Separation and partial characterization of chloroplast and cytoplasmic ribosomes from Euglena gracilis

Chloroplast and cytoplasmic ribosomes from Euglena graciliswere separated by centrifugation in zonal rotors. The particleswere characterized by their sedimentation rates as well as bytheir RNA components. Total extracts from green cells contained30S, 55S and 89S particles or their aggregates, depending uponthe Mg⁺⁺ concentration. Extracts from fractions enriched forchloroplasts contained essentially 30S and 55S particles, whilethe supernatant (obtained after sedimentation of the chloroplasts)contained predominantly 89S particles or aggregates of cytoplasmicribosomes. The 30S and 55S ribosomes contained RNA componentswhich were unique and distinct from those of the cytoplasmicribosomes. We were unable to detect 70S particles from the chloroplastpreparations. Under our conditions, chloroplast extracts yielded30S and 55S subunits or a series of rapidly sedimenting particles,possibly polysomes. Despite a variety of extraction techniques,we were unable to detect 70S particles from the chloroplasts. ¹This study was supported in part by grant No. HD 01787 fromthe U. S. Public Health Service. Journal paper of the New JerseyAgricultural Experiment Station (Received December 3, 1969; )     

Extraction and comparison of bean cytoplasmic and chloroplast ribosomes

1. In isolation of total ribosomes from Phaseolus leaves thehighest yield was obtained with an extraction medium containingHEPES buffer, low Mg²⁺-concentration (1 mM) and SH-group protectingagents. 2. Chloroplast and cytoplasmic ribosomes were obtained in aratio of 1 : 28 after selective isolation. 3. After freezing identical portions of cytoplasmic extractthe amount of ribosomes/ml extract dropped, but later increasedwith freezing time. 4. Ribosomal preparations showed UV-absorption spectra typicalfor RNA. Extinction ratios suggested that preparations werecontaminated with extraneous protein to a certain extent, purityincreasing after selective isolation, especially after freezing. 5. Protein patterns and sedimentation behaviour of cytoplasmicribosomes were identical regardless of addition or omissionof detergent in leaf exeracts. 6. After polyacrylamide gel electrophoresis of ribosomal proteins,29 bands of basic protein were found in cytoplasmic ribosomesand 22 in chloroplast ribosomes. Acidic proteins could not bedetected. Patterns in both extracts were different and highlyreproducible, resembling those recently reported. Tests forfive enzymes, highly active in the leaf tissue, gave negativeresults on the isolated ribosomal proteins. ¹Present address: Max Planck Institut für ExperimentelleMedizin, Arb.-Gr. Biochemie, D-34 Göttingen, Hermann-Rein-Straße3, West Germany. (Received August 10, 1968; )     

Characterization of cytoplasmic and chloroplast ribosomal proteins of Euglena gracilis.

Active cytoplasmic ribosone subunits 41 and 62S were prepared by treatment with 0.1 mM puromycin in the presence of 265 mM KCl. Active chloroplast subunits 32 and 49S were obtained after dialysis of chloroplast ribosomal preparations against 1 mM Mg(2+)-containing buffer. Proteins from these different ribosomal particles were mapped by two-dimensional gel electrophoresis in the presence of urea. The 41S small cytoplasmic ribosomal subunit contains 33-36 proteins, the 62S large cytoplasmic ribosomal subunit contains 37-43, the 32S small chloroplast ribosomal subunit contains 22-24, and the 49ts large chloroplast ribosomal subunit contains 30-34 proteins. Since some proteins are lost during dissociation of monosomes into subunits, the 89S cytoplasmic monosome would have 73-83 proteins and the 68S chloroplast monosome, 56-60. The amino acid composition of ribosomal proteins shows differences between chloroplast and cytoplasmic ribosomes.     

Activity of Euglena gracilis chloroplast ribosomes with procaryotic and eucaryotic initiation factors

A method that permits the preparation of Euglena gracilis chloroplast 30 S ribosomal subunits that are largely free of endogenous initiation factors and that are active in the binding of fMet-tRNA in response to poly(A, U, G), has been developed. These 30 S subunits have been tested for activity in initiation complex formation with initiation factors from both procaryotes and eucaryotes. We have observed that Escherichia coli IF-2 binds fMet-tRNA nearly as well to Euglena chloroplast ribosomal subunits as it does to its homologous subunits. Neither wheat germ eIF-2 nor Euglena eIF-2A can bind fMet-tRNA efficiently to Euglena chloroplast or E. coli 30 S subunits although both are active with wheat germ 40 S ribosomal subunits. Euglena chloroplast 68 S ribosomes will also bind the initiator tRNA. Both E. coli IF-2 and E. coli IF-3 stimulate this reaction on chloroplast ribosomes with approximately the same efficiency as they do on their homologous ribosomes. E. coli IF-1 enhances the binding of fMet-tRNA to the chloroplast 68 S ribosomes when either IF-2 or IF-3 is limiting. The chloroplast ribosomes unlike E. coli ribosomes show considerable activity over a broad range of Mg2+ ion concentrations.     

The chloroplast and cytoplasmic ribosomes of euglena: I. Stability of chloroplast ribosomes prepared by an improved procedure

A new isolation procedure has resulted in an improved yield of stable 68S chloroplast ribosomes from Euglena gracilis var. bacillaris. Chloroplasts are isolated by suspending the cells in buffer I (sorbitol, 250 mm; sucrose, 250 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm) and passing through a French press at less than 1500 pounds per square inch. The crude chloroplasts are purified by three washings with buffer II (sorbitol, 150 mm; sucrose, 150 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm). Stable 68S chloroplast ribosomes are obtained when the isolated chloroplasts are resuspended in ribosome buffer (tris-HCI, pH 7.6, 10 mm; magnesium acetate, 12 mm; KCI, 60 mm) containing spermidine, 0.5 mm; mercaptoethanol, 14 mm; sucrose, 8% (w/w), passed through a French press at 4000 pounds per square inch and extracted with either 0.1% (w/v) sodium deoxycholate or 1.0% (v/v) Triton X-100. At 0 to 4 C in ribosome buffer, the purified 68S chloroplast monosome forms a 53S particle while the 35S particle, an expected product of monosome dissociation, cannot be detected. Spermidine and mercaptoethanol prevent the formation of 53S particles from 68S monosomes. The purified 53S particles derived from 68S monosomes contain 23S RNA as well as a significant amount of 16S RNA, suggesting that this particle may not be a true ribosomal subunit.     

Formation of chloroplast ribosomes and ribosomal RNA in Euglena incubated with protein inhibitors

It has been shown previously (cf [26]) that virginiamycin M blocks temporarily chlorophyll formation and chloroplast multiplication; these effects are made permanent by virginiamycin S.Virginiamycin M inhibits reversibly the assembly of the 30S and 50S subunits of chloroplast ribosomes. The association of the two virginiamycin components, M and S, causes a permanent arrest of 70S ribosome formation. On the contrary the antibiotics have no apparent effect on the 87S cytoplasmic ribosomes.Formation of the 16S chloroplastic ribosomal RNA is also prevented by virginiamycin: in a transient manner by the M component, and in a permanent way by the association of M and S. Biosynthesis of the 26S and 21S cytoplasmic rRNAs does not undergo appreciable changes in the presence of the antibiotic.The alterations of rRNA synthesis by virginiamycin in eucaryotic organelles, thus, differ from those induced by the drug in procaryotes; these findings might have evolutionary implications. Moreover, they might explain the temporary bleaching caused by virginiamycin M, and the permanent bleaching effect produced by the two virginiamycin components.     

Comparative base compositions of chloroplast and cytoplasmic tRNAPhe''s from Euglena gracilis.

The nucleoside compositions of chloroplast and cytoplasmic tRNAPhe's from Euglena gracilis have been determined. The modified nucleoside compositions of these two tRNAs indicate that tRNAPheChl is more similar to procaryotic (E. coli) tRNAPhe than to either the Euglena cytoplasmic tRNAPhe or other eucaryotic cytoplasmic tRNAPhe's.     

Turnover of chloroplast and cytoplasmic ribosomes during gametogenesis in Chlamydomonas reinhardi

A number of novel observations on ribosomal metabolism were made during gametic differentiation of Chlamydomonas reinhardi. Throughout the gametogenic process the amount of chloroplast and cytoplasmic ribosomes decreased steadily. The kinetics and extent of such decreases were different for each of the two ribosomal species. Comparable rRNA degradation accompanied this ribosome degradation. Concurrent with the substantial ribosome degradation was the synthesis of rRNA, ribosomal proteins and the assembly of new chloroplast and cytoplasmic ribosomes throughout gametogenesis. The newly synthesized chloroplast ribosomes exhibited distinctively faster turnover than their cytoplasmic counterpart. Cytoplasmic ribosomes, pulse-labeled in early gametogenic stages, retained label until differentiation was nearly complete even though a net decrease in the level of cytoplasmic ribosomes continued, indicating that the newly synthesized cytoplasmic ribosomes were preferentially retained during differentiation. Hence the regulation of ribosome metabolism during gametogenesis contrasts with the conservation of ribosomes obtained during vegetative growth of C. reinhardi and other organisms. This unique pattern of ribosome metabolism suggests that new ribosome synthesis is necessary during gametogenesis and that some specific structural or functional difference relating to the development stage of the life cycle might exist between degraded and newly synthesized ribosomes.     

Euglena gracilis chloroplast ribosomes: improved isolation procedure and comparison of elongation factor specificity with prokaryotic and eukaryotic ribosomes

An improved method for the isolation of Euglena chloroplast ribosomes is described which presents a number of advantages over past procedures. First, ribosomes are prepared from whole cell extracts, thus bypassing the need to isolate intact chloroplasts and resulting in a 10-fold improvement in yield. Second, the inclusion of 40 mm Mg²⁺ in the preparation buffers, while stabilizing the chloroplast ribosomes, precipitates and, thereby, virtually eliminates the cytoplasmic 89 S ribosomes. Third, greater than 95% of the chloroplast ribosomes sediment at 68 S rather than as the damaged 53 S particle frequently generated in other preparation procedures. Fourth, even with a high-salt wash to remove endogenous factors, the chloroplast ribosomes still sediment at 68 S and are just as active in in vitro protein synthesis as are E. coli ribosomes. These ribosomes have been tested for activity with elongation factors from prokaryotes, eukaryotes, and the chloroplast itself, and the results have been compared to those obtained with E. coli and wheat germ ribosomes. The data may be summarized as follows: (a) Chloroplast ribosomes use E. coli$\text{EF}\text{-}\text{Tu}\text{Ts}$ and EF-G with the same efficiency as do E. coli ribosomes in protein synthesis, (b) E. coli and chloroplast ribosomes can use Euglena chloroplast EF-G to catalyze translocation, but wheat germ ribosomes cannot, (c) Wheat germ EF-1_H and EF-2 are highly active in polymerization with wheat germ ribosomes, but ribosomes from neither E. coli nor the chloroplast are able to recognize these factors, (d) All three types of ribosomes accept Phe-tRNA from E. coli EF-Tu although to differing degrees. However, neither chloroplast nor E. coli ribosomes recognize wheat germ EF-1_H for the binding of Phe-tRNA.     

Determination of the site of synthesis of some Euglena cytoplasmic and chloroplast ribosomal proteins.

Ribosomal protein synthesis during chloroplast development in Euglena gracilis has been studied by using inhibitors specific for either chloroplast or cytoplasmic protein syntheses. Fifty proteins of cytoplasmic and 39 of chloroplast ribosomes have been examined. Synthesis of all cytoplasmic ribosomal proteins is strongly inhibited by cycloheximide. Lincomycin (LIN) seems to have no effect on the synthesis of these proteins. In contrast, formation of 12 chloroplast ribosomal proteins is inhibited by cycloheximide (CHI), that of 9 by lincomycin, and that of 6 by both of these antibiotics; the technique used in this study did not permit definite determination of the sites of synthesis of the remaining proteins.