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 well 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.