Improvements are described in the preparation and in vitro conditions of an intact pea (Pisum sativum Progress No. 9) chloroplast system which provides high efficiency for translation of endogenous messenger RNA, using light as an energy source. High rates result in the incorporation into protein of up to 100 nanomoles tritiated leucine per milligram chlorophyll. These rates suggest extensive reinitiation, and repeated utilization of the messenger RNA that code for thylakoid proteins. Up to 39 radioactive thylakoid peptide bands were detected by fluorography after labeling with tritiated leucine.Protein synthesis by intact chloroplasts isolated from several species of plants has been studied to a considerable extent, and was reviewed recently (10). Much of the work has had the goal of identifying which of the numerous soluble and membrane proteins of the chloroplast are synthesized within the organelle on its own 70S ribosomes. However, the rates of protein synthesis, and especially of polypeptide chain initiation, achieved in this 'in organello' (32) synthesis must be adequate in order to meet this goal. This consideration is important because a low initiation rate can result in preferential translation ofmRNA with high rate constants for initiation (19,23). This would give a distorted picture of the synthetic capacities of the isolated chloroplasts. Thus, it is highly desirable, for studies in which translation products are examined, that the in vitro rate of initiation be as close as possible to the in vivo rate.Toward this goal, and in conjunction with related studies on ribosome binding to the green, thylakoid membranes (12, 41), we have sought to optimize most of the parameters involved in isolation of intact chloroplasts and in their reaction conditions, in order to increase rates of protein synthesis. These studies have resulted in obtaining rates two orders of magnitude higher than those reported in previous papers. Examination of labeled membrane polypeptides after denaturing gel electrophoresis shows up to 39 products of 'in organello' synthesis, or three to four times more than noted in earlier studies (9, 13-15, 40, 42 In the final adopted procedure, shoot tissue was homogenized in two batches of 20 g each with 47 ml of grinding buffer containing 350 mm sorbitol, 50 mm Hepes-KOH (pH 8.3 at 4°C), 2 mM EDTA, 1 mtM MgCl2, 1 mim MnCI2, 0.5% BSA, 2 mim EGTA,3 and 4 mm ascorbic acid using a Polytron homogenizer (Brinkmann Instruments) for 20 to 30 s, until all tissue was smaller than 1 mm2. The homogenate was filtered through eight layers of cheesecloth, and centrifuged for 3 min at 3,000 rpm in a Sorvall HB-4 rotor. The pellet was resuspended in a minimal volume (0.2-0.4 ml) of the grinding buffer and overlayered onto a 13-ml linear 25% to 92% Percoll gradient containing the same ingredients as the grinding buffer, 0.6 mm glutathione, and gradients of 0.7 to 2.7% (w/v) of polyethyleneglycol 4000 and 0.25 to 0.92% (w/v) of both BSA and Ficoll. The gradient tubes were centrifuged for 7 min at 9,000...

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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-'

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