PhotosynthesisResearch 45: 99-104, 1995. (~) 1995KluwerAcademicPublishers. Printedin the Netherlands. Regular paper

Turnover of the D1 protein and of Photosystem II in a mutant lacking Tyrz

Synechocystis 6803

W i m V e r m a a s 1, C a t h y M a d s e n 1, J i u j i a n g Yu 1, J a n i n e V i s s e r I , J a m e s M e t z 2, P e t e r J. N i x o n 2 & Bruce Diner 2 1 Department of Botany and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe AZ 85287-1601, USA; 2 Central Research and Development, Experimental Station, E.L du Pont de Nemours & Co., Inc., PO. Box80173, Wilmington DE 19880-0173, USA Received7 March 1995; acceptedin revisedform 19 May 1995

Key words: chlorophyll radicals, cyanobacteria, photosynthesis, photoinhibition, protein degradation, thylakoids

Abstract

The Photosystem II reaction center is rapidly inactivated by light, particularly at higher light intensity. One of the possible factors causing this phenomenon is the oxidized primary donor, P680 +, which may be harmful to Photosystem II because of its highly oxidizing nature. However, no direct evidence specificially implicating P680 + in photoinhibition has been obtained yet. To investigate whether P680 + is harmful to Photosystem II, turnover of the D1 protein and of the Photosystem II reaction center complex were measured in vivo in a mutant of the cyanobacterium Synechocystis sp. PCC 6803, in which the physiological donor to P680 +, Tyrz, was genetically deleted. In this mutant, D1 degradation in the light is an order of magnitude faster than in wild type. The most straightforward explanation of this phenomenon is that accumulation of P680 + leads to an increased rate of turnover of the Photosystem II reaction center complex, which is compatible with the hypothesis of destructive oxidation by P680 + that is damaging to the Photosystem II complex. Introduction

The Photosystem II (PS II) complex consists of more than a dozen integral membrane proteins, two of which are the reaction center proteins D1 and D2 (reviewed by Nixon et al. 1992; Erickson and Rochaix 1992; Vermaas 1993). It has long been recognized that the rate of synthesis and degradation of the D 1 protein in the light is rapid (Hoffman-Falk et al. 1982; Mattoo et al. 1984) and exceeds that of the other PS II components. The reason for the rapid turnover of the D1 protein in the light is generally thought to be light-induced damage of D1, followed by degradation of the damaged protein and replacement with a newly synthesized D1 copy (reviewed by Pr~isil et al. 1992; Kyle et al. 1987). Several possible mechanisms for light-induced D1 damage have been proposed. These include reactions involving QB (Ohad et al. 1990; Hundal et al. 1990; Kirilovsky et al. 1990), double-reduction and

protonation of QA (van Mieghem et al. 1989; Styring et al. 1990; Setlik et al. 1990; Vass and Styring 1993; Vass et al. 1992) followed by chlorophyll triplet formarion and subsequent reaction with oxygen to yield singlet oxygen (Durrant et al. 1990; Vass and Styring 1993; Vass et al. 1992), and the formation of highly oxidizing species (such as Tyrz°x and P680 +) in PS II (Callahan et al. 1986; Jegersch61d et al. 1990), possibly as a consequence of electron transport inhibition at the donor side caused by acidification of the lumen (Krieger et al. 1992; Krieger and Weis 1993). Even though highly oxidizing radicals formed at the donor side generally are considered to be likely contributors to light-induced D 1 damage, no clear evidence is yet available regarding the precise identity of the factor(s) that are most damaging to PS II. To evaluate the role of P680 + with respect to D1 damage in PS II, we chose a system in which Tyrz (a redox-active Tyr residue in D1 serving as the physi-

100 ological electron donor to P680) (Debus et al. 1988; Metz et al. 1989) is absent; in such a system P680 +, the most oxidizing species of PS II, is expected to accumulate, particularly at high light intensity. In addition, less oxidizing species (such as cytochrome b559°x and TyrD°x) resulting from side-reactions with P680 + may be formed as well. A suitable Tyrz-less system is conveniently provided by a Synechocystis mutant, in which Tyrz (Tyrl61 of the D1 protein) is replaced by a Phe residue. This mutant, Y161F, lacks the physiological electron donor to P680 +, causing the reduction of P680 + to occur primarily by a back reaction with QA or by extracting an electron from other sources (Metz et al. 1989). In the Y161F mutant, the back reaction between P680 + and QA occurs with a tl/2 of about 1 ms (Metz et al. 1989), and is somewhat slower than QA oxidation by QB (250--400 #s in Synechocystis 6803). Therefore, in the Y161F mutant in the light an accumulation of P680 + is expected to occur. Several methods exist to monitor D1 synthesis and turnover under in vivo conditions. One of the most generally applied methods is pulse-chase labeling using 35S-methionine or sulfate. This works well in vivo in Synechocystis 6803 (Yu and Vermaas 1990). Another option is to measure D 1 by western blotting in the presence of an inhibitor of protein synthesis (Yu and Vermaas 1993). A third possibility is to measure the amount of structurally intact PS II complexes; this can be done in vivo by monitoring atrazine-replaceable laC-diuron binding (Vermaas et al. 1990). In this study, we have utilized these methods in parallel, and show that in the Y161F mutant the turnover of the D1 protein and of structurally intact PS II is significantly faster than in wild type. The results presented here support the notion that the presence of P680 + can be a significant factor contributing to D1 damage.

Materials and methods

Growth conditions Wild type and the D1 mutant Y161F of Synechocystis sp. PCC 6803 were propagated in BG11 medium (Rippka et al. 1979) in the presence of 5 mM glucose at a light intensity of 50 #E m -2 s -1. For growth on plates, 10 mM TES/NaOH (pH 8.0) and 0.3% (w/v) sodium thiosulfate were added. Construction of Y 161F has been described in Metz et al. (1989). This mutant is resistant to chloramphenicol, spectinomycin, and kanamycin.

Pulse-chase using 35S-methionine Pulse-chase experiments utilizing 35S-methionine and logarithmically growing wild-type and mutant cells were carried out essentially as described in Yu and Vermaas (1990, 1993). Before the experiment, cultures of wild type and Y161F were harvested at an optical density at 730 nm (OD730) of 0.4-0.5; cells were washed and resuspended in the original volume of BG11 and glucose, but now with sulfate replaced by twice the concentration of nitrate. Subsequently, 50 #Ci 35S-methionine was added to the cells to a final concentration of 0.6 nM. The culture then was incubated for 5 min at 30 °C at normal light intensity (50 #E m -2 s -1) with gentle bubbling of air. After this incubation, 1.7 #M unlabelled methionine was added (time 0 of the pulse-chase experiment), and the culture was continued to be incubated under the same conditions as during the pulse. At various timepoints after the addition of cold methionine a cell sample was removed, was chilled on ice, and was harvested by centrifugation. Thylakoids were isolated from these cells in the presence of protease inhibitors as described in Yu and Vermaas (1990). SDS -PAGE and autoradiography was carried out as in Yu and Vermaas (1990, 1993).

Determination of the lifetime olD1 using an inhibitor of protein synthesis and western blotting To follow degradation of the D1 protein by a complementary method, at time = 0, 100 #g/ml of the protein synthesis inhibitor lincomycin was added to logarithmically growing cell cultures of wild type and the Y161F mutant (OD730 = 0.4--0.5). The cultures then were continued to be grown with gentle bubbling of air at 30 °C at a light intensity of 50 #E m -2 s -1. At various times after lincomycin addition, cell samples were removed, and after chilling and harvesting, thylakoids were isolated in the presence of protease inhibitors. The primary reason lincomycin was selected as inhibitor of protein synthesis is that Y161F is resistant to chloramphenicol. The amount of lincomycin added was sufficient to quantitatively block incorporation of asS-labeled methionine into protein (data not shown). The relative abundance of the D1 protein at various time points was estimated by immunodetection using a polyclonal antiserum raised against spinach D1. SDS-PAGE, blotting, and immunodetection were carded out as described (Yu and Vermaas 1993). The intensity of the immunoreaction was determined by densitometry.

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time (rain) Fig. 2. Relativeintensity of the D1 band as determinedby densitometry. For each Y161F lane shown in Fig. 1, the density of the D1 band was determined relative to the 15-17 kDa bands attributed to stable phycobilisomecomponents. Results

Pulse-chase labeling of thylakoids using 35S-methionine Fig. 1.

Autoradiogram of asS-labeled thylakoid proteins from the Y161F mutant separated by SDS-PAGE.Thylakoids were isolated from a Synechocystis culture that was pulse-labeled with 35S-methionine in the light, and to which unlabeled methionlne was added at time 0. The time of incubation of the culture with unlabeled methionine under normal culture conditions in the light before isolation of thylakoids has been indicated. The left lane contains pulse-labeled wild-type thylakoids. Per lane. 5 #g chlorophyll was loaded.

Quantitation of PS H complexes by diuron binding For ~4C-diuron binding measurements, cells from wild type and Y161F were harvested, washed, and resuspended in fresh BG11 medium with 5 mM glucose and 100 #g/ml lincomycin, to a final concentration of 100 #g/ml of chlorophyll. This mixture was incubated at 30 ° C at a light intensity of 5,000 #E m - 2 s - ] for 0-30 min. After incubation for various periods of time in the light and in the presence of lincomycin, samples were removed, chilled on ice, and used directly for diuronbinding assays. Measurements of atrazine-replaceable 14C-diuron binding at different diuron concentrations were carried out essentially as described in Vermaas et al. (1990).

A generally accepted method for monitoring turnover of the D1 protein is by pulse-chase experiments with radiolabeled amino acids (Hoffman-Falk et al. 1982; Mattoo et al. 1984). To measure whether deletion of Tyrz, leading to an inability to rapidly reduce P680 +, affected the lifetime of the D 1 protein, the time course of in vivo degradation of 35S-labeled proteins was followed. Figure 1 shows a comparison of the incorporation of 35S-methionine in thylakoid proteins of wild type and of the Tyrz-less mutant, Y161E One heavily labeled band is particularly prominent in the 31-32 kDa region. The identity of this band as the Synechocystis D1 protein has been well-documented (Yu and Vermaas 1990, 1993). In wild type Synechocystis 6803 the half-time of D1 degradation as determined by a subsequent chase is 2-3 h under normal growth conditions (data not shown here; see Yu and Vermaas 1990). However, in Y161F the D1 degradation in the light is much faster: after 15 mins, a considerable amount of the label in D1 already has disappeared (Fig. 1). If cells are kept in darkness from the beginning of the chase, the amount of radiolabeled D1 remains essentially constant over time (data not shown). However, in spite of the rapid light-dependent degradation of the D 1 protein, the D2, CP43, and CP47 bands, which are visible and which are running at about 36, 45, and 50 kDa in this gel system (Yu and Vermaas 1990), do not

102

Fig. 3. Westernblot of thylakoidproteins fromwild type and from the Y161F mutantprobed with polyclonalantiserumraised against spinachD1. Afteraddition of lincomycinto the cellculture, followed by incubationunder standardlight conditions for the time indicated, thylakoidswereisolated.The amountofthylakoidsloadedper laneis indicated on a chlorophyllbasis.For wildtype,the immunoresponse is shownto be approximatelylinearin the range indicated. degrade rapidly in the Y161F mutant in the light. This supports the concept that these proteins may remain stably assembled in a complex in the membrane while D1 is being replaced. Within the time period of the measurement, by far the most significant changes in labeling intensity during the chase are seen for the D 1 protein. For quantitation, densitometer readings of the intensity of the D1 band in the different lanes were recorded relative to bands in the 14-18 kDa region; these bands are due to phycobilisome components, and remain essentially stable during the time course of the experiment. The results are presented in Fig. 2. In the Y 161F mutant, after 15 min the amount of radiolabeled D 1 has decreased to about half.

D1 quantification using western blotting As an independent method to follow degradation of the D1 protein in the light, the steady-state concentration of the D1 protein was determined as a function of time in the absence of de novo protein synthesis in both the wild type and the Y161F mutant. For this purpose, thylakoids were isolated from cells that had been grown under standard conditions and exposed to 100 #g/ml lincomycin in the light (50 #E m -2 s -1) for different time periods. Lincomycin is a protein synthesis inhibitor. As indicated in Materials and methods, the amount of lincomycin used was sufficient to complete-

ly block protein synthesis under in vivo conditions. Subsequently, the amount of D1 in thylakoids was estimated from the immunoreaction with D 1 antisera. Under our experimental conditions, an approximately linear dose-immunoresponse curve was obtained for crossreactivity between D1 and antisera when up to 2 #g chlorophyll was loaded per lane (Fig. 3). Thus, one can quantitatively estimate the decrease in D 1 content of thylakoids in cells in the light in the absence of protein synthesis. In wild type, no significant variation was found in the amount of immunodetectable D1 upon a 30-min incubation with lincomycin. However, in the Y161F mutant the amount of immunodetectable D 1 decreases significantly upon a 30-min incubation (Fig. 3). Quantitation by densitometry showed an approximately four-fold decrease in the amount of D1 during the 30min incubation. This corresponds well with the rapid D1 degradation kinetics in Y161F measured with 35Smethionine.

Quantitation of functionally intact PS H reaction centers The rapid degradation of the D1 protein in the Y161F mutant should lead to an accelerated decrease in the number of functionally intact PS II reaction centers in the thylakoid membrane in the light when de novo synthesis of D1 is blocked. A simple way to monitor this is by 14C-diuron binding assays using cells exposed to different times of illumination in the presence of lincomycin. For practical reasons, the cell density in these diuron-binding experiments was approximately 20-fold more than that used for pulse-chase and immunoreaction experiments described above; to partially compensate for this increased cell concentration a higher light intensity was chosen (5 000 #E m -2 s - 1) for illumination. The results of the herbicide-binding experiments as a function of illumination time in the presence of lincomycin are shown in Table 1. In Y161F, after a 30min illumination the number of diuron-binding sites was decreased by about 35%, while the affinity of the remaining sites remained close to normal. After a onehour illumination the decrease in the number of sites was even more notable, while part of the remaining sites displayed a somewhat decreased diuron affinity. In wild type, no significant change could be seen in the number of herbicide-binding sites during this incubation time. In darkness, in Y161F and wild type essentially no change in either affinity or number of

103 Table 1. Diuron affinity and number of diuron binding sites on a chlorophyll basis in cells of wild type and the Y161F mutant after light treatment. The chlorophyll concentrationduring the illumination was 100 #g/ml. The light intensity was 5000 #E m-2 s -1. The experiment was carried out in the presence of lincomycin, and one set of results out of three replicates performed on different days has been presented here. The error associated with these measurements is 10-15 %.

SU~n

Iflumin~iontime(min)

KD(nM)ofdiuron

Chlorophyll/bounddiuronratio

Wild type

0 2 8 30 60

20 19 20 20 20

950 1000 940 1000 1020

Y161F

0 2 8 30 60

20 20 20 20 25

3800 4000 4300 5200 8000

diuron-binding sites was observed during a 60-min incubation (not shown). It is interesting to note that under conditions for diuron-binding experiments (at a 20-fold higher cell density and a 100-fold higher light intensity) the turnover rate of D1 is lower than under pulse-labeling conditions, indicating that D1 degradation rates are not simply determined by the light intensity / chlorophyll concentration ratio. In any case, the light-dependent loss in the number of herbicidebinding sites in the Y161F mutant thus was found to be much faster than in the wild type, again indicating an increase in the rate of light-dependent PS II (D1) degradation in the Y161F mutant.

Discussion A prominent feature of the Tyrz-less mutant Y161F as compared to wild type is that light-dependent turnover of the D1 protein is greatly accelerated. The half time of the D1 protein in Y161F under usual culture conditions is about 15 min, which is about an order of magnitude smaller than that of wild type. This indicates that removal of Tyrz very much destabilizes D1 in the light. The most probable cause for this phenomenon is an increased lifetime of P680 + in the Y161F mutant. However, other factors, such as conformational changes as a result of the introduced mutation, cannot be rigorously excluded. Judging from the estimated midpoint redox potential of the P680/P680 +

couple (Em,7about

+1.12 V (Klimov and Krasnovskii 1981)), P680 + is among the strongest oxidants found in nature. P680 + may oxidize nearby components (for example, amino acid residues or chlorophylls) leading to direct or indirect damage to the D1 protein. It is important to note in this respect that in the light the lifetime of the D2 protein, which is also thought to be in close contact with P680 +, is much longer than that of D1 in both wild type and the Y161F mutant. Moreover, the lifetime of the D2 protein in the light does not appear to be affected considerably by introduction of the Y161F mutation: the pulse-labeled D2 protein remained present at significant concentration 8 hours after the beginning of the chase (Fig. 1). Therefore, an increase in the P680 + concentration primarily leads to damage of the D 1 protein, and not to greatly increased turnover of D2. The considerable light-dependent decrease in D1 stability in the absence of Tyrz is evidence for P680 + as a potential contributor to light-induced degradation of D 1 and inactivation of the PS II complex. A relevant question, of course, is to what degree accumulation of oxidizing equivalents on the donor side is likely to occur under more or less physiological conditions. Under conditions where the water-splitting system is fully functional a significant concentration of P680 + will not build up as electron donation is rapid and redox equilibria on the donor side are very much in favor of P680 (rather than P680+). However, if indeed the water-splitting system can be inactivated under physio-

104 logical conditions, such as by acidification of the l u m e n (Krieger et al. 1992; Krieger and Weis 1993), then a c c u m u l a t i o n o f P680 + and/or Tyrz °x is likely e v e n under p h y s i o l o g i c a l conditions. Thus, accumulation of P 6 8 0 + m a y contribute significantly to light-dependent D1 t u r n o v e r in vivo. H o w e v e r , this does not take away the possibility of i n v o l v e m e n t o f other m e c h a n i s m s that introduce d a m a g e to the PS II c o m p l e x at higher light intensities.

Acknowledgements This w o r k was supported in part by grants f r o m the National Science Foundation ( D M B 90-58279) (to W V ) and f r o m the N R I C G P o f the U S Department o f Agriculture (to BD). This is publication number 217 f r o m the A r i z o n a State University Center for the Study o f Early E v e n t s in Photosynthesis.

References Callahan FE, Becker DW and Cheniae GM (1986) Studies on the photoactivation of the water-oxidizing enzyme. II. Characterization of weak-light photoinhibition of PS II and its light-induced recovery. Plant Physiol 82:261-269 Debus RJ, Barry BA, Sithole I, Babcock GT and Mclntosh L (1988) Directed mutagenesis indicates that the donor to P680+ in Photosystem II is tyrosine-161 of the D1 polypeptide. Biochemistry 27:9071-9074 Durrant JR, Giorgi LB, Barber J, Klug DR and Porter G (1990) Characterization of triplet states in isolated PS II reaction centres: Oxygen quenching as a mechanism for photodamage. Biochlm Biophys Acta 1017:167-175 Erickson JM and Rochaix J-D (1992) The molecular biology of Photosystem II. In: Barber J (ed) Topics in Photosynthesis, Vol 11, pp 101-177. Elsevier, Amsterdam Hoffman-Falk H, Mattoo AK, Marder JB, Edelman M and Ellis RJ (1982) General occurrence and structural similarity of the rapidly synthesized 32 000 Da protein of the chloroplast membrane. J Biol Chem 257:4583-4587 Hundal T, Aro E-M, Carlberg I and Andersson B (1990) Restoration of light induced Photosystem II inhibition without de novo protein synthesis. FEBS Lett 267:203-206 Jegersch61d C, Virgin I and Styring S (1990) Light-dependent degradation of the Dl-protein in Photosystem II is accelerated after inhibition of the water-splitting reaction. Biochemistry 29:61796186 Kirilovsky D, Ducruet JM and Etienne AL (1990) Primary events occurring in photoinhibition in Synechocystis6714 wild-type and an atrazine-resistant mutant. Biochim Biophys Acta 1020:87-93 Klimov VV and Krasnovskii AA (1981) Pheophytin as the primary electron acceptor in photosystem 2 reaction centres. Photosynthetica 15:592--609 Krieger A and Weis E (1993) The role of calcium in the pHdependent control of Photosystem II. Photosynth Res. 37: 117130

Krieger A, Moya I, and Weis E (1992) Energy-dependent quenching of chlorophyll-a-fluorescence: Effect of pH on stationary fluorescence and picosecond-relaxation kinetics in thylakoid membranes and Photosystem II preparations. Biochim Biophys Acta 1102: 167-176 Kyle DJ, Osmond CB and Amtzen CJ (eds) (1987) Topics in Photosynthesis, Vol 9. Elsevier, Amsterdam Mattoo AK, Hoffman-Falk H, Marder JB and Edelman M (1984) Regulation of protein metabolism: Coupling of photosynthetic electron transport to in vivo degradation of the rapidly metabolized 32-kilodalton protein of the chloroplast membranes. Proc Natl Acad Sci USA 81:1380-1384 Metz J, Nixon PJ, R6gner M, Brudvig GW and Diner BA (1989) Directed alteration of the D 1 polypeptide of Photosystem II: Evidence that tyrosine- 161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680. Biochemistry 28:6960-6969 Nixon PJ, Chisholm DA and Diner B A (1992) Isolation and functional analysis of random and site-directed mutants of Photosystem II. In: Shewry P and Gutteridge S (eds) Plant Protein Engineering, pp 93-141. Cambridge University Press, Cambridge Ohad I, Adir N, Koike H, Kyle DJ and Inoue Y (1990) Mechanism of photoinhibition in vivo. A reversible light-induced conformational change of reaction center II is related to an irreversible modification of the D1 protein. J Biol Chem 265:1972-1979 Pr~isil O, Adir N and Ohad I (1992) Dynamics of Photosystem II: Mechanisms of photoinhibition and recovery processes. In: Barber J (ed) Topics in Photosynthesis, Wol 11, pp 295-348. Elsevier, Amsterdam Rippka R, Deruelles J, Waterbury JB, Herdman M and Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111: 1-61 Setlik I, Allakhverdiev SI, Nedbal L, Setlikova E and Klimov VV (1990) Three types of Photosystem II photoinactivation. Photosynth Res 23:39-48 Styring S, Virgin I, Ehrenberg A and Anderson B (1990) Strong light inhibition of electron transport in Photosystem II. Impairment of the function of the first quinone acceptor QA. Biochim Biophys Acta 1015:269-278 Van Mieghem FJE, Nitschke W, Mathis P and Rutherford AW ( 1989) The influence of the quinone-iron acceptor complex on the reaction centre photochemistry of Photosystem II. Biochim Biophys Acta 977:207-214 Vass I and Styring S (1993) Characterization of chlorophyll triplet promoting states in Photosystem II sequentially induced during photoinhibition. Biochemistry 32:3334-3341 Vass I, Styring S, Hundal T, Kovnniemi A, Aro E-M and Andersson B (1992) Reversible and irreversible intermediates during photoinhlbition of Photosystem II. Stable reduced QA species promote chlorophyll triplet formation. Proc Natl Acad Sci USA 89:1408-1412 Vermaas W (1993) Molecular-biological approaches to analyze Photosystem II structure and function. Annu Rev Plant Physiol Plant Mol Bio144:457-481 Vermaas W, Charit6 J and Shen G (1990) QA binding in D2 contributes to the functional and structural stability of Photosystem II. Z Naturforsch 45c: 359-365 Yu J and Vermaas WFJ (1990) Transcript levels and synthesis of Photosystem II components in cyanobacterial mutants with inactivated Photosystem II genes. Plant Cell 2:315-322 Yu J and Vermaas WFJ (1993) Synthesis and turnover of Photosystem II reaction center polypeptides in cyanobacterial D2 mutants. J Biol Chem 268:7407-7413