrane toxicity study[END_REF]. This observation also further supports the hypothesis that uptake of free peptides might deviate significantly from that of fluoresceinyl labeled ones.

The routine use of carboxyfluorescein as a CPP cargo molecule provides a valuable tool for measurements of cellular uptake, however, it is not a biologically relevant cargo.

Therefore, to further investigate the potential of using CPPs to facilitate uptake of biologically relevant molecules, dsDNA was utilized as a model cargo for decoy-DNA or siRNA. All CPPs in this study can promote dsDNA uptake in a dose-dependent manner. Both penetratin and Tat are virtually nontoxic together with dsDNA at concentrations up to 50 µM. The presence of dsDNA seems to decrease the cytotoxic side effects caused by TP10, most probably due to electrostatic and hydrophobic interactions between the peptide and the DNA, which makes cellular membranes less exposed to the peptide.

Introducing proteins to cells offer a great therapeutic potential as many diseases are caused by deficient protein expression. The major obstacle in protein delivery to date is the poor bioavailability of these molecules. Unlike oligonucleotides, where several transfection reagents have been developed to facilitate the cellular uptake, few delivery vectors exist for proteins. CPPs have been utilized in numerous studies to convey bioactive proteins inside cells [START_REF] El-Andaloussi | Cell-penetrating peptides: mechanisms and applications[END_REF]. In most cases peptides have been recombinantly expressed from plasmids as a fusion with the protein, resulting in a conjugate of one peptide per protein [START_REF] Nagahara | Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration[END_REF][START_REF] Wadia | Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis[END_REF]. Some protein transduction experiments have been conducted with CPPs using the same co-incubation strategy as for oligonucleotides, resulting in non-covalent complexes of several peptides per protein [START_REF] Jain | Penetratin improves tumor retention of single-chain antibodies: a novel step toward optimization of radioimmunotherapy of solid tumors[END_REF][START_REF] Morris | A peptide carrier for the delivery of biologically active proteins into mammalian cells[END_REF]. Both these strategies have been successfully used to transport various proteins inside cells both in vitro and in vivo.

To get a comprehensive study of protein translocation by CPPs, two diverse methods to promote protein uptake were applied together with two proteins (streptavidin and avidin).

Streptavidin and avidin display, as mentioned in the introduction, similar and dissimilar properties making them suitable as models for whichever protein desired. Either, the CPPs were co-incubated with proteins, or biotinylated CPPs were pre-incubated with proteins to form stable CPP-protein complexes.

TP10 and penetratin was found to be the most potent vectors for protein delivery when co-incubated with the proteins (Fig 5a). This is not unexpected since they both have several hydrophobic residues that can contribute to protein complex formation. Tat on the other hand does not promote any protein uptake when utilizing co-incubation strategy. Since the Tat peptide is a highly cationic with few hydrophobic residues it might be less prone to form cell penetrating protein complexes. Tat is on the other hand a potent vector for protein uptake when conjugated to the protein through a stable linker. Then Tat is as efficient as TP10 to promote uptake of both avidin and streptavidin (Fig 5c). These results are in line with several other studies implying that uptake of fluoresceinylated Tat is nearly negligible [START_REF] Lundberg | Uptake Mechanisms of Cell-Penetrating Peptides Derived from the Alzheimer's Disease Associated Gamma-Secretase Complex[END_REF] while it is readily internalized when conjugated to a protein [START_REF] Wadia | Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis[END_REF]. One plausible explanation for this behavior could be that Tat utilizes different internalization routes depending on coupled cargo. Fluoresceinylated Tat might have the ability to pass directly through the plasma membrane as suggested recently in [START_REF] Tunnemann | Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells[END_REF], but the uptake is not high enough to be visualized. In contrast, when a large cargo has been attached to the peptide the uptake mechanism is changed to endocytosis, as suggested in [START_REF] El-Andaloussi | Induction of splice correction by cell-penetrating peptide nucleic acids[END_REF][START_REF] Wadia | Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis[END_REF]28]. Further, it seems that conjugating peptides to proteins is overall more effective than co-incubating peptides with proteins, at least when using above-mentioned proteins.

On the whole, TP10 presents itself as the most efficient CPP of the tested peptides to deliver different cargos. As with many other delivery vectors it seems to be a correlation between efficacy and toxicity at higher concentrations. Therefore, although TP10 is an effective transporter, it can not be administered to cells at as high concentrations as Tat or penetratin.

Penetratin displays virtually no membrane perturbation or long term toxicity up to 50 µM concentration, and the uptake yield is average compared to Tat and TP10 for all tested compounds. Tat coupled to fluorescein is as previously reported poorly taken up by tested cell lines compared to penetratin and TP10 [START_REF] Kramer | No entry for TAT(44-57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cellpenetrating peptides[END_REF]. However, when conjugated to protein, Tat translocation is increased dramatically. Consequently, our results emphasize that it is unfeasible to compare uptake of fluoresceinyl labeled CPPs and later use these results as a gauge for translocation efficacy for various cargos. Further, certain CPPs may be practical for use whith one cargo, but be insufficient to use as a vector for another. Cargos, such as dsDNA and proteins, seem to decrease both the acute and long term toxicity of CPPs. However, this is not true for all cargo molecules, since fluorescein moiety increases the toxicity of Tat (Fig 1d), and also TP10 displays completely different cytotoxic properties when conjugated to fluorescein (Fig 2b,c).

There are many impediments to overcome before CPPs can be used as efficient and safe pharmaceutical vectors. Obviously, the mechanism of entry must be resolved, but, additionally, it is important to use appropriate peptide vectors for desired cargos. We have shown that different peptides are suitable for different cargos and that the uptake yield of one cargo does not assure that the same CPP is effective in delivery for all bio-active molecules.

In addition we have also shown that the cytotoxicity of peptides is highly dependent on cargo used and cargo coupling position within the peptide. In conclusion, we believe that the results presented in this paper can serve as guidelines to select appropriate CPPs for specific cargos. TP10 reduces proliferation (i.e. mitochondrial activity) significantly at higher concentrations while Tat and penetratin have negligible effects on proliferation up to 50 µM concentrations. Overall, CHO cells are less sensitive to CPP treatment. 10 000 cells seeded in 96-well plates 48 h prior experiment were treated with the indicated peptide concentrations in 100 µl serum free media for 24 h and analyzed using the wst-1 assay.

Biochemical

  Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society Microscopy 200.000 HeLa cells/well were seeded one day prior to exposure with the above mentioned compounds in 12-well plates. Cells were treated for one hour and analyzed in an Olympus 1X70 microscope (with a DP50 camera) using the Wiewfinder Lite V1.0 software.Statistic analysesAll results represent the mean of at least three independent experiments done in quadruplicate (mean ±SEM, n=3) and statistics were calculated using Anova, Dunnett´s. **:p<0,01 *:p<0,05. Biochemical Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Fig. 1 a

 1 , b). Biochemical Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society as a labeled peptide already at 20 µM concentrations (Fig 2b). This difference shows the importance of measuring both acute membrane toxicity, as well as more long term toxic effects when studying CPPs, since the two does not necessarily correlate.

Biochemical

  Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1 .

 1 Figure 1. Both TP10 and penetratin are dose-dependently internalized to CHO (a) and HeLa

Figure 2 .

 2 Figure 2. TP10 labeled orthogonally with carboxyfluorescein displays several fold higher

Figure 3 .

 3 Figure 3. Apart from causing membrane perturbation and affecting mitochondrial activity,

Figure 4 .

 4 Figure 4. CPPs promote a dose-dependent internalization of fluoresceinyl labeled dsDNA. a)

Figure 5 .

 5 Figure 5. a) CPPs have the ability to deliver 60 kDa proteins in to HeLa cells either as non-

Fl 1 Fl 1 -: fluoresceinyl modification at αN 1 Fl 7 -: fluoresceinyl modification at εN 7 Biochemical

 11177 nucleobases in DNA. ¤ DNA was labelled at the 3´end with carboxyfluorescein. Fl is the abbreviation for fluoresceinyl. Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 1 . Sequences of cell-penetrating peptides and DNA.

 1

	Peptide	Sequence

Table 2 . Long-term toxic effects in HeLa and CHO cells after treatment with CPPs.

 2

		Tat				Pen			TP10
		HeLa		CHO		HeLa	CHO		HeLa	CHO
	µM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM
	1	96.5	4.5	97.0	1.5	91.3 6.8	112.2 3.8	94.3 7.6	90.4	3.1
	2	103.5 6.9	92.7	2.6	86.3 1.1	92.2	4.3	92.9 4.9	85.3	2.4
	5	94.8	8.1	87.5	3.6	89.7 6.8	92.7	2.0	85.1 5.9	87.1	3.1
	10 92.1	6.6	102.0 3.4	91.1 6.1	112.9 4.1	88.9 5.0	100.0 3.0
	20 100.2 6.2	93.5	4.8	86.2 4.5	127.7 5.6	49.7 1.4	76.2	2.3
	50 86.6	3.2	95.3	3.4	88.2 7.2	107.7 6.6	16.1 2.0	24.9	1.0

Biochemical Journal Immediate Publication. Published on 12 Jul 2007 as manuscript BJ20070507 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

The authors would like to thank the Swedish Research Council (VR-NT, VR-Med) for supporting this work.