Outcome and Prognostic Impact of Surgical Staging in Serous Tubal Intraepithelial Carcinoma: A Cohort Study and Systematic Review.

The optimal management of breast cancer susceptibility gene (BRCA)1/2 carriers with isolated serous tubal intraepithelial carcinoma (STIC) found at risk-reducing salpingo-oophorectomy (RRSO) is unclear. The prevalence of occult carcinoma and STIC in a consecutive series of BRCA1/2 carriers undergoing RRSO is reported. The outcome of staging procedures in BRCA1/2 carriers with isolated STIC at RRSO as well as the relationship between staging, chemotherapy treatment and risk of recurrence was assessed via a systematic review of the literature. Our series included 235 BRCA1/2 carriers who underwent RRSO. Federation of Gynaecology and Obstetrics stage IA carcinoma or STIC was found at RRSO in three (1.3%) and two (0.9%) patients, respectively. A systematic review of the literature included 82 BRCA1/2 carriers with isolated STIC found at RRSO. In 13/82 (16%) cases with STIC, staging was reported. In none of these cases staging revealed more advanced disease. Recurrent disease was found in four of 36 patients with reported follow-up. The estimated risk of recurrence in patients with isolated STIC at RRSO was about 11% (95% confidence interval 3-26%) after a median follow-up of 42 months (range 7-138). No recurrences were reported in those patients with STIC at RRSO who underwent staging or received chemotherapy. We found 1.3% occult carcinoma and 0.9% STIC at RRSO in our cohort of BRCA1/2 carriers. A systematic review of the literature suggests that additional treatment after RRSO, i.e. staging and/or chemotherapy, is associated with a lower risk of recurrence. However, data on staging and follow-up are limited.

Proteomics of the chloroplast: experimentation and prediction.

New technologies, in combination with increasing amounts of plant genome sequence data, have opened up incredible experimental possibilities to identify the total set of chloroplast proteins (the chloroplast proteome) as well as their expression levels and post-translational modifications in a global manner. This is summarized under the term 'proteomics' and typically involves two-dimensional electrophoresis or chromatography, mass spectrometry and bioinformatics. Complemented with nucleotide-based global techniques, proteomics is expected to provide many new insights into chloroplast biogenesis, adaptation and function.

Light is required for efficient translation elongation and subsequent integration of the D1-protein into photosystem II.

The light dependence of translation and successive assembly of the D1 reaction center protein into Photosystem II subcomplexes was followed in fully developed chloroplasts isolated from the dark phase of diurnally grown spinach. The incorporation of synthesized D1 protein into Photosystem II (PSII) was analyzed by fractionation of radiolabeled unassembled protein and PSII (sub)complexes on sucrose density gradients. The ribosomes with attached nascent chains were recovered as pellets in the same gradients, and nascent chains of the D1 protein were immunoprecipitated. The analysis showed that absence of light during translation leads to an increased accumulation of polysome-bound D1 translation intermediates, indicating that light is required for efficient elongation of the D1 protein. The accumulation of the D1 protein and CP43 decreased three-fold in darkness, whereas accumulation of the D2 reaction center protein was not affected by light. In addition, light was also required for efficient incorporation of the D1 protein into the PSII core complex. In darkness, the newly synthesized D1 protein accumulated predominantly as unassembled protein or in PSII subcomplexes smaller than 100 kDa.

Evidence for SecA- and delta pH-independent insertion of D1 into thylakoids.

Many nuclear-encoded proteins are targeted into chloroplast thylakoids by an azide sensitive Sec-related mechanism or by a delta pH-driven mechanism. In this report, the requirements for the integration of chloroplast-encoded thylakoid proteins have been analysed in pulse-labeled intact chloroplasts. We show that the integration of the photosystem II reaction centre protein, D1, continues in the absence of a delta pH and in the presence of azide. A range of other proteins are similarly targeted to thylakoids in the presence of azide, suggesting that the SecA-related mechanism is not widely used for the targeting of chloroplast-encoded proteins.

Complementation of bacterial SecE by a chloroplastic homologue.

The SecE protein is an essential component of the SecAYE-translocase, which mediates protein translocation across the cytoplasmic membrane in bacteria. In the thylakoid membranes of chloroplasts, a protein homologous to SecE, chloroplastic (cp) SecE, has been identified. However, the functional role of cpSecE has not been established experimentally. In this report we show that cpSecE in cells depleted for bacterial SecE (i) supports growth, (ii) stabilizes, just like bacterial SecE, the Sec-translocase core component SecY, and (iii) supports Sec-dependent protein translocation. This indicates that cpSecE can functionally replace bacterial SecE in vivo, and strongly suggests that the thylakoid membrane contains a SecAYE-like translocase with functional and structural similarities to the bacterial complex. This study further underscores the evolutionary link between chloroplasts and bacteria.

Real-time phase-contrast flow MRI of the ascending aorta and superior vena cava as a function of intrathoracic pressure (Valsalva manoeuvre).

OBJECTIVE: Real-time phase-contrast flow MRI at high spatiotemporal resolution was applied to simultaneously evaluate haemodynamic functions in the ascending aorta (AA) and superior vena cava (SVC) during elevated intrathoracic pressure (Valsalva manoeuvre). METHODS: Real-time phase-contrast flow MRI at 3 T was based on highly undersampled radial gradient-echo acquisitions and phase-sensitive image reconstructions by regularized non-linear inversion. Dynamic alterations of flow parameters were obtained for 19 subjects at 40-ms temporal resolution, 1.33-mm in-plane resolution and 6-mm section thickness. Real-time measurements were performed during normal breathing (10 s), increased intrathoracic pressure (10 s) and recovery (20 s). RESULTS: Real-time measurements were technically successful in all volunteers. During the Valsalva manoeuvre (late strain) and relative to values during normal breathing, the mean peak flow velocity and flow volume decreased significantly in both vessels (p < 0.001) followed by a return to normal parameters within the first 10 s of recovery in the AA. By contrast, flow in the SVC presented with a brief (1-2 heartbeats) but strong overshoot of both the peak velocity and blood volume immediately after pressure release followed by rapid normalization. CONCLUSION: Real-time phase-contrast flow MRI may assess cardiac haemodynamics non-invasively, in multiple vessels, across the entire luminal area and at high temporal and spatial resolution. ADVANCES IN KNOWLEDGE: Future clinical applications of this technique promise new insights into haemodynamic alterations associated with pre-clinical congestive heart failure or diastolic dysfunction, especially in cases where echocardiography is technically compromised.

Perceptions of team workers in youth care of what makes teamwork effective.

In youth care, little is known about what makes teamwork effective. What is known mostly reflects the view of managers in care organisations, as objective outcome measures are lacking. The objective of this article was to explore the views of youth care workers in different types of teams on the relative importance of characteristics of teamwork for its effectiveness. Q methodology was used. Fifty-one respondents rank-order 34 opinion statements regarding characteristics of teamwork. Individual Q sorts were analysed using by-person factor analysis. The resulting factors, which represented team workers' views of what is important for effective teamwork, were interpreted and described using composite rankings of the statements for each factor and corresponding team workers' explanations. We found three views of what makes teamwork effective. One view emphasised interaction between team members as most important for team effectiveness. A second view pointed to team characteristics that help sustain communication within teams as being most important. In the third view, the team characteristics that facilitate individuals to perform as a team member were put forward as most important for teamwork to be effective. In conclusion, different views exist on what makes a team effective in youth care. These views correspond with the different types of teams active in youth care as well as in other social care settings.

Paternal effects on seed mass in Arabidopsis thaliana.

Who is in control of seed size, and do some fathers sire bigger seeds than others? We used isogenic male-sterile genotypes of the Arabidopsis thaliana accessions Col and Ler. By fertilising flowers side-by-side with either pollen from the same accession ('self-pollination') or pollen from another accession (outcrossing), we compared, on the same mother plant, seed set of flowers that were very similar in resource status. Some paternal genotypes had a significant effect on seed mass, with the most extreme father siring seeds 15.3% heavier than seeds resulting from 'self-pollination'. There was no correlation between seed mass of paternal parents and the seeds they sired. We discuss the evolution of seed size as a tug-of-war between parent and offspring.

In vitro reconstitution of insertion and processing of cytochrome f in a homologous chloroplast translation system.

Using a homologous chloroplast translation system, we have reconstituted insertion and processing of the chloroplast-encoded thylakoid protein cytochrome f (pCytf). Cross-linking demonstrated that pCytf nascent chains when attached to the 70 S ribosome tightly interact with cpSecA, but this is strictly dependent on thylakoid membranes and a functional signal peptide. This indicates that cpSecA is only operative in pCytf biogenesis when it is bound to the membrane, most likely as part of the Sec translocon. No evidence for interaction between the 54-kDa subunit of the chloroplast signal recognition particle (cpSRP) and the pCytf nascent chain could be detected, suggesting that pCytf, in contrast to the polytopic D1 protein, does not require cpSRP for targeting. Insertion of pCytf occurred only co-translationally, resulting in processing and accumulation of both the processed signal peptide and the mature protein in the thylakoid. This co-translational membrane insertion and processing required a functional signal peptide and was inhibited by azide, demonstrating that cpSecA is essential for translocation of the soluble luminal domain. pCytf also associated post-translationally with thylakoids, but the soluble N-terminal domain could not be translocated into the lumen. This is the first study in which synthesis, targeting, and insertion of a chloroplast-encoded thylakoid membrane protein is reconstituted from exogenous transcripts and using the chloroplast translational machinery.

The quantum efficiency of photosystem II and its relation to non-photochemical quenching of chlorophyll fluorescence; the effect of measuring-and growth temperature.

The relation between the quantum yield of oxygen evolution of open photosystem II reactions centers (Φp), calculated according to Weis and Berry (1987), and non-photochemical quenching of chlorophyll fluorescence of plants grown at 19°C and 7°C was measured at 19°C and 7°C. The relation was linear when measured at 19°C, but when measured at 7°C a deviation from linearity was observed at high values of non-photochemical quenching. In plants grown at 7°C this deviation occurred at higher values of non-photochemical quenching than in plants grown at 19°C. The deviations at high light intensity and low temperature are ascribed to an increase in an inhibition-related, non-photochemical quenching component (qI).The relation between the quantum yield of excitation capture of open photosystem II reaction centers (Φexe), calculated according to Genty et al. (1989), and non-photochemical quenching of chlorophyll fluorescence was found to be non-linear and was neither influenced by growth temperature nor by measuring temperature.At high PFD the efficiency of overall steady state electron transport measured by oxygen-evolution, correlated well with the product of q N and the efficiency of excitation capture (Φexe) but it deviated at low PFD. The deviations at low light intensity are attributed to the different populations of chloroplasts measured by gas exchange and chlorophyll fluorescence and to the light gradient within the leaf.

Oxygen dependence of photoinhibition at low temperature in intact protoplasts of Valerianella locusta L.

Photoinhibition of photosynthesis in vivo is shown to be considerably promoted by O2 under circumstances where energy turnover by photorespiration and photosynthetic carbon metabolism are low. Intact protoplasts of Valerianella locusta L. were photoinhibited by 30 min irradiation with 3000 µmol photons · m(-2) · s(-1) at 4° C in saturating [CO2] at different oxygen concentrations, corresponding to 2-40% O2 in air. The photoinhibition of light-limited CO2-dependent photosynthetic O2 evolution increased with increasing oxygen concentration. The uncoupled photochemical activity of photosystem II, measured in the presence of the electron acceptor 1,4-benzoquinone, and maximum variable fluorescence, Fv, were strongly affected and this inhibition was closely correlated to the O2 concentration. The effect of O2 did not saturate at the highest concentrations applied. An increase in photoinhibitory fluorescence quenching with [O2], although less pronounced than in protoplasts, was also observed with intact leaves irradiated at 4° C in air. Initial fluorescence, Fo, was slightly (about 10%) increased by the inhibitory treatments but not influenced by [O2]. A long-term cold acclimation of the plants did not substantially alter the O2-sensitivity of the protoplasts under the high-light treatment. From these experiments we conclude that oxygen is involved in the photoinactivation of photosystem II by excess light in vivo.

Synthesis of reaction center proteins and reactivation of redox components during repair of photosystem II after light-induced inactivation.

The D1 reaction center protein in photosystem II (PSII) has a high turnover rate due to light-induced inactivation of the redox components. We have studied the reactivation kinetics of the redox components of PSII after strong illumination and compared these kinetics with the turnover of the D1 protein and translation kinetics of the plastid-encoded PSII core proteins in Chlamydomonas reinhardtii cells. Repair of PSII was to a large extent dependent on protein translation. During the first hours of repair, D1 translation was highly accelerated as compared to the other PSII core proteins. By addition of protein synthesis inhibitors during the recovery process, it was found that the time from protein synthesis to full reassembly and reactivation of the individual PSII complexes was about 55 +/- 10 min. Inactivation and reactivation of the redox components in PSII were followed by electron spin resonance and electron transport measurements. Combining the data shows that reactivation of the individual components proceeded together or shortly after one another. Thus, no accumulation of any partially active reactivation intermediate occurred. We conclude that the rate-limiting step of the repair cycle of PSII lies in the degradation and synthesis of the PSII reaction center proteins. Once stable synthesis of the PSII core proteins is achieved, reactivation of the redox components occurs very quickly.

Kinetic resolution of the incorporation of the D1 protein into photosystem II and localization of assembly intermediates in thylakoid membranes of spinach chloroplasts.

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.

Photoinhibition of photosystem II in vivo is preceded by down-regulation through light-induced acidification of the lumen: Consequences for the mechanism of photoinhibition in vivo.

The mechanism of photoinhibition of photosystem II (PSII) was studied in intact leaf discs of Spinacia oleracea L. and detached leaves of Vigna unguiculata L. The leaf material was exposed to different photon flux densities (PFDs) for 100 min, while non-photochemical (qN) and photochemical quenching (qp) of chlorophyll fluorescence were monitored. The 'energy' and redox state of PSII were manipulated quite independently of the PFD by application of different temperatures (5-20° C), [CO2] and [O2] at different PFDs. A linear or curvilinear relationship between qp and photoinhibition of PSII was observed. When [CO2] and [O2] were both low (30 µl · l(-1) and 2%, respectively), PSII was less susceptible at a given qp than at ambient or higher [CO2] and photoinhibition became only substantial when qp decreased below 0.3. When high levels of energy-dependent quenching (qE) (between 0.6 and 0.8) were reached, a further increase of the PFD or a further decrease of the metabolic demand for ATP and NADPH led to a shift from qE to photoinhibitory quenching (qI). This shift indicated that photoinhibition was preceded by down-regulation through light-induced acidification of the lumen. We propose that photoinhibition took place in the centers down-regulated by qE. The shift from qE to qI occurred concomitant with qP decreasing to zero. The results clearly show that photoinhibition does not primarily depend on the photon density in the antenna, but that photoinhibition depends on the energy state of the membrane in combination with the redox balance of PSII. The results are discussed with regard to the mechanism of photoinhibition of PSII, considering, in particular, effects of light-induced acidification on the donor side of PSII. Interestingly, cold-acclimation of spinach leaves did not significantly affect the relationship between qP, qE and photoinhibition of PSII at low temperature.

Synthesis and assembly of the D1 protein into photosystem II: processing of the C-terminus and identification of the initial assembly partners and complexes during photosystem II repair.

In previous studies [van Wijk, K. J., Bingsmark, S., Aro, E.-M., & Andersson, B. (1995) J. Biol. Chem. 270, 25685-25695; van Wijk, K. J., Andersson, B., & Aro, E.-M. (1996) J. Biol. Chem 271, 9627-9636], we have demonstrated that D1 protein synthesized in isolated chloroplasts and thylakoids is incorporated into the photosystem II (PSII) core complex. By pulse-chase experiments in these in vitro systems, followed by sucrose gradient fractionation of solubilized thylakoid membranes, it was shown that this assembly proceeded stepwise; first the D1 protein was incorporated to form a PSII reaction center complex (PSII rc), and through additional assembly steps the PSII core complex was formed. In this study, we have analyzed this assembly process in more detail, with special emphasis on the initial events, through further purification and analysis of the assembly intermediates by nondenaturing Deriphat-PAGE and by flatbed isoelectric focusing. The D2 protein was found to be the dominant PSII reaction center protein initially associating with the new D1 protein. This strongly suggests that the D2 protein is the primary "receptor" or stabilizing component during or directly after synthesis of the D1 protein. After formation of the D1-D2 heterodimer, cyt b559 became attached, whereas the psbI gene product was assembled as a subsequent step, thereby forming a PSII reaction center complex. Subsequent formation of the PSII core occurred by binding of CP47 and then CP43 to the PSII rc. The rapid radiolabeling of a minor population of a PSII core subcomplex without CP43 indicated that an association of newly synthesized D1 protein with a preexisting complex consisting of D2/cyt b55q/psbI gene product/CP47 was possibly occurring, in parallel to the predominant sequential assembly pathway. The kinetics of synthesis and processing of the precursor D1 protein were followed in isolated chloroplasts and were compared with its incorporation into PSII assembly intermediates. No precursor D1 protein was found in PSII core complexes, indicating either that incorporation into the PSII core complex facilitates the cleavage of the C-terminus or, more likely, that processing is more rapid than the assembly into the PSII core.

Transcriptional and translational adjustments of psbA gene expression in mature chloroplasts during photoinhibition and subsequent repair of photosystem II.

The D1 reaction centre protein of photosystem II (PSII), encoded by the plastid psbA gene, has the highest turnover rate of all thylakoid proteins, due to light-induced damage to D1. The expression of the psbA gene was studied in chloroplasts of fully developed pea (Pisum sativum L.) leaves during high-light photoinhibitory treatment and subsequent restoration of PSII function at low light. psbA transcript levels were determined and the translational activity was followed by in vivo pulse-labelling, by in vitro translations with intact chloroplasts, and by run-off translations on isolated thylakoid membranes. PSII photochemical efficiency was determined in vivo by monitoring the ratio of variable fluorescence to maximal fluorescence (F(V)/F(M)). Enhanced D1 synthesis in pea leaves, upon a shift first from darkness to growth light and subsequently to high light, was accompanied by a substantial increase in the total number of pshA transcripts and by the accumulation of psbA mRNA x initiation complexes on thylakoid membrane. This suggested that high-light illumination increased the transcriptional activity of the psbA gene in mature leaves, and that enhanced translational initiation of psbA mRNA was followed by docking of the initiation complexes to the thylakoid membrane. The high-light-induced increase in the number of thylakoid-associated psbA mRNA x initiation complexes, occurred in parallel with enhanced in vivo D1 synthesis. This, however, did not result in an enhanced accumulation of D1 translation products in in vitro run-off translations when pea leaves were shifted from growth light to high light. This may suggest that at high light only a portion of thylakoid-associated psbA mRNA can be under translational elongation at a given moment. When the leaves were shifted from high light to low light to allow repair of PSII, thylakoid-associated psbA mRNA was rapidly released from the membrane and the high-light-induced pool of psbA transcripts was degraded. The synthesis of the D1 protein decreased on the same time scale. However, the restoration of PSII photochemical function, measured as F(V)/F(M), took a substantially longer time. It is concluded that during changing light conditions, mature leaves are able to adjust psbA gene expression both at the transcriptional and at the translational level.

Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54.

The mechanisms of targeting, insertion and assembly of the chloroplast-encoded thylakoid membrane proteins are unknown. In this study, we investigated these mechanisms for the chloroplast-encoded polytopic D1 thylakoid membrane protein, using a homologous translation system isolated from tobacco chloroplasts. Truncated forms of the psbA gene were translated and stable ribosome nascent chain complexes were purified. To probe the interactions with the soluble components of the targeting machinery, we used UV-activatable cross-linkers incorporated at specific positions in the nascent chains, as well as conventional sulfhydryl cross-linkers. With both cross-linking approaches, the D1 ribosome nascent chain was photocross-linked to cpSRP54. cpSRP54 was shown to interact only when the D1 nascent chain was still attached to the ribosome. The interaction was strongly dependent on the length of the nascent chain that emerged from the ribosome, as well as the cross-link position. No interactions with soluble SecA or cpSRP43 were found. These results imply a role for cpSRP54 in D1 biogenesis.

Spectroscopic characterization of tyrosine-Z in histidine 190 mutants of the D1 protein in photosystem II (PSII) in Chlamydomonas reinhardtii. Implications for the structural model of the donor side of PSII.

EPR spectra attributed to the redox active tyrosine residues on the oxidizing side of photosystem II (TyrZ and TyrD) have almost identical line shapes, although the tyrosyl radicals differ in stability and redox characteristics. Strongly modified spectra of oxidized TyrD in site-directed mutants in a histidine residue, H189 on the D2 reaction center protein in the cyanobacterium Synechocystis 6803, support a structural model where H189 interacts closely, probably via a hydrogen bond, to TyrD (Tommos, C., Davidsson, L., Svensson, B., Madsen, C., Vermass, W., and Styring, S. (1993) Biochemistry 32, 5436-5441). To determine whether TyrZ and the corresponding histidine on the D1 protein (D1-H190) interacts similarly, we have generated His-Phe (H190F) and His-Tyr (H190Y) mutations in the C2 symmetry related H190 residue on the D1 reaction center protein by site-directed mutagenesis in Chlamydomonas reinhardtii. The H190F and H190Y mutants assemble photosystem II reaction centers capable of primary photochemistry but unable to oxidize water. We have obtained kinetic spectra of a flash-induced transient EPR signal that we assign to oxidized TyrZ in the D1-H190 mutants. The spectra are identical in line width (18-20 G) and hyperfine structure to the wild-type spectrum from oxidized TyrZ and exhibit decay kinetics (t 1/2 approximately 500 ms) typical for the TyrZ radical in managenese-depleted photosystem II membranes. However, both TyrZ and TyrD were oxidized with reduced (10-15%) quantum yield in these mutants, indicating that the kinetics of electron donation to P+680 were significantly modified as a result of the mutation. Thus, the altered kinetics of TyrZ in the mutants suggest that there is an interaction between TyrZ and His-190 on the D1 protein. However, unlike the situation on the D2 side, the presence of a hydrogen bond between TyrZ and H190 on the D1 protein is improbable.

Co-translational assembly of the D1 protein into photosystem II.

Assembly of multi-subunit membrane protein complexes is poorly understood. In this study, we present direct evidence that the D1 protein, a multiple membrane spanning protein, assembles co-translationally into the large membrane-bound complex, photosystem II. During pulse-chase studies in intact chloroplasts, incorporation of the D1 protein occurred without transient accumulation of free labeled protein in the thylakoid membrane, and photosystem II subcomplexes contained nascent D1 intermediates of 17, 22, and 25 kDa. These N-terminal D1 intermediates could be co-immunoprecipitated with antiserum directed against the D2 protein, suggesting co-translational assembly of the D1 protein into PS II complexes. Further evidence for a co-translational assembly of the D1 protein into photosystem II was obtained by analyzing ribosome nascent chain complexes liberated from the thylakoid membrane after a short pulse labeling. Radiolabeled D1 intermediates could be immunoprecipitated under nondenaturing conditions with antisera raised against the D1 and D2 protein as well as CP47. However, when the ribosome pellets were solubilized with SDS, the interaction of these intermediates with CP47 was completely lost, but strong interaction of a 25-kDa D1 intermediate with the D2 protein still remained. Taken together, our results indicate that during the repair of photosystem II, the assembly of the newly synthesized D1 protein into photosystem II occurs co-translationally involving direct interaction of the nascent D1 chains with the D2 protein.

In vitro synthesis and assembly of photosystem II core proteins. The D1 protein can be incorporated into photosystem II in isolated chloroplasts and thylakoids.

The D1 reaction center protein of the membrane-bound photosystem II complex (PSII) has a much higher turnover rate than the other PSII proteins. Thus, the D1 protein has to be replaced while the other PSII components are not newly synthesized. In this study, this D1 protein replacement into PSII complexes was followed in two in vitro translation systems: isolated chloroplasts and a homologous run-off translation system consisting primarily of isolated thylakoids with attached ribosomes. The incorporation of newly synthesized radiolabeled products into different (sub)complexes was analyzed by sucrose density gradient centrifugation of n-dodecyl beta -D-maltoside-solubilized thylakoid membranes. This analysis allowed us to follow the release of the nascent polypeptide chains from the ribosomes and identification of at least four assembly steps of the PSII complex, as shown below. (i) Both in isolated chloroplasts and in thylakoids, newly synthesized D1 protein is predominantly incorporated into existing PSII subcomplexes, indicating that synthesis and import of nuclear-encoded factors is not needed for D1 protein replacement. (ii) In chloroplasts, D1 protein incorporation into PSII core complexes is more efficient than during translation in isolated thylakoids. In the thylakoid translation system, a large percentage of radiolabeled D1 protein is found in smaller PSII subcomplexes, like PSII reaction center particles, and as unassembled protein in the membrane. This indicates that stromal factors are required in the replacement process of the D1 protein. (iii) Both in isolated chloroplasts and in thylakoids, the other PSII core proteins D2, CP43, and CP47 are also synthesized and released from the membrane-bound ribosomes, but incorporation into PSII complexes occurs to a much smaller extent than the D1 protein. Instead they accumulate predominantly as unassembled proteins in the thylakoid membrane. (iv) In chloroplasts, synthesis of the D1 protein seems to be adjusted according to the possibilities of incorporation into PSII complexes, while synthesis of the D2 protein, CP43, and CP47 is less regulated and their accumulation as unassembled protein in the membrane is abundant.