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

Abstract

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...

PsbN Is Required for Assembly of the Photosystem II Reaction Center in Nicotiana tabacum

“…This is supported by the localization of the PSII assembly factor HCF136 in stroma lamellae, polysome binding to exposed thylakoids, cotranslational assembly of D1, and the preferential appearance of distinct smaller PSII subcomplexes in stroma lamellae (Yamamoto et al, 1981;Meurer et al, 1998;Zhang et al, 1999). Initial assembly of thylakoid membrane complexes occurs through a number of intermediate steps, which follow a strict hierarchical and concerted order, and is to some extent also translationally regulated (Zhang et al, 2000;Choquet and Wollman, 2002).…”

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

“…Preparation of washed thylakoids by differential centrifugation and sucrose step gradients was described earlier (28). These are referred to as 'sucrose thylakoids.'…”

“…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.…”

“…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).…”

Characterization of Thylakoid-Derived Lipid-Protein Particles Bearing the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

“…Large subunit mRNA (rbcL) also has sequence motifs in common with the mRNAs of some chloroplastically encoded thylakoid proteins, including the /3-subunit of ATP synthase (atpB), the core complex of PSI (psaA), and the D1 protein of PSII (@A) (Kim et al, 1993). In addition, RLSU, like chloroplastically encoded thylakoid proteins, appears to be translated by thylakoid-bound polysomes associated with stromal lamellae and the outer margins of grana1 stacks (Yamamoto et al, 1981;Bhaya and Jagendorf, 1984;Hattori and Margulies, 1986;Klein et al, 1988).…”