BIOCHIMIE, 1982, 64, 1049-1058.

Proteolytic degradation of ribosomal proteins during isolation of ribosomes and ribosomal subunits from Tetrahymena. Marguerite CUNY, Michble MILET and Donal HAYES o.

(Regu le 23-6-1982, acceptg le 6-10-1982).

Laboratoire de Chimie cellulaire, Institut de Biologie Physico-Chimique, 13, rue Pierre-et-Marie-Curie, 75005 Paris, France.

R~sum6.

La m~thode d~crite pour la pr@aration des sous-unitds ribosomiques de T . <~ pyriformis >~ dite CGL (ph~noset non d~termin~), ne peut pas Otre appliqu~e h toutes les souches de ce proto- zoaire, les sous-unitds obtenues sub&sam une d~gradation protOolytique au cours de la prepa- ration. Seule l'addition d'iodo-acJtamide pendant l'extraction permet d'obtenir des sous-unit~s acti- ves. D'autre part, la composition prot~ique des ribosomes prepares en prdsence d'iodoac£tamide varie de fafon significative entre deux souches de ph~nosets dilf&ents.

Mots-el6s : ribosome / Tdtrahymena / prot~ases / iodoac~tamide.

Summary.

A preparation procedure previously used to isolate active ribosomal subunits Jrom an amicro- nucleate strain of Tetrahymena of undefined phe- noset (T. << pyriformis >,, CGL) yields inactive subunits when applied to other amicronucleate or to micronucleate strains of this protozoa.

Proteolytic degradation of a small number o] ribosomal proteins during preparation of riboso- mal subunits from these strains explains this results'. If cell extraction and ribosome isolation are carried out in the presence of iodoacetamide, proteolytic activity is inhibited and active riboso- mal subunits are obtained. Comparison of the protein complements o[ active ribosomal subunits prepared in the presence o[ iodoacetamide from three amicronucleate strains of Tetrahymena re- veals small but significant differences.

Key-words : ribosome / Tetrahymena / protease / iodoaeetamide.

Introduction.

Rodrigues Pousada and Hayes [1] reported the preparation of active ribosomal subunits from the amicronucleate Tetrahymena strain T. <~ pyri¢or- mis >> CGL and the electrophoretic characteriza- tion of their proteins. However application of the isolation procedure developped by these authors to micronucleate strains of Tetrahymena and to amicronucleate strains other than T. ¢ pyri[or- mis >> CGL yields inactive ribosomal subunits in most cases. Proteolytic degradation of ribosomal proteins, observed by Kristiansen and Kriiger [2] in experiments with an amicronuleate strain of

<) To whom all correspondence should be addressed.

Tetrahymena, offered a possible explanation for loss of ribosome activity. Experiments carried out with three amicronucleate strains of Tetrahymena, T. ~ pyrilormis ~> CGL, T. pyriformis ZGL and T. elliotti have verified this hypothesis. The re- sults, presented here, lead to the following con- clusions :

1. Proteolytic degradation of ribosomal pro- teins is both strain and growth temperature depen- dent. It occurs during the preparation of ribosomal subunits from T. elliotti but does not occur during their preparation from T. ~ pyriformis ~> CGL and T. pyriformis ZGL when cells are grown at 28°C. When cells grown at 31 ° C are extracted degrada-

1050 M. Cuny and coll.

t i on is o b s e r v e d in the case of T. elliotti and T. pyri[ormis Z G L , but n o t in tha t of T. << pyri- form& >> C G L .

2. T h i s p r o t e o l y t i c act ivi ty , w h i c h leads to iso- l a t i o n of pa r t i a l ly o r to t a l ly i n a c t i v a t e d r i b o s o m a l subun i t s can be e f f ic ien t ly inh ib i t ed b y ca r ry ing o u t r i b o s o m e p r e p a r a t i o n in t he p r e sence of 5 m M i o d o a c e t a m i d e .

3. R i b o s o m a l subun i t s o f T. elliotti and T. pyri- [ormis Z G L p r e p a r e d i n t h e p r e s e n c e Of i o d o a c e - t a m i d e a re ac t ive in vitro. Ana lys i s of the p r o t e i n c o m p l e m e n t s of ac t ive r i b o s o m a l subuni t s of T. elliotti shows tha t t hey d i f fer s l ight ly b u t s igni- f i can t ly f r o m those of ac t ive r i b o s o m a l subun i t s of T. << pyriJormis >> C G L and T. pyriformis Z G L .

4. A b s e n c e of p r o t e o l y t i c ac t iv i ty d u r i n g r ibo - s o m e p r e p a r a t i o n f r o m T. << pyriformis >> C G L is due to the p r e sence of a p r o t e a s e inh ib i to r in ex t rac t s of this s train.

vely growing cultures at cell densities between 7.5 × 104 and 9 × 105/ml and the mixtures were incubated with agitation at 28°C for 10 minutes. After addition of cold trichloroacetic acid (2 ml, 20 per cent w/v) the incubated culture samples were held for 10 min in an ice bath, followed by 20 minutes in a boiling water bath, before filtration on glassfiber filter discs. Material retained on the filters was washed with cold 5 per cent trichloroace- tic acid, and ether-ethanol (1:I, v / v ) ; filters were dried in air and their 14C content was measured.

Assay o] cellular RNA content.

Samples (2 ml) taken from growing cultures were added to 2 mt of ice-cold 20 per cent trichloroacetic acid. After standing in an ice bath for 10 min, the mixtures were centrifuged (5000 g, 5 rain) and the resulting pellets were washed twice by centrifugation with 5 per cent trichlo- roacetic acid and incubated at 37°C for 1 hour with 0.3 ml of 0.3 M KOH. The hydrolyzates were neutralized (30 ~1, 3 M HCI), protein and D N A were precipitated by addition of one volume of 20 per cent TCA and after centrifugation (5000 g, 5 min) the RNA content of the supernatant was assayed by measurement of A~0 ~m [5l.

M a t e r i a l s a n d M e t h o d s .

Chemicals.

Protease inhibitors, iodoacetamide, phenyl-methyl-sul- fonylfltioride, p-aminobenzamidine, and e-amino-n-caproic acid were supplied by Sigma Chemical Co, U.S.A. Cul- ture medium constituents and all other chemicals were as previously described ~[1, 3].

Uniformly labelled [14C]' L-lysine (s. act 4.33 GBq/ mmole), and [14C] r.-leucine (s. act. 2.59 GBq/mmole) were supplied by the Commissariat ~t l'Energie Atomique, France. ~

Strains and culture conditions.

Amicronucleate Tetrahymena strains T. elliotti belon- ging to phenoset B [4] and referred to here as EGL, and T. pyriformis GL << Zeuthen >> belonging to phenoset A [4] and referred to here as ZGL were obtained from the Culture Centre of Algae and Protozoa, Cambridge, England. (CCAP strain numbers 1630/ld, and 1630/lz). The strain called T. << pyriformis >~ CGL, the phenotype of which according to Borden et al. [4] has not been determined, has been described earlier [3]. Cells were grown as previously described [1], at 28°C or 31°C, as indicated. Cell densities were measured with a Coulter Counter.

Preparation of ribosomal subunits.

The methods described earlier [1] were used with .the following minor alterations :

a - - Cells were harvested at the stage of growth at which they display maximum in vivo protein synthetic activity. Different Tetrahymena strains reach .this stage at different culture densities (see legend figure 1).

b - - When used, protease inhibitors were added to cell suspensions before homogenization. The following con- centratious were used

iodoacetamide ~[6], 5 mM p-aminobenzamidine [7], 5 mM e-amino-n-caproic acid [8] 1 mM phenyl-methyl-sulfonyl-fluoride [9] 3 mM.

Because of its instability in aqueous media phenyl- methyl-sulfonyl-fluoride was added before ceU homogeni- zation and at 20 min intervals during the subsequent steps of ribosomal subunit preparation until the final seperation of subunits by sucrose gradient centrifugation.

c - - Ribosomal subunits were separated by ceutrifuga- tion on 10-30 per cent sucrose gradients prepared in 20 mM triethanolamine-HC1, pH 7.6, I O m M Mg acetate, 500 mM KCI, 6 mM ~-mercaptoethanol.

Ribosomal subunit reassociation and polyphenylalanine synthesis in vitro.

Previously described methods [3] were used.

Protein synthesis in growing cultures.

114C] lysine (185 kBq) and [14Cl leucine (185 kBq) were added together to 2 ml samples removed from acti-

Electrophoretic analysis of ribosomal proteins.

One dimensional SDS-polyacrylamide gel electropho- resis according to Adoutte-Panvier et al. [10] was used.

BIOCHIMIE, 1982, 64, n ° 11-12.

Proteolytic degradation of Tetrahymena ribosomes. 1051

Gel slabs were prepared by pouring a 5 per cent poly- acrylamide stacking gel onto a separation gel containing a 15-20 per cent linear polyacrylamide concentration gradient.

Preelectrophoresis of gels was omitted and electropho- resis was carried out in the cold room at 100 V for 4 hours followed by 600 V for 30 hours.

To prepare samples for analysis ribosomal subunits were incubated for 10 minutes at 65°C [11] in the ¢ sample buffer >> described by A. Adoutte-Panvier [10l supplemented wi~h 5 per cent v/v 13-mercaptoethanol, boi- led for 3 minutes and stored at room temperature over- night before loading onto gels. After electrophoresis gels were stained as described earlier [1].

Results.

PROTEIN SYNTHESIS IN GROWING CULTURES OF Tetrahymena.

Dietz et al. [12] have shown that the specific aminoacid incorporating activity of growing yeast cells varies during the exponential phase of culture growth, showing an initial increase, a maximum m mid exponential phase and a subsequent de- crease. Experiments were therefore carried out to detect possible variation of the aminoacid incorpo- ration activity of growing Tetrahymena. Since it is known that the cellular content of ribosomal R N A also varies inversely with the density of exponentially growing cultures of Tetrahymena [13 - 18]), the total R N A content of culture sam- pies was measured in addition to their protein synthesizing activity. The results of these deter- minations for strains CGL and EGL growing at 28 ° C are shown in figure 1. They show, as obser- ved earlier by Koroly and Conner [15] and by Rinaldy [18], that total R N A content per cell decreases uniformly throughout the part of the exponential growth phase examined here, but that protein synthesis varies widely during this period reaching a maximum at cell densities of 230000/ ml for strain CGL and 330000/mi for strain EGL. Similar results were obtained for strain ZGL growing at 28 ° C and for strains CGL, EGL and ZGL growing at 31 ° C. All ribosome preparations used in this study were isolated from cells harves- ted at the cell densities corresponding to maximum protein synthesis activity in vivo.

PROPERTIES OF ISOLATED RIBOSOMAL SUBUNITS.

A procedure has been described [1] for the preparation of active ribosomal subunits from

10 6

. . . . 3

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A

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FIG. l. - - Protein synthesis in growing Tetrahymena.

In vivo aminoacid incorporation activity and total cell content ef RNA were measured, as described in Materials and Methods, in samples taken from cultures of Tetra- hymena strains CGL, EGL and ZGL growing exponen- tially at 28°C and 31°C.

This figure shows the results obtained with s~rains CGL (solid symbols, continuous curves) and EGL (open sym- bols, dashed curves) growing at 28°C. t , O culture density. A, A in vivo protein synthesis. [14C] ]eucine + 114C]

lysine incorporated into hot TCA insoluble mate- rial, cts/min/ml of culture.

II, [] total RNA content of ceils.

Similar results were obtained with strain ZGL growing at 28°C and with the three strains growing at 31°C. Maximum in vivo protein synthesizing activity was observed at the following culture densities (cells/ml) : strain CGL, 28°C and 31°C ; 230 000. Strain ZGL, 28°C, 230000 ; 31°C, 200000. Strain EGL, 28°C 330000 ; 31 °C, 220 000.

T. << pyriformis >> CGL. However this method is not applicable to all Tetrahymena strains since attempts to use it to isolate ribosomal subunits from strain EGL gave only inactive products.

A possible explanation for this result was pro- vided when electrophoretic analyses of the protein complements of inactive ribosomal subunits of strain EGL showed that some of their proteins had been fragmented, presumably by proteolytic degradation. The effect of adding protease inhi- bitors to cell suspensions before homogenisation was therefore tested. The results of these experi- ments showed that iodoacetamide is an efficient inhibitor of proteloytic activity directed against ribosomal proteins in extracts of Tetrahymena,

BIOCHIM1E, 1982, 64, n ° 11-12.

1052 M. Cuny and coll.

i o d o a c e t a m i d e :

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FIG. 2. - - Reassociation of ribosomal subunits analyzed by sucrose gradient centrifugation.

a, b, c, d r ibosomal subuni ts f r o m T. ¢ pyriIormis ~> C G L grown at (a, b) 28°C, (c, d), 3 I°C.

e, f, g, h r ibosomal subuni ts f r o m T. pyriformis Z G L grown at (e, f) 28°C, (g, h) 31°C.

i, j, k, 1 r ibosomal subuni ts f r o m T. elliotti grown a t (i, j) 28°C, (k, 1) 31°C.

pannels a, c, e, g, i, k, ribosomes were isolated without i~nhibitar.

pannels b, d, f, h, j, 1, iodoacetamide 5 mM was present during extraction.

BIOCHIMIE, 1982, 64, n ° 11-12.

and that other inhibitors tested, (p-aminobenza- midine, E-amino-n-caproic acid, phenyl-methyl- sulfonyl-fluoride, results not shown) were much less effective.

REASSOCIATION OF RIBOSOMAL SUBUNITS.

Figure 2 shows the results of tests of the capa- city of ribosomal subunits of strains CGL, EGL, and ZGL prepared under various conditions to reassociate in vitro. The following conclusions can be deduced from these results :

1. Subunits prepared from strain CGL grown at 28°C reassociate completely whether isolated in the absence (figure 2 a) or presence (figure 2 b) of iodoacetamide. This result shows that reaction of ribosomal protein SH residues with iodoaceta- mide under the conditions used for ribosomal subunit preparation does not modify the reasso- ciation properties of the subunits.

2. Subunits prepared from strain CGL grown at 3 1 ° C reassociate incompletely (figure 2 c) when isolated in the absence of iodoacetamide but completely (figure 2 d) when isolated in its pre- sence.

3. The properties of subunits prepared from strains ZGL and CGL are very similar although the effect of cell growth at 3 1 ° C on subunit reassociation activity is more marked in the case of strain ZG L (compare figures 2 g, 2 c).

4. Subunits prepared in the absence of iodo- acetamide from strain EG L grown at 2 8 ° C or 3 1 ° C possess little ( 2 8 ° C figure 2 i ) or no (31 ° C, figure 2 k ) reassociation activity, but when isolated in the presence of iodoacetamide from the same cells reassociate efficiently (28 ° C, figure 2 j) or partially (3 ! ° C, figure 2 1).

PROTEIN SYNTHESIZING ACTIVITY OF RIBOSOMAL SUBUNITS.

The activities in polyphenylalanine synthesis in vitro of ribosomal subunits isolated in the presence and absence of iodoacetamide from cells grown at 28 ° C were tested, and the results agree with those of in vitro reassociation tests (see table I).

ELECTROPHORETIC ANALYSIS OF RIBOSOMAL SUBUNIT PROTEINS.

Although it possesses lower resolving power than the two-dimensional technique of Kaltschmidt and Wittmann [19] the one-dimensional electro-

Proteolytic degradation of T e t r a h y m e n a ribosomes. 1053

FIG. 3. - - Electrophoretic analysis of 40S subunit proteins on 15-20 per cent polyacrylamide gradient gels in the presence o] SDS.

Protein samples were prepared as described in Materials and Methods, from 1 A~0 ~ unit of 40S particles isolated, in the absence ( - - ) or in the presence ( + ) of iodoacetamide f rom strains T. <~ pyri]ormis >> CGL (lanes I, 2), T. elliotti EGL (lanes 3, 4) and T. pyri]ormis Z G L (lanes 5, 6) grown at 28°C and in the absence of iodoacetamide ( - - ) f rom cells of strain T. pyriform& ZGL grown at 31°C (lane 7 *).

Strains, growth temperature, and presence or absence of iodoacetamide during isolation of r ibosomal subunits are indicated above each gel lane. ---> Major differences in the electrophoretic patterns of proteins of 40S sub-

units of T. elliotti EGL prepared in the presence and absence of iodoace- tamide. major differences in the electrophoretic patterns of proteins of 40S sub- units of T. elliotti EGL and T. • pyriformis • C G L or T. pyriforrnis Z G L prepared in the presence of iodoacetamide.

• data in lane 7 and in lanes 1-6 are derived from two gel slabs in which protein migration was slightly different. Dotted lines link identical proteins in lanes 6 and 7 and indicate the relationship between the different migration patterns. Low mobili ty bands in the upper region of lane 7 are non riboso- mal proteins which occasionally contaminate preparations of 40S and 60S subunit proteins.

BIOCHIMIE, 1982, 64, n ° 11-12.

1 0 5 4 M . C u n y and coll.

FIG. 4. - - Electrophoretic analysis o] 60S subunit proteins on 15-20 per cent polyacrylamide gradient gels in the presence o] SDS.

Protein samples were prepared, as described in Materials and Methods, from 1 A~o ,m unit of 60S particles isolated, in the absence ( - - ) or in the presence ( + ) of iodoacetamide, from strains T. <~ pyriformis ~ CGL (lanes 1, 2), T. elliotti EG'L (lanes 3, 4) and T. pyri]ormis ZGL (lanes 5, 6) grown at 28°C and in the absence of iodoacetamide ( - - ) f rom cells of strain T. pyri- ]ormis ZGL grown at 31 °C (lane 7 *). ---> Major differences in the electrophoretic patterns of proteins of 60S sub-

units of T. elliotti EGL prepared in the presence and absence of iodoace- tamide.

• major differences in the electrophoretic patterns of proteins of 60S sub- units of T. elliotti EGL, and T. ¢ pyri]ormis >> CGL or T. pyri]ormis Z G L prepared in the presence of iodoacetamide.

* see legend to figure 3.

BIOCHIMIE, 1982, 64, n ° 11-12.

Proteolytic degradation of Tetrahymena ribosomes. 1055

TABLE I. Polyphenylalanine synthesis in vitro by mixtures o[ purified ribosomal subunits.

R i b o s o m a l s u b u n i t s l o d o a c e t a m i d e (14C) pheny la l an ine incorporation

(cpm above b a c k g r o u n d per 0.5 Ag6o nm r i b o s o m e s )

T. pyriformis Z G L -]- 5100 - - 3600

T. ~ pyriformis ~ C G L -~- 1600 - - 1700

T. elliotti E G L -~- 1600 - - 120

Incuba t ion sys tems conta ined 0.5 A m ., , uni ts o f e q u i m o l a r mix tu res of isolated 40S and 60S subuni t s p repared f r o m cells g rown at 28°C.

phoretic separation of ribosomal proteins used here (SDS-gel slabs containing a concentration gradient of polyacrylamide) resolves at least 20 components in total proteins of 40S and 60S ribosomal subunits of Tetrahymena and permits rapid and efficient comparison of large numbers of samples. When the one dimensional patterns of two protein samples were identical (strain CGL, 28 ° C and 31 ° C, strains CGL and ZGL, 28 ° C) they were reexamined by two-dimensional elec- trophoresis according to Kaltschmidt and Witt- mann [19]. In all such cases the two-dimensional patterns were also identical (results not shown).

In figures 3 and 4 proteins of ribosomal sub- units isolated in the presence and absence of iodo- acetamide from strains CGL, EGL and ZGL grown at 28 ° C are compared. It can be seen that the patterns of 40S and 60S proteins of strains CGL (lanes 1, figures 3 and 4), and ZGL (lanes 5, figures 3 and 4) are identical and are unchanged by isolation of ribosomal subunits in the presence of iodoacetamide (lanes 2 and 6, figures 3 and 4). In contrast, the 40S and 60S protein patterns of strain EGL contain smeared zones (~, lanes 3, figures 3 and 4) indicative of degradation pro- ducts. These zones disappear and new protein bands appear at positions corresponding to higher molecular weight components (~, lanes 4, figu- res 3 and 4) when ribosomal subunits are isolated in the presence of iodoacetamide. Similar analyses (results not shown) of proteins of ribosomal sub- units prepared in the presence or absence of iodo- acetamide from strains CGL and EGL grown at 31 ° C gave results identical to those in figures 3 and 4. In contrast, electrophoretic migration pat- terns of 40S and 60S ribosomal subunit proteins of strain ZGL grown at 31°C differ from the patterns observed for proteins prepared from cells

BIOCH1MIE, 1982, 64, n ° l 1-12.

of this strain grown at 28°C (compare lanes 7 with lanes 5 or 6 in figures 3 and 4). Disappea- rance of high molecular weight components and appearance of smeared zones (~ lanes 7, figures 3 and 4) is evident. These differences disappear when ribosomal subunits are prepared in the pre- sence of iodoacetamide from strain ZGL grown at 31 ° C and the electrophoretic patterns of 40S and 60S subunits proteins which are then observed are identical to those of proteins of ribosomal subunits isolated in the absence of iodoacetamide from strains CGL and ZGL grown at 28°C (results not shown). As mentioned earlier, the ef- fects of three further protease inhibitors, phenyl- methyl - sulfonyl fluoride, p - aminobenzamidine, and z-amino-n-caproic acid were examined in the present study. Although active products were not obtained when ribosomal subunits were prepared in the presence of any of these three compounds, analyses of the protein complements of these sub- units showed that phenyl-methyl-sulfonyl-fluoride and s-amino-n-caproic acid, but not p-amino- benzamidine, were partially effective as inhibitors of proteolysis of ribosomal proteins. Both com- pounds reduced, but did not eliminate, ribosomal protein degradation during extraction of cells of strain ZGL grown at 31 ° C, and e-amino-n- caproic acid was found to inhibit de~adation of 60S, but not of 40S subunit proteins during extraction of strain EGL. These findings suggest involvement of several proteases in ribosomal pro- tein degradation.

P O S S I B L E EXISTENCE OF A PROTEASE INHIBITOR

m STRAIN CGL.

An explanation for the absence of proteolytic activity directed against ribosomal proteins in extracts of strain CGL grown at 28 ° C or 31 ° C

1056 M. Cuny and coll.

was provided by the results of experiments in which cells of strains CGL, ZGL and EGL grown at 31 ° C were mixed before extraction. Compari- son of the migration patterns of 40S ribosomal proteins extracted from the CGL-EGL mixture (figure 5, lane 2) with those of 40S ribosomal proteins of strains EGL (figure 5, lane 1) and

played electrophoretic migration patterns which were the sums of those of ribosomal proteins of these two strains, i.e. proteolytic attack is not inhi- bited in extracts of mixtures of strains EGL and ZGL (results not shown). We conclude that strain CGL contains an inhibitor of the proteases invol- ved in ribosomal protein degradation.

FI6. 5. - - Analysis of 40S and 60S subunit proteins prepared in the absence of iodoaceta- mide from mixtures of strains CGL and EGL by electrophoresis on 15-17,5 per cent polyacryl- amide gradient gels in the presence of SDS.

Lane Subunit proteins

1 4OS, EGL 2 4OS, EGL -[- CGL 3 4OS, CGL 4 6OS, EGL 5 6OS, EGL --~ CGL 6 6OS, CGL

CGL (figure 5, lane 3) shows that degradation of 40S ribosomal proteins of strain EGL is inhibited in extracts of mixtures of the two strains. The same result is observed for 60S subunit proteins of strains CGL and EGL in lanes 4, 5, 6 of figure 5.

In contrast, ribosomal proteins prepared from mixtures of ceils of strains EGL and ZGL dis-

BIOCHIMIE, 1982, 64, ~a ° 11-12.

VARIATION OF THE RIBOSOMAL PROTEIN COMPLEMENTS OF AMICRONUCLEATE Tetrahymena STRAINS.

Electrophoretic analyses of ribosomal proteins isolated from the three amicronucleate Tetrahy- mena strains, T. <~ pyriformis >> CGL, T. pyriJor- mis ZGL and T. elliotti, grown at 28 ° C in the

Proteolyt ic degradation of Tetrahymena ribosomes. 1057

presence or absence of iodoacetamide, show that strains CGL and ZGL contain identical ribosomal protein complements (one-dimensional analyses, figures 3, 4 ; two-dimensional analyses, results not shown). These analyses also show that the ribosomal protein complement of strain EGL, grown at 28 ° C in the presence of iodoacetamide, differs from that of strains CGL and ZGL.

Comparison of the band patterns in lanes 4 (EGL + iodoacetamide) and 1, 2, 5 or 6 (CGL, ZGL --- iodoacetamide) of figures 3 and 4 reveals five major differences in 40S subunit proteins (4 in lane 4, figure 3) and six major differences in 60S subunit proteins (,q lane 4, figure 4). The fact that ribosomal subunits of strain EGL isola- ted in the presence of iodoacetamide are appa- rently fully functional (figure 2, table I) suggests that these differences are not artefacts of protease activity. However the presence of iodoacetamide resistant protease activity in extracts of strain EGL cannot be excluded.

Discussion.

Proteolytic degradation of ribosomal proteins was first observed during studies with rabbit reti- culocyte [20] and rat liver ribosomes [9 ; 21]. The effects were limited in extent and it was shown that degradation of rat liver ribosomal proteins could be inhibited by use of phenyl methyl-sulfo- nyl-fluoride [9] or N a -p-tosyl-L-lysine-chlorome- tyl-ketone [21]. More extensive proteolysis of ribosomal proteins was observed by Hallberg and Sutton by comparison of the protein complements of ribosomes isolated from exponentially growing and starved T. thermophi la BIV [22] (growth temp. 30 ° C) and by Kristiansen and Kriiger in similar experiments with a strain referred to as T. pyr i formis GL [2] (growth temp. 28°C). In both cases degradation of ribosomal proteins oc- curred when ribosomes were extracted from expo- nentially growing cells but not when cells were starved for 24 hours before extraction. The obser- vation that proteolytic activity is more pronounced in extracts of exponentially growing than of star- ved cells is discussed in detail by Kristiansen and Kriiger [2].

The results obtained in the present study with strain T. elliotti EGL agree with those found in Krlstiansen and Kriiger's earlier work with T. pyri- formis GL [2] ; those obtained with strains CGL

BIOCH1MIE, 1982, 64, n ° 11-12.

and ZGL show that proteolytic degradation of ribosomal proteins varies widely in extent in extracts of different amicronucleate strains of this protozoa and can also vary as a function of cell growth temperature (strain ZGL).

As judged by their protein synthetic activity in vitro, and the absence of obvious degradation pro- ducts in the electrophoretic distribution patterns of their proteins, ribosomes isolated in the pre- sence of iodoacetamide contain intact protein complements. However it should be noted :

a) That limited protease digestion of rat liver ribosomes in vitro can cause detectable changes in their protein complements without altering their in vitro protein synthesizing activity [23].

b) That our results do not exclude the possible existence of iodoacetamide resistant proteases in cell extracts. In all cases of proteolytic degradation of ribosomal proteins so far reported the number of proteins affected is small. For example exami- nation of results for Te t rahymena strain EGL in figures 3 and 4 shows that inhibition of proteo- lytic activity by iodoacetamide causes appearance of about three bands in the electrophoretic pat- terns of 40S subunit proteins and of only three or four bands in the 60S subunit pattern. Two- dimensional electrophoretic analyses of these pro- tein preparations show that the number of diffe- rent proteins which are protected against protease degradation by iodoacetamide is correspondingly small. Since ribosomal subunits containing degra- ded forms of these subsets of proteins are inactive it can be deduced that some or all of the protease sensitive proteins are required for ribosome func- tion. Analyses of the residual functions, if any, of ribosomal subunits prepared from T. elliotti and/ or T. thermophi la BIV in the absence of iodoace- tamide (tRNA binding, peptidyl transferase, etc.) are planned.

Acknowledgements.

The authors thank C. E. Sripati and A. Expert-Bezan~on for help]ul discussions.

Financial support has been provided by the Centre National de la Recherche Scienti]ique (E.R. 101), the DJl~gation d la Recherche Scienti]ique et Technique (A.C.C. n ° 7971045) and the North Atlantic Treaty Orga- nization (contrat n ° 1679).

74

1058 M. Cuny and coll.

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