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Experimental Cell Research 115 (1978) 207-219 DETERMINATION

OF THE SITE OF SYNTHESIS

OF SOME EUGLENA CHLOROPLAST

CYTOPLASMIC

RIBOSOMAL

AND

PROTEINS

GEORGES FREYSSINET’ Dkpartement de Biologie gPnbale et appliquee, Laboratoire associh au CNRS, Universitk Lyon I, 69621 Villeurbanne, France

SUMMARY 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.

Green Euglena cells contain three types of ribosomes. They are formed of RNA and proteins; cytoplasmic ribosomes are sensitive to cycloheximide (CHI) while chloroplast and mitochondrial ribosomes are sensitive to inhibitors of procaryotic translation, i.e., streptomycin, chloramphenicol, erythromycin and lincomycin (LIN) [l, 6, 12, 18, 431. In contrast to the knowledge of bacterial ribosomes, our understanding of organelle ribosome formation is still fragmentary. Although it is known that mitochondrial and chloroplast rRNAs are transcribed from the organelle’s genome [6, 33, 38, 411, the localization,,of coding and synthesis of ribosomal polypeptides remains to be elucidated. Nuclear DNA apparently codes for some of the mitochondrial and chloroplast ribosomal proteins [3, 23, 271while the organelle DNA would control the formation of the others [2,32,35]. It is not yet known, 14-781815

however, if the control directly affects the coding of the ribosomal polypeptides or that of an enzymatic system which would be able to modify a cytoplasmic preformed. structure. Finally, are the mRNAs translated on cytoplasmic ribosomes or on organelle ribosomes? Analysis of mitochondrial ribosomal proteins isolated by gel electrophoresis has led to the conclusion that the majority of the mitochondrial ribosomal proteins are synthesized in the cytosol, but the possibility is not excluded that some of them are formed in the mitochondria [28, 301.In addition, a recent analysis of mitochondrial ribosomal proteins from Neurospora indicates that at least one protein is synthesized in the mitochondria [25]. Similar studies with chloroplasts have been performed only with intact ribosomes 1 Present address: Institute for Photobiology, Brandeis University, Waltham, MA 02154, USA. Exp Cell Res II5 (1978)

208

G. Freyssinet

and conflicting results were obtained [9, 19, 22, 24, 391. In the present work we studied the formation of cytoplasmic and chloroplast ribosomal proteins during Euglena plastid morphogenesis. Greening cells were incubated with radioactive sulfate in the presence or absence of inhibitors of protein synthesis. The results obtained indicate that all of the cytoplasmic ribosomal proteins are formed within the cytosol while both the chloroplast and the cytosol participate in the formation of chloroplast ribosomal proteins. Preliminary reports of portions of this work have appeared elsewhere [ 11, 141. MATERIALS

AND METHODS

Growth conditions Klebs., strain Z Pringsheim, was erown in the dark on sulfur-deficient medium (SP,. [ills]). At the beginning of the stationary phase, ieli; were transferred to a 30 mM KC1 solution and illuminated 3 days later [ 131.Green cells were grown on NC1 medium [ 13) deficient in vitamin B1* 1121.

Euglena

gracilis

Conditions of incubation CHI (Sigma, 10 wg/ml) or LIN (Upjohn, 2 mg/ml) were added to treated cultures after 10 h of illumination. Four aliquots of Na,YjO, (carrier-free) were added to the cultures at 1 h intervals, the first addition being 0.5 h after the addition of inhibitors. Cells were harvested 3 h after the last addition of sulfate. Each culture received a total of 5&70 &i/ml. The conditions of culture and incubation were chosen for the following reasons: (a) growth in the dark was in a sulfurdeficient medium derived from a phosphorus-deficient medium PC,, r131)since. in the cultures thus obtained. chloro$asi de&lopmeht is enhanced compared with photoinduced cell division. Moreover we alreadv have information concerning the synthesis of Ehloroplast rRNA for such cells [16, 201; (b) radioactive sulfate was used as precursor because amino acid uptake is often low in cells grown in this medium r151. This prevented us from using the double labeling-technique and forced us to use larger amounts of radioactive sulfate because it is not a specific precursor for proteins and also the amount of sulfur-containine amino acids is low in ribosomal proteins c43], ereyssinet, unpublished results); (c) incubations started after 10 h of illumination. It is possible at this time to prepare chloroplast-enriched fractions for the extraction of chloroplast ribosomes. Net synthesis of chloroplast rRNA, thus formation of chloroplast ribosomes, is still present [20]; however, this formation is low compared Exp Cell Res 115 (1978)

with that of other chloroplast proteins. This obliged us to incubate cells for a long time. CHI and LIN have been previously used as effective inhibitors of chloroplast rRNA synthesis [ZO]and were found not to affect the uptake of the radioactive precursor [IS].

Buffers (I) 250 mM sorbitol, 250 mM sucrose, 2.5% (w/v) Ficoll, I mM magnesium acetate (Mg acetate), 0.01% (w/v) bovine serum albumin (BSA), 14 mM p-mercaptoethanol (P-m), 0.5 mM spermidine trihydrochloride (ST), 5 mM HEPES pH 7.6; (II) 150 mM sorbitol, 150 mM sucrose, 2.5% (w/v) Ficoll, 1 mM Mg acetate, 0.01% (w/v) BSA, 14 mM /3-m, 0.5 mM ST, 5 mM Hepes pH 7.6; (III) 10 mM Tris-HCl pH 7.6, 60 mM NH&I, 12 mM Mg acetate, 0.5 mM ST, 14 mM P-m; (IV) 8% sucrose (w/w) prepared in (III); (V) 10 mM Tris-HCl pH 7.6, 30 mM KCl, 2 mM Mg acetate, 14 mM P-m; (VI) 10 mM Tris-HCl pH 7.6, 100mM NH&I, 2 mM Mg acetate, 14 mM p-m.

Extraction of ribosomes and analysis of their proteins Cytoplasmic and chloroplast ribosomes were extracted following the flow chart shown in fig. 1. The technique used is a modification of methods already described [ 18, 371. Ribosomes were analyzed on sucrose density gradients, and 0.2 ml fractions were collected for the determination of radioactivity. Chloroplast ribosomes which were used for protein analysis were purified as follows: a 20000 g r&some supematant (see fig. 1) obtained from 2-3 liters of a greening culture incubated in the presence of Nh=SO, was mixed with 500-700 AzBO,,,,,of chloroplast ribosomes prepared from green cells as described in fig. 1 and loaded onto an exponential lo-45 % (w/v) sucrose gradient. After centrifugation at 29000 rpm for 16 h at 2°C in a Beckman Ti-14 zonal rotor, the gradient was displaced with 60% (w/v) sucrose and 8 ml fractions were collected. Fractions containing 68s chloroplast monosomes were pooled, dialyzed for 24 h against buffer III and the monosomes were recovered by an overnight centrifugation. Ribosomal proteins were extracted and analyzed as previously described [12, 181.

Radioactivity determinations Uptake and incorporation into whole cell proteins, ribosomal proteins or sucrose gradient fractions were determined as already described [lS, 181. The labeling of isolated ribosomal proteins was measured as follows: stained spots of the gel were cut out, immersed in 1 ml of 30% hydrogen peroxide in scintillation vials and incubated at WC ovemieht. Vials were cooled and 10 ml of scintillation cocktail (0.5 I Triton X-100, 8.5 g PPO and 150 mg POPOP/l toluene) was added to each vial. Radioactivity was then measured in a Packard 3375 scintilJ.ation counter at 70% efficiency. Background values were obtained by measuring the radioactivity of similar sized pieces

Ribosomal protein synthesis in Euglena

I Supernatant centrifuge 15000 g, 10 min discard pellet I Supernatant layer over a 2 ml cushion of 45 % (w/w) sucrose in buffer VI. Centrifuge 16 h 150000 g; discard supematant

Ribosomal pellet resuspend in buffer VI, keep suspension at 0°C for 24 h, then centrifuge 15000 g. 10 min. Layer supematant over a 2 ml cushion of 45 % (w/w) sucrose in buffer V, centrifuge 16 h, 150000 g, discard supematant Cytoplasmic ribosomal pellet

209

Harvested cells resuspend in buffer I(50 ml/15 1of culture); centrifuge 4 000 g, 5 min I Washed cells resuspend in buffer I (1 ml/g cells). French press at 1500 lbs/inZ and immediately dilute with buffer II (2 ml/g cells) I Cell lysate centrifuge lysate, 121g, 1.5 min; discard pellet then centrifuge supematant 700 g, 10 mitt Crude chloroplast pellet resuspend chloroplast in buffer II (2 ml/g cells); centrifuge suspension 121g, 0.5 mitt; discard pellet then centrifuge supematant 700 g, 10 mitt; discard supematant One-washed chloroplast pellet resuspend chloroplast in buffer IV (0.5 ml/g cells); centrifuge suspension 700 g, 10 min. Resuspend pellet in buffer IV (3 ml/g chloroplast). French press at 4000 lbs/in*. Add Na deoxycholate 0.1% (w/v) final cont. Shake 15 min, 2°C; centrifuge 20000 g. 15 min. Discard pellet Ribosome supernatant layer over a 2 ml cushion of 40% (w/w) sucrose in buffer III, centrifuge 16 h, 150000 g; discard supematant [ Chloroplast ribosomal pellet

1

Fig. 1. Flow chart for the isolation and puritication of cytoplasmic and chloroplast ribosomes.

of gel near the spots: it was about 250 dpm/spot for cytoplasmic ribosomal proteins, 40 dpmlspot for the cycloheximide treated culture and 30 dpmlspot for the chloroplast proteins. In order to determine the specific activity of chloroplast ribosomal proteins we measured the amount of protein present in each spot according to a moditication of the technique described by Fenner et al. [lo]: the dye was eluted from the cut spot by overnight incubation at 25°C with 1 ml of 25 % pyridine and the absorbance of the solution at 605 nm measured. In these conditions 80-90% of the radioactivity stay in the gel. For this reason we further treated the piece of the-gel with hydrogen peroxide as described above and the radioactivity in both, the pyridine and hydrogen peroxide solutions, was measured. The amount of protein was determined by reference to a standard curve. For that we prepared a&amide gels with increasing concentrations of bovine serum albumin; these gels were stained and destained as the slab gels. A known volume of each gel was then incubated with pyridine as described above. We obtained a linear relation between the absorbance at 605 nm and the protein concentration (fig. 2). We verified, by the staining technique of Bradford [S] which utilizes the principle of dye

binding and the classical method of Lowry et al. [29] as control, that bovine serum albumin and a mixture of total ribosomal proteins have, in solution, similar dye binding (MorlC & Freyssinet, unpublished observation). We can then assume that the dye-binding for one given ribosomal protein is the same whether the culture have been treated or not. This allows us to compare one protein of the control experiment with the same protein of the treated culture but does not permit comparison of several proteins of the same culture as differences in the dye binding of the various ribosomal proteins might exist. Protein content in the spots was between 0.5-4 pg. depending on the spot or the gel. In order to determine whether the effect of inhibitors on ribosomal protein synthesis is signiftcant we-tested our results by the Mann-Whitney test [21] whenever the number of repetitions in each treatment was sufticient (this was the case for most of the proteins). In a few cases, when this number was insufficient, we compared the results of the Mann-Whitney test on other proteins and the uercentage of labeling in treated and control cells; proteins were assumed significantly inhibited when this labeling represented 40% that of the control. Ribosomal proteins were then grouped into classes (see tables 2 and 3). Em Cell Res 115 (1978)

210

G. Freyssinet

0.6

0.4

Fig. 2. Abscissa: protein cont.; ordinate: Amsnm. Relationship between protein concentration in the gels and the absorbance at 605 nm after dye elution with 25 % pyridine. A known volume of gels containing increasing amounts of bovine serum albumin was treated as the slab gel (see Materials and Methods) and the absorbance at 605 nm of the pyridine solution was measured.

Fig. 3. Abscissa: ml migrated 4; ordinate: (left) cpm x10-5(x---x); (right)A,,,(-). Sucrose gradient sedimentation analysis of the cytoplasmic ribosomal pellet from untreated cells. Ribosomal pellet was resuspended in buffer V and layered over a 15-50 % (w/w) linear sucrose gradient prepared in buffer V. Gradient was centrifuged for 3 h at 33 000 rpm in a Beckman SW-36 rotor at 2°C. Fractions of 0.2 ml were collected for measurements of radioactivity.

proteins since it has been shown that a 3 h incubation following addition of precursor Analysis on intact ribosomes is sufficient for disappearance of native It is known that the culture conditions dur- polypeptide chains [22]. Moreover in our ing dark growth of Eugiena greatly in- system most of the sulfate is incorporated fluence chloroplast development [ 13, 161 into proteins 30 min after its addition to the and labeling of RNA [20] or proteins [15]. In cultures [ 151. The inhibition rates (table 1) expressed as the present work, the culture and incubation conditions were chosen for the reasons percent inhibition compared to control culdescribed in Material and Methods. tures, were calculated from the specific acSedimentation diagrams obtained with tivity of ribosomes either in the ribosomal cytoplasmic (fig. 3) or chloroplast (fig. 4) pellet (results expressed as dpm/A,,, ,,,,J, in ribosomal preparations correspond to those the case of cytoplasmic ribosomes, or in the previously described [X3, 371. It is to be sucrose gradient (results expressed as dpm/ noted that chloroplast preparations are con- surface unit of the peak) in the case of taminated by cytoplasmic ribosomes (89s) chloroplast ribosomes. Thus the radioacwhile no chloroplast particles are seen in tivity due to the cytoplasmic ribosomes sedimentation diagrams of cytoplasmic present in the chloroplast preparations was preparations. All ribosomal particles con- taken into account. The rates indicate that tain radioactivity from %O; (figs 3 and 4). CHI strongly inhibits cytoplasmic ribosome The major portion of this radioactivity formation while CHI and LIN both intershould correspond to structural ribosomal fere with chloroplast ribosome synthesis. RESULTS

Exp Cell Res 115 (1978)

Ribosomal protein synthesis in Euglena

211

Table 1. Effects of protein synthesis inhibitors on some parameters of Euglena metabolism during chloroplast development Inhibitors were added to greening cultures after 10 h of light; 0.5 h later, cultures received Na2’YS0, as described in Materials and Methods. Harvesting of the cells was performed 3 h after the last addition of sulfate. All the results are expressed as percent inhibition compared with the control culture; numbers in parentheses indicate S.D. Metabolism of Ti

Antibiotics Cycloheximide ( 10 &nl) Lincomycin (2 mglml)

Incorporation into cell proteins m

Chlorophyll synthesis (%I

Uptake (%I

98 (?3)

7 (&3)

85 (f9)

31(&9)

O(?l)

-7 (+38)

The rate of inhibition observed changes from one experiment to another. The reason of these changes is unknown, they might be due to variations in the physiological state of the cells. They are mainly found at the ribosome level (see table 1, S.D.). As controls we measured the inhibition of certain other parameters. We found that, under the experimental conditions used, LIN inhibits chlorophyll synthesis by 30 %. Previous results indicate that this phenomenon is due to the fact that incubation

Incorporation of ?I into ribosomes Cytoplasmic m

Chloroplast (%I

67 (_+21)

58 (? 19)

4 (f4)

35 (&26)

started after 10 h of illumination and is not peculiar to LIN [4, 8, 151. As with other prokaryotic type inhibitors [ 151,there is no visible effect of LIN on protein synthesis when measurements are done on whole cells. However, these inhibitors block 9095% of protein synthesis in isolated Euglena chloroplasts [ 1, 151. CHI strongly inhibits chlorophyll synthesis and sulfate incorporation into proteins. When LIN and CHI are added to the same culture, the inhibition observed is slightly higher than that observed with CHI alone but it does not

Fig. 4. Abscissa: ml migrated -+; ordinate: (left) cpmx lo5 (X * * . x); (right) AzM,, t-1. Sucrose density gradient analysis of a chloroplast preparation from Euglena. A 20000 g chloroplast ribosome supematant extracted from radioactive greening cells was mixed with 500Asso.,,&f chloroplast ribosomes extracted from nonradioactive green cells (see fig. 1) and loaded onto a 10-45 % (w/v) exponential sucrose gradient prepared in buffer III. The centrifugation was for 16 h at 29 000 rpm in a Beckman Ii-14 zonal rotor. Radioactivity was measured on a 0.2 ml aliquot of each 8 ml fraction collected. Results are expressed in cpm/l ml fraction. Exp Cell Res 115 (1978)

212

G. Freyssinet

Table 2. Radioactivity of proteins extracted from cytoplasmic ribosomes Spots were numbered (see fig. 5 A), cut out, and the radioactivity Cycloheximide spot no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 22: 23 24 25 E 28 29 :: 32 :: iz 37 :; 40 t:. 43 : it 48 49 50

Control” 1 (dpml spot) 2 725 14 847 876 388 809 1890 1 036 977 418 510 998 1 488 642 2 692 3601 3 568 2 342 1560 5 114 1 351 2 491 1209 2 107 2 311 830 2048 3 011 1043 2 381 4 055 1 886 2 530 3 1% 1717 3 350 1 104 1790 1 11.5 1 476 1231 324 695 1 150 1 626 1066 839 188 3 082 1 982 434

1 (dpml SPOO 441 I 732 404 180 413 321 104 236 50 46 161 107 36 125 116 110 218 132 229 53 129 1:: 163 184 300 486 177 364 1 114 264 0 123 270 261 329 121 101 137 80 91 175 131 125 39 80 2:: 288 98

(IO g/ml)b

2 16 12 46 46 51 17 10 24 12 9 16 7 5 4 3 3 9 8 4 4 5 7 6 2: 15 16 17 :: 14 0 4 16 3: ; 9 6 Z! 11 8 4 3; 8 14 22

3

measured. Spot 2, see caption to fig. 5 Lincomycin 1 (dpml spot)

f i I I I I I I I I I

11 406 905 458 790 577 1 722 921 1294 503 619 793 1004

: I I f

1 849 203 1 190 4706 1 918 839

I I I I I I I I i I I i i i I i I I i I i I I i I I I i

4 478 1 916 2706 2 569 2042 1 552 1 163 2 559 2647 1 686 2 507 9 071 3 363 788 845 1 910 39% 1 845 1 288 1997 3 185 1 340 313 749 I 360 1 486 1 276 1 032 827 1 559 1690 556

: I i

TA

814 557

107 890

900 039

TEl

100000

11 000

98 000

%

fip

0

Cell Res 115 (1978)

89 (+5)

2 (_+2)

(2 mg/ml)C 2

3

15 80 52 204 71 91 89 132 120 121 79 67 132 45 33 132 78 59 87 142 108 212 97 67 140 125 88 162 105 224 178 31 26 111 119 167 72 179 216 109 97 108 118 91 120 123 440 50 85 128

i 8 0 0 8 0 0 8 0 + i + 0 0 0 0 8 0 0 + 0 0 + 0 f + i I 0 00 0 0 + 8 0 0 0 : 00 0 0

Ribosomal protein synthesis in Euglena

213

reach 100%. This might be due to the ac- merizing)) activity. In all the experiments tivity of mitochondria which seem to be reported here, the labeling of this peak deresistant to inhibitors in Euglena. creases by 80-90% when one of the inhibitors is used. This served as a control of Analysis on isolated ribosomal the effect of inhibitors on cytoplasmic proteins (CHI) or chloroplast (LIN) protein synObservations concerning preparations of theses. This is in agreement with the obribosomes used for gel electrophoresis. servation of Diamond [8] which indicates Cytoplasmic ribosomes (carrier-free) were that, even if chlorophyll synthesis is inwashed with 100 mM NH&I and purified by hibited by 30-40%, RuDPCase activity is two high speed centrifugations through a inhibited by 80-90%; (c) 32S, 495, 68s and sucrose cushion (see fig. 1); these ribo- 100s chloroplast and 89s cytoplasmic ribosomes were used for gel electrophoresis somal particles. Discrepancies between without further purification. As the chloro- radioactivity and absorbance profiles are plast preparations obtained were con- due to differences between a 20000 g chlotaminated by cytoplasmic material we puri- roplast supematant of a greening culture, fied the chloroplast monosome by zonal radioactivity pattern, and a chloroplast centrifugation. On fig. 4 we represented the ribosomal preparation of green cells, most sedimentation diagram of the mixture: of the absorbance profile. Fractions cor20 000 g chloroplast supematant from a con- responding to the 68s peak were pooled and trol radioactive culture and cold chloroplast the chloroplast monosomes were recovered ribosomes from green cells. We can then as described in Materials and Methods. The distinguish, from the center of the rotor to analysis of the rRNA content of these puriits periphery: (a) soluble components pres- fied chloroplast monosomes indicates that ent in the 20000 g chloroplast supematant; they are free of any cytoplasmic contamina(6) a peak with a sedimentation rate close to tion [17]. Moreover, no cytoplasmic ribo17s and which contains RuDPCase (ribu- somal proteins are seen on two-dimensional lose biphosphate carboxylase EC4, 1.1, gel electrophoretograms of the purified 39.3-phospho-D-glycerate carboxylase (di- chloroplast monosomes (see fig. 6).

O1Means of four electrophoreses from two different experiments. b Means of three electrophoreses from three different experiments. c Means of four electrophoreses from three different experiments. As only one experiment can be done at a time, because of the zonal centrifugation, we have, for each experiment, used an aliquot of the culture as control. We then extracted and determined the specific activity of the ribosomes of this aliquot. The radioactivity of the treated culture was then corrected depending on that of the control. 1. These values, in dpmlspot, were obtained as follows: a,, a2 . . . al, . . . a, correspond to the gross counts measured from spots 1,2 . . . i, . . . n cut from one electrophoretogram of a control culture, and A corresponds to the background radioactivity. We calculated (a,‘=~+-A) and then the sum of a,’ (L;). This value was then brought to a constant TB= 100x 108dpm and used to calculate a;=~’ XTB&Z/. When several experiments were performed, we calculated the average value (61’)for each protein. TA corresponds to the average value of W;. In the case of treated cultures TB was corrected by the inhibition rate (%) observed in comparison with control cultures. In the case of cytoplasmic ribosomes, this inhibition rate was obtained from the ratio of the specific activity of the protein solution used for gel electrophoresis, numbers in parentheses indicate S.D. 2. Gives an idea of the effect of inhibitors: for each spot, the values indicated correspond to the ratio a;’ treated culture X lOO/u~control culture. 3. Proteins were grouped, following the Mann-Whitney test [21], into four classes: (+) proteins significantly activated at the 10% level; (0) proteins not significantly inhibited; (i) and (I) proteins significantly inhibited at the 10 and 5% level, respectively. Exp Cell Res 115 (1978)

214

G. Frey&net Characterization of proteins dimensional gel electrophoresis.

Fig. 5. Analysis of the cytoplasmic ribosomal proteins of greening Euglena on two-dimensional polyacryl-

amide gels. Cytoplasmic ribosomal proteins were extracted with acetic acid [18] and analyzed by twodimensional gel electrophoresis as previously described [12]. (a) Ordinnre: migration 2 D +. Schematic representation of the two-dimensional electrophoretogram. Spots were numbered successively horizontally, starting at the upper left of the gel. They were drawn according to their approximate original densities dotted enclosure, least dense; unbroken line enclosure, more dense; filled enclosure, most dense. Spot 2 represents the dark region of the gel: in this region, 10 spots can easily be seen on the gel but are difficult to identify on the photograph. (b) Photograph of the two-dimensional electrophoretogram. Exp Cell Res 115 (1978)

by

two-

The electrophoretogram of cytoplasmic ribosomal proteins shows at least fifty proteins (fig. 5). The study of numerous electrophoretograms lead to the following comments: (a) the stained zone at the upper part of the gel disappears when 14 mM P-mercaptoethanol is added to the protein solution just before its deposition onto the first dimensional gel; there is always a little radioactivity near spots in this region; (b) depending on the experiment 3-7 acidic proteins are visible in the gels [ 181, thus we cannot safely assume that these proteins are functionally part of the ribosome; (c) a few spots were due to more than one polypeptide, as when the electrophoresis was carried out for longer times, spot 30; for instance, was resolved into three polypeptides. We can then enumerate 70 spots [ 181. Finally, as the number of proteins in cytoplasmic ribosomes is high, a complete characterization is needed and is hampered by the unstable nature of purified Euglena ribosomes [7, 181.Accordingly, we have limited our study to the 50 spots which are easily visible on the gel shown in fig. 5. Fifty-seven spots are numbered on the electrophoretogram of chloroplast ribosomal proteins [12] of which 39 have been studied here (fig. 6). The others were not significantly labeled and corresponded either to acidic proteins faintly visible on the gel or to poorly resolved basic proteins. Analysis of labeling of ribosomal proteins

All the ribosomal proteins analyzed are significantly labeled in the control experiment (tables 2 and 3, col. 1). Thus all the proteins have at least one sulfur-containing amino acid. Measurements on intact ribosomes, without carrier, show that the la-

Ribosomal protein synthesis in Euglena

215

Table 3. Radioactivity of proteins extracted from chloroplast ribosomes

Groupe A

B

spot no. 5 6 14 19 21 22 28 29 32 36 42 44 3 3; 35 39

C

D

1; 50 55 7 13 27 38 45 46 1 ; 26 37 47 48 49 :: 56 57 TA TB %

ControP 1 (dpm/ PJ3Vd 448 412 178 777 216 264 301 142 133 69 241 223 50 226 189 377 213 171 211 245 165 840 616 214 263 594 606 41 45 2:: 237 248 117 189 40 99 131 126 26 782 10 000 0

Cycloheximide (10 @ml)b

Lincomycin (2 mg/ml)c

1 (dpd Iam

3

1 (dpml km

t i i

323 287 416 677

156 140 78 49 43 69 32 43 39 26 36 46 38 254 95 212 109 121 98 123 141 187 90 ZfJ 88 39 23 27 55 186 116 184 149 79 385; 147 253 12 548 4200 58

2 35 ii

2 72

i i i I i I 0

216 131

2;: 87 137 86 72 92

2:; 216 151 0

3:: 90 68 0

i 0 00

38 64 30 41 44

:i

0

65

it 22 15 39 38 15 6 56 64

0 I I I I I i 0

!E 163 190 137 89

4: 49 74 127 42 145 392 112 201

00 0 0 0 0 0 0 + 0 0

6 20 26 :A 29 38 15 21 70 112 50 56 51 71 46

1:: 197 223 113 332 264 133 49 103 190 118 151 133 17 307 6500 35

8 20 24 31 36 17 19 31 64 34 15 27 480 4% 138 129 111 54 42 55 475 119 115 106

3 0 0 0 0 8 8 0 + 00

I : I : I i i I I i I i i + + 0 0 8 0 0 + 0 0 0

LI Means of six electrophoreses from two different experiments. b Means of eight electrophoreses from three different experiments. c Means of nine electronhoreses from four different experiments, see also notes to table 2. d (dpm/pg protein). e Group A corresponds to proteins inhibited only by CHI, group B to proteins inhibited only by LIN, group C to proteins inhibited by both CHI and LIN, and group D to proteins for which we were not able to determine thesite of inhibition. 1. See table 2 for calculations. TB for the control culture is 100X 1oPdpm; the inhibition rate was taken from the average ratio of specific activity measured on whole ribosomes (table 1); for these ribosomes, direct measurements on protein solutions cannot be done since carrier ribosomes were added. Values are in dpm/pg protein. Calculations of specific activities of proteins were done after substracting the background indicated in Materials and Methods. 2. See table 2. Exp Cell Res 115 (1978) 3. See table 2. 14t-781815

216

G. Freyssinet

=0

5’0

@2D

Fig. 6. Analysis of the chloroplast ribosomal proteins of Eugkna on two-dimensional polyacrylamide gels. Chloroplasts monosomes recovered from the zonal centrifugation (fig. 4) were treated with RNase [18] and the proteins analyzed by two-dimensional gel electrophoresis [12]. (a) Or&are: migration 2D +. Schematic representation of the two-dimensional electrophoretogram. Significance of the enclosures is as in tig. 5; (b) photograph of the two-dimensional electrophoretogram. Exp Cell Res 115 (1978)

beling of chloroplast ribosomes is 70-100 % that of cytoplasmic ribosomes. The difference found here between the labeling of cytoplasmic ribosomal proteins and chloroplast ribosomal proteins (see tables 2 and 3, TA, col. 1) is due to the fact that chloroplast ribosomes were mixed with nonradioactive material in order to obtain enough proteins for the two-dimensional gel electrophoresis. The large range of radioactivity observed (tables 2 and 3, col. 1) might be due either to differences in the amount of sulfur-containing amino acids in the different ribosomal proteins or to the presence of precursor pools of various sizes. Results in tables 2 and 3, col. 2 and 3, give an idea of the effect of protein synthesis inhibitors on the labeling of ribosomal proteins. In order to determine whether this effect is significant, we tested our results by the Mann-Whitney test ([21], see Material and Methods). The results thus obtained can be summarized as follows. All the cytoplasmic ribosomal proteins are significantly inhibited when cultures are incubated in the presence of CHI (table 2). When cultures are treated with LIN, the majority of the proteins contain about the same radioactivity as does that of the control cultures. A few proteins are highly labeled, this might be a result of LIN inhibition on chloroplast protein synthesis, which may induce an increase in the specific activity of the cytoplasmic amino acid pools. Finally, a few proteins are inhibited; this unexpected result requires confirmation before the formulation of an hypothesis. It might be related with the fact that this type of inhibitor was found to modify the tumover of cytoplasmic rRNAs [26]. In the case of chloroplast ribosomes we can distinguish three types of proteins (table 3): (a) twelve proteins which are inhibited

Ribosomal protein synthesis in Euglena

217

only by CHI; (b) nine proteins inhibited only by LIN; and (c) six proteins inhibited by both inhibitors together. The technique used does not allow us to determine the site of inhibition for twelve proteins: the MannWhitney test indicates that the number of repetitions was insufficient for five proteins (nos 1, 2, 9, 52, 53); three proteins are significantly inhibited at the 20% level either by CHI (no. 26) or by LIN (nos. 48,49). No explanation has been found yet for the last four proteins (nos. 37, 47, 56, 57). Finally, as in the case of cytoplasmic ribosomal proteins, a few proteins exhibit a labeling that is higher in treated cultures than in controls.

scribed on intact ribosomes. Three points will be discussed here: (1) Direct influence of inhibitors on synthesis of ribosomal proteins: Results obtained in this study indicate that all the cytoplasmic ribosomal proteins studied are significantly inhibited by CHI while, in the case of chloroplast ribosomal proteins we were able to distinguish three groups, i.e., proteins inhibited only by CHI, proteins inhibited only by LIN and proteins inhibited by both LIN and CHI. Although our results do not enable us to say whether these inhibitors act directly on the translation of ribosomal polypeptides or on that of enzymatic systems which would affect their synthesis, we can assume that proteins inDISCUSSION hibited only by LIN or CHI are formed in Results obtained with intact ribosomes indi- the chloroplast or in the cytoplasm respeccate that both the cytosol and the chloro- tively. In such a case, all the cytoplasmic plast control chloroplast ribosome forma- ribosomal proteins studied would be syntion. They are in agreement with results of thesized in the cytosol whereas some chloEllis & Hartley [9] who observed an inhibi- roplast ribosomal proteins would be formed tion of chloroplast ribosome formation in the cytosol and others in the chloroplast. when detached etiolated apices were il- On the other hand, sensitivity of some riboluminated for 2 days in the presence of somal proteins to both inhibitors would inLIN. They disagree with those of Honey- dicate the presence of a dual control, one cutt & Margulies [22] who found no inhibi- compartment controlling the synthesis of tion of the labeling of chloroplast ribosomes the polypeptide itself, the other regulating when cultures of Chlamydomonas were in- its synthesis. This observation can be related to what cubated in the presence of chloramphenicol. This discrepancy might have two ori- has been found concerning the site of gins: (1) Ellis & Hartley [9] worked, as our- coding of these proteins, i.e., that some selves, with greening material whereas chloroplast ribosomal proteins would be Honeycutt & Margulies [22] used green coded by the nucleus while others would cells during growth phase; (2) Honeycutt & be coded, or at least closely controlled, by Margulies [22] employed chloramphenicol the chloroplast DNA [2,3,6,23,32, 351. In as chloroplast inhibitor whereas Ellis & the case of Euglena, results obtained with a Hartley [9] and ourselves used LIN, which streptomycin resistant mutant [ 121indicate should be, according to them, more specific that one chloroplast gene, at least, controls than chloramphenicol. In Eugfena this in- the formation of chloroplast ribosomes. hibitor is detoxified during incubation [42]. One can then ask whether, as for the subResults obtained with isolated ribosomal units of the RuDPCase, the ribosomal proproteins reinforce and clarify those de- teins coded by the nuclear DNA are formed Exp CellRes 115 (1978)

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in the cytosol while those coded by the chloroplast DNA are synthesized inside the chloroplast. Further studies will be needed to support this hypothesis. However, such experiments seem difficult to realize with Euglena as we do not yet have genetic markers for these proteins and as classical genetic studies are impossible due to the lack of a described sexual reproduction. (2) Indirect influence of inhibitors on ribosomal protein formation: It seems strange that a few chloroplast ribosomal proteins are apparently insensitive to the action of the inhibitors used. This phenomenon may have two origins: an underestimation of the labeling of these proteins in control experiments or an overestimation of the labeling of these proteins in treated cells. Two hypotheses will be formulated to explain that: (a) it might be a consequence of the technique used; the considerable variability found, on intact ribosome as well as on single ribosomal proteins between experiments might have induced a few errors; (b) it might also be related to secondary physiological effects. There might exist pools of ribosomal proteins especially since some chloroplast ribosomal proteins have to be transported. These pools might be of different sizes depending on the particular protein and might modify the intensity of labeling even if they are of small sizes. Moreover, we know that ribosomal proteins are subjected to maturation processes [36]. One can ask whether inhibitors would not act on one of these processes, reserve pools, transport or maturation of proteins, thereby modifying the apparent labeling of proteins independently of a direct action on their synthesis. (3) Indirect influences of CHI on synthesis of chloroplast proteins: Finally, it is very important to note that CHI inhibits the formation of chloroplast ribosomes. In Exp Cell Res 115 (1978)

fact, this inhibition can induce a decrease, and even a block, of protein synthesis within the organelle, especially during long-term incubation which is generally the case for studies on chloroplast development. This might then induce errors in the assigning of a localization of the site of synthesis of these proteins and might explain why there is a large discrepancy between results obtained from different groups by in vivo experiments, e.g. results on the site of formation of chloroplast enzymes. The complete inhibition of chloroplast development in Euglena [4, 341 induced by CHI might be partly due to the indirect effect of CHI on the protein synthesis apparatus of the chloroplast. In any case, results obtained by this technique must nevertheless be confirmed by other techniques, e.g., protein synthesis by isolated organelles. Attempts already carried out with isolated chloroplasts are, up to now, negative [40], and those with isolated mitochondria give conflicting results [6, 28, 311. This is primarily due to the fact that ribosomal protein synthesis is always low compared to that of other organelle proteins. Moreover, further analyses will have to specify the exact participation and time of addition of the ribosomal proteins to the chloroplast ribosome during its formation. I thank Dr V. Nigon for many stimulating discussions, Dr C. Gamier for calculation of the Mann-Whitney test, Miss A. M. Lorin for technical assistance, Dr J. Diamond, Dr F. Fong and Dr J. A. Schiff for careful reading of the manuscript. This work was partly supported by a grant from the French Commissariat a I’Energie Atomique.

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Received June 29, 1977 Revised version received November 25, 1977 Accepted February 22, 1978

Exp Cell Res I15 (1978)