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.     

Functional and mutational analysis of the light-harvesting chlorophyll a/b protein of thylakoid membranes

The precursor for a Lemna light-harvesting chlorophyll a/b protein (pLHCP) has been synthesized in vitro from a single member of the nuclear LHCP multigene family. We report the sequence of this gene. When incubated with Lemna chloroplasts, the pLHCP is imported and processed into several polypeptides, and the mature form is assembled into the light-harvesting complex of photosystem II (LHC II). The accumulation of the processed LHCP is enhanced by the addition to the chloroplasts of a precursor and a co-factor for chlorophyll biosynthesis. Using a model for the arrangement of the mature polypeptide in the thylakoid membrane as a guide, we have created mutations that lie within the mature coding region. We have studied the processing, the integration into thylakoid membranes, and the assembly into light-harvesting complexes of six of these deletions. Four different mutant LHCPs are found as processed proteins in the thylakoid membrane, but only one appears to have an orientation in the membrane that is similar to that of the wild type. No mutant LHCP appears in LHC II. The other two mutant LHCPs cannot be detected within the chloroplasts. We conclude that stable complex formation is not required for the processing and insertion of altered LHCPs into the thylakoid membrane. We discuss the results in light of our model.     

Light-Harvesting Chlorophyll a/b Complexes: Interdependent Pigment Synthesis and Protein Assembly

The biogenetic interdependence of light-harvesting chlorophyll (Chl) a/b proteins (LHCPs) and antenna pigments has been analyzed for two nuclear mutants of Chlamydomonas that have low levels of Chl b, neoxanthin, and loroxanthin. In mutant PA2.1, the apoprotein precursors (pLHCP II) of the major light-harvesting complex LHC II were synthesized at approximately wild-type rates, processed to their mature size, and rapidly degraded. Because the bulk of labile LHCP II in PA2.1 was soluble, a thylakoid integration factor apparently is defective in this strain. Chl a, Chl b, neoxanthin, and loroxanthin synthesis and accumulation were coordinately reduced in PA2.1, indicating that LHCP II play important regulatory or substrate roles in de novo synthesis of these pigments. Mutant GE2.27 is impaired principally in Chl b synthesis but nonetheless accumulated wild-type levels of all LHCPs. Topology studies of the GE2.27 LHCP II demonstrated that their insertion into thylakoids was incomplete even though they were not structurally altered. Thus, Chl b formation mediates conformational changes of LHCP II after thylakoid integration is initiated. GE2.27 also exhibited very low rates of neoxanthin synthesis and was unable to accumulate loroxanthin. Revertant GE2.27 strains with varying capacities for Chl b formation provided additional evidence that neoxanthin synthesis and accumulation are coupled with the final steps of LHCP II integration into thylakoids. We propose that biogenesis of LHC includes interdependent pigment synthesis/assembly events that occur during LHCP integration into the thylakoid membrane and that defects in these events account for the pleiotropic characteristics of many Chl b-deficient mutants.     

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.     

Spermidine levels and its relationship to DNA synthesis in outgrowing spores and vegetative cells of Bacillus subtilis

It is now established that the light-harvesting chlorophyll—protein complex (LHCP) of chloroplasts becomes phosphorylated in the light. In this study subfractionation of phosphorylated intact chloroplasts has been carried out to compare the phosphorylation of LHCP in non-appressed and appressed thylakoid regions. The results show around 10-times higher relative phosphorylation in the non-appressed regions than in the appressed ones. Since the non-appressed thylakoids also contain almost all photosystem 1, this region is likely to be the site for energy transfer from LHCP to photosystem 1 under phosphorylated conditions.     

Effects of pigment-deficient mutants on the accumulation of photosynthetic proteins in maize

We have monitored the accumulation of photosynthetic proteins in developing pigment-deficient mutants of Zea mays. The proteins examined are the CO₂-fixing enzymes, phoshoenolpyruvate carboxylase (E.C. 4.1.1.31) and ribulose-1,5-bisphosphate carboxylase (E.C.4.1.1.39), and three thylakoid membrane proteins, the light-harvesting chlorophyll a/b binding protein (LHCP) of photosystem II, the 65 kilodalton chlorophyll a binding protein of photosystem I and the alpha subunit polypeptide of coupling factor I. Using a sensitive protein-blot technique, we have compared the relative quantities of each protein in mutants and their normal siblings. Carboxylase accumulation was found to be independent of chlorophyll content, while the amounts of the thylakoid proteins increase at about the same time as chlorophyll in delayed-greening mutants. The relative quantity of LHCP is closely correlated with the relative quantity of chlorophyll at all stages of development in all mutants. Because pigment-deficient mutants are arrested at early stages in chloroplast development, these findings suggest that the processes of chloroplast development, chlorophyll synthesis and thylakoid protein accumulation are coordinated during leaf development but that carboxylase accumulation is controlled by different regulatory mechanisms. A white leaf mutant was found to contain low levels of LHCP mRNA, demonstrating that the accumulation of LHCP mRNA is not controlled exclusively by phytochrome.     

Assembly of the precursor and processed light-harvesting chlorophyll a/b protein of Lemna into the light-harvesting complex II of barley etiochloroplasts

When the in vitro synthesized precursor of a light-harvesting chlorophyll a/b binding protein (LHCP) from Lemna gibba is imported into barley etiochloroplasts, it is processed to a single form. Both the processed form and the precursor are found in the thylakoid membranes, assembled into the light-harvesting complex of photosystem II. Neither form can be detected in the stromal fraction. The relative amounts of precursor and processed forms observed in the thylakoids are dependent on the developmental stage of the plastids used for uptake. The precursor as well as the processed form can also be detected in thylakoids of greening maize plastids used in similar uptake experiments. This detection of a precursor in the thylakoids, which has not been previously reported, could be a result of using rapidly developing plastids and/or using an heterologous system. Our results demonstrate that the extent of processing of LHCP precursor is not a prerequisite for its inclusion in the complex. They are also consistent with the possibility that the processing step can occur after insertion of the protein into the thylakoid membrane.     

Early events in the import/assembly pathway of an integral thylakoid protein

The light-harvesting chlorophyll a/b protein (LHCP) is nuclear-encoded and must traverse the chloroplast envelope before becoming integrally assembled into thylakoid membranes. Previous studies implicated a soluble stromal form of LHCP in the assembly pathway, but relied upon assays in which the thylakoid insertion step was intentionally impaired [Cline, K., Fulsom, D. R. and Viitanen, P. V. (1989) J. Biol. Chem. 264, 14225-14232]. Here we have developed a rapid-stopping procedure, based upon the use of HgCl2, to analyze early events of the uninhibited assembly process. With this approach, we have found that proper assembly of LHCP into thylakoids lags considerably behind trans-envelope translocation. During the first few minutes of import, two distinct populations of mature-size LHCP accumulate within the chloroplast. One is the aforementioned soluble stromal intermediate, while the other is a partially (or improperly) assembled thylakoid species. Consistent with precursor/product relationships, both species reach peak levels at a time when virtually none of the imported molecules are correctly assembled. These results confirm and extend our previous interpretation, that upon import, preLHCP is rapidly processed to its mature form, giving rise to a soluble stromal intermediate. They further suggest that the stromal intermediate initially inserts into the thylakoid bilayer in a partially assembled form, which eventually becomes properly assembled into the light-harvesting complex.     

An amino-proximal hydrophobic domain in the major light-harvesting chlorophyll a/b-protein is essential for membrane integration and protein stability

The major light-harvesting chlorophyll a/b-protein (LHCP) of higher plant chloroplasts is a nuclearencoded, integral thylakoid membrane protein that binds photosynthetic pigments and occurs in situ in an oligomeric form. We have previously examined structural and functional domains of the mature apoprotein by use of mutant LHCPs and in vitro assays for uptake and insertion. Results presented here demonstrate the effects of several mutations in the amino terminal domain of the mature apoprotein. Deletion of amino acid residues 12–58 greatly affected import into chloroplasts, while deletion or alteration of the hydrophobic region E⁶⁵VIHARWAM⁷³ led to rapid degradation of the mutant LHCP. We suggest that this amino-proximal region is essential for the stability of the LHCP and its ability to integrate into the thylakoid membranes. A structural/functional relationship of this region to a previously examined hydrophobic carboxy-proximal domain [Kohorn and Tobin (1989), The Plant Cell 1, 159–166] is proposed.Abbreviations BSA bovine serum albumin faction V - ELIPs early light-inducible proteins - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - LHCP light-harvesting chlorophyll a/b-protein - LHC IIb light-harvesting complex associated with Photosystem II - pLHCP precursor to LHCP - Rubisco ribulose 1,5-biphosphate carboxylase-oxygenase - SDS-PAGE sodium dodecyl sulfate-poly-acrylamide gel electrophoresis     

Light-harvesting chlorophyll a/b-binding protein inserted into isolated thylakoids binds pigments and is assembled into trimeric light-harvesting complex.

The light-harvesting chlorophyll a/b-binding protein (LHCP) is largely protected against protease (except for about 1 kD on the N terminus) in the thylakoid membrane; this protease resistance is often used to assay successful insertion of LHCP into isolated thylakoids in vitro. In this paper we show that this protease resistance is exhibited by trimeric light-harvesting complex of photosystem II (LHCII) but not by monomeric LHCII in which about 5 kD on the N terminus of LHCP are cleaved off by protease. When a mutant version of LHCP that is unable to trimerize in an in vitro reconstitution assay is inserted into isolated thylakoids, it gives rise to only the shorter protease digestion product indicative of monomeric LHCII. We conclude that more of the N-terminal domain of LHCP is shielded in trimeric than in monomeric LHCII and that this difference in protease sensitivity can be used to distinguish between LHCP assembled in LHCII monomers or trimers. The data presented prove that upon insertion of LHCP into isolated thylakoids at least part of the protein spontaneously binds pigments to form LHCII, which then is assembled in trimers. The dependence of the protease sensitivity of thylakoid-inserted LHCP on the oligomerization state of the newly formed LHCII justifies caution when using a protease assay to verify successful insertion of LHCP into the membrane.     

A chlorophyll b-less mutant of Chlamydomonas reinhardii lacking in the light-harvesting chlorophyll a/b-protein complex but not in its apoproteins

The mutant pg 113, derived from Chlamydomonas reinhardii, arg₂⁻ mt⁺ (parent strain), completely lacks chlorophyll (Chl) b but is still able to grow under autotrophic conditions. The light-harvesting Chl a/b-protein complex (LHCP) is absent. This is shown (a) by the lack of the corresponding signal in the CD spectrum of thylakoids and (b) by the absence of the band of the LHCP after electrophoresis of partially solubilized thylakoid membranes on lithium dodecyl sulfate polyacrylamide gels. All the other chlorophyll-protein complexes are present. In spite of the absence of the LHCP, all the polypeptide components of this complex are present in the mutant in the same ratios as in the parent strain, although in slightly reduced amounts. The LHC apoproteins are synthesized, processed and transported into the thylakoid membrane of the mutant. Moreover, the phosphorylation of thylakoid membrane polypeptides, which is related to the regulation of the energy distribution between Photosystem I and II, is the same in the mutant and in the parent strain, indicating that phosphorylation is not dependent on the presence of Chl b. Electron micrographs of thin sections of whole cells show that there are stacked regions of thylakoids in both the mutant and the parent strain chloroplasts. However, in the mutant, stacks are located near the chloroplast envelope, while long stretches or sometimes circles of unstacked membranes are found in the interior, mostly around the pyrenoid.     

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.     

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.     

The major light-harvesting complex of Photosystem II: aspects of its molecular and cell biology

The light-harvesting complex of photosystem II (LHC II) contains one major (LHC IIb) and at least three minor chlorophyll-protein components. The apoproteins of LHC IIb (LHCP) are encoded by nuclear genes and synthesized in the cytoplasm as a higher molecular weight precursor(s) (pLHCP). Several genes coding for pLHCP have been cloned from various higher plant species. The expression of these genes is dependent upon a variety of factors such as light, the developmental stage of the plastids and the plant. After its synthesis in the cytoplasm, pLHCP is imported into plastids, inserted into thylakoids, processed to its mature form, and assembled into LHC IIb. The pathway of assembly of LHC IIb in the thylakoid membranes is currently being investigated in several laboratories. We present a model that gives some details of the steps in the assembly process. Many of the steps involved in the synthesis and assembly are dependent on light and the stage of plastid development.Abbreviations PS Photosystem - LHC II Light-harvesting complex of PS II - LHCP Apoproteins of LHC IIb - pLHCP Precursor of LHCP - PAGE Polyacrylamide gel electrophoresis     

Replacement of Histidines of Light Harvesting Chlorophyll a/b Binding Protein II Disrupts Chlorophyll-Protein Complex Assembly

Eukaryotic light harvesting proteins (LHCPs) bind pigments and assemble into complexes (LHCs) that channel light energy into photosynthetic reaction centers. The structures of several prokaryotic LHCPs are known and histidines are important for the binding of the associated pigments. It has been difficult to predict how the eukaryotic LHCPs associate with pigments as the structure of the major LHCP of photosystem II is not yet known. While each LHCPII binds approximately 13 chlorophylls the protein contains only three histidines, one in each putative transmembrane helix. Experiments that use isolated pea (Pisum sativum L.) chloroplasts and mutant LHCPII synthesized in vitro show that the substitution of either an alanine or an arginine for each histidine residue inhibits some aspect of LHCII assembly. The histidine of the first membrane helix, but not the second or third, may be involved in the transport across the chloroplast envelope. No histidine alone is essential for the insertion of LHCP into thylakoid membranes, yet arginine substitutions are more inhibitory than those of alanine. The histidine replacements have their most pronounced effect on the assembly of LHCP into LHCII.     

Regulation of the GTPase cycle in post-translational signal recognition particle-based protein targeting involves cpSRP43

The chloroplast signal recognition particle consists of a conserved 54-kDa GTPase and a novel 43-kDa chromodomain protein (cpSRP43) that together bind light-harvesting chlorophyll a/b-binding protein (LHCP) to form a soluble targeting complex that is subsequently directed to the thylakoid membrane. Homology-based modeling of cpSRP43 indicates the presence of two previously identified chromodomains along with a third N-terminal chromodomain. Chromodomain deletion constructs were used to examine the role of each chromodomain in mediating distinct steps in the LHCP localization mechanism. The C-terminal chromodomain is completely dispensable for LHCP targeting/integration in vitro. The central chromodomain is essential for both targeting complex formation and integration because of its role in binding the M domain of cpSRP54. The N-terminal chromodomain (CD1) is unnecessary for targeting complex formation but is required for integration. This correlates with the ability of CD1 along with the ankyrin repeat region of cpSRP43 to regulate the GTPase cycle of the cpSRP-receptor complex.     

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.     

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.     

Evidence for the role of the light-harvesting chlorophyll a/b protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a/b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a/b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II and PS II_β to the fluorescence induction kinetics. PS II characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

A chlorophyll b-less mutant of Chlamydomonas reinhardii lacking in the light-harvesting chlorophyll complex but not in its apoproteins

The mutant pg 113, derived from Chlamydomonas reinhardii, arg₂⁻ mt⁺ (parent strain), completely lacks chlorophyll (Chl) b but is still able to grow under autotrophic conditions. The light-harvesting Chl complex (LHCP) is absent. This is shown (a) by the lack of the corresponding signal in the CD spectrum of thylakoids and (b) by the absence of the band of the LHCP after electrophoresis of partially solubilized thylakoid membranes on lithium dodecyl sulfate polyacrylamide gels. All the other chlorophyll-protein complexes are present. In spite of the absence of the LHCP, all the polypeptide components of this complex are present in the mutant in the same ratios as in the parent strain, although in slightly reduced amounts. The LHC apoproteins are synthesized, processed and transported into the thylakoid membrane of the mutant. Moreover, the phosphorylation of thylakoid membrane polypeptides, which is related to the regulation of the energy distribution between Photosystem I and II, is the same in the mutant and in the parent strain, indicating that phosphorylation is not dependent on the presence of Chl b. Electron micrographs of thin sections of whole cells show that there are stacked regions of thylakoids in both the mutant and the parent strain chloroplasts. However, in the mutant, stacks are located near the chloroplast envelope, while long stretches or sometimes circles of unstacked membranes are found in the interior, mostly around the pyrenoid.