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Characterization of damage to the D1 protein of photosystem II under photoinhibitory illumination in non-phosphorylated and phosphorylated thylakoid membranes
J o u r n a l of
Photochemistry Photobiology and
ELSEVIER
J. Photochem.Photobiol. B: Biol. 48 (1999) 97-103
B:Biology
Characterization of damage to the D 1 protein of photosystem II under photoinhibitory illumination in non-phosphorylated and phosphorylated thylakoid membranes N. Mizusawa *' 1, N. Yamamoto, M. Miyao Laboratory of Photosynthesis, National Institute of Agrobiological Resources (N1AR), Kannondai, Tsukuba 305-8602, Japan
Received 13 August 1998; accepted 30 October 1998
Abstract
Thylakoid membranes retaining a high capacity to phosphorylate the D 1 protein of photosystem II but free from contamination by exogenous proteases have been prepared. Almost all the DI protein in this preparation is unphosphorylated, but it is almost completely phosphorylated by illumination in the presence of ATP. During photoinhibitory illumination of the thylakoid membranes, the D1 protein is cleaved in the DE loop to give rise to C-terminal fragments of 9.3 and 7.9 kDa in both the unphosphorylated and phosphorylated forms, though cleavage proceeds slightly more slowly in the phosphorylated form. Cleavage inside helix D giving rise to a C-terminal fragment of 16 kDa also occurs, albeit to a much lesser extent than that in the DE loop. When samples that have been illuminated with photoinhibitory light are kept in darkness for 2 h, the amount of the 9.3 kDa fragment selectively increases, irrespective of the phosphorylation state of the protein. This cleavage in darkness is not suppressed by protease inhibitors, an indication of the involvement of non-enzymatic reactions. No extra fragments of the D1 protein are detected either during or after photoinhibitory illumination, even when stromal proteins are present. These observations suggest that cleavage of the D1 protein proceeds via the same mechanism in both the phosphorylated and unphosphorylated forms in isolated thylakoid membranes. © 1999 Elsevier Science S.A. All rights reserved. Keywords: D1 Protein; Protein phosphorylation;Photoinhibition;PhotosystemII; Thylakoidmembranes
1. Introduction The D 1 protein of the reaction centre complex of photosystem II (PSII) has the highest turnover rate under illumination in vivo of all the proteins in the thylakoid membrane [ 1 ]. The rapid turnover of the protein has long attracted the attention of researchers, since this phenomenon is involved in the photoinhibition of photosynthesis [2]. Irrespective of extensive efforts, however, the detailed mechanism of the rapid turnover of the D1 protein is not yet fully understood. The mechanism of the initial cleavage of the protein under illumination is one of the most important problems to be solved. Numerous studies in vitro have been performed using isolated thylakoid membranes and PSII preparations since it was found in 1990 that cleavage of the D 1 protein can occur even in vitro when materials are illuminated with the strong light * Correspondingauthor. Tel.: + 81-298-38-7074;Fax: + 81-298-38-7073; E-mail: [email protected] ~Also at Bio-Oriented Technology Research Advancement Institution (BRAIN), Japan.
that causes photoinhibition of PSII (for review, see Ref. [ 3 ] ). It was demonstrated that cleavage in vitro occurred mainly in the loop that connects the membrane-spanning helixes D and E (the DE loop), giving rise to N-terminal and C-terminal fragments of 22-24 and 8-10 kDa, respectively [3]. This observation agreed with a previous finding that the cleavage in vivo under light conditions suitable for plant growth gave rise to a fragment of 23.5 kDa [4]. However, there is an argument that observations in vitro do not accurately reflect phenomena in vivo, since processes that operate under illumination in vivo do not occur in vitro. For example, formation of a pH gradient across the thylakoid membrane, lateral migration between the stroma and grana thylakoids of proteins, and phosphorylation of the PSII proteins cannot occur in isolated PSII sub-complexes. Even in isolated thylakoid membranes, stromal components are absent. In fact, photoinhibitory illumination in vitro differs from that in vivo in some respects, and phenomena that are not observed in vivo can occur: the cleavage of the D1 protein within or immediately adjacent to helix D to give rise to a fragment of about 16 kDa [5,6] and cross-linking reactions to form covalent
1011-1344/99/$ - see front matter © 1999Elsevier Science S.A. All rights reserved. PIISIO11-1344(99)00023-8
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adducts between the D1 and D2 proteins (the heterodimer) and between the D1 protein and the et subunit of cytochrome b559 ( the 41 kDa adduct) [ 7,8 ]. Furthermore, it has recently been proposed that the major cleavage site in vivo is not located in the DE loop [9]. Thus, there exists a gap between studies in vivo and in vitro. To understand the molecular mechanism of cleavage of the D1 protein, this gap has to be bridged by the use of experimental systems that can reproduce phenomena in vivo. It has been proposed that reversible phosphorylation of the D I protein has a regulatory function in its turnover [ 10-13] and, moreover, that cleavage occurs only after dephosphorylation of the D 1 protein that has been phosphorylated during illumination in vivo [9]. In this study, we prepared intact thylakoid membranes that retained a high capacity to phosphorylate the PSII core proteins but were totally free from contamination by proteins from cellular compartments other than the chloroplast. Using such thylakoid preparations, damage to the D 1 protein under photoinhibitory illumination was re-examined, focusing on the effects of phosphorylation on the damage.
Then, the chloroplasts were repurified by centrifugation through 40% (vol./vol.) Percoll in medium A at 3000g for 5 min and then washed twice with medium A by centrifugation at 2500g for 60 s and resuspension. To isolate thylakoid membranes, the thermolysin-treated intact chloroplasts were suspended in 5 mM MgC12 and 10 mM Hepes-NaOH (pH 7.8) at 1.0 mg Chl ml ~ and incubated in darkness at 0°C for 10 min. Then, the suspension was centrifuged at 10 000g for 10 rain. The resultant supernatant was taken as the stromal fraction and kept frozen in liquid nitrogen until use. Thylakoid membranes obtained as a pellet from the centrifugation were washed once with 10 mM NaC1, 5 mM MgCle, 0.1 M sucrose and 50 mM HepesNaOH (pH 7.8), washed twice with 10 mM NaCI, 5 mM MgClz, 0.4 M sucrose and 50 mM Hepes-NaOH (pH 7.8; medium B) by centrifugation at 10 000g for 5 min, finally suspended in the same medium and kept frozen in liquid nitrogen. All procedures were performed under dim light at 0-4°C. Chlorophyll was determined by the method of Amon [ 16].
2.2. Phospho~. lation treatment of thylakoid membranes
2. Experimental 2.1. Isolation of intact chloroplasts and thylakoid membranes Spinach intact chloroplasts were first isolated from leaf homogenates by differential centrifugation by the method of Nakatani and Barber [ 14] and purified further by Percoll density gradient centrifugation as follows. An intact chloroplast fraction obtained by differential centrifugation was pelleted by centrifugation at 2200g for 20 s, suspended in 0.1% (wt./vol.) bovine serum albumin, 0.33 M sorbitol and 30 mM Hepes-NaOH (pH 7.8) and layered onto a discontinuous gradient composed of 50 and 80% (vol./vol.) Percoll in the same medium. After centrifugation at 8500g for 15 min, the intact chloroplasts, which formed a band in the interface between 50 and 80% Percoll, were collected, diluted with five volumes of 0.33 M sorbitol and 30 mM Hepes-NaOH ( pH 7.8; medium A), and pelleted by centrifugation at 2500g for 60 s. They were then washed once with medium A by centrifugation and resuspension and finally suspended in the same medium at a chlorophyll (Chl) concentration of 1.0 mg ml ~. The intactness of the purified chloroplasts was confirmed by a light microscope. To remove proteins that had been adsorbed on the surface of the chloroplasts, the purified intact chloroplasts were treated with thermolysin as described previously [ 15 ] as follows, A suspension of the intact chloroplasts of 1.0 mg Chl ml - J was supplemented with thermolysin (from Bacillus thermoproteolyticus rokko, Sigma) and CaCI2 to give final concentrations of 50 gg ml ~and 2 mM, respectively. After an incubation in darkness at 0°C for 30 min, the suspension was supplemented with 10 mM EGTA to terminate digestion.
The thylakoid membranes were thawed in a water bath at 25°C, suspended in medium B at 0.4 mg Chl ml-~, and supplemented with 1/ 100 volume of 40 mM ATP to give a final concentration of 0.4 raM. After an incubation in darkness at 25°C for 10 rain, the suspension was illuminated with white light from a halogen lamp, which was passed through a layer of heat-reflecting filter, at 100 IxE m 2 s- ~and 25°C for 30 min. Then, the suspension was diluted with three volumes of medium B that also contained 10 mM NaF and 5 mM EDTA. The phosphorylated thylakoid membranes were collected by centrifugation at 18 000g for 5 min, washed once with medium B by centrifugation and resuspension, and finally suspended in the same medium. Control treatment was performed as above, but samples were illuminated in the absence of ATP.
2.3. Photoinhibitory treatment of thylakoid membranes Thylakoid membranes that had been subjected to the control or phosphorylation treatment were suspended in medium B at 0.1 mg Chl ml ~and incubated in darkness at 25°C for 10 min. Then, the suspension was illuminated with strong white light from a halogen lamp, which was passed through two layers of heat-reflecting filters, at 1000 txE m -~s J and 25°C. After illumination for the designated times, a portion of the suspension was withdrawn, supplemented with 10 mM NaF and 1/15 volume of a mixture of protease inhibitors (Protease Inhibitor Cocktail, Boehringer Mannheim) and assayed for the activity of PSII electron transport or immediately frozen in liquid nitrogen and kept at - 80°C for protein analyses by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).
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When indicated, the stromal fraction was included in the suspension of thylakoid membranes to give a ratio of protein to chlorophyll equivalent to that in the intact chloroplast.
a
b
NP P
NP P
c
NP P
2.4. A n a l y t i c a l p r o c e d u r e s
SDS-PAGE and subsequent immunoblotting were performed as described previously [ 17], except that the polyacrylamide concentration of the separation gel was 15%. For immunoblotting, two different antisera were used: anti-Dlc raised against a synthetic polypeptide that corresponded to the residue 333-344 of the D 1 protein of spinach (a generous gift from Dr T. Ono) and anti-phosphothreonine (antiphosphoThr; Zymed Laboratories). PSII electron transport activity was determined as the activity of photoreduction of 2,6-dichlorophenolindophenol (DCIP) in medium B that contained 2 mM NH4CI and 50 ~M DCIP at 25°C. The reduction of DCIP was determined spectrophotometrically from the absorbance change at 600 nm.
3. Results To obtain intact and active thylakoid membranes free from any contamination, we isolated thylakoid membranes from intact chloroplasts that had been purified by Percoll gradient centrifugation and subsequently treated with thermolysin. Thylakoid membranes thus prepared retained a high capacity to phosphorylate the PSII proteins under illumination with weak light in the presence of ATP (Fig. 1 ). The apoprotein ( s ) of the light-harvesting complex of PSII (LHCII), the DI and D2 proteins of the PSII reaction centre complex and the 43 kDa protein of the PSII core antenna were phosphorylated, as shown by immunoblotting with anti-phosphoThr. Phosphorylation of these proteins could also be detected as shifts of the Coomassie-stained bands in the gel. Phosphorylation of the PsbH protein was not detected by immunoblotting with anti-phosphoThr as reported previously [ 18]. Immunoblotting with anti-D l c revealed that more than 90% of the DI protein remained unphosphorylated in the control sample, while more than 90% became phosphorylated by the phosphorylation treatment. Hereafter, thylakoid membranes that had been illuminated in the presence and absence of ATP are designated as phosphorylated and non-phosphorylated thylakoid membranes, respectively. Fig. 2 compares inactivation of PSII and damage to the D1 protein under photoinhibitory illumination in the non-phosphorylated and phosphorylated thylakoid membranes. The inactivation proceeded in the same time courses in both types of the membranes as reported previously [ 12], and the activity was reduced by about 90% within 60 min (Fig. 2 ( A ) ) . The overall pattern of damage to the D1 protein was also identical in both cases (Fig. 2(B) ). As seen in panel (a), in which large amounts of sample were applied for detection of fragments, C-terminal fragments of the D1 protein of 16, 9.3 and 7.9 kDa and also the cross-linked products, namely, the
43* ~jDI* ~D1
LHClI*
'iiii ....
Fig. 1. Phosphorylationof the PSII core proteins and LHCI1apoproteinsby illumination of isolated thylakoidmembranes in the presence of ATP. Thylakoid membranes suspended in 10 mM NaC1,5 mM MgCI2,0.4 M sucrose and 50 mM Hepes-NaOH (pH 7.8) at 0.4 mg Chl ml ~were supplemented with 0.4 mM ATP and illuminated with white light at 100 p~Em : s ~at 25°C for 30 rain (phosphorylation treatment). Then, the suspension was diluted with three volumes of the same medium supplemented with 10 mM NaF and subjected to SDS-PAGE. (a) Polypeptide profiles after staining with Coomassie blue; (b) immunoblotprofiles with anti-Dlc; (c) immunoblot profiles with anti-phosphoThr. The amounts of sample applied for SDS-PAGE corresponded to 1 ~g Chl in panels (a) and (c) and 0.05 ~g Chl in panel (b). NP and P stand for non-phosphorylatedand phosphorylated thylakoid membranes that were illuminated in the absence and presence of ATP, respectively. Asterisks indicate phosphorylatedproteins. heterodimer and the 41 kDa adduct, were generated during the course of photoinhibitory illumination. It has been demonstrated that the 16 kDa fragment and the two fragments of 9.3 and 7.9 kDa originate from cleavage within helix D and that in the DE loop, respectively [19]. One difference between the non-phosphorylated and phosphorylated thylakoid membranes was that the appearance of the fragments and the cross-linked products was slightly slower in the phosphorylated membranes. Immunoblotting performed with small amounts of sample optimum for quantification of the D1 protein band (panel (b) of Fig. 2(B) ) indicated that the total level of the D 1 protein was reduced by photoinhibitory illumination in both the non-phosphorylated and phosphorylated thylakoid membranes, but that the extent of phosphorylation of the D1 protein remained unchanged during photoinhibitory illumination, although this was performed in the absence of NaF, an inhibitor of protein phosphatases. Polypeptide profiles of thylakoid membranes after staining with Coomassie blue remained unchanged under illumination for 60 min ( data not shown). This observation indicated that, unlike isolated PSII subcomplexes [ 17], unspecific damage to proteins did not occur in thylakoid membranes under the conditions for photoinhibitory illumination in this study. The presence of the stromal fraction during photoinhibitory illu-
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(A)
~ 2
o
100(
75
2s
n
°o
4o Illumination (min)
(B) a
NP
P
b
0 2.5 5 15 30 60 0 2.5 5 15 30 60 rain
NP 0 30
P 0 30 mln
HD 41
DI* D1 )
./DI* "~ D1
16 ::
i
~ 9.3 7.9
Fig. 2. Effects of photoinhibitory illumination on PSII electron transport activity and the DI protein in non-phosphorylated and phosphorylated thylakoid membranes. Non-phosphorylated (NP) and phosphorylated (P) thylakoid membranes suspended at 0.1 mg Chl ml ] were incubated in darkness at 25°C for 10 min, and then illuminated with strong white light at 1000 ixE m 2 s ~and 25°C for the designated times. (A) Activity of DCIP photoreduction: O, nonphosphorylated membranes; 0, phosphorylated membranes. Activities before photoinhibitory illumination were 230 IxmolDCIP mg t Chl h ~in both NP and P membranes. (B) Immunoblot profiles with anti-Dlc. Samples that corresponded to 1 and 0.05 Ixg Chl were applied for SDS-PAGE in panels (a) and (b), respectively. HD denotes the heterodimer of the D 1 and D2 proteins. mination did not affect the pattern of damage to the D 1 protein (Fig. 3). W e investigated if additional cleavage of the D1 protein could occur in darkness after photoinhibitory illumination. As shown in Fig. 4 ( A ) , an incubation in darkness for 2 h selectively increased the amount of the 9.3 kDa fragment, while the amount of the 7.9 kDa fragment was totally unaffected in both the non-phosphorylated and phosphorylated thylakoid membranes. These observations indicated that, among two different sites of cleavage in the DE loop, cleavage in one site occurred only under photoinhibitory illumination while cleavage in the other site could continue even after illumination. No extra fragments of the D1 protein were generated during the incubation in darkness. This was also the case when immunoblotting was performed with an antibody raised against the entire D1 protein of spinach (a generous gift of Dr M. Ikeuchi; data not shown). The increase in the 9.3 kDa fragment was not affected by the presence of the stromal fraction during the incubation.
To examine the involvement of proteases in the cleavage of the D1 protein in darkness, the phosphorylated thylakoid membranes that had been illuminated with photoinhibitory light were incubated in darkness in the presence of a mixture of protease inhibitors. This mixture contained inhibitors of serine and cysteine proteases and metalloproteases. As shown in Fig. 4 ( B ) , the protease inhibitors did not at all affect the increase in the 9.3 kDa fragment in darkness, an indication that the cleavage proceeded non-enzymatically. This was also the case in the non-phosphorylated thylakoid membranes (data not shown). An incubation in darkness after photoinhibitory illumination also affected the amount of the heterodimer, though its effects varied depending on the thylakoid membranes used: the amount of heterodimer was slightly decreased during the incubation in the experiments of Fig. 4 ( A ) , while it appeared rather to increase in the experiments of Fig. 4 ( B ) . It was found that the presence of the protease inhibitors during the incubation greatly increased the amount of heterodimer (Fig.
101
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NP
of Fig. 4(A), the digestion rate exceeded the generation rate, resulting in the decrease in heterodimer during incubation, while the opposite occurred in the experiments of Fig. 4 (B). The increase in the amount of heterodimer caused by the protease inhibitors did occur in the absence of the stromal fraction (Fig. 4(B) ). Therefore, it is likely that a thylakoidbound protease (s) is involved in the digestion.
P
0 30 60 0 3 0 6 0 ( m i n ) HD 41 DI*~ D1 '
4. Discussion
It has recently been reported that, unlike photoinhibitory illumination of thylakoid membranes and PSII preparations, photoinhibitory illumination of intact leaves gives rise to 20 and 18 kDa fragments of the D1 protein of C-terminal and N-terminal origin, respectively, and proposed that cleavage occurs only after dephosphorylation of the DI protein that has been phosphorylated during illumination [ 9]. To reproduce these observations in experiments in vitro, we first prepared thylakoid membranes that retained a high capacity to phosphorylate the D1 protein. By isolating thylakoid membranes from an intact chloroplast preparation of high purity, we could obtain preparations in which almost all the D1 protein was able to be phosphorylated by illumination in the presence of ATP (Fig. 1 ). Cleavage of the D1 protein was compared in two types of thylakoid membranes in which almost all the D 1 protein was either unphosphorylated or phosphorylated. It was found, however, that phosphorylation of the D 1 protein did not affect the overall pattern of damage to the D1 protein, though it slightly slowed down the course of damage: major cleavage
0.3 7.9 Fig. 3. Effects of the stromal fraction present during photoinhibitory illumination on the damage to the D1 protein. Non-phosphorylated (NP) and phosphorylated (P) thylakoid membranes were supplemented with the stromal fraction and illuminated with photoinhibitory light for the designated times, lmmunoblot profiles with anti-Dlc are shown. Samples that corresponded to 1 t-tg Chl were applied for SDS-PAGE. An arrow indicates a band of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), which cross-reacted with anti-Dlc.
4 ( B ) ) . This was also the case in the non-phosphorylated thylakoid membranes (data not shown). These observations suggested that the heterodimer was newly generated even after photoinhibitory illumination, but simultaneously, it was digested by a protease (s). Accordingly, the apparent amount of the heterodimer was determined by the relative rates of its generation and digestion. It is likely that, in the experiments
(A)
(B) NP
o ~ Dark R" ~ " +
P o ~ ~" d
Dark -+ Stroma R" R" - - "i" - - "1" Protean inhihitor8 o
Dark - - + Stroma
D
-HD
~
DI' D1 :
- 16
¢9.3 ~7.9 DI' D1
....
Fig. 4. Effects of an incubation in darkness after photoinhibitory illumination on the DI protein. Non-phosphorylated (NP) and phosphorylated (P) thylakoid membranes which had been illuminated with photoinhibitory light (PI) for 30 min were transferred to darkness and incubated further in darkness at 25°C for 2 h in the presence or absence of the designated additives, namely, the strom',d fraction and/or a mixture of protease inhibitors ( 1 / 15 volume; Protease Inhibitor Cocktail, Boehringer Mannheim). In experiments in (B), phosphorylated thylakoid membranes were used. Immunoblot profiles with anti-Dlc are shown. The sample amounts applied for SDS-PAGE in the upper and lower panels correspond to 1 and 0.05 Ixg Chl, respectively.
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N. Mizusawa et al./ J. Photochem. Photobiol. B: Biol. 48 (1999) 97-103
occurred in the DE loop and also cross-linked products were generated (Fig. 2(B) ). This was also the case when stromal proteins were present during photoinhibitory illumination (Fig. 3). According to the proposed mechanism of cleavage in vivo [9], our failure to reproduce these observations in vivo should result from slow dephosphorylation of the D1 protein in the thylakoid membranes used in this study. As shown in Fig. 2(B) and Fig. 4, dephosphorylation did not occur during photoinhibitory illumination. Even after photoinhibitory illumination, dephosphorylation was slow and it took about four hours to dephosphorylate about half the D 1 protein (data not shown). This rate of dephosphorylation, however, was almost comparable to that in intact leaves [ 13 ]. Accordingly, dephosphorylation during photoinhibitory illumination might be a prerequisite for reproduction of the observations in vivo, if dephosphorylation were to be concerned. The observations in Fig. 2(B) demonstrate that the D1 protein can be cleaved in the DE loop under photoinhibitory illumination. It is unlikely that the cleavage in the DE loop was a non-physiological event that resulted from severe photoinhibitory illumination, since photoinhibitory illumination for 60 rain did not substantially affect the polypeptide profiles of the thylakoid membranes used (data not shown). It is possible that, in vivo, fragments derived from cleavage in the DE loop were digested very rapidly and hence were barely detectable. Our observations also contrast with previous studies with isolated thylakoid membranes and PSII core complexes, which showed that the D 1 protein in the phosphorylated form was more resistant to degradation under illumination than that in the unphosphorylated form [ 11,12]. In the previous studies, however, disappearance of a band of the D1 protein of around 32 kDa was taken as an indication of degradation, but cleavage of the protein was not investigated. In addition, phosphorylated and unphosphorylated D1 proteins were compared in the same preparation in which only a fraction of the D1 protein was phosphorylated. We observed that, in thylakoid membranes, the presence of ATP resulted in phosphorylation of the unphosphorylated D 1 protein even after PSII electron transport was inactivated by about one-third by photoinhibitory illumination (data not shown). Therefore, it is possible that the rate of disappearance of the phosphorylated D1 protein could be underestimated if ATP was present during photoinhibitory illumination. During the course of experiments to survey conditions for dephosphorylation of the D1 protein, we found that the amount of the 9.3 kDa fragment of the DI protein was selectively increased in darkness after photoinhibitory illumination, while the 7.9 kDa fragment had been more abundant during photoinhibitory illumination. We previously proposed that the 9.3 kDa fragment originated from cleavage in the middle part of the DE loop and the 7.9 kDa fragment from cleavage in close proximity to the His272 that coordinates the non-haem iron [19]. This hypothesis was partly confirmed by microsequencing of fragments in which the N-
terminal sequence of the 9.3 kDa fragment matched that of the D1 protein from residue 257 (unpublished), which is located in a helix inside the DE loop (the de helix). Therefore, it is suggested that, during photoinhibitory illumination, cleavage occurred predominantly in close proximity to His272, while cleavage inside the d e helix became dominant in darkness. Protease inhibitors did not suppress the cleavage inside the d e helix in darkness, and it was also unaffected by the presence of the stromal proteins (Fig. 4). If the cleavage were to proceed via chemical reactions induced by active oxygen species as proposed previously [ 19,20], it is expected that cleavage in the proximity of His272 would proceed rapidly after attack by active oxygen species, while cleavage inside the d e helix would be trapped or proceed relatively slowly. We have previously demonstrated that the susceptibility of the D1 protein to cleavage after attack by active oxygen species depends on the conformation of the protein [20]. Since the DE loop of the D 1 protein has a flexible conformation, it is possible that cleavage reactions are initiated only when the conformation around the cleavage site becomes suitable for cleavage. Among products generated during photoinhibitory illumination, only the heterodimer appeared to be digested by a protease (s) (Fig. 4). Although the protease (s) involved has not been characterized yet, it might be the thylakoid-bound protease that digests proteins damaged by active oxygen species [21 ].
Acknowledgements The authors are grateful to Dr Taka-aki Ono of the Institute of Physical and Chemical Research (RIKEN), Japan, for his generous gift of an antibody, and to Ms Shizue Sudoh for her helpful assistance. This work was supported in part by a PROBRAIN grant from the Bio-Oriented Technology Research Advancement Institution (BRAIN) of Japan.
References [ 1] A.K.Mattoo,H. Hoffman-Falk,J.B. Marder,M. Edelman,Regulation of protein metabolism:couplingof photosyntheticelectron transport to in vivodegradationof the rapidlymetabolized32-kilodaltonprotein of the chloroplastmembranes,Proc. Natl. Acad. Sci. USA 81 (1984) 1380-1384. [2] D.J. Kyle, I. Ohad, C.J. Arntzen, Membrane protein damage and repair: selective loss of a quinone-protein function in chloroplast membranes,Proc. Natl. Acad. Sci. USA 81 (1984) 4070-4074. [3] E.-M. Aro, I. Virgin, B. Andersson, Photoinhibition of Photosystem II: inactivation,proteindamageand turnover,Biochim.Biophys.Acta 1143 (1993) 113-134. [4] B.M. Greenberg,V. Gaba, A.K. Mattoo, M. Edelman, Identification of a primaryin vivo degradationproduct of the rapidly-turningover 32 kD proteinof photosystem1I, EMBOJ. 6 (1987) 2865-2869. [5] J. De Las Rivas, B. Andersson, J. Barber, Two sites of primarydegradation of the Dl-protein induced by acceptoror donor side photo-
N. Mizusawa et al. / J. Photochem. Photobiol. B: Biol. 48 (1999) 97-103
[6]
[7]
[8]
[9]
[ 10]
[ 11]
[12]
[ 13]
inhibition in photosystem II core complexes, FEBS Lett. 301 (1992) 246-252. A.H. Salter, J. De Las Rivas, J. Barber, B. Andersson, On the molecular mechanism of light-induced D 1 protein degradation, in: N. Murata (Ed.), Research in Photosynthesis, vol+ IV, Kluwer, Dordrecht, 1992, pp. 395-402. R. Barbato, G. Friso, F. Rigoni, A. Frizzo, G.M. Giacometti, Characterization of a 41 kDa photoinhibition adduct in isolated photosystem II reaction centres, FEBS Lett. 309 (1992) 165-169, R. Barbato, G. Friso, M. Ponticos, J. Barber, Characterization of the light-induced cross-linking of the a-subunit of cytochrome b559 and the D1 protein in isolated photosystem II reaction centers, J. Biol. Chem. 270 (1995) 24032-24037. R. Kettunen, E. Tyystjiirvi, E.-M. Aro, Degradation pattern of photosystem II reaction center protein D1 in intact leaves. The major photoinhibition-induced cleavage site in D1 polypeptide is located amino terminally of the DE loop, Plant Physiol. 111 (1996) 11831190. F.E. Callahan, M.L. Ghirardi, S.K. Sopory, A.M. Mehta, M. Edelman, A.K. Mattoo, A novel metabolic torm of the 32 kDa-D1 protein in the grana-localized reaction center of Photosystem II, J. Biol. Chem. 265 (1990) 15357-15360. E.-M. Aro, R. Kettunen, E. Tyystj~vi, ATP and light regulate DI protein modification and degradation. Role of D 1* in photoinhibition, FEBS Lett. 297 (1992) 29-33. A. Koivuniemi, E.-M. Aro, B. Andersson, Degradation of the D 1- and D2-proteins of photosystem II in higher plants is regulated by reversible phosphorylation, Biochemistry 34 (1995) 16022-16029. E. Rintam~iki, R. Kettunen, E.-M. Aro, Differential DI dephosphorylation in functional and photodamaged photosystem II centers, J. Biol. Chem. 271 (1996) 14870-14875.
103
114] H.Y. Nakatani, J. Barber, An improved method for isolating chloroplasts retaining their outer membranes, Biochim. Biophys. Acta 461 (1977) 510--512. [ 15] K. Miyadai, T. Mae, A. Makino, K. Ojima, Characteristics ofribulose1,5-bisphosphate carboxylase/oxygenase degradation by lysates of mechanically isolated chloroplasts from wheat leaves, Plant Physiol. 92 (1990) 1215-1219. [16] D.I. Arnon, Copper enzyme in isolated chloroplasts: polyphenoloxidase in Beta vulgaris, Plant Physiol. 24 (1949) 1-15. [ 17] M. Miyao, Involvement of active oxygen species in degradation of the D1 protein under strong illumination in isolated subcomplexes of photosystem II, Biochemistry 33 (1994) 9722-9730. [18] E. RintamS.ki, M. Salonen, U.-M. Suoranta, I. Carlberg, I3. Andersson, E.-M. Aro, Phosphorylation of light-harvesting complex II and photosystem II core proteins shows different irradiance-dependent regulation in vivo. Application of phosphothreonine antibodies to analysis of thylakoid phosphoproteins, J. Biol. Chem. 272 (1997) 3047630482. [ 19] M. Miyao, M. Ikeuchi, N. Yamamoto, T. Ono, Specific degradation of the DI protein of photosystem II by treatment with hydrogen peroxide in darkness: implications for the mechanism of degradation of the D1 protein under illumination, Biochemistry 34 (1995) 1001910026. [20] K. Okada, M. Ikeuchi, N. Yamamoto, T. Ono, M. Miyao, Selective and specific cleavage of the D1 and D2 proteins of Photosystem II by exposure to singlet oxygen: factors responsible for the susceptibility to cleavage of the proteins, Biochim. Biophys. Acta 1274 (1996) 7379. [21] L.M. Casano, H.R. Lascano, V.S. Trippi, Hydroxyl radicals and a thylakoid-bound endopeptidase are involved in light- and oxygeninduced proteolysis in oat chloroplasts, Plant Ceil Physiol. 35 (1994) 145-152.
Photochemistry Photobiology and
ELSEVIER
J. Photochem.Photobiol. B: Biol. 48 (1999) 97-103
B:Biology
Characterization of damage to the D 1 protein of photosystem II under photoinhibitory illumination in non-phosphorylated and phosphorylated thylakoid membranes N. Mizusawa *' 1, N. Yamamoto, M. Miyao Laboratory of Photosynthesis, National Institute of Agrobiological Resources (N1AR), Kannondai, Tsukuba 305-8602, Japan
Received 13 August 1998; accepted 30 October 1998
Abstract
Thylakoid membranes retaining a high capacity to phosphorylate the D 1 protein of photosystem II but free from contamination by exogenous proteases have been prepared. Almost all the DI protein in this preparation is unphosphorylated, but it is almost completely phosphorylated by illumination in the presence of ATP. During photoinhibitory illumination of the thylakoid membranes, the D1 protein is cleaved in the DE loop to give rise to C-terminal fragments of 9.3 and 7.9 kDa in both the unphosphorylated and phosphorylated forms, though cleavage proceeds slightly more slowly in the phosphorylated form. Cleavage inside helix D giving rise to a C-terminal fragment of 16 kDa also occurs, albeit to a much lesser extent than that in the DE loop. When samples that have been illuminated with photoinhibitory light are kept in darkness for 2 h, the amount of the 9.3 kDa fragment selectively increases, irrespective of the phosphorylation state of the protein. This cleavage in darkness is not suppressed by protease inhibitors, an indication of the involvement of non-enzymatic reactions. No extra fragments of the D1 protein are detected either during or after photoinhibitory illumination, even when stromal proteins are present. These observations suggest that cleavage of the D1 protein proceeds via the same mechanism in both the phosphorylated and unphosphorylated forms in isolated thylakoid membranes. © 1999 Elsevier Science S.A. All rights reserved. Keywords: D1 Protein; Protein phosphorylation;Photoinhibition;PhotosystemII; Thylakoidmembranes
1. Introduction The D 1 protein of the reaction centre complex of photosystem II (PSII) has the highest turnover rate under illumination in vivo of all the proteins in the thylakoid membrane [ 1 ]. The rapid turnover of the protein has long attracted the attention of researchers, since this phenomenon is involved in the photoinhibition of photosynthesis [2]. Irrespective of extensive efforts, however, the detailed mechanism of the rapid turnover of the D1 protein is not yet fully understood. The mechanism of the initial cleavage of the protein under illumination is one of the most important problems to be solved. Numerous studies in vitro have been performed using isolated thylakoid membranes and PSII preparations since it was found in 1990 that cleavage of the D 1 protein can occur even in vitro when materials are illuminated with the strong light * Correspondingauthor. Tel.: + 81-298-38-7074;Fax: + 81-298-38-7073; E-mail: [email protected] ~Also at Bio-Oriented Technology Research Advancement Institution (BRAIN), Japan.
that causes photoinhibition of PSII (for review, see Ref. [ 3 ] ). It was demonstrated that cleavage in vitro occurred mainly in the loop that connects the membrane-spanning helixes D and E (the DE loop), giving rise to N-terminal and C-terminal fragments of 22-24 and 8-10 kDa, respectively [3]. This observation agreed with a previous finding that the cleavage in vivo under light conditions suitable for plant growth gave rise to a fragment of 23.5 kDa [4]. However, there is an argument that observations in vitro do not accurately reflect phenomena in vivo, since processes that operate under illumination in vivo do not occur in vitro. For example, formation of a pH gradient across the thylakoid membrane, lateral migration between the stroma and grana thylakoids of proteins, and phosphorylation of the PSII proteins cannot occur in isolated PSII sub-complexes. Even in isolated thylakoid membranes, stromal components are absent. In fact, photoinhibitory illumination in vitro differs from that in vivo in some respects, and phenomena that are not observed in vivo can occur: the cleavage of the D1 protein within or immediately adjacent to helix D to give rise to a fragment of about 16 kDa [5,6] and cross-linking reactions to form covalent
1011-1344/99/$ - see front matter © 1999Elsevier Science S.A. All rights reserved. PIISIO11-1344(99)00023-8
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adducts between the D1 and D2 proteins (the heterodimer) and between the D1 protein and the et subunit of cytochrome b559 ( the 41 kDa adduct) [ 7,8 ]. Furthermore, it has recently been proposed that the major cleavage site in vivo is not located in the DE loop [9]. Thus, there exists a gap between studies in vivo and in vitro. To understand the molecular mechanism of cleavage of the D1 protein, this gap has to be bridged by the use of experimental systems that can reproduce phenomena in vivo. It has been proposed that reversible phosphorylation of the D I protein has a regulatory function in its turnover [ 10-13] and, moreover, that cleavage occurs only after dephosphorylation of the D 1 protein that has been phosphorylated during illumination in vivo [9]. In this study, we prepared intact thylakoid membranes that retained a high capacity to phosphorylate the PSII core proteins but were totally free from contamination by proteins from cellular compartments other than the chloroplast. Using such thylakoid preparations, damage to the D 1 protein under photoinhibitory illumination was re-examined, focusing on the effects of phosphorylation on the damage.
Then, the chloroplasts were repurified by centrifugation through 40% (vol./vol.) Percoll in medium A at 3000g for 5 min and then washed twice with medium A by centrifugation at 2500g for 60 s and resuspension. To isolate thylakoid membranes, the thermolysin-treated intact chloroplasts were suspended in 5 mM MgC12 and 10 mM Hepes-NaOH (pH 7.8) at 1.0 mg Chl ml ~ and incubated in darkness at 0°C for 10 min. Then, the suspension was centrifuged at 10 000g for 10 rain. The resultant supernatant was taken as the stromal fraction and kept frozen in liquid nitrogen until use. Thylakoid membranes obtained as a pellet from the centrifugation were washed once with 10 mM NaC1, 5 mM MgCle, 0.1 M sucrose and 50 mM HepesNaOH (pH 7.8), washed twice with 10 mM NaCI, 5 mM MgClz, 0.4 M sucrose and 50 mM Hepes-NaOH (pH 7.8; medium B) by centrifugation at 10 000g for 5 min, finally suspended in the same medium and kept frozen in liquid nitrogen. All procedures were performed under dim light at 0-4°C. Chlorophyll was determined by the method of Amon [ 16].
2.2. Phospho~. lation treatment of thylakoid membranes
2. Experimental 2.1. Isolation of intact chloroplasts and thylakoid membranes Spinach intact chloroplasts were first isolated from leaf homogenates by differential centrifugation by the method of Nakatani and Barber [ 14] and purified further by Percoll density gradient centrifugation as follows. An intact chloroplast fraction obtained by differential centrifugation was pelleted by centrifugation at 2200g for 20 s, suspended in 0.1% (wt./vol.) bovine serum albumin, 0.33 M sorbitol and 30 mM Hepes-NaOH (pH 7.8) and layered onto a discontinuous gradient composed of 50 and 80% (vol./vol.) Percoll in the same medium. After centrifugation at 8500g for 15 min, the intact chloroplasts, which formed a band in the interface between 50 and 80% Percoll, were collected, diluted with five volumes of 0.33 M sorbitol and 30 mM Hepes-NaOH ( pH 7.8; medium A), and pelleted by centrifugation at 2500g for 60 s. They were then washed once with medium A by centrifugation and resuspension and finally suspended in the same medium at a chlorophyll (Chl) concentration of 1.0 mg ml ~. The intactness of the purified chloroplasts was confirmed by a light microscope. To remove proteins that had been adsorbed on the surface of the chloroplasts, the purified intact chloroplasts were treated with thermolysin as described previously [ 15 ] as follows, A suspension of the intact chloroplasts of 1.0 mg Chl ml - J was supplemented with thermolysin (from Bacillus thermoproteolyticus rokko, Sigma) and CaCI2 to give final concentrations of 50 gg ml ~and 2 mM, respectively. After an incubation in darkness at 0°C for 30 min, the suspension was supplemented with 10 mM EGTA to terminate digestion.
The thylakoid membranes were thawed in a water bath at 25°C, suspended in medium B at 0.4 mg Chl ml-~, and supplemented with 1/ 100 volume of 40 mM ATP to give a final concentration of 0.4 raM. After an incubation in darkness at 25°C for 10 rain, the suspension was illuminated with white light from a halogen lamp, which was passed through a layer of heat-reflecting filter, at 100 IxE m 2 s- ~and 25°C for 30 min. Then, the suspension was diluted with three volumes of medium B that also contained 10 mM NaF and 5 mM EDTA. The phosphorylated thylakoid membranes were collected by centrifugation at 18 000g for 5 min, washed once with medium B by centrifugation and resuspension, and finally suspended in the same medium. Control treatment was performed as above, but samples were illuminated in the absence of ATP.
2.3. Photoinhibitory treatment of thylakoid membranes Thylakoid membranes that had been subjected to the control or phosphorylation treatment were suspended in medium B at 0.1 mg Chl ml ~and incubated in darkness at 25°C for 10 min. Then, the suspension was illuminated with strong white light from a halogen lamp, which was passed through two layers of heat-reflecting filters, at 1000 txE m -~s J and 25°C. After illumination for the designated times, a portion of the suspension was withdrawn, supplemented with 10 mM NaF and 1/15 volume of a mixture of protease inhibitors (Protease Inhibitor Cocktail, Boehringer Mannheim) and assayed for the activity of PSII electron transport or immediately frozen in liquid nitrogen and kept at - 80°C for protein analyses by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).
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When indicated, the stromal fraction was included in the suspension of thylakoid membranes to give a ratio of protein to chlorophyll equivalent to that in the intact chloroplast.
a
b
NP P
NP P
c
NP P
2.4. A n a l y t i c a l p r o c e d u r e s
SDS-PAGE and subsequent immunoblotting were performed as described previously [ 17], except that the polyacrylamide concentration of the separation gel was 15%. For immunoblotting, two different antisera were used: anti-Dlc raised against a synthetic polypeptide that corresponded to the residue 333-344 of the D 1 protein of spinach (a generous gift from Dr T. Ono) and anti-phosphothreonine (antiphosphoThr; Zymed Laboratories). PSII electron transport activity was determined as the activity of photoreduction of 2,6-dichlorophenolindophenol (DCIP) in medium B that contained 2 mM NH4CI and 50 ~M DCIP at 25°C. The reduction of DCIP was determined spectrophotometrically from the absorbance change at 600 nm.
3. Results To obtain intact and active thylakoid membranes free from any contamination, we isolated thylakoid membranes from intact chloroplasts that had been purified by Percoll gradient centrifugation and subsequently treated with thermolysin. Thylakoid membranes thus prepared retained a high capacity to phosphorylate the PSII proteins under illumination with weak light in the presence of ATP (Fig. 1 ). The apoprotein ( s ) of the light-harvesting complex of PSII (LHCII), the DI and D2 proteins of the PSII reaction centre complex and the 43 kDa protein of the PSII core antenna were phosphorylated, as shown by immunoblotting with anti-phosphoThr. Phosphorylation of these proteins could also be detected as shifts of the Coomassie-stained bands in the gel. Phosphorylation of the PsbH protein was not detected by immunoblotting with anti-phosphoThr as reported previously [ 18]. Immunoblotting with anti-D l c revealed that more than 90% of the DI protein remained unphosphorylated in the control sample, while more than 90% became phosphorylated by the phosphorylation treatment. Hereafter, thylakoid membranes that had been illuminated in the presence and absence of ATP are designated as phosphorylated and non-phosphorylated thylakoid membranes, respectively. Fig. 2 compares inactivation of PSII and damage to the D1 protein under photoinhibitory illumination in the non-phosphorylated and phosphorylated thylakoid membranes. The inactivation proceeded in the same time courses in both types of the membranes as reported previously [ 12], and the activity was reduced by about 90% within 60 min (Fig. 2 ( A ) ) . The overall pattern of damage to the D1 protein was also identical in both cases (Fig. 2(B) ). As seen in panel (a), in which large amounts of sample were applied for detection of fragments, C-terminal fragments of the D1 protein of 16, 9.3 and 7.9 kDa and also the cross-linked products, namely, the
43* ~jDI* ~D1
LHClI*
'iiii ....
Fig. 1. Phosphorylationof the PSII core proteins and LHCI1apoproteinsby illumination of isolated thylakoidmembranes in the presence of ATP. Thylakoid membranes suspended in 10 mM NaC1,5 mM MgCI2,0.4 M sucrose and 50 mM Hepes-NaOH (pH 7.8) at 0.4 mg Chl ml ~were supplemented with 0.4 mM ATP and illuminated with white light at 100 p~Em : s ~at 25°C for 30 rain (phosphorylation treatment). Then, the suspension was diluted with three volumes of the same medium supplemented with 10 mM NaF and subjected to SDS-PAGE. (a) Polypeptide profiles after staining with Coomassie blue; (b) immunoblotprofiles with anti-Dlc; (c) immunoblot profiles with anti-phosphoThr. The amounts of sample applied for SDS-PAGE corresponded to 1 ~g Chl in panels (a) and (c) and 0.05 ~g Chl in panel (b). NP and P stand for non-phosphorylatedand phosphorylated thylakoid membranes that were illuminated in the absence and presence of ATP, respectively. Asterisks indicate phosphorylatedproteins. heterodimer and the 41 kDa adduct, were generated during the course of photoinhibitory illumination. It has been demonstrated that the 16 kDa fragment and the two fragments of 9.3 and 7.9 kDa originate from cleavage within helix D and that in the DE loop, respectively [19]. One difference between the non-phosphorylated and phosphorylated thylakoid membranes was that the appearance of the fragments and the cross-linked products was slightly slower in the phosphorylated membranes. Immunoblotting performed with small amounts of sample optimum for quantification of the D1 protein band (panel (b) of Fig. 2(B) ) indicated that the total level of the D 1 protein was reduced by photoinhibitory illumination in both the non-phosphorylated and phosphorylated thylakoid membranes, but that the extent of phosphorylation of the D1 protein remained unchanged during photoinhibitory illumination, although this was performed in the absence of NaF, an inhibitor of protein phosphatases. Polypeptide profiles of thylakoid membranes after staining with Coomassie blue remained unchanged under illumination for 60 min ( data not shown). This observation indicated that, unlike isolated PSII subcomplexes [ 17], unspecific damage to proteins did not occur in thylakoid membranes under the conditions for photoinhibitory illumination in this study. The presence of the stromal fraction during photoinhibitory illu-
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(A)
~ 2
o
100(
75
2s
n
°o
4o Illumination (min)
(B) a
NP
P
b
0 2.5 5 15 30 60 0 2.5 5 15 30 60 rain
NP 0 30
P 0 30 mln
HD 41
DI* D1 )
./DI* "~ D1
16 ::
i
~ 9.3 7.9
Fig. 2. Effects of photoinhibitory illumination on PSII electron transport activity and the DI protein in non-phosphorylated and phosphorylated thylakoid membranes. Non-phosphorylated (NP) and phosphorylated (P) thylakoid membranes suspended at 0.1 mg Chl ml ] were incubated in darkness at 25°C for 10 min, and then illuminated with strong white light at 1000 ixE m 2 s ~and 25°C for the designated times. (A) Activity of DCIP photoreduction: O, nonphosphorylated membranes; 0, phosphorylated membranes. Activities before photoinhibitory illumination were 230 IxmolDCIP mg t Chl h ~in both NP and P membranes. (B) Immunoblot profiles with anti-Dlc. Samples that corresponded to 1 and 0.05 Ixg Chl were applied for SDS-PAGE in panels (a) and (b), respectively. HD denotes the heterodimer of the D 1 and D2 proteins. mination did not affect the pattern of damage to the D 1 protein (Fig. 3). W e investigated if additional cleavage of the D1 protein could occur in darkness after photoinhibitory illumination. As shown in Fig. 4 ( A ) , an incubation in darkness for 2 h selectively increased the amount of the 9.3 kDa fragment, while the amount of the 7.9 kDa fragment was totally unaffected in both the non-phosphorylated and phosphorylated thylakoid membranes. These observations indicated that, among two different sites of cleavage in the DE loop, cleavage in one site occurred only under photoinhibitory illumination while cleavage in the other site could continue even after illumination. No extra fragments of the D1 protein were generated during the incubation in darkness. This was also the case when immunoblotting was performed with an antibody raised against the entire D1 protein of spinach (a generous gift of Dr M. Ikeuchi; data not shown). The increase in the 9.3 kDa fragment was not affected by the presence of the stromal fraction during the incubation.
To examine the involvement of proteases in the cleavage of the D1 protein in darkness, the phosphorylated thylakoid membranes that had been illuminated with photoinhibitory light were incubated in darkness in the presence of a mixture of protease inhibitors. This mixture contained inhibitors of serine and cysteine proteases and metalloproteases. As shown in Fig. 4 ( B ) , the protease inhibitors did not at all affect the increase in the 9.3 kDa fragment in darkness, an indication that the cleavage proceeded non-enzymatically. This was also the case in the non-phosphorylated thylakoid membranes (data not shown). An incubation in darkness after photoinhibitory illumination also affected the amount of the heterodimer, though its effects varied depending on the thylakoid membranes used: the amount of heterodimer was slightly decreased during the incubation in the experiments of Fig. 4 ( A ) , while it appeared rather to increase in the experiments of Fig. 4 ( B ) . It was found that the presence of the protease inhibitors during the incubation greatly increased the amount of heterodimer (Fig.
101
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NP
of Fig. 4(A), the digestion rate exceeded the generation rate, resulting in the decrease in heterodimer during incubation, while the opposite occurred in the experiments of Fig. 4 (B). The increase in the amount of heterodimer caused by the protease inhibitors did occur in the absence of the stromal fraction (Fig. 4(B) ). Therefore, it is likely that a thylakoidbound protease (s) is involved in the digestion.
P
0 30 60 0 3 0 6 0 ( m i n ) HD 41 DI*~ D1 '
4. Discussion
It has recently been reported that, unlike photoinhibitory illumination of thylakoid membranes and PSII preparations, photoinhibitory illumination of intact leaves gives rise to 20 and 18 kDa fragments of the D1 protein of C-terminal and N-terminal origin, respectively, and proposed that cleavage occurs only after dephosphorylation of the DI protein that has been phosphorylated during illumination [ 9]. To reproduce these observations in experiments in vitro, we first prepared thylakoid membranes that retained a high capacity to phosphorylate the D1 protein. By isolating thylakoid membranes from an intact chloroplast preparation of high purity, we could obtain preparations in which almost all the D1 protein was able to be phosphorylated by illumination in the presence of ATP (Fig. 1 ). Cleavage of the D1 protein was compared in two types of thylakoid membranes in which almost all the D 1 protein was either unphosphorylated or phosphorylated. It was found, however, that phosphorylation of the D 1 protein did not affect the overall pattern of damage to the D1 protein, though it slightly slowed down the course of damage: major cleavage
0.3 7.9 Fig. 3. Effects of the stromal fraction present during photoinhibitory illumination on the damage to the D1 protein. Non-phosphorylated (NP) and phosphorylated (P) thylakoid membranes were supplemented with the stromal fraction and illuminated with photoinhibitory light for the designated times, lmmunoblot profiles with anti-Dlc are shown. Samples that corresponded to 1 t-tg Chl were applied for SDS-PAGE. An arrow indicates a band of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), which cross-reacted with anti-Dlc.
4 ( B ) ) . This was also the case in the non-phosphorylated thylakoid membranes (data not shown). These observations suggested that the heterodimer was newly generated even after photoinhibitory illumination, but simultaneously, it was digested by a protease (s). Accordingly, the apparent amount of the heterodimer was determined by the relative rates of its generation and digestion. It is likely that, in the experiments
(A)
(B) NP
o ~ Dark R" ~ " +
P o ~ ~" d
Dark -+ Stroma R" R" - - "i" - - "1" Protean inhihitor8 o
Dark - - + Stroma
D
-HD
~
DI' D1 :
- 16
¢9.3 ~7.9 DI' D1
....
Fig. 4. Effects of an incubation in darkness after photoinhibitory illumination on the DI protein. Non-phosphorylated (NP) and phosphorylated (P) thylakoid membranes which had been illuminated with photoinhibitory light (PI) for 30 min were transferred to darkness and incubated further in darkness at 25°C for 2 h in the presence or absence of the designated additives, namely, the strom',d fraction and/or a mixture of protease inhibitors ( 1 / 15 volume; Protease Inhibitor Cocktail, Boehringer Mannheim). In experiments in (B), phosphorylated thylakoid membranes were used. Immunoblot profiles with anti-Dlc are shown. The sample amounts applied for SDS-PAGE in the upper and lower panels correspond to 1 and 0.05 Ixg Chl, respectively.
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N. Mizusawa et al./ J. Photochem. Photobiol. B: Biol. 48 (1999) 97-103
occurred in the DE loop and also cross-linked products were generated (Fig. 2(B) ). This was also the case when stromal proteins were present during photoinhibitory illumination (Fig. 3). According to the proposed mechanism of cleavage in vivo [9], our failure to reproduce these observations in vivo should result from slow dephosphorylation of the D1 protein in the thylakoid membranes used in this study. As shown in Fig. 2(B) and Fig. 4, dephosphorylation did not occur during photoinhibitory illumination. Even after photoinhibitory illumination, dephosphorylation was slow and it took about four hours to dephosphorylate about half the D 1 protein (data not shown). This rate of dephosphorylation, however, was almost comparable to that in intact leaves [ 13 ]. Accordingly, dephosphorylation during photoinhibitory illumination might be a prerequisite for reproduction of the observations in vivo, if dephosphorylation were to be concerned. The observations in Fig. 2(B) demonstrate that the D1 protein can be cleaved in the DE loop under photoinhibitory illumination. It is unlikely that the cleavage in the DE loop was a non-physiological event that resulted from severe photoinhibitory illumination, since photoinhibitory illumination for 60 rain did not substantially affect the polypeptide profiles of the thylakoid membranes used (data not shown). It is possible that, in vivo, fragments derived from cleavage in the DE loop were digested very rapidly and hence were barely detectable. Our observations also contrast with previous studies with isolated thylakoid membranes and PSII core complexes, which showed that the D 1 protein in the phosphorylated form was more resistant to degradation under illumination than that in the unphosphorylated form [ 11,12]. In the previous studies, however, disappearance of a band of the D1 protein of around 32 kDa was taken as an indication of degradation, but cleavage of the protein was not investigated. In addition, phosphorylated and unphosphorylated D1 proteins were compared in the same preparation in which only a fraction of the D1 protein was phosphorylated. We observed that, in thylakoid membranes, the presence of ATP resulted in phosphorylation of the unphosphorylated D 1 protein even after PSII electron transport was inactivated by about one-third by photoinhibitory illumination (data not shown). Therefore, it is possible that the rate of disappearance of the phosphorylated D1 protein could be underestimated if ATP was present during photoinhibitory illumination. During the course of experiments to survey conditions for dephosphorylation of the D1 protein, we found that the amount of the 9.3 kDa fragment of the DI protein was selectively increased in darkness after photoinhibitory illumination, while the 7.9 kDa fragment had been more abundant during photoinhibitory illumination. We previously proposed that the 9.3 kDa fragment originated from cleavage in the middle part of the DE loop and the 7.9 kDa fragment from cleavage in close proximity to the His272 that coordinates the non-haem iron [19]. This hypothesis was partly confirmed by microsequencing of fragments in which the N-
terminal sequence of the 9.3 kDa fragment matched that of the D1 protein from residue 257 (unpublished), which is located in a helix inside the DE loop (the de helix). Therefore, it is suggested that, during photoinhibitory illumination, cleavage occurred predominantly in close proximity to His272, while cleavage inside the d e helix became dominant in darkness. Protease inhibitors did not suppress the cleavage inside the d e helix in darkness, and it was also unaffected by the presence of the stromal proteins (Fig. 4). If the cleavage were to proceed via chemical reactions induced by active oxygen species as proposed previously [ 19,20], it is expected that cleavage in the proximity of His272 would proceed rapidly after attack by active oxygen species, while cleavage inside the d e helix would be trapped or proceed relatively slowly. We have previously demonstrated that the susceptibility of the D1 protein to cleavage after attack by active oxygen species depends on the conformation of the protein [20]. Since the DE loop of the D 1 protein has a flexible conformation, it is possible that cleavage reactions are initiated only when the conformation around the cleavage site becomes suitable for cleavage. Among products generated during photoinhibitory illumination, only the heterodimer appeared to be digested by a protease (s) (Fig. 4). Although the protease (s) involved has not been characterized yet, it might be the thylakoid-bound protease that digests proteins damaged by active oxygen species [21 ].
Acknowledgements The authors are grateful to Dr Taka-aki Ono of the Institute of Physical and Chemical Research (RIKEN), Japan, for his generous gift of an antibody, and to Ms Shizue Sudoh for her helpful assistance. This work was supported in part by a PROBRAIN grant from the Bio-Oriented Technology Research Advancement Institution (BRAIN) of Japan.
References [ 1] A.K.Mattoo,H. Hoffman-Falk,J.B. Marder,M. Edelman,Regulation of protein metabolism:couplingof photosyntheticelectron transport to in vivodegradationof the rapidlymetabolized32-kilodaltonprotein of the chloroplastmembranes,Proc. Natl. Acad. Sci. USA 81 (1984) 1380-1384. [2] D.J. Kyle, I. Ohad, C.J. Arntzen, Membrane protein damage and repair: selective loss of a quinone-protein function in chloroplast membranes,Proc. Natl. Acad. Sci. USA 81 (1984) 4070-4074. [3] E.-M. Aro, I. Virgin, B. Andersson, Photoinhibition of Photosystem II: inactivation,proteindamageand turnover,Biochim.Biophys.Acta 1143 (1993) 113-134. [4] B.M. Greenberg,V. Gaba, A.K. Mattoo, M. Edelman, Identification of a primaryin vivo degradationproduct of the rapidly-turningover 32 kD proteinof photosystem1I, EMBOJ. 6 (1987) 2865-2869. [5] J. De Las Rivas, B. Andersson, J. Barber, Two sites of primarydegradation of the Dl-protein induced by acceptoror donor side photo-
N. Mizusawa et al. / J. Photochem. Photobiol. B: Biol. 48 (1999) 97-103
[6]
[7]
[8]
[9]
[ 10]
[ 11]
[12]
[ 13]
inhibition in photosystem II core complexes, FEBS Lett. 301 (1992) 246-252. A.H. Salter, J. De Las Rivas, J. Barber, B. Andersson, On the molecular mechanism of light-induced D 1 protein degradation, in: N. Murata (Ed.), Research in Photosynthesis, vol+ IV, Kluwer, Dordrecht, 1992, pp. 395-402. R. Barbato, G. Friso, F. Rigoni, A. Frizzo, G.M. Giacometti, Characterization of a 41 kDa photoinhibition adduct in isolated photosystem II reaction centres, FEBS Lett. 309 (1992) 165-169, R. Barbato, G. Friso, M. Ponticos, J. Barber, Characterization of the light-induced cross-linking of the a-subunit of cytochrome b559 and the D1 protein in isolated photosystem II reaction centers, J. Biol. Chem. 270 (1995) 24032-24037. R. Kettunen, E. Tyystjiirvi, E.-M. Aro, Degradation pattern of photosystem II reaction center protein D1 in intact leaves. The major photoinhibition-induced cleavage site in D1 polypeptide is located amino terminally of the DE loop, Plant Physiol. 111 (1996) 11831190. F.E. Callahan, M.L. Ghirardi, S.K. Sopory, A.M. Mehta, M. Edelman, A.K. Mattoo, A novel metabolic torm of the 32 kDa-D1 protein in the grana-localized reaction center of Photosystem II, J. Biol. Chem. 265 (1990) 15357-15360. E.-M. Aro, R. Kettunen, E. Tyystj~vi, ATP and light regulate DI protein modification and degradation. Role of D 1* in photoinhibition, FEBS Lett. 297 (1992) 29-33. A. Koivuniemi, E.-M. Aro, B. Andersson, Degradation of the D 1- and D2-proteins of photosystem II in higher plants is regulated by reversible phosphorylation, Biochemistry 34 (1995) 16022-16029. E. Rintam~iki, R. Kettunen, E.-M. Aro, Differential DI dephosphorylation in functional and photodamaged photosystem II centers, J. Biol. Chem. 271 (1996) 14870-14875.
103
114] H.Y. Nakatani, J. Barber, An improved method for isolating chloroplasts retaining their outer membranes, Biochim. Biophys. Acta 461 (1977) 510--512. [ 15] K. Miyadai, T. Mae, A. Makino, K. Ojima, Characteristics ofribulose1,5-bisphosphate carboxylase/oxygenase degradation by lysates of mechanically isolated chloroplasts from wheat leaves, Plant Physiol. 92 (1990) 1215-1219. [16] D.I. Arnon, Copper enzyme in isolated chloroplasts: polyphenoloxidase in Beta vulgaris, Plant Physiol. 24 (1949) 1-15. [ 17] M. Miyao, Involvement of active oxygen species in degradation of the D1 protein under strong illumination in isolated subcomplexes of photosystem II, Biochemistry 33 (1994) 9722-9730. [18] E. RintamS.ki, M. Salonen, U.-M. Suoranta, I. Carlberg, I3. Andersson, E.-M. Aro, Phosphorylation of light-harvesting complex II and photosystem II core proteins shows different irradiance-dependent regulation in vivo. Application of phosphothreonine antibodies to analysis of thylakoid phosphoproteins, J. Biol. Chem. 272 (1997) 3047630482. [ 19] M. Miyao, M. Ikeuchi, N. Yamamoto, T. Ono, Specific degradation of the DI protein of photosystem II by treatment with hydrogen peroxide in darkness: implications for the mechanism of degradation of the D1 protein under illumination, Biochemistry 34 (1995) 1001910026. [20] K. Okada, M. Ikeuchi, N. Yamamoto, T. Ono, M. Miyao, Selective and specific cleavage of the D1 and D2 proteins of Photosystem II by exposure to singlet oxygen: factors responsible for the susceptibility to cleavage of the proteins, Biochim. Biophys. Acta 1274 (1996) 7379. [21] L.M. Casano, H.R. Lascano, V.S. Trippi, Hydroxyl radicals and a thylakoid-bound endopeptidase are involved in light- and oxygeninduced proteolysis in oat chloroplasts, Plant Ceil Physiol. 35 (1994) 145-152.