Reconstitution and pigment-binding properties of recombinant CP29

The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4), overexpressed in Escherichia coli, has been reconstituted in vitro with pigments. The recombinant pigment-protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for the homologous protein light-harvesting chlorophyll-a/b-binding protein of photosystem II (LHCII). The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native one, two additional forms of the complex could be obtained by increasing chlorophyll b content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, F. G. & Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146-150; Paulsen, H., Rümler, U. & Rüdiger, W. (1990) Planta (Heidelb.) 181, 204-211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII.     

Quenching of chlorophyll a fluorescence in the aggregates of LHCII: steady state fluorescence and picosecond relaxation kinetics

The protein composition, steady state and time-resolved fluorescence emission spectra were studied in solubilized and aggregated LHCII complexes, that were prepared according to two different isolation protocols: (1) by fractionation of cation-depleted thylakoid membranes using the non-ionic detergent Triton X-100 according to the procedure of Burke et al. [(1978) Arch. Biochem. Biophys. 187, 252-263] or (2) by solubilization with N-beta-dodecyl maltoside (beta-DM) of photosystem II (PSII) membrane fragments in the presence of cations [Irrgang et al. (1988) Eur. J. Biochem. 178, 207-217]. Based on the analysis of the decay-associated emission spectra measured at 10 and 80 K five long-wavelength chlorophyll species were identified in aggregated LHCII complexes. These five forms are characterized by emission maxima at 681.5, 683, 687, 695, or 702 nm. All of these forms were found in both types of LHCII preparations but the relative amounts and temperature dependency of these species were markedly different in the aggregated LHCII complexes isolated by the two procedures. It was found that these differences cannot be simply explained by effects due to using a less mild detergent as beta-DM or by an ionic influence of Ca2+. Biochemical analysis of the protein composition showed that beta-DM type LHCII consists of all the chlorophyll (Chl)binding proteins belonging to the antenna system of PSII except the CP29 type II gene product (CP29). In contrast, the Triton X-100-solubilized LHCII is highly depleted in CP26 (CP 29 type I gene product) and is contaminated by a variety of unidentified polypeptides. It is proposed that the aggregates of LHCII prepared using Triton X-100 acquire specific spectral and kinetic features due to interaction between the bulk of LHCII subunits and minor protein(s).     

Energy transfer in LHCII monomers at 77K studied by sub-picosecond transient absorption spectroscopy

Energy transfer from chlorophyll b (Chl b) to chlorophyll a (Chl a) in monomeric preparations of light-harvesting complex II (LHCII) from spinach was studied at 77 K using pump-probe experiments. Sub-picosecond excitation pulses centered at 650 nm were used to excite preferentially Chl b and difference absorption spectra were detected from 630 to 700 nm. Two distinct Chl b to Chl a transfer times, approximately 200 fs and 3 ps, were found. A clearly distinguishable energy transfer process between Chl a molecules occurred with a time constant of 18 ps. The LHCII monomer data are compared to previously obtained LHCII trimer data, and both data sets are fitted simultaneously using a global analysis fitting routine. Both sets could be described with the following time constants: 140 fs, 600 fs, 8 ps, 20 ps, and 2.9 ns. In both monomers and trimers 50% of the Chl b to Chl a transfer is ultrafast (<200 fs). However, for monomers this transfer occurs to Chl a molecules that absorb significantly more toward shorter wavelengths than for trimers. Part of the transfer from Chl b to Chl a that occurs with a time constant of 600 fs in trimers is slowed down to several picoseconds in monomers. However, it is argued that observed differences between monomers and trimers should be ascribed to the loss of some Chl a upon monomerization or a shift of the absorption maximum of one or several Chl a molecules. It is concluded that Chl b to Chl a transfer occurs only within monomeric subunits of the trimers and not between different subunits.     

Protein-protein interactions of the light-harvesting chlorophyll a/b protein. II. Evidence for two stages of cation independent association

In a previous paper, we observed a two-stage cation-independent association of the light-harvesting chlorophyll a/b protein from spinach chloroplasts based on concentration-dependent changes in the sedimentation coefficient. The two stages of association occurred between (2-4) and (4-7) mug/ml chlorophyll. In this paper, we provide further evidence for this association. This includes: (1) A decrease in the number of divalent cation binding sites in the second stage of association. (2) A corresponding decrease in the extent of the cation-dependent association. (3) A positive deviation from Beer's law for chlorophyll b for both stages of the cation-independent association and a positive deviation for chlorophyll a for the second stage of association only. (4) A change in the fluorescence emission of both chlorophyll a and b. The change for chlorophyll b was observed for both steps of association whereas that for chlorophyll a was observed for the second step of association only. Therefore, the first stage of association affects only chlorophyll b whereas the second stage alters the environment of both chlorophyll a and b. (5) In addition, divalent cations quenched chlorophyll fluorescence. However, the quenching which required 200-300 muM divalent cation for half-maximal effects was related neither to divalent cation binding nor to the divalent cation-induced association of the protein.     

Chlorophyll a formation in the chlorophyll b reductase reaction requires reduced ferredoxin

The reduction of chlorophyllide b and its analogue zinc pheophorbide b in etioplasts of barley (Hordeum vulgare L.) was investigated in detail. In intact etioplasts, the reduction proceeds to chlorophyllide a and zinc pheophorbide a or, if incubated together with phytyldiphosphate, to chlorophyll a and zinc pheophytin a, respectively. In lysed etioplasts supplied with NADPH, the reduction stops at the intermediate step of 7(1)-OH-chlorophyll(ide) and Zn-7(1)-OH-pheophorbide or Zn-7(1)-OH-pheophytin. However, the final reduction is achieved when reduced ferredoxin is added to the lysed etioplasts, suggesting that ferredoxin is the natural cofactor for reduction of chlorophyll b to chlorophyll a. The reduction to chlorophyll a requires ATP in intact etioplasts but not in lysed etioplasts when reduced ferredoxin is supplied. The role of ATP and the significance of two cofactors for the two steps of reduction are discussed.     

Heterogenous lipid distribution among chlorophyll-binding proteins of photosystem II in maize mesophyll chloroplasts

Photosystem II membrane fractions from dark-adapted mesophyll chloroplasts of maize were solubilized in different concentrations of dodecyl beta-D-maltoside. Chlorophyll-binding proteins from photosystem II were isolated either by ultracentrifugation on a sucrose gradient, or by flat bed isoelectric focusing and identified by gel electrophoresis analysis for their polypeptide composition. Lipid and fatty acid compositions were determined in complexes prepared by both methods and also in purified light-harvesting complex II, in minor chlorophyll a/b binding complexes 29, 26, 24, in photosystem II antennae (chlorophyll-protein complexes 43, 47) and in the photosystem II reaction centers chlorophyll-protein complexes. Comparative analysis of the results suggests that a true heterogeneity exists in the lipid class distribution among the different chlorophyll-protein complexes in this region of the photosynthetic membrane. Photosystem II core fractions prepared either by ultra-centrifugation on a sucrose gradient or by isoelectric focusing were found significantly enriched in monogalactosyldiacylglycerol; fractionation of the photosystem II core in its components showed that it was the chlorophyll-protein complexes 43 and 47 which were mainly responsible for this enrichment. One of them, the chlorophyll-protein complex 47, was found containing monogalactosyldiacylglycerol and having a very high level of saturated fatty acids. The minor chlorophyll a/b binding linkers (chlorophyll-protein complexes 24, 26 and 29) retain a largely higher amount of lipids than all other complexes and especially of highly unsaturated galactolipids. Concerning the main light-harvesting antenna (LHCII), it is demonstrated that phosphatidylglycerol is strongly linked to the complex if it cannot be detached at high detergent concentration, while many galactolipids (which nevertheless represent the major lipid classes) are lost. This main light-harvesting complex has been fractionated into several families by isoelectric focusing showing a marked difference in lipid and polypeptide composition. A spectacular increase in the phosphatidylglycerol content was observed in the fraction migrating near the anode and enriched in a 26-kDa polypeptide; but this result is difficult to interpret in physiological terms as it was shown that phosphatidylglycerol alone, because of its negative charge, also migrates toward the anode in isoelectric focusing.     

Membrane topography of the T domain of diphtheria toxin probed with single tryptophan mutants

The membrane insertion and translocation of diphtheria toxin, which is induced in vivo by low pH, is thought to be directed by the hydrophobic alpha-helices of its transmembrane (T) domain. In this study the structure of membrane-associated T domain was examined. Site-directed mutants of the T domain with single Trp residues were prepared at the two naturally occurring positions, 206 (near the N-terminal end of helix TH1) and 281 (within helix TH5), as well as at three residues in helix TH9, in which the substitutions F355W (near the N-terminal end of TH9), I364W (close to the center of TH9), and Y375W (near the C-terminal end of TH9) were made. All these mutants were found to undergo the low-pH-induced conformational change observed with wild-type T domain and insert into model membranes at low pH. The location of Trp residues relative to the lipid bilayer was characterized in model membrane vesicles by fluorescence emission and by quenching with nitroxide-labeled phospholipids. In TH9, residue 375 was shallowly inserted, residue 364 deeply inserted, and residue 355 located at an intermediate depth. Residues 206 and 281 exhibited moderately deep insertion. It was also found, in agreement with our previous study using bimane-labeled protein (Wang et al. (1997) J. Biol. Chem. 272, 25091-25098), that TH9 switches from a relatively shallowly inserted state to a more deeply inserted state when the concentration of the T domain in the membrane is increased or the thickness of the membrane bilayer is decreased. In particular, the depth of residue 355 was found to increase under the conditions giving deeper insertion. In contrast, residue 375 remained shallowly located in both states, as predicted from its location on the polar C-terminus of TH9. It is concluded that TH1 and TH5 insert into the lipid bilayer in both T domain conformations. In addition, Trp depths suggest that even in the shallowly inserted state there is a significant degree of insertion of TH9. These results suggest regions of the T domain in addition to the hydrophobic TH8/TH9 hairpin insert into membranes. Models for the structure of the membrane-inserted T domain are discussed.     

Spatial relationship of photosystem I, photosystem II, and the light-harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent-light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment-protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

On the role of Arg-210 and Glu-219 of subunit a in proton translocation by the Escherichia coli F0F1-ATP synthase

A strain of Escherichia coli was constructed which had a complete deletion of the chromosomal uncB gene encoding subunit a of the F0F1-ATP synthase. Gene replacement was facilitated by a selection protocol that utilized the sacB gene of Bacillus subtilis cloned in a kanamycin resistance cartridge (Ried, J. L., and Collmer, A. (1987) Gene (Amst.) 57, 239-246). F0 subunits b and c inserted normally into the membrane in the DeltauncB strain. This observation confirms a previous report (Hermolin, J., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 2815-2817) that subunit a is not required for the insertion of subunits b and c. The DeltauncB strain has been used to characterize mutations in Arg-210 and Glu-219 of subunit a, residues previously postulated to be essential in proton translocation. The aE219G and aE219K mutants grew on a succinate carbon source via oxidative phosphorylation and membranes from these mutants exhibited ATPase-coupled proton translocation (i.e. ATP driven 9-amino-6-chloromethoxyacridine quenching responses that were 60-80% of wild type membranes). We conclude that the aGlu-219 residue cannot play a critical role in proton translocation. The aR210A mutant did not grow on succinate and membranes exhibited no ATPase-coupled proton translocation. However, on removal of F1 from membrane, the aR210A mutant F0 was active in passive proton translocation, i.e. in dissipating the DeltapH normally established by NADH oxidation with these membrane vesicles. aR210A membranes with F1 bound were also proton permeable. Arg-210 of subunit a may play a critical role in active H+ transport that is coupled to ATP synthesis or hydrolysis, but is not essential for the translocation of protons across the membranes.     

The protease-protected 30 kDa domain of SecA is largely inaccessible to the membrane lipid phase

SecA binds to the inner membrane of Escherichia coli through low affinity lipid interactions or with high affinity at SecYEG, the integral domain of preprotein translocase. Upon addition of preprotein and nucleotide, a 30 kDa domain of SecYEG-bound SecA is protected from proteolysis via membrane insertion. Such protection could result from some combination of insertion into the lipid phase, into a proteinaceous environment or across the membrane. To assess the exposure of SecYEG-bound SecA to membrane lipids, a radiolabeled, photoactivatable and lipid-partitioning crosslinker, 3-trifluoromethyl-3-(m[125I]iodophenyl) diazirine benzoic acid ester, was incorporated into inner membrane vesicles. The 30 kDa domain of SecYEG-bound SecA, inserted into the membrane in response to translocation ligands, is 18-fold less labeled than SecY, which is labeled effectively. In contrast, incorporation of the purified 30 kDa SecA fragment into crosslinker-containing detergent micelles or addition of detergent to crosslinker-containing membranes bearing the protease-protected SecA domain readily allows for labeling of this domain. We propose that the protease-inaccessible 30 kDa SecA domain is shielded from the fatty acyl membrane phase by membrane-spanning SecYEG helices and/or is largely exposed to the periplasm.     

Mutant ATP-binding RNA aptamers reveal the structural basis for ligand binding

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced > or = 40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-SecA function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.     

Molecular cloning and functional expression of a water-soluble chlorophyll protein, a putative carrier of chlorophyll molecules in cauliflower

A cDNA for a water-soluble chlorophyll (Chl) protein (WSCP) from cauliflower (Brassica oleracea L. var botrys) was cloned and sequenced. The cDNA contained an open reading frame encoding 19 residues for a signal peptide and 199 residues for the mature form of WSCP. The sequence showed extensive homology to drought-stress-related, 22-kDa proteins in some Brassicaceae plants. Functional WSCP was expressed in Escherichia coli as a fusion protein with a maltose-binding protein (MBP). When the recombinant MBP-WSCP was incubated with thylakoid membranes, the MBP-WSCP removed Chls from these membranes. During this process, the monomer of the apo-MBP-WSCP successfully bound Chls and was converted into tetrameric holo-MBP-WSCP. The reconstituted MBP-WSCP exhibited absorption and fluorescent spectra identical to those of the native WSCP purified from cauliflower leaves. The Chl a/b ratio in native WSCP indicates a high content of Chl a, which was mainly due to the higher affinity of MBP-WSCP for Chl a. WSCP is the first example of a hydrophilic protein that can transfer Chls from thylakoid hydrophobic proteins. Possible functions of WSCP are discussed.     

Light-dependent chlorophyll a biosynthesis upon chlL deletion in wild-type and photosystem I-less strains of the cyanobacterium Synechocystis sp. PCC 6803

Part of the chlL gene encoding a component involved in light-independent protochlorophyllide reduction was deleted in wild type and in a photosystem I-less strain of Synechocystis sp. PCC 6803. In resulting mutants, chlorophyll biosynthesis was fully light-dependent. When these mutants were propagated under light-activated heterotrophic growth conditions (in darkness except for 15 min of weak light a day) for several weeks, essentially no chlorophyll was detectable but protochlorophyllide accumulated. Upon return of the chlL- mutant cultures to continuous light, within the first 6 h chlorophyll was synthesized at the expense of protochlorophyllide at a rate independent of the presence of photosystem I. Chlorophyll biosynthesized during this time gave rise to a 685 nm fluorescence emission peak at 77 K in intact cells. This peak most likely originates from a component different from those known to be directly associated with photosystems II and I. Development of 695 and 725 nm peaks (indicative of intact photosystem II and photosystem I, respectively) required longer exposures to light. After 6 h of greening, the rate of chlorophyll synthesis slowed as protochlorophyllide was depleted. In the chlL- strain, greening occurred at the same rate at two different light intensities (5 and 50 microE m-2 s-1), indicating that also at low light intensity the amount of light is not rate-limiting for protochlorophyllide reduction. Thus, in this system the rate of chlorophyll biosynthesis is limited neither by biosynthesis of photosystems nor by the light-dependent protochlorophyllide reduction. We suggest the presence of a chlorophyll-binding 'chelator' protein (with 77 K fluorescence emission at 685 nm) that binds newly synthesized chlorophyll and that provides chlorophyll for newly synthesized photosynthetic reaction centers and antennae.     

Synthesis in vitro of intrinsic membrane proteins by free, membrane-bound, and Golgi apparatus-associated polyribosomes from rat liver

A fraction of intrinsic membrane proteins was prepared from the major membranous cell components of rat liver by extraction of the membranes with KCl and deoxycholate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the compositions of the intrinsic protein fractions from rough and endoplasmic reticulum, smooth endoplasmic reticulum. Golgi apparatus, plasma membrane, and nuclear envelope were similar to each other but distinct from that of mitochondria. Among endomembranes, differences were in the ratios of protein constituents plus a few protein bands of Golgi apparatus and plasma membranes not found in endoplasmic reticulum or nuclear envelope. The abilities of total rough endoplasmic reticulum, polysomes released from rough endoplasmic reticulum, and free polysomes to incorporate amino acids into the intrinsic protein fraction were tested in vitro. Polysomes bound to endoplasmic reticulum has the greatest capacity to synthesize proteins of this fraction as shown by co-purification of radioactive products and by immunoprecipitation. Although the majority of the radioactive products synthesized by bound polysomes were distinct from those synthesized by free polysomes, certain radioactive products synthesized by free polysomes also co-purified with intrinsic membrane proteins. The results show no absolute segregation between free and bound polysomes in the synthesis of intrinsic membrane proteins. However, the majority of these proteins appear to be synthesized by polysomes bound to the endoplasmic reticulum. Several intrinsic proteins found in plasma membranes do not appear in rough endoplasmic reticulum. To determine where these proteins were synthesized, the ability of other endomembrane components to support in vitro incorporation of [14C]leucine into protein was examined. In contrast to plasma membranes, isolated Golgi apparatus fractions did incorporate [14C]leucine to an extent greater than could be explained by contamination with rough endoplasmic reticulum. Golgi apparatus in situ and isolated from rat liver have polyribosomes associated with a zone of cytoplasm at the Golgi apparatus periphery occupied by tubules and vesicles. The polysomes are not directly attached to membranes as with rough endoplasmic reticulum and may represent a special class of "Golgi apparatus-associated" polysomes. The polysomes, when associated with Golgi apparatus membranes, incorporated amino acids in vitro. The products synthesized in vitro were analyzed by treatment with KCl and deoxycholate and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Certain proteins synthesized by the Golgi apparatus-associated polysomes remained insoluble after the treatment with KCl and deoxycholate. The proteins synthesized by the Golgi apparatus fraction had mobilities similar to proteins in plasma membranes which were absent from endoplasmic reticulum, and which were relatively minor components of Golgi apparatus...     

Folding intermediates of a beta-barrel membrane protein. Kinetic evidence for a multi-step membrane insertion mechanism

The mechanism of folding and membrane insertion of integral membrane proteins, including helix bundle and beta-barrel proteins is not well understood. A key question is whether folding and insertion are coupled or separable processes. We have used the beta-barrel outer membrane protein A (OmpA) of Escherichia coli as a model to study the kinetics of folding and insertion into dioleoylphosphatidylcholine (DOPC) bilayers, as a function of temperature by gel electrophoresis, protease digestion, and fluorescence spectroscopy. OmpA was unfolded in 8 M urea solution (without detergent), and refolding and membrane insertion was initiated by rapid dilution of the urea concentration in the presence of phospholipid vesicles. In addition to the kinetically unresolved hydrophobic collapse in water, the time course of refolding of OmpA into DOPC bilayers exhibited three kinetic phases over a large temperature range. The first step was fast (k1 = 0.16 min-1) and not very dependent on temperature. The second step was up to two orders of magnitude slower at low temperatures (2 degrees C), but approached the rate of the first step at higher temperatures (40 degrees C). The activation energy for this process was 46 +/- 4 kJ/mol. A third slow process (k3 = 0.9 x 10(-2) min-1 at 40 degrees C) was observed at the higher temperatures. These results suggest that at least two membrane-bound intermediates exist when OmpA folds and inserts into lipid bilayers. We also show that both membrane-bound intermediates can be stabilized in fluid lipid bilayers at low temperatures. These intermediates share many properties with the adsorbed/partially inserted form of OmpA that was previously characterized in gel phase lipid bilayers [Rodionova et al. (1995) Biochemistry 34, 1921-1929]. Temperature jump experiments demonstrate, that the low-temperature intermediates can be rapidly converted to fully inserted native OmpA. On the basis of these and previous results, we present a simple folding model for beta-barrel membrane proteins, in which folding and membrane insertion are coupled processes which involve at least four kinetically distinguishable steps.