Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.     

The light-harvesting complex of photosystem I in Chlamydomonas reinhardtii: protein composition, gene structures and phylogenic implications

Light-harvesting chlorophyll a/b-binding proteins (LHCI) associated with photosystem I (PSI) and the genes encoding these proteins have been characterized in the unicellular green alga Chlamydomonas reinhardtii, extending previous studies of the PSII-LHCII [Teramoto et al. (2001) Plant Cell Physiol. 42: 849]. In order to assign LHCI proteins in the thylakoid membranes, the PSI-LHCI supercomplex that retains all of the major LHCI proteins was purified. Seven distinct LHCI proteins were resolved from the purified supercomplex by a high-resolution SDS polyacrylamide gel electrophoresis, and their N-terminal amino acid sequences were determined. One LHCI protein (band e) was newly found, although the other six LHCI proteins corresponded to those previously reported. Genomic clones encoding these seven LHCI proteins were newly isolated and the nucleotide sequences were determined. A comprehensive characterization of all members of Lhc gene family in this alga revealed that LHCI proteins are more highly diverged than LHCII, suggesting functional differentiation of the protein components in LHCI. Neighbor joining trees were constructed for LHC proteins from C. reinhardtii and those of Arabidopsis thaliana or Galdieria sulphuraria to assess evolutionary relationships. Phylogenetic analysis revealed that (1). green algal LHCI and LHCII proteins are more closely related to one another than to LHCI proteins in red algae, (2). green algae and higher plants possess seven common lineages of LHC proteins, and (3). Type I and III LHCI proteins are conserved between green algae and higher plants, while Type II and IV are not. These findings are discussed in the context of evolution of multiple diverse antenna complexes.     

Identification and Characterization of an Assembly Intermediate Subcomplex of Photosystem I in the Green Alga Chlamydomonas reinhardtii

Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12–15 core and 4–9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photo-oxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.     

Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP.

Cytosolically synthesized thylakoid proteins must be translocated across the chloroplast envelope membranes, traverse the stroma, and then be translocated into or across the thylakoid membrane. Protein transport across the envelope requires ATP hydrolysis but not electrical or proton gradients. The energy requirements for the thylakoid translocation step were studied here for the light-harvesting chlorophyll a/b protein (LHCP), an integral membrane protein, and for several thylakoid lumen-resident proteins: plastocyanin and OE33, OE23, and OE17 (the 33-, 23-, and 17-kDa subunits of the oxygen-evolving complex, respectively). Dissipation of the thylakoid protonmotive force during an in organello protein import assay partially inhibited the thylakoid localization of LHCP and OE33, totally inhibited localization of OE23 and OE17, and had no effect on localization of plastocyanin. We used reconstitution assays for LHCP insertion and for OE23 and OE17 transport into isolated thylakoids to investigate the energy requirements in detail. The results indicated that LHCP insertion absolutely requires ATP hydrolysis and is enhanced by a transthylakoid delta pH and that transport of OE23 and OE17 is absolutely dependent upon a delta pH. Surprisingly, OE23 and OE17 transport occurred maximally in the complete absence of ATP. These results establish the thylakoid membrane as the only membrane system in which a delta pH can provide all of the energy required to translocate proteins across the bilayer. They also demonstrate that the energy requirements for integration into or translocation across the thylakoid membranes are protein-specific.     

Movement of newly imported light-harvesting chlorophyll-binding protein from unstacked to stacked thylakoid membranes is not affected by light treatment or absence of amino-terminal threonines

In higher plants and algae, the transduction of captured light energy is highly regulated as excess excitation of photosystem II (PSII) reaction centers can be redirected to photosystem I (PSI) reaction centers. Models that attempt to explain this phenomenon involve light-harvesting chlorophyll-protein complexes (LHCII) that capture light energy and migrate between PSII and PSI. This report shows that in pea chloroplasts, the major protein component of LHCII, light-harvesting chlorophyll-binding protein (LHCP), can indeed migrate within the thylakoid membrane. We show, however, that although newly imported LHCP inserts into both stacked and unstacked thylakoid membranes, it then moves only from the unstacked, PSI-rich membranes to the stacked, PSII-rich membranes. The observed migration is not affected by light treatment that induces a redistribution of captured light energy (state I-state II transition) that previously was thought to induce LHCP to migrate in the opposite direction, from stacked to unstacked membranes. A mutation that removes the site of LHCP phosphorylation, the proposed trigger of state transitions, also has no effect on the integration and movement of LHCP, but does render LHCP more susceptible to proteolytic degradation. These results are not consistent with current models that deal with the short-term change in the distribution of light energy.     

Chloroplast Oxa1p homolog albino3 is required for post-translational integration of the light harvesting chlorophyll-binding protein into thylakoid membranes

Multiple sorting pathways operate in chloroplasts to localize proteins to the thylakoid membrane. The signal recognition particle (SRP) pathway in chloroplasts employs the function of a signal recognition particle (cpSRP) to target light harvesting chlorophyll-binding protein (LHCP) to the thylakoid membrane. In assays that reconstitute stroma-dependent LHCP integration in vitro, the stroma is replaceable by the addition of GTP, cpSRP, and an SRP receptor homolog, cpFtsY. Still lacking is an understanding of events that take place at the thylakoid membrane including the identification of membrane proteins that may function at the level of cpFtsY binding or LHCP integration. The identification of Oxa1p in mitochondria, an inner membrane translocase component homologous to predicted proteins in bacteria and to the albino3 (ALB3) protein in thylakoids, led us to investigate the potential role of ALB3 in LHCP integration. Antibody raised against a 50-amino acid region of ALB3 (ALB3-50aa) identified a single 45-kDa thylakoid protein. Treatment of thylakoids with antibody to ALB3-50aa inhibited LHCP integration, whereas the same antibody treatment performed in the presence of antigen reversed the inhibition. In contrast, transport by the thylakoid Sec or Delta pH pathways was unaffected. These data support a model whereby a distinct translocase containing ALB3 is used to integrate LHCP into thylakoid membranes.     

Light-Harvesting Chlorophyll a/b Protein : Membrane Insertion, Proteolytic Processing, Assembly into LHC II, and Localization to Appressed Membranes Occurs in Chloroplast Lysates

The apoprotein of the light-harvesting chlorophyll a/b protein (LHCP) is a major integral thylakoid membrane protein that is normally complexed with chlorophyll and xanthophylls and serves as the antenna complex of photosystem II. LHCP is encoded in the nucleus and synthesized in the cytosol as a higher molecular weight precursor that is subsequently imported into chloroplasts and assembled into thylakoids. In a previous study it was established that the LHCP precursor can integrate into isolated thylakoid membranes. The present study demonstrates that under conditions designed to preserve thylakoid structure, the inserted LHCP precursor is processed to mature size, assembled into the LHC II chlorophyll-protein complex, and localized to the appressed thylakoid membranes. Under these conditions, light can partially replace exogenous ATP in the membrane integration process.     

Requirement for three membrane-spanning alpha-helices in the post-translational insertion of a thylakoid membrane protein.

The insertion of a protein into a lipid bilayer usually involves a short signal sequence and can occur either during or after translation. A light-harvesting chlorophyll a/b-binding protein (LHCP) is synthesized in the cytoplasm of plant cells as a precursor and is post-translationally imported into chloroplasts where it subsequently inserts into the thylakoid membrane. Only mature LHCP is required for insertion into the thylakoid. To define which sequences of the mature protein are necessary and sufficient for thylakoid integration, fusion and deletion proteins and proteins with internal rearrangements were synthesized and incubated with isolated thylakoids and stroma. No evidence is found for the existence of a short signal sequence within LHCP, and, with the exception of the amino terminus and a short lumenal loop, the entire mature protein with consecutively ordered alpha-helices is required for insertion into thylakoid membranes. The addition of positive charges into stromal but not lumenal segments permits the insertion of mutant LHCPs into isolated thylakoids. Replacement of the LHCP transit peptide with the transit peptide from plastocyanin has no effect on LHCP insertion and does not restore insertion of the lumenal charge addition mutants.     

Integration of a chlorophyll-binding protein into Escherichia coli membranes in the absence of chlorophyll

The mechanism by which a protein integrates posttranslationally into a membrane can involve the composition of the membrane itself, domains within the inserting polypeptide, and a number of associating proteins. Some integral membrane proteins do not accumulate to normal levels when certain pigments are deficient, and this has been interpreted to mean that such proteins may be rapidly degraded when not in a correct complex. Alternatively, pigments could facilitate the movement of some proteins from an aqueous to a lipid environment. To determine whether chlorophyll is absolutely required for the membrane integration of the light-harvesting chlorophyll-binding protein (LHCP) of chloroplast thylakoid membranes, we have expressed LHCP in Escherichia coli that lacks photosynthetic pigments. LHCP is targeted to the bacterial inner membrane by the addition of a bacterial signal peptide and cannot be extracted from these membranes by NaOH, NaBr, or Na2HCO3 but is extracted by 0.2% Triton X-100. Treatment of isolated right-side-out and inside-out bacterial inner membrane vesicles with trypsin reveals that only the amino terminus of LHCP is exposed on the cytoplasmic face, and the remaining portion of the protein is inaccessible. Treatment of the inside-out vesicles with trypsin followed by alkaline extraction shows that LHCP is intrinsic to the membrane and is not anchored solely by the bacterial signal peptide. Chlorophyll, therefore, is not required for LHCP to integrate into a membrane, but in the absence of these pigments this process is observed to be inefficient.     

The L18 domain of light-harvesting chlorophyll proteins binds to chloroplast signal recognition particle 43

Chloroplast signal recognition particle (cpSRP) is a novel type of SRP that contains a homolog of SRP54 and a 43-kDa subunit absent from all cytoplasmic SRPs but lacks RNA. It is also distinctive in its ability to post-translationally interact with light-harvesting chlorophyll proteins (LHCP), hydrophobic proteins synthesized in the cytoplasm and targeted to the thylakoid via the stroma. LHCP integration into thylakoid membranes requires the two subunits of cpSRP, cpFtsY, GTP, and the membrane protein ALB3. It had previously been shown that the L18 domain, an 18-amino acid peptide between the second and third transmembrane domains, and a hydrophobic domain are required for interaction with cpSRP. In the present study we used a pull-down assay, with cpSRP43 or cpSRP54 fused to glutathione-transferase, to study interactions between cpSRP43, cpSRP54, LHCP, and cpFtsY. cpFtsY was not observed to form significant interactions with any of the proteins even in the presence of nonhydrolyzable GTP analogs. Our data indicate that cpSRP43 binds to the L18 domain, that cpSRP54 binds to the hydrophobic domain, and that LHCP and cpSRP54 independently bind to cpSRP43. These data confirm that the novel post-translational interaction between LHCP and cpSRP is mediated through binding to cpSRP43.     

Isolation of thylakoid membrane complexes from rice by a new double-strips BN/SDS-PAGE and bioinformatics prediction of stromal ridge subunits interaction

Thylakoid membrane complexes of rice (Oryza sativa L.) play crucial roles in growth and crop production. Understanding of protein interactions within the complex would provide new insights into photosynthesis. Here, a new "Double-Strips BN/SDS-PAGE" method was employed to separate thylakoid membrane complexes in order to increase the protein abundance on 2D-gels and to facilitate the identification of hydrophobic transmembrane proteins. A total of 58 protein spots could be observed and subunit constitution of these complexes exhibited on 2D-gels. The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp). Furthermore, the proteins separated from rice thylakoid membrane were identified by the mass spectrometry (MS). The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice. Using molecular dynamics simulation to explore the recognition mechanism of these subunits, we showed that salt bridge interactions between residues R19 of PsaC and E168 of PasD as well as R75 of PsaC and E91 of PsaD played important roles in the stability of the complex. This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy.     

A hydrophobic, carboxy-proximal region of a light-harvesting chlorophyll a/b protein is necessary for stable integration into thylakoid membranes.

Proteins synthesized as soluble precursors in the cytoplasm of eukaryotic cells often cross organellar membrane barriers and then insert into lipid bilayers. One such polypeptide, the light-harvesting chlorophyll a/b-binding protein (LHCP), must also associate with pigment molecules and be assembled into the photosystem II light-harvesting complex in the chloroplast thylakoid membrane. A study of the import of mutant LHCPs into isolated chloroplasts has shown that a putative alpha-helical membrane-spanning domain near the carboxy terminus (helix 3) is essential for the stable insertion of LHCP in the thylakoid. Protease digestion experiments are consistent with the carboxy terminus of the protein being in the lumen. This report also shows that helix 3, when fused to a soluble protein, can target it to the thylakoids of isolated, intact chloroplasts. Although helix 3 is required for the insertion of LHCP and mutant derivatives into the thylakoid, the full insertion of helix 3 itself requires additionally the presence of other regions of LHCP. Thus, LHCP targeting and integration into thylakoid membranes requires a complex interaction involving a number of different domains of the LHCP polypeptide.     

Disruption of thylakoid-associated kinase 1 leads to alteration of light harvesting in Arabidopsis.

To survive fluctuations in quality and intensity of light, plants and algae are able to preferentially direct the absorption of light energy to either one of the two photosystems, PSI or PSII. This rapid process is referred to as a state transition and has been correlated with the phosphorylation and migration of the light-harvesting complex protein (LHCP) between PSII and PSI. We show here that thylakoid protein kinases (TAKs) are required for state transitions in Arabidopsis. Antisense TAK1 expression leads to a loss of LHCP phosphorylation and a reduction in state transitions. Preferential activation of PSII causes LHCP to accumulate with PSI, and TAK1 mutants disrupt this process. Finally, TAKs also influence the phosphorylation of multiple thylakoid proteins.     

ATP stimulates signal recognition particle (SRP)/FtsY-supported protein integration in chloroplasts

The signal recognition particle (SRP) and its receptor (FtsY in prokaryotes) are essential for cotranslational protein targeting to the endoplasmic reticulum in eukaryotes and the cytoplasmic membrane in prokaryotes. An SRP/FtsY-like protein targeting/integration pathway in chloroplasts mediates the posttranslational integration of the light-harvesting chlorophyll a/b-binding protein (LHCP) into thylakoid membranes. GTP, chloroplast SRP (cpSRP), and chloroplast FtsY (cpFtsY) are required for LHCP integration into thylakoid membranes. Here, we report the reconstitution of the LHCP integration reaction with purified recombinant proteins and salt-washed thylakoids. Our data demonstrate that cpSRP and cpFtsY are the only soluble protein components required for LHCP integration. In addition, our studies reveal that ATP, though not absolutely required, remarkably stimulates LHCP integration into salt-washed thylakoids. ATP stimulates LHCP integration by a mechanism independent of the thylakoidal pH gradient (DeltapH) and exerts no detectable effect on the formation of the soluble LHCP-cpSRP-targeting complex. Taken together, our results indicate the participation of a thylakoid ATP-binding protein in LHCP integration.     

Absence of lutein, violaxanthin and neoxanthin affects the functional chlorophyll antenna size of photosystem-II but not that of photosystem-I in the green alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii double mutant npq2 lor1 lacks the beta, epsilon-carotenoids lutein and loroxanthin as well as all beta,beta-epoxycarotenoids derived from zeaxanthin (e.g. violaxanthin and neoxanthin). Thus, the only carotenoids present in the thylakoid membranes of the npq2 lor1 cells are beta-carotene and zeaxanthin. The effect of these mutations on the photochemical apparatus assembly and function was investigated. In cells of the mutant strain, the content of photosystem-II (PSII) and photosystem-I (PSI) was similar to that of the wild type, but npq2 lor1 had a significantly smaller PSII light-harvesting Chl antenna size. In contrast, the Chl antenna size of PSI was not truncated in the mutant. SDS-PAGE and Western blot analysis qualitatively revealed the presence of all LHCII and LHCI apoproteins in the thylakoid membrane of the mutant. The results showed that some of the LHCII and most of the LHCI were assembled and functionally connected with PSII and PSI, respectively. Photon conversion efficiency measurements, based on the initial slope of the light-saturation curve of photosynthesis and on the yield of Chl a fluorescence in vivo, showed similar efficiencies. However, a significantly greater light intensity was required for the saturation of photosynthesis in the mutant than in the wild type. It is concluded that zeaxanthin can successfully replace lutein and violaxanthin in most of the functional light-harvesting antenna of the npq2 lor1 mutant.     

Chloroplast FtsY, chloroplast signal recognition particle, and GTP are required to reconstitute the soluble phase of light-harvesting chlorophyll protein transport into thylakoid membranes.

The integration of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membrane proceeds in two steps. First, LHCP interacts with a chloroplast signal recognition particle (cpSRP) to form a soluble targeting intermediate called the transit complex. Second, LHCP integrates into the thylakoid membrane in the presence of GTP, at least one other soluble factor, and undefined membrane components. We previously determined that cpSRP is composed of 43- and 54-kDa polypeptides. We have examined the subunit stoichiometry of cpSRP and find that it is trimeric and composed of two subunits of cpSRP43/subunit of cpSRP54. A chloroplast homologue of FtsY, an Escherichia coli protein that is critical for the function of E. coli SRP, was found largely in the stroma unassociated with cpSRP. When chloroplast FtsY was combined with cpSRP and GTP, the three factors promoted efficient LHCP integration into thylakoid membranes in the absence of stroma, demonstrating that they are all required for reconstituting the soluble phase of LHCP transport.     

Biogenesis of a photosystem I light-harvesting complex. Evidence for a membrane intermediate.

CAB-7p is a chlorophyll a/b binding protein of photosystem I (PSI). It is found in light-harvesting complex I 680 (LHCI-680), one of the chlorophyll complexes produced by detergent solubilization of PSI. Two types of evidence are presented to indicate that assembly of CAB-7p into PSI proceeds through a membrane intermediate. First, when CAB-7p is briefly imported into chloroplasts or isolated thylakoids, we initially observe a fast-migrating membrane form of CAB-7p that is subsequently converted into PSI. The conversion of the fast-migrating form into PSI does not require stroma or ATP. Second, trypsin treatment of thylakoids containing radiolabeled CAB-7p indicates that there are at least two membrane forms of the mature 23-kD protein. The predominant form is completely resistant to proteolysis; a second form of the protein is cleaved by trypsin into 12- and 7-kD polypeptides. We interpret this to mean that the intermediate is a cleavable form that becomes protease resistant during assembly. This notion is supported by the observation that CAB-7p in LHCI-680 is largely cleaved by trypsin into 12- and 7-kD polypeptides, whereas CAB-7p in isolated PSI particles is trypsin resistant. In vitro, we generated a mutant form of CAB-7p, CAB-7/BgI2p, that was able to integrate into thylakoid membranes but was unable to assemble into PSI. The membrane form of CAB-7/BgI2p, like LHCI-680, was predominantly cleaved by trypsin into 12- and 7-kD fragments. We suggest that the mutant protein is arrested at an intermediate stage in the assembly pathway of PSI. Based on its mobility in nondenaturing gels and its susceptibility to protease cleavage, we suggest that the intermediate form is LHCI-680. We propose the following distinct stages in the biogenesis of LHCI: (a) apoprotein is integrated into the thylakoid, (b) chlorophyll is rapidly bound to apoprotein forming LHCI-680, and (c) LHCI-680 assembles into the native PSI complex.     

Photosynthetic functions and thylakoid membrane polypeptide composition in light-sensitive mutants of Chlamydomonas reinhardii

In this paper we compared the pigment composition, photochemical activity, chloroplast ultrastructure, thylakoid membrane polypeptide composition and ribosomal content of wild-type and seven light-sensitive mutants of Chlamydomonas reinhardii.All the mutants had low chlorophyll and carotenoid content compared to wild-type. Mutants lts-30 and lts-135 were also characterized by a complete absence of visible carotenoids, while mutant lts-19 was fully deficient in chlorophylls.In most mutants, the chloroplast fragment could not carry out any DCIP photoreduction and O₂ evolution was also blocked. The PSI/P₇₀₀/activity was decreased in most cases.The mutant strains contained mostly single lamellae in their plastids, that is the stacking capacity of the thylakoid membranes was very decreased or fully absent. In most cases the number of lamellae was also very low.The relative amounts of 70 S ribosomes were decreased in all of the mutants. The thylakoid membranes showed anomalies in the region of 24 000–30 000 dalton polypeptides. The common characteristic for them was the relatively higher amount of the 30 000 dalton polypeptide and considerably decreased level of the 27 000 and 24 000 dalton polypeptides relative to the wild-type. These polypeptides were probably constituents of the chlorophyll-protein complex II which has been suggested to be the light harvesting pigment complex for PSII. The polypeptide of 30 000 daltons is the precursor for the LHCP apoprotein (24 000 dalton protein). It may be that the lighstimulated conversion of this precursor into LHCP apoprotein was blocked in our pigment-deficient mutants.Abbreviations CPI Chlorophyll-protein complex I - PSI Photosystem I - PSII Photosystem II - LHCP Light-harvesting pigment complex - DCIP 2,6-dichlorophenolindophenol - RuDPC-ase Ribulose-1,5-biphosphate-carboxylase - SDS Sodium dodecyl sulfate - LIDS Lithium dodecyl sulfate - PAG Polyacrylamide gel - TKM buffer 25 mM Tris-HCl, pH 7.S; 25 mM KCl; 25 mM Mg acetate     

Immunological characterization of chlorophyll a/b-binding proteins of barley thylakoids

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCI) using a photosystem (PS) I preparation (PSI-200) wild-type from barley (Hordeum vulgare L. cv. Svaløf's Bonus) as the antigen. These antibodies cross-reacted with a minor light-harvesting chlorophyll a/b-protein of PSII (Chla/b-P1=CP29), but not with the major one, LHCII (=Chla/b-P2^**). Similarly, a monoclonal antibody to Chla/b-P1, elicited by a PSII preparation as the antigen, cross-reacted with LHCI, but not LHCII. This explains why an antigen consisting of LHCII, free of LHCI, but contaminated with Chla/b-P1, can elicit antibodies which cross-react with LHCI. Immunoblot assays showed that LHCI and Chla/b-P1 have at least two epitopes in common. Immunogold labelling of thin-sectioned wild-type thylakoids confirmed a preferential localisation of Chla/b-P1 in grana partition membranes and LHCI in stroma lamellae. The presence of LHCI was demonstrated in barley mutants lacking the PSI reaction centre (viridis-zb ⁶³) and chlorophyll b (chlorina-f2), and was correlated with the presence of long-wavelength (730 nm) fluorescence emission at 77 K. The mutant viridis-k ²³, which has a 77 K long-wavelength fluorescence peak at 720 nm, was shown by immune-blot assay to lack LHCI, although Chla/b-P1 was present.Abbreviations Chl-P chlorophyll-protein - CM Carlsberg Monoclonal - Da dalton - LHC light-harvesting complex - PAGE polyacrylamide gel electrophoresis - PSI, II photosystem I, II - PSI-200 PSI containing LHCI polypeptides - SDS sodium dodecyl sulphate     

A stromal protein factor maintains the solubility and insertion competence of an imported thylakoid membrane protein

The light-harvesting chlorophyll a/b protein (LHCP) is an approximately 25,000-D thylakoid membrane protein. LHCP is synthesized in the cytosol as a precursor and must translocate across the chloroplast envelope before becoming integrally associated with the thylakoid bilayer. Previous studies demonstrated that imported LHCP traverses the chloroplast stroma as a soluble intermediate before thylakoid insertion. Here, examination of this intermediate revealed that it is a stable, discrete approximately 120,000-D species and thus either an LHCP oligomer or a complex with another component. In vitro-synthesized LHCP can be converted to a similar form by incubation with a stromal extract. The stromal component responsible for this conversion is proteinaceous as evidenced by its inactivation by heat, protease, and NEM. Furthermore, the conversion activity coelutes from a gel filtration column with a stromal protein factor(s) previously shown to be necessary for LHCP integration into isolated thylakoids. Conversion of LHCP to the 120-kD form prevents aggregation and maintains its competence for thylakoid insertion. However, conversion to this form is apparently not sufficient for membrane insertion because the isolated 120-kD LHCP still requires stroma to complete the integration process. This suggests a need for at least one more stroma-mediated reaction. Our results explain how a hydrophobic thylakoid protein remains soluble as it traverses the aqueous stroma. Moreover, they describe in part the function of the stromal requirement for insertion into the thylakoid membrane.