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Reversible phosphorylation of chl a/b protein complex II (LHCII), the mobile light-harvesting antenna, regulates its association and energy transfer/dissipation to photosystem (PS) II or I (state transition). Excitation of LHCII induces conformational changes affecting the exposure of the phosphorylation site at the N-terminal domain to protein kinase(s) [Zer, H., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282; Zer, H., et al. (2003) Biochemistry 42, 728-738]. Thus, it was of interest to examine whether the pigment composition of LHCII affects the light-induced modulation of LHCII phosphorylation and state transition. To this end, we have used thylakoids of wild-type Chlamydomonas reinhardtii and xanthophyll deficient mutants npq1, lor1, npq2, npq1 lor1, and npq2 lor1. Phosphorylated protein bands P11, P13, and P17 are considered components of the mobile C. reinhardtii LHCII complex. The protein composition of these bands has been analyzed by mass spectrometry using Qtof-2 with a nanospray attachment. P11 and P13 contain C. reinhardtii light-harvesting chlorophyll a/b binding protein LhcII type I. P17 contains C. reinhardtii LhcII types III and IV. Illumination of isolated thylakoids inhibits the redox-controlled phosphorylation of polypeptide bands P13 and P17 and to a lower extent that of P11. The light-induced inhibition of LHCII phosphorylation and the state transition process are not influenced by extensive differences in the xanthophyll composition of the mutants. Thus, LHCII can be visualized as possessing two functionally distinct, independent domains: (i) the pigment binding transmembrane domain regulating the extent of energy transfer/dissipation and (ii) the surface-exposed phosphorylation site regulating the association of LHCII with PSII or PSI.

The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII–light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) proteins, PsbP and PsbQ, were completely lacking in ΔpsbL. In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence measurements and by the lack of PSII–LHCII supercomplexes. The ΔpsbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In contrast, very young, vigorously expanding leaves of all psbEFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were present and correctly assembled in all psbEFLJ operon mutants.

Light-harvesting complex II (LHCII) prepared from isolated thylakoids of either broken or intact chloroplasts by three independent methods, exhibits proteolytic activity against LHCII. This activity is readily detectable upon incubation of these preparations at 37 degrees C (without addition of any chemicals or prior pre-treatment), and can be monitored either by the LHCII immunostain reduction on Western blots or by the Coomassie blue stain reduction in substrate-containing "activity gels". Upon SDS-sucrose density gradient ultracentrifugation of SDS-solubilized thylakoids, a method which succeeds in the separation of the pigment-protein complexes in their trimeric and monomeric forms, the protease activity copurifies with the LHCII trimer, its monomer exhibiting no activity. This LHCII trimer, apart from being "self-digested", also degrades the Photosystem II (PSII) core proteins (D1, D2) when added to an isolated PSII core protein preparation containing the D1/D2 heterodimer. Under our experimental conditions, 50% of LHCII or the D1, D2 proteins are degraded by the LHCII-protease complex within 30 min at 37 degrees C and specific degradation products are observed. The protease is light-inducible during chloroplast biogenesis, stable in low concentrations of SDS, activated by Mg(2+), and inhibited by Zn(2+), Cd(2+), EDTA and p-hydroxy-mercury benzoate (pOHMB), suggesting that it may belong to the cysteine family of proteases. Upon electrophoresis of the LHCII trimer on substrate-containing "activity gels" or normal Laemmli gels, the protease is released from the complex and runs in the upper part of the gel, above the LHCII trimer. A polypeptide of 140 kDa that exhibits proteolytic activity against LHCII, D1 and D2 has been identified as the protease. We believe that this membrane-bound protease is closely associated to the LHCII complex in vivo, as an LHCII-protease complex, its function being the regulation of the PSII unit assembly and/or adaptation.

The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochromeb 6 /f complex in chloroplasts, is retarded in the stromal space after import into the chloroplast and only slowly translocated further into the thylakoid membrane system. As shown by the sensitivity to nigericin and to specific competitor proteins, thylakoid transport takes place by the ΔpH-dependent TAT pathway. The Rieske protein is an untypical TAT substrate, however. It is only the second integral membrane protein shown to utilize this pathway, and it is the first authentic substrate without a cleavable signal peptide. Transport is instead mediated by the NH2-terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of ΔpH/TAT-dependent transport signals. Furthermore, transport is affected by sodium azide as well as by competitor proteins for the Sec pathway in chloroplasts, demonstrating for the first time some cross-talk of the two pathways. This might take place in the stroma where the Rieske protein accumulates after import in several complexes of high molecular mass, among which the cpn60 complex is the most prominent. These untypical features suggest that the Rieske protein represents an intermediate or early state in the evolution of the thylakoidal protein transport pathways.

Kinetic studies of protein dephosphorylation in photosynthetic thylakoid membranes revealed specifically accelerated dephosphorylation of photosystem II (PSII) core proteins at elevated temperatures. Raising the temperature from 22°C to 42°C resulted in a more than 10-fold increase in the dephosphorylation rates of the PSII reaction center proteins D1 and D2 and of the chlorophyll abinding protein CP43 in isolated spinach (Spinacia oleracea) thylakoids. In contrast the dephosphorylation rates of the light harvesting protein complex and the 9-kD protein of the PSII (PsbH) were accelerated only 2- to 3-fold. The use of a phospho-threonine antibody to measure in vivo phosphorylation levels in spinach leaves revealed a more than 20-fold acceleration in D1, D2, and CP43 dephosphorylation induced by abrupt elevation of temperature, but no increase in light harvesting protein complex dephosphorylation. This rapid dephosphorylation is catalyzed by a PSII-specific, intrinsic membrane protein phosphatase. Phosphatase assays, using intact thylakoids, solubilized membranes, and the isolated enzyme, revealed that the temperature-induced lateral migration of PSII to the stroma-exposed thylakoids only partially contributed to the rapid increase in the dephosphorylation rate. Significant activation of the phosphatase coincided with the temperature-induced release of TLP40 from the membrane into thylakoid lumen. TLP40 is a peptidyl-prolyl cis-trans isomerase, which acts as a regulatory subunit of the membrane phosphatase. Thus dissociation of TLP40 caused by an abrupt elevation in temperature and activation of the membrane protein phosphatase are suggested to trigger accelerated repair of photodamaged PSII and to operate as possible early signals initiating other heat shock responses in chloroplasts.

Light induces conformational changes in the CP43 chl-a-protein antenna complex in isolated PS II core-complexes exposing phosphorylation site(s) to PS II core-associated protein kinase(s), to added ...

Light-dependent activation of thylakoid protein phosphorylation regulates the energy distribution between photosystems I and II of oxygen-evolving photosynthetic eukaryotes as well as the turnover of photosystem II proteins. So far the only known effect of light on the phosphorylation process is the redox-dependent regulation of the membrane-bound protein kinase(s) activity via plastoquinol bound to the cytochrome bf complex and the redox state of thylakoid dithiols. By using a partially purified thylakoid protein kinase and isolated native chlorophyll (chl) a / b light-harvesting complex II (LHCII), as well as recombinant LHCII, we find that illumination of the chl-protein substrate exposes the phosphorylation site to the kinase. Light does not activate the phosphorylation of the LHCII apoprotein nor the recombinant pigment-reconstituted complex lacking the N-terminal domain that contains the phosphothreonine site. The suggested light-induced conformational change exposing the N-terminal domain of LHCII to the kinase is evidenced also by an increase in its accessibility to tryptic cleavage after light exposure. Light activates preferentially the trimeric form of LHCII, and the process is paralleled by chl fluorescence quenching. Both phenomena are slowly reversible in darkness. Light-induced exposure of the LHCII N-terminal domain to the endogenous protein kinase(s) and tryptic cleavage occurs also in thylakoid membranes. These results demonstrate that light may regulate thylakoid protein phosphorylation not only via the signal transduction chain connecting redox reactions to the protein kinase activation, but also by affecting the conformation of the chl-protein substrate.

Summary Subunits CFo-I and CFo-II of ATP synthase in chloroplast thylakoid membranes are two structurally and functionally closely related proteins of bitopic membrane topology which evolved from a common ancestral gene. In higher plants, CFo-I still originates in plastid chromosomes (gene:atpF), while the gene for CFo-II (atpG) was phylogenetically transferred to the nucleus. This gene transfer was accompanied by the reorganization of the topogenic signals and the mechanism of membrane insertion. CFo-I is capable of integrating correctly as the mature protein into the thylakoid membrane, whereas membrane insertion of CFo-II strictly depends on a hydrophobic targeting signal in the transit peptide. This requirement is caused by three negatively charged residues at the N-terminus of mature CFo-II which are lacking from CFo-I and which have apparently been added to the protein only after gene transfer has occurred. Accordingly, the CFo-II transit peptide is structurally and functionally equivalent to typical bipartite transit peptides, capable of also translocating hydrophilic lumenal proteins across the thylakoid membrane. In this case, transport takes place by the Sec-dependent pathway, despite the fact that membrane integration of CFo-II is a Sec-independent, and presumably spontaneous, process.

We describe two related manganese-binding polypeptides with L-arginine metabolizing enzyme activity that can be detected as distinct components (designated PsbY-A1 and PsbY-A2, previously called L-AME) in membranes containing Photosystem II (PS II) from spinach. The polypeptides are bitopic and appear to exist in a heterodimeric form, but only in the chlorophyll a/b lineage of plants. Both proteins are encoded in the nucleus. In spinach and in Arabidopsis thaliana they are both derived from a single-copy gene (psbY) that is translated into a precursor polyprotein of approximately 20 kDa. The processing of the polyprotein is complex and includes at least four cleavage steps. Both polypeptides are exposed N-terminally to the lumenal and C-terminally to the stromal face of the thylakoid membrane.

The CtpA protein in the cyanobacterium Synechocystis 6803 is a C-terminal processing protease that is essential for the assembly of the manganese cluster of the photosystem II complex. When fused to different chloroplast-targeting transit peptides, CtpA can be imported into isolated spinach chloroplasts and is subsequently translocated into the thylakoid lumen. Thylakoid transport is mediated by the cyanobacterial signal peptide which demonstrates that the protein transport machinery in thylakoid membranes is functionally conserved between chloroplasts and cyanobacteria. Transport of CtpA across spinach thylakoid membranes is affected by both nigericin and sodium azide indicating that the SecA protein and a transthylakoidal proton gradient are involved in this process. Saturation of the Sec-dependent thylakoid transport route by high concentrations of the precursor of the 33-kDa subunit of the oxygen-evolving system leads to a strongly reduced rate of thylakoid translocation of CtpA which demonstrates transport by the Sec pathway. However, thylakoid transport of CtpA is affected also by excess amounts of the 23-kDa subunit of the oxygen-evolving system, though to a lesser extent. This suggests that the cyanobacterial protein is capable of also interacing with components of the deltapH-dependent route and that transport of a protein across the thylakoid membrane may not always be restricted to a single pathway.

The topology of the Rieske protein of the cytochrome b6/f complex in thylakoids from spinach chloroplasts was examined by protease protection experiments as well as polypeptide extraction assays using solutions of chaotropic salts or alkaline pH. While neither thermolysin nor trypsin cleave any of the Rieske protein when added to the stromal side of the thylakoid membrane, proteinase K is capable of removing approximately four residues from its NH2-terminus. The protein is resistant to membrane extraction by 0.1 M Na2CO3 or 2 M NaBr but is quantitatively released by 0.1 M NaOH. Treatment of thylakoids with 2 M NaSCN leads to extraction of variable amounts of the protein, depending on the presence or absence of sucrose in the medium which apparently stabilizes the cytochrome complex. From these results we conclude that the Rieske protein is an integral component of the cytochrome complex which spans the thylakoid membrane with a single hydrophobic segment and is anchored predominantly by electrostatic interactions.

Four distinct integration/translocation routes into/across thylakoid membranes have recently been deduced for nuclear-encoded polypeptides of the photosynthetic membrane. Corresponding information for the plastid-encoded protein complement is lacking. We have investigated this aspect with in-organello assays employing chimeric constructs generated with codoncorrect cassettes for genes of plastid-encoded thylakoid proteins, and appropriate transit peptides from six nuclear genes, representing three targeting classes, as a strategy. The three major plastid-encoded components of the cytochrome b 6f complex, namely pre-apocytochrome f, (including apocytochrome f, and pre-apocytochrome f lacking the C-terminal transmembrane segment), cytochrome b6, and subunit IV, which differ in the number of their transmembrane segments, were studied. Import into chloroplasts could be observed in all instances but with relatively low efficiency. Thylakoid integration can occurr post-translationally, but only components with secretory/secretory pathway (SEC)-route-specific epitopes were correctly assembled with the cytochrome complex, or competed with this process. Inhibitor studies were consistent with these findings. Imported cytochrome b6 and subunit IV operated with uncleaved targeting signals for thylakoid integration. The corresponding determinant for cytochrome f is its signal peptide; its C-terminal hydrophobic segment did not, or did not appreciably, contribute to this process. The N-termini of cytochrome b6 and subunit IV appear to reside on the same (lumenal) side of the membrane, consistent with the currently favored four-helix model for the cytochrome, but in disagreement with the topography proposed for both components. The impact of the findings for protein routing, including for applied approaches such as compartment-alien transformation, is discussed.

The isolated OEC23 (oxygen evolving complex) protein of Photosystem II can be affinity-labeled by ATP and GTP in the presence of UV light. Labeling is competed by ATP, AMP, and NAD as well as by the ATP and ATP-γ-S and 5′-p-Fluorosulfonylbenzoyl-adenosine (FSBA) and is inhibited by Dithiothreitol (DTT). The physiological significance of this finding and the possible implication of ATP and GTP binding in thylakoid membrane dynamics remains to be further investigated.

The D1 reaction center protein of the Photosystem II complex in green plants is synthesized with a short carboxyl-terminal extension. Proteolytic cleavage and removal of this extension peptide in the thylakoid lumen are necessary for the assembly of a manganese cluster that is essential for the oxygen evolution activity of Photosystem II. We have isolated cDNAs encoding CtpA, the carboxyl-terminal processing protease for the D1 protein, from two higher plants, spinach and barley. In each of these organisms, CtpA is encoded by a single copy nuclear gene, and its steady-state mRNA levels are light-regulated. The CtpA protein is detectable in etiolated material, and its level increases approximately 5-fold upon illumination. Moreover, the CtpA gene is expressed in shoot tissues and not in roots. In its precursor form, the CtpA protein harbors a bipartite transit sequence characteristic for thylakoid lumenal proteins. Cell fractionation studies demonstrated that CtpA is associated with thylakoid membranes and is resistant to treatments with thermolysin, consistent with its localization in the lumen of thylakoids. Comparisons of the sequence of the higher plant CtpA enzyme with those of other related carboxyl-terminal processing proteases suggest that these proteins constitute a new family of proteases.

Summary Nuclear-encoded proteins are targeted into and across the thylakoid membrane by a surprising variety of mechanisms. Distinct Sec- and ΔpH-dependent mechanisms have been shown to operate for lumenal proteins, and an integral membrane protein, LHCP, has been shown to insert via a signal recognition particle-dependent route. Integration of a further membrane protein, CFoII, requires neither soluble factors nor energy sources, prompting speculation of a spontaneous insertion mechanism. Although the requirements for soluble factors and energy sources have been determined in some detail, much less is known of the events taking place at the membrane surface. This report examines whether thylakoid proteins are involved in each of these pathways, by testing the effects of trypsin on the capacity of isolated thylakoids to import proteins. Because all of the pathways rely to some extent on the thylakoidal ΔpH, and a light-induced ΔpH is easily destroyed by proteolysis, the conditions under which reverse action of the ATP synthase in the dark generates a high ΔpH even after proteolysis of thylakoids have been established. This system is used to show that protease-sensitive thylakoidal import machinery is crucial for the ΔpH-, Sec- and signal recognition particle-dependent pathways, but not for integration of CFoII.

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 PQH2/PQ ratio prevailing in membranes of the plastocyanin-deficient mutant Ac208. 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.

A Sec-type system is responsible for the translocation of a subset of proteins across the thylakoid membrane in higher plant chloroplasts. Previous studies have suggested that the thylakoidal ΔpH plays a minor role in this translocation mechanism, but we show here that it can be essential for the translocation process, depending on the identity of the passenger protein and the concentration of ATP. Studies using chimeric proteins show that, whereas the presequence dictates the translocation pathway, the ΔpH requirement is dictated exclusively by the passenger protein; some passenger proteins are virtually ΔpH-independent whereas others are absolutely dependent. ΔpH requirement is not related to charge characteristics of the passenger proteins, ruling out an electrophoretic effect. Analysis of the 33-kDa photosystem II protein reveals an inverse relationship between ΔpH requirement and ATP concentration; import into isolated thylakoids is inhibited 14-fold by nigericin at moderate ATP concentrations, and totally inhibited when the ATP concentration is reduced to 2 μM. The results indicate that the roles of the ΔpH and ATP overlap and suggest that the ΔpH may be obligatory when the passenger protein is abnormally difficult to translocate, possibly due to the folding of the polypeptide chain. We compare the energetics of this system with those of prokaryotic systems from which the chloroplast system is believed to have evolved.

Thylakoid membranes of chloroplasts in higher plants harbor different pathways for the translocation of proteins. One of these routes is related to the prokaryotic Sec pathway, which mediates the secretion of particular proteins into the periplasmic space and involves the SecA protein as an essential component. We have isolated a full size cDNA of 3739 nucleotides encoding the SecA homologue from spinach. It contains an open reading frame of 1036 codons corresponding to a polypeptide with a calculated mass of 117 kDa. The deduced amino acid sequence shows between 43 and 49% identity to SecA proteins from bacteria and lower algae and 62% identity to SecA of the cyanobacterium Synechococcus sp. PCC7942. Compared with the Escherichia coli protein, spinach SecA carries an amino-terminal extension of approximately 80 residues. In organello experiments performed with the protein made in vitro by transcription of the cDNA and cell-free translation of the resulting RNA showed that this extension comprises a transit peptide that mediates the import of the protein into the chloroplast. The processed product of approximately 107 kDa accumulates predominantly in the stroma and to a lower extent associates with the thylakoid membrane. Comparably to E. coli, in which SecA activity can be inhibited by sodium azide, thylakoid translocation of a subset of lumenal proteins is sensitive to sodium azide in pea but not in spinach chloroplasts, suggesting that the latter contain an azide-resistant SecA variant.

Subunit 3 of photosystem I (PSI-3), the product of the nuclear psaF gene, is the docking protein for plastocyanin during photosynthetic electron transport in thylakoid membranes and is synthesized in the cytosol with a transit peptide that resembles structurally the bipartite targeting signals of hydrophilic, lumenal components such as plastocyanin. In organello import experiments performed with the authentic PSI-3 precursor and chimeric polypeptides consisting of residue-correct fusions of transit peptides and mature proteins derived from different plastid proteins demonstrate that the PSI-3 transit peptide is indeed capable of translocating proteins into the thylakoid lumen and that, conversely, mature PSI-3 depends on a bipartite transit peptide for its thylakoid transfer. Of the three recently described translocation/integration pathways for nucleus-encoded proteins carrying bipartite transit peptides that are distinct in their physiological requirements and strictly protein-specific, PSI-3, like plastocyanin and the 33-kDa protein of the oxygen-evolving complex, is translocated by a pathway that involves stromal factors but no proton gradient across the membrane. It is not affected by saturating amounts of the precursor for the 23-kDa protein of the oxygen-evolving complex that follows the latter route. Thylakoid translocation of PSI-3 is, however, impaired in the presence of sodium azide, which indicates that a homolog to the bacterial SecA protein might be involved in this process suggesting, thus, a prokaryote-like translocation pathway. The azide-sensitive factor appears to interact predominantly with the transit peptide of a precursor protein, since chimeras consisting of a presequence from an azide-resistant precursor and a mature part of an azide-sensitive polypeptide are still translocated in the presence of the inhibitor.

The translocation of plastocyanin across the thylakoid membrane in Pisum sativum has been studied in reconstitution assays and using chimeric constructs. The reconstitution assays demonstrate that plastocyanin translocation is absolutely dependent on the presence of a stromal factor(s) and nucleotide triphosphates (NTPs), whereas neither element is required for the translocation of the 23 or 16 kDa proteins of the oxygen-evolving complex. Previous studies had revealed that the transthylakoidal delta pH is essential for translocation of the 23 and 16 kDa proteins but unnecessary for plastocyanin translocation. The basis for these mechanistic differences has been tested by analysing the translocation of a chimeric construct consisting of the presequence of the 23 kDa protein linked to the mature plastocyanin sequence. This construct is efficiently imported into thylakoids in the absence of stromal extracts or NTPs and translocation across the thylakoid membrane within intact chloroplasts is totally inhibited by the uncoupler nigericin: the translocation requirements are thus identical to those of the pre-23 kDa protein and diametrically opposite to those of pre-plastocyanin. Transport across the thylakoid membrane of a second fusion protein, consisting of the presequence of the 16 kDa protein linked to mature plastocyanin, is also dependent on a delta pH. The data suggest that two distinct systems are involved in the translocation of proteins across the thylakoid membrane, with each system recognizing specific signals within the presequences of a subset of lumenal protein precursors.

Plastocyanin and the 16-, 23-, and 33-kDa polypeptides of the oxygen-evolving complex associated with photosystem II are hydrophilic, nuclear encoded components of the photosynthetic machinery that are all located in the lumen of thylakoid membranes. All four proteins are therefore imported into chloroplasts and, in addition, translocated across the thylakoid membrane. They share functionally equivalent, bipartite transit peptides, which are removed in two steps during or after import into the organelle and translocation across the thylakoid membrane, respectively. The transit peptides lack any homology at the sequence level but possess remarkably similar predicted secondary structures. We have studied the targeting potential of the authentic precursor molecules and all possible chimeric combinations generated by a specific, commonly applicable cassette system, which facilitates codon-correct reciprocal exchanges of transit peptides and mature parts. An unexpected specificity of import and sorting processes was found. All constructs can be imported into the organelle, though with greatly differing efficiency. On the other hand, the lumen-targeting parts are essential but not in all cases sufficient for correct intraorganellar routing. This implies that translocation across the thylakoid membrane appears not to depend merely on simple interactions of charged or hydrophobic regions between protein and membrane but requires an additional quality of information that includes the functional co-evolution of a transit peptide with its mature protein. Signaling and sorting appear to be essential at almost every step of the entire process for proper traffic regulation since distinct steps can be impaired (rate-limiting or arrested) in the individual combinations: the transfer across the envelope membranes (e.g. 16/PC) and the interaction with (e.g. 16/33) or the translocation across the thylakoid membrane (e.g. 33/23).

Summary The 20 kDa polypeptide, the apoprotein of the chlorophyll a/b antenna complex CP24 associated with photosystem II, is a remote relative of light-harvesting complex (LHC) apoproteins and thus a member of the extended cab gene family. LHC apoproteins are poly-topic integral components of the thylakoid membrane with probably three transmembrane segments which originate in nuclear genes and are made in the cytosol as precursors. They possess exclusively stroma-targeting transit peptides for import into the organelle and integrate into the thylakoid membrane via uncleaved hydrophobic domains of the mature protein. The CP24 apoprotein displays intriguing structural differences to LHC apoproteins with a potential impact on the routing and targeting processes during biogenesis. In particular, it lacks a pronounced second hydrophobic segment in the mature polypeptide chain found in LHCPs, and carries a transit peptide that is reminiscent of thylakoid-targeting transit peptides. We have used in organello assays with isolated intact chloroplasts and the authentic precursor of the 20 kDa apoprotein from spinach, or appropriate chimaeric polypeptides consisting of a transit peptide and the mature part of various nuclear-encoded thylakoid proteins of known location and targeting epitopes, in order to resolve the characteristics of its targeting properties, as well as to determine the contribution of the individual parts of the precursor molecule to its import and subsequent intra-organellar routing. Our experiments demonstrate that the transit peptide of the CP24 apoprotein is required only for the import of the protein into the organelle. All subsequent steps, such as the integration of the protein into the thylakoid membrane, binding of chlorophyll, assembly into the CP24 complex and migration to the grana lamellae, still take place if the authentic transit peptide is replaced by a targeting signal of a nuclear-encoded stromal protein.

The biogenesis of the lumenal 16 kDa protein of the photosynthetic oxygen-evolving complex was analysed using an assay for the import of proteins by isolated thylakoids. The precursor protein is imported with high efficiency in the light in both the presence and absence of stromal extract. Import is almost completely blocked in the dark or if the uncoupler nigericin is present in the light. The data indicate that transport across the thylakoid membrane is driven by a proton motive force in which the proton gradient is the dominant component, and that the full precursor protein can be transported across the thylakoid membrane without prior cleavage by the stromal processing peptidase.

The targeting of cytosolically synthesized proteins into the thylakoid lumen is mediated by an aminoterminal pre-sequence consisting of an "envelope transit" and a "thylakoid transfer" signal in tandem. We have investigated the structural characteristics of several thylakoid transfer signals by determining the intermediate sites at which the stromal processing peptidase cleaves to remove the transit sequences. Using this approach we have found that the thylakoid transfer signals of Silene pratensis plastocyanin, 23-kDa oxygen-evolving complex protein from wheat, and 33-kDa oxygen-evolving complex protein from wheat, are 25, 39, and 48 residues in length, respectively. All of the transfer signals contain hydrophobic core sequences and a "-3,-1" motif reminiscent of those found in signal sequences, but the amino-terminal regions of the transfer signals of the 23- and 33-kDa proteins are both longer and more highly charged. The net charge of each amino-terminal region of the transfer sequences is +1, including the amino-terminal amino group. In each case, the stromal processing peptidase cleaves immediately after a positively charged residue, but otherwise the cleavage sites exhibit no common elements of either primary or secondary structure.

We have isolated recombinant lambda gt11 phages which carry cDNA clones for the major light-harvesting chlorophyll a/b -binding proteins of photosystem I (LHCPI) and II (LHCPII), subunit II of photosystem I, a chlorophyll a/b -binding protein of photosystem II (CP24), the Rieske iron—sulphur protein of the cytochrome b 6 / f complex, and the 33, 23 and 16 kDa proteins of the water-oxidizing complex of photosystem II from Nicotiana tabacum . The nucleotide sequences of cDNA clones encoding the precursors for LHCPI and the FeS protein are presented. If tobacco or tomato seedlings, or seedlings of a phytochrome-deficient aurea mutant of tomato which lacks more than 95% of the phytochrome of the isogenic wild type, are kept in blue light, the transcript level of each of these genes is higher than in seedlings grown in red light suggesting the involvement of a blue—UVA-light photoreceptor. In the case of LHCPI, a 1 min blue-light pulse applied to red-light-grown seedlings is sufficient to increase the transcript levels to those present in blue-light-grown seedlings, whereas almost no increase is observed for transcripts encoding the FeS and 33 kDa proteins. If dark-grown tomato seedlings receive a single far-red-light pulse, significant stimulation is detected for LHCPI transcripts, whereas transcripts encoding the FeS and 33 kDa proteins are not stimulated. It is concluded that the lower light requirement for the increase in the LHCPI transcript level is not specific for one of the light-dependent signal transduction chains.

Thylakoid membrane proteins are phosphorylated by different enzymes, which are subject to different control mechanisms. Activation of the light harvesting complex (LHCII) kinase is signaled by the redox state of plastoquinone and the cytochrome b/f complex and modulated by the thiol reduction state. Phosphorylation of Photosystem II (PS II) proteins may involve kinase(s) associated with the PS II core complex that do not involve the cytochrome b/f complex. Exposure of the phosphoprotein phosphorylation site(s) to protein kinases is regulated by light-induced conformational changes. Thus, thylakoid protein phosphorylation is regulated at both the enzyme and substrate levels. Thylakoid protein dephosphorylation is also under regulatory control, involving interaction between an immunophilin and a membrane-bound phosphatase. The physiological significance of thylakoid protein phosphorylation is not fully understood. Phosphorylation of LHCII is suggested to have a dual role: i) regulation of the LHCII/PSII/PS I interaction, underlying the mechanism of energy transfer balance and ii) prevention of the light-induced aggregation of LHCII or LHCII-PS II complexes. The formation of such macrodomains may affect the dynamics of the thylakoid membrane, which requires unhindered lateral diffusion of integral protein complexes. Phosphorylation of PS II subunits appear to be essential for the repair of photodamage to its reaction center occurring during light stress conditions.

Biogenesis, maintenance, and adaptation of the thylakoid system in photosynthetic organelles are embodied in the genetic machinery of the plant cell. Plant cells are descendants of endosymbioses. Their genomes are compartmentalized and the result of a complex restructurating of the genetic potentials of (depending on the organism) three to five symbiotic partner cells during evolution that led to a common metabolism and a common inheritance in the entity. It appears that much of the thylakoid biogenesis and dynamics can be understood from the history of the plant cell. Inter-endosymbiotic genome restructuration included loss, gain, and intracellular transfer of genetic material. It was accompanied (i) by the generation of an integrated genetic system (rather than a nucleus and semiautonomous organelles) that in its entirety is spatiotemporally regulated, (ii) by a massive intermixing of structural genes, (iii) a fundamental change in expression signals in the entire system which included the evolution of an exquisite set of regulatory mechanisms that operate in concert with basically ancient regulatory circuits originating in the organelle ancestors, and (iv) by the establishment of nuclear regulatory dominance that is found at all levels of regulation. Most conspicuous are fundamental changes of the transcription machinery in all three subgenomes, in the establishment of an elaborate posttranscriptional RNA modification system in chloroplast that prokaryotes are largely lacking, and in sophisticated protein trafficking and assembly devices. The genetics of the photosynthetic machinery was fitted into the respective genetic programs for multicellular and terrestrial plants that developed new biochemistries including those for a spatiotemporal morphogenetic potential.

We describe the identification of a novel chloroplast protein, designated TCP34 (tetratricopeptide-containing chloroplast protein of 34 kDa) due to the presence of three tandemly arranged tetratricopeptide repeat (TPR) arrays. The presence of the genes encoding this protein only in the genomes of higher plants but not in photosynthetic cyanobacterial prokaryotes suggests that TCP34 evolved after the separation of the higher plant lineage. The in vitro translated precursor could be imported into intact spinach chloroplasts and the processed products showed stable association with thylakoid membranes. Using a specific polyclonal antiserum raised against TCP34, three protein variants were detected. Two forms, T(1) and T(2), were associated with the thylakoid membranes and one, S(1), was found released in the stroma. TCP34 protein was not present in etioplasts and appeared only in developing chloroplasts. The ratio of membrane-bound and soluble forms was maximal at the onset of photosynthesis. The high molecular mass thylakoid TCP34 variant was found in association with a transcriptionally active protein/DNA complex (TAC) from chloroplasts and recombinant TCP34 showed specific binding to Spinacia oleracea chloroplast DNA. Two TCP34 forms, T(1) and S(1), were found to be phosphorylated. An as yet unidentified phosphorelay signal may modulate its capability for plastid DNA binding through the phosphorylation state of the putative response regulator-like domain. Based on the structural properties and biochemical analyses, we discuss the putative regulatory function of TCP34 in plastid gene expression.

Redox-controlled, reversible phosphorylation of the thylakoid light harvesting complex II (LHCII) regulates its association with photosystems (PS) I or II and thus, energy distribution between the two photosystems (state transition). Illumination of solubilized LHCII enhances exposure of the phosphorylation site at its N-terminal domain to protein kinase(s) and tryptic cleavage in vitro [Zer et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282]. Here we report that short illumination (5-10 min, 15-30 micromol m(-2) s(-1)) enhances the accessibility of LHCII phosphorylation site to kinase(s) activity also in isolated thylakoids. However, prolonged illumination or higher light intensities (30 min, 80-800 micromol m(-2) s(-1)) prevent phosphorylation of LHCII in the isolated membranes as well as in vivo, although redox-dependent protein kinase activity persists in the illuminated thylakoids toward exogenous solubilized LHCII. This phenomenon, ascribed to light-induced inaccessibility of the phosphorylation site to the protein kinase(s), affects in a similar way the accessibility of thylakoid LHCII N-terminal domain to tryptic cleavage. The illumination effect is not redox related, decreases linearly with temperature from 25 to 5 degrees C and may be ascribed to light-induced conformational changes in the complex causing lateral aggregation of dephosphorylated LHCII bound to and/or dissociated from PSII. The later state occurs under conditions allowing turnover of the phospho-LHCII phosphate. The light-induced inaccessibility of LHCII to the membrane-bound protein kinase reverses readily in darkness only if induced under LHCII-phosphate turnover conditions. Thus, phosphorylation prevents irreversible light-induced conformational changes in LHCII allowing lateral migration of the complex and the related state transition process.

Chloroplast subfractions were monitored for sodium dodecyl sulfate-stable proteases. Nine distinct activities in the molecular mass range from 14 to 66 kD have been detected. Five of the proteases associated with thylakoid membranes belong to the serine and cysteine types of proteases. These activities could be preserved and purified by a two-step electrophoresis procedure.

Landsberg erecta x Columbia F8 recombinant inbred lines of Arabidopsis thaliana, arrayed BAC clones covering most of the genome, and databank sequence information were used to map the positions of 69 genes in the genome of A. thaliana. These genes encode all known constituents of the photosynthetic thylakoid membrane, some regulatory factors involved in its biogenesis, and the RNA polymerases of nuclear origin that operate in chloroplasts and mitochondria. Designations of novel genes are proposed. The data of these three approaches are generally consistent, although ambiguities have been noted for some genome segments and with gene duplications. For thylakoid multi-subunit structures, no positional clustering of genes has been found, not even for genes encoding different subunits of the same membrane complex. The genes of the lhc superfamily encoding antenna apoproteins and their relatives are a particularly intriguing example. The lack of positional clustering is consistent with phylogenetically independent gene translocations from the plastid (endosymbiont) to the nucleus. This raises the basic question of how independently translocated genes which acquired different promoter sequences and transit peptides were functionally integrated into common signal transduction chains.

Photosynthesis, the most fundamental biochemical process for maintenance of life and the only renewable energy source of our planet, is a highly regulated, but also one of the most light- and heat-sensitive processes in plants and cyanobacteria (Berry and Bjorkman 1980, Weis and Berry 1988, Havaux and Tardy 1996). Light, obviously, is not only useful, but can also cause problems, and even exert harm to a plant. In their natural habitat, plants are exposed to light intensity and quality changes that can lead to an imbalance between the excitation rates of the two photosystems, and thus diminish photosynthetic efficiency. Light can also induce photoinactivation of electron transport and damage both photosystems (Prasil et al 1992, Aro et al 1993, Sonoike 1995). Comparably, high temperatures dramatically inhibit carbon dioxide fixation (Berry and Bjorkman 1980, Feller et al 1998), and the heat tolerance limits of leaves, which possess surprisingly little heat capacity, can depend on the thermal sensitivity of photochemical reactions in thylakoid membranes (Berry and Bjorkman 1980, Weis and Berry 1988, Havaux and Tardy 1996). Even short exposure to increasing temperatures in the range of 40°C can cause a progressive destacking of stacked thylakoids (Gounaris et al 1984), a lateral redistribution of the membrane protein complexes (Sundby et al 1986) that constitute this membrane (Herrmann et al 1991), an increase in the ionic permeability and fluidity of the membrane (Havaux et al 1996, Tardy and Havaux 1997), rapid dephosphorylation and degradation of the photosystem II core protein Dl (Rokka et al 2000), and a release of extrinsic proteins of the photosystem II oxygen-evolving complex (Enami et al 1994).

Dephosphorylation of central photosynthetic proteins regulates their turnover in plant thylakoid membranes. A membrane protein phosphatase from spinach thylakoids was purified 13000-fold using detergent-engaged FPLC. The purified enzyme exhibited characteristics typical of eukaryotic Ser/Thr phosphatases of the PP2A family in that it was inhibited by okadaic acid (IC(50) = 0.4 nM) and tautomycin (IC(50) = 25 nM), irreversibly bound to microcystin-agarose, and recognized by a polyclonal antibody raised against a recombinant catalytic subunit of human PP2A. Furthermore, the anti-PP2A antibody inhibited protein dephosphorylation in isolated thylakoids. The phosphatase copurified with TLP40, a cyclophilin-like peptidyl-prolyl isomerase located in the thylakoid lumen. TLP40 could be released from the phosphatase immobilized on microcystin-agarose by high-salt treatment. Binding of cyclosporin A (CsA) to TLP40 led to thylakoid phosphatase activation, while cyclophilin substrates, prolyl-containing oligopeptides, inhibited protein dephosphorylation. This dephosphorylation could be modulated by CsA or oligopeptides only after the thylakoids had been ruptured to expose the lumenal membrane surface where the TLP40 is located. Regulation of the PP2A-like phosphatase at the outer thylakoid surface is likely to operate via reversible binding of TLP40 to the inner membrane surface. This is a first example of transmembrane regulation in which the activity of phosphatase is altered by the binding of a cyclophilin to a site other than the active one. We propose that signaling from TLP40 to the protein phosphatase coordinates dephosphorylation and protein folding, two processes required for protein turnover during the repair of photoinhibited photosystem II reaction centers.

Summary The plastid encoded RNA polymerase subunit genes rpoA, B and C1 of tobacco were disrupted individually by PEG-mediated plastid transformation. The resulting off-white mutant phenotype is identical for inactivation of the different genes. The mutants pass through a normal ontogenetic cycle including flower formation and production of fertile seeds. Their plastids reveal a poorly developed internal membrane system consisting of large vesicles and, occasionally, flattened membranes, reminiscent of stacked thylakoids. The rpo–material is capable of synthesising pigments and lipids, similar in composition but at lower amounts than the wild-type. Western analysis demonstrates that plastids contain nuclear-coded stroma and thylakoid polypeptides including terminally processed lumenal components of the Sec but not of the ΔpH thylakoid translocation machineries. Components using the latter route accumulate as intermediates. In striking contrast, polypeptides involved in photosynthesis encoded by plastid genes could not be detected by Western analysis, although transcription of plastid genes, including the rrn operon, by the plastid RNA polymerase of nuclear origin is found as expected. Remarkably, ultrastructural, sedimentation and Northern analyses as well as pulse experiments suggest that rpo–plastids contain functional ribosomes. The detection of the plastid-encoded ribosomal protein Rpl2 is consistent with these results. The findings demonstrate that the consequences of rpo gene disruption, and implicitly the integration of the two plastid polymerase types into the entire cellular context, are considerably more complex than presently assumed.

We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single‐copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin‐like C‐terminal segment of 20 kDa, a predicted N‐terminal leucine zipper and a potential phosphatase‐binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non‐appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl‐prolyl cis–trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light‐mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated.

A unique protein phosphorylation system regulated by light is present in the chloroplast thylakoid membrane (1,2). The light-induced phosphorylation occurs at the outer membrane surface on Thr residues of chlorophyll a/b-binding proteins and several subunits of photosystem II including the reaction centre proteins D1 and D2. This reversible phosphorylation regulates the balance of excitation energy between the two photosystems as well as the degradation and turnover of the central functional proteins of photosystem 11 (3). Very little is known about the enzymology of this reversible protein phosphorylation, in particular with respect to the control of thylakoid protein phosphatase activity. The enzymes involved is highly specific towards thylakoid phosphoproteins or mimicking phosphopeptides (4) and was proposed to be unrelated to conventional Ser/Thr phosphatases because of the failure to obtain inhibition by okadaic acid or microcystin in membrano experiments (5,6). In this work we demonstrate that upon purification, the intrinsic thylakoid membrane protein phosphatase reveals the properties characteristic of conventional eukaryotic protein phosphatases and that it is associated with and regulated by a recently discovered cyclophilin-like thylakoid lumenal prolyl isomerase of 40 kDa (TLP40) (7).

The main emphasis in photosynthesis research has so far been devoted to structural and functional aspects of electron/proton transport and light-harvesting. The progress has been significant, and the mechanism of photosynthetic energy conversion and the participating protein complexes can now be described at a very refined molecular level. In contrast, very little is known about the regulatory processes of photosynthesis, which maintain an optimal photosynthetic efficiency during fluctuating and stressful environmental conditions to the plant. The characterization of such regulatory or auxiliary processes, including the responsible enzymology and signal transduction mechanisms will be crucial topics for future photosynthesis research. Examples of such auxilary components or enzymes; are processing enzymes and chaperones involved in biogenesis of photosynthetic complexes [1], enzymes responsible for protein acylation [2] and isoprenylation [3], desaturases controlling membrane lipid composition and dynamics [4], stress induced proteins such as the ELIPs [5], as well as several enzymatic activities involved in various forms of regulatory proteolysis [6,7]. Protein phosphorylation is of importance for regulation of gene-expression and metabolic control and there is an increasing interest in thylakoid kinases and phosphatases [8].

The distribution of light energy between the two photosystems as well as the light-induced turnover of PSII proteins are regulated by the reversible phosphorylation of LHCII and the PSII-core proteins. The thylakoid protein kinase(s) is activated by a signal transduction system involving the interaction of reduced plastoquinone with the quinol oxidation site of the cytochrome bf complex [1]. Phosphorylation of the mobile pool of LHCII induces dissociation of this antenna from PSII and allows its interaction with the PSI in the stroma exposed membranes (state transition)[21. Dephosphorylation of LHCII by a membrane -bound phosphatase appears to be regulated by a cyclophilinlike protein located in the thylakoid lumen [1, 31. So far, it was assumed that the role of light in the above processes is only to drive the photosynthetic electron flow thus reducing plastoquinone and triggering the signal transduction system resulting in the protein kinase(s) activation. In this work we have used an in vitro reconstituted system consisting of partially purified, solubilized thylakoid protein-kinase(s) and isolated native as well as recombinant LHCII. We demonstrate that light induces conformational changes in the LHCII structure exposing the specific threonine phosphorylation site to the kinase activity, thus suggesting a regulation of protein phosphorylation also at the substrate level.

Reversible protein phosphorylation on serine and threonine residues plays a crucial role for the regulation of numerous cellular functions and signal transduction pathways. Control of this process is achieved by the modulation of the activities and substrate specificities of corresponding protein kinases and protein phosphatases. In thylakoids, a substantial number of proteins can be reversibly phosphorylated [I], and an intrinsic, redox-controlled kinase/phosphatase system is involved to adjust the energy distribution between the photosystems I and II [2]. Transient changes in light intensity and quality lead frequently to an imbalance of the excitation rates of the two photosystems which can result in a reduced photosynthetic efficiency. Plants harboring chlorophyll a/b antenna systems respond to this imbalance by modulating the association and energy transfer between the major light-harvesting antenna (LHCII) and the two photosystems via phosphorylation/dephosphorylation of LHCPs. This „state transition“ process is redox-controlled and activated by changes in the redox state of the thylakoid plastoquinone pool [3]. The exact mechanism of the phosphorylation-related reversible energy distribution of the LHCII antenna between the photosystems is not yet well understood. The signal transduction system appears to include a redox-controlled protein kinase of approx. 65 kDa molecular mass, the cytochrome b6f complex, induced conformational substrate (LHCII) changes (Zer et al., these Proceedings), and an immunophilin of the complex type (TLP40) located in the thylakoid lumen that appears to interact with the protein dephosphorylation signal transduction chain and to link it with protein degradation [4]. Attempts to isolate thylakoid-located kinase(s) and their cDNAs or genes have not been successful so far.

Two subunits of the fight-harvesting complex II (LHCII) and four of the photosystem II (PSII): the D1 and D2 core proteins, CP43 and a 9 kDa polypeptide constitute the principal phosphoproteins of the thylakoid membrane. Protein phosphorylation ensures the energy balance between the two photosystems [1] and regulates protein turnover during the repair of photoinhibitory damage to PSII [2,3]. Both thylakoid-bound [4–6] and soluble [7] protein phosphatases are present in chloroplasts, but only little is known about their specificity, location and regulation. In the present study we show that elevated temperature stimulates the thylakoid-bound protein phosphatase which dephosphorylates the reaction centre proteins D1 and D2. This activation is paralleled by a dissociation of the thylakoid cyclophilin TLP40 (thylakoid lumen PPIase of 40 kDa) [8, see also Andersson et al.; Vener et al. these Proceedings] from the inner thylakoid face, a possible regulator of thylakoid dephosphorylation.

Thylakoid membranes are specialized and highly versatile biomembranes, capable of converting solar energy to utilizable high-energy chemical metabolites. The invention of biological photosynthesis, which occurs in various forms in pro- and eukaryotes, ensured that life could survive; photosynthesis also provides the only renewable energy source of our planet. Thylakoid membranes are lipid bilayers into which several supramolecular protein complexes are intercalated in a vectorially oriented way. In oxygenic photosynthesis, they cooperate in the conversion of radiant energy together with mobile electron carriers in the stroma (or cytoplasm), lumen, and in the lipid bilayer.

Thylakoid membranes of chloroplasts are composed of approx. 75 polypeptide species. Nearly 60% originate in nuclear genes, the remainder in plastid genes. In order to localize representatives of the nuclear-encoded gene complement in a eukaryotic plant genome (sugar beet, Beta vulgaris L.), we have investigated the RFLP patterns of 21 cDNAs from spinach that code for thylakoid proteins or proteins peripherally associated with thylakoid membranes. Differences in gene dosage were noted between both related species. Polymorphism was found for 12 cDNA loci in a segregating sugar beet F2 population. These loci were mapped along with genomic RFLP, isozyme, and morphological markers, and shown to be distributed in six of the nine sugar beet linkage groups. The lack of positional clustering even of genes that encode components of the same supramolecular membrane assembly is commensurate with phylogenetically independent gene translocations from the plastid (endosymbiont), and raises the question of the functional integration of various translocated genes into common signal transduction chains.

We describe the isolation and characterization of cDNAs encoding the precursor polypeptide of the 6.1-kDa polypeptide associated with the reaction center core of the photosystem II complex from spinach. PsbW, the gene encoding this polypeptide, is present in a single copy per haploid genome. The mature polypeptide with 54 amino acid residues is characterized by a hydrophobic transmembrane segment, and, although an intrinsic membrane protein, it carries a bipartite transit peptide of 83 amino acid residues which directs the N terminus of the mature protein into the chloroplast lumen. Thylakoid integration of this polypeptide does not require a delta pH across the membrane, nor is it azide-sensitive, suggesting that the polypeptide chain inserts spontaneously in an as yet unknown way. The PsbW mRNA levels are light regulated. Similar to cytochrome b559 and PsbS, but different from the chlorophyll-complexing polypeptides D1, D2, CP43, and CP47 of photosystem II, PsbW is present in etiolated spinach seedlings.

The delta pH-driven and Sec-related thylakoidal protein translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH-dependent system contain a common twin-arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH-driven translocation process; substitution of the arg-arg by gln-gln or even arg-lys totally blocks translocation across the thylakoid membrane, and replacement by lys-arg reduces the rate of translocation by > 100-fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin-arg motif into a Sec-dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec-type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane protein does not affect the translocation pathway.

Steady-state mRNA levels for thylakoid proteins were analysed in spinach cotyledons under diurnally changing light conditions. Most fluctuate considerably throughout the day, while the levels of others show only low amplitude or no oscillation. Levels of mRNAs coding for proteins that belong to the same multiprotein complex generally oscillate in parallel and exhibit maxima that are specific for that complex: mRNAs for photosystem I proteins appear prior to those for photosystem II polypeptides and these again prior to mRNAs for the three polypeptides constituting the oxygen-evolving complex. For the mRNAs that change with high amplitudes (e.g. those for LHCP or the 20 kDa apoprotein of the CP24 complex) oscillations have also been found under constant conditions, indicating that a circadian oscillator is involved. Transgenic tobacco seedlings harbouring chimeric GUS gene fusions with 5'-flanking sequences from the spinach genes Lhcb, PsaF and AtpD (encoding a light-harvesting chlorophyll a/b apoprotein of photosystem II, subunit 3 of photosystem I and subunit delta of the plastid ATP synthase, respectively) confirm that the differences in the amplitudes as well as the timepoints of maximum mRNA accumulation are perceived via cis-regulatory elements upstream of the respective ATG codons.

Phosphorylation of chlorophyll a/b-binding proteins of the of photosystem II light-harvesting assembly controls the energy distribution between the two photosystems as well as the turnover of thylakoid membrane proteins. The LHCII kinase, suggested to be a 64 kDa protein, is light-regulated by a mechanism involving reduction of plastoquinone and the participation of the cytochrome b6/f complex. A cDNA encoding that protein has been isolated from a lambda gt11-based library made from spinach polyadenylated RNA using a two-step strategy involving screening by polyclonal monospecific antisera and plaque hybridization. The protein of 73.1 kDa molecular mass represents a precursor which contains a bipartite transit peptide of 101 amino acid residues (11.0 kDa) that directs the protein into the thylakoid lumen. It can be phosphorylated in vitro, and exhibits significant homology to plant polyphenol oxidases, not to kinases. The gene was therefore designated PpoA. Reinvestigation of components in the molecular mass range of 50–70 kDa disclosed five additional proteins which can accompany kinase-active cytochrome b6/f, photosystem II and AMS [1] preparations. Four of them can be phosphorylated in vitro; two with apparent molecular masses of 53 and 66 kDa are capable of phosphorylation, and represent new, yet unidentified proteins.

Etiolated lupine (Lupinus luteus L.) cotyledons respond in a highly sensitive manner to phytohormones and light. The effects of cytokinin, abscisic acid, gibberellic acid (GA3) and indolylacetic acid (IAA) have been studied at the ultrastructural, steady-state mRNA and protein levels using 15 gene-specific probes for plastid proteins and corresponding antisera. No effect was noted with GA3 and IAA. As in other systems, N6-benzylaminopurine (BAP) and abscisic acid (ABA) operated antagonistically. In both instances, the steady-state mRNA levels remained relatively unaffected for plastid-encoded polypeptides, but not for those nuclear-encoded genes that could be tested. On the other hand, synthesis and accumulation of proteins of nuclear and plastid origin varied significantly. Cytokinin strongly promoted the accumulation of cytochrome b 559 and subunit IV of the cytochrome b/f complex, while little effect was observed for cytochrome b 6, the β subunit of the chloroplast ATP synthase or the large subunit of ribulose-1,5-bisphosphate carboxylase. In etiolated seedlings the level of chlorophyll-binding proteins (the 43-kDa chlorophyll a protein of photosystem II and subunits I a, b of photosystem I) was below the level of detectability. Their accumulation in light was promoted by cytokinin and inhibited by ABA though to different extents. Cytochrome b 559 and the 33-kDa polypeptide of the water-oxidizing complex were not detectable in water-(control) and ABA-treated cotyledons. Cytokinin induced the synthesis of these proteins, even in darkness. These results indicate a protein-specific response to phytohormones, which can differ even for polypeptides belonging to the same membrane complex. They also suggest different modes of interaction between hormones and light, quite different phytohormone action in the two compartments, and demonstrate that phytohormones influence the biogenesis of the thylakoid membrane mainly posttranscriptionally.

We have analyzed plastid and nuclear gene expression in tobacco seedlings using the carotenoid biosynthesis inhibitor nor-flurazon. mRNA levels for three nuclear-encoded chlorophyll-binding proteins of photosystem I and photosystem II (CAB I and II and the CP 24 apoprotein) are no longer detectable in photobleached seedlings, whereas those for other components of the thylakoid membrane (the 33- and 23-kD polypeptides and Rieske Fe/S polypeptide) accumulate to some extent. Transgenic tobacco seedlings with promoter fusions from genes for thylakoid membrane proteins exhibit a similar expression behavior: a CAB-[beta]-glucuronidase (GUS) gene fusion is not expressed in herbicide-treated seedlings, whereas PC-, FNR-, PSAF-, and ATPC-promoter fusions are expressed, although at reduced levels. All identified segments in nuclear promoters analyzed that have been shown to respond to light also respond to photodamage to the plastids. Thus, the regulatory signal pathways either merge prior to gene regulation or interact with closely neighboring cis elements. These results indicate that plastids control nuclear gene expression via different and gene-specific cis-regulatory elements and that CAB gene expression is different from the expression of the other genes tested. Finally, a plastid-directing import sequence from the maize Waxy gene is capable of directing the GUS protein into the photodamaged organelle. Therefore, plastid import seems to be functional in photobleached organelles.

The CFoII subunit of the ATP synthase is an integral component of the thylakoid membrane which is synthesized in the cytosol with a bipartite, lumen-targeting presequence similar in structural terms to those of imported lumenal proteins such as plastocyanin. This presequence is shown to possess a terminal cleavage site for the thylakoidal processing peptidase, but no intermediate site for the stromal processing peptidase. The integration of CFoII into the thylakoid membrane of Pisum sativum has been analysed using in vitro assays for the import of proteins into intact chloroplasts or isolated thylakoids. Efficient integration into thylakoids is observed in the light and dark, and the integration process does not require the presence of either stromal extracts or nucleoside triphosphates. The uncoupler nigericin inhibits integration only very slightly, indicating that the thylakoidal delta pH does not play a significant role in the integration mechanism. In each of these respects, the requirements for CFoII integration differ notably from those determined for integration of the light-harvesting chlorophyll-binding protein of photosystem II. The integration mechanism also differs significantly from the two mechanisms involved in the translocation of lumenal proteins across the thylakoid membrane, since one of these processes requires the presence of stromal protein factors and ATP, and the other mechanism is dependent on the thylakoidal delta pH. This conclusion is reinforced by the finding that saturation of the translocation system for the precursor to the lumenal 23 kDa oxygen-evolving complex protein does not affect integration of CFoII into thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS)