Immunogold localization of LHC II proteins in chloroplasts with different degrees of grana stacking induced by SAN-9789.

The amount and distribution of proteins of the light-harvesting complex associated with photosystem II (PS II) were investigated using immunogold labelling of chloroplasts of wheat ( Triticum aestivum L. cv. Walde). The seedlings were grown in weak red light (16 mW m⁻²) after imbibition of grains with SAN-9789 (Norflurazon, 0.028 to 28 mg I⁻¹). Chloroplasts of these plants exhibited thylakoids with different degrees of stacking. Thylakoids of untreated plants grown in a greenhouse had most gold particles per unit membrane length in both appressed and non-appressed regions compared to red light grown plants. The ratios of labelling between appressed and non-appressed membranes were fairly constant in red light- and greenhouse-grown plants. The labelling densities were 2.5–3 times higher in the appressed thylakoids compared to the non-appressed thylakoids. However, at a SAN concentration of 2.8 mg I⁻¹ there was a sharp decrease in thylakoid appressions and in labelling density of both appressed and non-appressed membranes. The total amount of particles per chloroplast was also much lower as compared to that at lower SAN concentrations. Plants treated with the highest concentration of SAN (28 mg I⁻¹) contained chloroplasts devoid of normal grana structures. In these plastids, the thylakoids were elongated and single. The labelling density in these membranes was ca 50% of that observed at 2.8 mg I⁻¹. This paper thus supports earlier observations that proteins of the light-harvesting complex of PS II (LHC II) are mainly localized in the appressed regions of the grana membranes, and may be involved in the formation of grana.     

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.     

Expression of genes for plastid membrane proteins in barley under intermittent light conditions

Barley plants grown under intermittent light show a plastid membrane composition intermediate between those of etioplasts and chloroplasts. In particular protochlorophyll reductase disappears from the membranes whereas the 32000 protein, coded for by chloroplast DNA, becomes integrated into the membranes. The light-harvesting chlorophyll a/b protein does not accumulate within the membranes even after 11 d of development, while the corresponding mRNA can already be observed after 4 d and is translated under in vivo conditions.Abbreviations LHCP light-harvesting chlorophyll a/b protein - IL intermittent light - LD light-dark (12-h day) - EGTA ethyleneglycol-bis(oxy-ethylenenitrile)tetraacetic acid     

Import of nuclear-encoded proteins into carotenoid-deficient young etioplasts

Young etioplasts with different carotenoid contents were assayed for their ability to import in vitro synthesized nuclear-encoded proteins. The plastids were isolated from the basal 1. 5cm of dark-grown wheat seedlings developed from seeds imbibed with 4 different concentrations of Norflurazon. an inhibitor of the carotenoid biosynthesis. Plastids isolated from plants treated with the two highest concentrations. 2. 8 and 28 mg l⁻¹, of Norflurazon contained approximately 10 and 5% of the carotenoid contents, respectively, compared to the control. The total amounts of proteins in these plastids were approximately 68 and 60% compared to control plastids. Translocation assays employing the precursors of the small subunit of ribulose 1. 5-bisphosphate carboxylase/oxygenase (pSS), and the non-Photosynthetic heat-shock protein 21 (pHSP21), showed that the rate of protein import was considerably lower in plastids with low carotenoid contents. The amounts of imported, processed SS were 11 and 10% after 2. 8 and 28 mg 1⁻¹, respectively, compared to the control, whereas the amounts of HSP21 at these herbicide concentrations were 20 and 18%, respectively. The low apparent import in plastids of Norflurazon-treated leaves was not an effect of intraorganellar degradation of imported proteins, nor were there any differences in the amounts of processed, protease-protected protein when Norflurazon was added to the import reaction using control plastids. The low import capabilities are therefore discussed in relation to the possible role of the carotenoids in the translocation of cytosolically synthesized proteins into the plastidic compartment.     

Chloroplast photooxidation affects the accumulation of cytosolic mRNAs encoding chloroplast proteins in maize

Maize (Zea mays L.) seedlings were grown in the presence or absence of an herbicide, norflurazon (4-chloro-5-(methylamino)-2-(,,-trifluoro-m-tolyl)-pyridazinone), which prevents the accumulation of colored carotenoids. In the absence of carotenoids, plants grown in high light incur extensive photooxidative damage to their plastids, but relatively little damage elsewhere. Growth in very low light minimizes chlorophyll photooxidation and allows chloroplast development to proceed. We have previously reported that mRNA encoding light-harvesting chlorophyll a/b protein (LHCP) fails to accumulate in high-light-grown carotenoid-deficient seedlings, but accumulates normally in carotenoid-deficient seedlings grown in low light. Here we extend these results by examining the levels of translatable mRNAs encoding seven additional nuclear-encoded chloroplast proteins. When norflurazon-treated seedlings were grown in low light for 8 d and then transferred to high light for 24 h, three cytosolic mRNAs (plastocyanin, Rieske Fe–S protein, and the 33-kdalton (kDa) subunit of the photosystem II O₂-evolving complex) decreased to less than 1% the amount found in untreated seedlings. Two other mRNAs (NADP malic enzyme, EC 1.1.1.40, and the 23-kDa subunit of the photosystem II O₂-evolving complex) decreased significantly but not to levels as low as the first three. Levels of translatable mRNA for two other chloroplast proteins (pyruvate orthophosphate dikinase, EC 2.7.9.1, and ferredoxin NADP oxidoreductase, EC 1.18.1.2) were not reduced in nonflurazon-treated seedlings after 24 h in high light, but did not show the normal light-induced increase found in untreated plants. Photooxidative damage in the chloroplast thus affects the accumulation of a number of cytosolic mRNAs encoding proteins destined for the chloroplast.Abbreviations Da dalton - FNR ferredoxin NADP oxidoreductase - LHCP light-harvesting chlorophyll a/b-binding protein - poly(A)RNA polyadenylated RNA - PPDK pyruvate orthophosphate dikinase - PSII photosystem II - SDSPAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SSu small subunit (of ribulose-1,5-bisphosphate carboxylase)     

Correlation between pigment composition and apoproteins of the light-harvesting complex II (LHC II) in wheat (Triticum aestivum)

Seedlings of wheat (Triticum aestivum L. cv. Walde, Weibull) grown in continuous weak red light (16 mW m⁻²) with or without SAN-9789, contained significantly lower amounts of chlorophylls and carotenoids compared to untreated plants grown in a greenhouse. The Chl alb ratios were 3.6 in the greenhouse-grown plants, 5.1 in untreated and ca 16 in SAN-treated plants grown in weak red light, respectively. The main difference in polypeptide composition of thylakoids isolated from red light-grown plants, compared to those grown in the greenhouse, was a lower amount of proteins of the light-harvesting complex (LHC) II. The amount of apo-LHC and LHC were correlated to the xanthophyll to β-carotene ratios in these plants. The absence of grana and the absence of proteins of the light-harvesting complex 11 in SAN-treated plants, support the general dogma that these proteins are involved in the formation of grana. Since the amount of apo-LHC and LHC could be correlated to the presence of carotenoids as well as the chlorophylls, it is concluded that the carotenoids are necessary for the correct assembly and stabilization of the apoproteins of LHC II in the thylakoid membranes.     

Surface Charge Densities and Membrane Fluidities in Thylakoids with Different Degrees of Thylakoid Appression After Norflurazon Treatment

Wheat seedlings (Triticum aestivum L.) develop plastids (etioplasts and chloroplasts) which exhibit alterations in inner membrane organisation after treatment with Norflurazon (NF), an inhibitor of carotenoid biosynthesis. In dark-grown plants, it results in a decreased amount of partitions (contact zones) between prothylakoids. Under weak red radiation (WRR), plants contain chloroplasts devoid of grana. Using the fluorescent probe 9-amino acridine (9-AA), the average surface charge density of isolated prothylakoids (PTs) was –21.8±3.2 mC m^–2 and –27.4±2.6 mC m^–2 in the control and after treatment, respectively. Thylakoid membranes isolated from plants grown under WRR exhibited slightly more negative values, –23.5±2.9 mC m^–2 and –29.0±2.1 mC m^–2, in control and after NF treatment, respectively. The surface charge density of de-stacked thylakoids from greenhouse-grown untreated plants, containing extensive grana stacking, was –34.3±2.5 mC m^–2. Assays using the fluorescent probe of DPH (1,6-diphenyl-1,3,5-hexatriene) showed a higher polarisation value when incorporated into thylakoids from NF-treated plants compared to untreated plants grown under WRR. The highest polarisation value was found in untreated plants grown in the greenhouse. This indicates a lower rotation transition of the probe in the lipid environment of thylakoids after NF treatment, which can be interpreted as more rigid membranes. Hence the surface charge density and the mobility of membrane components may play a major role for the formation of partitions in dark-grown plants and in the formation of grana in plants grown under WRR.     

Photocontrol of phenylalanine ammonia-lyase in barley seedlings treated with pyridazinone inhibitors of chloroplast development

Seedlings imbibed for 48 hr in aqueous solutions of the pre-emergent herbicide Sandoz 6706, or its presumably active conversion product Norflurazon, grow into albino plants in white light. Neither herbicide has any effect on PAL in dark grown barley shoots. In white light, however, pretreatment with 100 μM herbicide causes an increase in barley shoot PAL of about 50% over that found in untreated plants. Barley root PAL is stimulated by 0.1 μM Sandoz 6706 but inhibited by higher concentrations. Mung bean primary leaves show dose responses similar to barley roots. The herbicides have no effect in continuous red light, yet blue light is as effective as white light in eliciting PAL responses. The results are discussed in relation to the subcellular distribution of PAL.     

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.     

Light-quality and irradiance-level control of light-harvesting complex of photosystem 2 in maize mesophyll cells. Evidence for a low fluence-rate threshold in blue-light reduction of mRNA and protein

Levels of pigment-proteins and mRNA coding for proteins associated with the light-harvesting complex of photosystem 2 (LHCP2) were reduced in maize ( Zea mays L. cv. OP Golden Bantum) plants grown for 14 days in 8.0 nmol m^-2s^-1 of blue light compared to those in plants grown under an equal irradiance of red light. At the same time, there was a small increase in steady state levels of mRNA for the Dl protein of PS2 (psbA) in blue-grown plants. The reduction of LHCP2 mRNA and the increase in psbA mRNA were observed in both 5- and 10-day-old blue-light-grown leaves, but the degree of reduction or increase was much greater in 10-day-old leaves. Maize grown under 6 different mixtures of blue and red light, each with a total irradiance level of 8.0 μmol m^-2 s^-1, showed the same degree of LHCP2 mRNA reduction relative to red light. This is different from the behavior of psbA which increased in a linear manner with increasing amounts of blue light. The amounts of Chi a and Chi b in these mixed-light samples were not significantly different froi those found in pure red light. This indicates that a low fluence level of blue light, even when combined with red light, is sufficient to reduce equilibrium levels of proteins and mRNA of LHCP2, and this reduction is independent of pigment formation. It also suggests that the mechanisms of blue-light regulation of mRNA may operate differently at the nuclear and chloroplast levels.     

An imported thylakoid protein accumulates in the stroma when insertion into thylakoids is inhibited

The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP) that is imported into chloroplasts and assembled into thylakoid membranes. Under appropriate conditions, either pLHCP or LHCP will integrate into isolated thylakoids. We have identified two situations that inhibit integration in this assay. Ionophores and uncouplers inhibited integration up to 70%. Carboxyl-terminal truncations of pLHCP also interfered with integration. A 22-residue truncation reduced integration to about 25% of control, whereas a 93 residue truncation completely abolished it. When pLHCP was imported into chloroplasts in the presence of uncouplers or when truncated forms of pLHCP were used, significant amounts of the imported proteins failed to insert into thylakoids and instead accumulated in the aqueous stroma. Accumulation of stromal LHCP occurred at uncoupler concentrations required to dissipate the trans-thylakoid proton electrochemical gradient and was enhanced at reduced levels of ATP. The latter effect may be a secondary consequence of a reduction in ATP-dependent degradation within the stroma. These results indicate that the stroma is an intermediate location in the LHCP assembly pathway and provide the first evidence for a soluble intermediate during biogenesis of a chloroplast membrane protein.     

Effect of light quality on chloroplast-membrane organization and function in pea

The effect of light quality during plant growth of chloroplast membrane organization and function in peas (Pisum sativum L. cv. Alaska) was investigated. In plants grown under photosystem (PS) I-enriched (far-red enriched) illumination both the PSII/PSI stoichiometry and the electrontransport capacity ratios were high, about 1.9. In plants grown under PSII-enriched (far-red depleted) illumination both the PSII/PSI stoichiometry and the electron-transport capacity ratios were significantly lower, about 1.3. In agreement, steady-state electron-transport measurements under synchronous illumination of PSII and PSI demonstrated an excess of PSII in plants grown under far-red-enriched light. Sodium dodecylsulfate polyacrylamide gel electrophoretic analysis of chlorophyll-containing complexes showed greater relative amounts of the PSII reaction center chlorophyll-protein complex in plants grown under farred-enriched light. Additional changes were observed in the ratio of light-harvesting chlorophyll a/b protein to PSII reaction center chlorophyll-protein under the two different light-quality regimes. The results demonstrate the dynamic nature of chloroplast structure and support the notion that light quality is an important factor in the regulation of chloroplast membrane organization and-function.Abbreviations and symbols Chl chlorophyll - CPa PSII reaction center chlorophyll protein complex - CPI PSI chlorophyll protein complex - FR-D light depleted in far-red sensitizing primarily PSII - FR-E light enriched in far-red sensitizing primarily PSI - LHCP PSII light-harvesting chlorophyll a/b protein complex - P ₇₀₀ primary electron donor of PSI - PSI, PSII photosystems I and II, respectively - Q primary electron acceptor of PSII     

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.     

Carotenoid-deficient young wheat etioplasts are able to bind precursor proteins on the plastid surface but are impaired in their translocation ability

Young carotenoid-deficient etioplasts, isolated from Norflurazon (NF)-treated wheat seedlings, were used to study the role of coloured carotenoids in the binding and import reactions of different nuclear-encoded plastid proteins. Plastids from control seedlings exhibited significantly higher import efficiencies than did plastids from NF-treated plants. Etioplasts containing normal levels of carotenoids imported approximately 2000 and 800 molecules per plastid of the precursors of the small Rubisco subunit (pSS) and the Rieske FeS protein (pFeS), respectively. Plastids from NF-treated plants imported approximately 100 and 70 pSS and pFeS molecules per plastid, respectively. In addition, a maximum binding capacity of NF-treated plastids of 1200 protein molecules per plastid was observed for both pSS and pFeS when assayed at 25°C: and a maximum binding capacity of approximately 1300 molecules per plastid was noted at 4°C. For control plastids, a similar amount of binding, or approximately 1400 protein molecules per plastid, could only be observed if import was inhibited by low ATP concentrations at 4°C. When these plastids were washed and transferred to conditions promoting import at 25°C and 10 mM Mg-ATP, close to 60% of the envelope-associated precursor protein molecules were imported. These results indicate that control and NF-treated young etioplasts contain similar amounts of binding sites for precursor proteins. However, only in the case of control plastids the binding was productive and lead to import and processing in the stroma upon transfer to conditions promoting import. Plastids isolated from wheat seedlings grown in weak red light and containing different amounts of carotenoids, were assayed for their ability to bind and import a protein with unusual import characteristics, the Chlamydomonas reinhardtii PsaF precursor of PSI (pPsaF) and transit peptide deletion constructs. The PsaF protein was imported in a transit peptide-dependent manner into control etioplasts, whereas import of pPsaF into young wheat etioplasts isolated from NF-treated plants was inhibited at low levels of plastid carotenoids.     

The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein

The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein (LHCP) is investigated in wild-type barley (Hordeum vulgare L.) and in the chlorophyll b-less mutant chlorina f2. In dark-grown plants a short red light pulse triggers the appearance of mRNA activity for the LHCP. While the accumulation of this mRNA is controlled by phytochrome (Apel (1979) Eur. J. Biochem. 97, 183–188), the red light treatment is not sufficient to induce the appearance of the LHCP within the membrane. Thus, at least one of the subsequent steps in the biosynthetic pathway leading to the assembly of the LHCP is controlled by light. The red light-induced mRNA is taken up into the polysomes during the subsequent dark period and is translated in vitro in a cell-free protein synthesizing system. However, an accumulation of the freshly synthesized polypeptide within the plant is not observed. The apparent instability of the polypeptide might be explained by the deficiency of chlorophyll in the red light-treated plants. In the chlorophyll b-less barley mutant chlorina f2 an accumulation of the freshly synthesized apoprotein of the LHCP can be observed in the light. Thus, chlorophyll a formation seems to be a light-dependent step which is required for the stabilization of the LHCP.Abbreviations mRNA messenger RNA - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecylsulfate - LHCP light-harvesting chlorophyll a/b protein     

The Regulation and Distribution of LHC-I and LHCP2 between the Photosystem I and Photosystem II

Evidences were provided in this paper that the relative distribution of chl-protein complexes of PSⅠ and PSⅡ could be regulated by Mg2+. addition of Mg2+ led to decrease in the amount of chl-protein complexes of PSⅠ and increase in the amount of chl-protein in complexes of PSⅡ. There was no effect of Mg2+ on the spectral property of LHCP1, but the addition of Mg2+ could change the spectral property of LHCP2 so that it became similar to that of the LHC-Ⅰ. CPIa2 was a complex of reaction centre of PSⅠ and LHC-I. LHC-I might be contacted specially with LHCP2 in chloroplast membranes. Addition of Mg2+ probably cansed the motion of LHC-I from PSⅠ to PSⅡ and became more closely connected with LHCP2. The relative amount of CPIa2, CPIa1, LHCP1 and LHCP2 in chloroplast membranes could be regulated by different light intensity. There were more CPIa2, LHCP1 and less LHCP2 in chloroplast membranes from the shade plant Malaxis monophyllos and sunflower grown under weak light, both of them lacked equally CPIa1. There were less CPIa2, LHCP1 and more LHCP2 in the sun plant spinach and sunflower grown under strong light, and they possessed equally CPIa1 chl-protein complexes. It is suggested that LHCP1 and LHCP2 are different light-harvesting Chl-protein complexes. The LHC-I and LHCP2 are mobile light-harvesting chl-protein complexes and shuttle back and forth between PSⅠ and PSⅡ They play an important role in the regulation and distribution of excitation energy between the two photosystems.     

Photooxidative destruction of chloroplasts and its consequences for expression of nuclear genes

Expression of nuclear genes involved in plastidogenesis is known to be controlled by light via phytochrome. Examples are the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase and the light harvesting chlorophyll a/b binding protein of photosystem II (LHCP). In the present study we show that, beside phytochrome, the integrity of the plastid is essential for the expression of the pertinent nuclear genes as measured at the level of translatable mRNA. When the plastids are severely damaged by photooxidation in virtually carotenoid-free mustard (Sinapis alba L.) seedling cotyledons (made carotenoid-free by the application of Norflurazon, NF), almost no SSU, no SSU precursor, LHCP and LHCP precursor can be detected by immunological assays, and almost no translatable mRNA of SSU and LHCP can be found, although the levels and rates of phytochrome-mediated syntheses of representative cytoplasmic, mitochondrial and glyoxisomal enzymes are not adversely affected and morphogenesis of the mustard seedling proceeds normally (Reiß et al. 1983; Planta 159, 518–528). Norflurazon per se has no effect on the amount of translatable mRNA of SSU and LHCP as shown by irradiation of NF-treated seedlings with far-red light (FR) which strongly activates phytochrome but does not cause photooxidation in the plastids. It is concluded that a signal from the plastid is required to allow the phytochrome-mediated appearance of translatable mRNA for SSU and LHCP. Seedlings not treated with NF show a higher level of translatable mRNA_LHCP in red light (RL) compared to FR, whereas the mRNA_SSU levels are the same in RL and FR. These facts indicate that the level of translatable mRNA_LHCP is adversely affected if the apoprotein is not incorporated into the thylakoid membrane.Abbreviations FR far-red light (3.5 W m^-2) - LHCP light harvesting chlorophyll a/b binding protein of photosystem II - LSU large subunit of RuBPCase - NF Norflurazon - RL red light (6.8 W m^-2) - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - SSU small subunit of RuBPCase - WL white light (28 W m^-2)     

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.