Absence of light-induced proton extrusion in a cotA-less mutant of Synechocystis sp. strain PCC6803.

cotA of Synechocystis sp. strain PCC6803 was isolated as a gene that complemented a mutant defective in CO2 transport and is homologous to cemA that encodes a chloroplast envelope membrane protein (A. Katoh, K.S. Lee, H. Fukuzawa, K. Ohyama, and T. Ogawa, Proc. Natl. Acad. Sci. USA 93:4006-4010, 1996). A mutant (M29) constructed by replacing cotA in the wild-type (WT) Synechocystis strain with the omega fragment was unable to grow in BG11 medium (approximately 17 mM Na+) at pH 6.4 or at any pH in a low-sodium medium (100 microM Na+) under aeration with 3% (vol/vol) CO2 in air. The WT cells grew well in the pH range between 6.4 and 8.5 in BG11 medium but only at alkaline pH in the low-sodium medium. Illumination of the WT cells resulted in an extrusion followed by an uptake of protons. In contrast, only proton uptake was observed for the M29 mutant in the light without proton extrusion. There was no difference in sodium uptake activity between the WT and mutant. The mutant still possessed 51% of the WT CO2 transport activity in the presence of 15 mM NaCl. On the basis of these results we concluded that cotA has a role in light-induced proton extrusion and that the inhibition of CO2 transport in the M29 mutant is a secondary effect of the inhibition of proton extrusion.     

Cloning of the cotA gene of Synechococcus PCC7942 and complementation of a cotA-less mutant of Synechocystis PCC6803 with chimeric genes of the two strains

cotA, a homologue of cemA that encodes a chloroplast envelope membrane protein, was cloned from Synechococcus PCC7942. The gene encodes a protein of 421 amino acids, which is similar in size to CotA of Synechocystis PCC6803 and CemA of liverwort and Chlamydomonas. There was significant sequence homology among these CotA and CemA in the C-terminal region but the homology was low in the N-terminal region. Sequencing of Synechococcus DNA in the cotA region revealed two other genes downstream of cotA, one of which is homologous to cobP and could be cotranscribed with cotA. A mutant (M48) was constructed by inactivating cotA in the wild-type (WT) Synechococcus. The mutant showed the same characteristics as the cotA-deletion mutant of Synechocystis (M29) and was unable to grow in a low sodium medium or at acidic pH under aeration with 3% CO₂in air (v/v). Synechococcus cotA did not comple-ment M29. Three chimeric cotA genes of the two cyanobacterial strains were constructed. One of these chimeric genes strongly and the other two weakly complemented the mutant.     

Inside-out membrane vesicles isolated from spinach thylakoids.

Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The sepration was obtained by partitionin an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.     

Immunological Evidence of Connexin-like Proteins in the Plasma Membrane of Vicia faba L.

Plasma membranes from three week old leaves of Vicia faba L. were enriched by aqueous two-phase partitioning to high purity. Plasma membrane proteins were immunoblotted with polyclonal, monospecific antibodies raised against mouse liver connexins (cx) 32 and 26. Immunostaining after treatments with cx 32 antibodies revealed the existence of a 29 kDa protein, clearly enriched in the plasma membrane fraction. An additional immunoreactive band of 20 kDa, possibly a degradation product of the 29 kDa protein, was found in the soluble fraction. When immunoblots were incubated with cx 26 antibodies, a 40 kDa band with a strong immunoresponse appeared, assumed to present the dimeric form of a 21 kDa, cx 26-like plant protein. The monomeric form could be only obtained when intact leaf material or mesophyll protoplasts from three week old plants were directly SDS-extracted. Furthermore, in young, one week old leaves, the monomer seems to exist in larger amounts, together with another crossreacting 35 kDa protein. The 29 kDa (cx 32-related) as well as the 40 kDa (cx 26-related) polypeptide is obviously located in the plasma membrane. The 40 kDa protein has to be considered as a new connexin-like plant protein.     

Inside-out membrane vesicles isolated from spinach thylakoids

Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The separation was obtained by partition in an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.     

Identification and localization of the CupB protein involved in constitutive CO2 uptake in the cyanobacterium, Synechocystis sp. strain PCC 6803

Antibody against cMyc cross-reacted strongly with the CupB protein tagged with His6-cMyc (HM) in thylakoid membrane of Synechocystis sp. strain PCC 6803 but only faintly with the cytoplasmic membrane fraction. The protein was not detected in the membranes of the DeltandhD4 and DeltandhF4 mutants in which CupB was tagged with HM. We concluded that a CupB complex containing NdhD4 and NdhF4 is largely, if not exclusively, confined to the thylakoid membrane. Both CupB and NdhH were detected in a fraction containing protein complexes of > 450 kDa, obtained after nickel column and gel filtration chromatography of the membranes solubilized with n-dodecyl-beta-maltoside.     

Fluorescent probes for non-invasive bioenergetic studies of whole cyanobacterial cells.

Fluorescent DeltapH and DeltaPsi indicators have been screened for the non-invasive monitoring of bioenergetic processes in whole cells of the cyanobacterium Synechocystis sp. PCC 6803. Acridine yellow and Acridine orange proved to be the best DeltapH indicators for the investigation of thylakoid and cytoplasmic membrane energization: While Acridine yellow indicated only cytosolic energization, Acridine orange showed signals from both the thylakoid lumen and the cytosol that could be separated kinetically. Both indicators were applied successfully to monitor cellular energetics, such as the interplay of linear and cyclic photosynthetic electron transport, osmotic adaptation and solute transport across the cytoplasmic membrane. In contrast, useful membrane potential indicators were more difficult to find, with Di-4-ANEPPS and Brilliant cresyl blue being the only promising candidates for further studies. Finally, Acridine yellow and Acridine orange could also be applied successfully for the thermophilic cyanobacterium Synechococcus elongatus. Different from Synechocystis sp. PCC 6803, where both respiration and ATP hydrolysis could be utilized for cytoplasmic membrane energization, proton extrusion at the cytoplasmic membrane in Synechococcus elongatus was preferentially driven by ATP hydrolysis.     

Disruption of the plastid ycf10 open reading frame affects uptake of inorganic carbon in the chloroplast of Chlamydomonas.

The product of the chloroplast ycf10 gene has been localized in the inner chloroplast envelope membrane (Sasaki et al., 1993) and found to display sequence homology with the cyanobacterial CotA product which is altered in mutants defective in CO2 transport and proton extrusion (Katoh et al., 1996a,b). In Chlamydomonas reinhardtii, ycf10, located between the psbI and atpH genes, encodes a putative hydrophobic protein of 500 residues, which is considerably larger than its higher plant homologue because of a long insertion that separates the conserved N and C termini. Using biolistic transformation, we have disrupted ycf10 with the chloroplast aadA expression cassette and examined the phenotype of the homoplasmic transformants. These were found to grow both photoheterotrophically and photoautotrophically under low light, thereby revealing that the Ycf10 product is not essential for the photosynthetic reactions. However, under high light these transformants did not grow photoautotrophically and barely photoheterotrophically. The increased light sensitivity of the transformants appears to result from a limitation in photochemical energy utilization and/or dissipation which correlates with a greatly diminished photosynthetic response to exogenous (CO2 + HCO3-), especially under conditions where the chloroplast inorganic carbon transport system is not induced. Mass spectrometric measurements with either whole cells or isolated chloroplasts from the transformants revealed that the CO2 and HCO3- uptake systems have a reduced affinity for their substrates. The results suggest the existence of a ycf10-dependent system within the plastid envelope which promotes efficient inorganic carbon (Ci) uptake into chloroplasts.     

Occurrence of a Photosystem II polypeptide in non-photosynthetic membranes of cyanobacteria

Cytoplasmic and thylakoid membranes have been purified from the cyanobacteria Anacystis nidulans R2 and Phormidium laminosum by sucrose density gradient centrifugation. Probing of Western blots of proteins from these purified membrane fractions with antibodies directed against the 33 kDa polypeptide of Photosystem II from pea indicates that this protein is present in both the thylakoid and cytoplasmic membranes, rather than just the thylakoid membranes. This has been confirmed by immunogold labelling of cells. Oxygen evolution assays have been used to show that the 33 kDa polypeptide is not assembled into a functional Photosystem II complex in the cytoplasmic membranes. This may be due to the absence of other Photosystem II components.     

Thylakoid protein phosphorylation is suppressed by 'free radical scavengers'. Correlation between PS II core protein degradation and thylakoid protein phosphorylation

Recent work has shown that the light-induced PS II core protein degradation, as monitored by immunostain reduction on Western blots, was stimulated even at low light during phosphorylation of thylakoid proteins in the presence of NaF, and that the thylakoid kinase inhibitor FSBA blocked completely the light- and ATP-stimulated degradation [Georgakopoulos and Argyroudi-Akoyunoglou (1997) Photosynth Res 53: 185–195]. To assess whether D1, D2 or both proteins are degraded, antibodies raised against D1/D2, or the D-E loop of D1 were used. Greatest immunostain reduction was observed with antibodies raised against D1/D2, immunostaining a 34 kDa protein on blots of 15% polyacrylamide-6 M urea gels, suggesting that the phosphorylation-induced degradation may be mainly directed against D2. To see how protein phosphorylation might be implicated in PS II core protein degradation we further tested the effect of free radical scavengers, on thylakoid protein phosphorylation. Active oxygen scavengers like n-propyl gallate, histidine, and imidazole, shown earlier to inhibit high light-induced D1 degradation, also suppressed the phosphorylation of thylakoid proteins; on the other hand, NaN3 and D-mannitol, known to stimulate light- induced D1 degradation did not suppress protein phosphorylation, whereas superoxide dismutase and catalase, known also to inhibit high light-induced D1 degradation, did not affect thylakoid protein phosphorylation. In addition, the ATP-induced degradation was also observed in the dark under conditions of kinase activation, and in the light under anaerobic conditions, that block light-induced degradation, whereas it was reduced in the absence of NaF, the phosphatase inhibitor. The results point to the involvement of a proteolytic system in PS II core protein degradation, which is active in its phosphorylated state.     

Development of Fe-deficiency responses in cucumber (Cucumis sativus L.) roots: involvement of plasma membrane H(+)-ATPase activity

One of the mechanisms through which some strategy I plants respond to Fe-deficiency is an enhanced acidification of the rhizosphere due to proton extrusion. It was previously demonstrated that under Fe-deficiency, a strong increase in the H(+)-ATPase activity of plasma membrane (PM) vesicles isolated from cucumber roots occurred. This result was confirmed in the present work and supported by measurement of ATP-dependent proton pumping in inside-out plasma membrane vesicles. There was also an attempt to clarify the regulatory mechanism(s) which lead to the activation of the H(+)-ATPase under Fe-deficiency conditions. Plasma membrane proteins from Fe-deficient roots submitted to immunoblotting using polyclonal antibodies showed an increased level in the 100 kDa polypeptide. When the plasma membrane proteins were treated with trypsin a 90 kDa band appeared. This effect was accompanied by an increase in the enzyme activity, both in the Fe-deficient and in the Fe-sufficient extracts. These results suggest that the increase in the plasma membrane H(+)-ATPase activity seen under Fe-deficiency is due, at least in part, to an increased steady-state level of the 100 kDa polypeptide.     

Anthroyl stearate as a fluorescent probe of chloroplast membranes.

1. A reversible light-induced enhancement of the fluorescence of a "hydrophobic fluorophore", 12-(9-anthroyl)-stearic acid (anthroyl stearate), is observed with chloroplasts supporting phenazine methosulfate, cyclic or 1,1'-ethylene-2,2'-dipyridylium dibromide (Diquat) pseudo-cyclic electron flow; no fluorescence change is observed when methyl viologen or ferricyanide are used as electron acceptors. The stearic acid moiety of anthroyl stearate is important for its localization and fluorescence response in the thylakoid membrane, since structural analogs of anthroyl stearate lacking this group do not show the same response. 2. This effect is decreased under phosphorylating conditions (presence of ADP, Pi, Mg2+), and completely inhibited by the uncoupler of phosphorylation NH4Cl(5-10mM), as well as the ionophores nigericin and gramicidin-D (both at 5 - 10(-8)M). The MgCl2 concentration dependence of the anthroyl stearate enhancement effect is identical to that previously observed for cyclic photophosphorylation, as well as for the formation of a "high energy intermediate". The anthroyl stearate fluorescence enhancement is inhibited by increasing concentrations of ionophores in parallel with the decrease in ATP synthesis, but is essentially unaffected by specific inhibitors (Dio-9 and phlorizin) of photophosphorylation; thus, it appears that anthroyl stearate monitors a component of the "high energy state" of the thylakoid membrane rather than a terminal phosphorylation step. 3. The light-induced anthroyl stearate fluorescence enhancement is suggested to monitor a proton gradient in the energized chloroplast because (a) similar enhancement can be produced by sudden injection of hydrogen ions in a solution of anthroyl stearate; (b) when the proton gradient is dissipated by gramicidin or nigericin light-induced anthroyl stearate fllorescence is eliminated; (c) when the proton gradient is dissipated by tetraphenylboron, light-induced anthroyl stearate fluorescence decreases, and (d) light-induced anthroyl stearate fluorescence change as a function of pH is qualitatively similar to that observed with other probes for a proton gradient (e.g. 9-aminoacridine). Furthermore, anthroyl stearate does not monitor H+ uptake per se because (a) the pH dependence of H+ transport is different from that of the anthroyl stearate fluorescence change, and (b) tetraphenylboron, which does not inhibit H+ uptake, reduces anthroyl stearate fluorescence. Thus, anthroyl stearate appears to be a useful probe of a proton gradient supported by phenazine methosulfate of Diquat catalyzed electron flow and is the first "non-amine" fluorescence probe utilized for this purpose in chloroplasts.     

The differential effects of short-time glutaraldehyde treatments on light-induced thylakoid membrane conformational changes, proton pumping and electron transport properties

A rapid quench technique utilizing the addition of excess buffer containing free amine groups (Tris, glycylglycine) to the reaction medium has enabled a detailed study of the time-course of glutaraldehyde inactivation on the spinach thylakoid membrane to be undertaken. The following light-induced parameters were inactivated in the sequence: slow transmittance changes (0–5 s) > coupling factor activity (5–20 s) > narrow angle 90° scattering changes (30–60 s). About 20% of PS II activity was lost by this treatment. No effect on activity, proton pumping and proton gradient formation was observed over the time-course studied. A consideration of these effects led to the proposal that the slow, light-induced transmittance changes reflect reversible thylakoid structural changes (unstacking, membrane flattening) in response to electron transport and the consequent proton pumping. The narrow angle 90° scattering changes were considered to reflect directly microconformational structural changes in response to the light-driven proton translocation as previously concluded from other workers.     

Light-induced cytoplasmic pH changes and their interrelation to the activity of the electrogenic proton pump in Riccia fluitans

In green thallus cells of the aquatic liverwort Riccia fluitans light-induced pH changes have been measured, using a turgor-resistant pH-sensitive microelectrode. (1) Light-off/-on causes oscillations of the cytoplasmic pH (pH_c), as well as of the membrane potential difference across the plasmalemma (ψ). Beside the well-known ψ_m changes, the first detectable pH_c change following light-off is a transient acidification of about 0.3 pH units, whereas light-on causes a transient alkalinization of roughly 0.4 pH units. (2) 1 μM DCMU eliminates these transients. (3) In the presence of 0.2 mM procaine, which alkalizes the cytoplasm to over pH 8, the light-induced ψ_m transients are enhanced, but are almost absent, if pH_c is acidified to 6.9 by 1 mM acetate. It is suggested that the transient light-induced changes in pH_c are caused by light-dependent proton translocation across the thylakoid membranes, and it is concluded that the subsequent changes in ψ_m are essentially the result of altered activities of the electrogenic proton pump in the plasmalemma, due to the observed fluctuations of its substrate, the proton.     

Characterisation of Zea mays L. plastidial transglutaminase: interactions with thylakoid membrane proteins

Chloroplast transglutaminase (chlTGase) activity is considered to play a significant role in response to a light stimulus and photo‐adaptation of plants, but its precise function in the chloroplast is unclear. The characterisation, at the proteomic level, of the chlTGase interaction with thylakoid proteins and demonstration of its association with photosystem II (PSII) protein complexes was accomplished with experiments using maize thylakoid protein extracts. By means of a specific antibody designed against the C‐terminal sequence of the maize TGase gene product, different chlTGase forms were immunodetected in thylakoid membrane extracts from three different stages of maize chloroplast differentiation. These bands co‐localised with those of lhcb 1, 2 and 3 antenna proteins. The most significant, a 58 kDa form present in mature chloroplasts, was characterised using biochemical and proteomic approaches. Sequential fractionation of thylakoid proteins from light‐induced mature chloroplasts showed that the 58 kDa form was associated with the thylakoid membrane, behaving as a soluble or peripheral membrane protein. Two‐dimensional gel electrophoresis discriminated, for the first time, the 58‐kDa band in two different forms, probably corresponding to the two different TGase cDNAs previously cloned. Electrophoretic separation of thylakoid proteins in native gels, followed by LC‐MS mass spectrometry identification of protein complexes indicated that maize chlTGase forms part of a specific PSII protein complex, which includes LHCII, ATPase and pSbS proteins. The results are discussed in relation to the interaction between these proteins and the suggested role of the enzyme in thylakoid membrane organisation and photoprotection.     

Antibodies to the photosystem I chlorophyll a + b antenna cross-react with polypeptides of CP29 and LHCII

A chlorophyll (a + b)--protein complex associated with photosystem I (PSI) was isolated from a larger PSI complex (CPIa) produced by electrophoresis of barley thylakoids solubilized with 300 mM octyl glucoside. It had an apparent Mr of 35,000-43,000 on 7.5% and 10% acrylamide gels respectively, and a chlorophyll a/b ratio of 2.5 +/- 1.5. Denaturation released four polypeptides migrating between 21-24 kDa. They were well separated from the polypeptides of the two photosystem II chlorophyll a + b antenna complexes: LHCII (25-27 kDa) and CP29 (28-29 kDa). In order to study the PSI antenna complex, antibodies were raised against highly purified CPIa. The antigen appeared to be pure when electrophoresed, blotted and reacted with its antiserum, i.e. anti-CPIa detected only the 64-66-kDa CPI apoprotein and the four 21-24 kDa antenna polypeptides. However, when blotted against the whole spectrum of thylakoid proteins, it cross-reacted with both LHCII and CP29 apoproteins. Removal of anti-CPI activity from the anti-CPIa did not affect these cross-reactions, showing that they were not due to antibodies directed against CPI. To show that the same antibody population was reacting with both the photosystem I and photosystem II antenna polypeptides, anti-CPIa was adsorbed onto highly purified CPIa on nitrocellulose. The bound antibody was eluted and used again in a Western blot against whole thylakoid proteins. This selected antibody population showed the same relative strength of reaction with photosystem I and photosystem II antenna polypeptides as the original antibody population had. Similar observations have been made with antibodies to the two photosystem II antenna complexes. We therefore conclude that there are antigenic determinants in common among the chlorophyll a + b binding polypeptides, and predict that there could be amino acid sequence similarities.     

Membranes of Rhodopseudomonas sphaeroides. VII. Photochemical properties of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes.

Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromotophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome (Em = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.     

Chloroplast envelope proteins are encoded by the chloroplast genome of Chlamydomonas reinhardtii.

To characterize envelope proteins encoded by the chloroplast genome, envelopes were isolated from Chlamydomonas reinhardtii cells labeled with [35S] sulfate while blocking synthesis by cytoplasmic ribosomes. One and two-dimensional gel electrophoresis of envelopes and fluorography revealed four highly labeled proteins. Two with masses of 29 and 30 kDa and pI 5.5 were absent from the stroma and thylakoid fractions, while the others at 54 kDa, pI 5.2 and 61 kDa, pI 5.4 were detected there in smaller amounts. The 29- and 30-kDa proteins were associated with outer envelope membranes separated from inner envelope membranes after chloroplast lysis in hypertonic solution. A 32-kDa protein not labeled by [35S]sulfate was found exclusively in the inner membrane fraction, suggesting the existence of a phosphate translocator in C. reinhardtii. To identify envelope proteins exposed on the chloroplast surface, isolated active chloroplasts were surface-labeled with 125I and lactoperoxidase. The 54-kDa, pI 5.2 protein as well as a protein corresponding to either of the 29- or 30-kDa proteins described above were among the labeled components. These results show that envelope proteins of C. reinhardtii are encoded by the chloroplast genome and two are located on the outer envelope membranes.