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ORCID ID: 0000-0003-2973-9514 (J.M.) The chloroplast-encoded low molecular weight protein PsbN is annotated as a photosystem II (PSII) subunit. To elucidate the localization and function of PsbN, encoded on the opposite strand to the psbB gene cluster, we raised antibodies and inserted a resistance cassette into PsbN in both directions. Both homoplastomic tobacco (Nicotiana tabacum) mutants ΔpsbN-F and ΔpsbN-R show essentially the same PSII deficiencies. The mutants are extremely light sensitive and failed to recover from photoinhibition. Although synthesis of PSII proteins was not altered significantly, both mutants accumulated only ;25% of PSII proteins compared with the wild type. Assembly of PSII precomplexes occurred at normal rates, but heterodimeric PSII reaction centers (RCs) and higher order PSII assemblies were not formed efficiently in the mutants. The ΔpsbN-R mutant was complemented by allotopic expression of the PsbN gene fused to the sequence of a chloroplast transit peptide in the nuclear genome. PsbN represents a bitopic trans-membrane peptide localized in stroma lamellae with its highly conserved C terminus exposed to the stroma. Significant amounts of PsbN were already present in dark-grown seedling. Our data prove that PsbN is not a constituent subunit of PSII but is required for repair from photoinhibition and efficient assembly of the PSII RC.

Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.

To investigate the nuclear-controlled mechanisms of [4Fe-4S] cluster assembly in chloroplasts, we selected Arabidopsis thaliana mutants with a decreased content of photosystem I (PSI) containing three [4Fe-4S] clusters. One identified gene, ACCUMULATION OF PHOTOSYSTEM ONE1 (APO1), belongs to a previously unknown gene family with four defined groups (APO1 to APO4) only found in nuclear genomes of vascular plants. All homologs contain two related motifs of approximately 100 amino acid residues that could potentially provide ligands for [4Fe-4S] clusters. APO1 is essentially required for photoautotrophic growth, and levels of PSI core subunits are below the limit of detection in the apo1 mutant. Unlike other Arabidopsis PSI mutants, apo1 fails to accumulate significant amounts of the outer antenna subunits of PSI and PSII and to form grana stacks. In particular, APO1 is essentially required for stable accumulation of other plastid-encoded and nuclear-encoded [4Fe-4S] cluster complexes within the chloroplast, whereas [2Fe-2S] cluster-containing complexes appear to be unaffected. In vivo labeling experiments and analyses of polysome association suggest that translational elongation of the PSI transcripts psaA and psaB is specifically arrested in the mutant. Taken together, our findings suggest that APO1 is involved in the stable assembly of several [4Fe-4S] cluster-containing complexes of chloroplasts and interferes with translational events probably in association with plastid nucleoids.

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.

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

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.

The interaction between plastocyanin and the intact cytochrome bf complex, both from spinach, has been studied by stopped-flow kinetics with mutant plastocyanin to elucidate the site of electron transfer and the docking regions of the molecule. Mutation of Tyr-83 to Arg or Leu provides no evidence for a second electron transfer path via Tyr-83 of plastocyanin, which has been proposed to be the site of electron transfer from cytochrome f. The data found with mutations of acidic residues indicate that both conserved negative patches are essential for the binding of plastocyanin to the intact cytochrome bf complex. Replacing Ala-90 and Gly-10 at the flat hydrophobic surface of plastocyanin by larger residues slowed down and accelerated, respectively, the rate of electron transfer as compared with wild-type plastocyanin. These opposing effects reveal that the hydrophobic region around the electron transfer site at His-87 is divided up into two regions, of which only that with Ala-90 contributes to the attachment to the cytochrome bf complex. These binding sites of plastocyanin are substantially different from those interacting with photosystem I. It appears that each of the two binding regions of plastocyanin is split into halves, which are used in different combinations in the molecular recognition at the two membrane complexes.

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.

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.

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.

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.

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.

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.

A preparation technique for high resolution field emission scanning electron microscopy of plant chromosomes is described. The technique was optimized to use standard squash preparations of mitotic and meiotic chromosomes from root tips of barley, wheat, and rye. After light microscopic observation and documentation, the same object can be investigated with a 100-fold higher resolution using a field emission scanning electron microscope. Tilting of the specimens provides a three-dimensional insight into chromosomal structures at different stages of condensation and decondensation. With this technique it was possible to document for the first time at high resolution structures such as the centromeric region, the spindle apparatus and the spindle fibre attachment region. The smallest unit of the DNA packed that can be resolved is a “beads on a string”-like structure of the nucleofilament (10–15 nm fibre).

A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.

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.

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.

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).

The cytochrome b6 polypeptide present in cytochrome b6/| preparations from spinach thylakoids is phosphorylated concomitantly with the autophosphorylation of the 64 kDa polypeptide identified as the redox-controlled LHCII kinase. The N-terminal sequence of the 64 kDa kinase and sequence analysis of cytochrome b6 indicate the existence of putative phosphorylation sites in both proteins.

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.

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.

The molecular recognition between plastocyanin and photosystem I was studied. Photosystem I and plastocyanin can be cross-linked to an active electron transfer complex. Immunoblots and mass spectrometric analysis of proteolytic peptides indicate that the two negative patches conserved in plant plastocyanins are cross-linked with lysine residues of a domain near the N-terminus of the PsaF subunit of photosystem I. Conversion of these negative to uncharged patches of plastocyanin by site-directed mutation D42N/E43Q/D44N/E45Q and E59Q/E60Q/D61N respectively, reveals the first patch to be essential for the electrostatic interaction in the electron transfer complex with photosystem I and the second one to lower the redox potential. The domain in PsaF, not found in cyanobacteria, is predicted to fold into two amphipathic alpha-helices. The interacting N-terminal helix lines up six lysines on one side which may guide a fast one-dimensional diffusion of plastocyanin and provide the electrostatic attraction at the attachment site, in addition to the hydrophobic interaction in the area where the electron is transferred to P700 in the reaction center of photosystem I. This two-step interaction is likely to increase the electron transfer rate by more than two orders of magnitude in plants as compared with cyanobacteria. Our data resolve the controversy about the function of PsaF.

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.

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.

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.

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)

Mutant plastocyanins with Leu at position 10, 90 or 83 (Gly, Ala and Tyr respectively in wildtype) were constructed by site-specific mutagenesis of the spinach gene, and expressed in transgenic potato plants under the control of the authentic plastocyanin promoter, as well as in Escherichia coli as truncated precursor intermediates carrying the C-terminal 22 amino acid residues of the transit peptide, i.e. the thylakoid-targeting domain that acts as a bacterial export signal. The identity of the purified plastocyanins was verified by matrix-assisted laser desorption/ionization mass spectrometry. The formation of a complex between authentic or mutant spinach plastocyanin and isolated photosystem I and the electron transfer has been studied from the biphasic reduction kinetics of P700+ after excitation with laser flashes. The formation of the complex was abolished by the bulky hydrophobic group of Leu at the respective position of G10 or A90 which are part of the conserved flat hydrophobic surface around the copper ligand H87. The rate of electron transfer decreased by both mutations to < 20% of that found with wildtype plastocyanin. We conclude that the conserved flat surface of plastocyanin represents one of two crucial structural elements for both the docking at photosystem I and the efficient electron transfer via H87 to P700+. The Y83L mutant exhibited faster electron transfer to P700+ than did authentic plastocyanin. This proves that Y83 is not involved in electron transfer to P700 and suggests that electron transfer from cytochrome f and to P700 follows different routes in the plastocyanin molecule. Plastocyanin (Y83L) expressed in either E. coli or potato exhibited different isoelectric points and binding constants to photosystem I indicative of differences in the folding of the protein. The structure of the binding site at photosystem I and the mechanism of electron transfer are discussed.

The levels of plastid gene transcripts are shown to be controlled by temperature in isolated pumpkin cotyledons. The temperature at which maximum transcript accumulation occurs varies between 38 and 42 degrees C for the genes studied (rbcL, psaA, psbA, psbB, psbC, psbD, psbE, atpA). Heat shock-induced transcript accumulation is transitory with a maximum after an approximately 3 h exposure at high temperatures. On the other hand, the accumulation of rbcL transcript was only moderately thermosensitive. A temperature increase to 46-48 degrees C induces a sharp decrease of transcript levels which correlates with damage to the plant. Relatively little correlation has been noted between RNA and protein patterns. However, there is a remarkable coincidence between temperature dependence of the accumulation of transcripts and the temperature dependence (28-46 degrees C) of the synthesis of chloroplast-located heat shock proteins indicating that both processes may be related.

Nucleotide sequence data for thylakoid membrane proteins, and calculation of the distribution of hydrophobic residues from these sequences, allows prediction of hydrophobic membrane-spanning helices and the position of attached redox groups. This provides a new perspective for considering the structure and function of the proteins. This review discusses the conclusions reached so far for several plant photosynthetic electron transport proteins. In cytochrome b 6 the arrangement of two hemes across the hydrophobic core of the membrane may illustrate a general structural principle for a transmembrane redox reaction.

Using lambda gt11 expression cloning and immunoscreening, cDNA-containing recombinant phages for subunits IV and V of the photosystem I reaction center were isolated, sequenced and used to probe Northern blots of polyadenylated RNA prepared from spinach seedlings. The mRNA sizes for both components are approximately 1000 and 850 nucleotides, respectively. The 968 nucleotide cDNA sequence and derived amino acid sequence for subunit IV predict a single open reading frame of 231 amino acid residues (25.4 kDa). Comparison with a 13-residue N-terminal amino acid sequence determined for subunit IV suggests a mature protein of 17.3 kDa (154 residues) and a transit sequence of 77 amino acids (8.1 kDa). The corresponding data for subunit V are 677 bp (cDNA), 167 residues for the precursor protein (18.2 kDa), 98 residues for the mature polypeptide (10.8 kDa) and 69 residues for the transit peptide (7.4 kDa). Secondary structure predictions indicate that both proteins possess greatly different transit sequences and that none is membrane-spanning.

The Oenothera plastome mutant Iα is deficient in ribulosebisphosphate carboxylase / oxygenase activity. Transcription-translation of the plastid DNA or translation of the plastid RNA of the mutant both give rise to a large subunit of this enzyme which has an M r of about 30 000, whereas that from the wild-type is 52 000. The mutant possesses mRNA of the same size as that of the wild-type, indicating that the lesion is a mutation within the coding region of the gene which causes premature termination of translation. The truncated polypeptide is expressed in vivo. The small subunit of the carboxylase is also expressed in vivo in the mutant but the free pool of processed protein is low. The data presented suggest that control mechanisms after transcription are involved in the biogenesis of ribulosebisphosphate carboxylase / oxygenase.

A second nonhomologous polypeptide in the thylakoid membrane cytochrome b-559 has been suggested by the finding of a smaller reading frame just slightly downstream from that corresponding to the 9 kDa cytochrome polypeptide that is dominant on a Coomassie-stained gel. This reading frame encoded a 39-residue polypeptide that was similar in having a central hydrophobic domain of 25–26 residues and a single His residue at the same position in the hydrophobic domain. The smallest polypeptide seen on SDS gels of the cytochrome was isolated by high-performance liquid chromatography (HPLC). The NH2-terminal sequence matched that of the downstream gene. The stoichiometry of the 2 gene products separated by HPLC was approx. 1:1, based on the molecular masses of 9.16 and 4.27 kDa calculated from the nucleotide sequence. It is concluded that the cytochrome contains both the 9.16 kDa (α) and 4.27 kDa (β) polypeptides. These data, the single His residue on each polypeptide, and the previous finding of a bis-histidine coordination, imply that the unit heme binding structure of the cytochrome is a heme cross-linked dimer. If the cytochrome contains a single heme, the dimer structure would be (αβ). If there are 2 hemes/cytochrome, the more likely structure would be (αβ)2, a tetramer consisting of 2 heme cross-linked hetero-dimers.

Analysis of a 0.6 kb fragment of the spinach plastic chromosome adjacent to the 3' end of the apocytochrome f gene has disclosed two uninterrupted reading frames of 83 and 39 triplets, the product of the first one being apocytochrome b-559. The first 27 predicted amino acid residues had been verified by protein sequence analysis and the molecular mass of 9390 Da derived from the amino acid sequence deduced here is close to that of the authentic protein. The two genes are transcribed by a bicistronic RNA in a direction opposite to that of cytochrome f, and their translation stop/potential ribosome binding sites overlap. Features of the two genes resembling those of bacterial genes include putative tetra- or pentanucleotide ShineDalgarno sequences, Pribnow boxes, ‘−35’ promotor consensus sequences and possibly a transcription termination region. Both gene structure and products of DNA- or RNA-programmed cell-free translation preclude that apocytochrome b-559 is made as a precursor. The amino acid sequence includes only one histidine residue located in a predicted secondary structure of strong hydrophobicity which indicates the intriguing possibility that more than one protein chain must cooperate in heme binding of this cytochrome.

Recombinant phages that encode the complete precursor polypeptide for the 22 kDa polypeptide associated with photosystem 11 have been serologically selected from two λgt11 expression libraries made from polyadenylated RNA or spinach seedlings. The cDNAs hybridize to a 1.3 kb RNA species. The precursor protein is comprised of 274 amino acid residues and carries an N-terminal transit peptide of probably 69 amino acid residues. The mature protein exhibits four, predicted transmembrane segments and is shown to be an integral component of photosystom 11 originating in a single-copy gene. The unique characteristics of this protein are: (1) it is the result of a gene-internal duplication of an ancestor with two membrane spans. (ii) a striking resemblance to LHC 1/11, CP24/CP29 apoproteins, and ELIPs, although it does not bind chlorophyll and is present in cyanobacteria, and, as these proteins, (iii) it integrates into the membrane with uncleaved routing signals that display remarkable resemblance to patterns found in bipartite transit pepticies.

In integral membrane proteins, regions that span the lipid bilayer alternate with regions that are exposed on either side of the membrane. For proteins from the plasma membrane of both prokaryotic and eukaryotic cells it has been shown that the exposed parts follow a ‘positve-inside rule’: on average, segments that are translocated across the membrane have a 2–4-fold lower frequency of positively charged residues than non-translocated segments. We now present an analysis of proteins from the thylakoid membrane of chloroplasts. It is shown that these proteins have the same charge asymmetry as has been reported for proteins from other membrane systems, with their more highly charged regions facing the stromal compartment.

In vitro reconstitution assays have been used in recent years to probe the mechanisms by which a variety of cytosolically synthesised proteins are transported across the thylakoid membrane within higher plant chloroplasts. The emerging data suggest that two distinct mechanisms operate. Translocation of a subset of lumenal proteins, namely the 23 kDa and 16 kDa proteins of the oxygen-evolving complex, and of the CFo2 protein (an integral membrane protein), requires only the presence of the thylakoidal delta pH. In contrast, two other lumenal proteins, the 33 kDa oxygen-evolving complex protein and plastocyanin, require also the presence of ATP and at least one stromal factor for efficient transport into isolated thylakoids to take place.