(Received for publication, September 25, 1995; and in revised form, December 20, 1995)
From the
The chloroplast-encoded D1 protein of photosystem II (PSII) has a much higher turnover rate than the other subunits of the PSII complex as a consequence of photodamage and subsequent repair of its reaction center. The replacement of the D1 protein in existing PSII complexes was followed in two in vitro translation systems consisting of isolated chloroplasts or isolated thylakoid membranes with attached ribosomes. By application of pulse-chase translation experiments, we followed translation elongation, release of proteins from the ribosomes, and subsequent incorporation of newly synthesized products into PSII (sub)complexes. The time course of incorporation of newly synthesized proteins into the different PSII (sub)complexes was analyzed by sucrose density gradient centrifugation.
Immediately after termination of translation, the D1 protein was found both unassembled in the membrane as well as already incorporated into PSII reaction center complexes, possibly due to a cotranslational association of the D1 protein with other PSII reaction center components. Later steps in the reassembly of PSII were clearly post-translational and sequential. Different rate-limiting steps in the assembly process were found to be related to the depletion of nuclear encoded and stromal components as well as the lateral migration of subcomplexes within the heterogeneous thylakoid membrane.
The slow processing of precursor D1 in the thylakoid translation system revealed that processing was not required for the assembly of the D1 protein into a PSII (sub)complex and that processing of the unassembled precursor could take place. The limited incorporation into PSII subcomplexes of three other PSII core proteins (D2 protein, CP43, and CP47) was clearly post-translational in both translation systems.
Radiolabeled assembly intermediates smaller than the PSII core complex were found to be located in the stroma-exposed thylakoid membranes, the site of protein synthesis. Larger PSII assembly intermediates were almost exclusively located in the appressed regions of the membranes.
Photosystem II (PSII) ()is a large multisubunit
protein complex in thylakoid membranes of oxygenic photosynthetic
organisms containing at least 25 different subunits (see (1) and (2) for reviews). In eukaryotic organisms,
nearly all proteins in the core of the complex are encoded by the
chloroplast genome, while the more peripheral proteins are nuclear
encoded, synthesized in the cytoplasm, imported into the chloroplasts,
and subsequently targeted to the thylakoid membrane (see (2, 3, 4, 5, 6) ). It is
assumed that most chloroplast-encoded membrane proteins are
cotranslationally inserted into the thylakoid membrane since the
ribosomes are bound to its stroma-exposed
regions(7, 8) . Synthesis of the chloroplast-encoded
membrane proteins therefore takes place on these unstacked,
stroma-exposed thylakoid regions (see (7) ), while the vast
majority of the functional PSII complexes are located in the grana, the
stacked thylakoid regions (see (9) and (10) ).
One
of the chloroplast-encoded proteins is the multiple membrane-spanning
D1 protein, which forms, together with the homologous D2 protein, the
PSII reaction center (e.g. see Refs. 3 and 11). This
heterodimer binds or contains all the essential redox components (P680,
pheophytin, non-heme iron, quinones, the redox-active tyrosines, and
accessory chlorophylls and carotenoids) that are needed to carry out
the light-driven reduction of plastoquinone. Isolated PSII reaction
center particles (11) consist of the D1-D2 heterodimer, two
cytochrome b subunits, a low molecular mass
protein (4.8 kDa; psbI gene product), and possibly a small
nuclear encoded protein (6.1 kDa; psbW gene
product)(12) . The PSII core complexes are larger and are
composed of the PSII reaction center surrounded by the chlorophyll a-binding proteins CP43 and CP47, an extrinsic 33-kDa protein,
and a number of smaller proteins (<10 kDa) of unknown function.
These particles contain manganese and can perform light-induced water
oxidation(10) .
A unique feature of PSII is that the turnover of the D1 protein is much higher than that of the other PSII proteins and is a consequence of photodamage of the PSII reaction center(13, 14, 15, 16) . To maintain the PSII complex in a functional state and to thereby avoid photoinhibition of photosynthesis, the damaged D1 protein must be replaced and is therefore synthesized at a much higher rate than all the other PSII proteins (e.g.(17, 18, 19) ). Degradation of some other PSII core proteins (especially the D2 protein) due to photodamage of the PSII complex has also been shown to occur, but the extent of this degradation is much lower than that of the D1 protein(13, 14, 15) .
This unique replacement of the D1 protein allows the study of the mechanism of cotranslational insertion of a membrane protein and its assembly into a pre-existing multisubunit and membrane-bound complex. At the same time, the ligation of several cofactors during translation and assembly can be addressed. It is expected that several features of this complicated turnover process have a general significance for the assembly of other membrane-bound protein complexes.
The D1 protein, encoded by the psbA gene, is assumed to be cotranslationally inserted into the thylakoid membrane (see (7) ). Recently, it was shown that in the presence of ATP, this insertion does not require a proton-motive force or SecA(20) .
Structural information on the organization of the PSII core complex is also important in understanding the possible ways in which the damaged D1 protein can be replaced. Several proposals have been made (e.g.(21) and (22) ) and are based on cross-linking studies (e.g. Refs. 23, 24, and 47), analysis of PSII deletion mutants (e.g. Refs. 2, 25, and 26), detergent treatments(11, 27) , and in recent years, crystallization studies(28, 29) . Most studies propose an organization of PSII in which the D1 protein is situated on the periphery of the core complex, with CP43 and CP47 primarily interacting with the D2 protein. A dimeric structure of PSII has been suggested in several studies(22, 28) , and in this case, the dimer must dissociate into two monomers to allow replacement of the D1 protein. Such monomerization under strong light conditions has been observed in vitro(30) . The monomers were found in the unstacked, stroma-exposed membranes, while the dimeric form was dominant in the stacked granal membranes(30) .
The mechanism
of replacement of the D1 protein in PSII has previously been studied in vivo in green
algae(19, 31, 32, 33) , Spirodela leaves(34) , and recently in vitro using isolated spinach chloroplasts and thylakoids(35) .
It was proposed that the D1 protein associates in the stromal membranes
with a PSII particle consisting of D2-cytochrome b-CP47 (31) or D2-cytochrome b
(34) . However, it has been
experimentally difficult to resolve assembly intermediates using in
vivo systems. Therefore, no direct proof for these hypotheses has
been given so far (for discussion, see (19) ).
In a recent study(35) , we developed an experimental system based upon analytical sucrose gradient centrifugation to follow the replacement of the D1 protein in isolated chloroplasts and to identify and to trap defined intermediate assembly stages. We have shown that in this in vitro system, the D1 protein could be assembled into PSII reaction center particles and core complexes. In contrast to the D1 protein, the other newly synthesized PSII core proteins (D2 protein, CP43, and CP47) accumulated in the thylakoid membranes predominantly as free unassembled proteins and in smaller (sub)complexes with a molecular mass of <100 kDa. It was also shown that the D1 protein could be synthesized and incorporated into existing PSII core complexes with a lower efficiency in a homologous run-off translation system (35) consisting of isolated thylakoids with bound ribosomes (see Refs. 7 and 36).
In this paper, we present a kinetic resolution of the assembly process of the D1 protein into existing PSII core complexes through application of pulse-chase experiments using our recently developed experimental system(35) . The complete pathway of D1 protein incorporation is followed from translation elongation and termination and subsequent release of the newly synthesized product from the ribosomes to sequential incorporation into PSII subcomplexes. Moreover, the localization of the different PSII assembly intermediates and unassembled PSII core proteins in the heterogeneous thylakoid membrane is shown. A scheme summarizing the pathway of incorporation of newly synthesized PSII core proteins and its kinetics is presented.
The purity of the different subthylakoid fractions was tested by immunodetection with antibodies against the D1 protein and PSI reaction center proteins and by comparing the relative amount of LHCII and CF1 in both granal and stromal membranes by Coomassie Brilliant Blue-stained SDS-PAGE (data not shown). The chlorophyll a/b ratio was >6.5 for the stromal membranes.
Figure 1:
Membrane-bound radiolabeled
proteins after translation in isolated chloroplasts and isolated
thylakoids. Labeling with [S]methionine was
carried out for 4 min, followed by a 10-min chase with an excess of
unlabeled methionine. After translation, the chloroplasts (C)
were lysed, and thylakoids (T) were washed repeatedly to
remove unincorporated radioactivity. Pausing intermediates are
indicated with i. Molecular mass standards are indicated in
kilodaltons.
Figure 2:
Assignment of PSII subcomplexes and
proteins in sucrose density gradients of n-dodecyl
-D-maltoside-solubilized thylakoid membranes. Prior to
loading on the sucrose gradients (0.1-1.0 M sucrose),
the thylakoid membranes (120 µg of chlorophyll) were solubilized
(at 0.5 mg of chlorophyll
ml
) in 1% n-dodecyl
-D-maltoside for 50 min on ice.
Sucrose gradients were centrifuged for 20 h at 180,000
g and fractionated from bottom to top into 20 equal fractions. The
assignment was determined by SDS-PAGE, followed by staining and
immunodetection on Western blots, 77 K chlorophyll fluorescence,
calibration of molecular mass by standard PSII preparations, and
molecular weight mass proteins as described (35) . Reaction
center particles contain D1-D2-cytochrome b
/psbI; small PSII core particles
contain D1-D2-cytochrome b
-psbI-CP43-CP47; and large PSII core
particles are small core particles with variable amounts of OEC33
(where OEC is oxygen-evolving complex), OEC23, and OEC17 + CP29
and low molecular mass polypeptides.
Using this recently developed experimental system, we
carried out a pulse-chase experiment in isolated chloroplasts in order
to follow the elongation and termination of translation, the release of
the nascent chains from the ribosomes, and the subsequent incorporation
steps of the newly synthesized proteins into PSII (sub)complexes. The
pulse time was kept as short as experimentally possible (5 min). After
this pulse, followed by different chase times, the thylakoids were
solubilized with n-dodecyl -D-maltoside, and
different PSII (sub)complexes were separated by sucrose density
gradient centrifugation as described(35) . To identify specific
newly synthesized proteins and their time course of incorporation into
complexes, autoradiograms of SDS-PAGE-separated proteins were prepared
from the sucrose gradients of each pulse-chase sample. The
autoradiograms of some of the gradients (2.5-, 10-, and 30-min chase
times) are shown in Fig. 3A. The sucrose gradient
fractions are numbered from the bottom (fraction 1) to the top
(fraction 20) of the gradients.
Figure 3:
Pulse-chase experiment in isolated
chloroplasts. Translation in isolated chloroplasts was carried out for
5 min and was stopped directly (no chase) or was followed by a chase of
2.5, 5, 10, 30, or 60 min with unlabeled methionine. After translation,
chloroplasts were lysed, and the thylakoid membranes were washed
repeatedly, solubilized in 1% n-dodecyl
-D-maltoside on ice, and subjected to sucrose density
gradient centrifugation. After fractionation of the sucrose gradient,
the fractions were precipitated in 10% trichloroacetic acid and run on
14% SDS-polyacrylamide gels containing 6 M urea. The fraction
numbers, 1-20, are indicated.
C-Labeled marker
proteins were run in the outer lanes of the gels. A,
autoradiograms of trichloroacetic acid-precipitated proteins in the
sucrose gradients after translation, followed by the indicated chase
times (2.5, 10, and 30 min). Gels were dried prior to exposure to film.
The sucrose gradient fractions are numbered from the bottom (fraction
1) to the top (fraction 20) of the tube. Molecular mass markers are
indicated. Arrows indicate low molecular mass proteins. B, quantification of the distribution of
S-labeled D1 protein (precursor and mature forms) (upper panel, black bars) and D2 protein (lower
panel, shaded bars) in the sucrose gradients after 5 min
of translation, followed by different chase times (0, 2.5, 5, 10, 30,
and 60 min). Quantification of newly synthesized protein was carried
out by scanning of the autoradiograms. To allow direct comparison of
the quantity of the two proteins, the intensity was corrected for the
number of methionine residues in each protein (the D1 and D2 proteins
contain 11 and 8 methionine residues, respectively). rc,
reaction center.
After a 2.5-min chase, the total amount of radiolabeled (p)D1 protein had tripled, accumulating as unassembled protein (fraction 15) and in reaction center complexes (fraction 12) (Fig. 3, A and B). As can be seen in the autoradiogram (Fig. 3A), a small amount of labeled D1 protein could now also be detected in fraction 10, representing smaller PSII core complexes. In the five bottom fractions, many bands could be observed, representing nascent chains still attached to ribosomal (sub)complexes (Fig. 3A). The majority of these bands are nascent chains of the D1 protein as judged by immunoprecipitation (data not shown).
After 5 min, the amount of radiolabeled D1 protein had nearly reached its maximum (data not shown). The newly synthesized D1 protein continued to accumulate predominantly as unassembled protein and in PSII reaction center complexes (in fractions 15 and 12, respectively) (data not shown).
After a 10-min chase, a clear incorporation of newly synthesized D1 protein into the ``small'' PSII core complexes and, to a lesser extent, into the ``large'' PSII core complexes (fractions 8-10 and 5-7, respectively) could be observed (Fig. 3, A and B). At the same time, the amount of unassembled labeled D1 protein (fractions 14 and 15) had diminished. With this increased chase time, several distinct translation intermediates (mainly of the D1 protein) between 15 and 24 kDa can be observed in the bottom fractions (Fig. 3B), but also in fractions 12-16. It should be noted that during these first 10 min of chase, the amount of nascent chains in the bottom fractions (fractions 1-5) decreased due to elongation, termination, and subsequent release from the ribosomes (Fig. 3A).
The process of
incorporation of D1 protein into the smaller and larger PSII core
complexes (fractions 8-10 and 5-7, respectively) continued
for up to 30 min of chase. After a 30-min chase (Fig. 3, A and B), the bulk of the D1 protein had been incorporated
into these PSII core complexes and into reaction center complexes of
140-215 kDa (fractions 11 and 12). Only
10% of the D1
protein remained unassembled (fractions 14 and 15). Nearly all nascent
chains had been released from the ribosomes, with only some higher
molecular mass proteins (>60 kDa) remaining on the ribosomes. A
longer chase time of 60 min did not change the distribution of
radioactivity significantly (data not shown), indicating that the
incorporation process was finished after 30 min.
These analyses were also carried out for CP43 and CP47. As can be observed from the autoradiograms (Fig. 3A), most of the synthesized CP43 and CP47 remained unassembled and accumulated in fractions 13 and 14. In the case of CP47, the synthesis rate was only half that of CP43, and no significant amount of CP47 was observed in the core complexes of fractions 5, 6, and 8-10.
The PSII core complex contains several low molecular mass chloroplast-encoded proteins (PsbE, PsbF, PsbH, PsbI, PsbJ, PsbK, PsbL, PsbM, and PsbN) (see (2) and (10) ), most of which contain one or more methionine residues. Indeed, at least three different, as yet unidentified, low molecular mass proteins below 10 kDa were found in sucrose density gradient fractions 5-16. Most these labeled products appeared in fractions 15 and 16 in the case of shorter chase times (Fig. 3A), and with increasing chase times (30 and 60 min), the small proteins were quite effectively chased into complexes in fractions 8-10 (Fig. 3A). Strikingly, these small proteins did not show a (transient) accumulation in the reaction center complexes in fractions 12 and 13, but instead appeared to be chased directly into the PSII core complexes.
Figure 4:
Pulse-chase run-off translation experiment
in isolated thylakoids. Translation in isolated thylakoids was carried
out for 5 min and was followed by a chase of 2.5, 5, 10, 30 or 60 min
with unlabeled methionine, or translation was stopped directly (no
chase). After translation, the thylakoid membranes were washed
repeatedly, solubilized in 1% n-dodecyl
-D-maltoside on ice, and subjected to sucrose density
gradient centrifugation. After fractionation of the sucrose gradients,
the fractions were precipitated in 10% trichloroacetic acid, and
proteins were separated by SDS-PAGE. A, autoradiograms (the
30-50-kDa region) of trichloroacetic acid-precipitated proteins
in the sucrose gradients after translation, followed by the indicated
chase times (0, 5, and 30 min). Gels were dried prior to exposure to
film. The fraction numbers, 1-20, are indicated.
C-Labeled marker proteins were run in the outer lanes of
the gels. B, quantification of the distribution of
S-labeled D1 protein (closed circles), D2 protein (open circles), and CP43 (closed triangles) in the
sucrose gradients after 5 min of translation without chase (upper
panel), after 5 min of chase (middle panel), and after 30
min of chase (lower panel). To allow direct comparison of the
quantity of the three proteins, the intensity was corrected for the
number of methionine residues in each protein. rc, reaction
center; arb., arbitrary.
The distribution of radiolabeled pD1 + D1, D2, and CP43 in different sucrose gradient fractions before the chase and after 2.5, 5, and 30 min of chase is shown in Fig. 4(A and B). A correction for the number of methionine residues in the protein sequences was carried out in order to be able to directly compare the amounts of the three synthesized core proteins (Fig. 4B). No quantification could be done for CP47 due to very low levels of labeling (synthesis rate of CP47 was at least 10 times lower than of the D1 protein).
Directly after the pulse (Fig. 4. A and B, upper panels), labeled pD1 + D1 accumulated predominantly as unassembled proteins (fractions 14 and 15) and in PSII subcomplexes of 130-160 kDa (fraction 12). A small amount of (p)D1 could also be found in the smaller PSII core complexes (fractions 8-10). On the other hand, CP43 and the D2 protein accumulated almost completely as ``free'' proteins, in fractions 13 and 14 and fractions 14 and 15, respectively. The total amount of synthesized D1 protein was more than twice that of the D2 protein and nearly six times that of CP43.
After a 2.5-min chase, (p)D1 protein could be observed in the small PSII core complexes (fractions 8 and 9) (data not shown), while very few other labeled products were apparent in fractions 1-10. The radiolabeled D2 protein was nearly exclusively located in fractions 14 and 15, while CP43 and CP47 were predominantly located in fractions 13 and 14 as unassembled proteins.
After a 5-min chase (Fig. 4, A and B, middle panels), D2,
CP43, and CP47 continued to be located as unassembled proteins, while a
considerable amount of D1 protein (17%) was integrated into the small
PSII core complexes in fractions 8-10. Interestingly, the ratio
between pD1 and D1 proteins in fractions 7-15 was quite constant
(0.5-0.6); thus, no specific accumulation of pD1 or D1
protein occurred in certain (sub)complexes, indicating that processing
was not a prerequisite for incorporation into PSII (sub)complexes, in
agreement with earlier studies using PSII mutants (29, 45) .
After a 30-min chase (Fig. 4, A and B, lower panels), the distribution of
these four PSII core proteins had changed. The radiolabeled D1 protein
was now quite evenly distributed among PSII core complexes (fractions
8-10), reaction center particles (fraction 12), and free protein
(fractions 14 and 15). No significant amounts of precursor D1 could now
be detected, indicating that processing was completed. Newly
synthesized D2 protein was predominantly found in reaction center
complexes, while 28% of this protein was incorporated into the
smaller PSII complexes. Interestingly, radiolabeled D2 protein was
nearly absent in fractions 10 and 11. Labeled CP43 (and CP47; data not
shown) was chased to a limited extent into PSII cores complexes, while
the major portion remained unassembled in the membrane. No significant
alterations occurred during longer chase periods (60 min) (data not
shown), indicating that after 30 min of chase, the ``end
situation'' for the assembly and incorporation of newly
synthesized proteins had been reached.
Figure 5:
Comparison of incorporation of newly
synthesized D1 protein into PSII (sub)complexes in thylakoid and
chloroplast translation systems. The data shown in Fig. 3and Fig. 4were used. Shown is the quantification of unassembled S-labeled D1 protein (fractions 14-16) and PSII core
complexes (fractions 5, 6, 8, and 9) after 0-60 min of chase for
translation in both chloroplasts (closed circles) and
thylakoids (open circles). The data are expressed in percent
of total D1 protein incorporation within each translation and sucrose
gradient.
To analyze the distribution of the different PSII assembly
intermediates over the stromal and granal membranes, thylakoid
membranes were fractionated into pure stromal and crude granal
membranes by differential centrifugation after digitonin
solubilization. The granal and stromal membranes were subsequently
solubilized in n-dodecyl -D-maltoside and then
subjected to sucrose gradient fractionation analysis.
The autoradiograms of the trichloroacetic acid-precipitated sucrose gradient fractions from both the granal and stromal membranes after translation in isolated thylakoids (10-min pulse followed by a 60-min chase) are shown in Fig. 6A, while Fig. 6B specifically shows the quantitative lateral distribution of the radiolabeled D1 protein. It can be observed that in the case of the granal membranes, the radiolabeled D1 protein was primarily located in PSII core complexes (fractions 5-10), while in the stroma-exposed membranes, most radiolabeled D1 protein was localized in smaller PSII complexes, such as PSII reaction centers (Fig. 6B). Furthermore, very little unassembled D1 protein was found in the granal membranes as compared with the stromal membranes, indicating that the D1 protein did only migrate from its site of synthesis in the stromal membranes to the granal membranes after it had been assembled together with other PSII subunits.
Figure 6:
Localization of radiolabeled complexes and
unassembled proteins in crude granal and stromal regions after
translation in isolated thylakoids. Translation was carried out for 10
min and was followed by a chase of 60 min with unlabeled methionine.
After translation, the thylakoids were fractionated into crude granal
and stromal regions by digitonin solubilization and differential
centrifugation. Granal and stromal regions were further solubilized in n-dodecyl -D-maltoside and spun on a sucrose
gradient. After fractionation of the sucrose gradient, the fractions
were precipitated in 10% trichloroacetic acid and run on 14%
SDS-polyacrylamide gels containing 6 M urea. A,
autoradiograms of trichloroacetic acid-precipitated proteins in the
sucrose gradients of granal regions (upper panel) and stromal
regions (lower panel). The fraction numbers, 1-20, are
indicated.
C-Labeled marker proteins were run in the outer
lanes of the gels. B, quantification of the distribution of
S-labeled D1 protein in PSII (sub)complexes in granal
regions (closed squares) and stromal regions (open
squares). The autoradiograms shown in A were scanned and
quantified. (arb., arbitrary).
Radiolabeled D2 protein, CP43, and CP47 were found to accumulate predominantly as free proteins (fractions 13 and 14) in the stromal membranes, while only small amounts of D2 and CP43 could be detected in small and large PSII core complexes (fractions 8 and 9 and fractions 4 and 5, respectively) in the granal membranes. A similar lateral distribution of labeled PSII subcomplexes was observed after translation in isolated chloroplasts (data not shown). However, incorporation of labeled proteins into PSII core complexes and lateral migration to the granal regions were slightly more efficient than in the isolated thylakoids.
Figure 7: Model for incorporation of newly synthesized D1 protein, D2 protein, and CP43 into PSII. A, mechanism of incorporation of D1 protein into PSII (sub)complexes. Both unassembled D1 protein and three distinct PSII assembly intermediates are distinguished. Directly after translation is terminated, the D1 protein either is located unassembled in the membrane or is immediately assembled into a PSII reaction center particle. The unassembled D1 protein is to a large extent (post-translationally) incorporated into a PSII reaction center. The mechanism for the fast incorporation of D1 into a PSII reaction center (PSIIrc) is either co- or post-translational (see ``Discussion''). The C-terminal processing of the precursor D1 protein (pD1) is not functionally related to the assembly process. Requirements for nuclear encoded (cytoplasmic) and stromal components are indicated. The assembly step from PSII reaction center particles to small core complexes involves lateral migration from stromal to granal regions. Dashed arrows indicate the requirement of components for the assembly process as observed by their depletion during the in vitro experiments. Unbroken arrows indicate the sequence of the assembly pathway. B, mechanism of incorporation of the D2 protein and CP43. Translation of the proteins is polycistronic, and incorporation into PSII subcomplexes is post-translational and sequential. Newly synthesized proteins initially accumulate as free protein (apoprotein or holoprotein) in the membrane. Subsequently, the proteins are incorporated stepwise. The step from PSII reaction center particles to small core complexes involves lateral migration.
The initial incorporation step seems to involve both a slow, clearly
post-translational incorporation and a faster, possibly cotranslational
incorporation of D1 protein into existing PSII complexes (Fig. 7A). From the kinetics, we observe that both
pathways occur in parallel ( Fig. 3and Fig. 4). In the
slow, post-translational pathway, the precursor D1 protein accumulates
first as a free unassembled protein after release from the ribosomes.
Subsequently, the labeled protein associates with a PSII reaction
center protein (e.g. the D2 protein) or with a small protein
subcomplex (e.g. a D1-less PSII reaction center) to form
transient PSII reaction center complexes, accumulating in sucrose
gradient fraction 12. The involvement of a post-translational
incorporation mechanism is particularly supported by the prominent
accumulation of unassembled D1 protein within the first 10 min of chase
time, followed by a relatively slow incorporation into the various PSII
(sub)complexes. This post-translational mechanism is likely to occur if
limited amounts of D2 protein or other PSII reaction center proteins
are available for direct assembly of a PSII reaction center. The
mechanism for the fast incorporation (Fig. 7A,
``fast mechanism'') could be a fast association of the
unassembled D1 protein with a small PSII subcomplex, but could also be
due to an interaction of the newly synthesized -helices of the D1
protein during translation (a cotranslational assembly) with a PSII
reaction center protein (e.g. the D2 protein) or a protein
subcomplex (a D1-less PSII reaction center). After termination of
translation, the D1 protein has already become part of PSII and is
consequently directly recovered in the fractions of the sucrose
gradients containing the reaction center particles. Preliminary
experimental support for a cotranslational interaction of the D1
nascent chain with the mature D2 protein is found in the relatively
high abundance of D1 translation intermediates in pseudopolysome
preparations in addition to substantial amounts of D2 protein. (
)Furthermore, this concept is supported by studies of PSII
deletion mutants that have shown that the D2 protein has a
translational control over the D1
protein(2, 25, 26) .
With longer chase times, the D1 protein was progressively found in the small and large PSII core complexes in which also the extrinsic proteins of the oxygen-evolving complex and some minor chlorophyll a/b-binding proteins became bound; the step from the PSII reaction center to small PSII core complexes involved the lateral migration from stromal to granal regions. These later steps of assembly of PSII core complexes are post-translational and sequential as judged from the kinetics of incorporation (Fig. 7A).
Although substantial amounts of the D2 protein, CP43, and CP47 were synthesized in both translation systems, only a small percentage of these proteins was incorporated into the PSII core complexes. Instead, they accumulated predominantly as free unassembled proteins and, in the case of the D2 protein, also in PSII reaction center complexes (Fig. 3B and Fig. 4B). During translation experiments with intact chloroplasts, slightly more D2 protein was incorporated into PSII cores and PSII reaction centers as compared with translation in isolated thylakoids, indicating that soluble stromal factors or chloroplast biosynthetic products slightly improve the incorporation of these two proteins. However, since the assembly of newly synthesized D2 protein, CP43, and CP47 is low even in isolated chloroplasts, the major limiting factor for their assembly process must be a cytoplasmic (nuclear encoded) component that is normally (in vivo) imported into the chloroplasts and that is freely available only to a limited extent in the chloroplast itself (see Fig. 7B). The accumulation of the D2 protein, CP43, and CP47 as unassembled subunits is therefore a reflection of their relatively low rate of light-induced damage and subsequent degradation (as compared with the D1 protein) (for discussion, see (35) ).
The PSII complex contains
several small (<10 kDa) chloroplast-encoded proteins (2) containing up to three methionine residues. Several small
proteins (especially of 4, 4.5, and 8 kDa) were synthesized in
both the chloroplast (Fig. 3A) and, to more limited
extent, thylakoid (data not shown) translation systems. The small
radiolabeled proteins were abundant in fractions 15 and 16 in the case
of the short chase times, and with increasing chase times (30 and 60
min), they were quite effectively chased into PSII core complexes.
Thus, the three small PSII proteins seemed to be actively synthesized
and incorporated into the PSII core, without transient accumulation in
PSII reaction center complexes. This suggests the possibility that in
addition to the D1 protein, some of these small proteins are also
replaced. To our knowledge, no previous study has been reported
concerning a high turnover of these small, chloroplast-encoded
proteins. Further identification and assessment of their turnover are
currently in progress.
The experimental system described in this paper has opened many new possibilities to study the assembly of PSII and possibly other thylakoid-bound complexes. Experiments are in progress to identify the nuclear encoded and stromal components needed for efficient incorporation of the D1 protein into the existing PSII core complexes and to characterize the initial assembly steps in molecular detail.