Biomedical subjects

I Ohad

Publications and source records attributed to I Ohad.

At least 37 records · Page 2Linked to original sources

PubMed1995

Activation/deactivation cycle of redox-controlled thylakoid protein phosphorylation. Role of plastoquinol bound to the reduced cytochrome bf complex.

Signal transduction via light-dependent redox control of reversible thylakoid protein phosphorylation has evolved in plants as a unique mechanism for controlling events related to light energy utilization. Here we report for the first time that protein phosphorylation can be activated without light or the addition of reducing agents by a transient exposure of isolated thylakoid membranes to low pH in darkness. The activation of the kinase after incubation of dark-adapted thylakoids at pH 4.3 coincides with an increase in the plastoquinol: plastoquinone ratio up to 0.25. However, rapid plastoquinol reoxidation ( < 1 min) at pH 7.4 contrasts with the slow kinase deactivation (t 1/2 = 4 min), which indicates that the redox control is not directly dependent on the plastoquinone pool. Use of inhibitors and a cytochrome bf-deficient mutant of Lemna demonstrate the involvement of the cytochrome bf complex in the low-pH induced protein phosphorylation. EPR spectroscopy shows that subsequent to the transient low pH treatment and transfer of the thylakoids to pH 7.4, the Rieske Fe-S center, and plastocyanin become reduced and are not reoxidized while the kinase is slowly deactivated. However, the deactivation correlates with a decrease of the EPR gz signal of the reduced Rieske Fe-S center, which is also affected by quinone analogues that inhibit the kinase. Our data point to an activation mechanism of thylakoid protein phosphorylation that involves the binding of plastoquinol to the cytochrome bf complex in the vicinity of the reduced Rieske Fe-S center.

Cytochrome b Group↗

PubMed1995

The 64 kDa polypeptide of spinach may not be the LHCII kinase, but a lumen-located polyphenol oxidase.

Phosphorylation of chlorophyll alb-binding proteins of the of photosystem II light-harvesting assembly controls the energy distribution between the two photosystems as well as the turnover of thylakoid membrane proteins. The LHCII kinase, suggested to be a 64 kDa protein, is light-regulated by a mechanism involving reduction of plastoquinone and the participation of the cytochrome b6lf complex. A cDNA encoding that protein has been isolated from a lambda gt11-based library made from spinach polyadenylated RNA using a two-step strategy involving screening by polyclonal monospecific antisera and plaque hybridization. The protein of 73.1 kDa molecular mass represents a precursor which contains a bipartite transit peptide of 101 amino acid residues (11.0 kDa) that directs the protein into the thylakoid lumen. It can be phosphorylated in vitro, and exhibits significant homology to plant polyphenol oxidases not to kinases. The gene was therefore designated PpoA. Reinvestigation of components in the molecular mass range of 50-70 kDa disclosed five additional proteins which can accompany kinase-active cytochrome b6lf, photosystem II and AMS [1] preparations. Four of them can be phosphorylated in vitro; two with apparent molecular masses of 53 and 66 kDa are capable of phosphorylation and represent new, yet unidentified proteins.

Amino Acid Sequence↗

PubMed1995

Photoinactivation of photosystem II induces changes in the photochemical reaction center II abolishing the regulatory role of the QB site in the D1 protein degradation.

The effect of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (diuron) binding at the secondary quinone (QB) binding site of reaction center II (RCII), on the high-light-induced degradation of the RCII proteins D1 and D2, and the core proteins CP43 and CP47 was investigated in vivo in Chlamydomonas reinhardtii. The degradation of the RCII-D2 and the CP43 proteins shows a short lag relative to that of the RCII-D1 protein. Diuron retards but does not prevent the degradation of RCII-D1, D2 and CP43 proteins. The degradation of the CP47 protein is not retarded by diuron. The RCII-D1 protein present in cells photoinactivated in the presence of diuron is subsequently degraded in cells transferred to low light or to darkness. The protein can be replaced (turnover) at least partially under both conditions. The RCII-D1 protein is not degraded during photoinactivation of a cytochrome-bf-defective mutant. Degradation occurs however when the cells are returned to low light permitting slow reoxidation of plastoquinol [Zer, H., Prasil, O. & Ohad, I. (1994) J. Biol. Chem. 269, 17,670-17,676]. Addition of diuron does not prevent the degradation of the protein at this stage. Tryptic digestion of the RCII-D1 protein is partially inhibited by diuron in isolated thylakoids [Trebst, A., Depka, B., Kraft, B. & Johanningmeier, U. (1988) Photosynth. Res. 18, 163-177] but not in thylakoids obtained from photoinactivated cells. We conclude that photoinactivation induces a series of sequential changes in RCII exposing the cleavage site of the RCII-D1 protein to degradation and abolishing the regulatory role of the QB site occupancy by plastoquinone or analog ligands on the cleavage process. The degradation of the RCII-D2 and CP43 proteins may be a secondary process following modification and/or loss of the RCII-D1 protein.

Animals↗

PubMed1995

An unusual organization of the genes encoding cytochrome b559 in Chlamydomonas reinhardtii: psbE and psbF genes are separately transcribed from different regions of the plastid chromosome.

The psbE and psbF genes encode the apoproteins of cytochrome b559, an essential component of the pigment protein complex photosystem II. Together with psbL and psbJ, these genes constitute a single operon in all photosynthetic organisms examined thus far. We have cloned and sequenced the psbE and psbF genes of the Chlamydomonas reinhardtii plastid genome. The predicted amino-terminal domains of both polypeptides are more basic than those of other organisms, and the sequence of the psbE gene product indicates a departure from the 'positive-inside' rule for the insertion of proteins in the thylakoid membrane. Northern blot analysis demonstrated that psbE is transcribed into a 0.3 kb mRNA, while transcription of psbF and psbL genes results in a 0.9 kb transcript. The splitting of the psbEFLJ operon into separate transcription units suggests a unique mechanism of regulation of expression of these genes in C. reinhardtii.

Amino Acid Sequence↗

PubMed1995

Oscillations of reaction center II-D1 protein degradation in vivo induced by repetitive light flashes. Correlation between the level of RCII-QB- and protein degradation in low light.

The D1 protein subunit of the photochemical reaction center II (RCII) turns over rapidly in oxygenic photosynthetic organisms exposed to the light. At high photon flux densities (PFD), photoinactivation of RCII precedes the degradation of the D1 protein. We found that the apparent quantum yield for the D1 protein degradation in Chlamydomonas cells is severalfold higher at low PFDs (10-100 mumol m-2 s-1) as compared to that observed at PFDs which induce photoinactivation of RCII (1.5-3 x 10(3) mumol m-2 s-1). Relative high levels of reduced RCII secondary plastoquinone acceptor, QB-, are induced in cells exposed to low PFDs as determined by thermoluminescence measurements. The probability of generating elevated levels of QB- which may recombine with the S2,3 oxidized states of the oxygen evolving complex decreases with increase in the light intensities at which consecutive double reduction of QB and exchange with the plastoquinone pool prevail. We have used light flashes to test if a correlation exists between the degradation of D1 protein and the relative level of QB-. D1 protein degradation could be induced in dark-incubated cells exposed to a series of 1.4 x 10(3) single light flashes given at intervals compatible with generation of elevated levels of QB- and its decay by charge recombination. Oscillations of the QB- level in cells exposed to 960-1440 series of 1 to several flashes correlated with oscillations of the D1 protein degradation in Chlamydomonas cells and in the Scenedesmus wild type but not in the LF-1 mutant lacking photosystem II donor side activity. In this mutant the "S state cycle" and QB- oscillations are abolished. We propose that the process of recombination of long lived RCII-QB- with the S2,3 states may involve damaging events related to the D1 protein degradation induced by light flashes or continuous low light in vivo.

Animals↗

PubMed1994

The ctpA gene encodes the C-terminal processing protease for the D1 protein of the photosystem II reaction center complex.

The D1 protein of the photosystem II (PSII) complex in the thylakoid membrane of oxygenic photosynthetic organisms is synthesized as a precursor polypeptide (pD1) with a C-terminal extension. Posttranslational processing of the pD1 protein is essential to establish water oxidation activity of the PSII complex. We have recently identified a gene, ctpA, a mutation in which resulted in a loss of PSII activity in the cyanobacterium Synechocystis sp. PCC 6803. To study the function of the CtpA protein, we inactivated the ctpA gene by inserting a kanamycin-resistance gene into its coding sequence. The resultant mutant strain, T564, had no PSII-mediated water oxidation activity, but it had normal cytochrome b6f and photosystem I activities. Measurements of thermoluminescence profiles and rates of reduction of 2,6-dichlorophenolindophenol indicated that PSII complexes in the mutant cells had functional reaction centers that were unable to accept electrons from water. Immunoblot analysis showed that D1, D2, CP47, CP43, and the alpha subunit of cytochrome b559, five integral membrane proteins of PSII, were present in T564 cells. Interestingly, the D1 protein in the mutant cells was 2 kDa larger than that in wild-type cells, due to the presence of a C-terminal extension. We conclude that the CtpA protein is a processing enzyme that cleaves off the C-terminal extension of the D1 protein. Interestingly, the CtpA protein shows significant sequence similarity to the interphotoreceptor retinoid-binding proteins in the bovine, human, and insect eye systems.

Algal Proteins↗

PubMed1994

Role of plastoquinol oxidoreduction in regulation of photochemical reaction center IID1 protein turnover in vivo.

The light-induced turnover of the D1 protein subunit of reaction center II (RCII) was investigated in Chlamydomonas reinhardtii y-1 (control) and D6, AC208, and B4 mutants lacking cytochrome b6/f, plastocyanin or photosystem I activity, respectively, and, thus, impaired in light-dependent plastoquinol (PQH2) oxidation. Charge recombination assayed by thermoluminescence measurements indicated similar RCII properties in control and mutant cells. The D1 protein is not degraded in the mutants during photoinactivation; however, RCII-D1 is irreversibly altered, and the protein is degraded when the cells are incubated in low light permitting slow reoxidation of the PQH2 pool. Photoinactivation precedes D1 degradation also in the control cells. Thus, in vivo under physiological conditions photoinactivation and "tagging" of RCII-D1 are resolved from the degradation process. RCII activity in photoinactivated cells may be recovered only following D1 degradation and replacement. Recovery may occur either in the light or dark in the absence of de novo chlorophyll synthesis. The degradation of the photoinactivated RCII-D1 protein is a prerequisite for the synthesis and stable integration of new D1 indicating that tagged D1 is still assembled in the inactive reaction centers. The physiological implication of these results is that oxidation of the PQH2 pool in photoinactivated cells affects RCII-D1 protein degradation and replacement, and, thus, D1 turnover in vivo is regulated by the turnover of PQ at the binding site of the secondary stable electron acceptor quinone of RCII.

Animals↗

PubMed1994

Reversible membrane association of heat-shock protein 22 in Chlamydomonas reinhardtii during heat shock and recovery.

The process of reversible membrane association of the nuclear-encoded heat-shock protein hsp22 in Chlamydomonas reinhardtii cells during recovery from heat stress has been investigated. hsp22 associates with a chloroplast membrane-enriched fraction, dissociates from the membranes during recovery from heat shock and rebinds during a subsequent heat-shock treatment in vivo. The protein remains in the cell soluble fraction for at least 22 h after heat-stress treatment. Dissociation of membrane-bound hsp22 occurs only at 25-38 degrees C and reassociation occurs only at the hsp22 induction temperature (38-42 degrees C). Hsp22 dissociation from the membrane fraction is not related to de novo protein synthesis in vivo and does not occur in vitro. Based on the derived amino acid sequence, hsp22 is not considered a typical chloroplast-associated heat-shock protein [Vierling, E. (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 579-620] and may be associated with the chloroplast envelope membrane. However, the reversible association of hsp22 with the chloroplast-enriched membrane fraction indicates similar properties to those of pea low-molecular-mass heat-shock proteins [Glaczinski, H. & Kloppstech, K. (1988) Eur. J. Biochem. 173, 579-583] and may be related to the transient response of the chloroplast to heat stress.

Animals↗

PubMed1993

Analysis of chimeric spinach/cyanobacterial CP43 mutants of Synechocystis sp. PCC 6803: the chlorophyll-protein CP43 affects the water-splitting system of Photosystem II.

Mutants of the cyanobacterium Synechocystis sp. PCC 6803 have been generated in which parts of psbC (the gene encoding the Photosystem II chlorophyll-protein CP43) have been replaced with the homologous gene fragment from spinach. Upon the replacement of all but the 3' 84 bp of the cyanobacterial psbC gene with the homologous fragment from spinach, an obligate photoheterotrophic mutant was generated. Two photoautotrophic derivatives of this mutant were made reincorporating 3' cyanobacterial sequences back into the spinach psbC gene of the mutant. These two mutants are similar to each other, carrying a chimeric CP43 with the N-terminal half from spinach. These mutants are photosynthetically active at a rate of about half that of wild type, which correlates with a decreased Photosystem II/chlorophyll ratio in these mutants. Thylakoids from the chimeric mutants contain a CP43 protein which migrates slightly more slowly on SDS-polyacrylmide gels than the native Synechocystis CP43. Interestingly, these mutants show significant shifts in thermoluminescence peaks, reflecting altered thermodynamic properties of the back reaction between the acceptor side and the water-splitting system. On the basis of the oscillations of these shifts with number of flashes, we conclude that S2 is stabilized and S3 is destabilized in these mutants. This represents evidence for an involvement of CP43 in events associated with water splitting.

Amino Acid Sequence↗

PubMed1993

Early light-inducible protein in pea is stable during light stress but is degraded during recovery at low light intensity.

The nuclear-encoded, thylakoid-bound early light-inducible protein (ELIP) reported to be related to the initial stages of chloroplast differentiation is synthesized in substantial amounts in leaves of mature plants exposed to light stress conditions (Adamska, I., Ohad, I., and Kloppstech, K. (1992b) Proc. Natl. Acad. Sci. U.S.A. 89, 2610-2613). Increase in ELIP content correlates with the photoinactivation of PSII, degradation of D1 protein, and changes in the level of pigments. Inhibition of phytoene desaturase and/or zeta-carotene desaturase during light stress drastically increases accumulation of the protein. ELIP mRNA is short-lived (t1/2 = 1 h). The thylakoid bound protein is stable in high light exposed leaves and is degraded only during recovery from light stress at low light intensity (40 microE/m2s). The lifetime of the protein during the recovery process increases with the extent of initial light stress condition. We propose that ELIP synthesis and degradation is related to the process of the plant response to light stress and recovery from photoinhibition.

Arabidopsis Proteins↗

PubMed1992

The redox-controlled light-harvesting chlorophyll a/b protein kinase. Deactivation by substituted quinones.

The deactivation of the redox-controlled light-harvesting chlorophyll a/b protein kinase of Acetabularia acetabulum and pea thylakoids was studied. Substituted benzoquinone, naphthoquinone, and anthraquinone analogs including mono-, di-, and trihalogenated and/or alkylated quinones, which are known to inhibit the cytochrome b6/f activity, deactivate the kinase in the dark, and prevent its activation in the light. Analogs halogenated at positions 2- or 3- are the most effective deactivators. Increasing the size of the alkyl side chain and/or the number of rings lowers the deactivation effect. The activated state of the pea kinase decays with a t1/2 of 15 min, while the Acetabularia enzyme retains its active state for at least 2 h. The midpoint potential for Acetabularia kinase activity in the dark is 120 +/- 10 mV and is compatible with the involvement of plastoquinone in the kinase activation via reduction of the cytochrome complex. Deactivation of kinase by the analogs inhibiting cytochrome b6/f complex activity and the kinase copurification with the cytochrome b6/f fraction obtained from the Acetabularia thylakoid further support this conclusion. These results indicate that the process of kinase activation/deactivation includes the binding of plastoquinol or quinone analogs by the cytochrome complex and its interaction with the kinase. We propose that the latter process may constitute the rate-limiting step controlling the kinase activation/deactivation kinetics.

Acetabularia↗

PubMed1992

UV light stress induces the synthesis of the early light-inducible protein and prevents its degradation.

ELIP is a nuclear-encoded protein localized in the thylakoid membranes. The protein is specifically induced by blue light in mature, light-grown plants (Adamska, I., Ohad, I., and Kloppstech, K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2610-2613), as well as in plants developed in the light in which pigment synthesis and plastid development were inhibited by the bleaching herbicide norflurazon. ELIP transcription and protein accumulation are induced also by UVA but not by UVB light. However, UVB light allows ELIP synthesis induced by superimposed white light. The protein is stable under light stress including UVA and UVB light, but it is rapidly degraded upon cessation of the light stress conditions. ELIP synthesis and integration into the chloroplast membranes is related neither to chloroplast translation activity nor to photosynthetic electron flow. Inhibition of carotenoid synthesis by fluridone, a bleaching herbicide which causes extensive damage to the photosynthetic apparatus, does not affect induction of ELIP transcription during light stress but greatly enhances ELIP accumulation. Based on these results it is proposed that ELIP turnover is related to the light stress and recovery process in plants.

Arabidopsis Proteins↗

PubMed1992

Synthesis of the early light-inducible protein is controlled by blue light and related to light stress.

The early light-inducible proteins (ELIPs) are expressed in developing plants in the first hours of the greening process. Here we report that strong light causing photoinhibition of photosynthesis also induces ELIP transcription and accumulation of the protein in mature green pea plants. Accumulation of ELIP transcript is induced in plants exposed to light intensities above 500 E/m2.s (E, einstein) and is maximal at approximately 1500 E/m2.s. The ELIP mRNA level increases in correlation with the degree of photoinhibition. The increase in ELIP level in the thylakoid membranes parallels the decrease in the amount of D1 protein of the photosystem II reaction center. Examination of ELIP induction as a function of light quality demonstrates that ELIP transcription is specifically induced by blue (410-480 nm) but not by red or far-red light. The level of blue light-induced ELIP transcript is significantly repressed by low-intensity red light. However, the accumulation of ELIP translation product is related to the total amount of blue and red light energy absorbed.

Journal Article↗

PubMed1992

Phosphorylation of cytochrome b6 by the LHC II kinase associated with the cytochrome complex.

The cytochrome b6 polypeptide present in cytochrome b6/f preparations from spinach thylakoids is phosphorylated concomitantly with the autophosphorylation of the 64 kDa polypeptide identified as the redox-controlled LHCII kinase. The N-terminal sequence of the 64 kDa kinase and sequence analysis of cytochrome b6 indicate the existence of putative phosphorylation sites in both proteins.

Amino Acid Sequence↗

PubMed1992

Temperature treatments of dark-grown pea seedlings cause an accelerated greening in the light at different levels of gene expression.

We have previously shown that heat-shock in the dark evokes photomorphogenesis-like effects and circadian rhythmicity at the level of mRNAs when applied to emerging pea plantlets during several consecutive days [15]. Here we extend these findings by showing that a temperature shift to 10 degrees C above average and a single heat-shock are sufficient for induction of circadian rhythmicity and changes in morphogenesis. The maximum response to a single heat-shock occurs at days 2 to 3 after sowing indicating additional developmental control of the response. An increasing number of heat-shock treatments intensifies the morphogenetic effect. The heat-shocked plantlets have an elevated level of the xanthophyll lutein in the dark. Upon illumination of heat-shocked plantlets accumulation of chloroplast pigments as well as that of individual thylakoid membrane proteins and their corresponding mRNAs occur much faster than in the etiolated controls. This is reflected in an accelerated formation of grana stacks. Therefore, heat-shock seems to evoke a responsiveness of plantlets similar to that obtained earlier by other authors using pre-illumination. The working hypothesis is put forward that induction or synchronization of circadian rhythmicity by either light or heat-shock might be sufficient to explain the observed morphogenetic changes.

Adaptation, Physiological↗

PubMed1991

The PQ/PQH2 ratio and occupancy of photosystem II-QB site by plastoquinone control the degradation of D1 protein during photoinhibition in vivo.

Photoinactivation of photosystem II (PSII) and light-dependent degradation of the reaction center II (RCII) protein D1 have been investigated in Chlamydomonas reinhardtii mutants D6, AC208, and B4 deficient in cytochrome b6/f, plastocyanin, and photosystem I (PSI) activity, respectively. These mutants possess active PSII and reduce plastoquinone (PQ) but cannot oxidize plastoquinol (PQH2) via light-dependent reduction of NADP. In light-exposed cells a high ratio PQH2/PQ and a low turnover of PQ/PQH2 at the RCII-QB site are maintained. In all mutants photoinactivation of RCII was slower as compared to the wild-type (wt) cells, and D1 degradation was drastically decreased. The degradation of D1 was also lower in the wt cells under anaerobic conditions and presence of ascorbate, while raising the concentration of dissolved oxygen increased the degradation of the D1 protein in the AC208 mutant. Photoinactivation and light-dependent degradation of the D1 protein were drastically increased in the Scenedesmus obliquus LF-1 mutant cells altered in its PSII manganese binding and thus unable to reduce PQ using water as an electron donor. Diuron inhibited the light-dependent degradation of D1 protein in both the LF-1 mutant and wt cells. Based on these results we propose that availability of PQ at the QB site is required for (i) the photoinactivation process of the RCII acceptor side followed by inactivation of the donor side leading to the generation of harmful cation radicals (Z+, P680+, chlz+) which damage the D1 protein, and (ii) the accessibility of the cleavage site of the damaged D1 protein to proteolytic degradation.

Animals↗

PubMed1990

Interaction between light harvesting chlorophyll-a/b protein (LHCII) kinase and cytochrome b6/f complex. In vitro control of kinase activity.

We have previously reported that the cytochrome b6/f complex may be involved in the redox activation of light harvesting chlorophyll-a/b protein complex of photosystem II (LHCII) kinase in higher plants (Gal, A., Shahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221, 205-210). The aim of this work was to establish whether a relation between the cytochrome b6/f and LHCII kinase activation can be demonstrated in vitro. Preparations enriched in cytochrome b6/f obtained from spinach thylakoids by detergent extraction and precipitation with ammonium sulfate followed by different procedures of purification, contained various amounts of LHCII kinase activity. Analysis of the cytochrome b6/f content and kinase activity of fractions obtained by histone-Sepharose and immunoaffinity columns, immunoprecipitation and sucrose density centrifugation, indicate functional association of kinase and cytochrome b6/f. Phosphorylation of LHCII by fractions containing both cytochrome b6/f and kinase was enhanced by addition of plastoquinol-1. LHCII phosphorylation and kinase activation could be obtained in fractions prepared by use of beta-D-octyl glucoside but not when 3-[(cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate was used as the solubilizing detergent. Kinase activity could be inhibited by halogenated quinone analogues (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2,3-diiodo-5-t-butyl-p-benzoquinone) known to inhibit cytochrome b6/f activity. However, kinase activity was inhibited by these analogues in all preparations including those which could not phosphorylate LHCII. We thus propose that the redox activation of LHCII phosphorylation is mediated by kinase interaction with cytochrome b6/f while the deactivation may be related to a distinct quinone binding site of the enzyme molecule.

Ammonium Sulfate↗

PubMed1990

Ubiquitin in Chlamydomonas reinhardii. Distribution in the cell and effect of heat shock and photoinhibition on its conjugate pattern.

Ubiquitin, a highly conserved 76-amino-acid protein, is involved in the response of many types of eukaryotic cells to stress but little is known about its role in lower plants. In the present study we have investigated the distribution of ubiquitin in the unicellular alga Chlamydomonas reinhardii as well as the effect of heat and light stress on its conjugation to cellular proteins. Immunoelectron microscopy shows that ubiquitin is located in the chloroplast, nucleus, cytoplasm, pyrenoid and on the plasma membrane. The location of ubiquitin within chloroplasts has not been observed previously. In immunoblots of whole cell extracts with an antibody to ubiquitin a prominent conjugate band with an apparent molecular mass of 29 kDa and a broad region of high-molecular-mass conjugates (apparent molecular mass greater than 45 kDa) were observed. Exposure of cells to a 41.5 degrees C heat shock in both the dark and light caused the disappearance of the 29-kDa conjugate and an increase in the high-molecular-mass conjugates. After step down to 25 degrees C the 29-kDa conjugate reappeared while the levels of high-molecular-mass conjugates decreased. In light, the recovery of the 29-kDa band was more rapid than in the dark. Photoinhibition alters the ubiquitin conjugation pattern similarly to heat shock, but to a lesser degree. These observations imply that, in Chlamydomonas, ubiquitin has a role in the chloroplast and in the response to heat and light stress.

Cell Membrane↗