Light-harvesting Complex II Binds to Several Small Subunits of Photosystem I
2004; Elsevier BV; Volume: 279; Issue: 5 Linguagem: Inglês
10.1074/jbc.m311640200
ISSN1083-351X
AutoresSuping Zhang, Henrik Vibe Scheller,
Tópico(s)Light effects on plants
ResumoMobile light-harvesting complex II (LHCII) is implicated in the regulation of excitation energy distribution between Photosystem I (PSI) and Photosystem II (PSII) during state transitions. To investigate how LHCII interacts with PSI during state transitions, PSI was isolated from Arabidopsis thaliana plants treated with PSII or PSI light. The PSI preparations were made using digitonin. Chemical cross-linking using dithio-bis(succinimidylpropionate) followed by diagonal electrophoresis and immunoblotting showed that the docking site of LHCII (Lhcb1) on PSI is comprised of the PSI-H, -L, and -I subunits. This was confirmed by the lack of energy transfer from LHCII to PSI in the digitonin-PSI isolated from plants lacking PSI-H and -L. Digitonin-PSI was purified further to obtain an LHCII·PSI complex, and two to three times more LHCII was associated with PSI in the wild type in State 2 than in State 1. Lhcb1 was also associated with PSI from plants lacking PSI-K, but PSI from PSI-H, -L, or -O mutants contained only about 30% of Lhcb1 compared with the wild type. Surprisingly, a significant fraction of the LHCII bound to PSI in State 2 was not phosphorylated. Cross-linking prior to sucrose gradient purification resulted in copurification of phosphorylated LHCII in the wild type, but not with PSI from the PSI-H, -L, and -O mutants. The data suggest that migration of LHCII during state transitions cannot be explained sufficiently by different affinity of phosphorylated and unphosphorylated LHCII for PSI but is likely to involve structural changes in thylakoid organization. Mobile light-harvesting complex II (LHCII) is implicated in the regulation of excitation energy distribution between Photosystem I (PSI) and Photosystem II (PSII) during state transitions. To investigate how LHCII interacts with PSI during state transitions, PSI was isolated from Arabidopsis thaliana plants treated with PSII or PSI light. The PSI preparations were made using digitonin. Chemical cross-linking using dithio-bis(succinimidylpropionate) followed by diagonal electrophoresis and immunoblotting showed that the docking site of LHCII (Lhcb1) on PSI is comprised of the PSI-H, -L, and -I subunits. This was confirmed by the lack of energy transfer from LHCII to PSI in the digitonin-PSI isolated from plants lacking PSI-H and -L. Digitonin-PSI was purified further to obtain an LHCII·PSI complex, and two to three times more LHCII was associated with PSI in the wild type in State 2 than in State 1. Lhcb1 was also associated with PSI from plants lacking PSI-K, but PSI from PSI-H, -L, or -O mutants contained only about 30% of Lhcb1 compared with the wild type. Surprisingly, a significant fraction of the LHCII bound to PSI in State 2 was not phosphorylated. Cross-linking prior to sucrose gradient purification resulted in copurification of phosphorylated LHCII in the wild type, but not with PSI from the PSI-H, -L, and -O mutants. The data suggest that migration of LHCII during state transitions cannot be explained sufficiently by different affinity of phosphorylated and unphosphorylated LHCII for PSI but is likely to involve structural changes in thylakoid organization. In oxygenic photosynthesis two photosystems, PSI 1The abbreviations used are: PSI and PSIIphotosystem I and photosystem IIChlchlorophyllDMβ-dodecyl maltosideDM-PSIPSI prepared after DM solubilizationDTSPdithio-bis(succinimidylpropionate)Fmmaximum fluorescent yieldL1photosystem I light (red light)L2photosystem II light (orange light)LHCII and LHCIlight-harvesting complex of photosystem II and photosystem I, respectivelyTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. and PSII, work in series to convert light energy into chemical energy. PSI is also involved in cyclic electron transport without the participation of PSII, and this process serves to produce additional ATP and to regulate the transthylakoidal proton gradient (1Munekage Y. Hojo M. Meurer J. Endo T. Tasaka M. Shikanai T. Cell. 2002; 110: 361-371Google Scholar), but in plants this is a minor part of the electron transport. In linear electron transport, PSI and PSII must operate with the same rate, but natural environmental conditions, such as the quality and quantity of light, are constantly fluctuating, and this may alter the balance between the two photosystems. The two photosystems have different absorption spectra, and therefore a change in light quality may favor one photosystem over the other. However, plants can balance the excitation energy distribution between the two photosystems via a mechanism known as state transitions, which was discovered more than 30 years ago (2Bonaventura C. Myers J. Biochim. Biophys. Acta. 1969; 189: 366-383Google Scholar, 3Murata N. Biochim. Biophys. Acta. 1969; 172: 242-251Google Scholar, 4Allen J.F. Science. 2003; 299: 1530-1532Google Scholar). If PSII is overexcited relative to PSI, the plastoquinone pool becomes overreduced, and this will activate a kinase that phosphorylates a mobile pool of light-harvesting complex II (LHCII), leading to the lateral movement of LHCII in favor of PSI. This is the so-called "State 2," in which the PSII antenna is smaller and the PSI antenna is larger than in State 1 (5Lunde C. Jensen P.E. Haldrup A. Knoetzel J. Scheller H.V. Nature. 2000; 408: 613-615Google Scholar, 6Allen J.F. Forsberg J. Trends Plant Sci. 2001; 6: 317-326Google Scholar). State 1 is obtained when PSI is preferentially excited, which leads to oxidation of the plastoquinone pool and inactivation of the LHCII kinase. The phospho-LHCII is then dephosphorylated by a redox-independent phosphatase and moves back to PSII. Although the phenomenon of state transitions has been recognized for a long time, there is still considerable uncertainty about the mechanism. 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 grana stacks (7Barber J. Annu. Rev. Plant Physiol. 1982; 33: 261-295Google Scholar, 8Bennett J. Biochem. J. 1983; 212: 1-13Google Scholar, 9Bennett J. Physiol. Plant. 1984; 60: 583-590Google Scholar). According to another model, the net movement of LHCII toward PSI in State 2 is caused by PSII with higher affinity for unphosphorylated LHCII and PSI with higher affinity for phospho-LHCII, therefore movement of phospho-LHCII is a question of molecular recognition (6Allen J.F. Forsberg J. Trends Plant Sci. 2001; 6: 317-326Google 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. A search for kinases involved in the phosphorylation of thylakoid proteins has been carried out by many workers for more than 20 years. A family of proteins, thylakoid-associated kinases, was identified as good candidates for LHCII kinases (10Snyders S. Kohorn B.D. J. Biol. Chem. 1999; 274: 9137-9140Google Scholar, 11Snyders S. Kohorn B.D. J. Biol. Chem. 2001; 276: 32169-32176Google Scholar). The antisense Arabidopsis plants with low amounts of thylakoid-associated kinase 1 showed a lower level of LHCII phosphorylation and were deficient in state transitions, but the phosphorylation of LHCII was distributed equally between PSII and PSI under white light, which was also the case in wild type plants (11Snyders S. Kohorn B.D. J. Biol. Chem. 2001; 276: 32169-32176Google Scholar). This indicates that the correlation between LHCII phosphorylation and state transition is complex. Recently, Depege et al. (12Depege N. Bellafiore S. Rochaix J.D. Science. 2003; 299: 1572-1575Google Scholar) reported a novel kinase in Chlamydomonas reinhardtii, thylakoid-associated serine-threonine protein kinase, and demonstrated that it is required for the phosphorylation of LHCII and for state transitions. photosystem I and photosystem II chlorophyll β-dodecyl maltoside PSI prepared after DM solubilization dithio-bis(succinimidylpropionate) maximum fluorescent yield photosystem I light (red light) photosystem II light (orange light) light-harvesting complex of photosystem II and photosystem I, respectively N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. LHCII consists of three different proteins, Lhcb1, Lhcb2, and Lhcb3. Lhcb1 and Lhcb2 are the most abundant and can form Lhcb1 homotrimers and Lhcb1/2 heterotrimers, which are believed to be a mobile complex (13Larsson U.K. Anderson J.M. Andersson B. Biochim. Biophys. Acta. 1987; 894: 69-75Google Scholar, 14Jackowski G. Kacprzak K. Jansson S. Biochim. Biophys. Acta. 2001; 1504: 340-345Google Scholar). Both proteins usually exist in several very similar isoforms, but specific isoforms are not well conserved between species, indicating that they probably are redundant rather than having specific functions. However, this is another unclear point because it is not known whether there are biochemical differences between the mobile and nonmobile LHCII apart from the reversible phosphorylation described above. Electron microscopy studies have revealed that PSII core complexes are found as dimers surrounded by LHCII trimers (15Boekema E.J. van Roon H. van Breemen J.F.L. Dekker J.P. Eur. J. Biochem. 1999; 266: 444-452Google Scholar, 16Boekema E.J. van Roon H. van Breemen J.F.L. Dekker J.P. Biochemistry. 1999; 38: 2233-2239Google Scholar, 17Boekema E.J. van Roon H. van Breemen J.F.L. Dekker J.P. J. Mol. Biol. 2000; 301: 1123-1133Google Scholar, 18Yakushevska A.E. Jensen P.E. Keegstra W. van Roon H. Scheller H.V. Boekema E.J. Dekker J.P. Eur. J. Biochem. 2001; 268: 6020-6028Google Scholar). There are specific binding sites for the trimers, but the number of trimers/PSII complex differs depending on species and growth conditions. Generally, strongly bound, intermediately bound, and loosely bound trimers can be recognized. In contrast to the situation with PSII, the binding site of LHCII on PSI is not known. PSI is composed of a multisubunit core complex (PSI core) and outer antenna, the light-harvesting complex I (LHCI). The PSI core complex in higher plants consists of 14 different subunits (PSI-A to PSI-L, PSI-N) (19Chitnis P.R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 593-626Google Scholar, 20Scheller H.V. Jensen P.E. Haldrup A. Lunde C. Knoetzel J. Biochim. Biophys. Acta. 2001; 1507: 41-60Google Scholar) and a recently discovered subunit PSI-O (21Knoetzel J. Mant A. Haldrup A. Jensen P.E. Scheller H.V. FEBS Lett. 2002; 510: 145-148Google Scholar). LHCI is composed of four different subunits of about 21–24 kDa, Lhca1 to Lhca4. The association between LHCII and PSI is relatively weak, and a stable complex is difficult to purify. Early studies (22Bassi R. Simpson D. Eur. J. Biochem. 1987; 163: 221-230Google Scholar) suggested that LHCII was bound to LHCI rather than directly to the PSI core, but this conclusion was based on the inability to reconstitute a LHCII·PSI complex using PSI devoid of LHCI-680 (composed of Lhca2 and Lhca3). We now know that preparation of a PSI complex devoid of LHCI is likely to cause the loss of additional small core subunits that were unknown at the time. Electron microscopy studies have revealed that all LHCI subunits bind at the side of PSI-F and PSI-J subunits of the PSI core complex (23Boekema E.J. Jensen P.E. Schlodder E. van Breemen J.F.L. van Roon H. Scheller H.V. Dekker J.P. Biochemistry. 2001; 40: 1029-1036Google Scholar). Arabidopsis plants lacking PSI-H were highly deficient in state transitions and have identical PSI antenna size in both States 1 and 2, whereas in wild type the antenna size of PSI was found to increase about 33% during transition to State 2 (5Lunde C. Jensen P.E. Haldrup A. Knoetzel J. Scheller H.V. Nature. 2000; 408: 613-615Google Scholar). Therefore we suggested that LHCII binds directly to the PSI core and that PSI-H is part of a docking site (5Lunde C. Jensen P.E. Haldrup A. Knoetzel J. Scheller H.V. Nature. 2000; 408: 613-615Google Scholar). Based on cross-linking and x-ray crystallography data, the PSI-H subunit is positioned close to PSI-L and PSI-I on the opposite side of the PSI core complex compared with PSI-F, PSI-J, and LHCI (24Jansson S. Andersen B. Scheller H.V. Plant Physiol. 1996; 112: 409-420Google Scholar, 25Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Google Scholar). However, until now, no direct biochemical evidence has ever been found for the association of LHCII to PSI during state transitions. To investigate how LHCII interacts with PSI during state transitions, we purified LHCII·PSI complex from wild type Arabidopsis in State 1 and State 2, and we also used mutants lacking the PSI-H, -L, -O, or -K subunits to determine the docking sites for LHCII. The results indicate that PSI-H, -L, -O, and -I all participate in forming the docking site. Furthermore, the results show that both phosphorylated and unphosphorylated LHCII are associated with PSI in State 2. Plant Materials—Arabidopsis thaliana (L.) Heyhn ecotype Col-0 was used for all experiments. Plants were grown in peat in a controlled environment Arabidopsis chamber (Percival AR-60L, Boone, IA) at a photosynthetic flux of 100–120 μmol photons m–2 s–1, 20 °C, 70% relative humidity. The photoperiod was 8 h. Transformants lacking specific PSI subunits were obtained by antisense or cosuppression and have been reported before (5Lunde C. Jensen P.E. Haldrup A. Knoetzel J. Scheller H.V. Nature. 2000; 408: 613-615Google Scholar, 26Naver H. Haldrup A. Scheller H.V. J. Biol. Chem. 1999; 274: 10784-10789Google Scholar, 27Jensen P.E. Gilpin M. Knoetzel J. Scheller H.V. J. Biol. Chem. 2000; 275: 24701-24708Google Scholar, 28Jensen P.E. Rosgaard L. Knoetzel J. Scheller H.V. J. Biol. Chem. 2002; 277: 2798-2803Google Scholar, PSI-O mutant). 2P. E. Jensen, A. Haldrup, S. P. Zhang, D. Leister, and H. V. Scheller, unpublished data. Light Treatment—Colored filters were used in the experiments to provide PSII light (L2) and PSI light (L1) essentially as described by Pfannschmidt et al. (29Pfannschmidt T. Nilsson A. Allen J.F. Nature. 1999; 397: 625-628Google Scholar). PSII light was obtained with an orange filter (Rosco, 105 orange, Teadon Aps, Stenløse, Denmark), and PSI light was obtained with a red filter (HT 027 medium red, LEE Filters, Andover, UK). Gray filter (209 neutral density, LEE Filters) was used to adjust the light coming through the orange filter to a level similar to that through the red filter (50–70 μmol photons m–2 s–1). The filters were mounted in a controlled environment chamber equipped with 400-watt Powertone HPI-T Plus lamps (Philips). Six-week-old wild type plants were exposed to PSI or PSII light for 1 h prior to harvesting of leaves. Plants lacking PSI-H, -L, -O, or -K subunits were treated with PSII light for 1 h. State Transitions in Leaves—State transitions were measured with a pulse amplitude modulation 101–103 fluorometer (Walz, Effeltrich, Germany) in the growth chamber equipped with filters as described above. Plants were dark-adapted for 30 min before the measurements. A detached leaf from a wild type plant was fixed to the light fiber, which was positioned so the leaf was horizontal and received the same irradiation as the plants used for preparation of thylakoids. Maximum fluorescence yield (Fm) was determined by exposing the leaf to a saturating flash (0.8 s, 6000 μmol photons m–2 s–1). The leaf was then exposed to orange light (which favors PSII, L2) for about 20 min, and the maximum fluorescence yield in State 2 (Fm2) was determined. The fiber with the leaf was then transferred rapidly to the red light (which favors PSI, L1), and after 20 min the maximum fluorescence yield (Fm1) was measured. The leaf was then exposed to orange light for another 20 min and finally kept in darkness. The difference of maximal fluorescence in State 1 and State 2 was calculated as Fm1/Fm2. The relative change in fluorescence was calculated as in Equation 1 (5Lunde C. Jensen P.E. Haldrup A. Knoetzel J. Scheller H.V. Nature. 2000; 408: 613-615Google Scholar, 30Haldrup A. Jensen P.E. Lunde C. Scheller H.V. Trends Plant Sci. 2001; 6: 301-305Google Scholar),Fr=((Fi'-Fi)-(Fii'-Fii))/(Fi'-Fi)(Eq. 1) where Fi and Fii designate fluorescence in the presence of PSI light in State 1 and State 2, respectively, and Fi′ and Fii′ designate fluorescence in the absence of PSI light in State 1 and State 2, respectively (see Fig. 1). Isolation of DM-PSI (β-Dodecyl Maltoside-solubilized PSI) and Digitonin-PSI—After plants were treated with light as described above, rosette leaves were rapidly harvested and frozen in liquid nitrogen. Thylakoids were prepared as described previously (31Haldrup A. Naver H. Scheller H.V. Plant J. 1999; 17: 689-698Google Scholar) except that all solutions contained 10 mm NaF to inhibit phosphatase. DM-PSI was isolated according to Jensen et al. (27Jensen P.E. Gilpin M. Knoetzel J. Scheller H.V. J. Biol. Chem. 2000; 275: 24701-24708Google Scholar). The sucrose gradients were prepared by freezing and thawing at 4 °C of 11 ml of 0.6 m sucrose, 20 mm Tricine-NaOH, pH 7.5, 0.06% DM, and 10 mm NaF. Digitonin-PSI was prepared by solubilizing thylakoids (0.5 mg of Chl/ml) with digitonin (final concentration 0.5% (w/v)) for 30 min at 4 °C with stirring, followed by centrifugation at 48,000 × g for 30 min. The supernatant was centrifuged at 257,000 × g for 1 h at 4 °C. The pellet was resuspended in 20 mm Tricine, pH 7.5, 20% glycerol, 10 mm NaF, and the samples (digitonin-PSI) were frozen in liquid nitrogen and stored at –80 °C until use. The Chl concentration and Chl a/b ratio were determined in 80% acetone (32Lichtenthaler H.K. Methods Enzymol. 1987; 148: 350-382Google Scholar). Preparation of LHCII·PSI Complex—For preparing pure LHCII·PSI complex, the digitonin-PSI preparations were diluted to 0.3 mg of Chl/ml in 20 mm Tricine, pH 7.5, 0.3% (w/v) DM, 10 mm NaF. Half of the preparation was treated with chemical cross-linker dithio-bis(succinimidylpropionate) (DTSP) on a shaker for 30 min at 22 °C in darkness as described previously (24Jansson S. Andersen B. Scheller H.V. Plant Physiol. 1996; 112: 409-420Google Scholar). After incubation with 0.015% (w/v) DTSP at room temperature for 30 min in darkness, the reaction was stopped by the addition of 1/7 volume of 10 mm Tris, pH 7.4, 1 mm EDTA. The DTSP-treated and the control samples were applied to sucrose gradients containing 0.06% (w/v) DM and centrifuged as described above. The LHCII bands and LHCII·PSI complex bands were collected with syringes, frozen in liquid nitrogen, and stored at –80 °C. Analysis of Phosphate Content—Phosphate content of LHCII in State 2 was analyzed according to Martensen (33Martensen T.M. Methods Enzymol. 1984; 107: 3-23Google Scholar). First the purified LHCII preparations were ashed to convert protein-bound phosphate to inorganic phosphate, then the inorganic phosphate was measured by absorbance at 660 nm in a microtiter plate reader after complexation of ammonium molybdate with malachite green. The standard curve was made with KH2PO4 (0–1 nmol). The content of phosphate in LHCII·PSI in State 2 was determined by immunoblotting using the phosphothreonine antibody and comparing with a dilution series of LHCII with known content of phosphate. SDS-PAGE and Immunoblotting—All the gels used were home-made 8–25% gradient gels prepared according to Fling and Gregerson (34Fling S.P. Gregerson D.S. Anal. Biochem. 1986; 155: 83-88Google Scholar), except that the first dimension of diagonal electrophoresis was on 12% homogeneous gels. All samples were loaded on Chl basis. Immunoblotting was carried out by transferring the electrophoretically separated proteins to nitrocellulose membranes followed by incubation with different polyclonal antibodies and detection with the ECL system (Amersham Biosciences). To determine the composition of cross-linked products on one-dimensional gels, the nitrocellulose strip for each lane was cut into two sections and incubated with two different antibodies. In this way the two sections could be aligned exactly. To determine the relative content of Lhcb1, Lhcb2, and phosphothreonine in the LHCII·PSI complexes each sample was electrophoresed together with a dilution series of known amounts of LHCII to ensure that the response on the immunoblots was in a linear range. Quantitation was carried out by scanning the x-ray films and analyzing them using the ImageQuant software (Molecular Dynamics). Polyclonal antibodies against PSI proteins were prepared in rabbits and have been described before (21Knoetzel J. Mant A. Haldrup A. Jensen P.E. Scheller H.V. FEBS Lett. 2002; 510: 145-148Google Scholar, 35Andersen B. Koch B. Scheller H.V. Physiol. Plant. 1992; 84: 154-161Google Scholar). Antibodies against Lhcb1 and Lhcb2 were kind gifts of S. Jansson, Umeå University, Sweden. Antibodies against phosphothreonine were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Diagonal Electrophoresis—The digitonin-PSI preparations (treated with PSII light) were diluted to 0.3 mg of Chl/ml in 20 mm Tricine, pH 7.5, 0.3% (w/v) DM, and 10 mm NaF. Chemical cross-linking with DTSP was carried out as described above. The cross-linked samples were mixed with 1 volume of nonreducing sample buffer (50 mm Na2CO3, 15% w/v sucrose, 2.5% (w/v) SDS), the solution was incubated for 20 min at room temperature, and loaded on the first dimension 12% gel. After electrophoresis, the lanes were cut out and incubated for 30 min in reducing sample buffer (50 mm Na2CO3, 15% sucrose, 2.5% SDS, and 50 mm dithiothreitol) to obtain complete reductive cleavage of the cross-linked products. The gel slice was placed on top of a second 8–25% gradient gel and reelectrophoresed. P700 Photooxidation Measurements—The photooxidation kinetics of P700 were monitored with the absorbance changes at 810 nm with 860 nm as reference (ΔA810–860) using the dual wavelength unit ED-P700DW of a PAM 101–103 fluorometer (Walz) as described by Bukhov et al. (36Bukhov N. Egorova E. Carpentier R. Planta. 2002; 215: 812-820Google Scholar) with some modifications. Actinic light from a KL1500 halogen lamp (Schott, Mainz, Germany) with different light intensities was controlled by an electronic shutter, which opened for 1 s with 40-s intervals. Digitonin-PSI preparations (15 μg of Chl) were diluted to a total volume of 500 μl in 50 mm Tricine buffer, pH 7.5, 0.01% (w/v) digitonin, and 0.5 mm ascorbate and 0.05 mm methyl viologen were added prior to measurements. Ascorbate was used to ensure complete reduction of P700 in the dark, and methyl viologen to prevent the back-reaction from PSI acceptors. The output signal from the fluorometer was passed to an oscilloscope where averaging of signals was performed. The half-life (t½) was calculated after fitting of exponential curves to the absorption curves. State Transitions in the Growth Chamber—To make sure that we chose the right light conditions to do the following experiments, we first tested whether the plants performed state transitions in the growth chamber. To induce state transitions, we equipped a growth chamber with orange and red filters for preferential excitation of PSII and PSI, respectively. The light intensity passing through the filters was about 50–70 μmol photons m–2 s–1, The plants were dark-adapted for 30 min before measuring the maximum fluorescence signal (Fm), and subsequently the leaves were exposed to orange light (L2, which favors PSII) for 20 min (Fig. 1). After the steady-state fluorescence level was reached, the maximal fluorescence in State 2 (Fm2) was determined. Then State 1 was induced by illuminating with red light (L1, which favors PSI) for 20 min, and the maximal fluorescence in State 1 (Fm1) was determined. To quantify state transitions we determined Fm1 and Fr (30Haldrup A. Jensen P.E. Lunde C. Scheller H.V. Trends Plant Sci. 2001; 6: 301-305Google Scholar). For wild type Arabidopsis plants, the value of Fm1/Fm2 was determined as 1.19 ± 0.03, and Fr as 0.86 ± 0.05 (± S.D., n = 4). The values are in good agreement with Haldrup et al. (30Haldrup A. Jensen P.E. Lunde C. Scheller H.V. Trends Plant Sci. 2001; 6: 301-305Google Scholar), who got the numbers 1.051 ± 0.005 and 0.851 ± 0.034 at a light intensity of 80 μmol photons m–2 s–1 using a conventional setup. This indicates that the right experimental conditions were established for performing state transitions in our growth chamber. Detergent Solubilization and Purification of PSI—From plants exposed to different light conditions we first used DM to solubilize thylakoids and then isolate PSI from sucrose gradients. As shown in Fig. 2A (lanes 1 and 2) the isolated PSI did contain all known higher plant PSI subunits but did not have any detectable LHCII attached both in State 1 and in State 2. The absence of LHCII in DM-PSI was confirmed by immunoblotting (data not shown). This demonstrates that the association between LHCII and PSI is weak and could be disrupted during solubilization with DM. Therefore, we used a milder solubilization with the nonionic detergent digitonin instead of DM and without running sucrose gradients. The resulting preparations were highly enriched in PSI and contained large amounts of LHCII (Fig. 2A, lanes 3 and 4). However, the preparations are obviously not pure PSI but also contain some PSII. Thus, we could not at this point determine how large a fraction of the LHCII was actually associated with PSI. However, it is clear that the LHCII isolated together with PSI was much more phosphorylated in State 2 than in State 1 (Fig. 2B). This result further confirmed that the reversible phosphorylation was functional under the chosen light conditions and that state transitions were induced. Chemical Cross-linking and Diagonal Electrophoresis—To determine whether LHCII in the digitonin-PSI preparation was associated with PSI and to investigate the possible docking sites of LHCII on PSI, we employed chemical cross-linking, diagonal electrophoresis, and immunoblotting. Lunde et al. (5Lunde C. Jensen P.E. Haldrup A. Knoetzel J. Scheller H.V. Nature. 2000; 408: 613-615Google Scholar) suggested that LHCII may bind to PSI-H and PSI-L. To test this possibility, digitonin-PSI was cross-linked with DTSP, subjected to SDS-PAGE under nonreducing conditions, and electroblotted to nitrocellulose membranes. Two cross-linked products could be clearly identified with an antibody against Lhcb1 (Fig. 3A, lane 1). One cross-linked product (Fig. 3, A and B, Δ) with an apparent molecular mass of ∼30 kDa, was most probably a product of PSI-I and Lhcb1 because an antibody against PSI-I identified a product of the same size. Previous work has shown that cross-linking products migrate with apparent molecular masses corresponding to the combined molecular masses of the individual proteins (24Jansson S. Andersen B. Scheller H.V. Plant Physiol. 1996; 112: 409-420Google Scholar), and 30 kDa is in excellent agreement with the combined molecular masses of Lhcb1 (25 kDa) and PSI-I (4 kDa). The other cross-linked product (Fig. 3A, *) had an apparent molecular mass of 55–60 kDa. Antibodies against PSI-I, PSI-L, and PSI-H reacted with a band of exactly the same size. The combined molecular mass of Lhcb1, PSI-I, PSI-H, and PSI-L is 57 kDa, and the data therefore indicate that the immunoreactive cross-linking product is a product of these four proteins. The previously described (24Jansson S. Andersen B. Scheller H.V. Plant Physiol. 1996; 112: 409-420Google Scholar) cross-linking products involving PSI-L+H (28 kDa), PSI-L+I (22 kDa), and PSI-H+I (14 kDa) are also clearly seen on the blot. With an antibody against Lhcb2 a cross-linking product with apparent molecular mass of around 50 kDa (▿) was observed (Fig. 3C). However, this product is clearly smaller than the cross-linking product involving PSI-I, -L, and -H, and it may correspond to an Lhcb2 dimer. A well known problem with using antibodies against cross-linked proteins is that epitopes may be lost because of the cross-linking (24Jansson S. Andersen B. Scheller H.V. Plant Physiol. 1996; 112: 409-420Google Scholar). However, with a cleavable cross-linker such as DTSP this problem can largely be overcome by electrophoresing the cleaved cross-linked products in a second dimension. To confirm the above conclusions further, the gel slices after the first dimension were therefore cut out, incubated with reducing sample buffer to cleave the cross-linked products completely, and reelectrophoresed. In the first dimension, cross-linked products will migrate approximately according to the combined apparent molecular mass, but after cleavage the previously linked proteins will migrate separately according to their normal apparent molecular masses and therefore form vertically aligned spots in the second dimension (24Jansson S. Andersen B. Scheller H.V. Plant Physiol. 1996; 112: 409-420Google Scholar). Proteins that are not cross-linked will migrate to the same position in both dimensions and thus form a diagonal in the final gel. The blot in Fig. 4A confirms that Lhcb1 formed two cross-linked products of 30 and 55–60 kDa. The 55–60-kDa product is clearly resolved into Lhcb1 (25 kDa) and PSI-L (14 kDa; note that the protein migrates faster than the actual molecular mass of 18 kDa). PSI-H (10 kDa) is also vertically positioned under Lhcb1 and PSI-L corresponding to 55–60 kDa in the first dimension (Fig. 4B), and the spots of PSI-L and PSI-H can be resolved into two cross-linking products. One might be Lhcb1/PSI-L/PSI-H, and another might be Lhcb1/PSI-L/PSI-H plus PSI-I (4 kDa). The presence of PSI-I in the 60-kDa product could not be confirmed in the second dimension, probably because of the lower sensitivity of the PSI-I antibody. The product of ∼30 kDa is seen to consist of large amounts of Lhcb1 to
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