The Thylakoid Lumen of Chloroplasts

The chloroplast compartment enclosed by the thylakoid membrane, the “lumen,” is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60–65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase. The chloroplast compartment enclosed by the thylakoid membrane, the “lumen,” is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60–65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase. The chloroplast is the photosynthetic organelle of green algae and higher plants. The chloroplast architecture comprises an envelope membrane, which encloses the soluble stroma as well as the highly specialized thylakoid membrane. The stromal compartment contains mainly the components of the Calvin cycle, which are required for the fixation of carbon dioxide. The thylakoids, on the other hand, carry out the light reactions of photosynthesis leading to the production of NADPH and ATP. The thylakoid membrane has a characteristic flat shape and is differentiated into appressed grana stacks and single non-appressed stroma-exposed lamellae. The inner surface of the thylakoid membrane encloses a narrow, continuous compartment, the lumen (1Andersson B. Barber J. Adv. Mol. Cell Biol. 1994; 10: 1-53Crossref Scopus (23) Google Scholar, 2Hall D.O. Rao K.K. Photosynthesis. 5th Ed. Cambridge University Press, Cambridge, UK1994Google Scholar). Electron microscopy studies of spinach thylakoids have suggested that the lumen is a densely packed space (3Weibull C. Albertsson P.-Å. J. Ultrastruct. Mol. Struct. Res. 1988; 100: 55-59Crossref Scopus (5) Google Scholar). No isolation method has so far been available for obtaining a high yield of pure thylakoid lumen. Thus, the present knowledge of the lumen from a compositional and functional point of view is fragmentary and is gathered from several independent approaches, addressing only single aspects of this compartment. By developing a technique for obtaining inside-out thylakoids, the investigation of the membrane surface of the lumenal side became possible (4Andersson B. Åkerlund H.-E. Biochim. Biophys. Acta. 1978; 503: 462-472Crossref PubMed Scopus (110) Google Scholar). This work contributed to the discovery of the extrinsic proteins PsbO, PsbP, and PsbQ (5Åkerlund H.-E. Jansson C. FEBS Lett. 1981; 124: 229-232Crossref Scopus (128) Google Scholar, 6Åkerlund H.-E. Jansson C. Andersson B. Biochim. Biophys. Acta. 1982; 681: 1-10Crossref Scopus (207) Google Scholar) that bind to the lumenal side of photosystem II and are thought to stabilize the water oxidizing complex (7Murata N. Miyao M. Trends Biochem. Sci. 1985; 10: 122-124Abstract Full Text PDF Scopus (152) Google Scholar, 8Vermaas W.F.J. Styring S. Schröder W.P. Andersson B. Photosynth. Res. 1993; 38: 249-263Crossref PubMed Scopus (75) Google Scholar). More recent studies have shown that these subunits of photosystem II occur also as soluble lumenal proteins (9Ettinger W.F. Theg S.M. J. Cell Biol. 1991; 115: 321-328Crossref PubMed Scopus (54) Google Scholar). This pool of unassembled PsbO, PsbQ, and PsbP was resistant to proteolytic degradation and was capable of assembling into photosystem II (10Hashimoto A. Yamamoto Y. Theg S.M. FEBS Lett. 1996; 391: 29-34Crossref PubMed Scopus (47) Google Scholar). Furthermore, it was found that during photoinhibitory conditions the extrinsic proteins were released from the membrane into the lumen (11Hundal T. Virgin I. Styring S. Andersson B. Biochim. Biophys. Acta. 1990; 1017: 235-241Crossref Scopus (83) Google Scholar,12Eisenberg-Domovich Y. Oelmüller R. Herrmann R.G. Ohad I. J. Biol. Chem. 1995; 270: 30181-30186Crossref PubMed Scopus (29) Google Scholar). Other important components of the thylakoid lumen are plastocyanin, the primary electron donor of photosystem I (13Haehnel W. Berzborn R.J. Andersson B. Biochim. Biophys. Acta. 1981; 637: 389-399Crossref Scopus (45) Google Scholar, 14Haehnel W. Annu. Rev. Plant. Physiol. 1984; 35: 659-693Crossref Google Scholar), and PsaN, a photosystem I subunit that is extrinsically bound to the lumenal side of the thylakoid membrane (15He W.-Z. Malkin R. FEBS Lett. 1992; 308: 298-300Crossref PubMed Scopus (20) Google Scholar). Recent investigations have revealed that polyphenol oxidases (16Sommer A. Ne'eman E. Steffens J.C. Mayer A.M. Harel E. Plant Physiol. 1994; 105: 1301-1311Crossref PubMed Scopus (110) Google Scholar, 17Sokolenko A. Fulgosi H. Gal A. Altschmied L. Ohad I. Herrmann R.G. FEBS Lett. 1995; 371: 176-180Crossref PubMed Scopus (39) Google Scholar) and violaxanthin de-epoxidase are also present in the thylakoid lumen (18Hager H. Holocher K. Planta. 1994; 192: 581-589Crossref Scopus (205) Google Scholar). Furthermore, the carboxyl-terminal processing protease for the D1 protein (19Inagaki N. Mori H. Fujita S. Yamamoto Y. Satoh K. Mathis P. Photosynthesis: From Light to Biosphere. 3. Kluwer Academic Publishers Group, Drodrecht, Netherlands1995: 783-786Google Scholar, 20Oelmüller R. Herrmann R.G. Pakrasi H.B. J. Biol. Chem. 1996; 271: 21848-21852Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and a processing protease for plastocyanin (21Kirwin P.M. Elderfield P.D. Williams R.S. Robinson C. J. Biol. Chem. 1988; 263: 18128-18132Abstract Full Text PDF PubMed Google Scholar) were found on the lumenal surface of the thylakoids, whereas chaperones may be located in the lumen (22Schlicher T. Soll J. FEBS Lett. 1996; 379: 302-304Crossref PubMed Scopus (38) Google Scholar). So far all lumenal proteins have been found to be nuclear-encoded and synthesized as precursors in the cytoplasm. These precursor proteins have characteristic amino-terminal bipartite transit peptides, which direct their import into the chloroplast stroma and across the thylakoid membrane into the lumen (23von Heijne G. Steppuhn J. Herrmann R.G. Eur. J. Biochem. 1989; 180: 535-545Crossref PubMed Scopus (910) Google Scholar, 24Robinson C. Klösgen R.B. Plant Mol. Biol. 1994; 26: 15-24Crossref PubMed Scopus (75) Google Scholar, 25Robinson C. Knott T.G. Andersson B. Salter A.H. Barber J. Molecular Genetics of Photosynthesis. IRL Press at Oxford University Press, Oxford1996: 145-159Google Scholar). On the basis of this property, bipartite transit peptides have become typical markers for lumenal proteins. However, not all chloroplast proteins encoded with such presequences are routed into the lumenal space. The PsbW protein and CFoII, for instance, are synthesized with bipartite transit peptides but have been shown to be integral proteins of the thylakoid membrane (26Michl D. Robinson C. Shackleton J.B. Herrmann R.G. Klösgen R.B. EMBO J. 1994; 13: 1317-1370Crossref Scopus (101) Google Scholar, 27Lorkovic Z.J. Schröder W.P. Pakrasi H.B. Irrgang K.-D. Herrmann R.G. Oelmüller R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8930-8934Crossref PubMed Scopus (83) Google Scholar, 28Shi L.-X. Schröder W.P. Photosynth. Res. 1997; 53: 45-53Crossref Scopus (13) Google Scholar). In this study we have developed a procedure by which a lumenal fraction can be isolated in a highly pure form from spinach thylakoids. We have carried out the first systematic characterization of this compartment, and we show that the thylakoid lumen contains a high concentration of proteins, among which at least 25 distinct polypeptides can be identified. Several have been characterized in terms of enzymatic activity or amino-terminal sequence. Spinach (Spinacia oleracea) was grown hydroponically for 6 weeks with alternating periods of 10 h light and 14 h darkness. The general approach applied consists of three principal steps as follows: (i), preparation of chloroplasts, (ii) purification of carefully washed thylakoids, and (iii) rupture of thylakoids by a Yeda press and isolation of the lumenal content Spinach were for in 10 and The was of and the was at The were in 10 for at and in of the The yield was intact of The were with 10 to a concentration of and in a The thylakoids were by for at and washed in the with of the following 10 to the soluble stromal to the fraction of used light complex and extrinsic thylakoid membrane to the thylakoids for Yeda press The thylakoid were in a of fragmentation to a concentration of of of The washed thylakoids were a Yeda press at a of 10 and for h at and The supernatant was from the and a the conditions to membrane The isolation procedure was on and the and thylakoid membranes were green The lumenal fraction was used or in The washed thylakoids of were in the presence of 10 for on in and The was by to concentration of and the thylakoids were washed with and These conditions were to the major of proteins on the stromal side of the thylakoid membrane, of the integral membrane proteins. were found to to membranes by a of the lumenal of the stromal lamellae. were as R.J. Biochim. Biophys. Acta. 1989; Scopus Google Scholar). of soluble proteins was carried out to Biochem. PubMed Scopus Google and that of membrane proteins was as S.M. Biochem. 1978; PubMed Scopus Google Scholar). The used was and of the were with a at using as the electron Malkin R. IRL Press at Oxford University Press, Scholar). was by the method in PubMed Scopus Google in and of were by using the from analyses proteins were a membrane in a The used were in the following spinach for Plant Genetics and Plant violaxanthin de-epoxidase University of plastocyanin University of PsbO, PsbP, PsbQ, the lumen fraction, and The was using in with an was using a and the from Molecular be to the for the lumenal with of the chloroplast and thylakoids, a was used for the lumenal was as follows: However, the of plastocyanin and in the lumenal fraction was determined from the the content in the washed thylakoids and their membrane was to E. Y. 1978; 53: and to the at the following from and spinach were used by P. and activity was determined by in the presence of Malkin R. IRL Press at Oxford University Press, Scholar). was to S. Biochem. Biophys. PubMed Scopus (65) Google of this during the lumenal all activity of polyphenol was determined at using the J.C. M. R. J. Biochem. 1995; PubMed Scopus Google Scholar). activity was using the as by at the following activity was using a and the in of of M. EMBO J. 1985; PubMed Scopus Google Scholar) as The activity of violaxanthin de-epoxidase was determined as in M. Åkerlund H.-E. Photosynth. Res. 1996; enzymatic were on for violaxanthin at 25 in the presence of was using the A. A. A. Biochem. PubMed Scopus Google Scholar) and the from activity was to P. P. Biochim. Biophys. Acta. 1990; PubMed Scopus Google were from membrane following by as P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The amino-terminal analyses were by P. I. of using an in the of and as well as were carried out using the for the Scholar). The of the present study was to a preparation of thylakoid lumen in a yield and purity high to it generally for this chloroplast compartment. The developed as in the of with the isolation of intact spinach chloroplasts. The were intact and an activity of of In the the were by and the thylakoids were and by several stromal proteins were by 10 and was used to and other thylakoid proteins. the thylakoids were in fragmentation these thylakoids an of of The washed thylakoids were by a Yeda and the released lumenal content was from the membrane by The fraction was of and a protein concentration of a yield of of protein from of spinach The during the of the preparation was by of the of the chloroplasts, the stromal fraction, the washed thylakoids, and the lumenal preparation The in the isolated were the and subunits of and the The subunits of and other soluble proteins were found in the stromal fraction The and other integral membrane proteins were recovered in the thylakoid fraction the soluble lumenal proteins released the of the washed thylakoids and of the membrane in were than 25 polypeptides in the lumenal Furthermore, the is different from that of the other chloroplast These polypeptides in from 14 to are 10 to and to 10 the polypeptides to the of in the of Furthermore, an investigation of the polypeptides of the lumenal fraction by H. G. Biochem. PubMed Scopus Google Scholar) revealed polypeptides in the of of the isolated thylakoid of subunit of and protein and protein and protein protein protein protein protein protein protein in a this general of the protein content of the isolated lumenal fraction, several different were used in to the three of the major polypeptides at and were identified as the extrinsic proteins PsbO, PsbP, and PsbQ of photosystem This is with the that a soluble unassembled pool of these proteins in the thylakoid lumen (9Ettinger W.F. Theg S.M. J. Cell Biol. 1991; 115: 321-328Crossref PubMed Scopus (54) Google Scholar). The major protein of the lumenal fraction, with the of was identified by as The proteins of the lumenal fraction were by amino-terminal protein This the of plastocyanin, PsbO, PsbP, and was identified as a with an of and polypeptides were at and I and was found to at of and of proteins from the thylakoid lumenal in a The of which is located on the stromal side of the thylakoid a of contamination in the isolated lumenal the washed thylakoids were by with to the Yeda press proteins, the be such proteolytic The conditions of were to degradation of of the protein on the stromal side of the thylakoid membrane and to membrane More could of the thylakoid membrane, as was A. Biochem. Biophys. Scopus Google Scholar). The of the of the lumenal fraction following and was by of the using the protein as an The from three different that of the polypeptides of the lumenal fraction were not that these proteins were located the thylakoid lumen. in were polypeptides protein in by whereas the was to which is with at the stromal surface of the thylakoid membranes (1Andersson B. Barber J. Adv. Mol. Cell Biol. 1994; 10: 1-53Crossref Scopus (23) Google Scholar, 2Hall D.O. Rao K.K. Photosynthesis. 5th Ed. Cambridge University Press, Cambridge, UK1994Google Scholar). The may have been the thylakoid surface during the Yeda press with the lumenal proteins in the supernatant following centrifugation. In for purity an immunoblot was to marker proteins associated with specific chloroplast plastocyanin as a soluble lumenal as a lumenal extrinsic the D1 protein of photosystem II as an integral membrane the for the thylakoid as the major stromal and as typical soluble stromal in 60–65% of the of plastocyanin and that of the extrinsic protein were recovered in the lumenal Furthermore, the lumenal fraction was of D1 protein the of thylakoid In less than 1% of the of the subunit of was present in the lumenal The major contamination was 10 and of this was found in the this of the protein content of the lumenal analyses of chloroplast marker thylakoid lumen membrane D1 stroma not in a not an of stromal as well as to possible chloroplast we for a of different marker These marker from the chloroplast from the and and the enzymes and shown in the contamination of the isolated lumenal fraction by was only of the activity of the isolated and that of was The activity of in the was of and that of spinach was of Furthermore, the activity of was of and that of the spinach was of on these the contamination of the lumenal fraction by soluble proteins was to be than The activity of was not in the lumenal of marker thylakoid lumen of violaxanthin stroma NADPH and of violaxanthin NADPH in a work suggested that polyphenol A. Fulgosi H. Gal A. Altschmied L. Ohad I. Herrmann R.G. FEBS Lett. 1995; 371: 176-180Crossref PubMed Scopus (39) Google Scholar) and violaxanthin de-epoxidase (18Hager H. Holocher K. Planta. 1994; 192: 581-589Crossref Scopus (205) Google Scholar) are located in the lumen of the spinach thylakoids. we the isolated lumenal fraction for the presence of these The specific activity of polyphenol from the to the washed thylakoids and a in the lumenal fraction The fraction of the polyphenol activity that with the thylakoid was of that present in the lumenal The presence of polyphenol in the lumenal fraction was also by amino-terminal protein sequencing of the with an of The specific activity of violaxanthin de-epoxidase in the lumenal fraction was of of which to of the for the spinach M. Åkerlund H.-E. Photosynth. Res. 1996; Scholar). In the immunoblot shown in that violaxanthin de-epoxidase was highly in the lumenal fraction as with the thylakoid In for other lumenal we the that photosystem II can W.P. Åkerlund H.-E. Biochim. Biophys. Acta. Scopus Google Scholar), and we the lumenal fraction for of the activity of the thylakoids was recovered in the lumenal fraction, which with work T. S. K. Plant Cell Physiol. 1984; Scholar) that suggested that are not present in this chloroplast compartment. The activity was not by of the thylakoids. the of the D1 protein of photosystem II during proteolytic in stromal and lumenal J. Andersson B. Trends Biochem. Sci. 1992; Full Text PDF PubMed Scopus Google Scholar). In processing of precursor proteins and the carboxyl-terminal of the D1 protein occur in the lumen C. Knott T.G. Andersson B. Salter A.H. Barber J. Molecular Genetics of Photosynthesis. IRL Press at Oxford University Press, Oxford1996: 145-159Google Scholar, 20Oelmüller R. Herrmann R.G. Pakrasi H.B. J. Biol. Chem. 1996; 271: 21848-21852Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The lumenal preparation was for protease The proteolytic specific activity in the chloroplast stroma and thylakoids was whereas the proteolytic activity in the lumenal fraction was only that in the thylakoids. The of the lumenal proteolytic activity was and However, in to the thylakoids this activity was in the of were found to this protease activity by the of not the proteolytic These the presence of The lumenal fraction was also for the presence of and The concentration was found to be than and activity could be lumenal proteins, amino-terminal sequencing of polypeptides from the lumenal fraction was their by This approach to the of polypeptides of the of and The protein to in the and However, the amino-terminal of the protein was to protein encoded by and The of and were an of the with the of The from A. contains of the and the Furthermore, the of a bipartite transit with a and the at the processing C. Klösgen R.B. Plant Mol. Biol. 1994; 26: 15-24Crossref PubMed Scopus (75) Google Scholar). shown in the amino-terminal of the protein was to a protein encoded by a from A. This also of contains a of a bipartite transit The at the processing is to the in the precursor of the PsbQ protein from spinach Furthermore, this is by a that is typical of thylakoid present on transit peptides of lumenal proteins. shown in the amino-terminal of the protein was to the of a protein from the to the this protein also contains a transit with a and at the processing the carboxyl-terminal of protein is highly to of the of from this This suggested that the protein from spinach and the protein were to cis/trans-isomerase. the spinach protein was and was and A. R. and B. EMBO in The is a high protein that is located in the thylakoid lumen. This was direct enzymatic of the activity of the spinach that is typical for G. B. K. T. 1989; PubMed Scopus Google Scholar). In the lumenal fraction from work was found to have a We have not been to the of the lumenal proteins. The thylakoid lumen a continuous space that is poorly characterized with the other chloroplast compartments. in the lumenal side of the thylakoid membrane has mainly on electron associated with the inner membrane the thylakoid lumen has also been as for the of the that and for the and by in the thylakoid membranes G. J. Biol. 1995; PubMed Scopus Google Scholar, G. J. Biol. 1996; PubMed Scopus Google Scholar). More with an of and of the photosynthetic the for enzymes B. Barber J. H. Trends in Scholar) in the lumenal space has become The present isolation of a thylakoid lumenal fraction high yield and a for of this chloroplast compartment. a to for thylakoids of of K. M. G. Biochim. Biophys. Acta. PubMed Scopus Google Scholar) and the yield of of lumenal protein of in the thylakoid the protein concentration in the lumenal space is to be higher than Thus, soluble proteins in the lumen are at to which is to that of the chloroplast the thylakoid lumen is of a densely packed of soluble as suggested from electron microscopy studies (3Weibull C. Albertsson P.-Å. J. Ultrastruct. Mol. Struct. Res. 1988; 100: 55-59Crossref Scopus (5) Google Scholar). The of polypeptides in the isolated lumenal fraction was of which to be identified. In to is for the thylakoid proteins only a of these are of The purity of the isolated lumenal fraction was high as judged by contamination of stromal and thylakoid integral membrane proteins. The was which is associated with the thylakoid surface and which could not be by is a protein that protein of G. J. Biol. Chem. Full Text PDF PubMed Google Scholar), B. Jansson C. U. Åkerlund H.-E. C. L. S. K. Molecular of the Scholar), and C. Schröder W.P. A. G. Andersson B. 1995; PubMed Scopus Google Scholar). The PsbO, PsbP, and PsbQ proteins and plastocyanin were found to be the major proteins present in the isolated lumenal The presence of extrinsic PsbO, PsbP, and PsbQ polypeptides is with the of an lumenal pool of these polypeptides (9Ettinger W.F. Theg S.M. J. Cell Biol. 1991; 115: 321-328Crossref PubMed Scopus (54) Google Scholar, A. Yamamoto Y. Theg S.M. FEBS Lett. 1996; 391: 29-34Crossref PubMed Scopus (47) Google Scholar), photoinhibitory conditions (11Hundal T. Virgin I. Styring S. Andersson B. Biochim. Biophys. Acta. 1990; 1017: 235-241Crossref Scopus (83) Google Scholar, Y. Oelmüller R. Herrmann R.G. Ohad I. J. Biol. Chem. 1995; 270: 30181-30186Crossref PubMed Scopus (29) Google Scholar). The of soluble in the isolated lumenal fraction was to the content of chloroplasts. This with the that of the of spinach in an unassembled in the thylakoid lumen (9Ettinger W.F. Theg S.M. J. Cell Biol. 1991; 115: 321-328Crossref PubMed Scopus (54) Google Scholar). The of violaxanthin and polyphenol as soluble polypeptides of the lumen work the of these enzymes in this compartment (16Sommer A. Ne'eman E. Steffens J.C. Mayer A.M. Harel E. Plant Physiol. 1994; 105: 1301-1311Crossref PubMed Scopus (110) Google Scholar, 17Sokolenko A. Fulgosi H. Gal A. Altschmied L. Ohad I. Herrmann R.G. FEBS Lett. 1995; 371: 176-180Crossref PubMed Scopus (39) Google Scholar, H. Holocher K. Planta. 1994; 192: 581-589Crossref Scopus (205) Google Scholar). Furthermore, the presence of a in the lumen may be important for of by photosystem II W.P. Åkerlund H.-E. Biochim. Biophys. Acta. Scopus Google Scholar). The of lumenal polyphenol is not from this (16Sommer A. Ne'eman E. Steffens J.C. Mayer A.M. Harel E. Plant Physiol. 1994; 105: 1301-1311Crossref PubMed Scopus (110) Google Scholar), it is not to a for The polyphenol be into the lumen it be from in the The of the three unassembled extrinsic proteins of photosystem II the of proteolytic in the thylakoid lumen. The present analyses as well as studies T. Y. Plant Cell Physiol. 1990; A. Altschmied L. Herrmann R.G. Plant Physiol. 1997; 115: PubMed Scopus Google Scholar) the presence of in this compartment. However, the protease activity of the lumenal fraction was only of that found in the thylakoid fraction, that of lumenal protease activity is bound to the inner thylakoid The lumenal protein is highly to the of the A. which a typical bipartite transit However, the three to the processing an for determined bipartite transit peptides of chloroplast proteins. be of to the specific import by which this protein is from the into the thylakoid lumen. The of lumenal chaperones (22Schlicher T. Soll J. FEBS Lett. 1996; 379: 302-304Crossref PubMed Scopus (38) Google Scholar) as well as for of lumenal proteins A. H. Ohad I. Physiol. Plant. 1997; 100: Google Scholar) have been This work not this view we could not show the presence of or activity in the lumen. The presence of in the thylakoid lumen We P.-Å. Albertsson and H.-E. Åkerlund of and for Plant Genetics and Plant for the of We the of