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Selective proteolytic dissociation of rabbit reticulocyte single ribosomes not attached to messenger RNA
719
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96745
SELECTIVE PROTEOLYTIC DISSOCIATION OF R A B B I T RETICULOCYTE SINGLE RIBOSOMES NOT ATTACHED TO MESSENGER RNA R A M O N V E L E Z * , N A N C Y L. F A R R E L L
AND M I C H A E L L. F R E E D M A N
Department o/ Medicine, New York University School o/ Medicine, New York, N . Y . zooi6 (U.S.A .) (Received O c t o b e r 26th, 197 o)
SUMMARY
Free and mRNA-bound ribosomes differ markedly in their susceptibility to mild proteolysis. Free ribosomes are dissociated into 6o-S and 4o-S subunits, while mRNA-bound ribosomes are completely protected. This protection can be reversed b y removing ribosomes from mRNA, and can be re-instated by allowing them to attach again. The resistance to proteolysis does not appear to be dependent upon the presence of nascent chains on the ribosome. This method has practical value as a means of determining if ribosomes have attached to mRNA and initiated globin chain synthesis.
INTRODUCTION
When the ribosomal component of rabbit reticulocytes is exposed to mild low temperature proteolytic conditions 1,3, a portion of the single 8o-S ribosomes dissociates into 6o-S and 4o-S subunits**. The polyribosomes under these same conditions are resistant and remain intact 1,~. On the basis of these observations, MALKIN AND RICH1 postulated that only those ribosomes not attached to mRNA are susceptible to nfild proteolysis. If this is the case, then mild proteolysis would be a valid method of differentiating between ribosomes attached and not attached to mRNA. However, there are at least three types of 8o-S ribosomes found in rabbit reticulocytes4: (I) those not attached to mRNA at the time of isolation, but still capable of associating with mRNA and initiating hemoglobin synthesis; (2) those not attached to mRNA and incapable of initiating hemoglobin synthesis, possibly as a result of damage to the ribosome in the maturation process; and (3)those attached to mRNA and synthesizing hemoglobin. An alternative explanation of the observed selective proteolysis, therefore, is that only damaged 8o-S ribosomes (type 2) are susceptible. The present study was undertaken in an attempt to evaluate the two alternative hypotheses. Our results favor the MALKIN AND RICH hypothesis, and indicate that mild proteolysis can in fact be used to distinguish free from mRNA-bound ribosomes. * P r e s e n t a d d r e s s : D e p a r t m e n t of Medicine, C o l u m b i a U n i v e r s i t y , College of P h y s i c i a n s a n d Surgeons, N e w York, N.Y., U.S.A. -- T h e d e s i g n a t i o n s 80 S, 60 S a n d 4 ° S are a d o p t e d a c c o r d i n g to t h e c o n v e n t i o n p r o p o s e d b y T s o AND VINOGRAD 8 to r e p r e s e n t t h e single ribosome, a n d t h e larger a n d s m a l l e r m a m m a l i a n r i b o s o m a l s u b u n i t s , respectively. T h e a c t u a l s e d i m e n t a t i o n coefficients were n o t d e t e r m i n e d .
Biochim. Biophys. Acta, 228 (1971) 719-727
720
R. VELEZ et al.
MATERIALSAND METHODS
Incubation o/intact cells and [ormation o] single ribosomes Reticulocyte-rich blood was collected in heparin by cardiac puncture of phenylhydrazine-treated rabbits. The cells were washed with an isotonic salt solution and incubated as previously described 5. The incubation was carried out for 30 rain at 37 °. The total volume of the incubation medium was 8.0 ml in a 25-ml erlenmeyer flask, with each flask containing I.O ml of reticulocytes. The incubation was stopped by placing the flask in ice. The cells were centrifuged at 600 × g and the supernatant fluid removed. The cells were washed, lysed, and the coarse particulate fraction was removed as previously described 5. Polyribosomes were disaggregated to form single ribosomes by incubating intact cells with 2. lO .-4 M 2,2'-bipyridine 6 or o.I M n-butanoF, or by the omission of tryptophan from the incubation medium s. When 2,2'-bipyridine was used, iron and transferrin were omitted.
Formation o[ polyribosomes not carrying nascenl chains Polvribosomes derived from intact cells carry nascent globin chains, while most single ribosomes do not 5. Puromycin, when added to intact cells in a final concentration of 2 mM, causes the premature release of all of the growing peptide chains from the ribosome 5,6,9. Polyribosomes not carrying nascent chains were formed, therefore, by incubating intact cells in the complete medium for ten minutes and then with puromycin (2 raM) for an additional IO rain at 37 °.
Cell-/ree incubation and ]ormation o] single ribosomes Ribosomes not attached to m R N A were formed in a cell-free system by allowing natural termination of globin synthesis in the absence of heme. Since heme is necessary for initiation, the result is a disaggregation of polyribosomes to form single ribosomes not attached to m R N A 5,1°,n. The cell-free system utilized was essentially t h a t previously described 5. Washed reticulocytes were lysed with an equal volume of water and the lysate cleared by centrifugation for 15 min at 25 ooo ×g. The medium contained o.15 ml of a Tris-NaC1 buffer, 0.90 ml of the cleared lysate, and 0.60 ml of a master mix, added in that order. The Tris-NaC1 buffer was prepared by adding I vol. of o.I M NaOH, 0.2 vol. of i M Tris, pH 7.4, and I vol. of o.I M HC1. The master mix contained the following ingredients in amounts to yield the individual final concentrations: KC1 (75 raM), MgC12 (2 raM), mercaptoethanol (6 raM), the 19 anfino acids reported present in rabbit hemoglobin 1~,13 in I/IO the concentration used for intact cell incubations s. ATP (1 raM), GTP (0.2 mM), creatine phosphate (15 mM), and creatine kinase (45 enzyme units/ml) were added as the energy source. The reticulocytes used in these cell-free studies were incubated first as intact cells either without tryptophan or in the complete medium as discussed above. After the cell-free incubation for 9° min at 34 °, the ribosomes were isolated and treated in the same fashion as those derived from intact cells.
Isolation and proteolytie treatment o] the ribosome-polyribosome component Ribosomes were removed from the large bulk of supernatant hemoglobin by centrifugation of the lysate at ioo ooo × g at 4 ° for 3 h over a cushion of 30 % sucrose.
Biochim. Biophys. Acta, 228 (1971) 719-727
PROTEOLYTIC DISSOCIA'IION OF SINGLE RIBOSOMES
721
T h e resulting a d h e r e n t pellet was rinsed g e n t l y w i t h s t a n d a r d buffer (o.oi M Tris, p H 7.4, o . o i M KC1 a n d o.oo15 M MgC12) a n d g e n t l y r e s u s p e n d e d in i . o ml of t h e same buffer. T h e res u s p en d e d ribosomes (2.5 rag) were m i x e d w i t h o.5 m g of pronase at a final v o l u m e of I.O m l at o ° for I h. T h e c o n c e n t r a t i o n of ribosomes was d e t e r m i n e d at 260 m/z, using an e x t i n c t i o n coefficient of 12 a b s o r b a n c e u n i t s / m g per ml ~. A f t e r proteolysis, t h e solution was centrifuged at 600 x g for IO m i n a n d a n y pellet was discarded.
Analysis o/ the ribosome-polyribosome component T h e r i b o s o m a l suspension was l a y e r e d on 36 m l of a 15-3o % (w/w) linear sucrose g r a d i e n t in s t a n d a r d buffer. A f t e r c e n t r i f u g a t i o n in a Spinco S W 27 swinging b u c k e t r o t o r at 4 ° for speeds a n d t im e s shown w i t h i n d i v i d u a l e x p e r i m e n t s , t h e grad i e n t was p u m p e d t h r o u g h the f l o w - t h r o u g h cell of B e c k m a n K i n t r a c V I I spectrop h o t o m e t e r to measure absorbance at 260 In#. In some e x p e r i m e n t s , t h e r i b o s o m a l suspension or t h e entire cell-free i n c u b a t i o n m e d i u m was placed d i r e c t l y on a sucrose g r a d i e n t , before p r o t e o l y t i c t r e a t m e n t , a n d c e n t ri fu g ed for 3 h at 25 ooo r e v . / m i n at 4 ° in t h e S W 27 rotor. Th e g r a d i e n t was analyzed, a n d t h e single ribosomes were s e p a r a t e d f r o m t h e polyribosomes. E a c h comp o n e n t was pelleted b y c e n t r i f u g a t i o n at IOO ooo x g at 4 ° for 3 h, a n d resuspended in s t a n d a r d buffer. T h e s e p a r a t e d 8o-S ribosomes an d polyribosomes were t h e n t r e a t e d w i t h pronase. Pronase, B grade, was o b t a i n e d f r o m t h e Calbiochemical Co. RESULTS
Proteolytic dissociation o~ single ribosomes/rom intact cells W h e n t h e r i b o s o m e - p o l y r i b o s o m e c o m p o n e n t isolated f r o m i n t a c t r a b b i t re t i cu l o cy t es was exposed at o ° to pronase for I h, t h e polyribosomes were n o t al t er ed
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Fig. i. Sucrose density gradient analysis of the ribosome-polyribosome component of rabbit reticulocytes exposed to mild proteolysis. The isolated ribosome-polyribosome component, derived from intact cells incubated in the complete medium, was re-suspended and incubated with and without pronase, as described in MATERIALSAND METHODS. Centrifugation in a 15-3 ° °/o sucrose density gradient was for 3 h at 25 ooo rev./min. , without pronase; - .-, with pronase. Fig. 2. Effect of pronase on 8o-S ribosomes derived from cells incubated in the complete medium. The isolated ribosome-polyribosome component was re-suspended and centrifuged in a 15-3o % sucrose density gradient for 3 h at 25 ooo rev./min. The fractions corresponding to the 8o-S ribosomes and subunits were collected and pelleted by centrifugation for 3 h at IOO ooo ×g. The pellet was re-suspended, incubated with and without pronase, and centrifuged in a sucrose density gradient for 16 h at 22 5o0 rev./min. , without pronase; - • -, with pronase.
Biochim. Biophys, Acta, 228
(1971) 719-727
722
1~. VELEZet al.
(Fig. I); except for some decrease in resolution of the peaks. In contrast, there was a decrease in the peak of 8o-S ribosomes. Centrifugation for a longer period of time demonstrated that a portion of the single ribosomes had been dissociated into material which sediments as 6o-S and 4o-S subunits (Fig. 2). Since the polyribosomes are resistant to these proteolytic conditions, the 8o-S ribosomes that are not dissociated are presumed to be those that are attached to m R N A (type 3). These observations confirm previous studies with reticulocytes 1,2 and sea-urchin eggs 14. I t is important to emphasize that these are mild low temperature conditions, as more vigorous and higher temperature proteolysis will also degrade polyribosomes 1. To determine if 8o-S ribosomes not attached to m R N A but still capable of initiating hemoglobin synthesis (type I) are dissociated into ribosomal subunits, b y these conditions, 8o-S ribosomes were obtained b y incubating intact reticulocytes with inhibitors of initiation, either 2,2'-bipyridine or n-butanol. Cells made iron deficient with the iron chelating agent, 2,2'-bipyridine, have been shown to be lacking in the initiation factor heme s which is necessary to prevent the formation of an inhibitor of globin chain initiation 5,~1. Butanol appears to inhibit initiation b y disorienting a lipid-protein complex necessary for the attachmePt of ribosomes to m R N A 7. With both of these methods of inhibiting initiation, translation is unaffected. The ribosomes already attached to m R N A are released after completing translation. Since they cannot re-initiate, the result is a disaggregation of the polyribosomes to form single ribosomes not attached to mRNA. There is no increase in ribosomal subunits 15. Both of these methods of obtaining single ribosomes are easily reversible in the intact cell, b y supplying iron-transferrin or heme to the bipyridine-treated cells 5, and by diluting the alcohol in the butanol-treated ones ~. Ribosomal re-attachment to m R N A following the removal of these inhibitors was prevented in these experiments b y maintaining the temperature at o °. When the 8o-S ribosomes obtained by these methods were exposed to pronase, there was complete dissociation into 6o-S and 4o-S subunits (Fig. 3). These ribosomes are fully capable of attaching to m R N A if the appropriate initiation conditions are present. The observation that they totally dissociate into subunits, therefore, suggests that ribosome damage is not required for susceptibility to mild proteolysis. Presumably, however, damaged ribosomes are also dissociated as no discernible peak of 8o-S ribosomes remained. I t is possible, however, the bipyridine or butanol treatments of the ribosomes alter their susceptibility to pronase without affecting their protein synthetic mechanism. The following experiment was performed to clarify this point. Intact cells previously treated with bipyridine or butanol were washed free of inhibitors and incubated for 30 min in the complete medium. Polyribosomes from such cells were found to be resistant to these proteolytic conditions. These results further indicate that susceptibility to proteolysis induced b y bipyridine or butanol is related to nona t t a c h m e n t to mRNA. Tryptophan deficiency results in a partial disaggregation of polyribosomes to form smaller aggregates and an increased amount of single ribosomes 8. Since tryptophan is found only at the amino-terminal end of both globin chains 1~A3, a deficiency of this amino acid results in a delay of translation at the 5'-nucleotide end of m R N ~. Ribosomes that complete translation are released and are unable to reinitiate protein synthesis, as ribosomes delayed in translation already are attached to m R N A proximal Biochim. Biophys. Acta, 228 (1971) 719-727
PROTEOLYTIC DISSOCIATION OF SINGLE RIBOSOMES
723
to the limiting codon. Therefore, one should find b o t h 8o-S ribosomes a t t a c h e d (type 3) a n d n o t a t t a c h e d (type I) to m R N A , as is t h e case of c o n t r o l cells (Fig. 2). This deficiency s t at e is reversible b y a d d i n g t r y p t o p h a n t o t h e i n c u b a t i o n m e d i u m s. I n a dd i t i o n , in t r y p t o p h a n - d e f i c i e n t cells, t h e r e is a slight increase in r i b o s o m a l subunits as c o m p a r e d to cells i n c u b a t e d in t h e c o m p l e t e m e d i u m , or w i t h b i p y r i d i n e or b u t a n o l 15. W i t h t r y p t o p h a n deficiency, t h e i n i t i a t i o n step itself is n o t affected, b u t it c a n n o t be expressed because of a m e c h a n i c a l block.
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Fig. 3- Effect of pronase on 8o-S ribosomes derived from iron-deficient cells. Polyribosomes were disaggregated to single ribosomes not attached to mlRNA by incubating intact cells with 2,2'-bipyridine as described in MATERIALSAND METHODS. The ribosomes were centrifuged in a sucrose density gradient, collected and pelleted as described in Fig. 2. The re-suspended pellet was incubated with and without pronase and centrifuged in a sucrose density gradient for 16 h at 22 50o rev./min. , without pronase; - • -, with pronase. Identical results were obtained with the single ribosomes derived from butanol-treated cells; all of the 8o-S ribosomes were dissociated into 6o-S and 4o-S subunits. Fig. 4. Effect of pronase on 8o-S ribosomes derived from tryptophan-deficient cells. These 8o-S ribosomes were isolated, centrifuged on a sucrose density gradient, collected and pelleted as described in Fig. 2. The pellet was re-suspended, incubated with and without pronase, and centrifuged in a sucrose density gradient for 16 h at 22 5o0 rev./min. , without pronase; . . . . . with pronase. W h e n t h e ribosomes f r o m t r y p t o p h a n - d e f i c i e n t reticulocytes were ex p o sed to these m i l d p r o t e o l y t i c conditions, t h e m a j o r i t y of t h e 80-S ribosomes dissocia t e d into subunits. H o w e v e r , a s m a ll p e a k of single ribosomes r em ai n ed , as was pred i c t e d (Fig. 4). This is a d d i t i o n a l e v i d e n c e t h a t these p r o t e o l y t i c conditions different i a t e b e t w e e n ribosomes a t t a c h e d a n d n o t a t t a c h e d to m R N A . F u r t h e r m o r e , i t supports t he hypothesis t h a t it is n o n - a t t a c h m e n t to m R N A which d e t e r m i n e s susceptibility to pronase r a t h e r t h a n t h e site where i n i t i a t i o n is inhibited. W h e n the t r y p t o p h a n deficiency was reversed b y th e a d d i t i o n of this a m i n o acid, t h e polyribosomes formed b y r e - a t t a c h m e u t of the ribosomes to m R N A were once again r esi st an t to pronase. I d e n t i c a l results were o b t a i n e d in all cases if either t h e isolated 80-S ribosome or t h e entire r i b o s o m e - p o l y r i b o s o m e c o m p o n e n t was exposed to pronase.
Resistance to proteolysis by polyribosomes not carrying nascent chains All t h e p o l y r ib o s o m e s described a b o v e h a v e carried n ascen t globin chains, as m e a s u r e d b y i n c o r p o r a t i o n of 14C-labelled a m i n o acids. It is possible, therefore, t h a t
Biochim. Biophys. Acta, 228 (1971) 719-727
R. VELEZet al.
724
the resistance to proteolysis is due to a stabilizing effect of the nascent chains. To test this, cells were incubated with 2 mM puromycin. This concentration of puromycin has been shown by WILLIAMSONAND S C H W E E T ~, and by one of us ~ to release all of the partially completed peptide chains from the ribosomes. The ribosome-polyribosome component derived from the puromycin-treated ceils was then exposed to pronase. The pol.vribosomes, without nascent chains, were still resistant to these proteolytic conditions (Fig. 5) while the single ribosomes were dissociated into subunits. The absence of nascent chains, as measured b y absence of incorporation of radioactive amino acids into the polyribosome fraction, was re-confirmed. In addition, there was some disaggregation of the polyribosomes to single ribosomes as previously describedU. The isolated polyribosome component, separated from the single ribosomes, was also exposed to pronase. These polyribosomes were likewise resistant to proteolysis. It appears, therefore, that it is attachment to mRMA rather than the presence of nascent chains that is responsible for resistance to proteolysis.
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F i g . 5- E f f e c t of p r o n a s e o n p o l y r i b o s o m e s d e r i v e d f r o m c e l l s w h e r e a l l n a s c e n t c h a i n s h a v e been removed by puromycin. The isolated ribosome-polyribosome component was re-suspended a n d i n c u b a t e d w i t h a n d w i t h o u t p r o n a s e . C e n t r i f u g a t i o n w a s for 3 h a t 25 ooo r e v . / m i n . - w i t h o u t p r o n a s e ; - - -, w i t h p r o n a s e . F i g . 6. D i s a g g r e g a t i o n of p o l y r i b o s o m e s t o s i n g l e r i b o s o m e s i n a cell-free s y s t e m w h e r e i n i t i a t i o n d o e s n o t o c c u r . C e l l - f r e e i n c u b a t i o n w a s c a r r i e d o u t a s d e s c r i b e d i n MKTERI&LS AND METHODS. T h e r i b o s o m e s w e r e i s o l a t e d b y c e u t r i f u g a t i o n of t h e cell-free i n c u b a t i o n m e d i u m a t i o o ooo × g t h r o u g h a c u s h i o n of 3 ° % s u c r o s e . T h e p e l l e t s w e r e r e - s u s p e n d e d a n d c e n t r i f u g e d i n a s u c r o s e d e n s i t y g r a d i e n t for 5 h a t 25 ooo r e v . / m i n . , i n c u b a t e d o n ice w i t h o u t a n e n e r g y s o u r c e ; - - -, i n c u b a t e d for 90 m i n a t 34 ° w i t h a n e n e r g y s o u r c e .
Proteolytic dissociation o/single ribosomes after cell-[ree incubation A small amount of the single ribosomes derived from control cells (Fig. 2) and tryptophan-deficient cells (Fig. 4) were resistant to proteolytic dissociation into subunits. Presumably, these are the single ribosomes attached to mRNA. If this is true, then after release of these ribosomes from mRNA, they should become susceptible to proteolysis. This was tested by allowing natural termination of globin chain synthesis in a cell-free system where initiation does not occur. After a 9o-min cell-free incubation at 34 °, the polyribosomes in control cell lysates completely disaggregated into single ribosomes (Fig. 6). Control lysates kept in the same medium, but without an energy source and on ice (Fig. 6) gave ultraviolet absorption profiles identical to those control ribosomes isolated from intact cells; they still contained a portion of 8o-S ribosomes that were resistant to proteolysis (Fig. 7). When the single ribosomes formed after the cell-free incubation at 34 ° were Biochim. Biophys. Acta, 228 (1971) 7 1 9 - 7 2 7
725
PROTEOLYTIC DISSOCIATION OF SINGLE RIBOSOMES
exposed to prouase, however, there was total dissociation into subunits (Fig. 7). A ribosome-containing lysate isolated from tryptophan-deficient cells was also incubated under these cell-free conditions. After the incubation at 34 ° there was total disaggregation of the polyaibosomes to single ribosomes, as described with the control cell lysates. The single ribosomes t h a t had been kept on ice without an energy source contained 8o-S ribosomes both resistant and susceptible to proteolysis (Fig. 8). The lysates incubated at 34 °, however, contained only susceptible single ribosomes. These studies demonstrate t h a t ribosomes resistant to proteolysis will become susceptible after release from attachment to mRNA. 0.25
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F i g . 7. P r o n a s e d i s s o c i a t i o n of 8 o - S r i b o s o m e s d e r i v e d f r o m c e l l - f r e e i n c u b a t i o n of l y s a t e s f r o m c o n t r o l cells. R i b o s o m e s w e r e i s o l a t e d b y c e n t r i f u g a t i o n of t h e c e l l - f r e e i n c u b a t i o n m e d i u m at ioo ooo ×g for 3 h through a cushion of 3° % sucrose. The pellets were re-suspended, incubated w i t h p r o n a s e , a n d c e n t r i f u g e d i n a s u c r o s e d e n s i t y g r a d i e n t f o r 16 h a t 22 o o o r e v . / m i n . - p r o n a s e t r e a t m e n t of r i b o s o m e s f r o m i n c u b a t i o n o n ice w i t h o u t a n e n e r g y s o u r c e ; - - -, p r o n a s e t r e a t m e n t of r i b o s o m e s f r o m i n c u b a t i o n f o r 9 0 r a i n a t 3 4 ° w i t h a n e n e r g y s o u r c e . F i g . 8. P r o n a s e d i s s o c i a t i o n o f 8 o - S r i b o s o m e s d e r i v e d f r o m c e l l - f r e e i n c u b a t i o n o f t r y p t o p h a n - d e f i c i e n t cells. R i b o s o m e s w e r e i s o l a t e d , r e - s u s p e n d e d , i n c u b a t e d w i t h c e n t r i f u g e d i n a s u c r o s e d e n s i t y g r a d i e n t a s d e s c r i b e d i n F i g . 7. - - , pronase r i b o s o m e s f r o m i n c u b a t i o n o n ice w i t h o u t a n e n e r g y s o u r c e ; - - -, p r o n a s e t r e a t m e n t from incubation for 90 min at 34 ° with an energy source.
lysates from pronase and treatment of of r i b o s o m e s
DISCUSSION
The experiments reported here confirm the hypothesis that mild low temperature proteolysis with pronase selectively dissociates rabbit reticulocyte ribosomes not attached to m R N A into ribosomal 6o-S and 4o-S subunits 1. Ribosomes attached to m R N A are resistant to this action whether or not they carry nascent globin chains. This technique m a y prove useful as a means of studying the initiation process of protein synthesis. A potential advantage of this method is that an early phase of initiation m a y be detected without having to rely upon translation and the formation of polyribosomes. These results with pronase are similar to those reported b y other investigators where high ionic strength buffers were found to dissociate selectively ribosomes not attached to m R N A ls-19. The ribosomes used were derived from cells other t h a n reticulocytes. I t appears that attachment to m R N A protects ribosomes against dissociation b y various methods. MARTIN AND HARTWELLle a]so found that the addition of yeast tRNA, supernatant, and polyuridylic acid to the ribosomal subunits derived from a temperaturesensitive yeast m u t a n t stabilized the resultant single ribosomes against subsequent Biochim. Biophys. Acta, 2 2 8 ( I 9 7 I) 7 1 9 - 7 2 7
726
R. VELEZ el al.
dissociation with KC1. As the authors p o i n t e d out, t h e y could not exclude t h a t the stabilization m i g h t be a result of small polypeptide formation. Our experiments with p u r o m y c i n indicate t h a t at least in the dissociation of r a b b i t reticulocyte ribosomes with pronase, n a s c e n t chains are not necessary. P u r o m y c i n seems to work b y competing with a m i n o a c y l - t R N A for peptide s y n t h e t a s e at the ribosomal enzyme site ~°-~ This appears to be also true in the i n t a c t r a b b i t reticulocyte, as p u r o m y c i n was shown to be specific for ribosomal sites bearing p e p t i d y l - t R N A ~. There is evidence, however, t h a t a m i n o a c y l - t R N A is still b o u n d to m R N A at the c o m p l e m e n t a r y ribosomal site "2,~s even t h o u g h p u r o m y c i n displaced it from the e n z y m e site. A d d i t i o n a l studies, therefore, are in progress to determine if t R N A a n d small a m o u n t s of s u p e r n a t a n t are necessary in the proteolytic dissociation of reticulocyte ribosomes. Recently, i n i t i a t i o n of globin chain synthesis in r a b b i t reticulocytes has been shown to require a n i n i t i a t i o n complex of 4o-S ribosomal s u b u n i t a t t a c h e d to m R N A 25, i n i t i a t i o n factors in'~7, a n d a p a r t i c u l a r m e t h i o n y l - t R N A~. One of the r e q u i r e m e n t s for the i n i t i a t i o n of globin chain synthesis t h e n is the dissociation of single ribosomes n o t a t t a c h e d to m R N A into ribosomal subunits. Indeed, a dissociation factor has been isolated from bacteria ~9,3° as well as r a b b i t reticulocyte ribosomes 31. Of interest is t h a t this factor is o b t a i n e d b y exposing ribosomes to high ionic s t r e n g t h salt solutions. I t is conceivable, therefore, t h a t the high ionic s t r e n g t h buffer dissociation of ribosomes 16-x9 is due to liberation of this dissociation factor. Similarly, the proteolytic dissociation t h a t we observed could be indirect, either b y liberating or a c t i v a t i n g dissociation factor. A n o t h e r possibility is t h a t the dissociation factor itself is a proteolytic enzyme. These possibilities are c u r r e n t l y being investigated.
ACKNOWLEDGEMENTS
This i n v e s t i g a t i o n was supported b y Public H e a l t h Service Research G r a n t No. AM 13532-Ol from the N a t i o n a l I n s t i t u t e of Arthritis a n d Metabolic Diseases. Michael L. F r e e d m a n is a Senior I n v e s t i g a t o r of the New York H e a r t Association.
REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17
L. I. 1VIALKINAND A. RICH, J. Mol. Biol., 26 (1967) 1329. M. L. FREEDMAN,M. HORI AND M. RABINOVITZ,Federation Proc., 27 (1968) 387. P- O. P. Tso AND J. VINOGRAD,Biochim. Biophys. Acta, 49 (1961) 113. L. LUZZATTO,J. ]BANKSAND P. A. MARKS,Biochim. Biophys. Acta, lO8 (I965) 434. M. RABINOVITZ,Yr. L. FREEDMAN, J. M. FISI~ER AND C. R. MAXWELL, Cold Spring Harbo~ Syrup. Quant. Biol., 34 (1969) 567. H. S. WAXMAN,IV[.L. FREEDMANANDM. RABINOVITZ,Biochim. Biophys. Acta, 145 (1967) 353 M. L. FREEDMAN,M. HORI A~D M. RABINOVITZ, Science 157 (1967) 323• M. HORI, J. M. FISHER AND M. RABINOVITZ,Science, 155 (1967) 83. A. R. WILLIAMSONAND R. SCHWEET,Nature, 202 (1965) 435. S. D. ADAMSON,E. HERBERTANDW. GODCHAUX,III, Arch. Biochem. Biophys., 125 (1968) 671 C. R. MAXWELLAND M. RABINOVITZ,Biochem. Biophys. Res. Commun., 35 (1969) 79. G. VON EHRENSTEIN, Cold Spring Harbor Syrup. Quant. Biol., 31 (1966) 705 . G. BRAUNITZER,J. S. BEST, U. FI.AMMAND B. SCHRANK, Z. Physiol. Chem., 347 (1966) 2o7. J. I~IATIGORSKY,Biochim. Biophys. Acla, 166 (1968) 142. M. L. FREEDMANAND M. RABINOVlTZ,Exptl. Cell Res., 60 (197o) 48o. T. E. MARTINAND I. G. WOOL, J. Mol. Biol., 43 (1969) 151. T. E. MARTIN,F. S. ROLLESTON,R. B. Low AND I. G. WOOL, J. Mol. Biol., 43 (1969) 135.
Biochim. Biophys. Acta, 228 (1971) 719-727
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22 23 24 25 26 27 28 29 3° 31
727
T. E. MARTIN AND L. H. HARTWELL, J. Biol. Chem., 245 (197 o) 15o 4. E. A. ZYLBER AND S. PENMAN, Biochim. Biophys. Acta, 2o 4 (197 o) 221. D. ~r. ALLEN AND P. C. ZAMECNIK, Biochim. Biophys. Acta, 55 (1962) 865. A. MORRIS, R. ARLINGIIAUS, S. FAVELUKES AND R. SCHWEET, Biochemistry, 2 (1963) lO84. F. O. WEITSTEIN AND H. NOLLS, J. Mol. Biol., I I (1965) 35. C. COURTSOGEORGOPOULOS, Biochemistry, 6 (1967) 17o 4. M. L. FREEDMAN, J. M, FISHER AND M. RABINOVITZ, J. Mol. Biol., 33 (1968) 315 . W . HOERZ AND K. S. MCCARTY, Proc. Natl. Acad. Sci. U.S., 63 (1969) 12o6. B. B. COHEN, Biochem. J., 115 (1969) 523 • P. M. PRICHARD, J. M. GILBERT, D. A. SHAFRITZ AND W. F. ANDERSON, Nature, (197 o) 511. D. B. WILSON, P r e s e n t e d a t 8th Intern. Congr. Biochem., Montreux, z97o, Abstr. (197 o) 196. A. R. SUBRAMANIAN, E. Z. RON AND B. D. DAVIS, Proc. Natl. Acad. Sci. U.S., 61 (r968) 761. E. G. BADE, N. S. GONZALEZ AND I. D. ALGRANATI, Proc. Natl. Acad. Sci. U. S., 64 (1969) 654. G. FAVELUKES, D. SORGENTINI, E. BARD AND C. MARTONE, P r e s e n t e d a t 8th Intern. Congr. Biochem., Montreux, z97o, Abstr. (197o).
Biochim. Biophys. Acta, 228 (1971) 719-727
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96745
SELECTIVE PROTEOLYTIC DISSOCIATION OF R A B B I T RETICULOCYTE SINGLE RIBOSOMES NOT ATTACHED TO MESSENGER RNA R A M O N V E L E Z * , N A N C Y L. F A R R E L L
AND M I C H A E L L. F R E E D M A N
Department o/ Medicine, New York University School o/ Medicine, New York, N . Y . zooi6 (U.S.A .) (Received O c t o b e r 26th, 197 o)
SUMMARY
Free and mRNA-bound ribosomes differ markedly in their susceptibility to mild proteolysis. Free ribosomes are dissociated into 6o-S and 4o-S subunits, while mRNA-bound ribosomes are completely protected. This protection can be reversed b y removing ribosomes from mRNA, and can be re-instated by allowing them to attach again. The resistance to proteolysis does not appear to be dependent upon the presence of nascent chains on the ribosome. This method has practical value as a means of determining if ribosomes have attached to mRNA and initiated globin chain synthesis.
INTRODUCTION
When the ribosomal component of rabbit reticulocytes is exposed to mild low temperature proteolytic conditions 1,3, a portion of the single 8o-S ribosomes dissociates into 6o-S and 4o-S subunits**. The polyribosomes under these same conditions are resistant and remain intact 1,~. On the basis of these observations, MALKIN AND RICH1 postulated that only those ribosomes not attached to mRNA are susceptible to nfild proteolysis. If this is the case, then mild proteolysis would be a valid method of differentiating between ribosomes attached and not attached to mRNA. However, there are at least three types of 8o-S ribosomes found in rabbit reticulocytes4: (I) those not attached to mRNA at the time of isolation, but still capable of associating with mRNA and initiating hemoglobin synthesis; (2) those not attached to mRNA and incapable of initiating hemoglobin synthesis, possibly as a result of damage to the ribosome in the maturation process; and (3)those attached to mRNA and synthesizing hemoglobin. An alternative explanation of the observed selective proteolysis, therefore, is that only damaged 8o-S ribosomes (type 2) are susceptible. The present study was undertaken in an attempt to evaluate the two alternative hypotheses. Our results favor the MALKIN AND RICH hypothesis, and indicate that mild proteolysis can in fact be used to distinguish free from mRNA-bound ribosomes. * P r e s e n t a d d r e s s : D e p a r t m e n t of Medicine, C o l u m b i a U n i v e r s i t y , College of P h y s i c i a n s a n d Surgeons, N e w York, N.Y., U.S.A. -- T h e d e s i g n a t i o n s 80 S, 60 S a n d 4 ° S are a d o p t e d a c c o r d i n g to t h e c o n v e n t i o n p r o p o s e d b y T s o AND VINOGRAD 8 to r e p r e s e n t t h e single ribosome, a n d t h e larger a n d s m a l l e r m a m m a l i a n r i b o s o m a l s u b u n i t s , respectively. T h e a c t u a l s e d i m e n t a t i o n coefficients were n o t d e t e r m i n e d .
Biochim. Biophys. Acta, 228 (1971) 719-727
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R. VELEZ et al.
MATERIALSAND METHODS
Incubation o/intact cells and [ormation o] single ribosomes Reticulocyte-rich blood was collected in heparin by cardiac puncture of phenylhydrazine-treated rabbits. The cells were washed with an isotonic salt solution and incubated as previously described 5. The incubation was carried out for 30 rain at 37 °. The total volume of the incubation medium was 8.0 ml in a 25-ml erlenmeyer flask, with each flask containing I.O ml of reticulocytes. The incubation was stopped by placing the flask in ice. The cells were centrifuged at 600 × g and the supernatant fluid removed. The cells were washed, lysed, and the coarse particulate fraction was removed as previously described 5. Polyribosomes were disaggregated to form single ribosomes by incubating intact cells with 2. lO .-4 M 2,2'-bipyridine 6 or o.I M n-butanoF, or by the omission of tryptophan from the incubation medium s. When 2,2'-bipyridine was used, iron and transferrin were omitted.
Formation o[ polyribosomes not carrying nascenl chains Polvribosomes derived from intact cells carry nascent globin chains, while most single ribosomes do not 5. Puromycin, when added to intact cells in a final concentration of 2 mM, causes the premature release of all of the growing peptide chains from the ribosome 5,6,9. Polyribosomes not carrying nascent chains were formed, therefore, by incubating intact cells in the complete medium for ten minutes and then with puromycin (2 raM) for an additional IO rain at 37 °.
Cell-/ree incubation and ]ormation o] single ribosomes Ribosomes not attached to m R N A were formed in a cell-free system by allowing natural termination of globin synthesis in the absence of heme. Since heme is necessary for initiation, the result is a disaggregation of polyribosomes to form single ribosomes not attached to m R N A 5,1°,n. The cell-free system utilized was essentially t h a t previously described 5. Washed reticulocytes were lysed with an equal volume of water and the lysate cleared by centrifugation for 15 min at 25 ooo ×g. The medium contained o.15 ml of a Tris-NaC1 buffer, 0.90 ml of the cleared lysate, and 0.60 ml of a master mix, added in that order. The Tris-NaC1 buffer was prepared by adding I vol. of o.I M NaOH, 0.2 vol. of i M Tris, pH 7.4, and I vol. of o.I M HC1. The master mix contained the following ingredients in amounts to yield the individual final concentrations: KC1 (75 raM), MgC12 (2 raM), mercaptoethanol (6 raM), the 19 anfino acids reported present in rabbit hemoglobin 1~,13 in I/IO the concentration used for intact cell incubations s. ATP (1 raM), GTP (0.2 mM), creatine phosphate (15 mM), and creatine kinase (45 enzyme units/ml) were added as the energy source. The reticulocytes used in these cell-free studies were incubated first as intact cells either without tryptophan or in the complete medium as discussed above. After the cell-free incubation for 9° min at 34 °, the ribosomes were isolated and treated in the same fashion as those derived from intact cells.
Isolation and proteolytie treatment o] the ribosome-polyribosome component Ribosomes were removed from the large bulk of supernatant hemoglobin by centrifugation of the lysate at ioo ooo × g at 4 ° for 3 h over a cushion of 30 % sucrose.
Biochim. Biophys. Acta, 228 (1971) 719-727
PROTEOLYTIC DISSOCIA'IION OF SINGLE RIBOSOMES
721
T h e resulting a d h e r e n t pellet was rinsed g e n t l y w i t h s t a n d a r d buffer (o.oi M Tris, p H 7.4, o . o i M KC1 a n d o.oo15 M MgC12) a n d g e n t l y r e s u s p e n d e d in i . o ml of t h e same buffer. T h e res u s p en d e d ribosomes (2.5 rag) were m i x e d w i t h o.5 m g of pronase at a final v o l u m e of I.O m l at o ° for I h. T h e c o n c e n t r a t i o n of ribosomes was d e t e r m i n e d at 260 m/z, using an e x t i n c t i o n coefficient of 12 a b s o r b a n c e u n i t s / m g per ml ~. A f t e r proteolysis, t h e solution was centrifuged at 600 x g for IO m i n a n d a n y pellet was discarded.
Analysis o/ the ribosome-polyribosome component T h e r i b o s o m a l suspension was l a y e r e d on 36 m l of a 15-3o % (w/w) linear sucrose g r a d i e n t in s t a n d a r d buffer. A f t e r c e n t r i f u g a t i o n in a Spinco S W 27 swinging b u c k e t r o t o r at 4 ° for speeds a n d t im e s shown w i t h i n d i v i d u a l e x p e r i m e n t s , t h e grad i e n t was p u m p e d t h r o u g h the f l o w - t h r o u g h cell of B e c k m a n K i n t r a c V I I spectrop h o t o m e t e r to measure absorbance at 260 In#. In some e x p e r i m e n t s , t h e r i b o s o m a l suspension or t h e entire cell-free i n c u b a t i o n m e d i u m was placed d i r e c t l y on a sucrose g r a d i e n t , before p r o t e o l y t i c t r e a t m e n t , a n d c e n t ri fu g ed for 3 h at 25 ooo r e v . / m i n at 4 ° in t h e S W 27 rotor. Th e g r a d i e n t was analyzed, a n d t h e single ribosomes were s e p a r a t e d f r o m t h e polyribosomes. E a c h comp o n e n t was pelleted b y c e n t r i f u g a t i o n at IOO ooo x g at 4 ° for 3 h, a n d resuspended in s t a n d a r d buffer. T h e s e p a r a t e d 8o-S ribosomes an d polyribosomes were t h e n t r e a t e d w i t h pronase. Pronase, B grade, was o b t a i n e d f r o m t h e Calbiochemical Co. RESULTS
Proteolytic dissociation o~ single ribosomes/rom intact cells W h e n t h e r i b o s o m e - p o l y r i b o s o m e c o m p o n e n t isolated f r o m i n t a c t r a b b i t re t i cu l o cy t es was exposed at o ° to pronase for I h, t h e polyribosomes were n o t al t er ed
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Fig. i. Sucrose density gradient analysis of the ribosome-polyribosome component of rabbit reticulocytes exposed to mild proteolysis. The isolated ribosome-polyribosome component, derived from intact cells incubated in the complete medium, was re-suspended and incubated with and without pronase, as described in MATERIALSAND METHODS. Centrifugation in a 15-3 ° °/o sucrose density gradient was for 3 h at 25 ooo rev./min. , without pronase; - .-, with pronase. Fig. 2. Effect of pronase on 8o-S ribosomes derived from cells incubated in the complete medium. The isolated ribosome-polyribosome component was re-suspended and centrifuged in a 15-3o % sucrose density gradient for 3 h at 25 ooo rev./min. The fractions corresponding to the 8o-S ribosomes and subunits were collected and pelleted by centrifugation for 3 h at IOO ooo ×g. The pellet was re-suspended, incubated with and without pronase, and centrifuged in a sucrose density gradient for 16 h at 22 5o0 rev./min. , without pronase; - • -, with pronase.
Biochim. Biophys, Acta, 228
(1971) 719-727
722
1~. VELEZet al.
(Fig. I); except for some decrease in resolution of the peaks. In contrast, there was a decrease in the peak of 8o-S ribosomes. Centrifugation for a longer period of time demonstrated that a portion of the single ribosomes had been dissociated into material which sediments as 6o-S and 4o-S subunits (Fig. 2). Since the polyribosomes are resistant to these proteolytic conditions, the 8o-S ribosomes that are not dissociated are presumed to be those that are attached to m R N A (type 3). These observations confirm previous studies with reticulocytes 1,2 and sea-urchin eggs 14. I t is important to emphasize that these are mild low temperature conditions, as more vigorous and higher temperature proteolysis will also degrade polyribosomes 1. To determine if 8o-S ribosomes not attached to m R N A but still capable of initiating hemoglobin synthesis (type I) are dissociated into ribosomal subunits, b y these conditions, 8o-S ribosomes were obtained b y incubating intact reticulocytes with inhibitors of initiation, either 2,2'-bipyridine or n-butanol. Cells made iron deficient with the iron chelating agent, 2,2'-bipyridine, have been shown to be lacking in the initiation factor heme s which is necessary to prevent the formation of an inhibitor of globin chain initiation 5,~1. Butanol appears to inhibit initiation b y disorienting a lipid-protein complex necessary for the attachmePt of ribosomes to m R N A 7. With both of these methods of inhibiting initiation, translation is unaffected. The ribosomes already attached to m R N A are released after completing translation. Since they cannot re-initiate, the result is a disaggregation of the polyribosomes to form single ribosomes not attached to mRNA. There is no increase in ribosomal subunits 15. Both of these methods of obtaining single ribosomes are easily reversible in the intact cell, b y supplying iron-transferrin or heme to the bipyridine-treated cells 5, and by diluting the alcohol in the butanol-treated ones ~. Ribosomal re-attachment to m R N A following the removal of these inhibitors was prevented in these experiments b y maintaining the temperature at o °. When the 8o-S ribosomes obtained by these methods were exposed to pronase, there was complete dissociation into 6o-S and 4o-S subunits (Fig. 3). These ribosomes are fully capable of attaching to m R N A if the appropriate initiation conditions are present. The observation that they totally dissociate into subunits, therefore, suggests that ribosome damage is not required for susceptibility to mild proteolysis. Presumably, however, damaged ribosomes are also dissociated as no discernible peak of 8o-S ribosomes remained. I t is possible, however, the bipyridine or butanol treatments of the ribosomes alter their susceptibility to pronase without affecting their protein synthetic mechanism. The following experiment was performed to clarify this point. Intact cells previously treated with bipyridine or butanol were washed free of inhibitors and incubated for 30 min in the complete medium. Polyribosomes from such cells were found to be resistant to these proteolytic conditions. These results further indicate that susceptibility to proteolysis induced b y bipyridine or butanol is related to nona t t a c h m e n t to mRNA. Tryptophan deficiency results in a partial disaggregation of polyribosomes to form smaller aggregates and an increased amount of single ribosomes 8. Since tryptophan is found only at the amino-terminal end of both globin chains 1~A3, a deficiency of this amino acid results in a delay of translation at the 5'-nucleotide end of m R N ~. Ribosomes that complete translation are released and are unable to reinitiate protein synthesis, as ribosomes delayed in translation already are attached to m R N A proximal Biochim. Biophys. Acta, 228 (1971) 719-727
PROTEOLYTIC DISSOCIATION OF SINGLE RIBOSOMES
723
to the limiting codon. Therefore, one should find b o t h 8o-S ribosomes a t t a c h e d (type 3) a n d n o t a t t a c h e d (type I) to m R N A , as is t h e case of c o n t r o l cells (Fig. 2). This deficiency s t at e is reversible b y a d d i n g t r y p t o p h a n t o t h e i n c u b a t i o n m e d i u m s. I n a dd i t i o n , in t r y p t o p h a n - d e f i c i e n t cells, t h e r e is a slight increase in r i b o s o m a l subunits as c o m p a r e d to cells i n c u b a t e d in t h e c o m p l e t e m e d i u m , or w i t h b i p y r i d i n e or b u t a n o l 15. W i t h t r y p t o p h a n deficiency, t h e i n i t i a t i o n step itself is n o t affected, b u t it c a n n o t be expressed because of a m e c h a n i c a l block.
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Fig. 3- Effect of pronase on 8o-S ribosomes derived from iron-deficient cells. Polyribosomes were disaggregated to single ribosomes not attached to mlRNA by incubating intact cells with 2,2'-bipyridine as described in MATERIALSAND METHODS. The ribosomes were centrifuged in a sucrose density gradient, collected and pelleted as described in Fig. 2. The re-suspended pellet was incubated with and without pronase and centrifuged in a sucrose density gradient for 16 h at 22 50o rev./min. , without pronase; - • -, with pronase. Identical results were obtained with the single ribosomes derived from butanol-treated cells; all of the 8o-S ribosomes were dissociated into 6o-S and 4o-S subunits. Fig. 4. Effect of pronase on 8o-S ribosomes derived from tryptophan-deficient cells. These 8o-S ribosomes were isolated, centrifuged on a sucrose density gradient, collected and pelleted as described in Fig. 2. The pellet was re-suspended, incubated with and without pronase, and centrifuged in a sucrose density gradient for 16 h at 22 5o0 rev./min. , without pronase; . . . . . with pronase. W h e n t h e ribosomes f r o m t r y p t o p h a n - d e f i c i e n t reticulocytes were ex p o sed to these m i l d p r o t e o l y t i c conditions, t h e m a j o r i t y of t h e 80-S ribosomes dissocia t e d into subunits. H o w e v e r , a s m a ll p e a k of single ribosomes r em ai n ed , as was pred i c t e d (Fig. 4). This is a d d i t i o n a l e v i d e n c e t h a t these p r o t e o l y t i c conditions different i a t e b e t w e e n ribosomes a t t a c h e d a n d n o t a t t a c h e d to m R N A . F u r t h e r m o r e , i t supports t he hypothesis t h a t it is n o n - a t t a c h m e n t to m R N A which d e t e r m i n e s susceptibility to pronase r a t h e r t h a n t h e site where i n i t i a t i o n is inhibited. W h e n the t r y p t o p h a n deficiency was reversed b y th e a d d i t i o n of this a m i n o acid, t h e polyribosomes formed b y r e - a t t a c h m e u t of the ribosomes to m R N A were once again r esi st an t to pronase. I d e n t i c a l results were o b t a i n e d in all cases if either t h e isolated 80-S ribosome or t h e entire r i b o s o m e - p o l y r i b o s o m e c o m p o n e n t was exposed to pronase.
Resistance to proteolysis by polyribosomes not carrying nascent chains All t h e p o l y r ib o s o m e s described a b o v e h a v e carried n ascen t globin chains, as m e a s u r e d b y i n c o r p o r a t i o n of 14C-labelled a m i n o acids. It is possible, therefore, t h a t
Biochim. Biophys. Acta, 228 (1971) 719-727
R. VELEZet al.
724
the resistance to proteolysis is due to a stabilizing effect of the nascent chains. To test this, cells were incubated with 2 mM puromycin. This concentration of puromycin has been shown by WILLIAMSONAND S C H W E E T ~, and by one of us ~ to release all of the partially completed peptide chains from the ribosomes. The ribosome-polyribosome component derived from the puromycin-treated ceils was then exposed to pronase. The pol.vribosomes, without nascent chains, were still resistant to these proteolytic conditions (Fig. 5) while the single ribosomes were dissociated into subunits. The absence of nascent chains, as measured b y absence of incorporation of radioactive amino acids into the polyribosome fraction, was re-confirmed. In addition, there was some disaggregation of the polyribosomes to single ribosomes as previously describedU. The isolated polyribosome component, separated from the single ribosomes, was also exposed to pronase. These polyribosomes were likewise resistant to proteolysis. It appears, therefore, that it is attachment to mRMA rather than the presence of nascent chains that is responsible for resistance to proteolysis.
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F i g . 5- E f f e c t of p r o n a s e o n p o l y r i b o s o m e s d e r i v e d f r o m c e l l s w h e r e a l l n a s c e n t c h a i n s h a v e been removed by puromycin. The isolated ribosome-polyribosome component was re-suspended a n d i n c u b a t e d w i t h a n d w i t h o u t p r o n a s e . C e n t r i f u g a t i o n w a s for 3 h a t 25 ooo r e v . / m i n . - w i t h o u t p r o n a s e ; - - -, w i t h p r o n a s e . F i g . 6. D i s a g g r e g a t i o n of p o l y r i b o s o m e s t o s i n g l e r i b o s o m e s i n a cell-free s y s t e m w h e r e i n i t i a t i o n d o e s n o t o c c u r . C e l l - f r e e i n c u b a t i o n w a s c a r r i e d o u t a s d e s c r i b e d i n MKTERI&LS AND METHODS. T h e r i b o s o m e s w e r e i s o l a t e d b y c e u t r i f u g a t i o n of t h e cell-free i n c u b a t i o n m e d i u m a t i o o ooo × g t h r o u g h a c u s h i o n of 3 ° % s u c r o s e . T h e p e l l e t s w e r e r e - s u s p e n d e d a n d c e n t r i f u g e d i n a s u c r o s e d e n s i t y g r a d i e n t for 5 h a t 25 ooo r e v . / m i n . , i n c u b a t e d o n ice w i t h o u t a n e n e r g y s o u r c e ; - - -, i n c u b a t e d for 90 m i n a t 34 ° w i t h a n e n e r g y s o u r c e .
Proteolytic dissociation o/single ribosomes after cell-[ree incubation A small amount of the single ribosomes derived from control cells (Fig. 2) and tryptophan-deficient cells (Fig. 4) were resistant to proteolytic dissociation into subunits. Presumably, these are the single ribosomes attached to mRNA. If this is true, then after release of these ribosomes from mRNA, they should become susceptible to proteolysis. This was tested by allowing natural termination of globin chain synthesis in a cell-free system where initiation does not occur. After a 9o-min cell-free incubation at 34 °, the polyribosomes in control cell lysates completely disaggregated into single ribosomes (Fig. 6). Control lysates kept in the same medium, but without an energy source and on ice (Fig. 6) gave ultraviolet absorption profiles identical to those control ribosomes isolated from intact cells; they still contained a portion of 8o-S ribosomes that were resistant to proteolysis (Fig. 7). When the single ribosomes formed after the cell-free incubation at 34 ° were Biochim. Biophys. Acta, 228 (1971) 7 1 9 - 7 2 7
725
PROTEOLYTIC DISSOCIATION OF SINGLE RIBOSOMES
exposed to prouase, however, there was total dissociation into subunits (Fig. 7). A ribosome-containing lysate isolated from tryptophan-deficient cells was also incubated under these cell-free conditions. After the incubation at 34 ° there was total disaggregation of the polyaibosomes to single ribosomes, as described with the control cell lysates. The single ribosomes t h a t had been kept on ice without an energy source contained 8o-S ribosomes both resistant and susceptible to proteolysis (Fig. 8). The lysates incubated at 34 °, however, contained only susceptible single ribosomes. These studies demonstrate t h a t ribosomes resistant to proteolysis will become susceptible after release from attachment to mRNA. 0.25
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1 2 3 4 5 6 7 8 DISTANCEFROMTOPOF TUBE(cm)
9
F i g . 7. P r o n a s e d i s s o c i a t i o n of 8 o - S r i b o s o m e s d e r i v e d f r o m c e l l - f r e e i n c u b a t i o n of l y s a t e s f r o m c o n t r o l cells. R i b o s o m e s w e r e i s o l a t e d b y c e n t r i f u g a t i o n of t h e c e l l - f r e e i n c u b a t i o n m e d i u m at ioo ooo ×g for 3 h through a cushion of 3° % sucrose. The pellets were re-suspended, incubated w i t h p r o n a s e , a n d c e n t r i f u g e d i n a s u c r o s e d e n s i t y g r a d i e n t f o r 16 h a t 22 o o o r e v . / m i n . - p r o n a s e t r e a t m e n t of r i b o s o m e s f r o m i n c u b a t i o n o n ice w i t h o u t a n e n e r g y s o u r c e ; - - -, p r o n a s e t r e a t m e n t of r i b o s o m e s f r o m i n c u b a t i o n f o r 9 0 r a i n a t 3 4 ° w i t h a n e n e r g y s o u r c e . F i g . 8. P r o n a s e d i s s o c i a t i o n o f 8 o - S r i b o s o m e s d e r i v e d f r o m c e l l - f r e e i n c u b a t i o n o f t r y p t o p h a n - d e f i c i e n t cells. R i b o s o m e s w e r e i s o l a t e d , r e - s u s p e n d e d , i n c u b a t e d w i t h c e n t r i f u g e d i n a s u c r o s e d e n s i t y g r a d i e n t a s d e s c r i b e d i n F i g . 7. - - , pronase r i b o s o m e s f r o m i n c u b a t i o n o n ice w i t h o u t a n e n e r g y s o u r c e ; - - -, p r o n a s e t r e a t m e n t from incubation for 90 min at 34 ° with an energy source.
lysates from pronase and treatment of of r i b o s o m e s
DISCUSSION
The experiments reported here confirm the hypothesis that mild low temperature proteolysis with pronase selectively dissociates rabbit reticulocyte ribosomes not attached to m R N A into ribosomal 6o-S and 4o-S subunits 1. Ribosomes attached to m R N A are resistant to this action whether or not they carry nascent globin chains. This technique m a y prove useful as a means of studying the initiation process of protein synthesis. A potential advantage of this method is that an early phase of initiation m a y be detected without having to rely upon translation and the formation of polyribosomes. These results with pronase are similar to those reported b y other investigators where high ionic strength buffers were found to dissociate selectively ribosomes not attached to m R N A ls-19. The ribosomes used were derived from cells other t h a n reticulocytes. I t appears that attachment to m R N A protects ribosomes against dissociation b y various methods. MARTIN AND HARTWELLle a]so found that the addition of yeast tRNA, supernatant, and polyuridylic acid to the ribosomal subunits derived from a temperaturesensitive yeast m u t a n t stabilized the resultant single ribosomes against subsequent Biochim. Biophys. Acta, 2 2 8 ( I 9 7 I) 7 1 9 - 7 2 7
726
R. VELEZ el al.
dissociation with KC1. As the authors p o i n t e d out, t h e y could not exclude t h a t the stabilization m i g h t be a result of small polypeptide formation. Our experiments with p u r o m y c i n indicate t h a t at least in the dissociation of r a b b i t reticulocyte ribosomes with pronase, n a s c e n t chains are not necessary. P u r o m y c i n seems to work b y competing with a m i n o a c y l - t R N A for peptide s y n t h e t a s e at the ribosomal enzyme site ~°-~ This appears to be also true in the i n t a c t r a b b i t reticulocyte, as p u r o m y c i n was shown to be specific for ribosomal sites bearing p e p t i d y l - t R N A ~. There is evidence, however, t h a t a m i n o a c y l - t R N A is still b o u n d to m R N A at the c o m p l e m e n t a r y ribosomal site "2,~s even t h o u g h p u r o m y c i n displaced it from the e n z y m e site. A d d i t i o n a l studies, therefore, are in progress to determine if t R N A a n d small a m o u n t s of s u p e r n a t a n t are necessary in the proteolytic dissociation of reticulocyte ribosomes. Recently, i n i t i a t i o n of globin chain synthesis in r a b b i t reticulocytes has been shown to require a n i n i t i a t i o n complex of 4o-S ribosomal s u b u n i t a t t a c h e d to m R N A 25, i n i t i a t i o n factors in'~7, a n d a p a r t i c u l a r m e t h i o n y l - t R N A~. One of the r e q u i r e m e n t s for the i n i t i a t i o n of globin chain synthesis t h e n is the dissociation of single ribosomes n o t a t t a c h e d to m R N A into ribosomal subunits. Indeed, a dissociation factor has been isolated from bacteria ~9,3° as well as r a b b i t reticulocyte ribosomes 31. Of interest is t h a t this factor is o b t a i n e d b y exposing ribosomes to high ionic s t r e n g t h salt solutions. I t is conceivable, therefore, t h a t the high ionic s t r e n g t h buffer dissociation of ribosomes 16-x9 is due to liberation of this dissociation factor. Similarly, the proteolytic dissociation t h a t we observed could be indirect, either b y liberating or a c t i v a t i n g dissociation factor. A n o t h e r possibility is t h a t the dissociation factor itself is a proteolytic enzyme. These possibilities are c u r r e n t l y being investigated.
ACKNOWLEDGEMENTS
This i n v e s t i g a t i o n was supported b y Public H e a l t h Service Research G r a n t No. AM 13532-Ol from the N a t i o n a l I n s t i t u t e of Arthritis a n d Metabolic Diseases. Michael L. F r e e d m a n is a Senior I n v e s t i g a t o r of the New York H e a r t Association.
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