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Protein synthesis in chloroplasts I. Light-driven synthesis of the large subunit of Fraction I protein by isolated pea chloroplasts

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Abstract

Intact isolated pea chloroplasts use light energy to incorporate labelled amino acids into protein. 25 % of this incorporation is present in a 150 000 × g chloroplast supernatant fraction. When this supernatant is analysed on sodium dodecyl sulphate polyacrylamide gels only one polypeptide is labelled. This polypeptide is the large subunit of Fraction I protein, a major protein constituent of the chloroplast. Identity of the soluble in vitro product with the large subunit of Fraction I protein was established by comparing a tryptic map of its [35S]methionine-labelled peptides with a tryptic map of the large subunit of Fraction I protein labelled in vivo with [35S]methionine. We conclude that only one of the many chloroplast soluble proteins, namely the large subunit of Fraction I protein, is synthesised on chloroplast ribosomes.

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... Using methods for isolating intact chloroplasts adapted from those of Walker, Ellis and co-workers demonstrated chloroplast protein synthesis by incorporation of radioactively labelled methionine into chloroplast polypeptides. Products of protein synthesis include the large subunit of the enzyme Rubisco ( Blair and Ellis, 1973 ) and a "peak D" membrane protein ( Eaglesham and Ellis, 1974 ), later identified as the D1 protein, or chloroplast psbA gene product, of the photochemical reaction center of one of the two pigment systems -photosystem II. Synthesis of D1 is lightdependent, and its rapid synthesis in the light is required for re-synthesis following its breakdown ( Ohad et al., 1984 ). ...
... Nine chloroplast genes were investigated by Pfannschmidt et al. (1999b ) with rbcL transcription being affected in the same way, and in the same direction, as psbA, though with a smaller amplitude of redox response. RbcL, also identified as a product of chloroplast protein synthesis ( Blair and Ellis, 1973 ), is the large subunit of the Rubisco, the enzyme catalyzing the primary carboxylation step of the Benson-Calvin cycle. RbcL is a membrane-extrinsic protein subunit and has a very slow turnover, in complete contrast to D1/psbA. ...
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Chloroplasts and mitochondria perform energy transduction in photosynthesis and respiration. These processes can be described in physico-chemical terms with no obvious requirement for co-located genetic systems, separated from those of the rest of the cell. Accordingly, biochemists once tended to regard endosymbiosis as untestable evolutionary speculation. Lynn Sagan's seminal 1967 paper “On the Origin of Mitosing Cells” outlined the evolution of eukaryotic cells by endosymbiosis of prokaryotes. The endosymbiont hypothesis is consistent with presence of DNA in chloroplasts and mitochondria, but does not assign it a function. Biochemistry and molecular biology now show that Sagan's proposal has an explanatory reach far beyond that originally envisaged. Prokaryotic origins of photosynthetic and respiratory mechanisms are apparent in protein structural insights into energy coupling. Genome sequencing confirms the underlying, prokaryotic architecture of chloroplasts and mitochondria and illustrates the profound influence of the original mergers of their ancestors’ genes and proteins with those of their host cells. Peter Mitchell's 1961 chemiosmotic hypothesis applied the concept of vectorial catalysis that underlies biological energy transduction and cell structure, function, and origins. Continuity of electrical charge separation and membrane sidedness requires compartments within compartments, together with intricate mechanisms for transport within and between them. I suggest that the reason for the persistence of distinct genetic systems within bioenergetic organelles is the selective advantage of subcellular co-location of specific genes with their gene products. Co-location for Redox Regulation – CoRR – provides for a dialogue between chemical reduction-oxidation and the action of genes encoding its protein catalysts. These genes and their protein products are in intimate contact, and cannot be isolated from each other without loss of an essential mechanism of adaptation of electron transport to change in the external environment.
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Chapter
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Biochemical studies on leaf proteins carried out by Wildman and Bonner (1947) revealed the presence of a major protein component having a large molecular size (18s) which was designated as fraction-1-protein. The ubiquitous distribution of this protein in green plant leaves and green algae, as determined by analytical ultracentrifugation and immunological precipitation methods, stimulated later studies on its enzymic nature (Dorner et al., 1958). The independent investigation of the path of carbon in photosynthetic CO2 fixation, together with that on the enzymic machinery of the reductive pentose phosphate cycle, led to the discovery of ribulose-1,5-bisphosphate (RuBP) carboxylase (E.C. 4.1.1.39; carboxydismutase) catalyzing the following key reaction [Eq. (1); Quayle et al., 1954; Weissbach et al., 1954, 1956; Chap. II.1, this vol.] $$ {\rm{RuBP + C}}{{\rm{O}}_2}{\rm{ }}{\rm{ 2}}\left( {{\rm{PGA}}} \right) $$ (1)
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Chapter
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Rubisco is responsible for net carbon dioxide fixation. Due to the high concentration of oxygen in the atmosphere and the relatively low concentration of carbon dioxide, Rubisco “misfires” frequently, splitting a molecule of ribulose bisphosphate rather than adding carbon to it. Evolution has worked to minimize this tendency, but the strategies have been varied, from slight changes in kinetic properties to wholesale re-organization of leaf anatomy. Rubisco consists of two types of subunits in higher plants, green algae, and certain cyanobacteria. The large (L) subunit is encoded in chloroplast DNA and the small (S) subunit in the nucleus. The discovery that Rubisco is encoded by genes in both the chloroplast and the nucleus of higher plants and green algae has motivated considerable research on the biogenesis and biochemistry of Rubisco. This article describes the role of my laboratory in the study of the assembly mechanism of this important enzyme in higher plants.
Chapter
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Eukaryotic plants contain three different protein-synthesizing systems, localized in the cell cytoplasm, in the mitochondrion, and in the chloroplast (1,2). Indeed it is possible to isolate from plant cells cytoplasmic, mitochondrial and chloroplastic ribosomes that differ in the sedimentation coefficient and in the rRNA composition (3) as well as in the ribosomal proteins (4). In addition different tRNA’s and aminoacyl-tRNA synthetases have been reported to be present in the cytoplasm, the chloroplast and the mitochondrion (5) and evidence has been presented suggesting the existence of cytoplasmic-, chloroplast- and mitochondrion-specific translation factors (6). Thus eukaryotic plants, whether unicellular or pluricellular, represent a unique material for many studies such as the regulation and the interrelations of the three protein-synthesizing systems, the comparative structure of the macromolecule s that catalyze the same reactions in the three sys-stems, the localization of the genetic information for these macro-molecules, etc.
Article
Amplification products of both large and small subunits of rubisco have been constructed with lichen thalli of Cladonia verticillaris and Evernia prunastri and their respective photobionts, using as primers oligonucleotides of Spinacea oleracea (large) and Arabidopsis thaliana (small) genes. Although a pattern of multiple bands appeared by the southern analysis of the large as well as the small subunits, hybridation experiments revealed that only one band exclusively hybridized, in both cases, with the corresponding amplified oligonucleotide.
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Development involves the spatial and temporal, selective regulation of gene expression and thus information about the photocontrol of gene expression is of central importance to our understanding of the cellular transduction of environmental stimuli leading to the initiation of developmental change during photomorphogenesis. The aim of this chapter is to review from a biochemical point of view our current understanding of the photocontrol of gene expression, paying particular attention to those systems where the underlying molecular mechanisms have been elucidated. Hence the treatment is not comprehensive but rather represents a critical consideration of selected experimental results and theoretical concepts currently under intense investigation and debate. In the last few years there have been a number of reviews related to the present topic and the reader is referred to these for historical details (Mohr 1974, Schopfer 1977, Zucker 1972).
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Article
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Chapter
The first convincing demonstration that chloroplasts contain RNA originated from the observation by Lyttleton (1962) that one of the two species of ribosomes which can be distinguished in leaf cells is actually localized within the chloroplasts. Confirmation of the presence of RNA in these organelles resulted from the direct analysis of purified chloroplasts, which showed that as much as 25% to 35% of the leaf cell RNA could be in the chloroplasts (Heber, 1963). The identification of chloroplast-specific ribosomal RNAs (rRNA) was followed by evidence for the existence of chloroplast-specific tRNAs and mRNAs. Chloroplasts are thus analogous to mitochondria in that they contain ribosomal and transfer RNA species that can be clearly differentiated from the corresponding cytoplasmic RNA species. That the RNA plays an active role in the functioning of chloroplasts follows from the observation that transfer of dark-grown plants to the light brings about a significant change in the level of certain of the chloroplast RNA species, and from the fact that isolated chloroplasts can synthesize protein. In this chapter an attempt is made to provide a general yet practical introduction to the subject of chloroplast RNA (both algal and higher plant), but an exhaustive review of the literature is not undertaken. Recent articles containing sections dealing with chloroplast RNA are: Ellis and Hartley, 1974; Kirk, 1970; Kirk and Tilney-Bassett, 1967; Loening, 1968; Parthier et al., 1975; Sager, 1972; Smillie and Scott, 1969; Tewari, 1971; Woodcock and Bogorad, 1971.
Chapter
It is a fundamental feature of the organisation of eukaryotic cells that they contain organelles possessing genetic systems additional to the one located in the nucleus. In the case of chloroplasts, the fact that these organelles contain both DNA and ribosomes was demonstrated first in 1962. It soon became apparent that both these components are present in significant quantities. Thus the chloroplast genome has the potential capacity for encoding about 125 proteins, each of molecular weight 50 000, whilst chloroplast ribosomes can represent up to 50% of the total ribosomal complement of leaves [1,2]. The existence of such quantities of chloroplast DNA and ribosomes prompts the question as to their roles in the formation of chloroplasts. Which genes are encoded in chloroplast DNA? Which proteins are synthesised by chloroplast ribosomes? I believe it is necessary to answer such simple direct questions before it is possible to tackle meaningfully the more interesting but far more complex question as to the molecular basis of chloroplast development.
Article
This chapter discusses nucleic acids of chloroplasts and mitochondria. Chloroplasts and mitochondria represent the two main energy transducers in eukaryotic cells. Chloroplast photosynthesis provides reducing power and energy to reduce carbon dioxide to carbohydrate. This important method of moving external energy into the biosphere represents the transducing force upon which animal life depends. Mitochondria, present in plant and animal cells alike, oxidize carbohydrates during cellular respiration, liberating energy. Both organelles contain electron transport chains and cytochromes, electron transport being coupled to ATP formation. These biochemical systems are localized on complex internal membrane systems unique to each organelle, the cristae in the case of mitochondria, and the thylakoids in the case of chloroplasts. The chloroplasts and mitochondria retain control over synthesis of several of their proteins via their own unique DNA and RNA molecules and protein-synthesizing machinery. As a result, organelle mutability can be separated from nuclear mutability. Other organelle-specific proteins require the cooperation of both nuclear and organelle genes for transcription and translation, indicating an interdependence of genetic systems within the plant cell in controlling the synthesis of informational macromolecules.
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Why should we be interested in chloroplast protein synthesis? There are two answers to this question. The major conceptual challenge in biology at the present time is to unravel the molecular basis of differentiation. The leaf is a highly differentiated tissue because of the presence of chloroplasts. Moreover, chloroplasts are easy to isolate, and contain massive amounts of ribulose bisphosphate carboxylase (or Fraction I protein), which catalyses the initial steps in both photosynthesis and photorespiration. The sheer abundance of this protein makes it ideal for studies on the control of protein synthesis, and it is no accident that the first reported in vitro translation of a specific messenger RNA for a plant enzyme produced the large subunit of Fraction I protein1. The second reason for being interested in chloroplast protein synthesis derives from the fact that chloroplasts represent an extranuclear genetic system. When it is realised that most, if not all, eukaryotic cells possess extranuclear genetic systems, the significance of this aspect of chloroplasts is seen to extend beyond photosynthesis and differentiation.
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Publisher Summary In higher plants, the process of photosynthesis occurs within specific membrane bounded organelles called “chloroplasts.” All the chloroplasts exhibit three major structural regions— namely, highly organized internal sac-like flat compressed vesicles called “thylakoids,” an amorphous background rich in soluble proteins called “stroma,” and a pair of outer membranes known as the “envelope.” The envelope essentially renders functional and structural integrity to the chloroplast. This chapter discusses the structure, isolation, chemical composition, and origin of the higher plant chloroplast envelope. The chapter examines the multiple functions of this important membranous system involved in the regulation of the inflow of raw materials for photosynthesis and the outflow of photosynthetic products. The chloroplast envelope of higher plants is a permanent structure and consists of two morphologically and topologically distinct membranes separated by a region about 10–20 nm thick, which appears electron-translucent. The structure of both envelope membranes is consistent with the lipid-globular protein mosaic model of membrane structure as proposed by Singer and Nicolson.
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Chloroplasts and mitochondria are subcellular bioenergetic organelles with their own genomes and genetic systems. DNA replication and transmission to daughter organelles produces cytoplasmic inheritance of characters associated with primary events in photosynthesis and respiration. The prokaryotic ancestors of chloroplasts and mitochondria were endosymbionts whose genes became copied to the genomes of their cellular hosts. These copies gave rise to nuclear chromosomal genes that encode cytosolic proteins and precursor proteins that are synthesized in the cytosol for import into the organelle into which the endosymbiont evolved. What accounts for the retention of genes for the complete synthesis within chloroplasts and mitochondria of a tiny minority of their protein subunits? One hypothesis is that expression of genes for protein subunits of energy-transducing enzymes must respond to physical environmental change by means of a direct and unconditional regulatory control-control exerted by change in the redox state of the corresponding gene product. This hypothesis proposes that, to preserve function, an entire redox regulatory system has to be retained within its original membrane-bound compartment. Colocation of gene and gene product for redox regulation of gene expression (CoRR) is a hypothesis in agreement with the results of a variety of experiments designed to test it and which seem to have no other satisfactory explanation. Here, I review evidence relating to CoRR and discuss its development, conclusions, and implications. This overview also identifies predictions concerning the results of experiments that may yet prove the hypothesis to be incorrect.
Chapter
The most important feature that distinguishes plants from animals is the possession of chloroplasts. These organelles are responsible for the generation of energy and reducing power used to fix CO2. They are also involved in the metabolism of nitrogen, sulphur, lipids, and some plant hormones. Questions concerning the origin, development, and function of chloroplasts have occupied plant scientists for much of the present century. It is now clear that these organelles arose during evolution by the development of an endosymbiotic relationship between free-living photosynthetic organisms and the ancestors of modern plant cells. Within the last 25 years we have moved from the discovery of chloroplast DNA to a complete description of the chloroplast genetic system using techniques of biochemistry and molecular biology. These studies have shown that the present-day chloroplasts are integrated harmoniously into the physiological and biochemical processes of plant cells and that this integration has involved the exchange of genetic information between different cell compartments.
Chapter
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Article
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Chapter
This chapter discusses proteins of the chloroplast. The mechanisms involved in transport and subsequent processing of cytoplasmically synthesized chloroplast proteins could provide a means by which either the chloroplast or nuclear genome maintains control over the polypeptide composition of the chloroplast. The phenomenon of posttranslational processing is not limited to products of nuclear–cytoplasmic origin. The major chloroplast-synthesized polypeptide of chloroplast thylakoid membranes (32K) is synthesized as a 34,000- to 35,000-MW precursor polypeptide; following insertion into the chloroplast thylakoid membrane, it is processed to 32,000 MW. The purpose of the processing step following insertion of the precursor into the membrane is unclear because of the lack of a known function for this polypeptide in the membrane. However, the precursor polypeptide is completely sensitive to proteolytic enzymes, whereas the processed 32K polypeptide is only partially sensitive. This suggests that processing may be necessary for proper structural integration of the polypeptide. The precursor itself may allow initial recognition and insertion of the newly synthesized polypeptide into the thylakoid membrane and facilitate its transition from the soluble to the membrane-bound phase.
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This chronologue seeks to document the discovery and development of an understanding of oligomeric ring protein assemblies known as chaperonins that assist protein folding in the cell. It provides detail regarding genetic, physiologic, biochemical, and biophysical studies of these ATP-utilizing machines from both in vivo and in vitro observations. The chronologue is organized into various topics of physiology and mechanism, for each of which a chronologic order is generally followed. The text is liberally illustrated to provide firsthand inspection of the key pieces of experimental data that propelled this field. Because of the length and depth of this piece, the use of the outline as a guide for selected reading is encouraged, but it should also be of help in pursuing the text in direct order.
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DNA and the machinery for gene expression have been discovered in chloroplasts during the 1960s. It was soon evident that the chloroplast genome is small, that many genes for chloroplast-localized proteins must reside in the nucleus, and that the expression of the genes in both cellular compartments must be coordinated. In the 1970s, the first evidence for plastid signals controlling nuclear gene expression was provided for plastid ribosome-deficient mutants. This review describes the discovery and the first studies on plastid-to-nucleus signaling. Today, many retrograde signals are known, which coordinate plastid and nuclear gene expression during the development of the organelle and in response to environmental changes. The nucleus receives information about the flux through the heme branch of the tetrapyrrole pathway, the expression of plastid genes, the metabolite stage in the organelle, and the efficiency of the photosynthetic electron flow. Singlet oxygen generated during light stress and breakdown products of carotenoids initiate signaling events in the organelle which alter nuclear gene expression. Operational signals permanently coordinate gene expression in both organelles. The biosynthesis of phytohormones like jasmonic, salicylic, and abscisic acids or cytokinins starts in the plastids, and these hormones became crucial players in coordinating plastid and nuclear gene expression under stress. Methylerythritol cyclodiphosphate, a biochemical intermediate of the methylerythritol phosphate pathway, alters the chromatin structure in the nucleus which in turn affects the expression of a particular subset of stress-inducible genes. Dual targeted proteins with plastid and nuclear locations participate in the interorganellar communication. We discuss our current knowledge about retrograde signaling and address open questions.
Chapter
The most important feature that distinguishes plants from animals is the possession of chloroplasts. These organelles are responsible for the generation of energy and reducing power used to fix CO2. They are also involved in the metabolism of nitrogen, sulphur, lipids, and some plant hormones. Questions concerning the origin, development, and function of chloroplasts have occupied plant scientists for much of the present century. It is now clear that these organelles arose during evolution by the development of an endosymbiotic relationship between free-living photosynthetic organisms and the ancestors of modern plant cells. Within the last 25 years we have moved from the discovery of chloroplast DNA to a complete description of the chloroplast genetic system using techniques of biochemistry and molecular biology. These studies have shown that the present-day chloroplasts are integrated harmoniously into the physiological and biochemical processes of plant cells and that this integration has involved the exchange of genetic information between different cell compartments.
Article
Although most previous studies on chloroplast (cp) DNA variation in plants have concentrated on systematics and evolution above the species level, intraspecific variation in cpDNA is common and has provided useful insights into population-level evolutionary processes. Polymerase chain reaction methods were used to examine restriction site and sequence variation in the chloroplast rpLI6 gene within and among populations of duckweed species (Spirodela and Lemna) from the southern and eastern United States. To our knowledge, the rpL16 region has not previously been used to investigate cpDNA variation in nature. While considerable restriction site and sequence variation were detected among species, no variation was found within populations of either of the two species (S. punctata and L. minor) selected for sequence analysis, and S. punctata showed no interpopulational variation. Two cpDNA haplotypes were identified in L. minor, with one haplotype restricted to three sites in Louisiana and the other found in all other populations sampled. This paucity of variation cannot be readily explained as the result of a low mutation rate. In general, group II introns appear to be subject to very little functional constraint, and extensive sequence differences have been found between species in the chloroplast rpL16 intron in particular. However, factors such as historical range expansions and contractions, founding effects, fluctuations in local population size, and natural selection may play a role in reducing cpDNA sequence variability in these species.
Article
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DNA and machinery for gene expression have been discovered in chloroplasts during the 1960s. It was soon evident that the chloroplast genome is relatively small, that most genes for chloroplast-localized proteins reside in the nucleus and that chloroplast membranes, ribosomes, and protein complexes are composed of proteins encoded in both the chloroplast and the nuclear genome. This situation has made the existence of mechanisms highly probable that coordinate the gene expression in plastids and nucleus. In the 1970s, the first evidence for plastid signals controlling nuclear gene expression was provided by studies on plastid ribosome deficient mutants with reduced amounts and/or activities of nuclear-encoded chloroplast proteins including the small subunit of Rubisco, ferredoxin NADP+ reductase, and enzymes of the Calvin cycle. This review describes first models of plastid-to-nucleus signaling and their discovery. Today, many plastid signals are known. They do not only balance gene expression in chloroplasts and nucleus during developmental processes but are also generated in response to environmental changes sensed by the organelles.
Article
Soluble proteins were extracted from pure green leaves, and from pure white leaves containing white plastids of the varieties “Mrs. Parker”, “Flower of Spring”, “Gnom”, and “Madame Salieron (BBC)” of Pelargonium zonale. The green plastids contain normal amounts of ribosomes; in the white leaves plastid ribosomes are absent. The proteins were electrophoretically separated on polyacrylamide gels. In extracts obtained from white leaves fraction I protein is absent (or only present in very small quantities). Not only the large subunit of fraction I protein is lacking; also the small subunit which is generally assumed to be synthesized on cytoplysmic ribosomes could not be detected.
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We present historic discoveries and important observations, related to oxygenic photosynthesis, from 1727 to 2003. The decision to include certain discoveries while omitting others has been difficult. We are aware that ours is an incomplete timeline. In part, this is because the function of this list is to complement, not duplicate, the listing of discoveries in the other papers in these history issues of Photosynthesis Research. In addition, no one can know everything that is in the extensive literature in the field. Furthermore, any judgement about significance presupposes a point of view. This history begins with the observation of the English clergyman Stephen Hales (1677–1761) that plants derive nourishment from the air; it includes the definitive experiments in the 1960–1965 period establishing the two-photosystem and two-light reaction scheme of oxygenic photosynthesis; and includes the near-atomic resolution of the structures of the reaction centers of these two Photosystems, I and II, obtained in 2001–2002 by a team in Berlin, Germany, coordinated by Horst Witt and Wolfgang Saenger. Readers are directed to historical papers in Govindjee and Gest [(2002a) Photosynth Res 73: 1–308], in Govindjee, J. Thomas Beatty and Howard Gest [(2003a) Photosynth Res 76: 1–462], and to other papers in this volume for a more complete picture. Several photographs are provided here. Their selection is based partly on their availability to the authors (see Figures 1-15). Readers may view other photographs in Part 1 (Volume 73, Photosynth Res, 2002), Part 2 (Volume 76, Photosynth Res, 2003) and Part 3 (Volume 80, Photosynth Res, 2004) of the history issues of Photosynthesis Research. Photographs of most of the Nobel-laureates are included in Govindjee, Thomas Beatty and John Allen, this volume. For a complementary time line of anoxygenic photosynthesis, see H. Gest and R. Blankenship (this volume).
Chapter
Fructose-1.6-bisphosphatase (FBPase; EC 3.1.3.11) is a key enzyme in the regulation of Calvin cycle, and it has been profusely studied from structural and regulatory points of view. Many authors have demonstrated that its activity rise after the dark-light transition is due, in addition to an increase in the stromal pH and Mg2+ concentration, to a light activation by reduction of essential -S-S- groups of the enzyme molecule. However, little attention has been paid to the possible existence of a second regulatory mechanism by light of FBPase activity, by way of a photoregulated induction of the enzyme synthesis, such it has been found for the NADP+-malate dehydrogenase (NADP+-MD)(Vidal, Gadal,1981), the 32–34 kilodalton polypeptide which shields the B acceptor of Phot.II (Steinback “et al”, 1981), and for the assembly of ribulose-1.5-bisphosphate carboxylase (Bloom “et al”, 1983).
Chapter
It is apparent that a major sub-set of heat shock proteins assist other polypeptides to maintain, or assume, a conformation required for their correct assembly into biologically active structures (Georgopoulos et al. 1973; Kochan and Murialdo 1983; Goloubinoff et al. 1989; Cheng et al. 1989; Ostermann et al. 1989; Bresnick et al. 1989) or localization (Deshaies et al. 1988; Chirico et al. 1988; Zimmermann et al. 1988; Bochkareva et al. 1988; Lecker et al. 1989). This group of proteins function as molecular chaperones, and they have been defined as proteins which assist the assembly of some oligomeric proteins, but are not components of the final structure (Ellis 1987; Ellis et al. 1989; Ellis and Hemmingsen 1989). One distinct group of related molecular chaperones are found in prokaryotes, mitochondria, and plastids, and are called chaperonins (Hemmingsen et al. 1988). In this chapter we outline the discovery and characterization of chaperonins in prokaryotic and eukaryotic organisms, and also describe recent data that show that these proteins have an important role in protein folding in cells.
Chapter
Rewriting the chapter more than 10 years after its original publication (Giles 1977) is not made easy by the exciting changes that have occurred in closely associated fields over that time. To a large degree the uptake of foreign DNA, using Agrobacterium tumefaciens as a vector has eclipsed organelle uptake studies in the past few years. This trend is understandable because of the increased flexibility and range of genetic manipulations possible using the Agrobacterium system, since it effectively allows the manipulation of the whole genome rather than the plastoms alone. Chloroplast uptake and exchanges, however, continue to have a useful role in studies involving nuclear-chloroplast interaction, the physiology of isolated plastids and experiments investigating the transfer of herbicide resistance, particularly to the triazines, between varieties and species.
Chapter
Two lessons I have learned during my research career are the importance of following up unexpected observations and realizing that the most obvious interpretation of such observations can be rational but wrong. When you carry out an experiment there is usually an expectation that the result will fall within a range of predictable outcomes, and it is natural to feel pleased when this turns out to be the case. In my view this response is a mistake. What you should be hoping for is a puzzling result that was not anticipated since with persistence and luck further experiments may uncover something new. In this article I give a personal account of how studies of the synthesis of proteins by isolated intact chloroplasts from pea leaves eventually led to the discovery of the chaperonins and the formulation of the general concept of the molecular chaperone function that is now seen to be a fundamental aspect of how all cells work.
Chapter
Ribulose-I, 5-bisphosphate carboxylase/oxygenase (E.4.1.1.39), often referred to as RuBPcase, has been extensively studied both at the structural and at the functional level becasue of its paramount role in the dark fixation of CO2 during photosynthesis. RuBPcase has been purified to electrophoretic homogeneity from a wide variety of plants. It is a multimeric enzyme (MW 550,000) and comprises eight large subunits (MW 55,000) and eight small subunits (MW 12,000-16,000) in higher plants. The carboxylase and oxygenase activities of the enzyme reside in its large subunit, while its small subunit has been assigned a regulatory role in the catalytic activity. The large subunit of RuBPcase is encoded in the chloroplast and translated in situ. The small subunit is encoded in the nucleus and synthesized in the cytoplasm as a precursor polypeptide with a “transit peptide” at the N'-terminus. The transit peptide is responsible for the transport of the small subunit into the chloroplast. The precursor small subunit is processed to maturity in the chloroplast by a stromal enzyme and is then assembled with the large subunit to form the holoenzyme. A high molecular weight binding protein (29 S) is associated with the large subunit and is crucial for the assembly of the large subunits. The transport and assembly of subunits into holoenzyme are energydependent processes.
Chapter
The major products of cytoplasmic protein synthesis in pea leaves correspond to polypeptide components of two chloroplast proteins. These polypeptides are the small subunit of ribulose-1,5- bisphosphate (RuBP) carboxylase and the constituent polypeptides of the chlorophyll-protein complex II (CPII) or light-harvesting chlorophyll a/b protein (Cashmore, 1976 and Schmidt et al., this volume). Both the small subunit and the CPII polypeptides are synthesized, on free cytoplasmic polyribosomes, as soluble precursors which function in the post-translational transport of the polypeptides from their site of synthesis into the chloroplast (Dobberstein et al., 1977; Cashmore et al., 1978; Highfield and Ellis, 1978; Apel and Kloppstech, 1978; Chua and Schmidt, 1978; Schmidt et al., this volume). From studies on the mode of inheritance of peptide variants it appears that these cytoplasmically synthesized polypeptides are encoded by nuclear genes (Kawashima and Wildman, 1972; Kung et al., 1972). In contrast, the large subunit of RuBP carboxylase is translated on chloroplast ribosomes (Blair and Ellis, 1973) and is encoded by the chloroplast genome (Coen et al., 1977).
Chapter
The studies of Schimper, Strasburger, and others in the 1880s demonstrated that chloroplasts in a sense have an existence of their own. Chloroplasts were found to proliferate by division of existing plastids and were passed on to daughter cells at the time of cell division. But more recent findings have indicated that the chloroplasts’ independence is quite limited. Plastids have their own DNA, which is quite distinct from nuclear DNA, and also have the ability to express the genetic information in their DNA. A question with much current interest, however, is the critical one of how much information chloroplast DNA actually contains. Although the total mass of DNA in a chloroplast is generally somewhat more than that in a bacterial cell, the genetic capacity of chloroplast DNA is much less than that of the bacterial chromosome. The reason for this paradox is that chloroplasts contain multiple copies of a relatively small molecule of DNA. As with mitochondria, the information in chloroplast DNA apparently is only that which is required, separate from the nucleus, to synthesize a few necessary functional proteins. However, even to accomplish the synthesis of these relatively few proteins, the chloroplast must contain its own ribosomes and complete machinery for protein synthesis. As work on the biosynthetic capabilities of the chloroplast proceeds, it is becoming quite clear that most of the plastid’s proteins and properties are determined by the nuclear genome and that most of its proteins are synthesized on cytoplasmic ribosomes.
Chapter
Ribulose-1, 5-bisphosphate carboxylase (RuBPCase) is the protein responsible for fixation of CO2 in photosynthetic organisms. In many higher plants RuBPCase also appears to serve as a storage protein that is hydrolyzed during leaf senescence (Huffaker et al., 1978). This provides a source of reduced N that can be transported to newly developing leaves or fruits (Dalling et al., 1976). RuBPCase is a large protein (MW ∿ 550 kD) consisting of 8 large subunits (50–57 kD) and 8 small subunits (13–15 kD). The large subunit is coded on chloroplast DNA and is synthesized within the chloroplast (Blair et al., 1973; Chan et al., 1972; Criddle et al., 1970; Kung, 1976). The small subunit is coded on nuclear DNA (Kung, 1976), synthesized as a precursor protein in the cytoplasm (Criddle et al., 1970; Highfield et al., 1978), and then processed at or in the chloroplast. The native protein is assembled in the chloroplast and the active enzyme is localized in the stroma. The synthesis of RuBPCase occurs predominantly during the greening of etiolated leaf tissue (Kleinkopf et al., 1970; Smith et al., 1974) or leaf expansion (Friedrich and Huffaker, 1980). The cellular concentration of RuBPCase (which can constitute 50–70% of the total soluble leaf protein) then remains nearly constant for several days; little or no apparent turnover takes place (Huffaker, 1979; Peterson et al., 1973). During senescence, protein is rapidly degraded, and RuBPCase is the predominant protein lost during the initial stages (Friedrich and Huffaker, 1980; Peterson and Huffaker, 1975). A1- though there is much data concerned with the synthesis of RuBPCase, information about the control of its degradation and turnover is lacking. Exo—and endoproteinases in green and senescing leaf tissue have been described (Dalling et al., 1976; Huffaker and Miller, 1978; Martin and Thimann, 1972; Peterson and Huffaker, 1975; Sopanen and Lauriere, 1976; Thomas, 1978; Wittenbach, 1978) but very little is known about their role in senescence or in normal cellular protein turnover.
Article
Polyadenylated RNA from leaves of pea (Pisum sativum) has been copied into DNA and cloned in the Escherichia coli plasmid pBR322. From these clones we have identified and sequenced DNA encoding the mRNA for the precursor of the small subunit of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase.
Article
Full-text available
1. The effects of changes in experimental conditions on the mobility of RNA in polyacrylamide-gel electrophoresis were investigated. 2. The linear relation between log(molecular weight) and electrophoretic mobility was shown to be independent within limits of salt or gel concentration. 3. The relative mobility of RNA with low content of guanylic acid and cytidylic acid residues was decreased in low-ionic-strength buffer. This was related to a small relative decrease in sedimentation coefficient. 4. However, Mg(2+) ion caused almost no increase in mobility although it was associated with large increases in sedimentation coefficient. This suggested opposing actions of Mg(2+) ion on the size and effective charge of the RNA. 5. It is concluded that the method provides a satisfactory measurement of molecular weight, which is almost independent of the nucleotide composition of RNA at moderate salt concentrations.
After isolated tobacco chloroplasts were incubated for protein synthesis and then centrifuged at 17 000×g, about one-half of the incorporated radioactivity was found to have been released into the 17 000×g supernatant, whereas the other half remained firmly associated with the chlorophyll-containing 17 000×g pellet.Of the radioactivity in the 17 000×g supernatant, 80% was in the form of nascent peptides associated with 70-S ribosomes and polysomes, and only 20% in the soluble region. However, there was no coincidence of the radioactivity with Fraction-I protein or other readily identifiable soluble chloroplast proteins as examined both by sucrose density gradient centrifugation and by polyacrylamide disc gel electrophoresis.The radioactivity of the 17 000×g chlorophyll-containing pellet could not be removed from the pellet by repeated washing with hypotonic media, but was released by the anionic detergent, sodium deoxycholate. Under conditions where the chloroplast protein/sodium deoxycholate weight ratio was approximately equal, there was insignificant solubilization of chlorophyll pigments from the membranes, but 30–40% of the radioactivity associated with the pellet was released, all of which was associated with released ribosomes. It is speculated that most of the radioactivity associated with the pellet could also be accounted for in the form of nascent peptides attached to membrane-bound ribosomes. The membrane-bound ribosomes were active in peptide synthesis in the absence of chloroplast mobile phase, although they exhibit a requirement for soluble enzymes for maximum activity.Evidence is presented to show that more than half of the ribosomes in tobacco chloroplasts are bound to thylakoid membranes.Isolated tobacco chloroplasts seem to be unlike isolated chloroplasts from other plants which have been reported to synthesize Fraction-I protein and structural protein of thylakoid membranes.
Tryptic peptides were resolved from the small subunit of highly purified Fraction I protein obtained from Nicotiana tabacum, Nicotiana glutinosa, Nicotiana glauca and four reciprocal, F1 hybrids: N. tabacum × N. glutinosa; N. tabacum × N. glauca. Information for synthesis of an extra N. tabacum peptide was transferred by pollen to N. glutinosa egg cells and therefore the Mendelian mode of inheritance signifies nuclear DNA as containing the code for the primary structure of the small subunit. Two differences in peptides between N. tabacum and N. glauca were also inherited in a Mendelian manner. Transfer of the new N. tabacum information to N. glauca egg cells also supressed synthesis of the N. glauca type of Fraction I protein.
Resolution of the tryptic peptides obtained from the large subunit of Fraction I proteins isolated from Nicotiana species indigenous to Australia revealed one peptide that was not found in Nicotiana species indigenous to the Western Hemisphere. The extra peptide appeared in the reciprocal F1 hybrids , only when N. gossei was the female parent. The maternal mode of inheritance requires chloroplast DNA to code for the sequence of amino acids in the large subunit, in contrast to a previous finding that nuclear DNA codes for the small subunit.
Conditions of pH and NaCl concentration were established whereby highly purified Fraction-I protein was crystallized. Identity of the crystalline material with Fraction-I protein was shown by ultraviolet absorption, analytical centrifugation, immunological properties, and Sephadex chromatography. The specific ribulose diphosphate carboxylase6 (3-phospho-d-glycerate carboxy-lyase (EC 4.1.1.39)) activity of crystallized Fraction-I protein was higher than either the purified Fraction-I protein used for crystallization or the protein which remained dissolved in the mother liquor. The crystals are composed of 12 faces, each being parallelograms, united to form 6 apices where 3 faces are joined and 8 apices where 4 faces are joined. The crystals show little birefringence. A model corresponding to the appearance of the crystals was constructed by combining an octahedron with a cube where 2/√2 was the ratio of the longer to shorter diameter, the angles between faces were 60°, and the acute angle of a face was 70.32° compared to the obtuse angle of 109.68°.
Article
The molecular weights of the two subunits of Fraction I protein have been estimated as 52,000 and 24,500 by Sephadex column chromatography, suggesting that the native protein is constructed from 8 large and 6 small subunits. On the basis of these numbers as well as the shape of the crystal of Fraction I protein, a model of the subunit structure of the protein is proposed.
Article
Preparations of ribulose diphosphate carboxylase (fraction-1 protein) from both soybean and spinach leaves catalyzed the formation of phosphoglycolate and 3-phosphoglycerate from ribulose 1,5-diphosphate in the presence of oxygen. A manometric assay was used, and the activity called ribulose diphosphate oxygenase. Fraction-1 protein was purified from spinach leaves by a two-step procedure involving DEAE-cellulose chromatography and sucrose density gradient centrifugation in a zonal rotor. The protein was electrophoretically homogeneous. The oxygenase and carboxylase activities co-purified, and other attempts to separate them were unsuccessful. However, the oxygenase was more stable than the carboxylase, and the activity ratio, oxygenase/carboxylase, increased from 0.25 in the crude extract to 0.59 in the final product. The oxygenase was also more stable than the carboxylase when the protein was stored as an (NH4)2SO4 precipitate. The pH optimum of the oxygenase activity was about 9.3-9.5, being much more alkaline than that of the carboxylase. No activity was observed in the absence of Mg2+ ions. A gas phase of 100% oxygen was not sufficient to saturate the oxygenase and the activity in air was 37% of that in pure oxygen. The Michaelis constant for ribulose 1,5-diphosphate was about 0.18 mM. The purified protein did not catalyze the oxygenation of several other phosphate esters. It is probable that, during photosynthesis, ribulose diphosphate is carboxylated and oxygenated by the same protein and that the oxygenating activity is responsible for the supply of phosphoglycolate, the first intermediate in the glycolate pathway of photorespiration.
Article
Apparently intact mitochondria from ovaries of Xenopus laevis can incorporate high molecular weight polynucleotides which then serve as templates for polypeptide synthesis on mitochondrial ribosomes.
Article
CHLOROPLASTS contain ribosomes which are distinct from those found in the cell cytoplasm1, but there has been no convincing identification of any of the proteins which these ribosomes presumably synthesize2. Chloroplasts, however, are clearly not genetically autonomous; for genetic studies of both higher plants and algae indicate that some chloroplast components are encoded in the nuclear DNA3, and it is unlikely that all the chloroplast proteins are made by chloroplast ribosomes. We are concerned here with the problem of identifying those proteins made by chloroplast ribosomes.Use of AntibioticsIn previous studies greening cells were treated with chloramphenicol and cycloheximide, which selectively inhibit protein synthesis on ribosomes from chloroplasts and the cytoplasm, respectively*.
Article
A wide variety of proteins have been shown to bind identical amounts of an amphiphile, sodium dodecyl sulfate, on a gram per gram basis at monomer equilibrium concentrations above 0.5 mM. The binding is independent of ionic strength and primarily hydrophobic in nature. Only the monomeric form of the amphiphile binds to proteins, not the micellar form. The application of these results to models for biological membranes and to gel electrophoresis in sodium dodecyl sulfate is discussed.
Article
Fraction I protein (RuDP carboxylase) consists of two subunits which differ in molecular weight and amino acid composition. Tobacco leaves were supplied C14O2 during 15 min of photosynthesis resulting in 8 of the amino acids of Fraction I protein becoming radioactive. The amount of C14 incorporation into the larger subunit was greater than into the smaller subunit. Resolution of the amino acids showed the specific radioactivity of each of the 8 amino acids was greater in the larger subunit compared to the smaller. Evidently, a large pool of smaller subunits exist or synthesis of Fraction I protein involves two ribosome sites and two DNA cistrons.
Article
Chloramphenicol specifically inhibited the synthesis of the large subunit of ribulosediphosphate carboxylase. Cycloheximide exerted a primary effect upon synthesis of the smaller subunit and influenced production of the larger subunit by rapidly inhibiting total protein synthesis.
Article
Young tobacco leaves contain two classes of ribosomes in about equal quantities. From ultracentrifugal analyses of chloroplast and cytoplasmic extracts, it is concluded that the 70 s class of ribosomes is located in the chloroplasts and the 80 s class in the cytoplasm. The 70 s ribosomes in a chloroplast extract are 10 to 20-fold more active in protein synthesis (200 to 300 μμmole [14C]valine incorporated/mg ribosome/30 minutes) than the 80 s ribosomes in a cytoplasmic extract. If the chloroplasts are disrupted by homogenization of leaves in a medium of low molarity, the 70 s ribosomes become mixed with the cytoplasm and are less active than the 70 s ribosomes in a chloroplast extract. Inactivation does not seem to result from nuclease action, or deficiencies in sRNA and/or activating enzymes or messenger RNA. Pelleting the ribosomes of a chloroplast extract markedly reduces their incorporating activity. In contrast, the 80 s ribosomes of a cytoplasmic extract are more active after purification. The 70 s and 80 s ribosomes have different magnesium ion requirements for maximum incorporating activity, the optimum concentration being 11 to 15 mM for 70 s ribosomes and 5 ml for 80 S ribosomes. At low concentrations of magnesium ions, most of the 70 s ribosomes dissociate into subunits of 50 s and 35 s, which reconstitute 70 s ribosomes on restoration of the magnesium. The 80 s ribosomes are resistant to dissociation in low concentrations of magnesium ions, but on prolonged dialysis dissociate into 58 s and 35 s particles, which do not reconstitute 80 s particles. 80 s ribosomes aggregate preferentially at high concentration of magnesium. The corrected sedimentation coefficients (S20.w0) of the 70 s and 80 s ribosomes are 69·9 and 82·0s, respectively.
Article
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Article
Amino acid incorporation into protein by chloroplasts from primary leaves of Phaseolus vulgaris L., var. Black Valentine is only partially inhibited by 400 mug/ml ribonuclease. The rate of incorporation, in the presence of ribonuclease, is progressively inhibited with time, and ceases after about half an hour. Preincubation of chloroplasts at 25 degrees , in the absence of ribonuclease, increases the inhibitory effect of ribonuclease on the initial rate of incorporation of amino acid into protein. Examination of electron micrographs of freshly prepared chloroplast suspensions shows that chloroplasts are largely intact. However, after incubation at 25 degrees for 1 hour the chloroplasts are disrupted, as indicated by loss of their stroma contents. It is concluded that the intact chloroplast membrane is relatively impermeable to ribonuclease. Amino acid incorporating activity probably becomes inhibited as the inside of the chloroplast is made accessible to ribonuclease by breakage of membranes during incubation at 25 degrees .
Article
Microsomal fractions isolated from sterile, aged disks of red beetroot incorporate leucine into protein when supplemented with the supernatant fraction, ATP, GTP, and KCl; the incorporation is sensitive to RNase and is not due to bacteria. The microsomal activity is inhibited by puromycin and cycloheximide but is virtually insensitive to both d-threo and l-threo-chloramphenicol, as predicted from physiological studies.Microsomes isolated from fresh disks have much lower incorporating ability than those from disks aged for 1 or 2 days; maximal activity occurs when the rate of protein synthesis by the intact disks is highest. The low activity of fractions from fresh disks is attributable to a deficiency in the microsomal fraction and not to the supernatant fraction; it is not due to a dissociable inhibitor. The RNA content of the microsomal fraction increases with aging and so the increase in incorporating ability may be due to a synthesis of messenger RNA induced by slicing, rather than to an activation of pre-existing messenger. These results support the view that the aging phenomenon involves a derepression of gene activity.
Article
The incorporation of uniformly labeled leucine-(14)C into protein by a chloroplast containing fraction from developing primary leaves of bean is reported. Chloroplasts, obtained from week old plants grown in darkness, and then illuminated with white light for 12 hours, were shown to be the principal sites of incorporating activity. Incorporation may continue for 2 hours. Rates of up to 50 mumumole leucine incorporated per mg protein per hour are observed when a 1 hour assay period is used. Incorporation is only partially sensitive to ribonuclease.
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