Peptidyl-puromycin synthesis by free and membrane-bound ribosomes

454

Biochimica et Biophysica Acta, 563 (1979) 454--465

© Elsevier/North-Holland Biomedical Press

BBA 99468 PEPTIDYL-PUROMYCIN SYNTHESIS BY FREE AND MEMBRANE-BOUND RIBOSOMES

LEROY KUEHL and WENDELL ROBISON Department of Biochemistry, University of Utah College of Medicine, Salt Lake City, UT 84108 (U.S.A.)

(Received August 16th, 1978) (Revised manuscript received January 16th, 1979) Key words: Peptidyl-transferase; Puromycin; Ribosome; (Membrane-bound)

Summary

The peptidyl transferase reaction, as measured by the formation of peptidylpuromycin, was compared for free ribosomes and ribosomes bound to two types of membrane, the endoplasmic reticulum and the outer nuclear membrane. In most respects the reaction catalyzed by the three types of ribosome was similar, demonstrating that interaction of the 60 S ribosomal subunit with the membrane has little effect on the functioning of peptidyl transferase, a 60 S protein. However, both the rate and extent of synthesis of peptidyl puromycin were lower for ribosomes bound to the nuclear membrane than for free or microsome-bound ribosomes. This difference appears to be a direct consequence of the ribosome-membrane interaction, since ribosomes stripped from the nuclear membrane could n o t be distinguished from the other classes o f ribosome.

Introduction

In eukaryotic cells polysomes may occur either free in the cytosol or bound to various intracellular membranes. Although a number of investigators have studied ribosome-membrane interactions (reviewed in Ref. 1--4), little attention has been given to the effect of membrane binding on the functioning of the ribosome, and those studies which have been done have been concerned primarily with a comparison of the overall rates of amino acid incorporation between free and membrane-bound ribosomes and have not been in good agreem e n t one with another. In the present paper we compare the peptidyl transferase reaction for free ribosomes and ribosomes attached to two types of mem-

455 brane, the endoplasmic reticulum and the outer membrane of the nuclear envelope. Experimental procedure Chemicals. [ M e t h o x y - 3 H ] P u r o m y c i n • 2 HC1 (3.82 Ci/mmol) was obtained from New England Nuclear. RNAase-free sucrose was purchased from Schwarz/ Mann. Electrophoretically purified, RNAase-free, pancreatic DNAase was a product of Worthington, as was three-times-crystallized pancreatic RNAase. Crystalline trypsin from bovine pancreas was from PL Biochemicals. Assays. Protein was determined by the method of Lowry et al. [5] as described previously [6]. For samples containing Triton X-100, 2.5% sodium dodecyl sulfate was added to reagent A [7]. RNA was precipitated from the samples to be assayed with 10% trichloroacetic acid (final concentration), washed three times with 5% trichloroacetic acid, and hydrolyzed by incubating for 1 h at 37°C with 0.3 M NaOH. The hydrolysates were neutralized with HCI, and the protein and DNA were precipitated with 10% trichloroacetic acid and sedimented. Aliquots of the resulting supernatants were taken for RNA determination by the method of Mejbaum [8]. DNA was determined in the pellets remaining after removal of the RNA. These were suspended in 5% trichloroacetic acid, and the DNA was hydrolyzed b y heating for 15 min at 90°C. The hydrolysates were centrifuged and aliquots o f the supernatants were taken for DNA determination by the method of Burton [9]. Mg, Ca and Mn concentrations were determined with a Jarrell-Ash Model 975 inductively coupled argon plasma spectrometer. Samples to be analyzed were solubilized with sodium dodecy] sulfate (0.3% final concentration) or were digested with H2SO4 at 190°C with the occasional addition of a drop of 30% H202 until the solutions were clear. Analysis of RNA on sucrose gradients. RNA was isolated by the method of Ritossa and Spiegelman [I0] and dissolvedin 0.01 M sodium acetate, pH 5.1. Portions (0.1--0.4 m]) of the RNA samples were layered on 10--40% linear sucrose gradients containing 0.1 M NaCl/1 mM EDTA/10 mM sodium acetate, pH 5.1 [11] and centrifuged at 0°C for 15 h at 156 000 Xgay in Beckman rotor SW 41. Gradients were analyzed at 260 nm using a Gilford Mode] 2000 recording spectrophotometer equipped with a flow cell of 0.2 cm light path. Animals. Male Holtzman albino rats weighing 250--350 g were kept on a schedule of alternating periods of light (14 h) and dark (10 h) and were fasted for 16--20 h prior to killing, which occurred early in the light period. Preparation of nuclear membranes. Rats were killed by a blow on the head, and the livers were removed and homogenized in 2 vols. of cold 0.25 M sucrose/Buffer I, pH 7.7 (Buffer I is 50 mM Tris/25 mM KCI/5 mM MgCI2; pH values of buffer solutions were measured at 22°C) by 60 strokes in a PotterElvehjem homogenizer rotating at 1700 rev./min. The clearance between the glass homogenizing vessel and the plastic pestle was 0.01 inch. The homogenate was filtered through three layers of cheesecloth, and centrifuged 15 min at 600 Xg to yield a nuclear pellet and a postnuclear supernate. The nuclear pellet

456 was suspended in approximately 2 vols. of 2.3 M sucrose/Buffer I, pH 7.7. Portions of this suspension having a volume of 28 ml were layered over 10 ml portions of 2.1 M sucrose/Buffer I, pH 7.7, and centrifuged in Beckman rotor SW 27 for 1 h at 63 000 × gay. The purified nuclei were washed twice with 0.25 M sucrose/1 mM MgC12 by suspension and centrifugation (15 min at 38 000 × g), and the washed nuclei were used to prepare nuclear membranes as described by Kay et al. [12], except that the DNAase digests were diluted into 0.1 mM MgC12 instead of water. Preparation of perinuclear ribosomes. Nuclei prepared by centrifugation through dense sucrose as described above were washed twice with 0.25 M sucrose/Buffer I, pH 7.7, and the washed pellets were used to prepare perinuclear ribosomes as described by Sadowski and Howden [13] except that cell sap was omitted from the Triton solutions. The final ribosomal pellets were rinsed with 0.1 MgCl2 and suspended in a small amount of buffer (usually Buffer I, pH 7.2). Preparation of rough microsomes. The postnuclear supernatant prepared as described above was centrifuged 10 min at 17 000 × g to yield a postmitochondrial supernatant. Step gradients containing 7 ml of 2.0 M sucrose/Buffer I, pH 7.7; 7 ml of 1.38 M sucrose/Buffer I, pH 7.7; and 12 ml of postmitochondrial supernatant were prepared and centrifuged in Beckman rotor Ti 60 for 20 h at 105 000 ×gay. The rough microsomes, which collected above the 2.0 M sucrose layer, were diluted with t w o volumes of 0.25 M sucrose/1 mM MgC12, and sedimented (15 min at 38 000 × g). The pellets were resuspended in the same solution and recentrifuged. The resulting microsomal pellets were then subjected to the same procedure used to isolate nuclear membranes from the nuclear pellets to insure that the history of both fractions was comparable. Isolation of ribosomes from the microsomal fraction. The microsomecontaining layers of the step gradients described above were collected and diluted with 2 vols. of Buffer I, pH 7.7. Sufficient 10% sodium deoxycholate was added to give a final concentration of 0.8%, and the resulting solutions were centrifuged in Beckman rotor Ti 60 for 80 min at 105 000 × ga,,. The ribosomal pellets were rinsed with 0.1 mM MgC12 and dissolved in a small a m o u n t of buffer (usually Buffer I, pH 7.2). Preparation of free ribosomes. The free ribosomes formed pellets in the step gradients used to prepare rough microsomes. These pellets were rinsed with 0.1 mM MgC12 and dissolved in a small amount of buffer (usually Buffer I, pH 7.2). Preparation of crude elongation factor II. Postnuctear supernatant (see above) was centrifuged in Beckman rotor Ti 50 for 90 min at 116 000 × ga~. The upper two-thirds of the supernatant was collected and brought to 80% saturation with ammonium sulfate (pH 7.5). The precipitate was collected, dissolved in Buffer I, pH 7.2/1 mM dithiothreitol and dialyzed overnight against several portions of the same solution. Peptidyl-[3H]puromycin synthesis. The transpeptidation reaction was compared for free and membrane-bound ribosomes by measuring conversion of [3H]puromycin to peptidyl-[3H]puromycin, as described by Pestka [14]. A standard reaction mixture contained the following components in 0.1 ml: 250 mM KCI; 5 mM MgC12; 50 mM Tris°HC1, pH 7.2; a b o u t 0.1 mg of free or

457 membrane-bound ribosomes; and 2 tiM [3H]puromycin. [~H]Puromycin was added last to start the reaction, which was usually allowed to proceed for 1 min at 22°C before being terminated by addition of 2 ml of cold 10% trichloroacetic acid. The assay for [3H]puromycin incorporation into trichloroacetic acid-precipitable material followed the method of Pestka [14] except that the reaction products were collected on Whatman glass-fibre filters (GF/C), the final two ethanol washes were performed after the filters had been removed from the filter apparatus and placed in counting vials, and the ethanol for the penultimate wash contained 100 t~M unlabelled puromycin. The washed filters were incubated for 30 min at 50°C with 0.5-ml portions of NCS reagent (Amersham/Searle) to solubilize the labelled peptidyl puromycin, and the samples were then made up to 20 ml with scintillation solution consisting of 4.0 g 2,5-diphenyloxazole (PPO) and 0.1 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene (POPOP) per 1 of toluene. Counting was done in a Packard Tri-Carb liquid scintillation spectrometer with automatic external standardization. Results

Characterization of subcellular fractions. RNA, protein and DNA contents of the various subcellular fractions used in the present investigation were determined and sucrose gradient analyses of the RNA extracted from these fractions were performed. Results of these analyses, which are in good agreement with those of other investigators, were used to estimate the rRNA content of each

freeribosomes

[ ~

0

~

membrones

I0 2O IncubatioTinm(rai e n)

3O

Fig. 1. T i m e c o u r s e of p e p t i d y l - [ 3 H ] p u r o m y c i n s y n t h e s i s , E a c h 0 . 1 - m l r e a c t i o n m i x t u r e c o n t a i n e d t h e f o l l o w i n g c o m p o n e n t s : 50 m M Tris-HCl, p H "1.2; 2 5 0 m M KCI; 5 mM MgCI 2 ; 4 ~M [ 3 H ] p u r o m y c i n ; free or membrane-bound ribosomes e q u i v a l e n t t o 1.23 A 2 6 0 u n i t s r R N A . I n c u b a t i o n s were performed at 2 2 ° C for the t i m e s i n d i c a t e d o n t h e figure.

458 ribosome and membrane preparation used in this investigation. Most fractions contained negligible DNA and little non-ribosomal RNA. The nuclear membrane fraction, however, had a DNA/RNA ratio of 1.32 ± 0.08 {Mean +_SE) and 21% of the RNA of this fraction moved to the 4--5 S region of the sucrose gradients compared with a value of 10% for free ribosomes. Per±nuclear ribosomes, also, had a significant content of DNA (DNA/RNA ratio = 0.13 ± 0.04) and 19% of the RNA of this fraction appeared in the 4--5 S region on sucrose gradients. Time course o f the reaction. Release of nascent polypeptide chains from free and membrane-bound ribosomes by [3H]puromycin as a function of time is illustrated in Fig. 1. Although the same a m o u n t of 18 S + 28 S rRNA was present in each reaction mixture of Fig. 1, less peptidyl-[3H]puromycin was formed by the nuclear membrane fraction than by endoplasmic reticulum or by free ribosomes. For ten preparations, the n u m b e r of pmol of peptidyl-[3H] puromycin formed per min per A260 unit of 18 + 28 S RNA under the reaction conditions given in the legend to Fig. 1 was 0.283 ± 0.122 for the nuclear membranes; 0.380 ± 0.123 for the microsomes; and the 0.432 +_0.168 for free ribosomes. The difference in activity between the nuclear membranes and either of the other two fractions was significant at P < 0.001 when the matched pair data were analyzed by Student's t-test. To control the possibility that this difference might have been due to a systematic overestimation of the ribosome concentration in the nuclear membrane fraction, membrane-bound and free ribosomes were also compared by pulse labelling rats with [14C]leucine in vivo, releasing the nascent polypeptide chains from the various ribosome classes in vitro with unlabelled puromycin, and determining the fraction of the total radioactivity released. Comparisons made by this method, which does not require that the concentration of ribosomes in each fraction be accurately known, confirmed that nascent polypeptide chains on the per±nuclear ribosomes were refractory to release by puromycin compared to those of free or endoplasmic reticulum bound ribosomes (data not shown). Effect o f treatment with hydrolytic enzymes. That preferential attack of nuclear-membrane bound ribosomes by proteolytic or nucleolytic enzymes during their preparation is not the explanation for their low activity in peptidyl-puromycin synthesis, is suggested by experiments in which free and membrane-bound ribosomes were incubated both in the presence and absence of exogeneous hydrolytic enzymes and the activities of the treated ribosomes were compared with those of unincubated controls. When ribosomes were suspended in the buffers used in their preparation and stored at 0°C for periods of up to a week, they gradually lost activity. However, under comparable incubation conditions the fractional loss of activity was similar for free ribosomes and both classes of membrane-bound ribosomes. Preincubation with DNAase or RNAase (1 yg/0.1 ml incubation mixture) did n o t affect the activity of the ribosomes. Preincubation with trypsin had no effect on either class of membrane bound ribosomes, but reduced the activity of the free ribosomes. In one experiment, for example, preincubation for 10 min at 22°C with 1 pg o f enzyme per 0.1 ml of incubation mixture reduced the activity of the free ribosomes to 28% of control. This effect is presumably due to digestion of the

459 nascent polypeptide chains which, in the case of membrane-bound ribosomes, are protected [15]. Effect of elongation factor II. Nascent polypeptides are reactive with puromycin only when they occupy the peptidyl site on the ribosome. To determine whether ribosomes bound to the nuclear membrane are less reactive because a greater proportion of their nascent polypeptide chains is in the aminoacyl site, an experiment was performed in which the various classes of ribosome were incubated with GTP and elongation factor II prior to treatment with puromycin (Table I, Expt. 1). The increase in puromycin reactivity following elongation factor II t r e a t m e n t was similar for the two membranebound classes of ribosome and much less than for free ribosomes. When ribosomes stripped from both types of membrane and free ribosomes were compared (Table I, Expt. 2), no significant difference was found, suggesting that the concentration of nascent polypeptide chains in the aminoacyl site was the same for ribosomes from each source and that differences between membranebound and free ribosomes are due to an inhibition of translocation resulting from the membrane-ribosome interaction. These experiments, which have been repeated several times, fail to support the hypothesis that ribosomes bound to the nuclear membrane are less reactive because a greater proportion of their nascent polypeptide chains is in the amino acyl site.

Activity of membrane-bound ribosomes after release from the membranes. When nuclear membrane-bound ribosomes are released from the membrane with Triton X-100 and sodium deoxycholate, they are as reactive to puromycin as are free ribosomes and ribosomes derived from the endoplasmic reticulum (Fig. 2), indicating that the lowered reactivity of ribosomes attached to the

TABLE I E F F E C T OF P R E T R E A T M E N T RIBOSOME CLASSES

W I T H E L O N G A T I O N F A C T O R II ON A C T I V I T I E S O F V A R I O U S

E a c h 0 . 1 - m l r e a c t i o n m i x t u r e c o n t a i n e d t h e f o l l o w i n g c o m p o n e n t s : 50 m M Tris-HCl, p H 7.2; 2 5 0 m M KC1; 5 m M MgC12; 36 m M 2 - m e r c a p t o e t h a n o l ; 0.36 m M G T P ; c r u d e e l o n g a t i o n f a c t o r II ( E F 2 ) ( a p p r o x . 1 m g t o t a l p r o t e i n ) ; 2 ~M [ 3 H ] p u r o m y c i n ; a n d u n b o u n d o r m e m b r a n e - b o u n d r i b o s o m e s c o n t a i n i n g 0 . 9 6 ( E x p t . 1) o r 0 . 8 2 ( E x p t . 2) A 2 6 0 u n i t s o f r R N A . E l o n g a t i o n f a c t o r II, G T P , a n d 2 - m e r c a p t o e t h a n o l w e r e o m i t t e d f r o m t h e c o n t r o l s a m p l e s . All c o m p o n e n t s o f t h e r e a c t i o n m i x t u r e e x c e p t [ 3 H ] p u r o m y c i n ( 2 0 pl) w e r e m i x e d a n d i n c u b a t e d 2 rain at 22°C. P u r o m y c i n w a s t h e n a d d e d a n d i n c u b a t i o n was c o n t i n u e d f o r 1 rain at 22°C b e f o r e t e r m i n a t i n g t h e r e a c t i o n . P r e i n c u b a t i o n w i t h e l o n g a t i o n f a c t o r II o r 2 - m e r c a p t o e t h a n o l a l o n e did n o t i n c r e a s e p e p t i d y l - [ 3 H ] p u r o m y c i n f o r m a t i o n o v e r c o n t r o l v a l u e s ; p r e i n c u b a t i o n w i t h G T P a l o n e r e s u l t e d in a small i n c r e a s e ( a v e r a g e 12%).

Peptidyl- [ 3 H] p u r o m y c i n formed (pmol)

P e r c e n t of control

Control

+EF2

Expt. 1 (ribosomes b o u n d to m e m b r a n e s ) Nuclear membranes Microsomes Free ribosomes

0.19 0.38 0.28

0.31 0.52 0.72

163 137 257

Expt. 2 (ribosomes stripped from membranes) Perinuclear ribosomes Microsomal ribosomes Free ribosomes

0.26 0.30 0.23

0.60 0.62 0.50

230 207 217

460

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InculxllioaTime(rain)

Fig. 2. T i m e c o u r s e o f p e p t i d y l - [ 3 H ] p u r o m y c i n synthesis by released ribosomes. Each 0.1-ml reaction m i x t u r e c o n t a i n e d t h e f o l l o w i n g c o m p o n e n t s : 5 0 m M T r i s - H C l , p H 7 . 2 ; 2 5 0 rnM KC1; 5 m M MgC12; 2 ~ M [ 3 H ] p u r o m y c i n ; free o r r e l e a s e d r i b o s o m e s e q u i v a l e n t t o 0 . 8 0 A 2 6 0 u n i t o f r R N A . I n c u b a t i o n s w e r e p e r f o r m e d a t 2 2 ° C f o r t h e t i m e s i n d i c a t e d o n t h e figure. P e r i n u c l e a r r i b o s o m e s , L~--~; m i c r o somal ribosomes, c ©; free r i b o s o m e s , ~ ~.

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Fig. 3. D o u b l e r e c i p r o c a l p l o t o f t r a n s p e p t i d a t i o n a c t i v i t y vs. p u r o m y c i n concer~tration. E a c h 0 . 1 - m l r e a c t i o n m i x t u r e c o n t a i n e d : 5 0 m M Tris-HCl, p H 7 . 2 , 2 5 0 m M K C I ; 5 m M MgCI 2 ; free o r m e m b r a n e b o u n d r i b o s o m e s e q u i v a l e n t t o 1 . 2 4 A 2 6 0 u n i t s o f r R N A ; a n d p u r o m y c i n as i n d i c a t e d ; i n c u b a t i o n w a s at 22°C. Usually four reaction mixtures were prepared for each substrate concentration and incubated a t t i m e s r a n g i n g f r o m 0 t o 2 . 0 r a i n . I n i t i a l v e l o c i t i e s d e r i v e d f r o m t h e r e s u l t i n g d a t a are p r e s e n t e d in t h e figure.

461 nuclear membrane is a consequence o f their interaction with the membrane rather than to a unique feature of the ribosomes themselves. Kinetics. Double reciprocal plots of puromycin concentration versus peptidyl puromycin synthesis (Fig. 3) demonstrate that the lower activity of perinuclear ribosomes as compared with free or endoplasmic reticulum-bound ribosomes is due to the lower V of the former class o f ribosome, the apparent Km values being the same for all ribosome classes. Effect of pH. pH vs. activity profiles were similar for free ribosomes and for both types of membrane-bound ribosome; in all three cases the pH o p t i m u m for the reaction was at 8.5 and activity was nearly abolished below pH 7 and above 10 (data n o t shown). Effect of monovalent cations. Free ribosomes and both classes of membraneb o u n d ribosome respond similarly to monovalent cations (Fig. 4), which they require for activity. Of the ions tested, potassium was most effective, sodum less active, ammonium only slightly effective, and lithium inactive in supporting peptidyl puromycin synthesis. The optimal concentration of sodium and ammonium chlorides was a b o u t 0.6 M, whereas the optimal potassium ion concentration had not yet been reached at 1.0 M, a concentration sufficient to begin dissociating ribosomal proteins. Effect of divalent cations. Peptidyl puromycin synthesis by both free and membrane-bound ribosomes is stimulated by Mg 2÷ and Mn 2÷ and inhibited by

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Fig. 4. E f f e c t o f m o n o v a l e n t c a t i o n s o n p e p t i d y l - p u r o m y c i n s y n t h e s i s , E a c h 0 . l - m l r e a c t i o n m i x t u r e c o n t a i n e d 5 0 m M Tris-HCI, p H 7 . 2 ; 5 m M MgCI 2 ; 2/~M [ 3 H ] p u r o m y c i n ; free or m e m b r a n e - b o u n d r i b o s o m e s e q u i v a l e n t t o 1 . 3 0 A 2 6 0 u n i t s o f r R N A ; a n d m o n o v a l e n t c a t i o n s as i n d i c a t e d . I n c u b a t i o n s w e r e f o r 1 m i n at 2 2 ° C . F r e e r i b o s o m e s (fr), ~ o; m i c r o s o m e s ( m ) , o o; n u c l e a r m e m b r a n e s ( n m ) , ~ A.

462

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Fig. 5. E f f e c t o f d i v a l e n t c a t i o n s o n p e p t i d y l - p u r o m y c i n s y n t h e s i s . E a c h 0 . 1 - m l r e a c t i o n m i x t u r e c o n t a i n e d 5 0 m M Tris-HC1, p H 7 . 2 ; 2 5 0 m M KC1; 2 ~tM [ 3 H ] p u r o m y c i n ; f r e e o r m e m b r a n e - b o u n d r i b o s o m e s e q u i v a l e n t t o 1 . 1 8 A 2 6 0 u n i t s o f r R N A ; a n d d i v a l e n t c a t i o n s as i n d i c a t e d . D u e t o t h e Mg 2+ p r e s e n t in t h e s u b c e l l u l a r f r a c t i o n s , t h e C a 2+ a n d M n 2+ s a m p l e s c o n t a i n e d i n a d d i t i o n t o t h e i n d i c a t e d i o n s , 0 . 1 m M MgC12. I n c u b a t i o n s w e r e f o r 1 m i n a t 2 2 ° C . F r e e r i b o s o m e ( f r ) , o ~; m i c r o s o m e s ( m ) , o o; nuclear membranes (nm), ~ ~.

Ca 2÷ (Fig. 5). However, the loss of peptidyl transferase activity u p o n decreasing the Mg 2÷ and Mn 2÷ concentration was observed to be much greater for free ribosomes than for ribosomes b o u n d to either the endoplasmic reticulum or to the nuclear membrane so that at low concentrations of divalent cations membrane-bound ribosomes were appreciably more active than u n b o u n d ones. The apparent resistance of membrane-bound ribosomes to lowered concentrations of divalent cations is artifactual. Nuclear membranes and rough endoplasmic reticulum isolated as described contain b o u n d magnesium, and a large percentage of this is released from the membranes under the incubation conditions used in the peptidyl transferase reaction (Table II), The magnesium released from the membranes in a standard reaction mixture is sufficient to yield a final concentration o f approximately 1 mM, a level sufficient to account

463 TABLE

II

CONCENTRATIONS

O F M g IN M E M B R A N E - B O U N D

AND

FREE

RIBOSOMES

In Expt. 2 one portion of each m e m b r a n e fraction was assayed without treatment. A second portion was incubated under conditions routinely e m p l o y e d to study peptidyl pttromycin synthesis except that [ 3H]p u r o m y c i n was omitted and the M g C I 2 concentration was 0.1 raM. Following incubation, the mixture was centrifuged 15 m i n at 38 0 0 0 × g to sediment the m e m b r a n e s : the supernatant and pellet were assayed separately. /~g Mg 2+ p e r m g r i b o s o m e s Expt. 1 Nuclear membranes Microsomes Free ribosomes Expt. 2 Nuclear membranes (untreated) Nuclear membranes (incubated) Supernatant Pellet Total Microsomes (untreated) Microsomes (incubated) Supematant Pellet Total Free ribosomes

23.0 17.8 5.0 24.8 21.9 5.0 26.9 16.2 13.7 1.1 14.8 2.8

for the differential effects of added Mg 2+ on free and membrane-bound ribosomes. As would be expected, ribosomes stripped from either the microsomes or nuclear membranes behave the same as free ribosomes with respect to Mg 2+ concentration (data n o t shown). The molar concentration of calcium in the membrane fractions is less than 5% that of magnesium; the concentration of manganese is negligible. Discussion

The results of the present investigation demonstrate that peptidyl puromycin formation by ribosomes b o u n d to membranes is generally similar to that catalyzed b y free ribosomes, although several minor differences were observed b e t w e e n the two ribosome classes. The resistance of endoplasmic reticulum b o u n d ribosomes to trypsin pretreatment is well known [15--17] and is due to the fact that polypeptide chains synthesized by these ribosomes are inserted into, and move vectorially across, the membrane into the lumen of the endoplasmic reticulum as synthesis proceeds. The observation that ribosomes attached to the nuclear envelope display a b o u t the same resistance to trypsin as do those b o u n d to the endoplasmic reticulum suggests that polypeptide chains synthesized b y perinuclear ribosomes become associated with the outer nuclear membrane in much the same way that polypeptides produced by endoplasmic-reticulum b o u n d ribosomes become associated with the microsomal membrane. This hypothesis is consistent with the observation o f Gorovsky [18] that nascent polypeptides associated with ribosomes b o u n d to the outer membrane o f isolated Tetra-

464

hymena nuclei remain associated with the nuclei upon completion or upon premature release by puromycin. Still another consequence of the membrane-ribosome interaction seems to be an inhibition of the translocation reaction catalyzed by exogenous elongation factor II. Further studies will be required to determine the significance of this observation. A consistent observation during the present investigation was that ribosomes bound to the nuclear membrane were less active in peptidyl puromycin synthesis than either free ribosomes or those bound to microsomes. The nuclear membrane-bound ribosomes are probably even less active than the data suggest since the isolated nuclear membranes are u n d o u b t e d l y contaminated with microsomes [19]. The lower activity of ribosomes bound to the nuclear membrane is a consequence of the binding, since ribosomes stripped from either the nuclear envelope or the endoplasmic reticulum have the same activity as free ribosomes. Kinetic experiments have failed to show a significant difference in the Km for puromycin between free ribosomes and the two membrane-bound ribosome classes. A hypothesis consistent with these observations is that a portion of the ribosomes which bind to the nuclear membrane are thereby inactivated. It might be suggested that all ribosomes bound to the nuclear membrane are refractory to puromycin and that the puromycin-reactive ribosomes in nuclear membranes preparations are due entirely to microsomal contamination. This, however, would require that, on the average, three-fourths of the ribosomes in the nuclear membrane preparations were due to microsomal contamination. On the basis of the electron microscopic and enzymic analyses of Kay et al. [ 12 ], this level of contamination appears highly unlikely. We have previously shown t h a t incorporation of labelled amino acids into nuclear proteins in vivo is less sensitive to inhibition by puromycin than is incorporation into cytoplasmic proteins [20,21]. This observation taken together with data of Gorovsky [18] suggesting that the polypeptides synthesized by perinuclear ribosomes are destined for transport into the nucleus suggests that the nascent polypeptide chains of perinuclear ribosomes might be particularly resistant to release by puromycin. The observations of the present paper are consistent with this hypothesis. Peptidyl puromycin synthesis by total polysomes from deoxycholate-treated homogenates of rat liver has been studied previously by Pestka and co-workers [14]. In general, the results which we have obtained using free ribosomes agree well with those of Pestka et al. However these authors reported t h a t maximal stimulation of peptidyl-puromycin synthesis by NaC1 was not achieved even at Na ÷ concentrations of 1.2 M, whereas we find a sharp optimum at about 0.6 M NaC1. Also, although our curve relating transpeptidation to magnesium ion concentration is superficially similar to t h a t obtained by Pestka et al., these authors obtained a straight line when their data were converted to a Hill plot, whereas the data of our Fig. 5 yield a Hill plot with a distinct negative curvature, especially at low Mg 2÷ concentrations.

Acknowledgements The authors thank Dr. Alan S. Peck for performing the metal analyses. This work was supported by National Institutes of Health Grant G M 13864.

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