Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts

Abstract

(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).

Synthesis of thylakoid membrane proteins by chloroplasts isolated from spinach. Cytochrome b559 and P700-chlorophyll a-protein.

“…In this study, we employed specific extractions and high-resolution gel electrophoresis techniques to characterize better and to determine the identities of some of the thylakoid polypeptides translated on ribosomes in isolated spinach chloroplasts. Our results support the general conclusions of other investigators who have either studied the synthetic activities of isolated plastids (6,13,20,21,33,43) or examined the synthesis of plastid proteins under rigorously controlled conditions of site-specific inhibition of protein synthesis in vivo (9) . More importantly, our results extend these earlier studies by identifying two specific, integral thylakoid polypeptides, the apoproteins of cytochrome b559 and the P700-chlorophyll a-protein (CPI), as translation products of the plastid .…”

“…Several of the polypeptides synthesized by isolated chloroplasts have been identified as specific proteins or as subunits of specific proteins . In the stromal fraction these include the large subunit of ribulose-1,5-bisphosphate carboxylase (3,6,33,43) and the elongation factors EF-G and EF-T (42); in the thylakoid fraction, these include the a, a, and e subunits of chloroplast coupling factor 1 (CFI) (23) and cytochrome f (12) . A 30,000-32,000 mol wt polypeptide of the thylakoids, also known as "peak D" (see, e .g., reference 13), is widely recognized as the most rapidly labeled polypeptide in the thylakoid fraction, but it has not been identified beyond its mobility on SDS gels.…”

Analysis of Euglena gracilis chloroplast DNA

“…Stained profiles (a,d) are aligned with the respective autoradiographs (b,c). From profile b it is evident that numerous thylakoid proteins are synthesized within the chloroplasts as expected from previous results with E. gracilis [9,22] and higher plants (cf. review [23] but the relative staining intensities do not parallel the intensities of the radioactive signal, suggesting that the turnover rates may vary considerably for these polypeptides.…”

“…We could argue, e.g., that it is a precursor of the strongly labelled 59 000 M r polypeptide observed in lane c, which comigrates with the large subunit of the purified Euglena gracilis RUBPase (not shown). Euglena RUBPase large subunit is a 59 000 M r polypeptide [24], which is synthesized in the chloroplast [22] and was shown to be synthesized in a chloroplast RNA directed wheat germ system [20]. A precursor form of the spinach RUBPase large subunit polypeptide was described in [25].…”

Preparation of Chloroplasts from Euglena Highly Active in Protein Synthesis

“…Salisbury et al (14) adapted the silica procedure to Euglena by combining Ludox gradients with the other components of the recipe of Vasconcelos et al ( 18 were still photosynthetically inactive (cf. 12,17). It became evident, moreover, that the integrity of the chloroplasts was imperfect; the plastids had less than the expected levels of ribulose-1,5-bisphosphate carboxylase and had lost most of their Cyt 552 (6, 7).…”