Biomedical subjects

I Ohad

Publications and source records attributed to I Ohad.

At least 55 records · Page 3Linked to original sources

PubMed1990

Light-dependent D1 protein synthesis and translocation is regulated by reaction center II. Reaction center II serves as an acceptor for the D1 precursor.

Light induces an irreversible modification of the photosystem II reaction center (RCII) affecting specifically one of its major components, the D1 protein (Ohad, I., Adir, N., Koike, H., Kyle, D. J., and Inoue, Y. I. (1990) J. Biol. Chem. 265, 1972-1979) which is degraded and replaced continuously (turnover). The turnover rate of D1 is related to light intensity. Evidence is presented showing that RCII translocates from the site of damage in the grana (appressed) domain of the chloroplast membranes to unappressed membrane domains where the D1 precursor protein (pD1) is translated and becomes integrated into RCII. Several forms of RCII (a, a*, and b) were identified on the basis of their electrophoretic mobility. pD1 was found only in the a and b forms in the unappressed membranes. Processing of pD1 occurs after its integration into RCII. Mature D1 appeared mostly in the a form of RCII and following its translocation to the appressed membrane domains also in the a* form. Thus the light intensity-dependent synthesis of D1 protein is related to the availability of modified RCII which serves as an acceptor for pD1. The shuttling of RCII between the two membrane domains may represent a control mechanism of thylakoid membrane protein synthesis.

Biological Transport↗

PubMed1990

Mechanism of photoinhibition in vivo. A reversible light-induced conformational change of reaction center II is related to an irreversible modification of the D1 protein.

The light-induced inactivation of the photochemical reaction center II (RCII) of oxygenic chloroplasts (photoinhibition) was investigated in cells and isolated thylakoids of the green alga Chlamydomonas reinhardtii. The process is resolved into a reversible conformational change followed by an irreversible modification of RCII D1 protein. The light-induced changes in vivo persisted in isolated thylakoids. The first step is characterized by (i) destabilization of the secondary acceptor semiquinone anion, Q-B, bound to the D1 protein. This is demonstrated by a reduction in the activation energy of S2,3Q-B charge recombination as measured by the thermoluminescence technique; and (ii) a rise in the intrinsic fluorescence and a decrease of the maximal fluorescence. Unoccupancy of the QB site by plastoquinone partially protected RCII against the light-induced destabilization of Q-B. The extent of charge separation (P+680Q-A) was not affected. However, the slow phase (microsecond) of P+680 dark reduction increased, and the amplitude of signal II was reduced by 20-30%, indicating that in a fraction of RCII, electron donation from Z to P+680 was impaired without losing primary photochemistry. This modification correlates with the irreversible change in D1 protein resulting in the formation of a trypsin-resistant fragment of 16 kDa detected in D1 isolated from light-exposed cells. The change in the Q-B stability could allow charge equilibration with QA and thus explain the rise in the intrinsic fluorescence level and reduction of electron flow to plastoquinone. The change in the lifetime of P+680 can account for further reduction in electron flow (photo-inhibition). The irreversible light-dependent modification of D1 may serve as the signal for its degradation and replacement by a newly synthesized molecule (turnover).

Chlamydomonas↗

PubMed1990

Light regulation of the 22 kd heat shock gene transcription and its translation product accumulation in Chlamydomonas reinhardtii.

Expression of the nuclear coded heat shock protein HSP22 in Chlamydomonas reinhardtii y-1 cells is light regulated at the level of transcript accumulation. In dark grown cells, containing a non-differentiated plastid, light has an additional regulatory effect on the accumulation of HSP22. When such cells are exposed to heat stress in the light, poly(A)+ RNA hybridizing with the HS22 probe is synthesized at levels comparable with those found in cells pre-illuminated for 3 h (greening) prior to the heat shock. However, this RNA is poorly translated in vitro and HSP22 does not accumulate in vivo. HS22 mRNA efficiently translated in vitro is induced in dark grown cells only when chloroplast differentiation has been initiated by exposure to the light for 3 h. In these cells HSP22 accumulates during heat shock. Inhibition of plastid translation activity during light-dependent chloroplast development prevents accumulation of HSP22 in vivo. However the HS22 mRNA formed in this case can be efficiently translated in vitro. Light requirement for the accumulation of HSP22 during heat stress is exhibited also by wild type C. reinhardtii cells which possess a differentiated chloroplast irrespective of the light conditions during cell growth. However dark grown wild type cells do not require pre-illumination for developing the ability to accumulate HSP22 during heat stress in the light.

Chlamydomonas↗

PubMed1990

Photoinactivation of photosystem II and degradation of the D 1 protein are reduced in a cytochrome b6/f-less mutant of Chlamydomonas reinhardtii.

The effect of unoccupancy of the QB site by plastoquinone on the photoinactivation of reaction center II in a Cyt b6/f-less mutant of Chlamydomonas reinhardtii, B6, was investigated. In these cells the oxidation of plastoquinol generated by electron flow via RC II to plastoquinone and thus the turnover of PQH2/PQ via the QB site are drastically reduced. Reaction center II of the mutant cells was resistant to photoinactivation relative to the control cells as demonstrated by measurements of light-induced destabilization of S2-QB- charge recombination, rise in intrinsic fluorescence and loss of variable fluorescence. These parameters relate to functions involving the reaction center II D 1 protein. The light-induced degradation of D 1 in the mutant cells was also considerably reduced, with a t1/2 value of 7 h as compared, under similar conditions, to about 1.5 h for the control cells. These results indicate that the photoinactivation of RC II and turnover of the D 1 protein are related and require occupancy of the QB site by PQ and its light-driven reduction.

Chlamydomonas↗

PubMed1990

Amino acid substitutions in the D1 protein of photosystem II affect QB- stabilization and accelerate turnover of D1.

Isogenic strains of Synechococcus PCC7942 were genetically engineered so that copy I of the gene psb A was mutated at specific sites. These mutations resulted in replacements of Ser 264 by Gly or Ala and of Phe 255 by Tyr or Leu in the D1 protein. The mutants were resistant to herbicides inhibiting electron transfer in photosystem II. All mutants exhibited alterations in the stability of QB- as demonstrated by a temperature downshift, to various extents, of the in vivo thermoluminescence emission. Measurements of the light-dependent turnover of D1 showed a marked decrease in the t 1/2 of this protein in the mutants as compared to wild-type, under low to medium light intensities. A correlation was found between the degree of perturbation in the QB- stability and the rate of acceleration in the turnover of D1. These data provide a direct evidence for the overlapping binding sites for the plastoquinone B and herbicides in the D1 protein. In addition these data indicate a close link between QB- destabilization in reaction center II and the mechanism controlling the light-dependent turnover of D1. Based on these results and previous work we suggest that destabilization of the semireduced quinone, facilitates a light-induced damage in D1 which triggers its degradation.

Amino Acid Sequence↗

PubMed1989

The nuclear-coded chloroplast 22-kDa heat-shock protein of Chlamydomonas. Evidence for translocation into the organelle without a processing step.

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine alpha-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.

Amino Acid Sequence↗

PubMed1988

Turnover of thylakoid photosystem II proteins during photoinhibition of Chlamydomonas reinhardtii.

The turnover of photosystem-II proteins during photoinhibition was analyzed in the green alga Chlamydomonas reinhardtii. Changes in the amount of photosystem II core complex polypeptides D1, D2, 44 kDa and 51 kDa, the antennae-CP-29 and light-harvesting-complex-II polypeptides and the water-oxidizing complex polypeptides of 30 kDa, 23 kDa and 16 kDa were monitored by a variety of techniques. Only the D1 and D2 polypeptides were found to turnover during photoinhibition when cells were exposed to ten fold photosynthesis-saturating light (2500 W/m2 for 90 min) at 25 degrees C. While 80% of photosystem-II activity was lost, a reduction of only 20% was observed in the total amount of D1 and D2 proteins. However, inhibition of chloroplast translation by chloramphenicol during photoinhibition resulted in the loss of about 60% of the D1 and 40% of the D2 proteins, as demonstrated by Western blotting and dot blotting of isolated thylakoids, quantitative analysis of immunogold-labeled whole-cell thin sections, and chase of radioactively prelabelled proteins during photoinhibition. We propose that the light-dependent turnover of the D1 protein is a protective mechanism against photoinhibition as far as the removal and replacement of D1 is compatible with the photoinactivation incurred by photosystem II. At light intensities at which the rate of D1 removal becomes limiting, loss of photosystem-II activity exceeds the turnover of D1 and the stability of the D2 protein is impaired as well.

Chlamydomonas↗

PubMed1988

Structure and biogenesis of Chlamydomonas reinhardtii photosystem I.

The photosystem I complex of the green alga Chlamydomonas reinhardtii was isolated and fractionated into its two subcomplex components: the core complex (CC I), which contained the reaction center (P-700) and had four polypeptide subunits, and the light-harvesting complex (LHC I) which contained four polypeptides of about 22, 25, 26 and 27 kDa. The 22-kDa apoprotein was isolated as a chlorophyll a and b binding protein. In the isolated photosystem I holocomplex, about ten copies of the 22-kDa LHC I apoprotein are present for each CC I unit. The 22-kDa polypeptide as well as the other three polypeptides of this complex and the subunit II of CC I are translated on 80S cytoplasmic ribosomes, and therefore are coded in the nucleus. During the greening process of the Chlamydomonas reinhardtii y-1 mutant the 22-kDa LHC I polypeptide, which cross-reacts with polyclonal antibodies raised against the Lemna gibba 20-kDa LHC I apoprotein, accumulates in thylakoids at a late stage of their development, and about 2-3 h after the LHC II and CC I subunit II polypeptides have accumulated. Accumulation of the 22-kDa protein during greening is inhibited by cycloheximide but not by chloramphenicol.

Chlamydomonas↗

PubMed1988

Role of the cytochrome b6.f complex in the redox-controlled activity of Acetabularia thylakoid protein kinase.

The regulation of the protein kinase activity responsible for the phosphorylation of the light-harvesting complex of photosystem II (LHCII) 27-kDa polypeptide involved in the State I-State II transitions in Acetabularia thylakoids was investigated. The LHCII kinase of isolated thylakoids retains its activity in absence of light-driven electron flow or reductants added in the dark. However, the kinase is reversibly inactivated by addition of oxidants in vitro or by far red (710 nm) light in vivo. Inhibitors of the quinol oxidase site of the cytochrome b6.f complex inactivate the LHCII kinase in the dark, and also in the light, or in presence of duroquinol when the plastoquinone pool is reduced. Inhibitors of the quinone reductase site of the b6.f complex have practically no effect in the dark and stimulate the kinase activity in the light. Based on these data and on our previous report, showing specific loss of LHCII kinase activity in a Lemna mutant lacking the cytochrome b6.f complex (Gal, A., Shahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221, 205-210), we propose that the activity of the LHCII kinase is regulated by the redox state of a cytochrome b6.f complex component(s) which responds to the balance of electron flow from photosystem II via the plastoquinone pool to photosystem I.

Acetabularia↗

PubMed1988

Structural properties of the D1 and surrounding photosystem II polypeptides as revealed by their interaction with cross-linking reagents.

Treatment of Chlamydomonas reinhardtii thylakoids with cross-linking reagents including glutaraldehyde causes polymerization of all thylakoid polypeptides, but not of the reaction center II polypeptide D1 unless the thylakoids are presolubilized by octyl beta-D-glucoside (Adir, N., and Ohad, I. (1986) Biochim. Biophys. Acta 850, 264-274). The results presented here show that this is a general property of D1 as it can be demonstrated in thylakoids of cyanophytes, Dasicladaceae, green algae, and C3 and C4 plants. Solubilization of the membranes by ionic detergents, deoxycholate, lauryl sucrose, or dodecyl beta-D-maltoside is not effective in inducing cross-linking of the D1 polypeptides by glutaraldehyde. The most effective alkyl glucosides were those with 7-9 carbon alkyl chains. The same behavior toward glutaraldehyde was exhibited by the unprocessed D1 precursor and by the palmitoylated D1 protein. Based on the refractility of the D1 protein to cross-linking reagents, a procedure was developed for its isolation from cross-linked thylakoids by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Isolated D1 retained its behavior toward cross-linking by glutaraldehyde and generated tryptic fragments similar to those obtained following trypsin treatment of intact thylakoids. Denaturation of isolated D1 protein by acetone facilitates cross-linking by glutaraldehyde and extensive degradation by trypsin. The photosystem II polypeptides are differentially cross-linked with increasing concentrations of glutaraldehyde, the most susceptible being the 28- and 23-kDa components of the light-harvesting chlorophyll a-b protein complex and the core complex 44- and 51-kDa polypeptides, and the least affected being the cytochrome b559, the D2 protein, and a 24-kDa component of the light-harvesting chlorophyll a-b protein complex. These results reflect the relative position and interaction of the photosystem II polypeptides within the complex and suggest that strong and specific hydrophobic interactions may be responsible for the tight and stable conformation of D1. This may be based mostly on the conserved amino acid sequences of D1 and possibly plays a role in the process of D1 integration and removal from the reaction center during its light-dependent turnover.

Aldehydes↗

PubMed1988

Evidence for protection by heat-shock proteins against photoinhibition during heat-shock.

The nuclear-coded 22 kd heat-shock protein (HSP-22) which is transported into the chloroplast and localized in the thylakoids was further characterized and found to be located in the grana lamellae (stacked thylakoids) as an extrinsic protein in the green alga Chlamydomonas reinhardtii. Inhibition of photosynthetic electron flow during heat-shock of Chlamydomonas cells was light-dependent, occurring at low-light intensities (<100 W/m) as compared with photoinhibition at 25 degrees C (>1000 W/m). The site of the damage was localized at the photosystem II (PS II) reaction center. The damage was drastically increased when heat-shock treatment was carried out in the presence of the 80S ribosomal translation inhibitor, cycloheximide (CHI). Pre-incubation of Chlamydomonas cells at 42 degrees C resulted in partial protection against photoinhibition during heat-shock, as compared with cells pre-incubated at 42 degrees C in the presence of CHI which, therefore, did not translate the heat-shock proteins. Analysis of the thylakoid polypeptides' pattern by SDS-PAGE revealed that during heat-shock in the light, thylakoid proteins became aggregated proportionally to the light intensity. Heat-shock in the presence of CHI enhanced the aggregation process which, at low light intensities, was specific to the PS II reaction center D1-protein. The results suggest that the chloroplasts HSPs prevent damage to the PS II reaction center during heat-shock in the light.

Journal Article↗

PubMed1986

Functional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts.

Phycobiliproteins obtained by dissociation of phycobilisomes were reassociated in vitro with intact thylakoids or isolated photosystems I and II preparations obtained from cyanophytes (prokaryotes) or green algae (eukaryotes) to form bound phycobilisome complexes. Energy transfer from Fremyella diplosiphon phycobiliproteins to chlorophyll a of reaction centers I and II was measured in: complexes containing intact thylakoids of the cyanophytes F. diplosiphon or Anacystis nidulans and the eukaryotic algae Euglena gracilis and mutants of Chlamydomonas reinhardtii; complexes containing isolated photosystem II particles of A. nidulans or C. reinhardtii; and complexes containing reaction center I of F. diplosiphon or C. reinhardtii. Energy transfer from phycoerythrin to chlorophyll a of photosystem II could be demonstrated in complexes containing phycobilisomes bound to cyanophyte thylakoids or isolated photosystem II particles of A. nidulans or C. reinhardtii. Bound phycobilisomes did not transfer energy to photosystem II within green algae thylakoids containing altered forms of light-harvesting chlorophyll a/b-protein complex (LHC) II antenna, reduced amounts of LHC II, or chlorophyll b, or chlorophyll b-less mutants, nor to chlorophyll a of photosystem I of intact thylakoids or isolated reaction centers. We conclude that phycobilisomes can form a specific and functional association with photosystem II particles of both cyanophytes and eukaryotic thylakoids. This interaction appears to be hindered by the presence of LHC II antenna in the eukaryotic thylakoids.

Chlamydomonas↗

PubMed1986

Photosystem I reaction centers from maize bundle-sheath and mesophyll chloroplasts lack subunit III.

Photosystem I reaction centers were isolated from mesophyll and bundle-sheath chloroplasts of the C4 maize plant. Both preparations were found to be free of chlorophyll b and to have the same spectral properties and chlorophyll/P700 ratio as photosystem I reaction centers isolated from C3 plants. Photosystem I reaction centers from both mesophyll and bundle sheath were found to consist of six subunits with apparent molecular masses of about 70 kDa, 20 kDa, 17 kDa, 16 kDa, 10 kDa and 8 kDa, corresponding to photosystem I reaction center subunits I, II, IV, V, VI and VII of spinach, as tested by their immunological cross-reactivity with antibody raised against the respective spinach subunits. No cross-reactivity was found with antibodies raised against subunit III of spinach, either in whole thylakoids or purified reaction centers of both bundle-sheath and mesophyll chloroplasts. It is concluded that photosystem I reaction centers of bundle-sheath and mesophyll thylakoids of maize are identical and lack the polypeptide corresponding to subunit III present in all C3 plants so far tested.

Centrifugation, Density Gradient↗

PubMed1986

Heat-shock protein synthesis in Chlamydomonas reinhardi. Translational control at the level of initiation of a poly(A)-rich-RNA coded 22-KDa protein in a cell-free system.

The effect of temperature on the in vitro translation of control and heat-shock poly(A)-rich RNA, obtained from Chlamydomonas reinhardi cells, incubated for 2 h at 25 degrees C respectively, was studied using the wheat-germ translation system. Incubation of the cells at 42 degrees C induces the synthesis of RNAs coding for several heat-shock proteins, including a 22-kDa major polypeptide as well as several proteins of 45-94 kDa, as demonstrated by run-off translation of polyribosomes isolated from intact cells. However, the high-molecular-mass heat-shock proteins are poorly translated in the wheat-germ system. The poly(A)-rich RNA coding for the 22-kDa heat-induced polypeptide has an apparent sedimentation coefficient higher than that expected from the molecular mass of its translation product, and was preferentially translated in vitro at temperatures above 31 degrees C as compared with pre-existing RNAs. Raising the temperature of translation, slightly inhibited (10%) the runoff translation of polyribosomes isolated from intact cells. However, when initiation was carried out in vitro for a short time at increasing temperatures and translation continued at 25 degrees C in the presence of aurintricarboxylic acid, the 22-kDa heat-shock polypeptides was preferentially translated. Aurintricarboxylic acid did not significantly inhibit incorporation of [35S]methionine when added to polyribosomes isolated from control or heat-shocked cells. From the above data we conclude that the translation of the 22-kDa heat-shock protein is controlled in vitro at the initiation level.

Cell-Free System↗

PubMed1986

Transient inactivation of the thylakoid photosystem II light-harvesting protein kinase system and concomitant changes in intramembrane particle size during photoinhibition of Chlamydomonas reinhardtii.

Light-dependent reduction of the plastoquinone pool regulates the activity of the thylakoid-bound protein kinase which phosphorylates the light harvesting chlorophyll a,b-protein complex (LHC II) and regulates energy distribution between photosystems II (PS II) and I (Staehelin, L. A., and C. J. Arntzen, 1983, J. Cell Biol., 97:1327-1337). Since reduction of plastoquinone by PS II is abolished in photoinhibited thylakoids due to loss of the secondary electron acceptor QB protein (Kyle, D. J., I. Ohad, and C. J. Arntzen, 1984, Proc. Natl. Acad. Sci. USA, 81:4070-4074), it was of interest to examine the activity of the LHC II protein kinase system during photoinhibition and recovery of PS II activity. The kinase activity was assessed both in vivo and in vitro in Chlamydomonas cells exposed to high light intensity (photoinhibition) and recovery at low light intensity. The kinase activity was progressively reduced during photoinhibition and became undetectable after 90 min. The inactive LHC II-kinase system could not be reactivated in vitro either by light or by reduction of the plastoquinone pool following addition of reduced duroquinone (TMQH2). The LHC II polypeptides were dephosphorylated in vivo when cells, prelabeled with [32P]orthophosphate before exposure to high light intensity, were transferred to photoinhibiting light in the presence of [32P]orthophosphate. In vivo recovery of the LHC II-kinase activity, elicited by the addition of TMQH2 to the assay system, did not require restoration of QB-dependent electron flow or de novo protein synthesis, either in the cytoplasm or in the chloroplast. Mild sonication of thylakoids isolated from photoinhibited cells restored the ability of the LHC II protein kinase system to be activated in vitro by addition to TMQH2. Restoration of the light-activated LHC-II kinase required recovery of QB-dependent electron flow. At the structural level, photoinhibition did not affect the ratio of grana/stroma thylakoids. A reduction of approximately 20% of the 11-17-nm intramembrane particles and an equivalent increase in the number of 6-10.5-nm particles was observed on the E-fracture faces of stacked thylakoid membranes. Similar but smaller changes were observed also on the E-fracture faces of unstacked thylakoid membranes (more 10-14-nm and less 6-9-nm particles) and P-fracture faces of stacked thylakoid membranes (more 6-8- and less 9.5-13-nm particles). All these structural changes were reversed to normal values during recovery of PS II activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Chlamydomonas↗

PubMed1985

Differentiation and development of bundle sheath and mesophyll thylakoids in maize. Thylakoid polypeptide composition, phosphorylation, and organization of photosystem II.

Photosynthetic electron flow, polypeptide pattern, presence of chlorophyll-protein complexes, and phosphorylation of thylakoid polypeptides have been investigated in differentiated mesophyll (M) and bundle sheath (B) thylakoids of the C4 plant Zea mays. The polypeptide pattern of M thylakoids and their photosynthetic electron flow are comparable to those of other green plants. B thylakoids exhibit only photosystem I (PSI) activity, contain only traces of the PSII light harvesting (LHCII) polypeptide, do not bind [3H] diuron, and lack polypeptides of the water-oxidation complex of PSII and the herbicide binding 32-kDa polypeptide, as detected by specific antibodies. However, B thylakoids possess a partially active PSII reaction center, as demonstrated by light-dependent reduction of silicomolybdate with 1,5-diphenylcarbazide (DPC) as an electron donor, and the presence of the PSII reaction center polypeptides of 44-47 kDa. Only one chlorophyll a-protein complex, corresponding to the PSI reaction center-core antenna, was detectable in B thylakoids, as opposed to chlorophyll a and chlorophyll a,b-protein complexes present in M thylakoids. The light-dependent, membrane-bound kinase activity present in M thylakoids could not be detected in B thylakoids which, nevertheless, contain a protein kinase able to phosphorylate casein. A total of 19 differences between the electrophoretic pattern of B and M thylakoid polypeptides were observed. The mRNA coding for the LHCII polypeptide is primarily, if not exclusively, localized in M cells. The development of PSII complex precedes that of PSI during the differentiation of B and M chloroplasts in expanding leaves of light-grown plants and during the greening of dark-grown etiolated seedlings. The differentiation of the maize leaf into cells programmed to form B or M chloroplasts does not require light. In light-grown plants, the differentiation of B and M thylakoids occurred progressively from the base of the leaf and was completed at 4-5 cm from the leaf base.

Cell Differentiation↗

PubMed1985

Light-dependent degradation of the Q(B)-protein in isolated pea thylakoids.

The 32 000-dalton Q(B)-protein of photosystem II (PS II) is rapidly damaged and removed from isolated pea thylakoids during incubation in the light resulting in a loss of photosynthetic electron flow through PS II. This in vitro photoinhibition is similar to that previously reported with intact Chlamydomonas cells. The damage occurs at a faster rate in vitro, however, due to the inability of isolated thylakoids to synthesize replacement Q(B)-protein. The removal of the damaged Q(B)-protein does not require any soluble components of the chloroplast stroma and is unaffected by the protease inhibitors phenyl-methylsulfonylfluoride or antipain. Unlike the effect of trypsin, no low mol. wt. membrane-bound or soluble fragments of the labelled Q(B)-protein could be identified either by autoradiography or immunologically using polyclonal antibodies specific for the Q(B)-protein. The lightinduced damage to the Q(B)-protein (indicated by a loss of Q(B) functional activity), preceded the removal of the protein from the membrane. We conclude that photodamage of the Q(B)-protein generates a conformational change which renders the protein susceptible to attack by a highly efficient, intrinsic membrane protease.

Journal Article↗

PubMed1985

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi.

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45 degrees C induces the expression of 10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36 degrees C to 42 degrees C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)RNAs endure in the cells at 42 degrees C up to 5 h but are no longer translated in vivo, whereas some poly(A)RNAs persist and are translated. As in pea, a poly(A)RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)RNA coding for the 22-kd HSP appears after 1 h at 36 degrees C. Its synthesis increases with the temperature of incubation up to 42 degrees C, although it decreases after 2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26 degrees C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.

Journal Article↗