Ayumi Tanaka, Yoshio Tanaka and Hideo Tsuji. Laboratory for Plant Ecological Studies, Faculty of Science, Kyoto University, Kyoto 606, Japan

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1 Plant Cell Physiol. 28(8): (1987) JSPP 1987 Resolution of Chlorophyll a/6-protein Complexes by Polyacrylamide Gel Electrophoresis: Evidence for the Heterogeneity of Light-Harvesting Chlorophyll a/6-protein Complexes Ayumi Tanaka, Yoshio Tanaka and Hideo Tsuji Laboratory for Plant Ecological Studies, Faculty of Science, Kyoto University, Kyoto 606, Japan P700-Chl a-protein complexes (CP1 and CP1*), Chl-protein complexes of PS II core (CPa-1 and CPa-2), light-harvesting Chi a/a-protein complexes (LHCPo and LHCPm) and of spinach thylakoids were resolved by SDS-polyacrylamide-gel electrophoresis (PAGE) under non-denaturing conditions. The LHCP oligomer purified by electrophoresis, had and 27-kDa polypeptides. CP1, and two LHCPs (LHCP-1 and LHCP-2) of spinach thylakoids were separated by a lithium dodecylsulfate (LDS) PAGE system with high resolution. The two LHCPs showed the same absorption spectrum on the gel. When LHCP oligomer was reelectrophoresed by this system it also gave LHCP-1, and LHCP-2. LHCP-1 had both and 27- kda polypeptides, but LHCP-2 had only 29.5 kda polypeptide. Both polypeptides seemed to bind Chi. The heterogeneity of LHCP was also observed with bean thylakoids. Key words: Barley Bean Chl-protein complex Light-harvesting Chi ajbprotein complex Spinach. Most of the Ch] molecules in thylakoids are bound to proteins in the form of CPs (Markwell et al. 1979). Light-harvesting Chi a/6-protein complexes of PS I (LHCPI) and of PS II (LHCPII) and are Chi a/i-protein complexes that harvest light and transfer their energy to the reaction centers (Thornber 1979, 1986). PAGE has been used to isolate these Chi a/6-protein complexes by a number of investigators. LHCPI migrated as the CP1-LHCPI complex (CPla) in PAGE (Argyroudi-Akoyunoglou 1984) or was isolated by mild dissociation of native PS I complex purified by sucrose gradient (Lam et al. 1984). was resolved by the octylglucoside/sds PAGE (Camm and Green 1980, 1981, Dunahay and Staehelin 1986) and the SDS-PAGE systems of Laemmli (Metz et al. 1983) and Machold and Meister (1979). In most of the PAGE systems used for the isolation of CPs, the monomeric form of LHCP (LHCPm) migrated as a single green band (Anderson et al. 1978) except in some cases (Green and Camm 1982). The heterogeneity of LHCP apoproteins was demonstrated with the sample purified by PAGE or gradient centrifugation. Anderson (1980) reported that LHCP isolated by SDS PAGE had major polypeptides of 23 and 25 kda. PAGE of LHCP precipitated with cations from Tritonsolubilized thylakoid membranes revealed the presence of four polypeptides (McDonnel and Staehelin 1980). Several polypeptide bands were found in the LHCP area on the gel after electrophoresis of thylakoid membranes or even "purified" LHCP. However, whether all these polypeptides actually bind Chi was not known. Dunsmuir (1985) showed that there are 16 genes Abbreviations: CP, Chl-protein complex; CP1, P700-Chl a-protein complex; CPa, Chl-protein complex of PS II core; LDS, lithium dodecylsulfate; PAGE, polyacrylamide-gel electrophoresis. 1537

2 1538 A. Tanaka, Y. Tanaka and H. Tsuji for LHCP apoproteins in petunia. In spite of much evidence for the heterogeneity of LHCP apoproteins, there is little evidence for the heterogeneity at the level of CP. Mullet (1983) obtained 29- and 27-kDa polypeptides by isoelectric focusing and showed that both polypeptides bound Chi. However, only the 29-k.Da polypeptide was found in a small amount and not at all in some preparations. Therefore, he inferred that it was an intermediate form of LHCP. Camm and Green (1981) obtained two green bands of LHCP monomer from the green alga Acetabularia. The apoproteins of the two LHCPs were 27 and 26 kda, respectively. They showed the LHCP heterogeneity also for spinach, but did not report on the corresponding apoproteins of the LHCPs. In the present study, using a high resolution PAGE system, we showed that LHCP could be resolved into two green bands at the position of the LHCP monomer. Spinach LHCP was made up of and 27-kDa polypeptides, both of which were thought to bind Chi. Materials and Methods Plant materials Spinach was purchased from a local market. Bean (Pkaseolus vulgaris L. cv. Yamashiro-kurosando-saito) and barley (Hordeum vulgare L. cv. Amagi Nijo) were grown under a cycle of 16 h light and 8 h dark at 25 C for 1-2 weeks. Chloroplast isolation Leaves were blended with 50 mm Na-Tricine (ph 8.0), 5 mm EDTA and 0.5 M sucrose. The homogenate was filtered through a layer of Miracloth and centrifuged at 10,000 x^ for 10 min. The green pellet was suspended in 5 mm EDTA (ph 8.0) and centrifuged at 10,000 x^ for 10 min (washed membranes). Isolation of CPs The SDS-PAGE system of Laemmli (1970) was used to separate CP with minor modifications. System I: Washed membranes were diluted with distilled water (thylakoid suspension). Thylakoid suspension (about 0.3 ml) was loaded on an equal volume of solubilizing buffer (0.3 M Tris-HCl (ph 8.8), 0.5% SDS and 10% glycerol in the final concentrations) in a small test tube. Next, thylakoid suspension and solubilizing buffer were mixed by shaking the test tube gently. As soon as the two solutions were made uniform, the shaking was stopped (within 10 s). Strong shaking and excess solubilization resulted in dissociation of oligomeric CPs and release of Chi from the apoproteins, especially for PS II core complexes (CPa-1 and CPa-2). For better resolution of and LHCP, dissociation of oligomeric CP had to be avoided because was not separated from the LHCPm band when LHCPm was dominant. Good resolution of CPs could be obtained without centrifugation after the solubilization procedure. In order to clearly separate, LHCP monomer, CPa-1 and CPa-2, the green bands should be sharp without bending or tailing. SDS/Chl ratio was about 10. No SDS was included in the separation gel. Stacking gel was omitted.' Solubilized thylakoids, 4-6 [A, were loaded on the gel which was 6 cm long and 0.6 cm in diameter. All procedures were done in an ice bath. Electrophoresis was carried out for 2-3 h at about 0.6 ma/tube in a cold room in the dark. System II: LDS PAGE of system II was employed to study LHCP heterogeneity. Thylakoid suspension was solubilized completely with 60 mm Na 2 CO 3, 60 mm DTT, 1% LDS and 12% sucrose, the LDS/Chl ratio being made about 20. The mixture was centrifuged at 10,000 Xjr for 20 min, and the supernatant was collected (solubilized thylakoids). The concentrations of acrylamide in the stacking and separation gels were 5 and 14%, respectively. The separation gel was 15 cm long and 0.6 cm in diameter. Better resolution of CPs was obtained with disc gels rather than slabs. The gel must have a flat surface for good resolution. A 400-ml buffer was provided each in upper and lower reservoir. Only four tubes were used because the use of a large number of tubes produced unsatisfactory results. Solubilized thylakoids, 4-8 (A, were loaded on the gel. Electrophoresis was carried out for about 20 h at 0.7 ma/tube in a cold room in the dark. The green bands on the gel should be clear without any bending or tailing after

3 Heterogeneity of light-harvesting Chl-protein complexes 1539 a long period of electrophoresis. When LHCP oligomer was further analyzed for its components, the green region of LHCPo on the gel obtained after PAGE of system I was excised and incubated with the solubilizing buffer (60 mm Na 2 CO 3, 60 mm DTT, 1% LDS and 12% sucrose) for 1 h at room temperature in the dark and loaded on the gel of system II. Other procedures were the same as for the thylakoid membranes. Polyacrylamide-gel electrophoresis of proteins Thylakoids or CP-containing gels were incubated with 60 mm Na 2 CO 3, 60 mm DTT, 2% LDS and 12% sucrose, and then heated at 100 C for 90 s. Electrophoresis was carried out according to Laemmli (1970) with stacking and separation gels of 5% and 14% polyacrylamide, respectively. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250. Apparent molecular weights were determined with known standards: Phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,000) and a-lactoalbumin (14,000). Measurement of absorption spectrum of CPs The absorption spectra of green bands on the gel were measured directly with a Hitachi 556 spectrophotometer. Chi determination The green bands on the gel were cut. Chi was extracted from the gel slices with 0.1% SDS and determined by fluorescence spectrophotometry in 80% acetone. Results CP1*, CP1, LHCPo, CPa-1, CPa-2, and LHCPm were separated from spinach chloroplasts by SDS PAGE of system I (Fig. 1). The results were similar to those obtained by the octylglucoside/sds-page system (Camra and Green 1980). The molecular weights of apoproteins of CPa-1, CPa-2 and were 49, 43 and 28kDa, respectively, and those of LHCPo were 29.5 and 27 kda (data not shown). The absorption maximum (Table 1), Chi a/b CPl CP1 LHCPo CPa-1 CPa-2 LHCPm FC C in CD.» > 03 (b (J C orba </) < J \ -CP1 U III LHCPo CPa-1 J H/ j ^ CPa-2 'j\ \.IHCPm 1 / Migration ( 0.5cm) FC

4 ism A. Tanaka, Y. Tanaka and H. Tsuji CP1 Table 1 Distribution of Chi among CPs in thylakoids of spinach and wavelengths of their absorption maxima CP CP1* CP1 LHCPo CPa-1 CPa-2 LHCPm FC Chi distribution Chi a/b Absorption maximum After electrophoresis (as in Fig. 1), green bands were excised and homogenized with 0.1% SDS and centrifuged. The pellet was reextracted with 0.1% SDS. To the combined supernatant, acetone was added to final concentration of 80%, and the mixture was centrifuged. Chi was determined by fluorescence spectrophotometry. Absorption spectrum of CPs on the gel was directly measured with a Hitachi 556 spectrophotometer.!lhcp-i LHCP-2 o C n i_ o en Wavelength ( nm) Fig. 2 Distribution of CPs after electrophoresis of system II and their absorption spectra. Thylakoid membranes of spinach were solubilized with 60 mm Na 2 CO 3, 60 mm DTT, 1% LDS and 12% sucrose and electrophoresed on disc gel by system II (high resolution system). Absorption spectrum of CPs on the gel was directly measured as in Table 1. Left, photograph of whole gel and amplification of the LHCP area; right, absorption spectra of CPs.

5 Heterogeneity of light-harvesting Chl-protein complexes 1541 ratio (Table 1) and protein composition of each green band coincided with previously reported results (Camm and Green 1980). This system allowed us to solubilize all the thylakoid membranes and minimize the amount of free Chi (FC) (Table 1). This system, therefore, is useful for studying the CP composition in the thylakoid. CPs of reaction centers of PS I (CPl* and CPl) and PS II (CPa-1 and CPa-2) accounted for 40 and 8% of the total Chi, respectively. LHCP (LHCPo and LHCPm) and, light-harvesting complexes of PS II, accounted for 35 and 10%, respectively. These results were generally consistent with Anderson's results (1980) except for CPa-1, CPa-2 and which are reported later using other methods (Camm and Green 1980). Chi a/b ratio of obtained with our system was low compared to that obtained with octylglucoside/sds PAGE by Camm and Green (1980). Contamination of with LHCP probably lowered the Chi a/b ratio in our system. The green band of LHCPm migrated as a single band as reported by other workers (Anderson et al. 1978). To examine the LHCP heterogeneity, we tried further separation of this LHCP band by a high-resolution-page system. The CP distribution pattern on the gel with PAGE of system II is shown in Fig. 2. CPl, and two LHCPs (LHCP-1 and LHCP-2) were separated on the gel. Oligomeric CPs (CPl*, LHCPo) dissociated into monomeric CPs with this system. Chi of T LHCPo CP1 ^ ^ ^ ^ LHCPo -LHCP-1 -LHCP-2 Fig. 3 Electrophoresis of CPs of thylakoid membranes and LHCPo by system II. After electrophoresis of thylakoid membranes by PAGE of system I (Fig. 1), LHCPo-containing gel was excised and incubated with 60 mm Na 2 CC>3, 60 mm DTT, 1% LDS and 12% sucrose for 1 h at room temperature in the dark. The gel slice was put on the gel of system II and electrophoresed. CPs of thylakoid membranes were separated by system II as in Fig. 2. Left, photograph of the whole gel; right, amplification of the LHCP area; T, thylakoids; LHCPo, LHCP oligomer.

6 **-»«# A. Tanaka, Y. Tanaka and H. Tsuji A B C D T LHCP-1 LHCP-2 Fig. 4 PAGE of thylakoid membranes and purified LHCP. LHCPo was reelectrophoresed as in Fig. 2. After that, portions A, B, C, and D were excised from the gel. The gel slices were incubated with 60 mm Na 2 CO 3, 60 mm DTT, 2% LDS and 12% sucrose, then heated at 100 C for 90 s, and put on the slab gel. Electrophoresis was carried out according to Laemmli (16). Thylakoid membranes were electrophoresed on the same gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250. CPa-1 and CPa-2 was released from their apoproteins during solubilization and electrophoresis. FC was eluted from the gel into the lower reservoir buffer. Absorption spectra of LHCP-1 and LHCP-2 were the same. showed a typical absorption spectrum which was different from those of LHCPs. LHCPo was isolated by PAGE of system I and then reelectrophoresed by system II (see Materials and Methods). LHCPo became dissociated and resolved into LHCP-1 and LHCP-2 (Fig. 3). was not a constituent of LHCPo. In order to investigate the apoproteins of the two LHCPs, we used LHCPo as a starting material because the LHCPo area was not contaminated with other polypeptides. After the second electrophoresis of LHCPo, the gels were excised as described in Fig. 4 and subjected to PAGE of the Laemmli system to analyze their apoprotein composition. Gel slices B and D contained LHCP-1 and LHCP-2, respectively. Gel slices A and C contained a small amount of Chi due to tailing of the LHCPs. LHCP-1 had and 27-kDa polypeptides, but LHCP-2 only the 29.5 kda polypeptide (Fig. 4). The same results

7 Heterogeneity of light-harvesting Chl-protein complexes 1543 LHCPo CPl Barley - heat heat LHCP LHCP-1 ~ LHCP Kef' 27 Kd~- Fig. 5.Apo LHCP J-'>po r'-apo LHCP in U c o / CPl CPl LHCP LHCP Bean Spinach Migration Fig. 6 Fig. 5 Electrophoretic pattern of polypeptides of LHCPo and thylakoid membranes. LHCPo obtained as in Fig. 1 was electrophoresed by system II as in Fig. 2, and thylakoid membranes were analyzed as in Fig. 4. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R heat, Thylakoids or gel slices containing LHCPo were heated before loading on the gel of system II. heat, Without heat treatment. Fig. 6 Comparison of patterns of thylakoid CPs among barley (upper), bean (middle) and spinach (lower). CPs were isolated by PAGE of system II as in Fig. 2, and gels were scanned at 675 nm. Amplification of the LHCP area is shown at the right of each tracing. were obtained when thylakoid membranes were used as starting material (data not shown). When non-heated LHCPo was reelectrophoresed with system II, four bands were found after staining (Fig. 5). Two of them located at the positions of LHCP-1 and LHCP-2 which had Chi before staining. Small amounts of and 27-kDa polypeptides were presumably derived from partial dissociation of LHCP during electrophoretic procedures. When the samples were heated, only and 27-kDa polypeptides were found on the gel. Apoproteins migrated faster than their native forms, especially in the case of the 27-kDa polypeptide. This was probably due to the release of Chi from CP. Delepelair and Chua (1979) reported that native CPs have faster mobilities compared to their apoproteins. In the present study, however, CPl,, LHCP-1 and LHCP-2 migrated more slowly than their respective proteins. When denatured thylakoid preparation was electrophoresed by system II, a polypeptide band of 29 kda was observed adjacent to the band of an LHCP apoprotein of 29.5 kda after staining. We also examined the LHCP heterogeneity in bean and barley (Fig. 6). Two main and V.

8 1544 A. Tanaka, Y. Tanaka and H. Tsuji several additional minor green bands of LHCP and one band of were separated from bean, but only one LHCP and one were resolved from barley. Discussion To study CP components and their relative amounts in tissues, the PAGE system requires that: (i) all the thylakoid membranes are solubilized and migrate into the gel; (ii) as many kinds of CPs as possible are resolved on the gel; (iii) release of Chi from apoproteins is minimized. Although thylakoid membranes were solubilized completely and the amount of FC was small in Anderson' SDS-PAGE system (Anderson 1980), two CPs of PS II and were not resolved. On the other hand, octylglucoside/sds PAGE was a useful system for separating CPs, although membrane solubilization was not complete and a considerable amount of the Chi was released and migrated as FC. We separated and two CPs of PS II, CPa-1 and CPa-2, by the SDS- PAGE system of Laemmli. Membrane solubilization was 100%, because only little Chi remained on the top of the gel after electrophoresis when solubilized thylakoid membranes were loaded without centrifugation. FC was less than 10% of the total Chi. With this system, and LHCPm did not clearly separate as with the octylglucoside/sds-page system. The lower Chi a/b ratio of with our system was probably due to contamination with LHCP. When the electrophoresis period was prolonged, and LHCPm were clearly resolved. However, a large amount of Chi migrated as FC in this case. Many investigators (Dos Santos and Hall 1982, Metz et al. 1983) have separated CPs with Laemmli's system, in which only CP1, and LHCPm are resolved. Therefore, the discrepancy between the results of early workers and ours was probably due to the difference in solubilizing conditions and the period of electrophoresis. We succeeded in separating the LHCP monomer into two green bands that had similar absorption spectra on the gel. Green and Camm (1982) obtained similar patterns when sucrose gradient fractions of octylglucoside extract from spinach chloroplasts were subjected to SDS- PAGE. The following facts suggest that both and 27-kDa polypeptides bind Chi. (i) The and 27-kDa polypeptides migrated with Chi on the gel with a non-denaturing PAGE system (Fig. 4 and 5). (ii) LHCP-1 and LHCP-2 migrated slowly compared to the Chl-depleted proteins, presumably due to the binding of Chi to apoproteins. (iii) LHCPo was found to have two polypeptides. Whether the 29.5-kDa polypeptide found in each of the LHCP-1 and LHCP-2 areas arose from different molecular species is not known. We can not exclude the possibility that the 29.5-kDa polypeptide of the LHCP-1 fraction was merely a contaminant from LHCP-2. In the area of LHCPm four polypeptides were resolved after electrophoresis of spinach thylakoid membranes with our system. The and 27-kDa polypeptides were LHCP apoproteins and 28 kda was a apoprotein. The 29-kDa polypeptide did not seem to bind Chi, because its amount and migration distance were not changed by heat treatment. We succeeded in separating the LHCP monomer into two green bands in spinach and two major and several faint green bands in bean. However, as only one LHCP was resolved in barley under the same conditions, the extent of heterogeneity of LHCP may vary among plant species. There may be two possible explanations for there being only a single band of LHCP found in barley. The first is that there is only one LHCP apoprotein or a very small amount of additional apoproteins in barley. Only one polypeptides were observed after reelectrophoresis of LHCPo in barley. Camm and Green (1980) reported that barley LHCP purified by PAGE had one polypeptide, although other investigators (Burke et al. 1979) reported finding three apoproteins in the LHCP preparation purified by sucrose gradient and cation precipitation. The other possible explanation for the finding of only a single band is that some LHCP comigrated on the gel because of their similar mobility. Further study is needed to establish whether or not the heterogeneity of LHCP is different from species to species.

9 Heterogeneity of light-harvesting Chl-protein complexes 1545 References Anderson, J. M. (1980) P-700 content and polypeptide profile of chlorophyll-protein complexes of spinach and barley thylakoids. Biochim. Biophys. Ada 591: Anderson, J. M., Waldrom, J. G. and Thorne, S. W. (1978) Chlorophyll-protein complexes of spinach and barley thylakoids: Spectral characterization of six complexes resolved by an improved electrophoretic procedure. FEBS Lett. 92: Argyroudi-Akoyunoglou, J. (1984) The 77K fluorescence spectrum of the photosystem I pigment-protein complex CPIa. FEBS Lett. 171: Burke, J. J., Steinback, K. E. andarntzen, C. J. (1979) Analysis of the light-harvesting pigment-protein complex of wild type and a chlorophyll-a-less mutant of barley. Plant Physiol. 63: Camm, E. L. and Green, B. R. (1980) Fractionation of thylakoid membranes with the nonionic detergent octyl-/3- D-glucopyranoside: Resolution of chlorophyll-protein complex II into two chlorophyll-protein complexes. Plant Physiol. 66: Camm, E. L. and Green, B. R. (1981) Widespread distribution of some minor chlorophyll-protein complexes in some plants and algae. Plant Physiol. 67: Delepelair, P. and Chua, N.-H. (1979) Lithium dodecyl sulfate/polyacrylamide gel electrophoresis of thylakoid membranes at 4 C: Characterizations of two additional chlorophyll a-protein complexes. Proc Nad. Acad. Sci. USA 76: Dos Santos, C. P. and Hall, D. O. (1982) Thylakoid polypeptides of light and dark aged chloroplasts. Plant Physiol 70: Dunahay, T. G. and Staehelin, L. A. (1986) Isolation and characterization of some minor chlorophyll a/a-protein complex (CP24) from spinach. Plant Physiol. 80: Dunsmuir, D. (1985) The petunia chlorophyll a/b binding protein genes: A comparison ofcab genes from different gene families. Nucl. Acids Res. 13: Green, B. R. and Camm, E. L. (1982) The nature of the light-harvesting complex as denned by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Biochim. Biophys. Ada 681: Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: Lam, E., Ortiz, W., Mayfield, S. and Malkin, R. (1984) Isolation and characterization of a light-harvesting chlorophyll a/b protein complex associated with photosystem I. Plant Physiol 74: Machold, O. and Meister, A. (1979) Resolution of the light-harvesting chlorophyll a/6-protein of Vicia Faba chloroplasts into two different chlorophyll-protein complexes. Biochim. Biophys. Ada 546: Markwell, J. P., Thornber, J. P. and Boggs, R. T. (1979) Higher plant chloroplasts: Evidence that all the chlorophyll exists as chlorophyll-protein complexes. Proc. Natl. Acad. Sci. USA 76: McDonnel, A. and Staehelin, L. A. (1980) Adhesion between liposomes mediated by the chlorophyll a/b lightharvesting complex isolated from chloroplast membranes. J. Cell Biol. 84: Metz, J. G., Miles, D. and Rutherford, W. (1983) Characterization of nuclear mutants of maize which lack the cytochrome f/b-563 complex. Plant Physiol. 73: Mullet, J. (1983) The amino acid sequence of the polypeptide segment which regulates membrane adhesion (grana stacking) in chloroplasts. /. Biol. Chem. 258: Thornber, J. P. (1979) Chlorophyll-proteins: Light-harvesting and reaction center components of plants. Annu. Rev. Plant. Physiol. 26: Thornber, J. P. (1986) Biochemical characterization and structure of pigment-proteins of photosynthetic organism. In Encyclopedia of Plant Physiology. Vol. 19 Photosynthesis III. Edited by Staehelin, L. A. and Arntzen, C.J. pp Springer-Verlag, DEU. (Received August 5, 1987; Accepted September 17, 1987)

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