Structure of methemerythrin at 5 A resolution.

Rabbit globin messenger RNA was labelled in vitro with ¹²⁵I to specific activities in the range 20 to 200 × 10⁶ cts/min per μg. This ¹²⁵I-labelled mRNA bound to rabbit reticulocyte ribosomes with the kinetics and sensitivity to inhibitors expected from its participation in the normal process of the initiation of protein synthesis. Furthermore, when modified in 25% of its cytidine residues with unlabelled iodide, the mRNA coded for the same series of initiation peptides as did the unmodified mRNA. Using the techniques of RNA fingerprinting, the binding reaction was shown to select against contaminants and against “globin mRNA” molecules which lack a particular oligonucleotide implicated in the initiation process. When the ¹²⁵I-labelled mRNA was bound to ribosomes, both the initiating 40 S subunits and the 80 S ribosomes protected a fraction of the mRNA from digestion by pancreatic ribonuclease. Fingerprint analysis showed that highly specific regions of the mRNA were protected by the 40 S subunits and 80 S ribosomes and that these two protected regions were not identical.     

Biosynthetic cause of in vivo acquired thermotolerance of photosynthetic light reactions and metabolic responses of chloroplasts to heat stress

Thermotolerance of photosynthetic light reactions in vivo is correlated with a decrease in the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and an increased incorporation into thylakoid membranes of saturated digalactosyl diacylglycerol species. Although electron transport remains virtually intact in thermotolerant chloroplasts, thylakoid protein phosphorylation is strongly inhibited. The opposite is shown for thermosensitive chloroplasts in vivo. Heat stress causes reversible and irreversible inactivation of chloroplast protein synthesis in heat-adapted and nonadapted plants, respectively, but doe not greatly affect formation of rapidly turned-over 32 kilodalton proteins of photosystem II. The formation on cytoplasmic ribosomes and import by chloroplasts of thylakoid and stroma proteins remain preserved, although decreased in rate, at supraoptimal temperatures. Thermotolerant chloroplasts accumulate heat shock proteins in the stroma among which 22 kilodalton polypeptides predominate. We suggest that interactions of heat shock proteins with the outer chloroplast envelope membrane might enhance formation of digalactosyl diacylglycerol species. Furthermore, a heat-induced recompartmentalization of the chloroplast matrix that ensures effective transport of ATP from thylakoid membranes towards those sites inside the chloroplast and the cytoplasm where photosynthetically indispensable components and heat shock proteins are being formed is proposed as a metabolic strategy of plant cells to survive and recover from heat stress.     

Controlled proteolysis of nascent polypeptides in rat liver cell fractions. II. Location of the polypeptides in rough microsomes

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.     

Changes in protein synthesis induced in tomato by chilling

Impaired chloroplast function is responsible for nearly two-thirds of the inhibition of net photosynthesis caused by dark chilling in tomato (Lycopersicon esculentum Mill.). Yet the plant can eventually recover full photosynthetic capacity if it is rewarmed in darkness at high relative humidity. As a means of identifying potential sites of chilling injury in tomato, we monitored leaf protein synthesis in chilled plants during this rewarming recovery phase, since changes in the synthesis of certain proteins might be indicative of damaged processes in need of repair. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins pulse labeled with [³⁵S]methionine revealed discrete changes in the pattern of protein synthesis as a result of chilling. A protein of M_r = 27 kilodaltons (kD), abundantly synthesized by unchilled plants, declined to undetectable levels in chilled plants. Reillumination restored the synthesis of this protein in plants rewarmed for 8 hours. Peptide mapping analysis showed the 27 kD protein to be the major chlorophyll a/b binding protein of the photosystem II light-harvesting complex (LHCP-II). The identity of this protein was confirmed by its immunoprecipitation from leaf extracts by a monoclonal antibody specific for the major LHCP-II species. While chilling abolished the synthesis of the major LHCP-II species, it also induced the synthesis of an entirely new protein of M_r = 35 kD. The protein was synthesized on cytoplasmic ribosomes, and two-dimensional polyacrylamide gel electrophroesis showed it to exist as a single isoelectric species. This chilling-induced 35 kD protein is structurally distinct from the 27 kD LHCP-II and appears to be synthesized specifically in response to low temperature. While the 35 kD protein was found not to be associated with the chloroplast thylakoid membrane, chilling did cause selective changes in thylakoid membrane protein synthesis. The synthesis of two unidentified proteins, M_r = 14 and 41 kD, and the β-subunit of the chloroplast coupling factor were substantially reduced after chilling. These losses may provide clues as to the causes of the overall reduction in net photosynthesis caused by chilling.     

Adaptation of the Photosynthetic Apparatus to Irradiance in Dunaliella tertiolecta: A Kinetic Study

The time course of adaptation from a high to a low photon flux density was studied in the marine chlorophyte Dunaliella tertiolecta. A one-step transition from 700 to 70 micromole quanta per square meter per second resulted in a reduction of doubling rate from 1.1 to 0.4 per day within 24 hours, followed by a slower accumulation of photosynthetic pigments, light harvesting antenna complexes, Photosystem II reaction centers and structural lipids that constitute the thylakoid membranes. Photoregulated changes in the biochemical composition of the thylakoid proteins and lipids were functionally accompanied by decreases in the minimal photosynthetic quantum requirement and photosynthetic capacity, and an increase in the minimal turnover time for in vivo electron transport from water to CO₂. Analysis of de novo synthesis of thylakoid membranes and proteins indicates that a high light to low light transition leads to a transient in carbon metabolism away from lipid biosynthesis toward the synthesis of the light harvesting antenna protein complexes, accompanied by a slower restoration rate of reaction centers and thylakoid membranes. This pattern of sequential synthesis of light harvesting complexes followed by reaction centers and membranes, appears to optimize light harvesting capabilities as cells adapt to low photon flux densities.     

Synthesis of soluble, thylakoid, and envelope membrane proteins by spinach chloroplasts purified from gradients.

Intact spinach chloroplasts that had been purified on gradients of silica sol incorporated [³⁵S]methionine into soluble and membrane-bound products, using light as the sole energy source. The labeled chloroplasts were lysed osmotically and fractionated on a discontinuous gradient of sucrose into the soluble fraction and the thylakoid and envelope membranes. About 29% of the radioactivity in the chloroplast was recovered in the soluble fraction, 59% in the thylakoid membranes, and 0.1% in the envelope membranes. The products of protein synthesis in the different fractions, as well as in the whole chloroplast, were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. There were two zones of radioactivity in the gels of the soluble fraction, the major zone coincident with the large subunit of ribulose diphosphate carboxylase at a molecular weight of about 50,000. The thylakoid membranes contained five labeled polypeptides, the most active having a molecular weight of about 31,000. The envelope membranes contained a major radioactive component of a molecular weight of about 50,000 and two other minor components.     

A MAJOR POLYPEPTIDE OF CHLOROPLAST MEMBRANES OF CHLAMYDOMONAS REINHARDI : Evidence for Synthesis in the Cytoplasm as a Soluble Component

Electrophoresis of thylakoid membrane polypeptides from Chlamydomonas reinhardi revealed two major polypeptide fractions. But electrophoresis of the total protein of green cells showed that these membrane polypeptides were not major components of the cell. However, a polypeptide fraction whose characteristics are those of fraction c (a designation used for reference in this paper), one of the two major polypeptides of thylakoid membranes, was resolved in the electrophoretic pattern of total protein of green cells. This polypeptide could not be detected in dark-grown, etiolated cells. Synthesis of the polypeptide occurred during greening of etiolated cells exposed to light. When chloramphenicol (final concentration, 200 µg/ml) was added to the medium during greening to inhibit chloroplastic protein synthesis, synthesis of chlorophyll and formation of thylakoid membranes were also inhibited to an extent resulting in levels of chlorophyll and membranes 20–25% of those found in control cells. However, synthesis of fraction c was not affected by the drug. This polypeptide appeared in the soluble fraction of the cell under these conditions, indicating that this protein was synthesized in the cytoplasm as a soluble component. When normally greening cells were transferred from light to dark, synthesis of the major membrane polypeptides decreased. Also, it was found that synthesis of both subunits of ribulose 1, 5-diphosphate carboxylase was inhibited by chloramphenicol, and that synthesis of this enzyme stopped when cells were transferred from light to dark.     

Responsibility of phosphatidylglycerol for biogenesis of the PSI complex

Phosphatidylglycerol (PG) ubiquitous in thylakoid membranes of photosynthetic organisms was previously shown to contribute to accumulation of chlorophyll through analysis of the cdsA⁻ mutant of a cyanobacterium Synechocystis sp. PCC6803 defective in PG synthesis (SNC1). Here, we characterized effects of manipulation of the PG content in thylakoid membranes of Synechocystis sp. PCC6803 on the photosystem complexes to specify roles of PG in biogenesis of thylakoid membranes. SNC1 cells with PG deprivation in vivo, together with the chlorophyll decrease, exhibited a decline not in PSII, but in PSI, at the complex level as well as the subunit levels. On the other hand, the decrease in the PSI complex was accounted for by a remarkable decrease in the PSI trimer with an increase in the monomer. These symptoms of SNC1 cells were complemented in vivo by supplementation of PG. Besides, a reduction in the PG content of thylakoid membranes isolated from the wild type in vitro on treatment with phospholipase A2 (PLA2), similar to the PG-deprivation in SNC1 in vivo, brought about a decrease in the trimer population of PSI with accumulation of the monomer. These results demonstrated that PG contributes to the synthesis and/or stability of the PSI complex for maintenance of the cellular content of chlorophyll, and also to construction of the PSI trimer from the monomer at least through stabilization of the trimerized conformation.     

Protein metabolism. 3. Relationship between albumin metabolism and elevated liver protein synthesis in the guinea pig infected with Trichostrongylus colubriformis

Studies of the incorporation of ¹⁴C-l-leucine into polypeptides by isolated liver ribosomes from guinea pigs confirmed previous in vivo studies that showed that Trichostrongylus colubriformis infection results in an elevated hepatic protein synthesis.The increased rate of protein synthesis was associated with the membrane-bound ribosomes that synthesize the circulating plasma proteins. Inappetance of infected animals was not resposible for the increased rate of synthesis by the membrane-bound ribosomes, but it was found that undernourishment may stimulate synthesis by free ribosomes.Plasma albumin turnover rate and loss into the intestine were both significantly increased in infected guinea pigs. It was concluded that these changes stimulated protein synthesis by membrane-bound ribosomes.The importance of elevated liver protein synthesis and loss of plasma protein in gastrointestinal nematode infections is discussed.     

An improved purification method and a further characterization of the 33-kilodalton protein of spinach chloroplasts

The 33-kDa protein was purified in a high yield from thylakoid membranes of spinach chloroplasts. The extinction coefficient and A^1%_1cm value at 276 nm of the protein were 22000 M^?1·cm^?1 and 6.8, respectively. The 33-kDa protein and a polypeptide appearing at 32 kDa in the SDS-polyacrylamide gel electrophoresis of thylakoid membranes were compared by peptide mapping after limited proteolysis. This indicates that the 32-kDa band is entirely due to the 33-kDa protein. The molar ratio of chlorophyll to the 33-kDa protein in the chloroplasts was estimated to be 300. This suggests that one photosynthetic unit possesses one or two molecules of the 33-kDa protein.     

Movement of ribosomes over messenger RNA in polysomes of rel + and rel - Escherichia coli strains

The in vitro movement of ribosomes over messenger RNA was studied in both the presence and the absence of protein synthesis. For this purpose, labeled polysomes were extracted from rel⁺ and rel^? strains of Escherichia coli grown in the presence of radioactive uracil and incubated in a cell-free system containing tRNA, amino acids, soluble enzymes and a source of energy. The gradual conversion of the labeled polysomes into monosomes and ribosomal subunits was followed by subjecting the reaction mixture to sucrose gradient sedimentation after various incubation times and measuring the radioactivity present in the three relevant ribosomal fractions.It was found that when the conditions of incubation allow protein synthesis to occur, polysomes extracted from rel⁺ and rel^? cells are converted mainly into free monosomes, which can be made to dissociate into subunits by high-sodium or low-magnesium ion concentrations. Under conditions in which protein synthesis cannot occur because a mutant aminoacyl-tRNA synthetase has been rendered inactive, polysome conversion still occurs, though to a reduced extent. When the products of such residual run-off are examined, however, a difference is manifest between polysomes extracted from rel⁺ and from rel^? strains: whereas the polysomes from the rel^? strain are still converted into free monosomes even in the absence of protein synthesis, the polysomes from the rel⁺ strain are now converted mainly into subunits. It can be inferred, therefore, that ribosomes from rel^? bacteria, but not those from rel⁺ bacteria, continue movement over messenger RNA in the absence of protein synthesis.Studies of mixed extracts from rel^? and rel⁺ bacteria have shown that the character of the run-off process does not depend on the source of tRNA and soluble enzymes; the proportions of monosomes and subunits among the run-off products formed in the absence of protein synthesis depend only on the source of the polysomes. It is suggested that the mutation of the rel gene alters the functional architecture of ribosomes.     

EFFECTS OF CHLORAMPHENICOL ON CHLOROPLAST AND MITOCHONDRIAL ULTRASTRUCTURE IN OCHROMONAS DANICA

The effect of chloramphenicol (CAP) on cell division and organelle ultrastructure was studied during light-induced chloroplast development in the Chrysophyte alga, Ochromonas danica. Since the growth rate of the CAP-treated cells is the same as that of the control cells for the first 12 hr in the light, CAP is presumed to be acting during that interval solely by inhibiting protein synthesis on chloroplast and mitochondrial ribosomes. CAP markedly inhibits chloroplast growth and differentiation. During the first 12 hr in the light, chlorophyll synthesis is inhibited by 93%, the formation of new thylakoid membranes is reduced by 91%, and the synthesis of chloroplast ribosomes is inhibited by 81%. Other chloroplast-associated abnormalities which occur during the first 12 hr and become more pronounced with extended CAP treatment are the presence of prolamellar bodies and of abnormal stacks of thylakoids, the proliferation of the perinuclear reticulum, and the accumulation of dense granular material between the chloroplast envelope and the chloroplast endoplasmic reticulum. CAP also causes a progressive loss of the mitochondrial cristae, which is paralleled by a decline in the growth rate of the cells, but it has no effect on the synthesis of mitochondrial ribosomes. We postulate that one or more chloroplast ribosomal proteins are synthesized on chloroplast ribosomes, whereas mitochondrial ribosomal proteins are synthesized on cytoplasmic ribosomes.     

Ribosome-membrane association in fat body tissues from reproductively active females of Leucophaea maderae

Polysomal and microsomal profiles from fat body tissues of Leucophaea analysed by a combination of equilibrium sedimentation and sedimentation velocity centrifugations on sucrose density gradients revealed that microsomes from egg-maturing females are considerably more dense than those from allatectomized (reproductively inactive) females or from males. This greater density is conferred on the microsomes of reproductively active females by the binding of many more ribosomes (polysomes) to the membranes. Treatment of the microsomes with 500 mM K⁺ or 1 mM puromycin resulted in a removal of only a few ribosomes and polysomes from the membranes, as is documented with the electron microscope. Incubation of microsomes with a combination of 500 mM K⁺ and 1 mM puromycin resulted in a complete degranulation of the membranes. Such microsomes attained a density similar to those of the inactive tissues. From the available evidence it is concluded that the female specific protein (vitellogenin), which is induced by the juvenile hormone, is synthesized on ergastoplasmic membranes and released into the cisternae as is known for the synthesis of exportable proteins in several vertebrate systems.     

Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis

Maternal mRNP particles were isolated from the postribosomal supernatant fluid of unfertilized sea urchin eggs. They were translated in a cell-free system derived from unfertilized eggs. The translation of these particles required the presence of 12 mM MgCl₂, which is considered very high. The same high Mg²⁺ requirement was observed when mRNP particles were translated in a cell-free system from morula embryos. In contrast, mRNA extracted from mRNP particles is translated at 3 mM MgCl₂. This concentration of Mg²⁺ is known to be optimal for initiation of mRNA translation. Likewise, a rabbit globin mRNA is faithfully translated into α and β globin chains in a cell-free system from eggs at 3, but not at 12, mM MgCl₂. The translational products directed by mRNP or by mRNA derived from mRNP were examined in two gel systems and were found to be very similar. In both cases, histones were identified as part of the translational product. This indicated that the translation of mRNP in high Mg²⁺ is not due to nonspecific binding of these particles to ribosomes. The rates of globin synthesis in a cell-free system derived from eggs is comparable to that of morula ribosomes and to that reported for translation of globin with mouse liver and reticulocyte ribosomes, indicating that unfertilized sea urchin egg ribosomes do not possess a translational inhibitor and that no deficiency in initiation factors for mRNA translation could explain the low rate of protein synthesis in unfertilized sea urchin eggs.     

An electron microscopic analysis of pathways for bacteriophage T4 DNA recombination

The interaction between ribosomes of Bacillus stearothermophilus and the RNA genomes of R17 and Qβ bacteriophage has been studied. Whereas Escherichia coli ribosomes can initiate the synthesis of all three RNA phage-specific proteins in vitro, ribosomes of B. stearothermophilus were previously shown to recognize only the A (or maturation) protein initiation site of f2 or R17 RNA. Under these same conditions, a Qβ region is bound and protected from nuclease digestion. Qβ RNA, however, does not direct the synthesis of any formylmethionyl dipeptide in the presence of B. stearothermophilus ribosomes, nor does the binding of either this Qβ region or the R17 A protein initiation site to these ribosomes show the same fMet-tRNA requirement for recognition of initiator regions as that previously established with E. coli ribosomes. Analysis of a 38-nucleotide sequence in the protected Qβ region reveals no AUG or GUG initiator codon. These observations suggest that messenger RNA may be recognized and bound by B. stearothermophilus ribosomes quite independently of polypeptide chain initiation.Binding experiments using R17 RNA and mixtures of components from B. stearothermophilus and E. coli ribosomes confirm the conclusion drawn by Lodish (1970a) that specificity in the selection of authentic phage initiator regions by the two species resides in the ribosomal subunit(s). However, anomalous attachment of B. stearothermophilus ribosomes to R17 RNA, which is observed upon lowering the incubation temperature of the binding reaction, is clearly a property of the initiation factor fraction. The results are discussed with respect to current ideas on the role of ribosomes and initiation factors in determining the specificity of polypeptide chain initiation.     

Mutants of Sweetclover (Melilotus alba) Lacking Chlorophyll b: Studies on Pigment-Protein Complexes and Thylakoid Protein Phosphorylation

Mutants of sweetclover (Melilotus alba) with defects in the nuclear ch5 locus were examined. Using thin-layer chromatography and absorption spectroscopy, three of these mutants were found to lack chlorophyll (Chl) b. One of these three mutants, U374, possessed thylakoid membranes lacking the three Chl b-containing pigment-protein complexes (AB-1, AB-2, and AB-3) while still containing A-1 and A-2, Chl a complexes derived from photosystems I and II, respectively. Complete solubilization and denaturation of the thylakoid proteins from this mutant revealed very little apoprotein from the Chl b-containing light-harvesting complexes, the major thylakoid proteins in normal plants. The normal and mutant sweetclover plants had active thylakoid protein kinase activities and numerous polypeptides were labeled following incubation with [γ-³²P]ATP. With the U374 mutant, however, there was very little detectable label co-migrating with the light-harvesting complex apoproteins on polyacrylamide gels. The Chl b-deficient chlorina-f2 mutant of barley (Hordeum vulgare) also had an active protein kinase activity capable of phosphorylating numerous polypeptides, including ones migrating with the same mobility as the light-harvesting complex apoproteins. These results indicate that the sweetclover mutants may be useful systems for studies on the function and organization of Chl b in thylakoid membranes of higher plants.     

Dissociation of ribosomes and seed germination

Ribosomes from rice embryos (Oryza sativa) were dissociated into ribosomal subunits in vitro by systematic reduction of the Mg²⁺ concentration. Ribosomes from imbibed (28 C) embryos were more easily dissociated than those from nonimbibed embryos. This was not observed with ribosomes from either imbibed, nonviable embryos, or from embryos imbibed at 0 C. Ribosomes from embryos which had been imbided and subsequently dehydrated resembled ribosomes from nonimbibed embryos in their resistance to dissociation. The change in the resistance to dissociation was essentially complete after the first 20 minutes of imbibition at 28 C, and accompanied activation in vivo of protein synthesis as determined by amino acid incorporation in vitro. Ribosomes from either imbibed or nonimbibed embryos could be dissociated into subunits by 0.5 m KCl. These subunits were separated by density gradient centrifugation, and, if recombined, were active for polyphenylalanine synthesis in vitro. The individual subunits prepared from nonimbibed embryos could be replaced by the corresponding subunit fraction from imbibed embryos without loss of capacity to support polyphenylalanine synthesis. The change in the ease of dissociation of ribosomes appears to be a physiological process, and its possible relationship to the initiation of protein synthesis during seed germination is discussed.     

Thylakoid membranes: the translational site of chloroplast DNA-regulated thylakoid polypeptides

Stromal ribosomes and those bound to thylakoid membranes were prepared from intact spinach chloroplasts which were purified on Percoll gradients. The products of read-out translation of these ribosomes supplemented with an Escherichia coli extract were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Striking similarity was found between the polypeptides labeled in the read-out translation of the chloroplastic ribosomes and those synthesized in isolated chloroplasts. Among the polypeptides translated on thylakoid-bound ribosomes, apoprotein of chlorophyll-protein complex I, alpha and beta subunits of coupling factor 1, and 32,000-Da membrane polypeptide were identified from their mobility on the polyacrylamide gel. The large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and other several stromal proteins were translated exclusively from stromal ribosomes. However, when the translation was programmed in cell-free systems from either E. coli, wheat germ, or rabbit reticulocytes by RNAs isolated separately from stroma and thylakoids, no qualitative difference was found between the products from those RNAs. These results suggest that thylakoid-bound ribosomes are the main sites of synthesis of thylakoid proteins and stromal-free ribosomes are that of stromal proteins, and that thylakoids and stroma contain mRNAs for the stromal and the thylakoid proteins, respectively, in a form not functioning in the chloroplasts.     

In Vivo and In Vitro Protein Phosphorylation Studies on Ochromonas danica, an Alga with a Chlorophyll a/c/Fucoxanthin Binding Protein

The phosphorylation of thylakoid membranes in the Chromophyte alga Ochromonas danica was studied in whole cells and in vitro. Protein kinase activity was observed in the thylakoid fraction, and several membrane-bound polypeptides were found to be phosphorylated. The thylakoid protein kinase demonstrated several unusual regulatory properties. Both the polypeptides that were phosphorylated and the rate of protein phosphorylation were independent of illumination. Protein kinase activity was also unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron. The kinase activity was inhibited under strong reducing conditions. Whole cells labeled with ³²PO₄³⁻ were converted to light states I and II by pre-illumination favoring photosystem I or photosystem II, respectively. Analysis of the phosphoproteins from cells in state I and state II showed that no changes in phosphorylation accompanied the change in energy redistribution.