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Reversible disaggregation by NaF of membrane-bound polyribosomes of mouse myeloma cells in tissue culture
453
BIOCI-IIMICA ET BIOPHYSICA ACTA
BBA 97217
R E V E R S I B L E D I S A G G R E G A T I O N BY N a F OF MEMBRANE-BOUND POLYRIBOSOMES OF MOUSE MYELOMA CELLS IN T I S S U E C U L T U R E I. B L E I B ] E R G * , M. Z A U D E R E R
AND (7. B A G L I O N I
Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139 (U.S.A.) (Received J a n u a r y 7th, 1972)
SUMMARY
Approximately one-quarter of the ribosomes of P-3 mouse myeloma cells in tissue culture are bound to membranes of the endoplasmic reticulum. Incubation with N a F causes polyribosome disaggregation; at the same time approximately 25 % of the membrane-bound ribosomes are released from membranes. After reversing the inhibition of N a F free ribosomes become associated with membranes until approximately the same proportion of free and membrane-bound ribosomes found in untreated cells is reestablished. During this time these cells resume the normal synthesis and secretion of immunoglobulin. This takes place even when the synthesis of RNA is completely inhibited. These experiments have suggested that the association of a class of ribosomes with membranes is dependent on their entry into polyribosomes active in protein synthesis whereas attachment of the other ribosomes occurs independently of their participation in protein synthesis.
INTRODUCTION
Plasma cells are highly specialized cells devoted to the synthesis and secretion of immunoglobulins. Plasma cell tumors (myelomas) have been induced in BALB/c mice and cell lines from several tumours have been adapted to grow in tissue culture 1. These cells provide a unique material for the study of immunoglobulin synthesis, since almost every line secretes a unique immunoglobulin or an immunoglobulin chain 1. The synthesis of proteins secreted by mammalian cells occurs on ribosomes bound to the membranes of the endoplasmic reticulum ~. The study of membranebound ribosomes m a y thus provide information on the molecular events which lead to the localization of ribosomes and of a specific class of messenger RNAs, those coding for proteins secreted by the cell, in the endoplasmic reticulum. We have investigated the behaviour of membrane-bound ribosomes in the P-3 tissue culture line of mouse myeloma. Cells of the P-3 line secrete an IgG immunoglobulin; these cells have been shown to be remarkably stable under tissue culture conditions for an extended period of time 3. We have attempted to establish whether membrane-bound ribosomes can be reversibly released from membranes of the endoplasnfic reticulum in the presence of the inhibitor of protein synthesis N a F 4. This * P r e s e n t a d d r e s s : T e l - H a s h o m e r Hospital, R a m a t - G a n . Israel.
Biochim. Biophys. Acta, 269 (1972) 453-464
454
I. BLEIBERG et al.
compound causes polyribosome disaggregation and decreases the level of free ribosomal subunits in mammalian cells 5. The results obtained suggest that a portion of the membrane-bound ribosomes can recycle between the pool of free ribosomes and those associated with membranes, and that after the disaggregation and reassembly of polyribosomes at least a part of the specificity of membrane-associated protein synthesis is restored. Related studies in HeLa cells have recently been published by Rosbach and Penman 6.
MATERIALS AND METHODS
Cell culture Mouse myeloma cells of the P-3 line were obtained from Dr M. Cohn of the Salk Institute for Biological Research. The cells were grown in Dulbecco's medium (GIBCO, New York) supplemented with IO °/o horse serum. The cell line was propagated in petri dishes in a CO 2 incubator. Large quantities of cells were grown in roller bottles.
Cell incubation To uniformly label RNA or protein I nCi/ml of E14Cluridine or io nCi/ml of ~14CI proline, respectively, were added for 2o-24 h directly to growing cells. For all other incubations the cells were collected at a density of 3 " lO5-5 . lO5 ceils per ml, concentrated by centrifugation for 5 rain at 200 × g, resuspended in the same medium at a density of i • lOs cells per ml and incubated in a rotary shaker under 5 % CO~.
Cell/ractionation At the end of an incubation the cells were collected by centrifugation, resuspended in I ml of NaCI-Tris-MgC12 buffer (o.oi M NaC1, o.oi M Tris-HC1, pH 7.4, and 1. 5 mM MgC12) per 2 • lO7 cells and homogenized by 15 strokes of a loose fitting Dounce homogenizer. Nuclei were spun down in 5 rain at 400 ×g. A membrane fraction was prepared from the postnuclear supernatant by a 3o-min centrifugation at 80 o o o × g through a continuous 15-3o % sucrose gradient in NaC1-Tris-MgC12 buffer 6. In some cases this procedure was modified by substituting a I5 and 30 °/'o sucrose step gradient. Control studies showed this did not affect membrane recoveries. Membrane-bound polyribosomes were prepared by resuspending the membrane pellet in o.5-1 ml of NaC1 Tris-MgC12 buffer and adding the detergent sodium deoxycholate to a final concentration of 1 % . Free polyribosomes were prepared from the postnuclear supernatant by sedimenting mitochondria and membranes at 27 ooo × g for 5 min3.
Gradient analysis Polyribosomes were analyzed by sucrose density gradient centrifugation as indicated in the legends. The absorbance at 260 nm was monitored in a Gilford continuously recording spectrophotometer. The fractions collected were precipitated with 5 % trichloroacetic acid onto cellulose nitrate filters and counted in a liquid scintillation counter. RNA has been analyzed in sucrose gradients made up in o.oi M Tris-HC1, pH 7.4, o.I M NaC1, I mM ethylenediamine tetraacetic acid and o.5 0'o sodium dodecyl sulphate (sodium dodecyl sulphate buffer). Biochim. Biophys. Acta, 269 (1972) 453-464
MEMBRANE-BOUND
455
POLYRIBOSOME S
Immunoglobulin analysis The protein secreted b y P-3 cells has been analyzed by gel filtration on Sephadex G-Ioo. The protein was precipitated with (NH4),SO 4 between 30 and 55 % saturation, redissolved in o.I M NaC1, o.oi M Tris-HC1 buffer, p H 7.4, and applied to a 2.4 cm × 60 cm equilibrated with the same buffer. 2-ml fractions were collected and counted as described above. The protein secreted by P-3 cells was also identified b y immunoprecipitation with a rabbit antiserum prepared with purified P-3 immunoglobulin (a kind gift of Dr Donato Cioli and Dr David Schubert); the antigenantibody complexes were then precipitated b y goat anti-rabbit immunoglobulin serum. The equivalence between the rabbit anti-P3 serum and the goat anti-rabbit was established b y conventional immunochemical techniques s.
RESULTS
Release o] membrane-bound ribosomes accompanying polyribosome disaggregation The distribution of free and membrane-bound ribosomes in myeloma cells varies somewhat between different lines 3. We have made a careful estimate of percent membrane-bound ribosomes in P-3 cells b y comparing total 28-S ribosomal RNA in the cytoplasm and in the membrane fraction and nuclear pellet, which was presumed to be contaminated with large membrane fragments. The data are summarized in Table I and indicate that 27 % of total cellular ribosomes are membrane associated. Kimmel 3 has observed that ribosomes associated with membranes are predominantly in polyribosomes, and that the majority of single 8o-S ribosomes are present in the free cytoplasmic fraction. We have confirmed this result as shown in
TABLE I DISTRIBUTION OF 28-S rR1NIA IN CONTROL CELLS AND IN CELLS INCUBATED WITH ~ a F 55 ° ml of cells were concentrated to i • lO 6 cells/ml as described in Materials and Methods. Approx. one-third served as control and the r e m a i n d e r were incubated w i t h 15 mM N a F . After 9o min t h e control and half the lXlaF-treated cells were r e m o v e d and cell e x t r a c t s were prepared. The rem a i n i n g cells were w a s h e d three times w i t h fresh m e d i u m and resuspended at the initial concent r a t i o n in fresh medium. I n c u b a t i o n was t h e n continued for 9o rain, the ce]ls were r e m o v e d and processed as the first t w o samples. A p a r t of the postnuclear s u p e r n a t a n t was t a k e n for the estim a t i o n of total cytoplasmic 28-S r R N A (see below). M e m b r a n e s were p r e p a r e d from the postnuclear s u p e r n a t a n t as described in Materials and Methods. I n addition, the nuclear pellet w a s resuspended in NaC1-Tris-MgC1 a buffer and made o. 5 % in the detergent nonidet P4 o to solubilize a n y large m e m b r a n e fragments. Nuclei were pelleted again 5 rain at 400 × g and the s u p e r n a r a n t was analyzed for ribosomal RNA. Microscopic e x a m i n a t t o n of the cell h o m o g e n a t e showed t h a t no whole cells remained after homogenization (for this e x p e r i m e n t homogenization was b y IO strokes of a t i g h t Dounce homogenizer). To estimate 28-S ribosomal RNA, samples were m a d e o. 5 % in sodium dodecyl s u l p h a t e and r u n in 5-2o % sucrose gradients in sodium dodecyl s u l p h a t e buffer. The area of the p e a k of 28-S ribosomal R N A was m e a s u r e d w i t h a planimeter. ~ihe area of total cytoplasmic 28-S ribosomal R N A chosen for normalization of all three samples w a s 248 cm 2.
Control 9o min N a F 9o min incubation after N a F
Total cytoplasm (normalized, cm 2)
Membrane fraction
Nuclear pellet
(cm2)
(cm2)
248.0 (ioo %) 248.0 (ioo %) 248.0 (IOO %)
55.2 (22.2 %) 42.0 (17.o %) 53.3 (21.5 %)
11.9 (4.8 %) 9.5 (3.8 %) 9.1 (3.7 %)
Biochim. Biophys. Acta, 269 (1972) 453-464
45b
i. BLEIBERG et al.
Figs IA and ID. It m a y be noted that these profiles differ somewhat from other profiles we have reportedT; this difference is thought to be due to the relatively long incubation at high cell concentrations for these experiments. It has been reported b y others that nutritional deficiencies cause polyribosome disaggregation and optimal nutritional conditions cause recruitment of ribosomes into polyribosomes 9. Since the relative proportion of 8o-S ribosomes and polyribosomes is dependent on the physiological conditions in mammalian cells, it seemed possible that 8o-S ribosomes free in the cytoplasm could be recruited into membrane-bound polyribosomes and that vice versa membrane-bound polyribosomes can release 8o-S ribosomes into the free pool upon disaggregation. We have therefore investigated the behaviour of membrane-bound ribosomes in cells incubated with the inhibitor of protein synthesis NaF. This compound has been shown to cause polyribosome disaggregation in rabbit reticulocytes 4 and H e L a cells ~. Incubation of P-3 cells with 15 mM N a F causes disaggregation of both free and membrane-bound polyribosomes (Fig. I). A large amount of 8o-S ribosomes is found bound to membranes after such an incubation with N a F (Fig. i). It was thus evident that the attachment of a large proportion of ribosomes to the membranes of the endoplasmic reticulum is not dependent upon the association of ribosomes into polyribosomes active in protein synthesis.
0
•
15
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0
E
~
d
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e
! 1:
Bottom
Top
Fig. I. Free and m e m b r a n e - b o u n d polyribosomes of control inyeloma cells; cells incubated with NaF; and cells which were incubated with NaF, washed and reincubated in fresh medium. The c o n d i t i o n s of i n c u b a t i o n are t h e s a m e as d e s c r i b e d in t h e l e g e n d t o T a b l e I, e x c e p t t h a t i n c u b a t i o n w i t h N a F was for 60 rain i n s t e a d of 90 rain. F o r each s a m p l e free p o l y r i b o s o m e s w e re p r e p a r e d f r om a p p r o x , i / 4 of t h e p o s t n u c l e a r s u p e r n a t a n t , a f t e r r e m o v i n g m e m b r a n e s b y s e d i m e n t a t i o n a t 27 ooo × g for 5 min. M e m b r a n e - b o u n d p o l y r i b o s o m e s w e re p r e p a r e d from a p p r o x . 2/5 of t h e pos t n u c l e a r s u p e r n a t a n t b y p u r i f y i n g m e m b r a n e s t h r o u g h a c o n t i n u o u s sucrose g r a d i e n t as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . P o l y r i b o s o m e s were d i s p l a y e d b y c e n t r i f u g a t i o n for 3.5 h a t 26 ooo r e v . / m i n in 15-4 ° % sucrose g r a d i e n t s in NaC1 Tris-MgC1, buffer. The a b s o r b a n c e profile a t 260 n m is shown, a,b,c, free p o l y r i b o s o m e s ; d,e,f, b o u n d p o l y r i b o s o m e s , a,d, c o n t r o l cells; b,e, cells i n c u b a t e d w i t h I5 m.Xl N a F for 6c m m ; c,f, cells i n c u b a t e d w i t h i 5 mM N a F for 60 mi n, w a s h e d a n d r e m c u b a t e d for 9 ° rain in fresh m e d i u m .
Biochim. Biophys. Acta, 269 (1972) 453 464
457
MEMBRANE-BOUND POLYRIBOSOMES
In order to establish whether some ribosomes are released from membranes during the incubation with NaF, the recovery of membrane-bound 28-S ribosomal RNA was compared before and after incubation with NaF. The results shown in Fig. 2 are normalized for recovery of total cytoplasmic 28-S ribosomal RNA. As is seen in Table I there was no difference in the membrane contamination of the nuclear pellet for these samples. We conclude, therefore, that approx. 25 % of the membraneassociated ribosomes are released during the incubation with NaF. o
28S
I8S
b
28S
[ O~ E 8 02
OI
Bottom
18S
c
1
28S
18S
1
/ Top
Fig. 2. M e m b r a n e - b o u n d r i b o s o m a l R N A of (a) control cells, (b) cells i n c u b a t e d w i t h N a F for 90 rain, a n d (c) cells i n c u b a t e d w i t h BIaF, t h e n w a s h e d a n d r e i n c u b a t e d in fresh m e d i u m for 90 min. E x p e r i m e n t a l details are g i v e n in t h e legend to T a b l e I. C e n t r i f u g a t i o n w a s t h r o u g h 5-20 % sucrose g r a d i e n t s in s o d i u m dodecyl s u l p h a t e b u f f e r for 13 h a t 24 ooo r e v . / m i n in t h e S W 27 rotor, t h e a b s o r b a n c e profile a t 260 n m is s h o w n . S a m p l e s were n o r m a l i z e d for e q u i v a l e n t recoveries of 28-S r i b o s o m a l R N A in t h e t o t a l c y t o p l a s m i c e x t r a c t .
Attachment o/ribosomes to membranes accompanying polyribosome re]ormation Inhibition of protein synthesis and polyribosome disaggregation caused b y N a F in reticulocytes is reversible, if the inhibitor is washed away and the cells are reincubated in fresh medium 4. We have thus investigated whether polyribosomes reform in P-3 cells incubated with NaF, washed and reincubated in fresh medium, and whether free ribosomes became associated with membranes under these conditions. The gradient analysis (Fig. I) showed almost complete reformation of polyribosomes. The same amount of ribosomes that were released from membranes during the incubation with NaF, became associated again with membranes (Fig. 2 and Table I). We have established that polyribosome reformation under these conditions does not require the synthesis of RNA. When P-3 cells were incubated with NaF, washed and reincubated in fresh medium containing 5/,g/ml of actinomycin D, polyribosomes were reassembled as well as in the absence of actinomycin (not shown).
Resumption o/ immunoglobulin secretion [ollowing reversal o/ NaF inhibition The membrane-bound polyribosomes of myeloma cells are specialized in the synthesis of immunoglobulins that are secreted b y these cells into the culture medium 1. We have investigated whether P-3 cells incubated with N a F and washed, synthesize immunoglobulin when reincubated in flesh medium. This m a y indicate that the membrane-bound polyribosomes that have reformed in these cells have recovered their normal biosynthetic activity. One culture of control P-3 cells was thus incubated with [14CJproline and another culture of cells, treated with N a F Biochim. Biophys. Acta, 269 (1972) 453-464
458 TABLE
I. BLEIBERG et al. Ii
CELLULAR PROTEIN AND IMMUNOGLOBULIN SYNTHESIS BY CONTROL I~IYELOM.~. CELLS AND BY CELLS INCUBATED WITH N a F AND WASHED F o r e a c h e x p e r i m e n t 5 " IO~ cells w e r e i n c u b a t e d w i t h 5 # C i / m l of [ D H j p r o l i n e . A n a l i q u o t o[ t h e cell s u s p e n s i o n w a s t a k e n a f t e r 2 a n d 4 h. T h e cells w e r e c e n t r i f u g e d a n d r e s u s p e n d e d i n E a r l e ' s s a l i n e . A n e q u a l f r a c t i o n o f cells a n d o f c u l t u r e m e d i u m w a s p r e c i p i t a t e d w i t h 5 % t r i c h l o r o a c e t i c a c i d a n d c o u n t e d . A , c o n t r o l cells; B , cells i n c u b a t e d w i t h 15 m M N a F f o r 6 0 miD, w a s h e d t h r e e times with fresh medium and reincubated in fresh medium containing [aHlproline. The cpln per 5 " lO5 c e l l s a n d a n e q u i v a l e n t v o l u m e of c u l t u r e m e d i u m a r e r e p o r t e d .
Incubation time (h)
Cells
Culture medium
.4
B
A
B
2 4
25 2 0 0 43 5 ° 0
18 3 0 0 33 4 ° 0
1650 335 °
135o 3200
and washed, was incubated with ~aHlproline. (This amino acid is not present in the tissue culture medium used and it provides a convenient protein label.) The ratio of protein secreted into the tissue culture medium to total protein synthesized was found to be similar for the two cultures (Table II). The protein secreted was precipitated from the culture medium with (NH4)2SOa between 30 and 55 % saturation and chromatographed on Sephadex G-Ioo (Fig. 3) or used in a precipitin test with a rabbit antiserum directed against the immunoglobulin secreted by P-3 tumors (see Materials and Methods for details). By these two criteria the protein secreted by control cells and by cells incubated with NaF, washed and reincubated, were found to be indistinguishable. This suggested that the membrane-bound polyribosomes reformed synthesize the same protein as polyribosomes of control cells and that the same m R N A which is associated with membrane-bound polyribosomes of control cells, is associated with polyribosomes reformed after N a F treatment.
i
-
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I00
50
5
I0
15
- 1000
:~
- 500
i
20
FRACTIONS
F i g . 3. G e l f i l t r a t i o n a n a l y s i s of t h e p r o t e i n s e c r e t e d i n t o t h e c u l t u r e m e d i u m b y c o n t r o l n l y e l o m a cells and by cells treated with NaF, washed and reincubated in fresh medium. The control cells w e r e i n c u b a t e d f o r 20 h w i t h IO n C i / m l of [ z 4 C ] p r o l i n e a t a n i n i t i a l c o n c e n t r a t i o n of 3 " lO5 c e l l s / m l . A t t h e e n d of t h e i n c u b a t i o n ttle c e l l s w e r e s p u n d o w n a n d t h e c u l t u r e m e d i u m u s e d a s a s o u r c e of l a C - l a b e l e d i m m u n o g l o b u l i n . T h e c u l t u r e m e d i u m o t s a m p l e B d e s c r i b e d i n T a b l e I I w a s u s e d t o p r e p a r e t h e p r o t e i n s e c r e t e d b y c e l l s i n c u b a t e d w i t h 15 m M N a F , w a s h e d a n d r e i n c u b a t e d f o r 4 h w i t h [ s I - [ ] p r o l i n e . P r o t e i n in t h e c u l t u r e m e d i u n l w a s c o n c e n t r a t e d b y p r e c i p i t a t i o n w i t h (NH4)~SO4 a n d a n a l y z e d a s d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s .
Biochim. Biophys. Acta, 2 6 9 (1972) 4 5 3 - 4 6 4
459
MEMBRANE-BOUND POLYRIBOSOMES
Behaviour o/ nascent polypeptide chains o/ membrane-bound polyribosomes o / N a F treated cells The experiments repolted above show that only a portion of the membrane associated ribosomes are released from membranes upon polyribosome disaggregation. Similar results have been reported b y Rosbach and Penman s who have used puromycin to disaggregate polyribosomes of H e L a cells. As will be indicated below our results differ somewhat from theirs, particularly as pertains to the association of messenger RNA (mRNA) with membranes after polyribosome disaggregation. These experiments suggest that bound ribosomes are heterogenous and that only some bound ribosomes can be reversibly dissociated from membranes. One possible source of heterogeneity m a y be the association of peptidyl-tRNA with ribosomes. In particular, since Redman and Sabatini 1° have shown that nascent peptides of membranebound ribosomes pass through the membranes into the cisternae of the endoplasmic 28S
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FRACTIONS
Fig. 4. A n a l y s i s of t h e R N A p r e s e n t in m e m b r a n e s p r e p a r e d f r o m control cells a n d f r o m cells t r e a t e d w i t h ~ a F . T h e c o n d i t i o n s of t h e i n c u b a t i o n a n d t h e f r a c t i o n a t i o n of m e m b r a n e s are described u n d e r t h e legend of T a b l e I I I . T h e R ~ A of m e m b r a n e s w a s a n a l y z e d on 5 - 2 0 % s u c r o s e g r a d i e n t s c o n t a i n i n g s o d i u m dodecyl s u l p h a t e buffer (see Materials a n d M e t h o d s ) . (a) Control cells, m e m b r a n e s p r e p a r e d in NaC1-Tris-MgC1, buffer; (b) control cells, m e m b r a n e s t r e a t e d w i t h IO m M E D T A ; (c) cells i n c u b a t e d w i t h BIaF, m e m b r a n e s p r e p a r e d in N a C 1 - T r i s - M g C l , b u f f e r ; (d) cells i n c u b a t e d w i t h N a F , m e m b r a n e s t r e a t e d w i t h i M KC1. Solid liue, absorbanc.-, a t 260 n m ; d o t t e d line, c p m of *H.
Biochim. Biophys. Acta. 269 (1972) 453-464
I. BLEIBERG et al.
460
reticulum, it is possible that they may act as an anchor to ribosomes. Even though chain completion takes place in reticulocytes incubated with NaF and the nascent chains are released from ribosomes 4, it seemed necessary to prove that this is the case for myeloma cells also. TABLE III RELEASE
OF POLYRIBOSOMES
FROM
MEMBRANES
BY
I M
KC1
AND
EDTA
36o ml of cells were i n c u b a t e d for 20 h w i t h I nCi/ml of [14C]uridine. The cells were t h e n collected b y centrifugation a n d r e s u s p e n d e d at I • IOe cells/ml. I # g / m l of e t h i d i u m b r o m i d e and 0.o 4/~g/ ml of a c t i n o m y c i n D were added and the ceils were i n c u b a t e d 3 ° rain before adding 4 #Ci/ml of [3H]uridine. After 3 h t w o - t h i r d s of the cells were w i t h d r a w n , and the remaining cells i n c u b a t e d 60 min w i t h 15 mM N a F . P o s t n u c l e a r s u p e r n a t a n t w a s p r e p a r e d from b o t h samples; aliquots were t h e n mixed w i t h an equal v o l u m e (1.5 ml) of NaC1-Tris-MgCl~ buffer or of 2 M KC1, 8 mM MgCl~ and 50 mM Tris containing E D T A or p u r o m y c i n as indicated above. The samples were applied to step gradients m a d e u p of one layer of 7.5 ml of 3 ° % sucrose in NaC1-Tris-MgC12 buffer a n d 7.5 ml of 15 % sucrose in either NaC1-Tris-MgC12 buffer or in a I : i dilution of the 2 M KC1 solution. The m e m b r a n e s were sedimented for 3 ° min a t 27 ooo r e v . / m i n in r o t o r SV~T27, r e s u s p e n d e d in 0. 5 ml of s o d i u m dodecyl s u l p h a t e buffer (see Materials and Methods), applied to 5-20 % sucrose gradients and centrifuged 16 h at 21 ooo rev./min. F r a c t i o n s were collected and c o u n t e d (the gradients of samples a, b, c, and d are s h o w n in Fig. 4; the 14C c p m have n o t been reported). The c p m of the first 16 fractions collected are s h o w n u n d e r 8H cpm; these corres p o n d to high molecular weight R N A (including m R N A ) which is synthesized in the presence of low levels (0.04 l,g/ml) of a c t i n o m y c i n D.
Incubation
Membrane preparation in:
14C cpm % remaining membrane associated 28 S 28 S
3H cpm % remaining membrane associated mRNA
Control
NaC1-Tris-MgC12 buffer
IOO (3185 cpm)
ioo (2o 959 cpm)
Control
NaC1-Tris-MgC12 b u f f e r + IO mM E D T A MaC1 Tris-MgCl 2 buffer i M KC1 I M KC1 I M KC1+o.33 mM puromycin
NaF NaF Control Control
ioo (1532 cpm)
46.4 74.4 14. 5 49.7
16.2 74 .6 18.2 49 .6
61.8 96.6 40.5 56.8
i 1.2
23.5
39.8
T A B L E IV RECOVERY
OF MEMBRANE
PROTEIN
AFTER
SALT OR
EDTA
TREATMENT
200 ml of cells were c o n c e n t r a t e d to I . IOe cells/ml and incubated IOO rain with o.i #Ci/ml L-[14C]proline (220 mCi/mM). P o s t n u c l e a r s u p e r n a t a n t w a s p r e p a r e d and equal aliquots were t r e a t e d w i t h N a C I - T r i s - M g C l , buffer (control), i M KC1 or io mM E D T A . M e m b r a n e s w e r e pelleted t h r o u g h a 15-3 ° % sucrose gradient in NaC1-Tris-MgC1, buffer. The s u p e r n a t a n t was collected from the b o t t o m in 4-ml aliquots A, B, C to the u p p e r m o s t 6 ml (D). The pellet was rinsed and r e s u s p e n d e d in s o d i u m dodecyl s u l p h a t e buffer. The trichloroacetic acid-precipitable c p m are s h o w n for each aliquot.
Pellet B o t t o m 4 ml (A) 4 ml (B) 4 ml (C) Top 6 ml (D)
Control (cpm)
2 M KCl (cpm)
20 m M E D T A (cpm)
2 943 IiO 635 252 16 561
2 399 89 149 236 16 289
2 724 lO3 186 218 17 988
Biochim. Biophys. Aeta, 269 (1972) 453-464
MEMBRANE-BOUND POLYRIBOSOMES
461
We have made use for this purpose of the recent observation of Sabatini et al. 11 who have reported that solutions containing I M KC1 release from the membranes of rat liver endoplasmic reticulum ribosomes that do not contain peptidyl-tRNA. These authors used puromycin in vitro to obtain complete release of peptidyl-tRNA. We reasoned that if nascent chains axe completed and released in cells incubated with NaF, treatment of membranes obtained from these cells with I M KC1 should release all the ribosomes. We have thus isolated membranes of the endoplasmic reticulum from control cells and from cells incubated with N a F directly and after treatment with I M KC1. The membranes were spun through a layer of sucrose and dissolved in sodium dodecyl sulphate buffer for the analysis of the RNA on sucrose gradients (Fig. 4)It is known that high salt concentrations solubilize protein and lipid components of the cell membranes TM. We were concerned that this might lead to the formation of small membrane fragments which would not be fully recovered in the pellet of the sucrose gradients. However, examination of the protein distribution in these gradients (Table IV) showed that the sedimentation properties of the membranes were not significantly altered. As expected there was less protein in the membrane pellet after treatment with salt, but the same proportion of protein was found in the body of the gradient (bottom two-thirds) as for control cells. In addition protein recovery in the membrane pellet was down b y only 18 %. The results shown in Fig. 4 and Table I I I indicate that I M KC1 releases almost all the ribosomes from membranes of cells incubated with NaF. This suggests that these ribosomes do not carry any peptidyl-tRNA and that they remain associated to membranes even after completion and release of the nascent peptide chains. When membranes from control cells were treated with I M KC1 approx. 50 % of the ribosomes were released (Table I I I ) . This finding was somewhat surprising, since we expected that only 8o-S ribosomes would be released b y this treatment and the 8o-S ribosomes account only for approx. 20 °/o of the membrane-bound ribosomes of control cells (Fig. I). It appears thus that some membrane-associated ribosomes can be released from membranes b y treatment with KC1 solutions. This finding has been confirmed in several experiments and will be t h e subject of a separate report (M. Zauderer and C. Baglioni, unpublished). Other authors have presented evidence for the heterogeneity of membranebound ribosomes. Sabatini et al. 13 working with liver cells, and Attardi et al. 14 and Rosbash and Penman ~ with H e L a cells have shown that the magnesium-chelating agent ethylenediamine tetracetic acid (EDTA) dissociates membrane-bound ribosomes, releasing all the small ribosomal subunits and approx. 50 % of the large ribosomal subunits into the supernatant. We have therefore investigated the effect of E D T A on membrane-bound ribosomes of P-3 cells and, in agreement with these authors, found that 50 % of the large ribosomal subunits remain attached to membranes after treatment with E D T A (Table I I I and Fig. 4). Behavior o] m R N A
o] membrane-bound polyribosomes
The synthesis of ribosomal RNA can be completely inhibited in mammalian cells by incubating them with low concentrations of actinomycin D 15. Under these conditions the synthesis of m R N A and t R N A is only slightly inhibited and the labeled RNA species found associated with polyribosomes is predominantly m R N A le. To Biochim. Biophys. Acta, 269 (1972) 453-464
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I. BLEIBERG et al.
further facilitate the study of membrane-associated m R N A we have preincubated cells with I #g/ml of ethidium bromide. At this concentration ethidium bromide inhibits the synthesis of mitochondrial RNA 17, a major contaminant of the endoplasmic reticulum in our membrane fraction. We have investigated the release of m R N A from membrane-bound polyribosomes of cells incubated with N a F and of control cells in the same experiment in which we studied the release of ribosomes from membranes in the presence of I M KC1 (Fig. 4, Table III). rRNA was the major species labeled during incubation for 20 h with E14CJuridine; m R N A was then labeled during a 3-h pulse in the presence of low levels of actinomycin D and ethidium bromide. It can be seen that NaF, while releasing 25 o/,/o of the ribosomal RNA associated with membranes, releases little or no membrane-associated m R N A (Table I I I ) . This contrasts with the effects of EDTA, previously reported b y Rosbash and Penman 6 and confirmed here, and of I M KC1 in releasing approx. 4 ° °/o of the m R N A fraction. These points will be discussed in the next section.
DISCUSSION Approximately 25 ~)o of the ribosomes of the P-3 line of myeloma cells are associated with membranes of the endoplasmic reticulum. These membrane-bound ribosomes are predominantly in polyribosomes, since most of the 80-S ribosomes and ribosomal subunits are found free in the cytoplasm. We have shown that a portion of tile membrane-bound ribosomes are released from membranes upon polyribosome disaggregation promoted b y NaF. Upon removal of NaF, polyribosomes reform and the same proportion of membrane-bound ribosomes is found as in controls cells. Polyribosomes reform even when RNA synthesis is inhibited by actinomycin D. This indicates that m R N A is preserved during polyribosome disaggregation and that it can be reutilized when N a F is removed. The m R N A of the reaggregated polyribosomes of NaF-treated cells is presumably the same as that of the corresponding membrane-bound polyribosomes of control cells since both cells synthesize and secrete immunoglobulin at a similar rate. Previous studies have shown that immunoglobulins are synthesized b y membranebound polyribosomes TM, although possibly not exclusively b y these polyribosomes 19. The observation that upon polyribosome disaggregation only a portion of membrane-bound ribosomes is released from membranes suggests that these ribosomes m a y be functionally heterogeneous. Association with membranes of the releasable ribosomes seems to depend on their participation in protein synthesis, whereas association of the non-releasable ribosomes is independent of their participation in protein synthesis. One possible source of functional heterogeneity of membrane-bound ribosomes originates from their involvement in the synthesis of different proteins. Only approximately IO % of the protein synthesized b y P-3 cells is immunoglobulin (see Table II) whereas 25 % of the ribosomes active in protein synthesis are associated with membranes. Assuming that the rate of peptide-chain elongation is similar for free and bound ribosomes, it follows that a large proportion of membrane-associated ribosomes are involved in the synthesis of proteins other than immunoglobulins. These proBiochim. Biophys. Acta, 269 (1972) 453-464
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teins are not secreted, since the only secretory product of myeloma cells is immunoglobulin. Membrane-bound ribosomes have been found in cells that do not secrete any protein, like H e L a cells. In these cells as well, two classes of bound ribosomes have been differentiated on the basis of their behavior when polyribosomes are dissociated in the intact cell b y puromycin or in cell extracts by ribonuclease or E D T A e. Furthermore, it has been shown in H e L a cells that the two classes of membrane ribosomes are organized in separate classes of polyribosomes ~1. It remains to be established in myeloma cells whether one or both of these two classes of polyribosomes is involved in the synthesis of immunoglobulin. It would also be of interest to determine whether other non-secretory proteins might be specifically synthesized on membrane-bound polyribosomes. We have studied the association of presumptive m R N A with membrane obtained from intact cells incubated with N a F or of cellular membrane fractions treated with E D T A or with puromycin and I M KC1. These treatments result in polyribosome breakdown and it was of interest to follow the fate of m R N A under these conditions. Labeling of non-ribosomal RNA was obtained b y incubating myeloma cells with radioactive uridine in the presence of low concentrations of actinomycin D. Mitochondrial RNA synthesis was inhibited by treatment with ethidium bromide. It m a y be that not all the RNA synthesized in a 3-h pulse under these conditions is mRNA. In particular we have observed a peak of RNA sedimenting faster than 28-S ribosomal RNA which appears to be too large for a normal cellular mRNA. This peak is particularly clear in Fig. 4 b, and has been shown to migrate more slowly than 28-S rRNA in a polyacrylamide gel. I t is likely, however, that much of the labeled RNA is m R N A since it is polyribosome associated; it is rapidly hyclrolyzed by treatment of the polyribosomes with i #g/ml of ribonuclease (5 rain at 2o°C); and after tleatment with E D T A less than IO °/o of the labeled RNA is recovered in the region where polyribosomes would sediment (unpublished observations). E D T A is known to dissociate polyribosomes into ribosomal subunits and ribonucleoprotein particles containing m R N A that sediment around 2o~6o S 2°. Polyribosome disaggregation caused by N a F does not lead to release of m R N A from membrane-bound ribosomes (Table III). This might explain the observation that upon removal of N a F m R N A can be reutilized for the assembly of polyribosomes active in immunoglobulin synthesis, and that the secretion of immunoglobulin is then normal. We do not know, of course, that if m R N A had been released this same reconstruction would not have been possible. These results with N a F differ from those obtained when membrane polyribosomes were disaggregated b y treatment with E D T A or I M KC1 in that (I) significantly fewer ribosomes were released from association with the membranes by N a F (25% v e r s u s 5o%), and (2) no m R N A was released from membranes after N a F treatment as compared to 4 ° ~o release b y E D T A or I M KC1. In other experiments it was found that if treatment with E D T A followed incubation with N a F the same number of ribosomes remained membrane-bound as when control cells were treated with EDTA. I t appears, therefore, that N a F removes a part of the same ribosomes which are sensitive to EDTA. Vesco and Colombo 22 have reported that m R N A of H e L a cells sediments predominantly around 95 S after incubation of these cells Biockim. Biophys. Aaa, 269 (1972) 453-464
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with NaF. We have found that similar complexes form in myeloma cells incubated with N a F (unpublished). It is possible that in the form of such a complex some of the EDTA-sensitive ribosomes and all of the m R N A remain associated with the membranes after polyribosome disaggregation by NaF. We have suggested elsewhere 7 a model for the assembly of membrane polyribosomes. In terms of that model m R N A destined to be localized on membranebound polyribosomes is initially associated with the membranes by forming a complex with a specific membrane-bound ribosome. The observation that N a F releases some membrane ribosomes without releasing m R N A , is not inconsistent with this model since all of the m R N A m a y be associated with some of the remaining ribosomes. We might add, however, that there m a y very well be significant differences in the mechanisms by which the two classes of membrane-bound polyribosomes are assembled. These experiments on m R N A are of a rather preliminary nature; they are based on the assumption that polyribosome-associated R N A labeled in the presence of low levels of actinomycin D can be equated to mRNA, However, only isolation of a discrete species of R N A and proof of its informational role conclusively identifies it as m R N A . W e have recently shown (unpublished) that histone m R N A is synthesized by myeloma cells in the presence of o.0 4 #g/ml of actinomycin D; it thus seems possible that other species of m R N A as well are synthesized by cells incubated in this concentration of antibiotic. Experiments aimed at demonstrating the synthesis of immunoglobulin m R N A are currently in progress. ACKNOWLEDGEMENTS
This investigation has been supported by grant AI o 8 i i 6 of the National Institutes of Health, Bethesda, Md. One of us (I.B) was a postdoctoral fellow of the International Cancer Research Agency. REFERENCES M. Cohn, Cold Spring Harb. Syrup. Quant. Biol., 32 (I967) 2 I I . 2 P. Siekevitz a n d G. G. P a l a d e , J. Biophys. Biochem. Cytol., 7 (196o) 619. 3 C. B. I f i m m e l , Biochim. Biophys. Acta, i 8 2 (I969) 36I. 4 P. A. Marks, E. R. B u r k a , F. M. Conconi, W . Perl a n d R. A. Rifkind, Proc. Natl. Acad. Sci. U.S., 53 (1965) 1437. 5 B. Colombo, C. Vesco a n d C. Baglioni, Proc. Natl. Acad. Sci. U.S., 61 (1968) 651. 6 M. R o s b a s h a n d S. P e n m a n , J. Mol. Biol., 59 (I97 x) 227. 7 C. ]3aglioni, I. Bleiberg a n d M. Zauderer, Nature, New Biol., 232 (i97 I) 8. 8 E. A. IZabat a n d M. M. ~ a y e r , Experimental Irnmunochemistry, Thomas, Springfield, [11., I961. 9 13. L. M. H o g a n a n d A. K o r n e r , Biochim. Biophys. Acta, 169 (I968b) I39. io C. 1V[. R e d m a n a n d D. D. Sabatini, Proc. Natl. Acad. Sci. U.S., 56 (I966) 608. 1i D . D . Sabatini, G. Blobel, Y. N o n o m u z a a n d M. R. A d e l m a n , in F. C l e m e n t i a n d E. T r a b u c c h i , i2 13 14 15 16 17 18 19 20 21 22
Proceeding of the Ist International Symposium on Cell Biology and Cytopharmacology, Raven Press, New York, I969. C. D. Mitchell a n d D. J. H a n a h a n , Biochemistry, 5 (1966) 51. D. D. Sabatini, Y. T a s h i r o a n d G. E. P a l a d e , J. Mol. Biol., 19 (1966) 503. B. A t t a r d i , G. Cravioto a n d G. A t t a r d i , J . Mol. Biol., 44 (I969) 47. R. P. P e r r y , Proc. Nail. Acad. Sci. U.S., 48 (1962) 2179. S. P e n m a n , C. Vesco a n d M'. P e n m a n , J. Mol. Biol., 34 (1968) 49. E. Zylber, C. Vesco a n d S. P e n m a n , J. Mol. Biol., 44 (I969) 195. P. Vassalli, Proc. Natl. Acad. Sci. U.S., 58 (1967) 2117. B. L i s o w s k a - B e r n s t e i n , M. E. L a m m a n d P. Vassali, Proc. Natl. Acad. Sci. U.S., 66 (197 o) 425, R. P. P e r r y a n d D. E. Kelly, J. Mol. Biol., 35 (1968) 37. M. R o s b a s h a n d S. P e n m a n , J. Mol. Biol., 59 (1971) 243. C. Vesco a n d B. Colombo, J. Mol. Biol., 47 (197 °) 335.
Biochim. Biophys. Acta, 269 (1972) 453-464
BIOCI-IIMICA ET BIOPHYSICA ACTA
BBA 97217
R E V E R S I B L E D I S A G G R E G A T I O N BY N a F OF MEMBRANE-BOUND POLYRIBOSOMES OF MOUSE MYELOMA CELLS IN T I S S U E C U L T U R E I. B L E I B ] E R G * , M. Z A U D E R E R
AND (7. B A G L I O N I
Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139 (U.S.A.) (Received J a n u a r y 7th, 1972)
SUMMARY
Approximately one-quarter of the ribosomes of P-3 mouse myeloma cells in tissue culture are bound to membranes of the endoplasmic reticulum. Incubation with N a F causes polyribosome disaggregation; at the same time approximately 25 % of the membrane-bound ribosomes are released from membranes. After reversing the inhibition of N a F free ribosomes become associated with membranes until approximately the same proportion of free and membrane-bound ribosomes found in untreated cells is reestablished. During this time these cells resume the normal synthesis and secretion of immunoglobulin. This takes place even when the synthesis of RNA is completely inhibited. These experiments have suggested that the association of a class of ribosomes with membranes is dependent on their entry into polyribosomes active in protein synthesis whereas attachment of the other ribosomes occurs independently of their participation in protein synthesis.
INTRODUCTION
Plasma cells are highly specialized cells devoted to the synthesis and secretion of immunoglobulins. Plasma cell tumors (myelomas) have been induced in BALB/c mice and cell lines from several tumours have been adapted to grow in tissue culture 1. These cells provide a unique material for the study of immunoglobulin synthesis, since almost every line secretes a unique immunoglobulin or an immunoglobulin chain 1. The synthesis of proteins secreted by mammalian cells occurs on ribosomes bound to the membranes of the endoplasmic reticulum ~. The study of membranebound ribosomes m a y thus provide information on the molecular events which lead to the localization of ribosomes and of a specific class of messenger RNAs, those coding for proteins secreted by the cell, in the endoplasmic reticulum. We have investigated the behaviour of membrane-bound ribosomes in the P-3 tissue culture line of mouse myeloma. Cells of the P-3 line secrete an IgG immunoglobulin; these cells have been shown to be remarkably stable under tissue culture conditions for an extended period of time 3. We have attempted to establish whether membrane-bound ribosomes can be reversibly released from membranes of the endoplasnfic reticulum in the presence of the inhibitor of protein synthesis N a F 4. This * P r e s e n t a d d r e s s : T e l - H a s h o m e r Hospital, R a m a t - G a n . Israel.
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compound causes polyribosome disaggregation and decreases the level of free ribosomal subunits in mammalian cells 5. The results obtained suggest that a portion of the membrane-bound ribosomes can recycle between the pool of free ribosomes and those associated with membranes, and that after the disaggregation and reassembly of polyribosomes at least a part of the specificity of membrane-associated protein synthesis is restored. Related studies in HeLa cells have recently been published by Rosbach and Penman 6.
MATERIALS AND METHODS
Cell culture Mouse myeloma cells of the P-3 line were obtained from Dr M. Cohn of the Salk Institute for Biological Research. The cells were grown in Dulbecco's medium (GIBCO, New York) supplemented with IO °/o horse serum. The cell line was propagated in petri dishes in a CO 2 incubator. Large quantities of cells were grown in roller bottles.
Cell incubation To uniformly label RNA or protein I nCi/ml of E14Cluridine or io nCi/ml of ~14CI proline, respectively, were added for 2o-24 h directly to growing cells. For all other incubations the cells were collected at a density of 3 " lO5-5 . lO5 ceils per ml, concentrated by centrifugation for 5 rain at 200 × g, resuspended in the same medium at a density of i • lOs cells per ml and incubated in a rotary shaker under 5 % CO~.
Cell/ractionation At the end of an incubation the cells were collected by centrifugation, resuspended in I ml of NaCI-Tris-MgC12 buffer (o.oi M NaC1, o.oi M Tris-HC1, pH 7.4, and 1. 5 mM MgC12) per 2 • lO7 cells and homogenized by 15 strokes of a loose fitting Dounce homogenizer. Nuclei were spun down in 5 rain at 400 ×g. A membrane fraction was prepared from the postnuclear supernatant by a 3o-min centrifugation at 80 o o o × g through a continuous 15-3o % sucrose gradient in NaC1-Tris-MgC12 buffer 6. In some cases this procedure was modified by substituting a I5 and 30 °/'o sucrose step gradient. Control studies showed this did not affect membrane recoveries. Membrane-bound polyribosomes were prepared by resuspending the membrane pellet in o.5-1 ml of NaC1 Tris-MgC12 buffer and adding the detergent sodium deoxycholate to a final concentration of 1 % . Free polyribosomes were prepared from the postnuclear supernatant by sedimenting mitochondria and membranes at 27 ooo × g for 5 min3.
Gradient analysis Polyribosomes were analyzed by sucrose density gradient centrifugation as indicated in the legends. The absorbance at 260 nm was monitored in a Gilford continuously recording spectrophotometer. The fractions collected were precipitated with 5 % trichloroacetic acid onto cellulose nitrate filters and counted in a liquid scintillation counter. RNA has been analyzed in sucrose gradients made up in o.oi M Tris-HC1, pH 7.4, o.I M NaC1, I mM ethylenediamine tetraacetic acid and o.5 0'o sodium dodecyl sulphate (sodium dodecyl sulphate buffer). Biochim. Biophys. Acta, 269 (1972) 453-464
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POLYRIBOSOME S
Immunoglobulin analysis The protein secreted b y P-3 cells has been analyzed by gel filtration on Sephadex G-Ioo. The protein was precipitated with (NH4),SO 4 between 30 and 55 % saturation, redissolved in o.I M NaC1, o.oi M Tris-HC1 buffer, p H 7.4, and applied to a 2.4 cm × 60 cm equilibrated with the same buffer. 2-ml fractions were collected and counted as described above. The protein secreted by P-3 cells was also identified b y immunoprecipitation with a rabbit antiserum prepared with purified P-3 immunoglobulin (a kind gift of Dr Donato Cioli and Dr David Schubert); the antigenantibody complexes were then precipitated b y goat anti-rabbit immunoglobulin serum. The equivalence between the rabbit anti-P3 serum and the goat anti-rabbit was established b y conventional immunochemical techniques s.
RESULTS
Release o] membrane-bound ribosomes accompanying polyribosome disaggregation The distribution of free and membrane-bound ribosomes in myeloma cells varies somewhat between different lines 3. We have made a careful estimate of percent membrane-bound ribosomes in P-3 cells b y comparing total 28-S ribosomal RNA in the cytoplasm and in the membrane fraction and nuclear pellet, which was presumed to be contaminated with large membrane fragments. The data are summarized in Table I and indicate that 27 % of total cellular ribosomes are membrane associated. Kimmel 3 has observed that ribosomes associated with membranes are predominantly in polyribosomes, and that the majority of single 8o-S ribosomes are present in the free cytoplasmic fraction. We have confirmed this result as shown in
TABLE I DISTRIBUTION OF 28-S rR1NIA IN CONTROL CELLS AND IN CELLS INCUBATED WITH ~ a F 55 ° ml of cells were concentrated to i • lO 6 cells/ml as described in Materials and Methods. Approx. one-third served as control and the r e m a i n d e r were incubated w i t h 15 mM N a F . After 9o min t h e control and half the lXlaF-treated cells were r e m o v e d and cell e x t r a c t s were prepared. The rem a i n i n g cells were w a s h e d three times w i t h fresh m e d i u m and resuspended at the initial concent r a t i o n in fresh medium. I n c u b a t i o n was t h e n continued for 9o rain, the ce]ls were r e m o v e d and processed as the first t w o samples. A p a r t of the postnuclear s u p e r n a t a n t was t a k e n for the estim a t i o n of total cytoplasmic 28-S r R N A (see below). M e m b r a n e s were p r e p a r e d from the postnuclear s u p e r n a t a n t as described in Materials and Methods. I n addition, the nuclear pellet w a s resuspended in NaC1-Tris-MgC1 a buffer and made o. 5 % in the detergent nonidet P4 o to solubilize a n y large m e m b r a n e fragments. Nuclei were pelleted again 5 rain at 400 × g and the s u p e r n a r a n t was analyzed for ribosomal RNA. Microscopic e x a m i n a t t o n of the cell h o m o g e n a t e showed t h a t no whole cells remained after homogenization (for this e x p e r i m e n t homogenization was b y IO strokes of a t i g h t Dounce homogenizer). To estimate 28-S ribosomal RNA, samples were m a d e o. 5 % in sodium dodecyl s u l p h a t e and r u n in 5-2o % sucrose gradients in sodium dodecyl s u l p h a t e buffer. The area of the p e a k of 28-S ribosomal R N A was m e a s u r e d w i t h a planimeter. ~ihe area of total cytoplasmic 28-S ribosomal R N A chosen for normalization of all three samples w a s 248 cm 2.
Control 9o min N a F 9o min incubation after N a F
Total cytoplasm (normalized, cm 2)
Membrane fraction
Nuclear pellet
(cm2)
(cm2)
248.0 (ioo %) 248.0 (ioo %) 248.0 (IOO %)
55.2 (22.2 %) 42.0 (17.o %) 53.3 (21.5 %)
11.9 (4.8 %) 9.5 (3.8 %) 9.1 (3.7 %)
Biochim. Biophys. Acta, 269 (1972) 453-464
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i. BLEIBERG et al.
Figs IA and ID. It m a y be noted that these profiles differ somewhat from other profiles we have reportedT; this difference is thought to be due to the relatively long incubation at high cell concentrations for these experiments. It has been reported b y others that nutritional deficiencies cause polyribosome disaggregation and optimal nutritional conditions cause recruitment of ribosomes into polyribosomes 9. Since the relative proportion of 8o-S ribosomes and polyribosomes is dependent on the physiological conditions in mammalian cells, it seemed possible that 8o-S ribosomes free in the cytoplasm could be recruited into membrane-bound polyribosomes and that vice versa membrane-bound polyribosomes can release 8o-S ribosomes into the free pool upon disaggregation. We have therefore investigated the behaviour of membrane-bound ribosomes in cells incubated with the inhibitor of protein synthesis NaF. This compound has been shown to cause polyribosome disaggregation in rabbit reticulocytes 4 and H e L a cells ~. Incubation of P-3 cells with 15 mM N a F causes disaggregation of both free and membrane-bound polyribosomes (Fig. I). A large amount of 8o-S ribosomes is found bound to membranes after such an incubation with N a F (Fig. i). It was thus evident that the attachment of a large proportion of ribosomes to the membranes of the endoplasmic reticulum is not dependent upon the association of ribosomes into polyribosomes active in protein synthesis.
0
•
15
)5
0
E
~
d
°:!
e
! 1:
Bottom
Top
Fig. I. Free and m e m b r a n e - b o u n d polyribosomes of control inyeloma cells; cells incubated with NaF; and cells which were incubated with NaF, washed and reincubated in fresh medium. The c o n d i t i o n s of i n c u b a t i o n are t h e s a m e as d e s c r i b e d in t h e l e g e n d t o T a b l e I, e x c e p t t h a t i n c u b a t i o n w i t h N a F was for 60 rain i n s t e a d of 90 rain. F o r each s a m p l e free p o l y r i b o s o m e s w e re p r e p a r e d f r om a p p r o x , i / 4 of t h e p o s t n u c l e a r s u p e r n a t a n t , a f t e r r e m o v i n g m e m b r a n e s b y s e d i m e n t a t i o n a t 27 ooo × g for 5 min. M e m b r a n e - b o u n d p o l y r i b o s o m e s w e re p r e p a r e d from a p p r o x . 2/5 of t h e pos t n u c l e a r s u p e r n a t a n t b y p u r i f y i n g m e m b r a n e s t h r o u g h a c o n t i n u o u s sucrose g r a d i e n t as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . P o l y r i b o s o m e s were d i s p l a y e d b y c e n t r i f u g a t i o n for 3.5 h a t 26 ooo r e v . / m i n in 15-4 ° % sucrose g r a d i e n t s in NaC1 Tris-MgC1, buffer. The a b s o r b a n c e profile a t 260 n m is shown, a,b,c, free p o l y r i b o s o m e s ; d,e,f, b o u n d p o l y r i b o s o m e s , a,d, c o n t r o l cells; b,e, cells i n c u b a t e d w i t h I5 m.Xl N a F for 6c m m ; c,f, cells i n c u b a t e d w i t h i 5 mM N a F for 60 mi n, w a s h e d a n d r e m c u b a t e d for 9 ° rain in fresh m e d i u m .
Biochim. Biophys. Acta, 269 (1972) 453 464
457
MEMBRANE-BOUND POLYRIBOSOMES
In order to establish whether some ribosomes are released from membranes during the incubation with NaF, the recovery of membrane-bound 28-S ribosomal RNA was compared before and after incubation with NaF. The results shown in Fig. 2 are normalized for recovery of total cytoplasmic 28-S ribosomal RNA. As is seen in Table I there was no difference in the membrane contamination of the nuclear pellet for these samples. We conclude, therefore, that approx. 25 % of the membraneassociated ribosomes are released during the incubation with NaF. o
28S
I8S
b
28S
[ O~ E 8 02
OI
Bottom
18S
c
1
28S
18S
1
/ Top
Fig. 2. M e m b r a n e - b o u n d r i b o s o m a l R N A of (a) control cells, (b) cells i n c u b a t e d w i t h N a F for 90 rain, a n d (c) cells i n c u b a t e d w i t h BIaF, t h e n w a s h e d a n d r e i n c u b a t e d in fresh m e d i u m for 90 min. E x p e r i m e n t a l details are g i v e n in t h e legend to T a b l e I. C e n t r i f u g a t i o n w a s t h r o u g h 5-20 % sucrose g r a d i e n t s in s o d i u m dodecyl s u l p h a t e b u f f e r for 13 h a t 24 ooo r e v . / m i n in t h e S W 27 rotor, t h e a b s o r b a n c e profile a t 260 n m is s h o w n . S a m p l e s were n o r m a l i z e d for e q u i v a l e n t recoveries of 28-S r i b o s o m a l R N A in t h e t o t a l c y t o p l a s m i c e x t r a c t .
Attachment o/ribosomes to membranes accompanying polyribosome re]ormation Inhibition of protein synthesis and polyribosome disaggregation caused b y N a F in reticulocytes is reversible, if the inhibitor is washed away and the cells are reincubated in fresh medium 4. We have thus investigated whether polyribosomes reform in P-3 cells incubated with NaF, washed and reincubated in fresh medium, and whether free ribosomes became associated with membranes under these conditions. The gradient analysis (Fig. I) showed almost complete reformation of polyribosomes. The same amount of ribosomes that were released from membranes during the incubation with NaF, became associated again with membranes (Fig. 2 and Table I). We have established that polyribosome reformation under these conditions does not require the synthesis of RNA. When P-3 cells were incubated with NaF, washed and reincubated in fresh medium containing 5/,g/ml of actinomycin D, polyribosomes were reassembled as well as in the absence of actinomycin (not shown).
Resumption o/ immunoglobulin secretion [ollowing reversal o/ NaF inhibition The membrane-bound polyribosomes of myeloma cells are specialized in the synthesis of immunoglobulins that are secreted b y these cells into the culture medium 1. We have investigated whether P-3 cells incubated with N a F and washed, synthesize immunoglobulin when reincubated in flesh medium. This m a y indicate that the membrane-bound polyribosomes that have reformed in these cells have recovered their normal biosynthetic activity. One culture of control P-3 cells was thus incubated with [14CJproline and another culture of cells, treated with N a F Biochim. Biophys. Acta, 269 (1972) 453-464
458 TABLE
I. BLEIBERG et al. Ii
CELLULAR PROTEIN AND IMMUNOGLOBULIN SYNTHESIS BY CONTROL I~IYELOM.~. CELLS AND BY CELLS INCUBATED WITH N a F AND WASHED F o r e a c h e x p e r i m e n t 5 " IO~ cells w e r e i n c u b a t e d w i t h 5 # C i / m l of [ D H j p r o l i n e . A n a l i q u o t o[ t h e cell s u s p e n s i o n w a s t a k e n a f t e r 2 a n d 4 h. T h e cells w e r e c e n t r i f u g e d a n d r e s u s p e n d e d i n E a r l e ' s s a l i n e . A n e q u a l f r a c t i o n o f cells a n d o f c u l t u r e m e d i u m w a s p r e c i p i t a t e d w i t h 5 % t r i c h l o r o a c e t i c a c i d a n d c o u n t e d . A , c o n t r o l cells; B , cells i n c u b a t e d w i t h 15 m M N a F f o r 6 0 miD, w a s h e d t h r e e times with fresh medium and reincubated in fresh medium containing [aHlproline. The cpln per 5 " lO5 c e l l s a n d a n e q u i v a l e n t v o l u m e of c u l t u r e m e d i u m a r e r e p o r t e d .
Incubation time (h)
Cells
Culture medium
.4
B
A
B
2 4
25 2 0 0 43 5 ° 0
18 3 0 0 33 4 ° 0
1650 335 °
135o 3200
and washed, was incubated with ~aHlproline. (This amino acid is not present in the tissue culture medium used and it provides a convenient protein label.) The ratio of protein secreted into the tissue culture medium to total protein synthesized was found to be similar for the two cultures (Table II). The protein secreted was precipitated from the culture medium with (NH4)2SOa between 30 and 55 % saturation and chromatographed on Sephadex G-Ioo (Fig. 3) or used in a precipitin test with a rabbit antiserum directed against the immunoglobulin secreted by P-3 tumors (see Materials and Methods for details). By these two criteria the protein secreted by control cells and by cells incubated with NaF, washed and reincubated, were found to be indistinguishable. This suggested that the membrane-bound polyribosomes reformed synthesize the same protein as polyribosomes of control cells and that the same m R N A which is associated with membrane-bound polyribosomes of control cells, is associated with polyribosomes reformed after N a F treatment.
i
-
'
I00
50
5
I0
15
- 1000
:~
- 500
i
20
FRACTIONS
F i g . 3. G e l f i l t r a t i o n a n a l y s i s of t h e p r o t e i n s e c r e t e d i n t o t h e c u l t u r e m e d i u m b y c o n t r o l n l y e l o m a cells and by cells treated with NaF, washed and reincubated in fresh medium. The control cells w e r e i n c u b a t e d f o r 20 h w i t h IO n C i / m l of [ z 4 C ] p r o l i n e a t a n i n i t i a l c o n c e n t r a t i o n of 3 " lO5 c e l l s / m l . A t t h e e n d of t h e i n c u b a t i o n ttle c e l l s w e r e s p u n d o w n a n d t h e c u l t u r e m e d i u m u s e d a s a s o u r c e of l a C - l a b e l e d i m m u n o g l o b u l i n . T h e c u l t u r e m e d i u m o t s a m p l e B d e s c r i b e d i n T a b l e I I w a s u s e d t o p r e p a r e t h e p r o t e i n s e c r e t e d b y c e l l s i n c u b a t e d w i t h 15 m M N a F , w a s h e d a n d r e i n c u b a t e d f o r 4 h w i t h [ s I - [ ] p r o l i n e . P r o t e i n in t h e c u l t u r e m e d i u n l w a s c o n c e n t r a t e d b y p r e c i p i t a t i o n w i t h (NH4)~SO4 a n d a n a l y z e d a s d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s .
Biochim. Biophys. Acta, 2 6 9 (1972) 4 5 3 - 4 6 4
459
MEMBRANE-BOUND POLYRIBOSOMES
Behaviour o/ nascent polypeptide chains o/ membrane-bound polyribosomes o / N a F treated cells The experiments repolted above show that only a portion of the membrane associated ribosomes are released from membranes upon polyribosome disaggregation. Similar results have been reported b y Rosbach and Penman s who have used puromycin to disaggregate polyribosomes of H e L a cells. As will be indicated below our results differ somewhat from theirs, particularly as pertains to the association of messenger RNA (mRNA) with membranes after polyribosome disaggregation. These experiments suggest that bound ribosomes are heterogenous and that only some bound ribosomes can be reversibly dissociated from membranes. One possible source of heterogeneity m a y be the association of peptidyl-tRNA with ribosomes. In particular, since Redman and Sabatini 1° have shown that nascent peptides of membranebound ribosomes pass through the membranes into the cisternae of the endoplasmic 28S
18S
,,,,l
t
0
28S
18S
il
°8-i" i
,~
,
b
1
2000
i~
,
os
\
o,
:i
\ /'
0.2 j
!
1500
/~\o,,
1000
\
~
f
', ~x~o"°/
500
E
O
~0 fM <
c 0.8
"]
o, -
0.4
7 ~4
\
E ck
d
'i
_~000
50O
i
~, !
I0oo i
Too
Bottom
FRACTIONS
Fig. 4. A n a l y s i s of t h e R N A p r e s e n t in m e m b r a n e s p r e p a r e d f r o m control cells a n d f r o m cells t r e a t e d w i t h ~ a F . T h e c o n d i t i o n s of t h e i n c u b a t i o n a n d t h e f r a c t i o n a t i o n of m e m b r a n e s are described u n d e r t h e legend of T a b l e I I I . T h e R ~ A of m e m b r a n e s w a s a n a l y z e d on 5 - 2 0 % s u c r o s e g r a d i e n t s c o n t a i n i n g s o d i u m dodecyl s u l p h a t e buffer (see Materials a n d M e t h o d s ) . (a) Control cells, m e m b r a n e s p r e p a r e d in NaC1-Tris-MgC1, buffer; (b) control cells, m e m b r a n e s t r e a t e d w i t h IO m M E D T A ; (c) cells i n c u b a t e d w i t h BIaF, m e m b r a n e s p r e p a r e d in N a C 1 - T r i s - M g C l , b u f f e r ; (d) cells i n c u b a t e d w i t h N a F , m e m b r a n e s t r e a t e d w i t h i M KC1. Solid liue, absorbanc.-, a t 260 n m ; d o t t e d line, c p m of *H.
Biochim. Biophys. Acta. 269 (1972) 453-464
I. BLEIBERG et al.
460
reticulum, it is possible that they may act as an anchor to ribosomes. Even though chain completion takes place in reticulocytes incubated with NaF and the nascent chains are released from ribosomes 4, it seemed necessary to prove that this is the case for myeloma cells also. TABLE III RELEASE
OF POLYRIBOSOMES
FROM
MEMBRANES
BY
I M
KC1
AND
EDTA
36o ml of cells were i n c u b a t e d for 20 h w i t h I nCi/ml of [14C]uridine. The cells were t h e n collected b y centrifugation a n d r e s u s p e n d e d at I • IOe cells/ml. I # g / m l of e t h i d i u m b r o m i d e and 0.o 4/~g/ ml of a c t i n o m y c i n D were added and the ceils were i n c u b a t e d 3 ° rain before adding 4 #Ci/ml of [3H]uridine. After 3 h t w o - t h i r d s of the cells were w i t h d r a w n , and the remaining cells i n c u b a t e d 60 min w i t h 15 mM N a F . P o s t n u c l e a r s u p e r n a t a n t w a s p r e p a r e d from b o t h samples; aliquots were t h e n mixed w i t h an equal v o l u m e (1.5 ml) of NaC1-Tris-MgCl~ buffer or of 2 M KC1, 8 mM MgCl~ and 50 mM Tris containing E D T A or p u r o m y c i n as indicated above. The samples were applied to step gradients m a d e u p of one layer of 7.5 ml of 3 ° % sucrose in NaC1-Tris-MgC12 buffer a n d 7.5 ml of 15 % sucrose in either NaC1-Tris-MgC12 buffer or in a I : i dilution of the 2 M KC1 solution. The m e m b r a n e s were sedimented for 3 ° min a t 27 ooo r e v . / m i n in r o t o r SV~T27, r e s u s p e n d e d in 0. 5 ml of s o d i u m dodecyl s u l p h a t e buffer (see Materials and Methods), applied to 5-20 % sucrose gradients and centrifuged 16 h at 21 ooo rev./min. F r a c t i o n s were collected and c o u n t e d (the gradients of samples a, b, c, and d are s h o w n in Fig. 4; the 14C c p m have n o t been reported). The c p m of the first 16 fractions collected are s h o w n u n d e r 8H cpm; these corres p o n d to high molecular weight R N A (including m R N A ) which is synthesized in the presence of low levels (0.04 l,g/ml) of a c t i n o m y c i n D.
Incubation
Membrane preparation in:
14C cpm % remaining membrane associated 28 S 28 S
3H cpm % remaining membrane associated mRNA
Control
NaC1-Tris-MgC12 buffer
IOO (3185 cpm)
ioo (2o 959 cpm)
Control
NaC1-Tris-MgC12 b u f f e r + IO mM E D T A MaC1 Tris-MgCl 2 buffer i M KC1 I M KC1 I M KC1+o.33 mM puromycin
NaF NaF Control Control
ioo (1532 cpm)
46.4 74.4 14. 5 49.7
16.2 74 .6 18.2 49 .6
61.8 96.6 40.5 56.8
i 1.2
23.5
39.8
T A B L E IV RECOVERY
OF MEMBRANE
PROTEIN
AFTER
SALT OR
EDTA
TREATMENT
200 ml of cells were c o n c e n t r a t e d to I . IOe cells/ml and incubated IOO rain with o.i #Ci/ml L-[14C]proline (220 mCi/mM). P o s t n u c l e a r s u p e r n a t a n t w a s p r e p a r e d and equal aliquots were t r e a t e d w i t h N a C I - T r i s - M g C l , buffer (control), i M KC1 or io mM E D T A . M e m b r a n e s w e r e pelleted t h r o u g h a 15-3 ° % sucrose gradient in NaC1-Tris-MgC1, buffer. The s u p e r n a t a n t was collected from the b o t t o m in 4-ml aliquots A, B, C to the u p p e r m o s t 6 ml (D). The pellet was rinsed and r e s u s p e n d e d in s o d i u m dodecyl s u l p h a t e buffer. The trichloroacetic acid-precipitable c p m are s h o w n for each aliquot.
Pellet B o t t o m 4 ml (A) 4 ml (B) 4 ml (C) Top 6 ml (D)
Control (cpm)
2 M KCl (cpm)
20 m M E D T A (cpm)
2 943 IiO 635 252 16 561
2 399 89 149 236 16 289
2 724 lO3 186 218 17 988
Biochim. Biophys. Aeta, 269 (1972) 453-464
MEMBRANE-BOUND POLYRIBOSOMES
461
We have made use for this purpose of the recent observation of Sabatini et al. 11 who have reported that solutions containing I M KC1 release from the membranes of rat liver endoplasmic reticulum ribosomes that do not contain peptidyl-tRNA. These authors used puromycin in vitro to obtain complete release of peptidyl-tRNA. We reasoned that if nascent chains axe completed and released in cells incubated with NaF, treatment of membranes obtained from these cells with I M KC1 should release all the ribosomes. We have thus isolated membranes of the endoplasmic reticulum from control cells and from cells incubated with N a F directly and after treatment with I M KC1. The membranes were spun through a layer of sucrose and dissolved in sodium dodecyl sulphate buffer for the analysis of the RNA on sucrose gradients (Fig. 4)It is known that high salt concentrations solubilize protein and lipid components of the cell membranes TM. We were concerned that this might lead to the formation of small membrane fragments which would not be fully recovered in the pellet of the sucrose gradients. However, examination of the protein distribution in these gradients (Table IV) showed that the sedimentation properties of the membranes were not significantly altered. As expected there was less protein in the membrane pellet after treatment with salt, but the same proportion of protein was found in the body of the gradient (bottom two-thirds) as for control cells. In addition protein recovery in the membrane pellet was down b y only 18 %. The results shown in Fig. 4 and Table I I I indicate that I M KC1 releases almost all the ribosomes from membranes of cells incubated with NaF. This suggests that these ribosomes do not carry any peptidyl-tRNA and that they remain associated to membranes even after completion and release of the nascent peptide chains. When membranes from control cells were treated with I M KC1 approx. 50 % of the ribosomes were released (Table I I I ) . This finding was somewhat surprising, since we expected that only 8o-S ribosomes would be released b y this treatment and the 8o-S ribosomes account only for approx. 20 °/o of the membrane-bound ribosomes of control cells (Fig. I). It appears thus that some membrane-associated ribosomes can be released from membranes b y treatment with KC1 solutions. This finding has been confirmed in several experiments and will be t h e subject of a separate report (M. Zauderer and C. Baglioni, unpublished). Other authors have presented evidence for the heterogeneity of membranebound ribosomes. Sabatini et al. 13 working with liver cells, and Attardi et al. 14 and Rosbash and Penman ~ with H e L a cells have shown that the magnesium-chelating agent ethylenediamine tetracetic acid (EDTA) dissociates membrane-bound ribosomes, releasing all the small ribosomal subunits and approx. 50 % of the large ribosomal subunits into the supernatant. We have therefore investigated the effect of E D T A on membrane-bound ribosomes of P-3 cells and, in agreement with these authors, found that 50 % of the large ribosomal subunits remain attached to membranes after treatment with E D T A (Table I I I and Fig. 4). Behavior o] m R N A
o] membrane-bound polyribosomes
The synthesis of ribosomal RNA can be completely inhibited in mammalian cells by incubating them with low concentrations of actinomycin D 15. Under these conditions the synthesis of m R N A and t R N A is only slightly inhibited and the labeled RNA species found associated with polyribosomes is predominantly m R N A le. To Biochim. Biophys. Acta, 269 (1972) 453-464
462
I. BLEIBERG et al.
further facilitate the study of membrane-associated m R N A we have preincubated cells with I #g/ml of ethidium bromide. At this concentration ethidium bromide inhibits the synthesis of mitochondrial RNA 17, a major contaminant of the endoplasmic reticulum in our membrane fraction. We have investigated the release of m R N A from membrane-bound polyribosomes of cells incubated with N a F and of control cells in the same experiment in which we studied the release of ribosomes from membranes in the presence of I M KC1 (Fig. 4, Table III). rRNA was the major species labeled during incubation for 20 h with E14CJuridine; m R N A was then labeled during a 3-h pulse in the presence of low levels of actinomycin D and ethidium bromide. It can be seen that NaF, while releasing 25 o/,/o of the ribosomal RNA associated with membranes, releases little or no membrane-associated m R N A (Table I I I ) . This contrasts with the effects of EDTA, previously reported b y Rosbash and Penman 6 and confirmed here, and of I M KC1 in releasing approx. 4 ° °/o of the m R N A fraction. These points will be discussed in the next section.
DISCUSSION Approximately 25 ~)o of the ribosomes of the P-3 line of myeloma cells are associated with membranes of the endoplasmic reticulum. These membrane-bound ribosomes are predominantly in polyribosomes, since most of the 80-S ribosomes and ribosomal subunits are found free in the cytoplasm. We have shown that a portion of tile membrane-bound ribosomes are released from membranes upon polyribosome disaggregation promoted b y NaF. Upon removal of NaF, polyribosomes reform and the same proportion of membrane-bound ribosomes is found as in controls cells. Polyribosomes reform even when RNA synthesis is inhibited by actinomycin D. This indicates that m R N A is preserved during polyribosome disaggregation and that it can be reutilized when N a F is removed. The m R N A of the reaggregated polyribosomes of NaF-treated cells is presumably the same as that of the corresponding membrane-bound polyribosomes of control cells since both cells synthesize and secrete immunoglobulin at a similar rate. Previous studies have shown that immunoglobulins are synthesized b y membranebound polyribosomes TM, although possibly not exclusively b y these polyribosomes 19. The observation that upon polyribosome disaggregation only a portion of membrane-bound ribosomes is released from membranes suggests that these ribosomes m a y be functionally heterogeneous. Association with membranes of the releasable ribosomes seems to depend on their participation in protein synthesis, whereas association of the non-releasable ribosomes is independent of their participation in protein synthesis. One possible source of functional heterogeneity of membrane-bound ribosomes originates from their involvement in the synthesis of different proteins. Only approximately IO % of the protein synthesized b y P-3 cells is immunoglobulin (see Table II) whereas 25 % of the ribosomes active in protein synthesis are associated with membranes. Assuming that the rate of peptide-chain elongation is similar for free and bound ribosomes, it follows that a large proportion of membrane-associated ribosomes are involved in the synthesis of proteins other than immunoglobulins. These proBiochim. Biophys. Acta, 269 (1972) 453-464
MEMBRANE-BOUND POLYRIBOSOMES
463
teins are not secreted, since the only secretory product of myeloma cells is immunoglobulin. Membrane-bound ribosomes have been found in cells that do not secrete any protein, like H e L a cells. In these cells as well, two classes of bound ribosomes have been differentiated on the basis of their behavior when polyribosomes are dissociated in the intact cell b y puromycin or in cell extracts by ribonuclease or E D T A e. Furthermore, it has been shown in H e L a cells that the two classes of membrane ribosomes are organized in separate classes of polyribosomes ~1. It remains to be established in myeloma cells whether one or both of these two classes of polyribosomes is involved in the synthesis of immunoglobulin. It would also be of interest to determine whether other non-secretory proteins might be specifically synthesized on membrane-bound polyribosomes. We have studied the association of presumptive m R N A with membrane obtained from intact cells incubated with N a F or of cellular membrane fractions treated with E D T A or with puromycin and I M KC1. These treatments result in polyribosome breakdown and it was of interest to follow the fate of m R N A under these conditions. Labeling of non-ribosomal RNA was obtained b y incubating myeloma cells with radioactive uridine in the presence of low concentrations of actinomycin D. Mitochondrial RNA synthesis was inhibited by treatment with ethidium bromide. It m a y be that not all the RNA synthesized in a 3-h pulse under these conditions is mRNA. In particular we have observed a peak of RNA sedimenting faster than 28-S ribosomal RNA which appears to be too large for a normal cellular mRNA. This peak is particularly clear in Fig. 4 b, and has been shown to migrate more slowly than 28-S rRNA in a polyacrylamide gel. I t is likely, however, that much of the labeled RNA is m R N A since it is polyribosome associated; it is rapidly hyclrolyzed by treatment of the polyribosomes with i #g/ml of ribonuclease (5 rain at 2o°C); and after tleatment with E D T A less than IO °/o of the labeled RNA is recovered in the region where polyribosomes would sediment (unpublished observations). E D T A is known to dissociate polyribosomes into ribosomal subunits and ribonucleoprotein particles containing m R N A that sediment around 2o~6o S 2°. Polyribosome disaggregation caused by N a F does not lead to release of m R N A from membrane-bound ribosomes (Table III). This might explain the observation that upon removal of N a F m R N A can be reutilized for the assembly of polyribosomes active in immunoglobulin synthesis, and that the secretion of immunoglobulin is then normal. We do not know, of course, that if m R N A had been released this same reconstruction would not have been possible. These results with N a F differ from those obtained when membrane polyribosomes were disaggregated b y treatment with E D T A or I M KC1 in that (I) significantly fewer ribosomes were released from association with the membranes by N a F (25% v e r s u s 5o%), and (2) no m R N A was released from membranes after N a F treatment as compared to 4 ° ~o release b y E D T A or I M KC1. In other experiments it was found that if treatment with E D T A followed incubation with N a F the same number of ribosomes remained membrane-bound as when control cells were treated with EDTA. I t appears, therefore, that N a F removes a part of the same ribosomes which are sensitive to EDTA. Vesco and Colombo 22 have reported that m R N A of H e L a cells sediments predominantly around 95 S after incubation of these cells Biockim. Biophys. Aaa, 269 (1972) 453-464
464
i. BLEIBERGet al.
with NaF. We have found that similar complexes form in myeloma cells incubated with N a F (unpublished). It is possible that in the form of such a complex some of the EDTA-sensitive ribosomes and all of the m R N A remain associated with the membranes after polyribosome disaggregation by NaF. We have suggested elsewhere 7 a model for the assembly of membrane polyribosomes. In terms of that model m R N A destined to be localized on membranebound polyribosomes is initially associated with the membranes by forming a complex with a specific membrane-bound ribosome. The observation that N a F releases some membrane ribosomes without releasing m R N A , is not inconsistent with this model since all of the m R N A m a y be associated with some of the remaining ribosomes. We might add, however, that there m a y very well be significant differences in the mechanisms by which the two classes of membrane-bound polyribosomes are assembled. These experiments on m R N A are of a rather preliminary nature; they are based on the assumption that polyribosome-associated R N A labeled in the presence of low levels of actinomycin D can be equated to mRNA, However, only isolation of a discrete species of R N A and proof of its informational role conclusively identifies it as m R N A . W e have recently shown (unpublished) that histone m R N A is synthesized by myeloma cells in the presence of o.0 4 #g/ml of actinomycin D; it thus seems possible that other species of m R N A as well are synthesized by cells incubated in this concentration of antibiotic. Experiments aimed at demonstrating the synthesis of immunoglobulin m R N A are currently in progress. ACKNOWLEDGEMENTS
This investigation has been supported by grant AI o 8 i i 6 of the National Institutes of Health, Bethesda, Md. One of us (I.B) was a postdoctoral fellow of the International Cancer Research Agency. REFERENCES M. Cohn, Cold Spring Harb. Syrup. Quant. Biol., 32 (I967) 2 I I . 2 P. Siekevitz a n d G. G. P a l a d e , J. Biophys. Biochem. Cytol., 7 (196o) 619. 3 C. B. I f i m m e l , Biochim. Biophys. Acta, i 8 2 (I969) 36I. 4 P. A. Marks, E. R. B u r k a , F. M. Conconi, W . Perl a n d R. A. Rifkind, Proc. Natl. Acad. Sci. U.S., 53 (1965) 1437. 5 B. Colombo, C. Vesco a n d C. Baglioni, Proc. Natl. Acad. Sci. U.S., 61 (1968) 651. 6 M. R o s b a s h a n d S. P e n m a n , J. Mol. Biol., 59 (I97 x) 227. 7 C. ]3aglioni, I. Bleiberg a n d M. Zauderer, Nature, New Biol., 232 (i97 I) 8. 8 E. A. IZabat a n d M. M. ~ a y e r , Experimental Irnmunochemistry, Thomas, Springfield, [11., I961. 9 13. L. M. H o g a n a n d A. K o r n e r , Biochim. Biophys. Acta, 169 (I968b) I39. io C. 1V[. R e d m a n a n d D. D. Sabatini, Proc. Natl. Acad. Sci. U.S., 56 (I966) 608. 1i D . D . Sabatini, G. Blobel, Y. N o n o m u z a a n d M. R. A d e l m a n , in F. C l e m e n t i a n d E. T r a b u c c h i , i2 13 14 15 16 17 18 19 20 21 22
Proceeding of the Ist International Symposium on Cell Biology and Cytopharmacology, Raven Press, New York, I969. C. D. Mitchell a n d D. J. H a n a h a n , Biochemistry, 5 (1966) 51. D. D. Sabatini, Y. T a s h i r o a n d G. E. P a l a d e , J. Mol. Biol., 19 (1966) 503. B. A t t a r d i , G. Cravioto a n d G. A t t a r d i , J . Mol. Biol., 44 (I969) 47. R. P. P e r r y , Proc. Nail. Acad. Sci. U.S., 48 (1962) 2179. S. P e n m a n , C. Vesco a n d M'. P e n m a n , J. Mol. Biol., 34 (1968) 49. E. Zylber, C. Vesco a n d S. P e n m a n , J. Mol. Biol., 44 (I969) 195. P. Vassalli, Proc. Natl. Acad. Sci. U.S., 58 (1967) 2117. B. L i s o w s k a - B e r n s t e i n , M. E. L a m m a n d P. Vassali, Proc. Natl. Acad. Sci. U.S., 66 (197 o) 425, R. P. P e r r y a n d D. E. Kelly, J. Mol. Biol., 35 (1968) 37. M. R o s b a s h a n d S. P e n m a n , J. Mol. Biol., 59 (1971) 243. C. Vesco a n d B. Colombo, J. Mol. Biol., 47 (197 °) 335.
Biochim. Biophys. Acta, 269 (1972) 453-464