Biochemistry of Protein Synthesis in Plants

Boulter, D.; Ellis, R. John; Yarwood, A.

doi:10.1111/j.1469-185x.1972.tb00971.x

Cited by 101 publications

(18 citation statements)

References 366 publications

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“…One such antibiotic is chloramphenicol, a known inhibitor of protein synthesis on chloroplast ribosomes in both higher plants and algae (see ref. 32). Surprisingly, however, analysis of an S17 fraction prepared from chloramphenicol-treated cells revealed a large reduction of 70S subunits relative to 80S subunits as compared to the S17 of the control sample (compare Fig.…”

Section: Methodsmentioning

confidence: 87%

Attachment of Chloroplast Polysomes to Thylakoid Membranes in Chlamydomonas reinhardtii

Chua

Blobel

Siekevitz

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

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Treatment of synchronous cultures of Chlamydomonas reinhardtii with chloramphenicol at 4 hr after the beginning of the light phase led to a preferential loss of 70S ribosomes from the 17,000 X gmax supernate. The "lost" 70S ribosomes were found associated with a thylakoid membrane fraction prepared from the 17,000 X gm. pellet. Electron microscopic examinations of this fraction revealed that the 70S ribosomes were bound to the unstacked regions of the thylakoid membranes as polygonal penta-and hexamers. These bound ribosomes were only released by treatment with 500 mM KCI and puromycin, suggesting that both ionic interactions and nascent peptide chains were involved in the ribosome-membrane attachment. Since growth of the thylakoid membranes occurs in the light, it is suggested that bound chloroplast ribosomes function in the synthesis of thylakoid membrane proteins.Eukaryotic ribosomes (80 S) occur in two distinct states within the cytoplasm of animal cells: they are either free or bound to the membranes of the endoplasmic reticulum (1, 2). The relative distribution of the two ribosome classes greatly depends on the cell type. Thus, cells active in the synthesis of secretory proteins contain a large proportion of bound ribosomes (3, 4), whereas cells synthesizing proteins primarily for intracellular use have most of their ribosomes in the free state (5,6). In a limited number of cases, it has been shown that the free and membrane-bound ribosomes are indeed involved in the synthesis of different classes of proteins (3,(7)(8)(9)(10)(11), in agreement with the generalization mentioned above. There is, however, indirect evidence that in mammalian hepatocytes the bound ribosomes synthesize not only secretory proteins but also proteins of the endoplasmic reticulum membrane (12-16).Since chloroplasts contain their own ribosomes (70 S), and since their is evidence that these ribosomes synthesize some of the proteins of the thylakoid membranes (17-20), the question arises as to whether these ribosomes also exist as two distinct classes within the chloroplast. In this paper, we demonstrate that in synchronous cultures of Chlamydomonas reinhardtii a detectable fraction of the chloroplast ribosome population becomes bound to the thylakoid membranes during the light phase of the cell cycle at the time when growth of the thylakoid membranes occurs. The correlation suggests that these bound ribosomes are involved in synthesis of thylakoid membrane proteins. Experiments were performed when the cultures reached a cell density of 1 to 2 X 106 cells per ml. Each culture was divided into two equal portions 4 hr after the beginning of the light phase. To one sample, chloramphenicol was added from a stock solution of 100 mg of chloramphenicol per ml of ethanol to give a final concentration of 100 yg/ml, the control sample receiving an equivalent amount of ethanol (final concentration, 0.1%). Both samples were further incubated in the light for 1 hr, which brought the cultures to the 5th hr of the light phase. At this time, t...

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Section: Methodsmentioning

confidence: 87%

Attachment of Chloroplast Polysomes to Thylakoid Membranes in Chlamydomonas reinhardtii

Chua

Blobel

Siekevitz

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

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“…

(4,6,(15)(16)(17). The formation and function of a completely functional chloroplast must result from the interactions between the chloroplast genome and the nuclear genome.

The most direct means of identifying the biosynthetic activities of chloroplasts is to study their ability to incorporate labeled precursors in vitro.

…”

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confidence: 99%

Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts

Vasconcelos¹

1976

Plant Physiol.

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(4,6,(15)(16)(17). The formation and function of a completely functional chloroplast must result from the interactions between the chloroplast genome and the nuclear genome.The most direct means of identifying the biosynthetic activities of chloroplasts is to study their ability to incorporate labeled precursors in vitro. Several researchers (3,8,12,18) have found that chloroplasts incorporated labeled amino acids into soluble and membrane-bound proteins. Ellis and co-workers (2, 9-11) were particularly successful in demonstrating the synthesis of discrete proteins by pea chloroplasts through analysis on SDSpolyacrylamide gels. More recently, Morgenthaler and Mendiola-Morgenthaler (20) reported similar success with spinach chloroplasts which had been purified on gradients of silica. They also showed that the chloroplast preparation synthesized the two large subunits of coupling factor CF1 and possibly the smallest inhibitory subunit (19).I have been concerned with preparing chloroplasts from Euglena with similar capabilities, in order to achieve better controls over growth and development than can be obtained with spinach and pea. I have extended an earlier observation (21) that chloroplasts can be obtained from Euglena which are structurally intact and active in light-driven protein synthesis. I have now fractionated the labeled chloroplasts into soluble, thylakoid, and envelope fractions (22), and identified the newly synthesized poly- as a "chase," and the tubes were placed in ice after an additional 5-min incubation. The chloroplast suspensions were diluted 5-fold with sorbitol-Tricine medium, and the chloroplasts pelleted by centrifugation at 3,000 rpm. The chloroplasts were then washed twice in fresh medium, osmotically shocked, resuspended in Tricine-KCI buffer, pH 6.8 (25 mm Tricine-12.5 mM KCI) at 4 C, stirred with a magnetic bar for 20 min, and homogenized with 10 strokes of a TenBroek homogenizer.Subfractionation of Chloroplasts and Analysis of Polypeptides by Electrophoresis on SDS-Polyacrylamide Gels. Subfractionation of chloroplasts into soluble, thylakoid, and envelope fractions, and the subsequent analyses of these fractions by SDS-gel electrophoresis were as described previously (22). For measuring radioactivity, the gels were frozen in Dry Ice and sliced sequentially into 1-mm sections. The slices were placed in scintillation vials and solubilized by incubation with 0.1 ml of 30% H202 at 35 to 45 C overnight. After cooling, 7 ml of Aquasol (New England Nuclear) were added to each vial. Radioactivity was measured in a Beckman liquid scintillation counter.To minimize bacterial contamination, sterile media and glassware were used throughout the experiments.Miscellaneous Analyses. Chlorophyll was determined in 80% acetone according to Bruinsma's (7) modification of the method of Arnon (1).

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“…The observation that UV radiation stimulates the efflux of 'Rb+ from plant cells is important because Rb+ is moved in and out of plant cells like K+, and numerous studies, both with animal and plant systems, have shown the need for the proper concentrations of K+ and Mg2" for in vitro protein synthesis (1,2,14,19).…”

Section: Methodsmentioning

confidence: 99%

Ultraviolet Radiation-stimulated Efflux of 86-Rubidium from Cultured Tobacco Cells

Wright¹,

Murphy²

1978

Plant Physiol.

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Ultraviolet (254 -) radiation stimulated the efflux of MRb+ from liquidcultured tobacco (Nkotiana tabacum) cells; it did not stimulate the movement of mannitol or 2-deoxyglucose. These results indicate that the efflux of 'Rb+ is not to a generalized disruption of membrane structure.Previous studies have shown that UV radiation disrupts many plant cell functions, including photosynthesis, respiration, active transport, RNA and protein synthesis (10,15,16,21 (12,17,18) and that the K+ from the regular culture medium competed with the ssRb+ for uptake by the cells. The higher concentration of Ca2+ present in the incubation medium may also have promoted the absorption of ssRb+ (11,20). Fifty ml of cells were shaken in incubation medium for 24 hr, then placed in a beaker with sufficient MRb+ so that the cells would take up 0.08 to 0.27 nmol of ssRb+ (3.1-10.9 nCi)/ml of cells after 9 to 10 hr of labeling. The wide range of seRb+ taken up was probably due to differences in the densities of the cell suspensions. A typical cell suspension had a packed cell volume of about 0.04 ml/ml of suspension. To measure uptake, samples of cells were removed from the beaker, filtered on glass filter papers, and washed for about 15 sec with 30 ml of incubation medium which was supplemented with 20 mm KCI. The filtered cells were mixed with a dioxane-based scintillation fluid and counted in a liquid scintillation spectrometer (15). Cells were charged with 2-deoxy-D[l-3HJglucose (specific activity 21 Ci/mmol), and uptake was measured, essentially as described above, except that the cells were washed with unsupplemented incubation medium.Determination of MRb+ and 12-3HIDeoxyglucose Efflux. For the 86Rb+ studies, about 10 ml of labeled cells were filtered, washed for about 10 min with 200 ml of incubation medium supplemented with 20 mM KCI, and finally washed for about 5 min with 50 ml of incubation medium containing no K+ or Rb+. For the [2-3H] deoxyglucose experiment, 15 ml of labeled cells were washed for about 10 min with 75 ml of the unsupplemented incubation medium. In both experiments, the washed cells were transferred to 20 ml of incubation medium which was then subdivided into two 10-ml fractions, one for the control and the other for UV irradiation. At various times after irradiation, l-ml samples of cells plus medium were filtered and the filtrates were collected.One-quarter-ml aliquots of the filtrates were then mixed with the dioxane-based scintillation fluid for counting.

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Biochemistry of Protein Synthesis in Plants

Cited by 101 publications

References 366 publications

Attachment of Chloroplast Polysomes to Thylakoid Membranes in Chlamydomonas reinhardtii

Attachment of Chloroplast Polysomes to Thylakoid Membranes in Chlamydomonas reinhardtii

Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts

Ultraviolet Radiation-stimulated Efflux of 86-Rubidium from Cultured Tobacco Cells

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