A Method for Enzymic Extraction and the Measurement of Chloroplast RNA

Some of the chloroplast 70S ribosomes are seen in electron micrographs to be associated with the thylakoid membranes (5, 9, 19, 28). At least some of these ribosomes must be associated with mRNA, since washed thylakoids are capable of synthesizing proteins when supplemented with soluble factors (1,2,8,20). Retention in the thylakoids of the newly synthesized peptides provided evidence that the proteins synthesized were discharged into the membranes (2,20). From this, it seemed likely that the ribosomes active in vitro were tightly bound to the thylakoids by a nascent peptide chain. The presence of this type of linkage was demonstrated more directly for Chlamydomonas (5) and for pea (28), using puromycin-induced release of the ribosomes in the presence of high salt concentrations (0.5 to 1.0 M KCI) as a criterion.The nascent peptide linkages between ribosomes and the thy-'

Section: Discussionmentioning

confidence: 94%

“…We used the convenient and rapid enzymic method for measuring total RNA (10) to estimate the number of ribosomes bound to thylakoids, because there is no simple, quantitative method to obtain this estimate more directly. For this procedure to be valid, however, the total RNA has to consist very largely of the rRNA of 70S ribosomes.…”

Section: Discussionmentioning

confidence: 99%

Light-Induced Increase in the Number and Activity of Ribosomes Bound to Pea Chloroplast Thylakoids in Vivo

Fish¹,

Jagendorf²

1982

“…The amount of RNA remaining in these incubated, washed membranes was measured using the enzymic method described earlier (10).…”

Section: Methodsmentioning

confidence: 99%

Bound Ribosomes of Pea Chloroplast Thylakoid Membranes: Location and Release in Vitro by High Salt, Puromycin, and RNase

Yamamoto¹,

Burke²,

Autz³

et al. 1981

Self Cite

The mode of attachment of 70S ribosomes to thylakoid membranes from pea leaves was studied by determining the proportion of the bound RNA which was released by various incubation conditions. The results supported a model in which several classes of bound ribosomes could be distinguished:(a) very tightly bound, not released by any conditions yet tested (20% of the total); (b) monomeric ribosomes attached by electrostatic interaction with the membranes (30 to 40% of the total) and released by high salt; and (c) polysomes, with some of the ribosomes attached by a combination of electrostatic interactions and insertion of the nascent polypeptide chain into the membrane. These required a combination of puromycin and high salt for release. Other ("hanging") ribosomes of the polysomes were inferred to be attached through mRNA but not actually attached to the membranes directly; they could be released by RNase under low salt conditions, as weDl as by puromycin plus high salt.To obtain these results, chloroplasts had to be prepared in media containing 0.2 molar Tris at pH 8.5. Using Tricine buffers at pH 7.5 yielded thylakoid membranes whose ribosomes were removed almost completely by high salt alone; these showed no response to puromycin. However, pH 7.5 had to be used in all cases for ribosome dissociation in high salt media, as the ribosome structure appeared to be degraded by high salt at pH 8.5, and release then occurred without the need for puromycin.The kinetics of ribosome release by high salt showed a rapid initial phase with a half-life of 20 seconds. The extent of release by high salt was very dependent on the temperature of the incubation. Plotting the data according to the Arrhenius interpretation shows a significant break at about 15 C, with apparent activation energy of 20 kilocalories per mole below that temperature and 5 kilocalories per mole above that temperature.This result suggests that membrane fluidity might be an important factor permitting release of ribosomes under high salt conditions. Electron microscope pictures of the washed thylakoids showed polysomes closely associated with the outer membranes of grana stacks, and with the stroma lamellae. Following digitonin treatment of the membranes and centrifugation, fractions enriched in Photosystem I and presumed stroma lamellae were also enriched in bound RNA.Whereas many of the chloroplast 70S ribosomes are free in the stroma, others appear to be closely associated with the thylakoid ' Supported in part by Grant PCM-77-18839 membranes and are likely to be specifically involved with the synthesis of membrane proteins. The association has been observed in electron microscope studies of Phaseolus leaves (8) and of Chlamydomonas (5). Even after isolated thylakoid membranes are washed repeatedly, ribosomes remain bound (1,5,9,15,16,30) and proteins synthesized by bound polysomes remain as part of the membrane system (1,4,7,19). Estimates of the bound ribosomes as a per cent of the total number of plastid 70S ribosomes, measured in vitro, rang...

“…Proteins were electrophoresed using the Laemmli buffer system (16) RNA content of thylakoids was estimated by the method of Fish and Jagendorf (10).…”

mentioning

confidence: 99%

Optimal Conditions for Translation by Thylakoid-Bound Polysomes from Pea Chloroplasts

Bhaya

Jagendorf²

1984

ABSTRACIPolysomes bound to washed thylakoids from pea Pisum sativum cv Progress No. 9 chloroplasts are capable of protein synthesis when supplemented with amino acids, ATP and a regenerating system, GTP, and soluble factors required for translation. The extent of protein synthesis in previous reports, however, was quite low when compared to in organeilo transltion. By systematic testing of parameters in the isolation of thylakoids and reaction mixture components we have been able to establish more optimal conditions. Incorporation of 2 to 10 nanomoles of leucine per milligram chlorophyll in a 20-minute reaction period is now possible, representing a 10-to 60-fold increase over amounts previously reported. Autoradiographs of solubilized, electrophoresed membranes show about 30 discrete labeled polypeptides which remain associated with the thylakoid membranes.There are two populations of ribosomes within chloroplastthose free in the stroma, and those bound to the thylakoid membranes (20,27). Further, it is known that a large fraction of the bound ribosomes are in the form of polysomes attached at least in part by nascent peptide chains (3,20,21,27). These attached polysomes can continue protein synthesis when supplied with ATP, GTP, amino acids, and soluble factors (enzymes and tRNAs) for translation, although at quite low rates (3, 21) compared to those found in organello (12,22). Before investigating the nature of polypeptides synthesized by attached polysomes, we felt it important to optimize the conditions for translation, since a distorted picture of the extent and relative abundance of specific polypeptides may result from nonoptimal reaction rates (17