©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Deletion of the PEST-like Region of Photosystem Two Modifies the Q-binding Pocket but Does Not Prevent Rapid Turnover of D1 (*)

Peter J. Nixon (§) , Josef Komenda (¶) , James Barber , Zsuzsanna Deak (1), Imre Vass (1), Bruce A. Diner (2)

From the (1)Photosynthesis Research Group, Wolfson Laboratories, Biochemistry Department, Imperial College of Science, Technology and Medicine, London, SW7 2AY, United Kingdom, theBiological Research Centre, Hungarian Academy of Sciences, P. O. Box 521, H-6701 Szeged, Hungary, and the (2)Central Research and Development Department, E. I. Du Pont de Nemours & Company, Experimental Station, P. O. Box 80173, Wilmington, Delaware 19880-0173

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The rapid turn-over of the D1 polypeptide of the photosystem two complex has been suggested to be due to the presence of a ``PEST''-like sequence located between putative transmembrane helices IV and V of D1 (Greenberg, B. M., Gaba, V., Mattoo, A. K. and Edelman, M.(1987) EMBO J. 6, 2865-2869). We have tested this hypothesis by constructing a deletion mutant (226-233) of the cyanobacterium Synechocystis sp. PCC 6803 in which residues 226-233 of the D1 polypeptide, containing the PEST-like sequence, have been removed. The resulting mutant, PEST, is able to grow photoautotrophically and give light-saturated rates of oxygen at wild type levels. However electron transfer on the acceptor side of the complex is perturbed. Analysis of cells by thermoluminescence and by monitoring the decay in quantum yield of variable fluorescence following saturating flash excitation indicates that Q, but not Q, is destabilized in this mutant. Electron transfer on the donor side of photosystem two remains largely unchanged in the mutant.

Turnover of the D1 polypeptide as examined by pulse-chase experiments using [S]methionine was enhanced in the PEST mutant compared to strain TC31 which is the wild type control. We conclude that the PEST sequence is not absolutely required for turn-over of the D1 polypeptide in vivo although deletion of residues 226-233 does have an effect on the redox equilibrium between Q and Q.


INTRODUCTION

The photosystem two (PSII)()complex catalyzes the light-induced oxidation of water to molecular oxygen. Although over 20 subunits are associated with this complex (Erickson and Rochaix, 1992), primary photochemistry occurs within a minimal reaction center complex composed of a heterodimer of the D1 and D2 polypeptides (Tang et al., 1990). A unique property of the D1 polypeptide is its high rate of turnover in vivo compared to other PSII components (Edelman and Reisfeld, 1978; Wettern et al., 1983). It is believed that this turnover represents a repair cycle to replace D1 that has been damaged or in the process of being irreparably damaged (Ohad et al., 1984). The nature of this photoinhibitory damage is uncertain, but in vivo one possible mechanism may be the action of singlet oxygen generated by aberrant chlorophyll triplet states within PSII (Telfer et al., 1994; reviewed by Barber and Andersson, 1992). The signaling mechanisms involved in the turnover of D1, as well as the enzymology of D1 degradation, are poorly understood.

Specific D1 cleavage products have been difficult to detect in vivo probably because they are degraded extremely rapidly. However, when cytoplasmic protein synthesis was inhibited, Greenberg and co-workers(1987) detected an N-terminal 23.5-kDa D1 fragment which, if resulting from degradation of the D1 polypeptide, rather than a paused intermediate in translation (Kim et al., 1991, 1994; Taniguchi et al., 1993), would place a cleavage site in the region between putative transmembrane helices IV and V on the stromal side of the complex. This suggestion has been strengthened considerably by Cánovas and Barber(1993) who recently detected a light-induced 10-kDa C-terminal D1 fragment in photoinhibited wheat leaves. This cleavage site maps to a region of D1 that is thought to be involved in binding the secondary quinone electron acceptor, Q, and various herbicides.

Greenberg et al.(1987) also identified within the Q-binding region of D1 a sequence between residues Argand Arg that is particularly rich in glutamate, serine, and threonine residues which they speculated was important for turnover of D1. Despite the absence of proline, they suggested that this sequence of D1 may constitute a so-called ``PEST-region'' (regions containing proline (P), glutamate (E), serine (S), and threonine (T) bordered by positively charged residues) which had been proposed previously to be an important structural signal for the rapid degradation of proteins (Rogers et al., 1986), a concept that has gained some experimental support from studies on ornithine decarboxylase (Loetscher et al., 1991).

In this paper we describe the construction and characterization of a deletion mutant of the cyanobacterium Synechocystis sp. PCC 6803 in which the PEST-like sequence of D1, which is encoded by the psbA gene, has been removed. To achieve this a modified psbA3 gene lacking the PEST-like sequence was restored to the chromosome of a psbA-deletion strain of Synechocystis sp. PCC 6803 in which the three members of the psbA gene family have been removed (Nixon et al., 1992). Although we have detected modifications to electron transfer on the acceptor side of the PSII complex, turnover of D1 is enhanced in this mutant compared to the control strain.


MATERIALS AND METHODS

Growth and manipulation of the cyanobacterium Synechocystis sp. PCC 6803 was performed as described previously (Nixon et al., 1992). All cyanobacterial strains described in this paper are derivatives of the glucose-tolerant strain originally isolated by Williams(1988).

The PEST mutation was constructed in vitro in the psbA3 gene by oligonucleotide-mediated site-directed mutagenesis using single-stranded DNA obtained from plasmid pTC3 as the template (Nixon et al., 1992). A 37-base oligonucleotide (5`-AACCTCCTCCTTGGTGCG-CAACTACGGTTACAAATTC-3`) was designed to remove eight codons(226-233) and introduce an additional mutation to create a new FspI restriction site (underlined sequence) which would help screening for the mutant plasmid. The mutant plasmid was used to transform the psbA triple deletion strain TD41 as described before (Nixon et al., 1992) to generate the PEST mutant. The presence of the correct mutation within PEST was confirmed by sequencing single-stranded DNA generated by asymmetric PCR using genomic DNA as the template (Nixon et al., 1992).

Photoautotrophic growth rates were determined by monitoring the absorbance at 730 nm of 100-ml cultures grown at 32 °C in 250-ml flasks bubbled with air. Fluorescent light was used at an intensity of approximately 50-70 µEms at the vessel surface. The initial absorbance of the cultures were 0.003 or 0.03 and growth was exponential up to an absorbance of 1.5.

Rates of relaxation of the chlorophyll fluorescence yield following actinic flashes were measured in whole cells using a flash detection spectrophotometer (Nixon and Diner, 1992). F is the fluorescence yield when PSII is ``open'' and capable of photochemistry whereas F is the maximum yield of fluorescence obtained when the PSII reaction centers are ``closed'' (reviewed in Krause and Weis, 1991). The relative PSII concentration/cell (OD of 0.9) was determined by measuring the variable chlorophyll fluorescence (F-F) in the presence of of NHOH plus DCMU as described by Nixon and Diner(1992). Cells in BG-11 medium plus 5 mM glucose were made 50 mM in HEPES/KOH pH 7.5, 0.3 mM in p-benzoquinone and 0.3 mM in KFe(CN) and incubated for 10 min in the dark prior to the start of the measurement. A fresh sample was used for each actinic-detecting flash interval in the experiment shown in Fig. 3, while a single sample was used for all of the time points of the experiment shown in Fig. 6. The data from two to three samples were typically averaged.


Figure 3: Relaxation of the variable fluorescence yield (F-F), normalized to F, after each of a series (1.67 Hz) of five saturating flashes given to whole cells of TC31 (filled-in circles and mutant PEST (squares). Measurements are shown over a 5-ms range starting at 50 µs.




Figure 6: Relaxation of variable fluorescence yield (F-F) resulting from charge recombination between Q and the donor side of PSII following single saturating flash excitation of whole cells of TC31 (squares) and the PEST mutant (circles) in the presence of 30 µM DCMU.



Light-saturated rates of oxygen evolution were determined from whole cells (3-10 µg/ml of chlorophyll) suspended in BG-11 medium using a Hansatech DW2 oxygen electrode and a light intensity of 3000 µEms. Sodium bicarbonate was used at 10 mM, and the artificial quinone acceptors 2,5-dimethylbenzoquinone (DMBQ) or 2,6-dichlorobenzoquinone (DCBQ) were used at 1 mM in the presence of 3 mM KFe(CN).

Thermoluminescence signals were measured using a home-built apparatus as described previously (Vass et al., 1981) from cells resuspended at 100 µg/ml chlorophyll. The cells were first illuminated with continuous white light for 30 s at 20 °C. After 5 min dark adaptation at 20 °C, a single saturating flash was given at +5 °C (in the absence of DCMU) or at -10 °C (in the presence of DCMU), which was followed by a fast cooling to -40 °C.

The photoinhibition experiments using intact cells were performed using the protocol of Komenda and Barber.()Cells were grown with bubbling until late exponential phase (8 µg/ml chlorophyll), harvested, and resuspended to a chlorophyll concentration of 25 µg/ml. Cells were stirred in flat glass dishes at 32 °C and subjected to heat-filtered white light of approximately 100 (low light conditions) or 1000 (high light conditions) µEms. For inhibition of protein synthesis, lincomycin at 100 µg/ml was added.

The pulse-chase labeling of cells using [S]methionine was performed using the conditions developed by Komenda and Barber. For the pulse, washed cells were resuspended in a sulfur-depleted BG-11 medium (BG-11-S) in which MgSO, ZnSO, and CuSO had been substituted by MgCl, ZnCl, and Cu(NO). The chlorophyll concentration was adjusted to 25 µg/ml, and the cell suspension was exposed to white light (200 µEms) for 1 h at 32 °C. Radiolabeled L-[S]methionine (>1000 Ci/mmol, Amersham International plc) was added to a final activity of 1 µCi/ml and the light treatment continued for a further 30 min. For the chase, the labeled cells were spun down and resuspended in the same volume of BG-11-S but now containing cold methionine at a final concentration of 2 mM. Cells were subjected to either high or low light treatment as described above, and 10-ml aliquots were withdrawn at appropriate time intervals for preparation of thylakoid membranes.

Thylakoid membranes were isolated from cells (equivalent to 250 µg of chlorophyll) that were first collected by centrifugation at 4 °C, washed once with buffer A (10 mM MES/NaOH, pH 6.5, 5 mM ethylenediaminetetraacetate, 1 mM phenylmethanesulfonyl fluoride, 2 mM -aminocaproic acid, and 1 mM benzamidine) and resuspended in 0.4 ml of buffer A. The same volume of glass beads (150-212 µm diameter) was added, and the cells were broken in an Eppendorf tube by vortexing twice for 90 s using a Whirlimixer (Fisons Scientific Equipment, Loughborough, Leicestershire, United Kingdom) with a 1-min interval on ice. Unbroken cells, cell debris, and thylakoid membranes were decanted off by repeated washing with buffer A. The decanted material was centrifuged in a microfuge at 900 g for 30 s to remove unbroken cells and debris. The supernatant was centrifuged at 15,000 g for 15 min in order to pellet the thylakoids which were finally resuspended in 50 mM Tris-HCl, pH 7.5, 1 M sucrose, and stored at -80 °C. The isolation procedure was carried out in a cold room at 4 °C with samples kept on ice.

Thylakoid membrane proteins were resolved by SDS-polyacrylamide gel electrophoresis on 10-17% polyacrylamide resolving gels containing 6 M urea and 0.1% SDS. The stacking gel contained 6% acrylamide. The electrophoresis buffer system described by Laemmli (1977) was used. Thylakoid membranes containing 5 µg of chlorophyll were loaded per lane, and protein was visualized using Coomassie Blue G. Autoradiography was performed by exposing dried gels to Hyperfilm -max. (Amersham International plc) at room temperature for 2-3 days. Quantification of radiolabeled D1 was done using a Hirschmann Elsript 400 densitometer.

Immunoblotting was performed according to the methods described by Shipton and Barber(1991) using a D1 antiserum which was raised against a peptide corresponding to the last 29 amino acid residues of precursor D1 from pea.


RESULTS

Construction of the PEST Deletion Mutant

Fig. 1shows a folding model of the D1 polypeptide of Synechocystis sp. PCC 6803 (from residues 190 to 344) based on the structural analogy with the L-subunit of purple non-sulfur photosynthetic bacteria (Michel and Deisenhofer, 1988). Because Glufound in tobacco D1 is replaced by a tyrosine residue in Synechocystis, the PEST-like region is located between residues Gluand Ser. To test the functional significance of these residues, a Synechocystis mutant (designated PEST) carrying a deletion 226-233 in the psbA3 gene was constructed (``Materials and Methods''). The psbA1 and psbA2 genes are absent in this strain (Nixon et al., 1992) so that only the mutant phenotype is observed. Fig. 2shows an autoradiogram of a sequencing gel confirming that the correct mutation was engineered in strain PEST.


Figure 1: Folding model of the D1 polypeptide of Synechocystis sp. PCC 6803 showing the location of the PEST sequence. Putative transmembrane helices IV and V are shown together with possible locations for P680, the non-heme iron, Fe, and quinone Q. The region in D1 that is deleted in the PEST mutant (residues 226-233 inclusive) is indicated in bold.




Figure 2: Sequencing gel autoradiogram confirming the deletion of the PEST sequence (residues 226-233 inclusive) from the psbA3 gene in strain PEST. Single-stranded DNA containing the psbA3 gene was amplified from the genomic DNA of strains TC31 and PEST using asymmetric PCR and sequenced as described under ``Materials and Methods.'' For each strain, the DNA sequence of the sense strand is shown together with the decoded amino acid sequence.



The PEST Sequence Is Not Required for PSII Activity

The PEST mutant was found to grow photoautotrophically (doubling time of 17 h) and show light-saturated oxygen evolution (150-220 µmol of O(mg of Chl)h) at similar rates to strain TC31, which is the WT control in these experiments (Nixon et al., 1992). Quantification of the total yield of variable fluorescence from whole cells, which is a measure of PSII concentration (Chu et al., 1994), indicated that the level of PSII in the PEST mutant was >80% of levels in TC31. These results clearly establish that the PEST region is not required for assembly of an active PSII complex or for photoautotrophic growth under the conditions employed.

Deletion of the PEST Sequence Impairs Electron Flow between Q and Q

Electron transport between Q and Q was examined in the PEST mutant by monitoring the decay in the quantum yield of fluorescence following a series of five saturating flashes given to dark-adapted cells (Fig. 3) (Nixon and Diner, 1992). Following the first flash the initial fast phase is thought to represent electron transfer between Q and Q (Nixon and Diner, 1992). In the PEST mutant the kinetic of the fast phase is approximately the same as that observed in TC31 although the amplitude is reduced. The most dramatic difference between PEST and TC31 lies in the proportion of slow phase to fast phase. The slow phase is thought to represent the binding, and subsequent reduction, of PQ to centers in which the Q site is devoid of quinone and also charge recombination between the donor and acceptor sides of the complex within centers that either do not bind Q during the lifetime of the charge separated state or do not transfer an electron from Q to Q (or Q). The ratio of slow phase to fast phase therefore gives an indication of the apparent equilibrium constant, K = [QQ]/([Q] + [QQ]), for the reaction QQ = QQ. This is in turn related to the intrinsic equilibrium constant, K = [QQ]/[QQ], by the equation K/K = [PQ]/(K + [PQ]) where Kis the dissociation constant, K = ([Q] + [PQ])/[QQ], for the binding of plastoquinone to the Q site. The increased proportion of slow phase to fast phase in the PEST mutant is therefore consistent with a decreased occupancy of the Q site and/or a decrease in the midpoint redox potential of the Q/Q couple relative to the Q/Q couple so that K is reduced.

Thermoluminescence Measurements of PEST Indicate a Destabilization of Q

Photosynthetic oxygen evolution occurs at a cluster of four manganese ions within PSII following the accumulation of four oxidizing equivalents (reviewed in Debus, 1992). The oxidation state of the cluster is designated by S where n is the number of positive charges stored. The thermally activated recombination of positive charges stored in the redox states S and S of the water-oxidizing complex with electrons of the reduced quinone electron acceptors (Q and Q) of PSII results in the emission of radiation called thermoluminescence (TL) (reviewed in Vass and Inoue, 1992). The peak temperature of a TL component is indicative of the energetic stability of separated charge pair. As a general rule, the higher the peak temperature of TL the greater the stabilization, whereas the TL intensity is proportional to the amount of recombining charges (Vass et al., 1981).

In dark-adapted thylakoids or PSII-enriched membranes, illumination with one flash results in a single TL band at approximately 30 °C, called the B band, which arises from the recombination of the SQ charge pair (Rutherford et al., 1982; Demeter and Vass, 1984). If electron transfer is blocked between Q and Q, e.g. by the addition of the herbicide DCMU, the main TL component, called the Q band, arises from SQ recombination at around 5-15 °C (Rutherford et al., 1982; Demeter et al., 1982).

Fig. 4shows the TL curves from TC31 and PEST cells. For TC31, the main TL peak after single-flash excitation, the B band, is at around 26-28 °C (Fig. 4a, solid line). Small shoulders are also observed at 40 and 55 °C. The origin of the 40 °C band is not yet clear. The 55 °C band is most likely the so-called C band, which has been assigned to charge recombination between TyrDQ in thylakoid membranes and PSII-enriched membranes isolated from spinach (Demeter et al. 1993; Johnson et al., 1994). This component is not induced in TC31 as a result of the single saturating flash, but rather by the preceding preillumination (30 s continuous light) which was used to ensure reproducible initial conditions for the measurements (data not shown). In the presence of DCMU, the main component, the Q band, appears at around 13-15 °C (Fig. 4a, dashed line). Under these conditions the C band is also enhanced in agreement with its origin from Q. The small shoulder observed at 3 °C is an artifact resulting from the melting of the sample and is discussed by Vass et al.(1991). These TL characteristics of TC31 are very similar to those observed previously in WT cells of Synechocystis 6803 (Mayes et al., 1993; Vass et al., 1992), although the intensity of the 40 °C component, seen in the absence of DCMU, is generally smaller in the WT than in TC31.


Figure 4: Flash-induced thermoluminescence curves from cells of TC31 (a) and PEST (b) at 100 µg Chl ml after excitation with one flash either at 5 °C without any addition (solid line) or at -10 °C in the presence of 10 µM DCMU (dashed curves).



For the PEST mutant, the position of the B band is somewhat lower, at about 22-24 °C, than observed in TC31 (Fig. 4b, solid line). This shows that the energetic stability of the SQ charge pair is decreased in the mutant relative to TC31. In contrast, the position of the Q band is practically the same in the mutant and TC31 (Fig. 4, dashed lines), indicating that the stability of the SQ charge pair is the same in the two strains. Since the S state is the common recombination partner in the the two charge pairs, it follows that the modification observed in the PEST mutant affects the stability of the electron on Q and decreases the energy difference between the Q/Q and Q/Q redox couples leading to a decrease in the equilibrium constant for the reaction QQ = QQ, as indicated from the fluorescence measurements.

The Donor Side Is Unaffected in the PEST Mutant

A variety of measurements indicate that the electron transfer reactions on the donor side of PSII are relatively unperturbed in PEST. Fig. 5shows that the flash-induced oscillation of oxygen evolution from PEST is similar to TC31 with a characteristic periodicity of four and maximum yield on the third flash. The signals observed on the first and second flashes have been observed previously in Synechocystis 6803 and ascribed to the light-induced inhibition of respiration (Boichenko et al., 1993). One difference between the two oxygen oscillations is the somewhat higher degree of dampening in the mutant cells, which is probably caused by impaired Q to Q (or Q) electron transfer on the acceptor side of the complex. A similar conclusion has also been reached from analysis of a number of herbicide-resistant mutants (Etienne et al., 1990).


Figure 5: Flash-induced oscillation of oxygen evolution from cells of TC31 (A) and PEST (B) at 300 µg Chlml. Oxygen yield was measured from a sequence of exciting flashes of frequency 1 Hz. The relative amplitude of the flash oxygen signal in PEST is 110-120% of that in TC31.



That normal electron transfer occurs on the donor side of PSII in PEST is also indicated in Fig. 3which shows that there is no significant quenching of fluorescence at 50 µs after each of a series of five flashes given to whole cells. A perturbation in electron transfer that allowed the photoaccumulation of the oxidised primary electron donor, P680, would cause a progressive quenching of fluorescence at early time points through the flash series (Nixon and Diner, 1992). The PEST mutant does, however, show some quenching of fluorescence on the first flash compared to TC31, which then remains fairly constant throughout the flash series. This may indicate the presence of a subpopulation of PSII centers in PEST which shows a slower reduction of P680.

Fig. 6shows the decay in the quantum yield of fluorescence from cells of PEST and TC31 following single flash excitation in the presence of the herbicide DCMU. This decay monitors the rate of charge recombination within PSII between S and Q. No difference in rate was observed between the two strains. Because this rate is dependent on the concentration of P680 following equilibration on the donor side of PSII (Nixon and Diner, 1992) this result indicates that the energetics of the redox components on the donor side are relatively unperturbed in the S state of PSII in the PEST mutant.

A PSII Repair Cycle Operates in the PEST Mutant

Fig. 7shows that when cells of TC31 or the PEST mutant are exposed to relatively intense white light (1000 µEms) there is no loss of PSII activity as judged by oxygen evolution using bicarbonate as the ultimate electron acceptor. The slight stimulation in activity observed with TC31 and PEST is not due to an increase in cell density or to an evaporation artifact as the chlorophyll concentration of the sample was constant throughout the experiment. In the presence of lincomycin at 100 µg/ml, however, there is a light-induced loss of activity in both strains with a t of approximately 60 min. Under these conditions protein synthesis is blocked and so loss of PSII activity is a monitor of the damage to PSII that is normally repaired by de novo protein synthesis. The similarity in the decline of oxygen evolution between TC31 and the PEST mutant indicates that PSII in the two strains is equally susceptible to photoinactivation. That there is no decline of PSII activity in PEST in the absence of lincomycin means that protein synthesis and assembly of PSII is able to match PSII inactivation under these conditions.


Figure 7: Effect of high light treatment on the rate of oxygen evolution from whole cells of TC31 (A) or PEST (B) assayed in the presence of 10 mM NaCO. Cells were treated with high light (1000 µEm s) in the absence (closed symbols) or presence of lincomycin (open symbols) as described under ``Materials and Methods.'' 100% rate of oxygen evolution is 180 µmol Omg Chlh for TC31 and 220 µmol Omg Chlh for PEST.



A Possible Light-induced Modification of the Q Site in PEST

Fig. 8shows the results of a similar experiment to that shown in Fig. 7except that oxygen evolution from cells was assayed in the presence of artificial electron acceptors that accept electrons from PSII directly. DMBQ is thought to accept electrons from ``Q-active'' reaction centers where regular electron transfer occurs between Q and Q whereas dichlorobenzoquinone (DCBQ) accepts electrons from all reaction centers including ``Q-inactive'' ones (Graan and Ort, 1986). For TC31, in the absence of lincomycin, the absolute rates of oxygen evolution in the presence of DMBQ and DCBQ were similar to that observed with bicarbonate and again were stimulated slightly by the light treatment (Fig. 8A). When lincomycin was added to the cell suspension of TC31, oxygen evolution decreased with approximate half-times of 40 min with DMBQ and 60 min with DCBQ (Fig. 8A).


Figure 8: Effect of high light treatment on the rate of oxygen evolution from whole cells of TC31 (A) or PEST (B) when assayed in the presence of ferricyanide and the artificial electron acceptors DMBQ (squares) or DCBQ (circles). Cells were treated with high light (1000 µEm s) in the absence (closed symbols) or presence of lincomycin (open symbols) as described under ``Materials and Methods.'' With DCBQ the 100% rates of oxygen evolution are approximately 140 µmol Omg Chlh for TC31 and 210 µmol Omg Chlh for PEST. With DMBQ the 100% rates of oxygen evolution are approximately 180 µmol Omg Chlh for TC31 and 200 µmol Omg Chlh for PEST.



In contrast to TC31 in the presence of DCBQ and DMBQ there, was a rapid decline in oxygen evolution in the PEST mutant even in the absence of lincomycin (Fig. 8B). Under these conditions oxygen evolution in the presence of bicarbonate is unaffected (Fig. 7B). This inhibition is more acute with DMBQ than DCBQ. At longer time points oxygen evolution is stable in the absence of lincomycin but further decreases in its presence (Fig. 8B). These results suggest that there is a photoinduced modification in PEST that alters the ability to reduce certain exogenous quinones, possibly arising from structural changes within the Q region.

Turnover of D1 in PEST

Pulse-chase experiments using [S]methionine were used to assess the rate of turnover of D1 in PEST and TC31 under low (100 µEms) and high light conditions (1000 µEms). The D1-labeled band was identified by immunoblotting and by its absence in a psbA triple deletion strain of Synechocystis (not shown). As expected D1 from PEST shows a lower apparent molecular weight than in TC31 ( Fig. 9and Fig. 10). In low light the turnover of D1 is accelerated in the PEST mutant compared to TC31 where D1 is relatively stable (Fig. 9A). In high light, turnover of D1 is similar in PEST and TC31 (Fig. 9B). The difference in turnover rates in low light is also reflected in the greater degree of labeling during the pulse period of D1 in PEST compared to D1 in TC31. Fig. 9also shows that under both high and low light conditions there is significant turnover of the D2 polypeptide in both TC31 and PEST.


Figure 9: Comparison of D1 turnover in TC31 and PEST under (A) low (100 µEms) or (b) high (1000 µEms) irradiance during the chase period. Upper panels, aliquots were taken at the times indicated during the chase and thylakoid membranes isolated as described under ``Materials and Methods.'' Thylakoid proteins were subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. Lower panels, densitometric quantitation of radiolabeled D1 in TC31 (closed circles) and PEST (closed squares).




Figure 10: Steady-state levels of D1 protein in TC31 and PEST following high light treatment. Upper panel, the thylakoid membranes analyzed in Fig. 9A were subjected to immunochemical analysis as described under ``Materials and Methods.'' The band indicated by * is present in the psbA triple deletion strain, TD41, and is therefore not related to D1. The extent of migration of prestained molecular weight markers is also shown. Lower panel, densitometric quantification of the amount of immunodetectable D1 in TC31 (filled circles) and PEST (filled squares) after normalization to the band labeled * in the upper panel.



Steady-state Levels of D1 in TC31 and PEST Mutant following High Light Treatment

The steady-state level of the D1 polypeptide in TC31 was assessed by immunoblotting (Fig. 10). To correct for variations in loading, the intensity of the D1 band in each sample was compared to the band in Fig. 10designated * which is present in the psbA triple deletion strain, TD41, hence unrelated to D1, and which appears to be stable to light treatment. For TC31 D1 was found to be stable during high light treatment. In contrast levels of D1 in the PEST mutant decreased slightly (Fig. 10). In the experiment shown in Fig. 10, the PEST mutant also appears to contain more immunodetectable D1 than TC31. On an overdeveloped blot, the formation of high molecular mass D1-related bands with apparent molecular masses of 38 and 65 kDa was observed in the high light-treated PEST cells but not in TC31 or in a psbA triple deletion strain (not shown). This observation is akin to the situation observed in vitro when isolated PSII reaction centers consisting of D1, D2, cytochrome b559 and PsbI protein are subjected to high light treatment (Shipton and Barber, 1990). In this complex, protein oxidation and cross-linking induced by singlet oxygen is the probable cause of such effects. Deletion of the PEST sequence may therefore be accelerating the formation of such reactive species in PSII or enhancing the susceptibility of PSII to attack.


DISCUSSION

The rapid turnover of the D1 polypeptide in vivo has been suggested by Greenberg and co-workers(1987) to arise from the presence of a PEST-like sequence in D1 located between positively charged residues at positions 225 and 238. We have tested the importance of this PEST-like region by constructing a deletion strain of Synechocystis (226-233) that lacks this sequence.

Surprisingly, the removal of these 8 amino acids has only minor effects on PSII function. PEST contains approximately WT levels of PSII, evolves oxygen, and grows photoautotrophically at WT rates. This indicates that the extrinsic loop connecting putative helices IV and V of D1 can accommodate substantial changes in structure. Our results are in agreement with a recent mutational analysis of this extrinsic loop of D1 by Kless and co-workers(1994). They showed that the deletion of up to 5 residues in the region from residue 229 to 251 of D1, which is thought to contribute little to Q binding, has little effect on photoautotrophic growth. In contrast removal of residues in the region from residue 254 to 270 which is considered to be more closely associated with Q has dramatic effects on the accumulation of PSII (Kless et al., 1994). For D2 the situation is likely to be similar (Kless et al., 1992).

A Possible Interaction between the PEST-like Sequence and PsbH

Electron transfer between Q and Q is, however, modified in PEST consistent with its proximity in primary structure to the Q-binding site (Ohad and Hirschberg, 1992). The kinetic of the fast phase of the decay in the quantum yield of fluorescence in the absence of DCMU reveals that the rates of electron transfer between Q and Q and between QQ and Q are not drastically altered (Fig. 3). However, the greater proportion of slow phase compared to fast phase can be interpreted as either reflecting partial occupancy of the Q site by plastoquinone (e.g. by an increase in the dissociation constant for PQ at the Q site) or a perturbation in the equilibrium constant, K, for the reaction QQ = QQ so that QQ is favored. This latter interpretation is more likely based on the thermoluminescence measurements. Very similar observations have been reported for herbicide-resistant mutants (Demeter et al., 1985; Etienne et al., 1990; Gleiter et al., 1992) as well as a psbH deletion mutant of Synechocystis (Mayes et al., 1993). The PsbH protein (also known as the 9- or 10-kDa phosphoprotein in higher plants) co-purifies with the PSII core complex, and its phosphorylation is thought to modulate electron transfer between Q and Q (Packham, 1988).

A role for the PEST sequence in defining the binding properties of the Q site is also suggested by the photoinactivation experiments shown in Fig. 8which indicated that PSII activity as measured by oxygen evolution was dependent on the type of electron acceptor used. No decline of activity was observed with the natural plastoquinone electron acceptor, as judged by the use of bicarbonate as the added terminal electron acceptor, whereas a strong inhibition was observed using DMBQ and less with DCBQ. The reason behind this differential effect of the quinones is uncertain but is possibly related to light-induced conformational changes near the Q site which have little effect on Q function but drastic effects on the binding and efficacy of the artificial quinones. As yet there is no information available on the nature of the binding sites for DCBQ and DMBQ in PSII.

It could be argued that the rate of oxygen evolution obtained in the presence of bicarbonate is not a suitable assay of PSII activity as this rate may be largely determined by electron transfer steps beyond PSI. In contrast the quinones, DCBQ and DMBQ, which are directly reduced by PSII should be a more direct measure of activity. However, it should be noted that the the light-saturated rates of oxygen evolution from cells of TC31 or PEST using each of the three electron acceptors (DCBQ, DMBQ, or bicarbonate) were very similar (see legend of Fig. 8). This is rather unexpected if oxygen evolution in the presence of bicarbonate was limited by electron transfer downstream of PSII. In addition there is no lag in the decline of oxygen evolution from cells of TC31 or PEST when photoinhibited in the presence of lincomycin and assayed using bicarbonate as the ultimate electron acceptor (Fig. 7).

This same differential response to added quinone has also been observed recently for a psbH mutant of Synechocystis()further reinforcing the similarity of the PEST and psbH mutants with regard to Q activity. One model to explain these similarities is that the PEST sequence of D1 is required for optimal binding of PsbH to PSII, possibly through a direct interaction. It is important to note that the psbH mutant is more sensitive to photoinhibition than the PEST mutant (Mayes et al., 1993) in terms of the ability to grow photoautotrophically. This would suggest that although PEST and psbH show some similarities in phenotype, particularly with regard to Q activity, the absence of PsbH has a more profound effect on the PSII repair cycle.

The PEST-like Sequence Is Not Required for Turnover of D1

We have shown that a D1 repair cycle is operative in PEST. This clearly establishes that the PEST-like region is not absolutely required for turnover of D1. This conclusion is in line with a recent preliminary analysis of a number of mutants in the PEST-like region which failed to find evidence for a PEST activity (Kless et al., 1994). However, unlike the PEST mutant described here, none of the mutants analyzed by Kless and co-workers(1994) had completely lost the PEST-like sequence. In contrast another mutant in the PEST-like region, E229D, was found to show a slower rate of D1 degradation under high light conditions (Tyystjärvi et al., 1994). In view of the work presented in this paper, we would suggest that this is not the result of a diminution of a PEST activity but instead reflects the fact that a number of structural features, many probably subtle, contribute to the rate of degradation of D1.

D2 Turnover Is Enhanced in TC31 and PEST Compared to WT

Pulse-labeling experiments such as that shown in Fig. 9have revealed some interesting differences between the TC31 (and derivatives) and WT strains of Synechocystis PCC 6803. The most obvious is that in TC31 the D2 protein seems to be turning over far more rapidly than D1 both at high and low light intensities (see Fig. 9). The reason for this must be related to the genetic background of TC31. Only one of the three psbA genes, psbA3, is functional in these strains (Nixon et al., 1992). The other two, psbA1 and psbA2, have been deleted and replaced by antibiotic resistance cassettes conferring resistance to chloramphenicol and kanamycin, respectively (Nixon et al., 1992). In addition a tetracycline resistance cassette has been inserted 53 base pairs downstream of the stop codon of psbA3 so as to identify the transformant (Nixon et al., 1992). The presence of this cassette appears to reduce psbA3 expression. Recent experiments indicate that the steady-state level of psbA3 mRNA in TC31 is 10-30% that in WT Synechocystis 6803 (Dalla Chiesa, 1994) and that the rate of synthesis of D1 in TC31 as measured in pulse experiments is also reduced to 10-30% of WT levels. In contrast the rate of synthesis of D1 in a strain which lacks psbA2 but retains psbA3 as well as the inactive psbA1 gene is indistinguishable from WT under high light conditions (Dalla Chiesa, 1994). This reduction in the rate of D1 synthesis in TC31 is not detrimental to photoautotrophic growth under normal conditions as TC31 has the same growth rate as WT (Nixon and Diner, 1992). The rapid turnover of D2 in TC31 may therefore reflect the fact that D1 synthesis is unable to match D2 synthesis so that excess D2 which is not incorporated into a PSII complex is rapidly degraded in a manner similar to that observed in psbA deletion strains (Dalla Chiesa, 1994).

In the PEST mutant the D2 protein continues to turn over rapidly in response to low and high light treatments, although at a slower rate than that observed in TC31, but as shown in Fig. 9, the D1 protein is now turning over at a rate greater than that in TC31. The reason for the increase in D1 turnover in PEST is unclear, but the results show that deletion of the putative PEST region in the D1 protein does not prevent the turnover of the D1 protein. However, a role for the PEST-like sequence in optimizing the turnover of D1 cannot yet be totally ruled out.

The PEST Hypothesis

The significance of PEST regions in the degradation of proteins remains unclear. Early studies in which a C-terminal PEST sequence of the rapidly degraded enzyme ornithine decarboxylase was either removed, thus stabilizing the enzyme (Ghoda et al., 1989), or fused to a normally stable enzyme to destabilize the fusion protein (Loetscher et al., 1991) provided some support for the PEST hypothesis. However, other work with the same enzyme has shown that a single amino acid replacement (Miyazaki et al., 1993) or a deletion (Rosenberg-Hasson et al., 1991) which do not affect the PEST sequence also stabilize the enzyme. Therefore, the presence of a PEST sequence does not in itself act as a degradation signal; instead it may act to destabilize the structure to give the protein a propensity to unfold leading to proteolytic degradation. It is also possible that the PEST-dependent pathway may not be the only avenue by which a particular protein is degraded (Loetscher et al., 1991).


FOOTNOTES

*
This research was supported by the Royal Society (to P. J. N.), the Biotechnology and Biological Sciences Research Council and Research Institute of Innovative Technology for the Earth (to J. B.), and the United States Department of Agriculture (to B. A. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 011-594-5269; Fax: 071-594-5267.

Present address: Institute of Microbiology, Sector of Autotrophic Microorganisms, Opatovicky mlyn, 379 01 Trebon, Czech Republic.

The abbreviations used are: PSII, photosystem II; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; MES, 4-morpholineethanesulfonic acid; PCR, polymerase chain reaction; TL, thermoluminescence; PQ, plastoquinone; Q, primary plastoquinone electron acceptor; Q, secondary plastoquinone electron acceptor; DMBQ, 2,5-dimethylbenzoquinone; DCBQ, 2,6-dichlorobenzoquinone; WT, wild type.

J. Komenda, and J. Barber, manuscript in preparation.

J. Komenda and J. Barber, personal communication.


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