Investigation of the Lateral Light-induced Migration of Photosystem II Light-harvesting Proteins by Nano-high Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry
2005; Elsevier BV; Volume: 280; Issue: 32 Linguagem: Inglês
10.1074/jbc.m504998200
ISSN1083-351X
AutoresAnna Maria Timperio, Lello Zolla,
Tópico(s)Photoreceptor and optogenetics research
ResumoThis study reports a detailed analysis of the light-induced lateral migration of the photosystem II (PSII) antennae between appressed and non-appressed thylakoid membranes. The relative PSII antennae that migrated to stroma lamellae were readily established on the basis of peak areas of the separated stroma proteins in the ultraviolet chromatograms. Phosphorylation was predicted by intact molecular mass measurements, and this was confirmed by immunoblotting. When thylakoid membrane and chloroplasts were illuminated at 100 μE m–2s–1, light-harvesting complex type II (Lhcb2) was the first PSII antenna to migrate, preferentially in phosphorylated form. However, the amount of Lhcb2 that migrated decreased after the first 20 min when the total amount of the three different Lhcb1 isoforms (1.1, 1.2, and 1.3) reached maximum. Lhcb1.1 was always found in the unphosphorylated form and migrated later than the other two isoforms, although the latter were also found to have low levels of phosphorylation. At the same time, major antennae on the grana were not found to be phosphorylated, whereas Lhcb4 showed a significant increase in molecular mass. At higher light intensity Lhcb2 migration was negligible, whereas migration of Lhcb1 isoforms was little changed, increasing in irradiated chloroplasts. Because there was no significant phosphorylation at high light intensity, and yet pigments were found to have significantly increased on the stroma lamellae, it may be that pigments play a role in migration and that, in fact, there is no direct correlation between phosphorylation and migration. We hypothesize that the Lhcb1 isoforms expressed by the multigene families play a role in plant adaptation. This study reports a detailed analysis of the light-induced lateral migration of the photosystem II (PSII) antennae between appressed and non-appressed thylakoid membranes. The relative PSII antennae that migrated to stroma lamellae were readily established on the basis of peak areas of the separated stroma proteins in the ultraviolet chromatograms. Phosphorylation was predicted by intact molecular mass measurements, and this was confirmed by immunoblotting. When thylakoid membrane and chloroplasts were illuminated at 100 μE m–2s–1, light-harvesting complex type II (Lhcb2) was the first PSII antenna to migrate, preferentially in phosphorylated form. However, the amount of Lhcb2 that migrated decreased after the first 20 min when the total amount of the three different Lhcb1 isoforms (1.1, 1.2, and 1.3) reached maximum. Lhcb1.1 was always found in the unphosphorylated form and migrated later than the other two isoforms, although the latter were also found to have low levels of phosphorylation. At the same time, major antennae on the grana were not found to be phosphorylated, whereas Lhcb4 showed a significant increase in molecular mass. At higher light intensity Lhcb2 migration was negligible, whereas migration of Lhcb1 isoforms was little changed, increasing in irradiated chloroplasts. Because there was no significant phosphorylation at high light intensity, and yet pigments were found to have significantly increased on the stroma lamellae, it may be that pigments play a role in migration and that, in fact, there is no direct correlation between phosphorylation and migration. We hypothesize that the Lhcb1 isoforms expressed by the multigene families play a role in plant adaptation. The main constituents of higher plants for the harvesting of solar energy and the light-driven electron transport of photosynthesis are contained in two main complexes of the thylakoid membranes: photosystem I and photosystem II (PSI 1The abbreviations used are: PSI and II, photosystem I and II; LHC I and II, light-harvesting complex I and II; HPLC, high performance liquid chromatography; ESI, electrospray ionization; MS, mass spectrometry; RP, reversed phase; MES, 4-morpholineethanesulfonic acid. and PSII, respectively). Photosystem I is localized in the stroma lamellae and peripheral membranes of the grana, whereas Photosystem II is localized mainly in grana appressions. Each photosystem is composed of several chlorophyll-protein complexes, most of which function as antennae that capture visible light (1Hankamer B. Barber J. Boekema E.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 641-671Crossref PubMed Scopus (296) Google Scholar). These are called light-harvesting chlorophyll a/b pigment proteins (LHCI and LHCII, respectively, or Lhca and Lhcb). The major light-harvesting proteins of PSII have been designated Lhcb1, Lhcb2, and Lhcb3 (1Hankamer B. Barber J. Boekema E.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 641-671Crossref PubMed Scopus (296) Google Scholar, 2Rogner M. Boekema E.J. Barber J. Trends Biochem. Sci. 1996; 21: 44-49Abstract Full Text PDF PubMed Google Scholar) on the basis of the nomenclature for the genes encoding these proteins (3Jansson S. Pichersky E. Bassi R. Green B.R. Ikeuchi M. Melis A. Simpson D.J. Spangfort M. Staehelin L.A. Thornber J.P. Plant Mol. Biol. Rep. 1992; 10: 242-248Crossref Scopus (171) Google Scholar), whereas other less abundant proteins have been called minor antenna proteins and designated Lhcb4, Lhcb5, and Lhcb6. LHCII can form Lhcb1 homotrimers and Lhcb1/2 heterotrimers, which are believed to be a mobile complex (4Larsson U.K. Anderson J.M. Andersson B. Biochim. Biophys. Acta. 1987; 894: 69-75Crossref Scopus (68) Google Scholar, 5Jackowski G. Kacprzak K. Jansson S. Biochim. Biophys. Acta. 2001; 1504: 340-345Crossref PubMed Scopus (54) Google Scholar). Recently it has been demonstrated that Lhcb1 antenna proteins can exist in several very similar isoforms (6Huber C.G. Timperio A.M. Zolla L. J. Biol. Chem. 2001; 276: 45755-45761Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Because specific isoforms are not well conserved between species, it is not clear whether they are redundant rather than having specific functions. The PSI antenna proteins are made up of four different chlorophyll/carotenoid binding polypeptides (Lhca1–4) that are organized into dimers (7Chitnis P.R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 593-626Crossref PubMed Scopus (207) Google Scholar, 8Sheller H.V. Jensen P.E. Haldrup A. Lunde C. Knoetzel J. Biochim. Biophys. Acta. 2001; 1507: 41-60Crossref PubMed Scopus (170) Google Scholar). To optimize photosynthetic performance and to avoid damage when exposed to excess light, plants must balance the excitation of the two photosystems (9Allen J.F. Bennett J. Steinback K.E. Arntzen C.J. Nature. 1981; 291: 25-29Crossref Scopus (518) Google Scholar, 10Allen J.F. Biochim. Biophys. Acta. 1992; 1098: 275-335Crossref PubMed Scopus (727) Google Scholar, 11Allen J.F. Forsberg L. Trends Plant Sci. 2001; 6: 317-326Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). When photosystem II is favored, a mobile pool of light-harvesting complex II moves from photosystem II to photosystem I. The dissociation of a mobile subpopulation of the phospho-LHCII from PSII and its subsequent lateral migration from the appressed grana membranes to the PSI containing stroma-exposed membranes (12Kyle D.J. Staehelin L.A. Arntzen C.J. Arch. Biochem. Biophys. 1983; 222: 527-541Crossref PubMed Scopus (169) Google Scholar) are thought to be important for balancing the excitation energy, protection against photodestruction of PSII, and acclimation of the photosynthetic apparatus in plants (12Kyle D.J. Staehelin L.A. Arntzen C.J. Arch. Biochem. Biophys. 1983; 222: 527-541Crossref PubMed Scopus (169) Google Scholar). This short term and reversible redistribution is known as a state transition. It is associated with changes in the phosphorylation of light-harvesting complex II, but the regulation is complex. Two models have been proposed to explain the movement of LHCII. According to one model, alteration in the surface charge upon phosphorylation leads to structural changes of the thylakoid membrane and results in the movement of phospho-LHCII away from the grana stacks (13Barber J. Annu. Rev. Plant Physiol. 1982; 33: 261-295Crossref Google Scholar, 14Bennett J. Biochem. J. 1983; 212: 1-13Crossref PubMed Scopus (189) Google Scholar, 15Bennett J. Physiol. Plant. 1984; 60: 583-590Crossref Scopus (55) Google Scholar). The addition of negatively charged phosphate groups to the LHCII complex causes it to dissociate from PSII and favors its migration out of the appressed regions of the grana and into the stroma lamellae, where PSI centers are located (16Andersson B. Akerlund H.E. Jergil B. Larson C. FEBS Lett. 1982; 149: 181-184Crossref Scopus (55) Google Scholar). This indicates that the correlation between LHCII phosphorylation and state transition is complex. According to another model, the net movement of LHCII toward PSI in State 2 is caused by PSII having a higher affinity for unphosphorylated LHCII and PSI a higher affinity for phospho-LHCII; therefore, movement of phospho-LHCII is a question of molecular recognition (11Allen J.F. Forsberg L. Trends Plant Sci. 2001; 6: 317-326Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Although the models differ in the way of explaining state transitions, they both involve phosphorylation of LHCII as a prerequisite for the initiation of State 1-State 2 transitions. The purpose of the present work was to better characterize and quantify which type of chlorophyll proteins, comprising the isoforms, are involved in the migration and consequently in the regulation of energy distribution. For this purpose both spinach chloroplasts and thylakoid membrane were subjected to varying light stress over different time periods. Stroma and grana lamellae were then isolated using digitonin, and their protein composition was analyzed by nano-HPLC coupled with mass spectrometry. Through this technique it has been possible to identify and quantify which type of Lhcb1 isomer is involved in the migration and whether it exists in the phosphorylated form. Differences were observed at low and high light intensity. Chemicals—Reagent-grade sodium chloride, magnesium chloride, sorbitol, N-tris(hydroxymethyl)methylglycine (Tricine), tris-(hydroxymethyl)aminomethane (Tris), natrium fluoride, trifluoroacetic acid, methanol, ethanol, formamide, as well as HPLC-grade water and acetonitrile, were obtained from Carlo Erba (Milan, Italy). Digitonin was obtained from Sigma, acrylamide, N,N′ methylene-bis-acrylamide, and all other reagents for SDS-PAGE were purchased from Bio-Rad. Plant Material and Growth Conditions—Hydroponic cultures of spinach (Spinacia oleracea L.) were grown in a greenhouse at a photon flux density of 400 μmol m–2 s–1 with 10-h light/14-h dark rhythm at 25 °C. Whole leaves were used in the experiments. Isolation of Intact Chloroplasts—Well developed spinach leaves were used to isolate intact chloroplasts using the method of Walker et al. (17Walker D.A. Cerovic Z.G. Robinson S.P. Methods Enzymol. 1987; 148: 145-157Crossref Scopus (67) Google Scholar) with minor modifications. Briefly, chloroplast isolation procedures were carried out at 4 °C. Spinach leaves (10 g), grown under normal light conditions and harvested in the middle of the photoperiod, were chopped using a blender in 50 ml of extraction buffer (0.3 mm sorbitol, 50 mm HEPES/KOH, pH 7.9, 5 mm MgCl2, 2 mm isoascorbate). The homogenates were then filtered through 100-mm meshes, followed by centrifugation at 1006 × g for 3 min. To enrich for chloroplasts, the pellets were resuspended in 2 ml of extraction buffer and precipitated again at 10006 × g for 7 min. Chloroplasts were further purified by isopycnic centrifugation using 50% Percoll gradients (18Douce R. Joyard J. Edelman M. Hallick R. Chua N.-H. Methods in Chloroplast Molecular Biology. Elsevier, Amsterdam1982: 239-256Google Scholar). Isolation of Chloroplast Thylakoids—Chloroplast thylakoid membranes (PSII membranes) were isolated from spinach leaves according to the method of Berthold (19Berthold D.A. Babcock G.T. Yocum C.F. FEBS Lett. 1981; 134: 231-234Crossref Scopus (1651) Google Scholar) with the following modifications. Leaves were powdered in liquid nitrogen and subsequently homogenized in an ice-cold 20 mm Tricine pH 7.8 buffer containing 0.3 m sucrose and 5.0 mm magnesium chloride (B1 buffer). The homogenization was followed by filtration through one layer of Miracloth (Calbiochem) and centrifuged at 4,000 × g for 10 min at 4 °C. Pellets were suspended in B1 buffer and centrifuged as above. This second pellet was resuspended in 20 mm Tricine pH 7.8 buffer containing 70 mm sucrose and 5.0 mm magnesium chloride (B2 buffer) and centrifuged at 4,500 × g for 10 min. Pellets containing the thylakoid membranes were then resuspended in 50 mm MES pH 6.3 buffer containing 15 mm sodium chloride and 5 mm magnesium chloride (B3 buffer) at 2.0 mg of chlorophyll/ml. The concentration of chlorophyll was determined according to the method described by Porra (20Porra R.J. Thompson W.A. Kriedemann P.E. Biochim. Biophys. Acta. 1989; 975: 384-394Crossref Scopus (4696) Google Scholar). Light Treatments of the Chloroplast and Thylakoid Membranes— Migration of PSII antennae was induced in spinach thylakoid membranes and in chloroplasts, at 0.2 mg of chlorophyll/ml, by exposure to 100, 500, and 1000 μE m–2 s–1 from a metal halide HQI-T 250W daylight lamp that served as a light source in a temperature-controlled glass cuvette at 4 °C with gentle stirring. Chloroplasts were gently resuspended in an incubation buffer containing 330 mm sorbitol, 20 mm Tricine (pH 8), 6.6 mm MgCl2, 1 mm Na2HPO4, whereas isolated thylakoids were resuspended in 20 mm Tricine (pH 8), 100 mm sorbitol, 5 mm MgCl2, 1 mm ATP, and both were irradiated as described above. Separation of Grana/Stroma Lamellae—Thylakoid membranes and chloroplasts were resuspended to a concentration of 200 μg of Chl/ml and 55 μg of Chl/ml, respectively, in 20 mm Tricine (pH 7.8), 0.1 m sorbitol (0.3 m for chloroplast), 10 mm NaCl, 5 mm MgCl2, 10 mm NaF. Recrystallized digitonin (1% in water) was added to the stirred membranes to give a final concentration of 0.4%. The 2-min detergent treatment was terminated by a 10-fold dilution of the sample with resuspension buffer at 0 °C. Differential centrifugation according to Anderson and Boardman (21Anderson J.M. Boardman N.K. Biochim. Biophys. Acta. 1966; 112: 403-410Crossref Scopus (248) Google Scholar) yielded pellets following 1,000 × g for 10 min, 10,000 × g for 30 min, 40,000 × g for 30 min, and 144,000 × g for 60 min. The centrifuged fractions were injected directly onto the column without any further sample pretreatment. Nano-High Performance Liquid Chromatography and Electrospray Mass Spectrometry—Liquid chromatography was carried out at 200 nl/min using an Ultimate nano-HPLC (LC-Packings, a Dionex Company). The column was interfaced with an ion trap mass spectrometer Esquire 3000 plus (Bruker Daltonik). The UV absorbance was monitored at 280 nm. Samples were introduced onto the capillary column by a sample injection valve with a 1-μl sample loop. The proteins were separated in a reversed-phase capillary column packed with 5-μm porous butyl silica particles (Vydac Protein C-4, 15 cm × 180 μm ID; The Separations Group, Inc., Hesperia, CA). All solutions were filtered through a membrane filter (type FH 0.5-μm; Millipore) and degassed by sparging with helium during use. The Vydac C-4 columns were preequilibrated with 10% (v/v) aqueous acetonitrile solution containing 0.05% trifluoroacetic acid, and samples were eluted using a gradient consisting of a first linear gradient from 10 to 40% (v/v) acetonitrile in 15 min, followed by a second gradient segment from 40 to 90% (v/v) acetonitrile in 60 min. At the end of the run, the column was flushed with 100% acetonitrile for 3 min. This post-run gradient was used to ensure that hydrophobic contaminants of proteins were eluted from the column by the second gradient segment up to 95% acetonitrile. The ESI-MS was performed on an ion trap mass spectrometer (Esquire 3000 plus; Bruker Daltonik). For analysis with pneumatically assisted ESI, an electrospray voltage of 3–4 kV and a nitrogen sheath gas flow were employed. The temperature of dry gas was set to 300 °C. The scan range was 500–2000 atomic mass unit. Fine tuning for ESI-MS of proteins was accomplished by infusion of 3.0 μl/min of a 0.4-pmol/μl solution of cytochrome c or a 6.9-pmol/μl solution of carbonic anhydrase in 0.050% aqueous trifluoroacetic acid solution containing 20% acetonitrile (v/v). Polyacrylamide Gel Electrophoresis—To determine protein composition, the samples separated by HPLC were dried and solubilized in 4% lauryl sulfate, 120 mm dithiothreitol, and 120 mm Tris/HCl, pH 8.45, 5 m urea and then run in SDS-PAGE of uniform polyacrylamide concentration (13%) with the Tris/Tricine system (22Schagger H. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar), using an Invitrogen model V16 vertical gel electrophoresis system. Gels were fixed and stained for 2 h in a 5:1:4 (v/v) methanol-glacial acetic acid-water mixture, containing 0.1% (w/v) Coomassie Brilliant Blue. For silver staining, gels were fixed in 50% (v/v) methanol-water and 10% (v/v) ethanol-water solutions, stained with 0.1% (w/v) silver nitrate-water solution, and developed in 3.5% (w/v) aqueous sodium carbonate containing 0.05% (v/v) formamide. Detection of Thylakoid Phosphoproteins by Polyclonal Thr(P) Antibody—Following electrophoresis, the polypeptides were transferred to an Immobilon-P membrane (Millipore), which was blocked with 1% bovine serum albumin (fatty acid; Sigma). Phosphoproteins were immunodetected using an Immun-Lite assay kit (Bio-Rad). Three different commercial antibodies to phosphothreonine were tested: rabbit polyclonal antibody and monoclonal antibody (Zymed Laboratories Inc. and mouse monoclonal anti-phosphothreonine (Sigma). Pigment Determination by High Performance Liquid Chromatography—Thylakoid membrane or chloroplasts were frozen in liquid N2 immediately after sampling. Pigments were extracted from samples under low and high light by grinding in liquid N2 followed by grinding in degassed 100% acetone at 0–4 °C. On occasion a small amount of NaHCO3 (∼0.1 g·g–1 plant material) was added to plant material before extraction to ensure no acidification of the extract occurred; however, this had no influence on carotenoid composition. The pigment extracts were filtered through a 0.2-mm membrane filter and applied immediately to an HPLC column. Pigment composition was determined by HPLC by using a Waters Nova-Pak C18 radial compression column according to Johnson et al. (23Johnson G.N. Scholes J.D. Horton P. Young A.J. Plant Cell Environ. 1993; 16: 681-686Crossref Scopus (78) Google Scholar). Additionally a Spherisorb ODS-1 column (5-mm particle size, 250 × 4.6 mm ID) was used based on the method by Gilmore and Yamamoto (24Gilmore A.M. Yamamoto H.Y. J. Chromatogr. 1991; 543: 137-145Crossref Scopus (412) Google Scholar). Pigment concentrations were calculated from the respective peak areas at 440 nm. Before injection, however, samples were normalized on the basis of chlorophyll a and b concentration evaluated by spectrophotometric absorption. To investigate qualitatively and quantitatively which type of PSII antenna protein is involved in light-induced migration and over what time period this occurs, spinach thylakoid membrane or chloroplasts were illuminated at different light intensities. The analysis consisted of separating proteins by nano-reversed-phase liquid chromatography (nano-RP-HPLC), interfaced to mass spectrometry (MS) with an electrospray (ESI) ion source (nano-RP-HPLC ES-MS) to identify them. Both thylakoids and chloroplasts were irradiated at different light intensities in the presence of the phosphatase inhibitor NaF, in order to block dephosphorylation during sample manipulation, and then assayed for phosphorylation capacity (25Rintamaki E. Salonem M. Suaranta U.M. Carlberg I. Andersson B. Aro E.M. J. Biol. Chem. 1997; 272: 30476-30482Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). The actual distribution of the various forms of PSII complexes in the thylakoid membrane was obtained using digitonin, which causes specific and rapid (<3 min) cleavage of the thylakoid membrane (26Timperio A.M. Huber C.G. Zolla L. J. Chromatogr. A. 2004; 1040: 73-81Crossref PubMed Scopus (12) Google Scholar) into grana and stroma lamellae. Recent studies that used this technique found that grana membranes were strongly enriched in PSII dimers and PSII-LHCII supercomplexes, whereas only a small amount of PSI antenna proteins and traces of PSI complexes were present (26Timperio A.M. Huber C.G. Zolla L. J. Chromatogr. A. 2004; 1040: 73-81Crossref PubMed Scopus (12) Google Scholar). In the stroma thylakoids, the PSII complexes as well as the various free LHCII subassemblies were in the minority, whereas the PSI complexes and ATP synthase were the dominating membrane protein complexes (27Aro E.-M. Msuorsa A. Rokka Y. Allahverdiyeva V. Paakkarinen A. Saleem N.B. Rintamaki E. J. Exp. Bot. 2004; 29: 1-10Google Scholar, 28Danielsson R. Albertsson P.-A. Mamedov F. Styring S. Biochim. Biophys. Acta. 2004; 1608: 53-61Crossref PubMed Scopus (117) Google Scholar). The stroma lamellae from leaves kept in darkness overnight were found to contain a small amount of PSII Lhcb1 antennae, estimated as 10% of the Lhca3 present; the latter was chosen as an internal reference (26Timperio A.M. Huber C.G. Zolla L. J. Chromatogr. A. 2004; 1040: 73-81Crossref PubMed Scopus (12) Google Scholar), whereas Lhcb2 levels were negligible. Thus the heavier grana membranes were easily and quickly separated from the lighter stroma membranes, which were recovered from the supernatant. Grana and stroma lamellae were separately injected onto a capillary C4 column and chromatographed using a linear water-acetonitrile elution gradient in trifluoroacetic acid. Because antenna proteins from PSI have different elution times than PSII antennae, it was easy to determine the relative amount of each. Further confirmation of protein identity in each UV trace was obtained by deconvolution analysis of each reconstruction ion current chromatogram peak (26Timperio A.M. Huber C.G. Zolla L. J. Chromatogr. A. 2004; 1040: 73-81Crossref PubMed Scopus (12) Google Scholar). This method allowed rapid separation and identification of stroma and grana lamellae proteins with few manipulations, while the relative distributions of the chlorophyll-protein complexes derived from appressed (grana) and non-appressed (stroma) regions, before and after illumination of leaves, were determined from the area underlying each chromatographic peak. Illumination at 100 μE m–2s–1—In a pilot investigation thylakoids were irradiated at 100 μE m–2s–1 for 20 min in the presence of ATP 0.4 mm to obtain the maxima antenna migration, in accordance with work reported by Rintamaki (25Rintamaki E. Salonem M. Suaranta U.M. Carlberg I. Andersson B. Aro E.M. J. Biol. Chem. 1997; 272: 30476-30482Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Fig. 1 compares the chromatogram recorded by nano-HPLC at 280 upon injection of stroma proteins extracted from control or light-stressed thylakoids. A significant increase was recorded in peaks corresponding to Lhcb1 (Lhcb1.1, Lhcb1.2, and Lhcb1.3, respectively) after illumination. Although leaves kept in darkness overnight contained a small amount of Lhcb1 (∼10%) (26Timperio A.M. Huber C.G. Zolla L. J. Chromatogr. A. 2004; 1040: 73-81Crossref PubMed Scopus (12) Google Scholar), the amount increased significantly upon illumination. Interestingly, a new peak appeared near Lhca3, which deconvolution analysis (see later) showed to be PSII Lhcb2. It is worth emphasizing that it was barely detected in stroma lamellae kept in darkness overnight. Fig. 2 shows the reconstructed ion current that was recorded simultaneously with the UV trace upon injection of the stroma membrane fraction. As previously shown (26Timperio A.M. Huber C.G. Zolla L. J. Chromatogr. A. 2004; 1040: 73-81Crossref PubMed Scopus (12) Google Scholar, 27Aro E.-M. Msuorsa A. Rokka Y. Allahverdiyeva V. Paakkarinen A. Saleem N.B. Rintamaki E. J. Exp. Bot. 2004; 29: 1-10Google Scholar), most of the UV peaks had a corresponding peak in the reconstructed ion chromatograms, so it was possible to identify each protein by deconvolution analysis of ESI spectra and determine whether it had undergone any chemical modifications. Deconvolution analysis of recontruction ion current (RIC) spectra corresponding to Lhcb2 (Fig. 2C, inset) showed that two proteins were present with a difference in molecular mass of 80 Da (24,760 and 24,839, respectively). These are probably the phosphorylated and unphosphorylated forms of Lhcb2, because the difference in molecular mass corresponds to a single phosphorylation. Interestingly, with deconvolution analysis of ESI spectra it is possible to estimate approximately the amount of phosphorylated protein present as a percentage of the total protein by comparing the intensity of deconvolution of each protein. In the case of Lhcb2, it was ∼70% phosphorylated (see Fig. 2C, inset), suggesting that most of this protein migrated in this form. Regarding the Lhcb1, reversed phase of Lhcb1 differentiated two peaks, the first one containing the Lhcb1.1 isomeric form and the second one the isomers Lhcb1.2 and Lhcb1.3, respectively (29Walcher W. Timperio A.M. Zolla L. Huber C.G. Anal. Chem. 2003; 75: 6775-6780Crossref PubMed Scopus (16) Google Scholar). Deconvolution analysis of reconstruction ion current spectra revealed that Lhcb1.1 (Fig. 2A, inset) was not phosphorylated upon illumination, whereas both Lhcb1.2 and Lhcb1.3 had phosphorylated forms (Fig. 2B, inset), displaying the characteristic increase in molecular mass of 80 Da (Lhcb1.2: 24.936.7 and 25.019; Lhcb1.3: 25.006 and 25.083). However, in contrast to Lhcb2, the phosphorylated form of both Lhcb1.2 and Lhcb1.3 is <10% of the total protein (see Fig. 2B, inset). To confirm that these 80-Da mass increases actually represent protein phosphorylation, the stroma proteins were run on semipreparative RP-HPLC in a butyl silica column to separate the main peaks, which were collected, lyophilized, and probed with phosphothreonine antibody. The latter was performed by dissolving fractions containing the major antennae Lhcb2, or Lhcb1.1, or Lhcb1.2 and Lhcb1.3, or Lhcb4 in 120 mm TRIS/HCl pH 8.45 buffer containing 120 mm dithiothreitol, 5 m urea, and 4% (w/v) SDS, which were then analyzed by SDS-PAGE urea according to the method reported by Schagger (22Schagger H. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar) (Fig. 3A). Following electrophoresis, the gels were either silver stained or transferred to nitrocellulose and incubated with a phosphothreonine antibody (Fig. 3B). As expected, the antibody did not bind to Lhcb1.1, whereas a signal was detected for Lhcb2 and the two isomers Lhcb1.2 and Lhcb1.3; therefore, measuring differences in intact molecular mass appears to be a reliable way of identifying proteins and their post-translational phosphorylation. This analysis also confirmed previous observations of relative phospho-protein concentrations derived from immunoblots of total thylakoid membrane (30Hou C.-X. Rintamaki E. Aro E.-M. Biochemistry. 2003; 42: 5828-5836Crossref PubMed Scopus (25) Google Scholar) and intact molecular mass measurements in the present study, because the Lhcb2 (70% phosphorylated) band stained much more intensely with Coomassie than the Lhcb1.2 and Lhcb1.3 bands (10%). Only traces of PSII Lhcb3 were found on illuminated stroma lamellae, and no minor antenna proteins have ever been detected. We then went back to the grana lamellae, which were present in the bottom of centrifuged thylakoid after digitonin treatment, and analyzed them as above. Comparison of HPLC chromatograms from control or stressed grana did not reveal any significant differences in terms of peak number and relative stoichiometry (data not shown). This was partly because the sample was strongly diluted before injection onto a column, so it was difficult to differentiate a small decrease in the total amount of major antennae that migrated, and also because very little of the major antennae migrate anyway. Moreover, in stressed grana the deconvolution analysis of ESI spectra showed that only the minor antenna Lhcb4 or CP29 (Fig. 4D, inset) had an increased (∼35% of total protein) molecular mass of 110 Da, suggesting the presence of posttransductional modifications on the apoprotein. Immunoblotting of isolated Lhcb4 (see Fig. 3B) revealed the presence of phosphorylation, indicating that the apoprotein at this light illumination contains only one phosphorylation and some other post-translational modification(s). Time Course of Antennae Migration—To collect information about the time course of antennae migration, stroma lamellae from thylakoid membrane illuminated at 100 μE m–2s–1 were analyzed after different periods of illumination. Fig. 5A shows the chromatograms recorded, whereas Fig. 5B summarizes the evaluation of the percentage of migration of a given substance that has been performed using the abundancy of the corresponding chromatographic peaks. However, the absolute values of the abundancy cannot be directly compared, because of the different response of the detector in different runs. Therefore, the absolute abundancy has been scaled by a normalizing factor obtained by comparing the absolute abundancy in different runs of PSI Lhca2 whose concentration remains practically unchanged after the “stressing” procedure. It can be seen that after the onset of illumination Lhcb2 and all Lhcb1 isoforms started to migrate. Lhcb2 reached its maximum conce
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