Determination of Photosystem II Subunits by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry
2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês
10.1074/jbc.m008081200
ISSN1083-351X
AutoresIldikò Szabó, Roberta Seraglia, Fernanda Rigoni, Pietro Traldi, Giorgio M. Giacometti,
Tópico(s)Photoreceptor and optogenetics research
ResumoPhotosystem II of higher plants and cyanobacteria is composed of more than 20 polypeptide subunits. The pronounced hydrophobicity of these proteins hinders their purification and subsequent analysis by mass spectrometry. This paper reports the results obtained by application of matrix-assisted laser desorption/ionization mass spectrometry directly to isolated complexes and thylakoid membranes prepared from cyanobacteria and spinach. Changes in protein contents following physiopathological stimuli are also described. Good correlations between expected and measured molecular masses allowed the identification of the main, as well as most of the minor, low molecular weight components of photosystem II. These results open up new perspectives for clarifying the functional role of the various polypeptide components of photosystems and other supramolecular integral membrane complexes. Photosystem II of higher plants and cyanobacteria is composed of more than 20 polypeptide subunits. The pronounced hydrophobicity of these proteins hinders their purification and subsequent analysis by mass spectrometry. This paper reports the results obtained by application of matrix-assisted laser desorption/ionization mass spectrometry directly to isolated complexes and thylakoid membranes prepared from cyanobacteria and spinach. Changes in protein contents following physiopathological stimuli are also described. Good correlations between expected and measured molecular masses allowed the identification of the main, as well as most of the minor, low molecular weight components of photosystem II. These results open up new perspectives for clarifying the functional role of the various polypeptide components of photosystems and other supramolecular integral membrane complexes. Photosystem II is a pigment-protein complex of the thylakoid membrane of higher plants, eukaryotic algae, and cyanobacteria. It catalyzes light-induced electron transfer from water to plastoquinone, with associated production of molecular oxygen. PSII 1The abbreviations used are: PSIIphotosystem IIRCIIisolated reaction center of PSII comprising D1, D2, α and β subunits of cytochrome b559, and PsbI proteinsHPLChigh performance liquid chromatographyESIelectrospray ionizationMSmass spectrometryMALDImatrix-assisted laser desorption/ionizationMes4-morpholineethanesulfonic acidSDS-PAGESDS-polyacrylamide gel electrophoresisMWmolecular weight consists of a large complex with a number of polypeptide components, most of which are integral membrane proteins. A number of extrinsic proteins are also associated with it at the membrane surface. The entire set of electron transfer cofactors, including chlorophyll a, pheophytina, plastoquinones, and non-heme iron, is associated with the D1/D2 heterodimer. These two proteins, together with the α and β subunits of cytochrome b559 and the PsbI protein, constitute the so-called reaction center II (RCII), which is the smallest PSII subparticle still able to perform light-induced charge separation (1Nanba O. Satoh K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 109-112Crossref PubMed Google Scholar). Two large integral membrane proteins, CP43 and CP47, each coordinating a number (12Barbato R. Friso G. Giardi M.T. Rigoni F. Giacometti G.M. Biochemistry. 1991; 30: 10220-10226Crossref PubMed Scopus (53) Google Scholar, 13Douglas S.E. Curr. Opin. Genet. Dev. 1998; 8: 655-661Crossref PubMed Scopus (168) Google Scholar, 14Shimada H. Sugiura M. Nucleic Acids Res. 1991; 19: 983-995Crossref PubMed Scopus (198) Google Scholar, 15Irrgang K.D. Shi L.X. Funk C. Schroeder W.P. J. Biol. Chem. 1995; 270: 17588-17593Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) of chlorophylla molecules, and several low molecular mass (<10 kDa) polypeptides are constituents of the PSII core complex, which is very similar in higher plants and prokaryotic cyanobacteria.During the last decade, considerable progress has been made in our understanding of the organization of polypeptides constituting the reaction center of PSII, but the topology and functional role of the small protein subunits are still largely unknown. Some of them are universal, whereas others are present only in cyanobacteria (PsbU and PsbV) or only in higher plants (e.g. PsbP, PsbQ, PsbS) (2Barber J. Nield J. Morris E.P. Zheleva D. Hankamer B. Physiol. Plant. 1997; 100: 817-827Crossref Google Scholar).Investigation of the structural and functional roles of the various PSII subunits requires, as a preliminary step, a suitable method to detect them in the thylakoid membrane or purified subparticle preparations. The detection and study of stimuli-induced modifications of several PSII components have mainly been based on the use of polyclonal antibodies. However, these are often not available and, when available, are time-consuming to use.A different technique, capable of accurately detecting even small amounts of the various PSII subunits in an integrated system, could be of great help in studying the function of these proteins. Mass spectrometry has recently been used for structural biology studies, also in the field of photosynthesis. A reverse phase high performance liquid chromatography (HPLC) purification system has been developed for the separation of PSII reaction center proteins, and the molecular masses of the resulting purified, intact proteins have been determined by electrospray ionization mass spectrometry (ESI-MS) (3Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 3935-3943Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 4Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 5Sharma J. Panico M. Shipton C.A. Nilsson F. Morris H.R. Barber J. J. Biol. Chem. 1997; 272: 33158-33166Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6Zheleva D. Sharma J. Panico M. Morris H.R. Barber J. J. Biol. Chem. 1998; 273: 16122-16127Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). These studies demonstrate that ESI-MS is indeed a useful technique for studying hydrophobic membrane proteins also. Whereas ESI-MS allows highly accurate measurement of the molecular mass, it suffers from the important limitation that it cannot be applied in the presence of detergents, and the proteins, free of salts and detergents prior to MS analysis, must be purified (7Barnidge D.R. Dratz E.A. Jesaitis A.J. Sunner J. Anal. Biochem. 1999; 10: 1-9Crossref Scopus (44) Google Scholar). This paper reports the results obtained by MALDI mass spectrometry for rapid determination of non-purified proteins of photosynthetic complexes isolated from spinach and cyanobacteria.MATERIALS AND METHODSMALDI Mass SpectrometryMeasurements were performed on a REFLEX time-of-flight instrument (Bruker-Franzen Analytik, Bremen, Germany) equipped with a SCOUT ion source operating in positive linear mode. Ions, formed by a pulsed UV laser beam (nitrogen laser, λ = 337 nm), were accelerated to 25 kV. The use of this beam did not result in degradation of photosynthetic proteins during measurements. Sinapinic acid (saturated solution in acetonitrile:water (50:50 v:v)) was used as a matrix. Samples, at chlorophyll concentrations of 9–250 μg/ml, were diluted 2- to 10-fold with a 0.1% trifluoroacetic acid aqueous solution. 5 μl of the diluted sample solution was mixed with the same volume of matrix solution, and 1 μl of the resulting mixture was deposited on a stainless steel sample holder and allowed to dry before introduction into the mass spectrometer.Because of the wide mass range to be examined, two different instrumental conditions were used. For low molecular masses (2,000–20,000 Da), pulsed ion extraction was carried out applying a voltage of 22.3 kV for 300 ns to the second grid. External mass calibration was carried out using [M + H]+ ions of bovine insulin and horse myoglobin at m/z 5734 and 16952, respectively, and the corresponding doubly charged species atm/z 2868 and 8476, respectively. For high molecular masses (20,000–80,000 Da), pulsed ion extraction was used applying a voltage of 21.9 kV for 400 ns to the second grid. External mass calibration was carried out using [M + H]+ ions of horse myoglobin and bovine serum albumin at m/z16952 and 66431, respectively, and the doubly charged species of bovine serum albumin at m/z 33216. The GuessProt algorithm was used to determine the predicted molecular masses of the PSII subunits.Preparation of PSII Particles and Isolated RCII from SpinachPSII core complexes from spinach were prepared according to Ghanotakis et al. (8Ghanotakis D.F. Demetriou D.M. Yocum C.F. Biochim. Biophys. Acta. 1987; 891: 15-21Crossref Scopus (221) Google Scholar), and isolation of RCII was performed as described in Ref. 9Chapman D.J. Gounaris K. Barber J. Rogers L.J. Methods in Plant Biochemistry. Academic Press, London1990: 171-193Google Scholar.Preparation of Thylakoid Membranes and PSII from PSI-less SynechocystisThylakoids were prepared using a modified version of the procedure of Mayes et al. (10Mayes S.R. Dubbs J.M. Vass I. Hideg E. Nagy L. Barber J. Biochemistry. 1993; 32: 1454-1465Crossref PubMed Scopus (82) Google Scholar). Briefly, cells were cultured as previously described (11Barbato R. Polverino de Laureto P. Rigoni F. De Martini E. Giacometti G.M. Eur. J. Biochem. 1995; 234: 459-465Crossref PubMed Scopus (28) Google Scholar), harvested by centrifugation (2000 × g for 5 min at 4 °C), and resuspended in a breaking buffer containing the following: 20 mm Mes, 5 mm MgCl2, 5 mm CaCl2, 1 mm benzamidine, 1 mm aminocaproic acid, and 25% glycerol (pH 6.35 with NaOH). Cells were broken on ice using a bead beater (Biospec) by application of 15 30-s cycles at 5-min intervals. Unbroken cells were eliminated (5-min centrifugation at 2000 × g), and thylakoids were pelleted at 140,000 × g for 30 min. Pellets were then resuspended in 50 mm Mes and 20 mm sodium pyrophosphate (pH 6.5). Isolated thylakoids were solubilized with 2% (w/v) dodecyl maltoside for 30 min and loaded on a continuous sucrose gradient containing 25 mm Mes, 0.5 m sucrose, 10 mm NaCl, 5 mm CaCl2, and 0.03% dodecyl maltoside (pH 6.5). Three bands, i.e. carotenoids, cytochrome b6/f, and PSII, separated out after 17 h of centrifugation at 35,000 rpm. The PSII band was collected and concentrated in Centricon-100 tubes.Preparation of RC47 from SynechocystisRC47 particles, containing the PSII reaction center (RCII) and the chlorophylla internal antenna CP47 but lacking the other chlorophylla internal antenna, CP43, were prepared from PSI-lessSynechocystis following the procedure described in Szabò et al. 2I. Szabò, F. Rigoni, M. Bianchetti, D. Carbonera, F. Pierantoni, R. Seraglia, and G. M. Giacometti, Submitted for publication. Briefly, thylakoid membranes from a PSI-less mutant strain ofSynechocystis were solubilized in 10% dodecyl maltoside for 30 min, subjected to anion exchange chromatography, and eluted with a linear 0–200 mm gradient of MgSO4. The elution profile comprised a single peak for RC47 at about 110 mmMgSo4.SDS-PAGESDS-polyacrylamide gel electrophoresis in the presence of urea (6 m) was performed as described previously (12Barbato R. Friso G. Giardi M.T. Rigoni F. Giacometti G.M. Biochemistry. 1991; 30: 10220-10226Crossref PubMed Scopus (53) Google Scholar).Irradiation ConditionsPSII samples at 90 μg/ml chlorophyll were irradiated in a cuvette with a 1-cm light path using a visible light intensity of 700 μmol of photons m−2 s−1 and a UV-B light intensity of 5 μmol of photons m−2s−1. A Vilbert-Lourmat 215M lamp was used as a UV-B source, wrapped in cellulose diacetate foil (0.15 mm thick) to screen out any UV-C component emitted by the UV-B source.RESULTSPSII Core and RCII Complexes from SpinachThe representative MALDI spectrum of a PSII core complex from spinach in the low molecular mass range (3000–6000 Da) is shown in Fig.1 A. At least 11 peaks are detected, and some of them, identifiable as known PSII subunits, are listed in Table I. It is well known that PSII core complexes of higher plants contain several low molecular weight polypeptides, as demonstrated by analysis of the plastidial genome (13Douglas S.E. Curr. Opin. Genet. Dev. 1998; 8: 655-661Crossref PubMed Scopus (168) Google Scholar, 14Shimada H. Sugiura M. Nucleic Acids Res. 1991; 19: 983-995Crossref PubMed Scopus (198) Google Scholar). Morris and co-workers (3Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 3935-3943Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 4Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 6Zheleva D. Sharma J. Panico M. Morris H.R. Barber J. J. Biol. Chem. 1998; 273: 16122-16127Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) studied polypeptides purified from preparations of PSII reaction center (RCII) and the RC47 complex of higher plants (pea and spinach, respectively) by HPLC purification/separation followed by ESI-MS analysis. RC47 is a PSII subcomplex, resulting from detergent-induced dissociation of internal antenna CP43 from the PSII core complex. To better identify the MALDI peaks of Fig. 1 A and to compare our results with those reported by Sharma et al. (3Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 3935-3943Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 4Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), the RCII from spinach was prepared and analyzed by MALDI.Table IProposed identification of measured m/z peaks in PSII or RCII from spinachPutative PSII subunit (spinach)Expected mass of unprocessed precursorMeasured mass from spinach PSII or RCII (this work)Measured mass from pea or spinach RCII or RC47 (3–6)psbTc3,8223,8533,849.6 sp.psbI4,1684,2014,195.5 sp.PsbK6,9144,2964,292.1 sp.psbL4,3664,3694,365.5 sp.β cytochromeb5594,3674,4124,409.1 sp.PsbW14,1775,9285,927.4 sp.α cytochromeb5599,2559,2609,255.1 sp.D138,81937,99838,040.9 peaD239,37639,37939,456.1 pea Open table in a new tab MALDI mass spectra for low and high molecular masses of RCII are shown in Fig. 1, B and C, respectively. SDS-PAGE analysis and the absorption spectra of the PSII core and RCII preparations, generally used to identify and characterize the various PSII subparticles obtained by detergent extraction, are shown in Fig.2. A small amount of light-harvesting complex II (LHCII) still present in the preparations is observable in both the SDS-PAGE and the 20–30-kDa range of the MALDI spectra (data not shown).Figure 2A, SDS-PAGE of PSII core complex (lane 1) and PSII reaction center (lane 2) from spinach; 1.5 μg of chlorophyll/lane (silver-staining). B, absorption spectrum of reaction center. Same preparations as in Fig.1.View Large Image Figure ViewerDownload (PPT)Table I compares our data with the expected mass values of unprocessed precursors of PSII components and with the results obtained by ESI-MS (3Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 3935-3943Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 4Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 5Sharma J. Panico M. Shipton C.A. Nilsson F. Morris H.R. Barber J. J. Biol. Chem. 1997; 272: 33158-33166Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6Zheleva D. Sharma J. Panico M. Morris H.R. Barber J. J. Biol. Chem. 1998; 273: 16122-16127Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). As may be seen, MALDI data on subunit composition and MW values in intact spinach PSII cores and RCII complexes closely match those obtained by Morris and co-workers (3Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 3935-3943Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 4Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 5Sharma J. Panico M. Shipton C.A. Nilsson F. Morris H.R. Barber J. J. Biol. Chem. 1997; 272: 33158-33166Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6Zheleva D. Sharma J. Panico M. Morris H.R. Barber J. J. Biol. Chem. 1998; 273: 16122-16127Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) on polypeptides purified from RCII and RC47. However, in the case of RCII in the low molecular mass region, we clearly detected an additional peak atm/z 5928, assigned to the PsbW protein. Accordingly, this protein has recently been suggested (15Irrgang K.D. Shi L.X. Funk C. Schroeder W.P. J. Biol. Chem. 1995; 270: 17588-17593Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) to be the sixth component of the RCII complex prepared from higher plants. PsbW has been detected by ESI-MS only in spinach RC47 (6Zheleva D. Sharma J. Panico M. Morris H.R. Barber J. J. Biol. Chem. 1998; 273: 16122-16127Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) but not in pea RCII (3Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 3935-3943Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). A peak at m/z 5928 assigned to PsbW is also visible in the spectrum of the PSII core complex (Fig.1 A). In the high molecular mass region, as expected for RCII, two main peaks are clear at m/z 37,998 and 39,379, attributed to D1 and D2 proteins, respectively (Fig.1 C).Altogether, these results show that, in particular in the low molecular mass range, direct measurements on PSII and RCII preparations are quite accurate and that MW values are comparable with those of isolated proteins obtained by ESI-MS.PSII Core Complexes from CyanobacteriaStudy of the structural-functional role of the subunit components of supramolecular complexes has been greatly advanced by the development of molecular genetic techniques such as the generation of mutants carrying site-specific modifications or gene deletions. In this respect, the identification of an easily transformable photosynthetic organism, with high frequency of homologous recombination, able to survive and propagate without functional photosystems, has been of great help. The cyanobacterium Synechocystis sp PCC 6803 fulfils these requirements, and, moreover, its whole genome has been sequenced. However, preparation of purified PSII subparticles and their isolated polypeptide components is even more difficult from cyanobacteria than from higher plants. A method to reveal and identify PSII components in cyanobacteria quickly, with no need for extended, time-consuming purification, would be useful, and it was for this reason that we focused our attention on Synechocystis.PSII cores were isolated from a PSI-less mutant cyanobacterium. Part of the psaAB operon coding for the core complex of PSI was deleted from the genome of Synechocystis wild-type strain (16Shen G. Boussiba S. Weermas W.F.J. Plant Cell. 1993; 5: 1853-1863Crossref PubMed Scopus (182) Google Scholar). PSI-less cells have approximately six times more PSII than do wild-type cells, as estimated on a chlorophyll basis. The MALDI spectra of Fig. 3 were obtained from two different preparations. In the 20,000–70,000 m/zrange, seven main peaks were detected at m/z55276 ± 424, 51470 ± 9, 39268 ± 37, 38049 ± 17, 34399 ± 9, 31030 ± 7, and 25750 ± 9 (n = 16 spectra from 8 different preparations). Each MALDI measurement was highly reproducible for the same sample, and good reproducibility was observed in the m/z values of the peaks for samples deriving from different preparations (compareA and B of Fig. 3). However, the relative intensity of the peaks depended on sample preparation, due to the presence of varying amounts of interfering materials, such as salts and lipids (17Xiang F. Beavis R.C. Org. Mass Spectrom. 1993; 28: 1424-1428Crossref Scopus (69) Google Scholar).Figure 3MALDI spectra of two distinct preparations of PSII core complex from Synechocystis. m/z values are indicated in A; assignment of the main peaks is shown in B.View Large Image Figure ViewerDownload (PPT)Molecular weight values obtained by MALDI were compared with protein masses calculated from the nucleotide sequences of PSII components. Good correlations between calculated and measured values revealed the following correspondence between measured m/zvalues and known PSII components: 55,276 m/z, CP47 (molecular mass of unprocessed precursor, 55,902 Da); 51,470 m/z, CP43 (molecular mass of unprocessed precursor, 51,760 Da); 39,268 m/z, D2; 38,049m/z, D1 (Fig. 3 B). It is worth noting that the two latter values are very close to those ascribed to the predominant form of D2 and D1 purified from pea by reverse phase HPLC and detected by ESI-MS (see above). Concerning the other peaks detectable in the spectra of Fig. 3, that at m/z25,750 may correspond to the doubly charged species of the ion atm/z 51,470. The peaks atm/z 31,030 and 34,399 may represent subunits of ATP synthase (predicted masses of 30,698 and 34,605 Da, respectively). Alternatively, the peak at m/z 31,030 may correspond to the phycobilisome 32-kDa linker peptide (predicted mass of 30,797 Da). Clear-cut identification of these peaks would require the use of mutants lacking these proteins.It is worth noting that the m/z peaks assigned to the apoproteins of the two internal antennae, CP43 (m/z 51,460) and CP47 (m/z55,276), are reproducibly very different in their amplitude, despite the fact that both components have a 1:1 stoichiometric ratio to the reaction center. This may be related to the different ionization probability of the two subunits when embedded in the PSII core complex. It might be speculated that CP43, which is less closely associated with the core and is located in a more external position with respect to CP47 (18Barber J. Biochim. Biophys. Acta. 1998; 1365: 269-277Crossref PubMed Scopus (92) Google Scholar), offers a higher cross-section for ionization to incoming laser photons. To confirm unequivocally the assignment of them/z peaks to the internal antennae, we prepared an RC47 particle from the Synechocystis PSII core. This PSII subparticle, obtained by dissociating the CP43 subunit by partial detergent solubilization, is the cyanobacterial analogue of the RC47 obtained from higher plants by other groups (6Zheleva D. Sharma J. Panico M. Morris H.R. Barber J. J. Biol. Chem. 1998; 273: 16122-16127Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). An immunoblot of its polypeptide components is shown in Fig.4 C. Whereas the D1, D2, and CP47 subunits of the PSII core are left unchanged in the RC47 particle (Fig. 4 C, lanes 2, 4, and8), only a tiny amount of CP43 is contained in the latter (lane 6). If the proposed assignments are correct, we expect the m/z peak at 51,470 to disappear from the MALDI spectrum of the RC47 particle. Fig. 4 compares the spectrum of RC47 (B) with that of the core complex (A). The expectation is fully matched, because the m/zpeak at 51,470 is highly reduced with respect to the intensity of the D1 and D2 peaks. It may be noted that the peak at 55,276, attributed to CP47, maintains approximately the same relative intensity. Therefore, an eventual shadowing action on CP47 must be exerted by core proteins other than CP43, in accordance with the recent finding that the two internal antennae are on opposite sites of the D1-D2 heterodimer, rather than sequentially located within the PSII core (20Hankamer B. Morris E.P. Barber J. Nat. Struct. Biol. 1999; 6: 560-564Crossref PubMed Scopus (116) Google Scholar). It may also be noted that detergent treatment on sample A to produce RC47 (Fig.4 B) also eliminates the impurities giving rise tom/z peaks in the range of 25–35 kDa.Figure 4MALDI spectra of PSII core complexes (A) and RC47 subparticle (B) fromSynechocystis. Immunoblots (C) of PSII core complexes (lanes 1, 3, 5, and7) and RC47 (lanes 2, 4, 6, and 8). Lanes 1 and 2, 3and 4, 5 and 6, and 7 and8 were probed with antibodies specific for D1, D2, CP43, and CP47, respectively. PSII samples were loaded at 0.25 μg of chlorophyll.View Large Image Figure ViewerDownload (PPT)MALDI Spectrum of High MW Subunits of PSII Core Complex in Light Stress ConditionsIt is known that the D2 and, to an even greater extent, D1 proteins of higher plants and cyanobacteria are characterized by high turnover and that their degradation rate is increased by UV-B radiation (21Friso G. Barbato R. Giacometti G.M. Barber J. FEBS Lett. 1994; 339: 217-221Crossref PubMed Scopus (56) Google Scholar, 22Giacometti G.M. Barbato R. Chiaramonte S. Friso G. Rigoni F. Eur. J. Biochem. 1996; 242: 799-806Crossref PubMed Scopus (18) Google Scholar, 23Mate Z. Sass L. Szekeres M. Vass I. Nagy F. J. Biol. Chem. 1998; 273: 17439-17444Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 24Vass I. Kirilovsky D. Perewoska I. Mate Z. Nagy F. Etienne A.L. Eur. J. Biochem. 2000; 267: 2640-2648Crossref PubMed Scopus (28) Google Scholar, 25Barbato R. Bergo E. Szabò I. Dalla-Vecchia F. Giacometti G.M. J. Biol. Chem. 2000; 275: 10976-10982Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) or an excess of photosynthetic active radiation (26Greenberg B.M. Gaba V. Canaani O. Malkin S. Mattoo A.K. Edelman M. Proc. Natl. Acad. Sci. U. S. A. 1987; 86: 6617-6620Crossref Scopus (265) Google Scholar, 27Barbato R. Friso G. de-Laureto P.P. Frizzo A. Rigoni F. Giacometti G.M. FEBS Lett. 1992; 311: 33-36Crossref PubMed Scopus (29) Google Scholar, 28Aro E.M. Virgin I. Andersson B. Biochim. Biophys. Acta. 1993; 1143: 113-134Crossref PubMed Scopus (1892) Google Scholar, 29Kettunen R. Tyystjarvi E. Aro E.M. Plant Physiol. 1996; 111: 1183-1190Crossref PubMed Scopus (53) Google Scholar). The usual way to reveal these phenomena is to measure the D1 and D2 contents of the thylakoid membrane after exposure to radiation by SDS-PAGE and immunoblotting and then to search for the generated fragments by the same methods. The following experiment aimed at checking whether the MALDI technique could be used as an alternative, direct method to study the phenomenon. Fig.5 shows an example of this application. PSII isolated from PSI-less organisms was left untreated (Fig. 5 A) or was irradiated with visible light (B) or UV-B light (C) for 30 min. The intensity of each peak was measured in the three samples deriving from the same preparation and expressed as a percentage of the intensity of the CP43 peak (m/z 51,470), which is known to be unaffected by the light intensities used here (30Rajagopal S. Murthy S.D. Mohanty P. J. Photochem. Photobiol. B Biol. 2000; 54: 61-66Crossref PubMed Scopus (34) Google Scholar). Whereas the relative intensities of most of the peaks did not change upon exposure to radiation, those corresponding to D1 and D2 (m/z 38,048 and 39,268, respectively) showed marked variations. In particular, the intensities of the D1 peak decreased by 63% in visible light-illuminated PSII and by 13% in the UV-B-treated sample. The intensities of the D2 peak decreased by 45 and 35%, respectively. It is worth stressing the point that the three spectra (Fig. 5, A–C) were obtained from aliquots of the same sample, thus ensuring that evaluation of the peak areas was accurate enough to make quantitative comparisons significant. An unidentified peak at m/z 31,071 was also found to be slightly affected by both kinds of irradiation, suggesting that this protein is involved in the stress response.Figure 5MALDI spectra of PSII core complexes fromSynechocystis: untreated sample (A) and samples irradiated with visible (B) and UV-B light (C). See “Materials and Methods” for irradiation conditions.View Large Image Figure ViewerDownload (PPT)Low MW Components of PSII from CyanobacteriaBecause the low MW subunits of cyanobacteria and spinach PSII differ in their predicted masses, the low molecular mass region of the MALDI spectra of PSII core complex prepared from the PSI-less strain of Synechocystiswas examined (Fig. 6). Detected peaks were tentatively identified with the main known subunits, as listed in Table II.Figure 6MALDI spectrum in the low MW region of PSII core complex isolated from Synechocystis.View Large Image Figure ViewerDownload (PPT)Table IIProposed identification of m/z peaks of low MW subunits of PSII from SynechocystisPutative subunitCalculated mass of unprocessed precursor (Synechocystis PCC6803)Measured mass from PSII of cyanobacteria (n = 7)PsbM38823920 ± 2PsbY42014219 ± 0.5PsbI43064342.5 ± 1.6PsbL44734481 ± 1β cytochromeb55948024805.5 ± 0.8PsbK51125180 ± 0.5PsbH69856991 ± 2α cytochrome b55993179329 ± 1.2 Open table in a new tab The identity of a few peaks is still unresolved. For example, the nature of the peaks at m/z 3570 and 8170 is unknown, whereas the peak at m/z 8008 may correspond either to a phycobilisome 7.8-kDa linker peptide or to a subunit of ATP synthase (calculated masses are 7805 and 7968 Da, respectively).In an intermediate molecular mass region (data not shown), one peak atm/z 12,474 may correspond to the psbW precursor (unprocessed protein mass of 12,590 Da), and another peak atm/z 14,468 may correspond to cytochromec550, al
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