From the
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 (
Turnover of the D1 polypeptide as examined
by pulse-chase experiments using [
The photosystem two (PSII)
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
Greenberg et al.(1987) also identified within the
Q
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.
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
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
µE
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
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.
The pulse-chase labeling of cells using
[
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
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
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.
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
S
Fig. 4shows
the TL curves from TC31 and
Fig. 6shows the
decay in the quantum yield of fluorescence from cells of
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 (
Surprisingly, the removal of these 8 amino acids has only
minor effects on PSII function.
A
role for the PEST sequence in defining the binding properties of the
Q
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
This same differential response to added quinone has also been
observed recently for a
In the
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.
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
.
(
)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.
, and various herbicides.
-binding region of D1 a sequence between residues
Arg
and 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).
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).
m
s
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.
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 NH
OH 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
K
Fe(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
µEm
s
.
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 K
Fe(CN)
.
(
)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) µE
m
s
.
For inhibition of protein synthesis, lincomycin at 100 µg/ml was
added.
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 µE
m
s
)
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.
-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.
-max. (Amersham International
plc) at room temperature for 2-3 days. Quantification of
radiolabeled D1 was done using a Hirschmann Elsript 400 densitometer.
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 Glu
and
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
Electron transport
between Q and 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
=
[Q
Q
]/([Q
]
+ [Q
Q
]), for
the reaction Q
Q
=
Q
Q
. This is in turn related
to the intrinsic equilibrium constant, K
=
[Q
Q
]/[Q
Q
],
by the equation K
/K
= [PQ]/(K
+
[PQ]) where K
is the
dissociation constant, K
=
([Q
] +
[PQ])/[Q
Q
],
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
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 SPEST Indicate a
Destabilization of
Q
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).
Q
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 S
Q
recombination at around 5-15 °C (Rutherford et
al., 1982; Demeter et al., 1982).
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
TyrD
Q
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 S
Q
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 S
Q
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
Q
Q
=
Q
Q
, as indicated from the
fluorescence measurements.
The Donor Side Is Unaffected in the
A variety of measurements indicate that the electron
transfer reactions on the donor side of PSII are relatively unperturbed
in PEST
Mutant
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 Chl
ml
. 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
.
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
Fig. 7shows that when cells of TC31 or the PEST Mutant
PEST
mutant are exposed to relatively intense white light (1000
µE
m
s
) 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
O
mg
Chl
h
for TC31 and 220 µmol O
mg
Chl
h
for
PEST.
A Possible Light-induced Modification of the Q
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 Site in
PEST
-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 O
mg
Chl
h
for TC31 and 210 µmol
O
mg
Chl
h
for
PEST. With DMBQ the 100% rates of oxygen evolution are
approximately 180 µmol O
mg
Chl
h
for TC31 and 200 µmol
O
mg
Chl
h
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
Pulse-chase
experiments using [PEST
S]methionine were used to
assess the rate of turnover of D1 in
PEST and TC31 under low (100
µE
m
s
) and high
light conditions (1000
µE
m
s
). 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
µE
m
s
) or (b) high (1000
µE
m
s
) 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
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
following High Light Treatment
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.
226-233) that lacks this
sequence.
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 Q
Q 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 Q
Q
=
Q
Q
so that
Q
Q
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).
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.
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).
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
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).
PEST Compared to
WT
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).
, primary
plastoquinone electron acceptor; Q
, secondary plastoquinone
electron acceptor; DMBQ, 2,5-dimethylbenzoquinone; DCBQ,
2,6-dichlorobenzoquinone; WT, wild type.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.