(Received for publication, June 16, 1995; and in revised form, September 14, 1995)
From the
The nuclear-encoded proteins of the oxygen-evolving complex
(OEC) of photosystem II are bound on the lumenal side of the thylakoid
membrane and stabilize the manganese ion cluster forming the
photosystem II electron donor side. The OEC proteins are released from
their binding site(s) following light-induced degradation of reaction
center II (RCII)-D1 protein in Chlamydomonas reinhardtii. The
kinetics of OEC proteins release correlates with that of RCII-D1
protein degradation. Only a limited amount of RCII-D2 protein is
degraded during the process, and no loss of the core proteins CP43 and
CP47 is detected. The release of the OEC proteins is prevented when the
photoinactivated RCII-D1 protein degradation is retarded by addition of
3-(3,5-dichlorophenyl)-1,1-dimethylurea or by a high PQH/PQ
ratio prevailing in membranes of the plastocyanin-deficient mutant
Ac
. The released proteins are not degraded but persist in
the thylakoid lumen for up to 8 h and reassociate with photosystem II
when new D1 protein is synthesized in cells exposed to low light, thus
allowing recovery of photosystem II function. Reassociation also occurs
following D1 protein synthesis in darkness when RCII activity is only
partially recovered. These results indicate that (i) the D1 protein
participates in the formation of the lumenal OEC proteins binding
site(s) and (ii) the photoinactivation of RCII-D1 protein does not
alter the conformation of the donor side of photosystem II required for
the binding of the OEC proteins.
The nuclear encoded 33-, 23-, and 16-kDa polypeptides of the
oxygen-evolving complex (OEC) ()stabilize the manganese
cluster of photosystem II (PSII) (reviewed by Ghanotakis and
Yocum(1990) and Vermaas et al.(1993)). These proteins are
synthesized in the cytosol and are translocated across the chloroplast
outer envelope to the lumenal space of the thylakoid membranes (Brock et al., 1995). The translocation process which involves
participation of bipartite transit sequences and processing of the
precursor proteins, has been previously investigated in detail
(reviewed in Robinson and Klösgen(1994)). The OEC
proteins are bound to PSII on the lumenal side of the thylakoid
membrane and can be released from the binding site(s) by high Tris
concentration at alkaline pH as well as rebind under appropriate
conditions including presence of chloride and calcium ions and light
(Vermaas et al., 1993; Tamura et al., 1989). The
amount of these proteins in mature differentiated thylakoids is not
stoichiometric with the PSII content and apparently an excess of the
OEC proteins exists in the form of a free pool (Ettinger and Theg,
1991).
The OEC proteins are present in thylakoids of etiolated barley (Hashimoto et al., 1993) as well as in Chlamydomonas mutant cells lacking a functional PSII (Rochaix and Erickson, 1988). The Chlamydomonas y-1 mutant does not synthesize chlorophyll and lacks thylakoid membranes when grown in darkness. In these cells as well as in the wild type, OEC proteins accumulate in the dark and their levels vary only slightly during greening (Malnoe et al., 1988).
While the levels of the OEC proteins seem to be independent of their functional association with an assembled PSII unit, it is possible that the unbound proteins turn over at rates higher than those of the proteins associated with the thylakoid membrane (Palomares et al., 1993). Results of in vitro experiments in which isolated thylakoids were exposed to photoinactivation and degradation of the RCII-D1 protein demonstrated release of the bound manganese (Virgin et al., 1988; Hundal et al., 1990) in agreement with the fact that the carboxyl end of the RCII-D1 protein is involved in the stabilization of the manganese cluster of PSII (Metz et al., 1986; Diner et al., 1988). Under these conditions the OEC proteins dissociated from the remaining components of PSII concomitant with the manganese release. Thus the question arises what is the fate of the OEC proteins during the process of light-induced photoinactivation of PSII and degradation of the RCII-D1 protein in vivo. If the free OEC proteins turn over faster than those associated with a functional PSII, one would expect an increase in the turnover of the OEC proteins following degradation of the RCII-D1 protein in vivo. Furthermore, it is not clear whether the release of the OEC proteins occurs only as a result of the D1 protein degradation and disassembly of the RCII or the release is already induced by an alteration of the RCII donor side following irreversible photoinactivation of photosystem II. In the present work we have investigated the fate of the OEC proteins during the process of photoinactivation, RCII-D1 protein degradation, and recovery of activity of the PSII complex in control and mutants of the unicellular green alga Chlamydomonas reinhardtii. The results demonstrate that the level of the OEC proteins remains constant during photoinactivation and subsequent degradation of the RCII-D1 protein in the absence of cytosolic protein translation activity. The OEC proteins remain bound to the lumenal side of the membranes following irreversible photoinactivation of photosystem II, but are released from the binding site(s) following degradation of the RCII-D1 protein and reassociate with the membrane binding site(s) following synthesis and assembly of new RCII-D1 protein.
Cells were harvested in the late exponential
phase of growth. The cells were washed by centrifugation in fresh
growth medium and were resuspended in the same medium at a final
chlorophyll concentration of 30 µgml
.
Photoinhibition and recovery were carried out as described before (Zer
and Ohad 1995). To prevent chloroplast protein synthesis,
chloramphenicol (D-threo form; Sigma) was added as
indicated at a final concentration of 200 µg
ml
(Schuster et al., 1988). When used, cycloheximide which
completely inhibits cytosolic protein synthesis in Chlamydomonas (Schuster et al., 1988) was added at a final
concentration of 2 µg
ml
and
3-(3,5-dichlorophenyl)-1,1-dimethyl urea (DCMU) was added at 10
µM final concentration.
Figure 1:
Correlation between the light-induced
degradation of RCII-D1 protein and the release of OEC proteins from the
lumenal binding site of the membranes. Cells were photoinhibited at
2500 µmolm
s
for
4.5 h in the presence of chloramphenicol. Samples of thylakoid
membranes (lanes A) and of total cell proteins (lanes
B) from control cells (C) and photoinhibited cells (PI) were separated by SDS-PAGE. Immunoblots were incubated
with anti-OEC-33, anti-OEC-23, anti-OEC-16, anti-D1, or
anti-plastocyanin (PC) antibodies.
Measurements of the OEC-33 content of isolated thylakoid as a function of photoinhibition time show similar kinetics for the degradation of the RCII-D1 protein and loss of OEC-33 from the membrane fraction (Fig. 2). These results indicate that under conditions inducing light dependent degradation of the RCII-D1 protein, OEC-33, and presumably the other OEC proteins are released from the binding site(s) on the lumenal side of the PSII complex (Hundal et al., 1990).
Figure 2:
Kinetics of light induced degradation of
RCII-D1 protein and the dissociation of OEC-33 from PSII. Cells were
photoinhibited for 3 h at 2000
µmolm
s
in the
presence of chloramphenicol. Samples were taken at 0.75, 1.5, and 3 h.
The samples were assayed by immunoblotting using anti-D1 and
anti-OEC-33 polyclonal antibodies. Densitometric quantification,
average of three independent experiments are shown.
, D1
protein;
, OEC-33 protein. The correlation coefficient between
the degradation of the D1 protein and the release of OEC-33 is
0.99.
In the experiments described above we have used chloramphenicol as an inhibitor of chloroplast translation activity so as to prevent de novo synthesis of the RCII-D1 protein during the light-induced photoinactivation process. However, it is possible that the observed release of OEC-33 from the thylakoid binding site(s) could be due to inhibition of synthesis of other chloroplast translated proteins. To examine this possibility we have used a C. reinhardtii temperature sensitive mutant, T44, that is unable to accumulate newly synthesized PSII-core proteins at the non permissive temperature (37 °C), but is not otherwise impaired in chloroplast translation activity (Reisman et al., 1986). This allows us to study the effect of the degradation of RCII-D1 protein and possibly other photosystem II core components without inhibiting all chloroplast translation activity. When this mutant grown at the permissive temperature (25 °C) is exposed to photoinhibitory conditions at 37 °C in the absence of chloroplast translation inhibitors, a significant amount of the RCII-D1 and to some extent also the RCII-D2 protein is degraded, while practically no loss of the core proteins CP47 and CP43 is detected. However, no net loss of the RCII-D1 and D2 proteins is detected in the control y-1 cells in which the degraded proteins are rapidly replaced (turnover, Schuster et al., 1988). The OEC-33 content of thylakoids isolated from control y-1 and the T44 cells exposed to photoinhibitory light at 37 °C was significantly reduced only in the T44 cells (Fig. 3). These results indicate that the release of OEC-33 from the membrane binding site(s) is related primarily to the degradation of the RCII-D1 protein in agreement with the data shown in Fig. 2.
Figure 3:
Correlation between dissociation of OEC-33
and light-induced degradation of RCII proteins in the ts T44 mutant
cells. Chlamydomonas y-1 and T44 cells were photoinhibited at
2000 µmolm
s
for
1.5 h at 37 °C without the addition of chloramphenicol. The level
of CP47, CP43, OEC-33, D2, and D1 protein was assayed in thylakoid
membranes isolated from control (C) and photoinhibited (PI) cells by immunodecoration.
Figure 4:
Photoinactivation of RCII-D1 protein is
not sufficient to induce dissociation of OEC-33 from PSII if the
degradation of the D1 protein is prevented by an increase in the ratio
PQH/PQ. y-1 and Ac
cells were photoinhibited
at 2500 µmol
m
s
for 5 h with the addition of chloramphenicol. Samples were taken
at times as indicated, and membrane preparations were assayed for the
presence of OEC-33 and D1 proteins by immunodecoration (Panel
A); Panel B, densitometric quantification of the
immunoblots: squares, D1; triangles, OEC-33; closed symbols, y-1; open symbols, AC
.
Photoinactivation was measured as loss of variable fluorescence in
whole cells and was more than 90% after 3 h of light exposure (not
shown) as reported before (Zer et al.,
1994).
Figure 5:
OEC-33 does not dissociate from the
thylakoid membranes obtained from cells photoinactivated in presence of
DCMU. Cells were photoinhibited at 1800
µmolm
s
for 9 h in
the presence of chloramphenicol, with or without addition of DCMU.
Samples were taken at times as indicated, and thylakoid membranes were
isolated and assayed by immunodecoration for the content of OEC-33 and
D1 proteins (Panel A). Panel B, densitometric
quantification of the immunoblots: squares, D1 protein; triangles, OEC-33; closed and open figures represent cells incubated in the absence or presence of DCMU,
respectively. Photoinactivation was more than 90% after 3 h of light
exposure as assayed by measurements of variable
fluorescence.
Figure 6:
Reassociation of OEC-33 with PSII requires
chloroplast protein synthesis. Chlamydomonas y-1 cells were
photoinhibited at 2000
µmolm
s
for 4 h (PI) in the presence of chloramphenicol. At the end of
photoinhibition the cells were washed and incubated for recovery at 25
µmol
m
s
for 8 h.
Recovery was carried out in the presence or absence of chloramphenicol (Cap) or cycloheximide (Chi). Thylakoid membranes
were isolated, and the OEC-33 and D1 protein content was assayed by
immunodecoration. C, control.
It has been reported that the degraded RCII-D1 protein can also be replaced by newly synthesized protein in cells allowed to recover from photoinhibitory light treatment in darkness (Zer et al., 1994; Gong and Ohad, 1995). However, the RCII reassembled under such conditions recover only partially photosynthetic electron flow activity (Huse and Nilsen, 1989; Gong and Ohad, 1995). To test whether OEC-33 may reassociate with the inactive RCII reassembled in the dark, cells were photoinactivated in high light in presence of chloramphenicol and then washed free of the inhibitor and allowed to recover in low light or in the dark for up to 8 h. The OEC-33 protein content of the thylakoid fraction isolated at the end of the photoinhibitory treatment and after 4 and 8 h of recovery, respectively, was determined. The results show that, in cells allowed to recover photosynthetic electron flow in low light, significant amounts of RCII-D1 protein were synthesized and OEC-33 reassociated with the thylakoid membranes (Fig. 7). OEC-33 reassociated with the thylakoid membranes also in cells in which RCII-D1 protein was synthesized in darkness and in which photosynthetic electron flow was only partially recovered (Fig. 7B).
Figure 7:
Association of OEC-33 with the thylakoid
membranes following recovery from photoinhibition in the dark or low
light. Cells were photoinhibited at 2500
µmolm
s
for 3 h (PI). At the end of photoinhibition the cells were washed and
incubated for recovery at low light (LL) or in the dark for 4
or 8 h as indicated. Panel A, immunoblots of membrane
preparations. Panel B, photosynthetic activity measured by
variable fluorescence kinetics (F
/F
) as
described under ``Materials and Methods.''
, recovery
in low light;
, recovery in the dark.
In this work we took advantage of the fact that the thylakoid membranes of the green alga C. reinhardtii are temporarily opened during cell breakage and release the free soluble proteins located in the lumenal space of the membrane as demonstrated by the release of plastocyanin. Plastocyanin release occurs irrespective of the light treatment to which the cells have been subjected prior to breakage. The release of this protein loosely bound to the membrane, which participates in electron transfer as a free mobile carrier (Haehnel et al., 1989) is expected if the thylakoid vesicles are broken open during the process of cell homogenization. The OEC proteins were present in the lumenal space of the thylakoids before the mechanical breakage of the cells as indicated by immunogold localization of the OEC-33 polypeptide in both cells fixed prior to or after the high light treatment (not shown). This finding supports the proposed explanation of the release of the free OEC proteins as being the result of transient mechanical rupture of the thylakoids. However, the OEC proteins are freed from the binding site(s) only in thylakoids in which the RCII-D1 protein has been degraded, but not in those in which the PSII is not affected by a high light treatment. Similarly, the OEC proteins are not released from thylakoids in which the PSII has been photodamaged and irreversibly inactivated while the degradation of the RCII-D1 protein was prevented as it is the case in the mutants defective in plastoquinol oxidation (Zer et al., 1994), or in wild type cells photoinactivated in the presence of DCMU (Gong and Ohad, 1991; Zer and Ohad, 1995).
The results presented in this work
demonstrate that the RCII-D1 protein is involved in the reversible
association of the OEC proteins with the donor side of the PSII complex
exposed on the lumenal thylakoid surface. This conclusion is supported
by the fact that the kinetics of OEC-33 dissociation from the lumenal
side of the thylakoids follows closely that of the light induced
degradation of the RCII-D1 protein. Under the conditions used in this
work to induce the degradation of the RCII-D1 protein, only partial
degradation of the RCII-D2 protein was detected. It was shown that
similarly, CP47 and CP43, chlorophyll-binding proteins of the PSII
core, are also relatively stable during photoinactivation. Furthermore,
under the conditions of light-induced degradation of RCII-D1 protein in vivo the RCII disassembly is only partial and the CP43 and
RCII-D2 protein as well as the cytochrome b subunits remain associated (Adir et al., 1990). Thus it
appears that the binding of the OEC protein on the donor side of PSII
is related to the presence of an assembled PSII core complex containing
the RCII-D1 protein as part of the complex.
The results demonstrating that the process of photodamage in itself is not sufficient to cause the release of the OEC proteins from the binding site, indicate that the organization of the core polypeptides segments forming the PSII donor side on the lumenal face of the thylakoid membranes is not sufficiently altered to induce the dissociation of the OEC proteins and presumably also of the manganese cluster forming the oxygen evolving complex. It was previously reported that the photoinactivated RCII-D1 protein must be degraded before newly synthesized precursor pD1 protein can be integrated and stabilized in a reassembled PSII, indicating that the photoinactivated RCII-D1 protein in this case is still assembled in a core complex (Adir et al., 1990; Zer et al., 1994). The results of this work indicate that the photoinactivated RCII-D1 protein, which has lost it ability to host a functional acceptor side (Gong and Ohad, 1995; Zer and Ohad, 1995), retains its structural organization in the complex maintaining the OEC binding site(s) of the PSII on the donor side. Thus the reassociation of OEC proteins with the thylakoid membranes during recovery from photoinhibition corresponds to synthesis of new RCII-D1 protein and its reassembly in a PSII complex.
Newly synthesized RCII-D1 protein does not accumulate in differentiated chloroplasts unless the preexisting RCII-D1 protein in RCII is degraded during light exposure. However, newly synthesized D1 protein is stabilized and accumulates in darkness following preceding photodamage of RCII and ensuing degradation of the RCII-D1 protein in darkness (Zer and Ohad, 1995; Gong and Ohad 1995). However, in this case the reassembled PSII is not functional in electron transfer from water to plastoquinone. The lack of the reactivation of PSII recovered in darkness is not directly related to the binding of manganese, which was shown to require light following in vitro dissociation of the manganese and OEC proteins due to Tris washing at high pH (Tamura et al., 1989). This conclusion is based on the fact that reactivation of electron transfer following resynthesis and integration of RCII-D1 protein in the dark is prevented in both wild type Chlamydomonas and Scenedesmus cells which can form active photosynthetic membranes during growth in darkness as well as in the Scenedesmus LF-1 mutant (Gong and Ohad 1995) in which the manganese cluster is not assembled due to the lack of processing of the precursor pD1 protein. However, the LF-1 cells contain a partially active PSII capable of charge separation and electron transfer from the secondary donor Yz to PQ (Taylor et al., 1988; Guenther et al., 1990). This activity is not recovered in the photoinactivated LF-1 mutant cells following synthesis of the RCII-D1 protein in the darkness (Gong and Ohad, 1995). OEC-33 reassociates with the lumenal side of the membranes following recovery of the degraded RCII-D1 protein proportional to the amount of the new RCII-D1 protein synthesized during the recovery process irrespective of whether the recovery of RCII occurred in the light or in darkness. This indicate that binding of the OEC-33 per se does not require light in this case. The fact that the inactive complex is assembled in a way sufficient to bind OEC-33 indicates that the light requirement for the activation of the PSII assembled in the darkness following photoinactivation and RCII-D1 protein degradation is not necessarily related to the process of manganese and OEC-33 protein binding. The role of light in this process remains to be elucidated.
The question arises as to what may be the control system preventing additional OEC proteins synthesis during the high light treatment and the recovery process. The data we have presented here do not exclude de novo synthesis and turnover of a small amount of OEC proteins below the detection level by immunoassays. However, it is clear that no significant amounts of the OEC proteins are synthesized nor degraded during the photoinhibition and recovery process. Preliminary results indicate that the level of the OEC-33 message is not significantly altered during the above processes. Thus the control mechanism preventing excessive accumulation of the OEC translation products remains to be established.
The persistence of the OEC-33 proteins during the turnover of the RCII-D1 protein may fulfill an important physiological function. During the process of light-induced turnover of the RCII-D1 protein as well as during recovery from severe photoinhibition and massive degradation of the protein, it is essential that newly synthesized precursor pD1 protein may become part of a functional complex containing the OEC proteins as soon as it is integrated and processed (Prasil et al., 1992). In the absence of OEC protein binding, the in vivo reassembled PSII will lack the activity of the donor side, resulting in rapid photoinactivation and loss of its RCII-D1 protein at rates considerably faster than in normal functional PSII (Wang et al., 1992; Ohad et al., 1994; Rova et al., 1994; Gong and Ohad, 1995). Thus, the persistence of the OEC proteins during the turnover of the RCII-D1 protein ensures fast reactivation and recovery, i.e. maintenance of photosynthetic activity even in high light, exposed cells as long as the rate of the precursor pD1 protein synthesis matches that of the RCII-D1 protein degradation.