Multiple Evidence for Nucleotide Metabolism in the Chloroplast Thylakoid Lumen

Multiple evidence for nucleotide metabolism in the chloroplast thylakoid lumen

Cornelia Spetea*†, Torill Hundal*, Bjo¨ rn Lundin*‡, Mounia Heddad§, Iwona Adamska§¶, and Bertil Andersson*§

*Division of Cell Biology and ‡Department of Physics and Measurement Technology, Linko¨ping University, SE-581 85 Linko¨ping, Sweden; and §Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden

Communicated by Olle E. Bjorkman, Carnegie Institution of Washington, Stanford, CA, December 9, 2003 (received for review June 12, 2003) The apparatus of photosynthetic energy conversion in chloroplasts activity without any conclusive results (12), and none of the is quite well characterized with respect to structure and function. proteins identified through proteomics have shown to have any Light-driven electron transport in the thylakoid membrane is conventional nucleotide-binding motifs (10). Furthermore, coupled to synthesis of ATP, used to drive energy-dependent transport of nucleotides across the thylakoid membrane has not metabolic processes in the stroma and the outer surface of the been considered, in contrast to mitochondrial membranes, thylakoid membrane. The role of the inner (luminal) compartment where an ATP͞ADP carrier (AAC) has been extensively studied of the thylakoids has, however, remained largely unknown al- (13, 14). though recent proteomic analyses have revealed the presence of Chloroplast metabolism has mainly been associated with ATP, up to 80 different proteins. Further, there are no reports concern- but more recent results also point to the physiological signifi- ing the presence of nucleotides in the thylakoid lumen. Here, we cance of GTP. Thus, requirement for stromal GTP has been bring three lines of experimental evidence for nucleotide-depen- reported for the integration of light-harvesting complex proteins dent processes in this chloroplast compartment. (i) The thylakoid into the thylakoid membrane (15) and the proteolytic removal of lumen contains a protein of 17.2 kDa, catalyzing the transfer of the the PSII reaction center D1 protein during repair of photoin- ␥-phosphate group from ATP to GDP, proposed to correspond to hibitory damage to this photosystem (16). the nucleoside diphosphate kinase III. (ii) The 33-kDa subunit of In this work, we provide several lines of evidence for the photosystem II, bound to the luminal side of the thylakoid mem- occurrence of nucleotides in the thylakoid lumen. Using a brane and associated with the water-splitting process, can bind combination of molecular biology, biochemical approaches, and GTP. (iii) The thylakoid membrane contains a nucleotide transport database searches, GTP synthesis by a luminal nucleoside system that is suggested to be associated with a 36.5-kDa nucle- diphosphate kinase (NDPK, EC 2.7.4.6.) could be demonstrated otide-binding protein. Our results imply, against current dogmas, along with the observation that the OEC33 protein associated that the thylakoid lumen contains nucleotides, thereby providing with the water-splitting reaction of PSII can bind GTP. In unexpected aspects on this chloroplast compartment from a met- addition, we provide evidence for a transthylakoid nucleotide abolic and regulatory perspective and expanding its functional transporter that is suggested to be associated with a 36.5-kDa signiﬁcance beyond a pure bioenergetic function. nucleotide-binding protein, which shows immunological crossreactivity with an antibody against the mitochondrial AAC he thylakoid membrane of chloroplasts is the site for the protein. photosynthetic electron transport coupled to ATP synthesis T Materials and Methods (1). This membrane is surrounded by the soluble stroma, which Plant Material. contains the enzymes involved in CO2 fixation, and it encloses Arabidopsis (A. thaliana L. cv. Columbia), pea the luminal space. In contrast to the other chloroplast compart- (Pisum sativum), and spinach (Spinacea oleracea) were grown ments, the thylakoid lumen has been considered to have a limited hydroponically (17). In this work, spinach was the main material; functional significance for the photosynthetic process and mainly however, where indicated, pea and Arabidopsis were used. Intact viewed as a sink for protons from a chemio-osmotic perspective. pea chloroplasts were isolated on a Percoll gradient (18). Intact Until recently the protein composition of the thylakoid lumen mitochondria from Arabidopsis leaves were purified as in ref. 19. was thought to be very simple and dominated by three extrinsic Thylakoid membranes, PSII membranes, and PSII core com- photosystem (PS) II proteins and plastocyanin (2). In the last few plexes were isolated as described (16). The preparations were years, however, many different categories of proteins have been finally resuspended in 50 mM Hepes-NaOH (pH 7.4), 400 mM found in the thylakoid lumen by applying biochemical and sucrose, 5 mM MgCl2, and 15 mM NaCl (buffer A), supple- ͞ proteomic approaches, pointing to several unexpected functions mented with 5 mM CaCl2 and 0.01% (wt vol) dodecyl maltoside for this chloroplast compartment. Thus, the thylakoid lumen has for the core particles. The OEC33 protein was isolated from PSII PLANT BIOLOGY been found to contain chaperones (3), immunophilins (4), membranes by washing with 50 mM Mes-NaOH (pH 6.0), 400 ⅐ carbonic anhydrases (5), violaxanthin deepoxidases (6), peroxi- mM sucrose, and 1.5 M NaCl followed by 20 mM Tris HCl (pH ϫ dases (7), and proteases (8). Furthermore, systematic mass 9.0) and 1 M KCl. After centrifugation at 40,000 g, the spectrometric analyses after two-dimensional electrophoretic supernatant was concentrated, and the buffer was exchanged for separation of luminal preparations combined with prediction of 50 mM Hepes-KOH (pH 7.4), 2 mM MgCl2,and5mMKCl transit peptides estimated the existence of Ϸ80 different thyla- (buffer B). Thylakoid lumen was isolated by mechanical disrup- koid luminal proteins of Arabidopsis thaliana (9, 10), out of which ture of thylakoids in 30 mM sodium phosphate (pH 7.4), 100 mM only half have been assigned a putative function. This considerably more complex view of the thylakoid lumen ͞ Ј raises several questions of mechanistic and physiological nature Abbreviations: AAC, ATP ADP carrier; 8-N3ATP, 8-azido-adenosine 5 -triphosphate; 8-N3GTP, 8-azido-guanosine 5Ј-triphosphate; Chl, chlorophyll; DCMU, 3-(3,4-dichloro- related to chloroplast function and regulation. One of these phenyl)-1,1-dimethylurea; NDPK, nucleoside diphosphate kinase; OEC33, 33-kDa subunit questions concerns the presence of nucleotides in such a poten- of photosystem II; PS, photosystem. tially multifunctional cellular compartment as particularly sug- †To whom correspondence should be addressed. E-mail: [email protected]. gested by indications for luminal chaperones (3, 9) and ATP ¶Present address: Department of Physiology and Plant Biochemistry, University of Kon- binding to luminal proteins (11). On the other hand, luminal stanz, DE-784 57 Konstanz, Germany. preparations have been tested for presence of ATP and ATPase © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308164100 PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1409–1414 Downloaded by guest on September 29, 2021 sucrose, 5 mM MgCl2, and 1 mM DTT (buffer C) as in ref. 12. Import of NDPKIII into Isolated Chloroplasts. The ndpkIII gene from Stroma was prepared by lysis of chloroplasts in 20 mM sodium- Arabidopsis was amplified by PCR using a CD4-13 ␭-ZipLox Ј phosphate (pH 7.4), 5 mM MgCl2, and 1 mM DTT followed by cDNA library. Specific PCR primers (5 -ATGAGCTCT- separation from the thylakoid and envelope membranes by CAAATCTGCAGATC-3Ј and 5Ј-TTAGTTGTCACCATA- centrifugation at 10,000 ϫ g and 140,000 ϫ g, respectively. GAGCC-3Ј) were designed based on the sequence present in the Subfractionation of thylakoids was performed by the digitonin EST cDNA database (tentative consensus, TC127858). The PCR method (20). Chlorophyll (Chl) concentration was measured cycling profile (30 cycles) was denaturation at 94°C for 1 min, according to Arnon (21), and protein concentration as described annealing at 60°C for 1 min, and extension at 72°C for 1 min. The in ref. 22. A correction factor of 0.895 should be applied to obtain amplified 717-bp fragment (including stop codon) correspond- Chl concentration values according to Porra (23). ing to the coding region of NDPKIII cDNA was ligated into a pCR2.1 vector by using the TA cloning kit (Invitrogen), and the Light Treatments. Thylakoids and PSII core complexes diluted to insert sequence was verified by sequencing (CyberGene, Hud- 0.3 mg of Chl per ml in buffer A were illuminated in an ELISA dinge, Sweden). For in vitro transcription, NDPKIII cDNA was plate (50 ␮l final volume), with visible light (1,500 ␮mol photons subcloned into a pGEM3Z plasmid (Promega) by using EcoRI mϪ2⅐sϪ1) by using a KL2500 lamp (Schott) for 30 min on ice. restriction sites. Plasmid pGEM3Z containing NDPKIII insert Bam Where indicated, 3-(3,4-dichlorophenyl)-1,1-dimethylurea was linearized with the restriction enzyme HI and used for in vitro transcription with T7-RNA polymerase as described (25). (DCMU) 50 ␮M was added. Ј In vitro translation was performed in a wheat germ lysate The radiolabeled nucleotides 8-azido-guanosine 5 -triphos- 35 ␣ 32 Ј (Promega) in the presence of [ S]methionine (Amersham Phar- phate ([ - P]8-N3GTP) and 8-azido-adenosine 5 -triphosphate ␥ 32 macia). In vitro import into isolated intact pea chloroplasts ([ - P]8-N3ATP) (ICN) were incubated with dark-control or was performed according to ref. 18. After import, chloroplasts preilluminated samples on ice for 3 min before photolabeling. ␮ were separated into stroma and membrane fractions and sub- The samples were exposed for 90 s to UV-C light (20 mol jected to SDS͞PAGE before exposure of gels to x-ray film or Ϫ2⅐ Ϫ1 photons m s ) supplied with a UVG-11 lamp (254 nm, phosphorimager (Molecular Dynamics) plates. Merck) at a distance of 3 cm. For nucleotide competition studies, the samples were preincubated with 10- to 20-fold higher con- Immunoprecipitation of Luminal NDPK and Antibody Inhibition of centrations of various nonlabeled nucleotides. After photolysis, Nucleotide Transport into Thylakoid Lumen. A rabbit antibody was the labeling was stopped by the addition of Laemmli sample raised against the GATFPQKSEPGTIR peptide of the Arabi- buffer (24). dopsis NDPKIII (Innovagen, Lund, Sweden). The thylakoid Isolated PSII core complexes (0.1 mg Chl͞ml) photolabeled lumen from Arabidopsis (150 ␮g of protein, 250-␮l volume) was ␮ ␣ 32 ⅐ with 50 M[ - P]8-N3GTP were incubated with 0.8 M Tris HCl incubated with the NDPKIII antibody (1:50 dilution) overnight (pH 8.5) for 30 min on ice. Released proteins were precipitated at 4°C. The bound proteins were precipitated with Protein G in 80% (vol͞vol) acetone and solubilized in Laemmli sample Sepharose Fast Flow (Amersham Pharmacia) for1hat22°C and buffer. analyzed by SDS͞PAGE. Isolated OEC33 protein (0.35 ␮g) was preincubated with Thylakoids (3 ␮g of Chl, 10-␮l final volume) were incubated increasing concentrations of GTP or ATP in buffer B (25 ␮l final with buffer A, an antibody raised against the beef heart mito- volume) and photolabeled with 10 ␮M[␣-32P]GTP (Amersham chondrial AAC or preimmune serum (0.1 mg of protein per ml) Pharmacia) for 5 min on ice. The protein was precipitated in 80% for1honiceindarkness, followed by centrifugation to remove acetone and solubilized in Laemmli sample buffer. unbound proteins. Thylakoids were resuspended in buffer A and For studies on nucleotide transport, preilluminated intact assayed for NDPK activity. thylakoids were photocrosslinked with 100 ␮M nonlabeled Protein Analysis. ͞ 8-N3ATP, and unbound nucleotides were washed away. The SDS PAGE and Western blotting were per- thylakoids were further incubated with [␥-32P]ATP (Amersham formed as described (16, 24), and radioactively labeled proteins Pharmacia) and GDP, and the production of GTP was assayed were detected by phosphorimaging. Antibodies were raised as described below. against the spinach OEC33, spinach NDPKII, Arabidopsis NDPKIII, and beef mitochondrial AAC protein. Assay of NDPK Activity. Ten microliters of reaction mixture con- ␮ ␥ 32 ␮ ϭ Database Searches. Homology search using BLAST was performed taining 0.2 Ci [ - P]ATP (1 Ci 37 kBq), 10 nmol ATP, 10 ͞ nmol GDP, and 2 ␮g of thylakoid luminal protein or 0.2 ␮gof in Swiss-Prot (www.expasy.org sprot) and TIGR (www.arabidopsis.org) databases. Prediction for transit peptides was per- stromal protein were incubated at 35°C in buffer C. The opti- formed by using the TARGETP and SIGNALP programs (www. mum pH for the NDPK activity was determined in 50 mM cbs.dtu.dk͞services). acetate-KOH (pH 5.0), Mes-KOH (pH 5.5–6.0), or Hepes-KOH (pH 7.0–7.5) containing 5 mM MgCl2 and1mMDTT.TheKm Results value for GDP was determined by measuring the formation of Detection and Characterization of NDPKIII Activity. ␥ 32 NDPK is a [ - P]GTP from increasing concentrations of GDP with 1 mM ubiquitous enzyme that catalyzes the transfer of the ␥-phosphate ␥ 32 [ - P]ATP. Similarly, the Km value for ADP was determined group of 5Ј-triphosphate nucleosides to 5Ј-diphosphate nucleo- ␥ 32 by assaying the formation of [ - P]ATP from increasing con- sides. In plants, several isoenzymes have been purified from ␥ 32 centrations of ADP with 1 mM [ - P]GTP (Amersham spinach cytosol (NDPKI) (26), spinach and pea chloroplasts Pharmacia). For studies on nucleotide transport, NDPK activity (NDPKII and -III) (26–28), and pea mitochondria (mtNDPK) was measured in thylakoid membranes in a reaction mixture (29). Among the chloroplast proteins, only NDPKII has been ␮ containing 3 g of Chl incubated at 22°C. In all cases, the precisely assigned to the stroma (27, 28). enzymatic reaction was stopped by the addition of 2 ␮lof4M To test for NDPK activity, a lumen preparation from spinach formic acid and 8 ␮l of ice-cold buffer B. Five microliters of the chloroplasts was incubated with [␥-32P]ATP in the presence or resulting mixture was applied to a poly(ethyleneimine)-cellulose absence of GDP followed by separation of the nucleotides by plate (Merck), and the reaction products were separated by TLC TLC. As shown in Fig. 1A, production of GTP could be detected with 0.75 M KH2PO4 (pH 3.65) as elution buffer, and detected in samples incubated in the presence of GDP. The NDPK activity by phosphorimaging. in the lumen was 600 ␮mol of GTP per mg protein per min as

1410 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308164100 Spetea et al. Downloaded by guest on September 29, 2021 Furthermore, Western blotting with an antibody against the stromal NDPKII was performed. A band detected in the stromal fraction had a molecular mass of 18.5 kDa, typical for NDPKII, whereas a band in the luminal fraction showed a distinctly lower molecular mass of 17.2 kDa, indicating the presence of NDPKIII in this compartment (Fig. 1B). Database searches for a homologue to the stromal NDPKII revealed the presence of a potential NDPKIII in the genome of Arabidopsis (At4g11010). The putative protein (Swissprot O49203) contains 238 amino acids and an N-terminal transit peptide (amino acids 1–85), with an intermediate cleavage site typical for proteins located in the thylakoid lumen (Fig. 1C). The calculated molecular mass for the precursor, intermediate, and mature forms are 25.7, 22.5, and 17.1 kDa, respectively. The mature NDPKIII shows 50% identity to NDPKII, explaining the immunological crossreactivity.

Fig. 1. Characterization of NDPK activity in isolated thylakoid lumen. (A) Cloning of the ndpkIII Gene. To exclude the possibility that the Thylakoid lumen (0.2 ␮g of protein per ␮l) was incubated with [␥-32P]ATP (20 NDPKIII band was the result of mitochondrial contamination, nCi͞␮l), 1 mM ATP, and 1 mM GDP in buffer C (pH 7.4) for the indicated periods intact chloroplasts and mitochondria from Arabidopsis were of time at 35°C. The nucleotides were separated by TLC followed by phosphor- isolated and tested for the presence of NDPKIII by using the imaging. (B) Western blot of stroma (S) and lumen (L) fractions with anti- heterologous NDPKII antibody. Consistent with the results NDPKII. (C) Deduced amino acid sequence of the NDPKIII precursor from presented in Fig. 1B, NDPKIII was detected only in chloroplasts Arabidopsis (SwissProt O49203). The predicted two-step cleavage sites for together with the 18.5-kDa band of NDPKII (Fig. 2A). targeting into chloroplast and thylakoid lumen are marked by black arrows. To clone the At4g11010 gene, a 717-bp cDNA sequence was amplified by PCR and encodes the protein with the deduced amino acid sequence presented in Fig. 1C. Using an in vitro compared with 6,600 ␮mol of GTP per mg protein per min in the transcription and translation assay, the radioactively labeled stroma, indicating a lower abundance of the enzyme in the precursor (pNDPKIII) and its processed form (NDPKIII) were lumen. The optimum pH for the luminal NDPK activity was 6.0 immunoprecipitated with the NDPKII antibody. Two radioac- whereas the pH dependence for the stromal enzyme was broad, tively labeled bands with molecular mass corresponding to with a high activity up to pH 8.0 (not shown), which is consistent pNDPKIII and NDPKIII could be detected by SDS͞PAGE with a location of the former enzyme in the more acidic lumen. (Fig. 2B). ␮ The calculated Km for GDP of the luminal NDPK was 27.4 M, Furthermore, in vitro import of radioactively labeled pNDP- which is lower than the previously reported values for other KIII into isolated intact chloroplasts was performed followed by NDPKs (26). When incubating isolated lumen with [␥-32P]GTP separation into stroma and thylakoid membrane fractions. The and ADP, ATP was produced in the reversible enzymatic results demonstrated that the labeled NDPKIII was present ␮ reaction. The calculated Km value for ADP was 89.05 M, which inside trypsin-treated chloroplasts (Fig. 2C). During the import is 4-fold higher than the Km for GDP, indicating a preference of assay, the 25-kDa pNDPKIII was processed to a 17-kDa NDP- the luminal NDPK for GDP. KIII, representing the mature form located in the thylakoid PLANT BIOLOGY

Fig. 2. Localization of NDPKIII in the thylakoid lumen of Arabidopsis.(A) Western blot with anti-NDPKII of isolated chloroplasts (C) and mitochondria (M). As references, the distribution of the major chlorophyll a͞b-binding protein of PSII (LhcbII) and the F1␤ subunit of the mitochondrial ATP-synthase complex (F1␤-ATP) are shown. (B) Immunoprecipitation of in vitro transcribed and translated NDPKIII (TL, translation products) in the presence (ϩ) and absence (Ϫ)of anti-NDPKII. (C) In vitro import of radioactively labeled NDPKIII translation products (TL) into isolated pea chloroplasts followed by separation into stroma (S) and thylakoid (T) fractions. pNDPKIII and NDPKIII, the precursor and processed forms of NDPKIII, respectively. (D) Arabidopsis thylakoid lumen was incubated with buffer C (Ϫ), preimmune (PI), or the NDPKIII antibody, and NDPK activity was measured in the corresponding unprecipitated fractions as in Fig. 1.

Spetea et al. PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1411 Downloaded by guest on September 29, 2021 Fig. 3. Photoaffinity labeling of thylakoids (T) or PSII core complexes (PSII) 32 32 with 25 ␮M[␣- P]8-N3GTP (A) and 5 ␮M[␥- P]8-N3ATP (B). Samples diluted to 0.3 mg͞ml Chl in buffer A were first dark-incubated (Ϫ) or preilluminated (ϩ) on ice for 30 min, followed by incubation with the photoprobe for 3 min in darkness. The samples were photolysed with UV-C light for 90 s on ice and subjected to SDS͞PAGE and phosphorimaging. (C) Identification of the GTP- binding protein as the OEC33. PSII core complexes were photolabeled with 50 32 ␮M[␣- P]8-N3GTP as described above followed by alkaline Tris-washing, centrifugation, and separation of proteins by SDS͞PAGE in the pellet (Pel.) and the supernatant (Sup.). The phosphorimage in the 33-kDa region and the corresponding Western blot with anti-OEC33 antibody are shown.

fraction. A possible processing intermediate of 22 kDa was also Fig. 4. Photolabeling of the OEC33 and the 36.5-kDa proteins. (A) PSII core detected in the thylakoid membrane, which is in accordance with complexes were preilluminated and photolysed with the indicated concen- 32 a two-step cleavage necessary for a translocation into the luminal trations of [␣- P]8-N3GTP followed by SDS͞PAGE and phosphorimaging (In- space. set). The 32P incorporation into the OEC33 band was quantiﬁed, and the At a later stage, a polyclonal antibody against Arabidopsis maximal value was set as 100%. (B) The photolabeling of the OEC33 protein ␮ ␣ 32 NDPKIII could be obtained and used in immunoprecipitation with 25 M[ - P]8-N3GTP was carried out in preilluminated PSII core complexes in the presence of 250 ␮M nonlabeled nucleotides. The photolabeling experiments to further verify the luminal location of this isoen- 32 of the 36.5-kDa protein with 7 ␮M[␥- P]8-N3ATP was carried out in preillu- zyme. As shown in Fig. 2D, the NDPK activity in the unprec- minated thylakoids in the presence of 200 ␮M nonlabeled nucleotides. Values ipitated fraction of the lumen was reduced to 40% whereas no represent the percentage of remaining photolabeling compared with the effect on the activity was observed by using the preimmune value obtained in the absence of nonlabeled nucleotides (n.d., not deter- serum. Western blotting with the NDPKIII antibody detected a mined). (C) Phosphorimage of SDS͞PAGE containing isolated OEC33 protein band of 17 kDa in the precipitated protein fraction (not shown). photolabeled with 10 ␮M[␣-32P]GTP in the presence of the indicated concentrations of nonlabeled GTP. Photoaffinity Labeling of Thylakoid Proteins. To identify potential GTP-binding proteins, dark-control and 30-min preilluminated ␣ 32 DCMU. Furthermore, the 36.5-kDa protein bound ATP to a thylakoids were incubated with [ - P]8-N3GTP, which, after UV-activation, binds covalently to polypeptides carrying nucle- much higher extent than GTP. Thylakoid subfractionation stud- otide-binding sites. Two major radiolabeled bands with molec- ies showed that the 36.5-kDa protein was mainly located in the stroma-exposed membranes. ular masses of 36.5 and 33 kDa could be detected (Fig. 3A, T). ␮ Although the labeling of the 36.5-kDa protein was present The photolabeling of OEC33 was saturated at 40–50 M ␣ 32 already in the dark-control and enhanced 4-fold by preillumi- [ - P]8-N3GTP in preilluminated PSII core complexes (Fig. ␮ nation, the 33-kDa band was detected only in preilluminated 4A). From Scatchard plots, an apparent Kd of 22.8 M and one samples. After photolabeling of isolated PSII core complexes GTP-binding site on the OEC33 protein were calculated. Fur- with [␣-32P]8-N GTP (Fig. 3A, PSII) the 33-kDa but not the thermore, based on the amount of Chl loaded per gel lane, 3 3 ͞ 36.5-kDa band was observed, and the labeling was 4-fold en- nmol GTP mg Chl was determined. Assuming that in PSII core hanced by preillumination. As compared with thylakoids, the complexes 1 mg of Chl corresponds to 5 nmol PSII, the obtained ͞ ͞ 33-kDa band was 10-fold stronger labeled in isolated PSII core value is 0.6 nmol GTP nmol PSII, i.e., 1 GTP 2 PSII. particles, suggesting that this protein is less accessible to GTP in To further define the specificity of the GTP binding to the intact membranes. Furthermore, the labeling of the 33-kDa OEC33 protein, competition experiments were performed in protein was inhibited by DCMU, suggesting a connection to preilluminated PSII core complexes photolabeled with 25 ␮M ␣ 32 photosynthetic electron transport. The photolabeled 33-kDa [ - P]8-N3GTP (Fig. 4B). Under our experimental conditions, protein was released by alkaline Tris-washing and crossreacted the nonspecific binding was 31%, as determined from photola- with the antibody against the luminal OEC33 protein (Fig. 3C) beling in the presence of 2.5 mM GTP. This value was sub- (30). Furthermore, sequence analysis of the Coomassie-stained stracted from the percentage remaining labeling in the presence 33-kDa band demonstrated that the first seven amino acids, of various competitors (Fig. 4B). Both GTP and GDP reduced EGGKRLT, correspond to the amino terminus of the OEC33 the level of radioactive 8-N3GTP photoinsertion into the OEC33 protein. protein to Ϸ20% whereas GMP, ATP, and CTP did not compete ␥ 32 When photolabeling experiments with [ - P]8-N3ATP were with this binding. performed, only the 36.5-kDa protein was detected. This labeling Notably, isolated OEC33 protein can also bind GTP. To was 3- to 4-fold stimulated by light (Fig. 3B) but not affected by determine its binding constant for GTP, photolabeling experi-

1412 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308164100 Spetea et al. Downloaded by guest on September 29, 2021 ments were performed with [␣-32P]GTP by using isolated OEC33 protein in the presence of increasing concentrations of nonlabeled GTP (Fig. 4C). The calculated value from logarith- ␮ mic plots for Kd was 8.1 M, which is significantly lower than the one determined for the interaction between 8-N3GTP and the OEC33 in situ. Increasing concentrations of nonlabeled ATP could also prevent photolabeling of isolated OEC33 protein but ␮ with an apparent Kd of 179.28 M, demonstrating a higher binding specificity to GTP than to ATP. Trypsination of the photolabeled pure OEC33 protein indicated a location of the GTP-binding site on the N-terminal part of the protein. ␥ 32 To investigate the specificity of [ - P]8-N3ATP binding to the 36.5-kDa protein, saturation and competition studies were carried out in thylakoid membranes. The calculated Kd values for the interaction revealed the existence of two types of binding sites. In preilluminated thylakoids, the binding of ATP was much ϭ ␮ tighter (Kd 7.7 M) than in the samples kept in the dark (Kd ϭ 225 ␮M). On the basis of the amount of Chl loaded per gel lane, we determined that membranes corresponding to 1 mg of Chl can bind 4.2 pmol 8-N3ATP with high affinity, indicating a 600-fold lower abundance of the 36.5-kDa protein as compared Fig. 5. Characterization of nucleotide transport across thylakoid membrane. with the PSII complex. Competition experiments in preillumi- (A) Thylakoids (0.3 ␮g͞␮l Chl) were incubated with [␥-32P]ATP (20 nCi͞␮l), nated thylakoids indicated that the 36.5-kDa protein can bind 1 mM ATP, and 1 mM GDP in buffer A (pH 7.4) for the indicated periods of time various nucleotides with different affinities in vitro, with ATP at 25°C, and the nucleotides were separated and detected as described in Fig. and ADP as the preferred ones (Fig. 4B). 1. After 45 min, one sample was centrifuged, and the pellet (Pel.) and supernatant (Sup.) were processed separately. In a competition experiment, preilluminated thylakoids were ﬁrst photolabeled with 100 ␮M 8-N3ATP, unbound Nucleotide Transport Across the Thylakoid Membrane. The evidence nucleotides were washed away, and the formation of [␥-32P]GTP was mea- for nucleotide metabolism and GTP—protein interactions in the sured as described above. The numbers indicate the percentage of remaining thylakoid lumen suggested the existence of a system for transt- labeling as compared with the [␥-32P]GTP produced in the 45-min dark- hylakoid nucleotide transport. sample. (B) Western blot with an antibody against the beef mitochondrial To test whether externally added nucleotides could be trans- AAC protein of thylakoids (T), chloroplast envelope (E), or beef heart mito- ported across the thylakoid membrane, we took advantage of the chondria (M). The bands detected at 28 and 36.5 kDa correspond to the luminal NDPKIII activity. [␥-32P]ATP was added to isolated mitochondrial AAC protein and the thylakoid nucleotide-binding protein, intact thylakoids and incubated in darkness or light. After this respectively, whereas the lower minor bands are potential degradation products. (C) Thylakoids were preincubated with buffer A (Ϫ), preimmune (PI), or incubation, the thylakoids were washed and reisolated, and the AAC antibody followed by measurement of [␥-32P]GTP formation in the lumen ␥ 32 lumen was analyzed for the presence of [ - P]GTP. As shown as described in A. in Fig. 5A, GTP could be formed via the NDPK activity by using externally added ATP as a substrate. Furthermore, [␥-32P]GTP was predominantly found in the washed thylakoid membrane To verify that the 36.5-kDa protein is connected to the fraction (87%) whereas only 13% were detected in the super- observed transthylakoid transport system, thylakoids were pre- natant. This experiment provided a strong evidence that ATP incubated with the AAC antibody or preimmune serum followed was transported across the thylakoid membrane to be enzymat- by measurement of GTP formation in the lumen. The AAC ically metabolized to GTP in the luminal space. Under the antibody reduced the activity to 22% as compared with 72% in present experimental conditions, we were not able to show any the case of the preimmune serum (Fig. 5C), giving support that stimulation of the nucleotide transport by light. the 36.5-kDa protein is the thylakoid nucleotide transporter. The nucleotide transport activity was also analyzed in the presence of a possible competitive inhibitor (Fig. 5A). Preillu- Discussion minated intact thylakoids were photolabeled with 8-N3ATP, Using a combination of enzymatic assays, Western blotting, and unbound nucleotides were washed away, and the thylakoids were import studies, we show that the thylakoid lumen contains a incubated with [␥-32P]ATP and GDP as described above. The nucleotide-metabolizing enzyme, NDPKIII, preferentially in- level of [␥-32P]GTP produced in the lumen decreased to 64% as volved in GTP synthesis. We have also detected two thylakoid PLANT BIOLOGY compared with thylakoids without crosslinked 8-N ATP, cor- 3 proteins of 33 kDa and 36.5 kDa, respectively, that bind nucle- roborating the existence of a transthylakoid nucleotide transporter involving a nucleotide-binding site at the outer thylakoid otides. The 33-kDa GTP-binding protein was identified as the surface. OEC33 subunit of the PSII complex whereas the 36.5-kDa The evidence for a transthylakoid nucleotide transport system preferentially binds ATP and shows crossreactivity with an prompted questions with respect to protein components in- antibody against the mitochondrial AAC protein. These results volved. Western blotting experiments using an antibody against bring experimental evidence for nucleotide-dependent processes the AAC protein from beef heart mitochondria revealed a major in the thylakoid lumen and transport of nucleotides across the band of 36.5 kDa and a minor band of 17.2 kDa in spinach membrane. Although the functional implication of these unex- thylakoids (Fig. 5B). The phosphorimage of Fig. 3A revealed a pected discoveries remain to be elucidated, our results suggest close correspondence between the labeled 36.5-kDa protein and that the thylakoid lumen may have a more significant role in the immunologically crossreacting polypeptide. Notably, no metabolism and cellular signaling than has been considered so crossreaction was found in the chloroplast envelope. In the far. control experiments with beef heart mitochondria (Fig. 5B) and Among the two NDPK detected in spinach chloroplasts, only spinach mitochondria (not shown), bands with molecular masses one (NDPKII) has been shown to be located in the stroma (27, of 28 and 17.5 kDa, corresponding to the intact AAC protein and 28). NDPKIII has previously been found in crude chloroplast its degradation product, respectively, were detected. extracts at a very low abundance. NDPKIII has a molecular mass

Spetea et al. PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1413 Downloaded by guest on September 29, 2021 of 17 kDa and prefers GDP rather than ADP as phosphate its dependence on photosynthetic transport needs to be further acceptor (26). The data provided in this work demonstrate that elucidated. NDPKIII is located in the thylakoid lumen, despite previous One potential candidate for a protein responsible for the interpretations on its cellular location (27, 29). By using a transthylakoid transport is the detected 36.5-kDa nucleotide- proteomic approach, it has been suggested that the Arabidopsis binding protein, as supported by two observations: (i) this NDPKIII may be located in the mitochondrial intermembrane thylakoid protein crossreacted with an antibody against the space (31). However, the presented import studies clearly show mitochondrial AAC and (ii) the AAC antibody inhibits the that the pNDPKIII of Arabidopsis is targeted to the thylakoid transthylakoid nucleotide transport activity, suggesting a rela- lumen involving two-step processing, and no NDPKIII could be tion to the mitochondrial carrier family (PROSITE PS00215). traced in mitochondria by Western blotting. Homology search with the sequence of the bovine AAC protein The luminal NDPKIII uses ATP to synthesize GTP (Fig. 1), (SwissProt P02722) combined with prediction for chloroplast the nucleotide known to control the conformation of a wide targeting peptides in the Arabidopsis database have provided variety of GTP-binding proteins. In this study, we bring unex- several potential candidates for further investigation. pected evidence for GTP-binding to the OEC33 subunit of PSII. In contrast to mitochondrial membranes containing an abun- dant AAC protein exporting the produced ATP into the cytosol, The extrinsic location of OEC33 at the luminal side of the the thylakoid membrane requires most likely low amounts of thylakoid membrane explains the lack of accessibility of the transporter sufficient to supply the lumen with ATP, consistent [␣-32P]8-N GTP in intact dark-control thylakoids (Fig. 3A). 3 with the low abundance of the 36.5-kDa protein. Furthermore, However, its labeling can be detected in preilluminated thyla- the plant and animal mitochondrial AAC proteins share simi- koids possibly due to the light-dependent transport of GTP larities but also differences with respect to the mechanism of across the membrane. The OEC33 protein does not contain nucleotide transport (13, 14). In animal mitochondria, the typical GTP-binding motifs similarly to previous reports on transport is limited to ATP and ADP and is driven by the tubulin (PROSITE PS00017). The OEC33 protein is important membrane potential whereas plant mitochondrial carriers also for the functional integrity of the manganese cluster, which can transport GDP and GTP and do not depend on a membrane catalyzes photosynthetic water-splitting (30). The discovery of potential (32). To what extent the molecular mechanism of GTP binding to the OEC33 protein points to additional func- nucleotide transport across the thylakoid membrane is similar to tions for this protein, e.g., participation in signal transduction the one in plant mitochondria remains to be investigated. associated with the thylakoid membrane, possibly connected to Taken together, our results imply that nucleotides are trans- the GTP-dependent turn-over of the D1 protein (16). ported across the thylakoid membrane and that there is a The luminal NDPK activity and GTP binding to the OEC33 nucleotide metabolism in the luminal space. These findings protein suggest that nucleotides must be transported from the expand the functional significance of the thylakoid lumen by chloroplast stroma across the thylakoid membrane into the adding potentially new metabolic and regulatory aspects that luminal space. Our experiments involving thylakoid membranes remain to be elucidated. supplied with radioactively labeled ATP and the subsequent recovery of labeled GTP in the thylakoid lumen (Fig. 5A) We thank G. von Heijne (Stockholm University) for valuable discussions provide experimental evidence for that notion. Notably, the and the Arabidopsis Biological Resource Center at Ohio State University binding of GTP to the OEC33 protein after the addition of for providing the Arabidopsis cDNA library (CD4-13). Antibodies raised against the mitochondrial AAC and the stromal NDPKII were kindly nucleotides to the outside of the thylakoid membrane was found provided by J. Houstek (University of Prague) and J. Soll (Ludwig- to be both light and DCMU sensitive, suggesting a connection to Maximilians University, Munich), respectively. This work was supported photosynthetic electron transport. We do not yet understand the by the Swedish Research Council, the Carl Tryggers Foundation, and the mechanism behind this transthylakoid transport, and particularly Graduate Research School in Genomics and Bioinformatics.

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