Regulation of accumulation of the major thylakoid polypeptides in Chlamydomonas reinhardtii y-1 at 25 degrees C and 38 degrees C

The amount of messenger RNA (mRNA) for polypeptides of the chlorophyll a/b-protein complex of thylakoid membranes in etiolated and greening cells of Chlamydomonas reinhardtii y-1 was examined by immunoprecipitation and electrophoresis of products of in vitro translation to determine at which stage production of these polypeptides is regulated. Cells grown 4 d in the dark at 25 degrees C contained small amounts of translatable mRNA for the major membrane polypeptides. Exposure of these etiolated cells to light, under conditions in which the membrane polypeptides accumulated, resulted in a significant increase in the quantity of the mRNA. In contrast, when etiolated cells were incubated for 1-2 h in the dark at 38 degrees C, translation assays indicated that mRNA for the membrane polypeptides became abundant. Moreover, the quantity of the mRNA did not increase when these cells subsequently were exposed to light. Therefore, at 38 degrees C the cellular level of the polypeptides is not regulated by synthesis of mRNA. The in vitro synthesized polypeptides, which were precipitated with antibodies prepared against the purified thylakoid polypeptides, had apparent molecular weights of 31,500 and 30,000. The corresponding immunoprecipitated polypeptides made in vivo had apparent molecular weights of 29,500 and 26,000. Thus, the membrane polypeptides are made as precursors. No net accumulation of the polypeptides occurred in cells in the dark at 38 degrees C, but immunoreactive polypeptides the size of the mature membrane components were labeled during incubation of cells with [14C]acetate in the dark. These results indicated that the mRNA was translated in the dark, but since the polypeptides did not accumulate, the products of translation were probably degraded. We conclude from our experiments that at 25 degrees C production of the polypeptides is regulated by the level of translatable mRNA in the cells. At 38 degrees C, however, the accumulation of the polypeptides is controlled by posttranslational processes.     

Polypeptides of chloroplastic and cytoplastic origin required for development of Photosystem II activity,and chlorophyll-protein complexes,in Euglena gracilis Z chloroplast membranes

The development of photosynthetic activity and synthesis of chloroplast membrane polypeptides was studied during greening of Euglena gracilis Z in alternate light-dark-light cycles. The results show: (a) The development of both Photosystem II and Photosystem I can be dissociated from chlorophyll synthesis. (b) Most of the polypeptides required for development of Photosystem I are already synthesized during the initial light period (10–12 h); the further rise in Photosystem I activity in the dark is not inhibited by cycloheximide nor by chloramphenicol. (c) The development of Photosystem II requires continuous de novo synthesis of polypeptides and is inhibited by chloramphenicol. The water-splitting activity already present at the end of the first light period decays in the presence of chloramphenicol while that of 1,5-diphenylcarbazide oxidation is only partially retained. The activity can be repaired in the absence of chlorophyll synthesis and is correlated with the de novo synthesis of polypeptides of 50 000–60 000 daltons. The synthesis of these polypeptides and associated repair of Photosystem II activity is not inhibited by cycloheximide. (d) The chloroplast membranes can be resolved into about 40 distinct polypeptides, among them several in the molecular weight range 50 000–60 000, 20 000–35 000 and 10 000–15 000, which are major membrane constitutents. (e) The synthesis of two major polypeptides (M_r = 20 000–30 000) required for the formation of chlorophyll-protein complex(es) containing chlorophyll a and traces of chlorophyll b (CPII?) is light-dependent and cycloheximide-inhibited. It is concluded that the synthesis and addition to the growing membrane of chlorophyll and polypeptides required for the formation of Photosystem II and Photosystem I complexes can be dissociated in time. The H₂O-splitting enzyme(s) and possibly other components of Photosystem II complex are of chloroplastic origin and turn over in the dark while at least some of the chlorophyll binding polypeptides are of cytoplastic origin and their synthesis is light-controlled.     

Changes in thylakoid polypeptide phosphorylation during membrane biogenesis in Chlamydomonas reinhardii y-1

Thylakoid polypeptide phosphorylation has been studied in vivo and in vitro during plastid differentiation in Chlamydomonas reinhardii y-1. Pulse labeling cells at different stages of greening with [³²P]orthophosphate revealed differences in the pattern of protein phosphorylation. In the early phase of greening the 44–47 kDa reaction center II polypeptides were labeled but the 22–24 kDa polypeptides of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHC) were not. Later in the greening, coinciding with the formation of the antenna of Photosystem I and membrane stacking, the converse was found. Furthermore, the 22–24 kDa polypeptides of grana lamellae were less labeled than the same polypeptides found in the corresponding stroma lamellae. Polypeptides in the molecular mass range of 32–34 kDa were phosphorylated at all stages following the onset of greening. Dark-grown cells did not incorporate ³²P in vivo or in vitro into the polypeptides present in the residual thylakoids. Similarly, cells greened in the presence of chloramphenicol, in which the synthesis of reaction centers is inhibited, showed no light-stimulated phosphorylation in vitro. However, the residual 32–34 kDa and 44–47 kDa polypeptides found in thylakoids of these cells were phosphorylated in vivo, whereas the LHC polypeptides synthesized in the presence of chloramphenicol were not. Phosphorylation of the LHC polypeptides (22–24 kDa) in these cells occurred if new reaction center polypeptides and all antennae components were formed, following removal of the inhibitor and further incubation of the cells in the light. Phosphorylation of LHC polypeptides was not resumed if active reaction centers were formed in the absence of complete restoration of all antenna components (incubation in the dark or light with addition of cycloheximide). It is concluded that phosphorylation is correlated with the thylakoid polypeptide content and organization.     

Phosphorylation of thylakoid proteins during chloroplast biogenesis in greening etiolated and light-grown wheat leaves

Phosphorylation of polypeptides in isolated thylakoids was examined during chloroplast biogenesis in greening etiolated wheat leaves and 4 day-old wheat leaves grown under a diurnal light regime. At early stages of plastid development standard thylakoid preparations were heavily contaminated with nuclear proteins, which distorted the polypeptide phosphorylation profiles. Removal of contamination from membranes by sucrose density centrifugation demonstrated that the major membrane phosphoprotein in etioplasts was at 35 kDa. During etioplast greening a number of phosphoproteins appeared, of which the 25–27 kDa apoproteins of the light-harvesting chlorophylla/b protein complex associated with photosystem II (LHCII) became the most dominant. At the early stages of thylakoid development found at the base of the 4-day-old light grown leaf the LHCII apoproteins were evident as phosphoproteins; however the major phosphoprotein was polypeptide atca. 9kDA. Phosphorylation of both the LHCII apoproteins and the 9 kDa polypeptide in these thylakoids was not light-dependent. In the older thylakoids isolated from the leaf tip the LHCII apoproteins were the major phosphoproteins and their phosphorylation had become light-regulated; however phosphorylation of the 9 kDa polypeptide remained insensitive to light.     

Accumulation of Chlorophyll a/b-Binding Polypeptides in Chlamydomonas reinhardtii y-1 in the Light or Dark at 38 degrees C : Evidence for Proteolytic Control

The kinetics of accumulation of light harvesting chlorophyll (Chl) a/b-binding polypeptides (LHCPs) in thylakoid membranes were analyzed during greening of Chlamydomonas reinhardtii y-1 at 38°C. Initial accumulation of LHCPs in thylakoid membranes was linear; LHCP precursors or polypeptides in transit within the chloroplast stroma were not detected. The rate of accumulation in the light was at least five-fold greater than that in the dark. The relatively small amount of LHCPs that accumulated in the dark was integrated properly in the membrane, as judged by the pattern of cleavage in vitro by exogenous proteases, and did not turn over at a significant rate in vivo. The kinetic data suggested that in y-1 cells either translation of LHCP mRNA was inhibited in the dark or newly synthesized polypeptides were degraded concurrently with transport into the chloroplast unless rescued by Chl. LHCPs accumulated in cells of the Chl b-deficient strain pg-113 at the same rate in the dark or the light at 38°C, an indication that light did not affect translation of LHCP mRNA. Membrane-associated LHCPs in pg-113 cells were completely degraded, in contrast to those in y-1 cells, by exogenous proteases, which suggested that pg-113 cells are deficient in a proteolytic activity. A peptidase was recovered from y-1 cells in a membrane fraction with a buoyant density slightly less than that of thylakoid membranes. Although a role for this activity in degradation of LHCPs has not been established, the specific activity of this peptidase in pg-113 cells was only 10 to 15% of the level in y-1 cells.     

CHANGES IN CHEMICAL COMPOSITION OF THYLAKOID MEMBRANES DURING GREENING OF THE y-1 MUTANT OF CHLAMYDOMONAS REINHARDI

Two fractions of thylakoid membranes (TMF) have been isolated from disrupted (French press) algal cells by using a discontinuous sucrose gradient. TMF-II consists mostly of thylakoid membranes still partially organized in grana; it contains also fragments of chloroplast envelope, pyrenoid tubules, and starch granules; thus it amounts to a fraction of chloroplast fragments which have lost practically all matrix components. TMF-I consists of smaller chloroplast fragments and is contaminated to a larger extent than TMF-II by other subcellular components, primarily mitochondria. TMF-II accounts for about 12% of the protein and 30% of the chlorophyll of the whole cell; it contains cytochrome 554 and carotenoids in the same ratio to chlorophyll as the latter, and shows photosystems I and II activities but lacks enzymatic activities characteristic of the dark reactions. During the greening of the y-1 mutant of Chlamydomonas, TMF's have been isolated over a range of chlorophyll concentrations from 5 to 25 µg/10⁷ cells. The results showed that during this period the ratios of chlorophyll to cytochrome 554 and of chlorophyll to carotenoids, and the relative concentrations of individual carotenoids were continuously changing. The findings support the view that during greening, thylakoid membranes are produced by multistep assembly.     

In Vitro Processing of Precursors of Thylakoid Membrane Proteins of Chlamydomonas reinhardtii y-1

Studies of in vitro processing of precursors of the major chlorophyll a/b-binding polypeptides of Chlamydomonas reinhardtii y-1 were undertaken to define the precursor-product relationships. Analysis of translates, prepared from C. reinhardtii poly(A)-rich RNA in a rabbit reticulocyte lysate system, which were incubated with the soluble fraction from C. reinhardtii cells, showed that the 31,500 relative molecular mass (M_r) precursor was converted to the M_r 29,500 thylakoid membrane polypeptide whereas the M_r 30,000 precursor was converted to the M_r 26,000 product. Furthermore, the M_r 31,500 polypeptide, when bound to antibodies, was not processed to the mature polypeptide of M_r 29,500, although the presence of antibodies did not prevent the precursor of M_r 30,000 from being converted to the mature M_r 26,000 polypeptide. The mature fraction of M_r 26,000, was separated into two bands corresponding to polypeptides 16 and 17 in the electrophoretic system of Chua and Bennoun (1975 Proc Natl Acad Sci USA 72: 2175-2179).

Processing activity was present in the soluble fraction obtained from cells grown in the light or in the dark. Therefore, processing of the precursor polypeptides does not appear to be involved in the regulation by light of the accumulation of these polypeptides in thylakoid membranes.

     

Plastid Development in Pisum sativum Leaves during Greening : I. A Comparison of Plastid Polypeptide Composition and in Organello Translation Characteristics

Changes in plastid polypeptide composition during greening of etiolated peas were investigated by two-dimensional gel electrophoresis. One hundred of the more than 250 polypeptides which could be detected upon silver staining were followed during plastid development. Thirty-nine polypeptides decreased in abundance on a per organelle basis. Twentythree of the 46 polypeptides which increased in abundance upon greening could be identified as proteins of the thylakoid membrane. The changes in proteins observed during greening of etiolated leaves corresponded largely to those observed during normal leaf expansion. The origin of some of the polypeptides was traced back by comparing the two-dimensional gels of plastid proteins with in organello translation products and with polypeptides which had been synthesized in vitro from poly(A⁺) mRNA preparations and posttranslationally imported by chloroplasts. Some polypeptides were specifically identified in two-dimensional gels by Western blot analysis.     

Composition and biosynthesis of thylakoid membrane polypeptides in the red alga Cyanidium caldarium: Comparison with the thylakoid polypeptide composition of higher plants and cyanobacteria

The polypeptide composition of thylakoid membranes of the red alga Cyanidium caldarium was studied by PAGE in the presence of lithium dodecyl sulfate. The thylakoid membranes were shown to contain 65 polypeptides with mol wt from 110 to 10 kDa. PS I isolated from C. caldarium cells is composed of at least 5 components, one of which is the chlorophyll-protein complex with mol wt of 110 kDa typical of higher plants. Cyt f, c ₅₅₂, b ₆ and b ₅₅₉ were identified. Inhibition of carotenoid biosynthesis with norflurazon caused no changes in the polypeptide composition of thylakoid membranes of the algae grown in dark. The suppression of the biosynthesis rate of some thylakoid polypeptides in the algae grown with norflurazon in light is a result of membrane photodestruction. Thylakoid membranes from C. caldarium cells are more similar in the number of protein components to thylakoid membranes from cells of the cyanobacterium Anacystis nidulans than to those of higher plants (Pisum sativum), which was proved by immune-blotting assays: Thylakoid membranes of the red alga and cyanobacteria contain 28 homologous polypeptides, while thylakoid membranes of the alga and pea, only 15.Abbreviations CD circular dichroism - CP chlorophyll-protein complex - LDS lithium dodecyl sulfate - NF norflurazon     

Synthesis of chloroplast membrane polypeptides during synchronous growth of Chlamydomonas reinhardtii

The synthesis of the major chloroplast membrane polypeptides has been studied during synchronous growth of Chlamydomonas reinhardtii. Under these conditions, chlorophyll is synthesized during the latter part of the light period and cell division takes place during the dark period. The profile of the chloroplast membrane polypeptides of C. reinhardtii has been well characterized and shown to contain two major classes by size (Hoober, J. 1970. J. Biol. Chem. 245:4327). Polypeptides of group I have a mol wt range of 50,000–55,000 daltons. The second region consists of at least three polypeptide groups, IIa, IIb, and IIc, having mol wt of 40,000, 31,000, and 27,000 daltons, respectively. The synthesis of these polypeptides has been measured using a double-labeling technique and a computer-aided statistical analysis. The rate of labeling of group I polypeptides is highest during the early light period and decreases after 6 h of growth. Group IIa is labeled from the beginning of the light period, but little synthesis of IIb occurs before 3 h, and significant amounts of label are not found in IIc before 5 h of growth. After approximately 8 h of light, groups IIb and IIc are synthesized at rates significantly greater than those of the other membrane polypeptides. The synthesis of the major polypeptide groups ceases in the dark. We conclude that the biosynthesis of the chloroplast membranes is a sequential or stepwise process.     

The fluidity of chloroplast thylakoid membranes and their constituent lipids: A comparative study by ESR

Comparative measurements were made of the fluidity of chloroplast thylakoids, total membrane lipids and polar lipids utilizing the order parameter and motion of spin labels.No significant differences were found in the fluidity of membranes or total membrane lipids from a wild type and a mutant barley (Hordeum vulgare chlorina f₂ mutant) which lacks chlorophyll $\text{b}$ and a 25 000 dalton thylakoid polypeptide. Redistribution of intrinsic, exoplasmic face (EF) membrane particles by unstacking thylakoid membranes in low salt medium also had no effect on membrane fluidity. However, heating of isolated thylakoids decreased membrane fluidity.The fluidity of vesicles composed of membrane lipids is much greater than that of the corresponding membranes. Fluidity of the membranes, however, increased during greening indicating that the rigidity of the membranes, compared with that of total membrane lipids, is not caused by chlorophyll or its associated peptides. It is concluded that the restriction of motion in the acyl chains in the thylakoids is not caused by chlorophyll or the major intrinsic polypeptide but by some other protein components.     

A chloroplast-associated fatty acid synthetase system in Euglena

Fatty acid synthetase activity in etiolated Euglena gracilis strain Z is independent of added ACP and associated with a high-molecular-weight complex of the type found in yeast. Cells grown in the dark and then greened by illumination in a resting medium develop a second enzyme system which is dependent on added ACP and generally resembles the corresponding E. coli and plant enzymes. Cycloheximide has no effect on the appearance of the ACP-dependent fatty acid synthetase in greening cells whereas chloramphenicol causes complete inhibition at concentrations which decrease chlorophyll synthesis by 66%. An induction of the ACP-dependent fatty acid synthetase in the absence of chloroplast development occurs on exposure of dark-grown cells to doses of ultraviolet light which selectively affect proplastid nucleoprotein. This enzyme induction by ultraviolet light is inhibited by chloramphenicol. The protein synthesis machinery of the chloroplast appears to be responsible, either directly or indirectly, for the appearance of the ACP-dependent fatty acid synthetase of Euglena.     

Periodic variations in the ratio of free to thylakoid-bound chloroplast ribosomes during the cell cycle of Chlamydomonas reinhardtii

The ratio of free to thylakoid-bound chloroplast ribosomes in Chlamydomonas reinhardtii undergoes periodic changes during the synchronous light-dark cycle. In the light, when there is an increase in the chlorophyll content and synthesis of thylakoid membrane proteins, about 20-30% of the chloroplast ribosomes are bound to the thylakoid membranes. On the other hand, only a few or no bound ribosomes are present in the dark when there is no increase in the chlorophyll content. The ribosome-membrane interaction depends not only on the developmental stage of the cell but also on light. Thus, bound ribosomes were converted to the free variety after cultures at 4 h in the light had been transferred to the dark for 10 min. Conversely, a larger number of chloroplast ribosomes became attached to the membranes after cultures at 4 h in the dark had been illuminated for 10 min. Under normal conditions, when there was slow cooling of the cultures during cell harvesting, chloroplast polysomal runoff occurred in vivo leading to low levels of thylakoid-bound ribosomes. This polysomal runoff could be arrested by either rapid cooling of the cells or the addition of chloramphenicol or erythromycin. Each of these treatments prevented polypeptide chain elongation on chloroplast ribosomes and thus allowed the polyosomes to remain bound to the thylakoids. Addition of lincomycin, an inhibitor of chain initiation on 70S ribosomes, inhibited the assembly of polysome-thylakoid membrane complex in the light. These results support a model in which initiation of mRNA translation begins in the chloroplast stroma, and the polysome subsequently becomes attached to the thylakoid membrane. Upon natural chain termination, the chloroplast ribosomes are released from the membrane into the stroma.     

LIGHT-HARVESTING COMPLEX AF'OPROTEINS IN CYTOPLASMIC VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Cells of Chlamydomonas reinhardtii Dangeard strain cw15arg7A contain electron-opaque material, often in the form of large granules, within cytoplasmic vacuoles. Immunoelectron microscopy with antibodies to polypeptide 11, a component of the major light-harvesting chlorophyll (Chl) a/b-protein complex (LHCII,) of thylakoid membranes, revealed the presence of LHCII Polypeptides within the chloroplast and in vacuolar material in cells grown in the light. Vacuolar material was also heavily immunodecorated in dark-grown cells that did not synthesize Chl. Accumulation of LHCII polypeptides was further studied in greening and light-grown cells of a pale green mutant, deficient in LHCII, that was derived from cu15arg7A by insertional mutagenesis. Light-grown cells of this mutant strain contained relatively few thylakoid membranes and synthesized LHCII polypeptides at a low rate. However, cytoplasmic vacuoles were immunoreactive. Appearance of mature-sized LHCII polypeptides in vacuoles suggested that these proteins were partially translocated across the envelope but not retained by the chloroplast without assembly of LHCII.     

Events surrounding the early development of Euglena chloroplasts. 16. Plastid thylakoid polypeptides during greening

Using sulfolipid to locate plastid thylakoid membranes in gradients from dark-grown resting cells it has been possible to study the plastid thylakoid membrane polypeptides of Euglena gracilis var. bacillaris undergoing light-induced chloroplast development. All plastid thylakoid bands seen in dark-growing wild-type cells and in mutant W₃BUL in which plastid DNA is undetectable, are observed to increase in amount during plastid development. Others, which are undetectable in dark-grown wild-type and W₃BUL increase greatly during plastid development and appear to be those associated with pigment-protein complexes. The data obtained from experiments where the polypeptides were labeled with ³⁵S during development, either continuously or in pulses, were consistent with these findings. Cycloheximide strongly inhibited the increases in amount in all bands and chloramphenicol or streptomycin produced a lower level of inhibition in all bands indicating tight control of the formation of each plastid membrane constituent by the others. The formation of a polypeptide band of 25 000 molecular weight, thought to be a part of a pigment-protein complex of the thylakoid, and chlorophyll synthesis were inhibited identically by these antibiotics.     

Events surrounding the early development of Euglena chloroplasts. 15. Origin of plastid thylakoid polypeptides in wild-type and mutant cells

Techniques are described for the isolation of plastid thylakoid membranes from light-grown and dark-grown cells of Euglena gracilis var. bacillaris, and from mutants affecting plastid development. These membranes, which have minimal contamination with other cell fractions, are localized in sucrose gradients by using the thylakoid membrane sulfolipid as a specific marker. The plastid thylakoid membrane polypeptides isolated from these membranes were separated on SDS polyacrylamide gels and yielded patterns containing 30–40 polypeptides. Light-grown strain Z gave patterns identical with bacillaris. Since the plastid thylakoid polypeptide patterns obtained from dark-grown wild-type cells and from a bleached mutant W₃BUL in which plastid DNA is undetectable are identical, it appears that the proplastid thylakoid polypeptides of wild-type cannot be coded in plastid DNA and are probably coded in nuclear DNA. The plastid thylakoid polypeptide patterns obtained from various dark-grown mutants are identical to those obtained from dark-grown wild-type cells. Light-grown mutants, making large but abnormal chloroplasts, show a correlation between the amount of chlorophyll formed and the amount of a plastid thylakoid polypeptide thought to be associated with one of the pigment-protein light-harvesting complexes. Treatment with SAN 9789 (4-chloro-5-(methyl-amino)-2-(α,α,α,-trifluoro-m-tolyl)-3-(2H(pyridazinone) known to block carotenoid synthesis at the level of phytoene, causes a progressive loss of all plastid thylakoid polypeptides during growth in darkness and results in the establishment of a new, lower steady-state level of sulfolipid. At least ten of the plastid thylakoid polypeptides become labeled when isolated chloroplasts are supplied with radioactive amino acids; of these six are undectable in W₃BUL and are, therefore, candidates for coding by plastid DNA.     

Polypeptides of a Light-Harvesting Complex of the Diatom Phaeodactylum tricornutum Are Synthesized in the Cytoplasm of the Cell as Precursors

A light-harvesting fucoxanthin-chlorophyll a/c-protein complex has been isolated from the diatom Phaeodactylum tricornutum by detergent extraction of thylakoid membranes coupled with sucrose density gradient centrifugation. The isolated complex was devoid of photochemical activity and displayed spectral characteristics consistent with light harvesting function. It has three major polypeptides of apparent molecular weights 18,000, 19,000, and 19,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Using protein synthesis inhibitors, these polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes. Antibodies raised to a mixture of the 19,000 and 19,500 dalton components of the complex were used to demonstrate structural similarity among the three polypeptide components. Immunoprecipitation from primary translation products synthesized in a reticulocyte lysate system primed with P. tricornutum poly(A) RNA, indicates that the polypeptide components are synthesized as precursors 3,000 to 5,000 daltons larger than the mature polypeptides.