In vitro exchange of ribosomal subunits between free and membrane-bound ribosomes

J. Mol. Bid

(1973) 74, 415438

In vitro Exchange of Ribosomal Subunits between Free and Membrane-bound Ribosomes DOMINICA BOECIESE,GIJNTER BLOBEL mu DAVID D. SABATINI~ The Rockefeller University, New York, N.Y. 10021, U.X.A. (Received 7 July 19Y2) Studies on the distribution of isotopically labeled ribosomal subunits between free and membrane-bound ribosomes from rat liver showed that, upon release of nascent polypeptides in vitro, the small subunits of membrane-bound ribosomes could exchange with small subunits derived from free polysomes. However, under the same conditions, the large subunits of membrane-bound ribosomes did not exchange efficiently with large subunits derived either from free or bound polysomes; instead, the addition of large subunits caused a transfer of microsomal small subunits into a newly formed pool of free monomers. The small subunit exchange required a macromolecular fraction of the cell sap, was stimulated by ATP or GTP, and occurred at low concentrations of magnesium ions. Sodium dodecyl sulfate, polyacrylamide gel eleotrophoresis revealed close similarities between the protein complement of subunits from free and membranebound ribosomes, with the exception of one protein band which was more intense in free large subunits.

1. Introduction Ribosomes consist of two unequal subunits which undergo cyclic dissociation and association during protein synthesis. The existence of a subunit cycle was implicit in the finding that the small ribosomal subunit of bacterial ribosomes first partioipates in the formation of an initiation complex with messenger RNA (Nomura t Lowry, 1967; Ghosh & Khorana, 1967), and only subsequently joins the large subunit to form an active monomer (Nomura et al., 1967). Direct evidence for the operation of the subunit cycle, in viva and in vitro, in bacteria (Kaempfer et al., 1968; Kaempfer, 1968) as well as in eukaryotic cells (Kaempfer, 1969; Ceccarini et al., 1970; Jacobs-Lorena & Baglioni, 1970; Howard et al., 1970; Palvey & Staehlin, 1970) was obtained by subunit exchange experiments which demonstrated the formation of ribosome hybrids from differently labeled ribosome populations. In many eukaryotic cells a large proportion of cytoplasmic ribosomes are bound to membranes of the endoplasmic reticulum (Palade, 1956). In hepatocytes it has been shown that approximately 75% of the ribosomes are membrane-bound (Blobel & Potter, 1967a) and that the binding is mediated by the large ribosomal subunits (Sabatini et al., 1966 ; Shelton & Kuff, 1966 ; Florendo, 1969). The similarities in structural and metabolic properties (Loeb et al., 1967 ; Talal & Kaltreider, 1968; t Present address: New York University, N.Y. 10016, U.S.A. 2s

School of Medicine, 550 First Avenue, New York, 415

416

D. BORGESE,

G. BLOBEL

AND

D. D.

SABATINI

Tanaka et al., 1970) of free and membrane-bound ribosomes suggest that in the cell, ribosomes may change between the free and the bound state. However, the possibility that the two types of ribosome are interchangeable has not been tested directly in vitro. In order to elucidate this question, we have investigated whether the subunits of membrane-bound ribosomes undergo a dissociation-association reaction similar to that of free ribosomes, and whether the large subunit membrane complex undergoes a comparable reaction. To this purpose, we have conducted in vitro exchange experiments under conditions where there is little net detachment of ribosomes from membranes even after release of nascent chains by puromycin (Adelman et al., 1970, 1973b). The results show that upon release of nascent polypeptides small subunits of membrane-bound ribosomes become exchangeable; a comparable exchange for large subunits, however, could not be demonstrated.

2. Materials and Methods (a) General All solutions were prepared using deionized distilled water, were Millipore-filtered (0.45 pm for most, 1.2 pm for cone. sucrose stock solutions) and were stored in the cold. The notation TriseHCl will be used for Tris*HCl, pH 7.6 at 20°C. Tris/K/Mg buffer is 50 m&r-Tris*HCl, 25 mm-KCl, 5 mm-MgCl,, High-salt buffer is 50 mm-Tris*HCl, 500 mMKCl, 5 mM-MgCl,. All centrifugations, unless otherwise specified, were carried out in an IEC B60 centrifuge (International Equipment Co., Needham Heights, Mass.). (b) Fractionation (i) Rough microsomes

and labeling

of liver

cells

and free polysomes

These were prepared by a method recently developed by Adelman et al. (1973a). The procedure involves the separation, by discontinuous sucrose gradient centrifugation, of free polysomes, smooth and rough miorosomes from a post-mitochondrial supernatant containing more than 50% of the membrane-bound ribosomes of the tissue. Rough microsomes, collected from the discontinuous sucrose gradient, were sedimented (15 min, 35,000 revs/m& A211 rotor) after dilution with 6 vol. of a modified high-salt buffer (containing 10 mM-MgCl, and 0.25 M-SUCrOSe). This high-salt wash was introduced to obtain a microsome fraction containing only tightly bound ribosomes. The pellets of washed rough microsomes were resuspended in 0.25 M-sucrose/Tris/K/Mg buffer (15 to 2.0 mg RNA/ml). Samples of the suspension (1 ml) were diluted with 2 vol. of glycerol and stored at - 20°C for up to two months. Before use, the suspensions were diluted with 2 vol. Tris/K/Mg buffer, and the microsomes recovered by oentrifugation (15 min, 40,000 revs/min, A321 rotor). (ii) The G50 supwnatant

fraction

This was used for amino acid incorporation in vitro and was obtained from 1:2 w/v homogenates prepared in 0.25 M-sucrose/Tris/K/Mg buffer. The high-speed supernatant, obtained after two centrifugations (20 min, 25,000 revs/mm, A211 rotor: 2 h, 44,000 revs/ min, A21 1 rotor), was passed through a G50 Sephadex column equilibrated with Tris/K/Mg buffer containing 1 mm-dithiothreitol. The material excluded from the column (G50 supernatant fraction) was collected and stored in l-ml fractions (~14 mg protein/ml) at -80°C for up to 2 weeks. (iii)

Ribosomal subunits

These were prepared from free polysomes (Blobel & Sabatini, 1971) or rough microsomes (Adelman et al., 1970) as previously described. Microsomes or polysome suspensions were incubated for 10 mm at 37’C with puromycin (low3 M) in a modified high-salt buffer containing 2.5 mM-IV@&. Fractions (1.65 ml) were layered onto 33-ml linear sucrose

EXCHANGE

BETWEEN

FREE

AND

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417

gradients (5% to ZO%T), containing high-salt buffer, which were centrifuged (5 h, 25,000 revs/rain, SBllO rotor) at 20°C. The gradients were withdrawn from the top by an Auto Densiflow probe (Buchler Instruments, Fort Lee, N.J.) connected to a peristaltic pump and passed through a U.V. analyzer (LKB Uvicord II, type 8303A; LKB-Produkter AB, Bromma-1, Sweden) attached to a recorder. The fractions corresponding to the 40 S and 60 S subunit peaks were collected separately, diluted 1: 1 with Tris/K/Mg buffer and sedimented by an overnight centrifugation at 3°C and 40,000 revs/min in a Spin00 no. 40 rotor. The pellets were rinsed with deionized water and stored for up to a month at - 80°C. (iv) Free polysomes

and rough microsomes labeled in the ribosomal RiVA

These were prepared from rats which received 200 to 250 &!i of 3H-labeled erotic acid intraperitoneally 36 to 40 h before saoritlce. The specific radioactivity in ribosomes was 6 x lo5 to 1.2 x lo6 disints/min/mg RNA. We will refer to ribosomes and rough microsomes labeled in this way as 3H-labeled rough microsomes or 3H-labeled ribosomes.

(c) Polyacrylamide

gel electrophoreeis

Electrophoresis of ribosomal proteins was carried out in acrylamide, sodium dodecyl sulfate gels (12.5% aorylamide; ratio of acrylamide to bisaorylamide = 30 : 0.8) according to Maize1 (1971). A vertical electrophoresis cell (E-C Apparatus Corp., St. Petersburg, Fla.) provided with eight slots was used with slabs 3 mm thick. After eleotrophoresis the gels were stained in Coomassie brilliant blue. (d) Subunit exchange The exchange was followed either (a) by adding 3H-labeled small or large subunits to rough microsomes or (2) by adding non-labeled subunits to 3H-labeled rough microsomes. Since in case (1) a transfer of radioactivity from free subunits to rough microsomes was assayed, we will refer to the exchange detected in this way as exchange-in. Conversely, we will refer to the exchange detected in case (2) as exchange-out. Incubation was carried out either in a medium optimal for amino acid incorporation or in a minimal exchange medium. The amino acid incorporation medium contained in one ml : 1 pm01 ATP, 0.5 pm01 GTP, 10 pm01 phosphoenolpyruvate, 5 ~1 pyruvate kinase, 25 ~1 ammo acid supplement (12.5 ~1 of a soln of essential amino acids at molarities 10 times those described by Eagle (1959) except for leucine, which was present at 0.14 mM, plus 12.5 ~1 of a soln of non-essential amino acids, each at 1.0 m@, 50 &i n-[4,5-3H]leucine, 150 CJ. (~2 mg protein) G50 supernatant fraction, 150 pmol NH&Cl, 20 pm01 Tris.HCl, 5 pm01 MgClz, 0.15 ~01 dithiothreitol, 250 pm01 sucrose, and rough microsomes corresponding to 0.5 to I.0 mg RNA. The composition of minimal exchange medium is described in Results, section (b). After incubation for 10 mm at 37”C, the subunit distribution was determined by sucrose density gradient analysis in the SB283 rotor. All gradients were run at 20°C to avoid the formation of subunit aggregates in the cold. Sucrose concentrations as well as centrifugation conditions were chosen so that microsomes were either banded isopycnically in the lower part of the gradient or sedimented into a pellet. The latter procedure was followed when a better resolution between subunits and monomers was desired. Details of gradient analysis are given in the Figure legends. Profiles of the optical density at 254 run throughout the gradients were recorded as described in section (a) (iii), above. The effluent from the monitoring system was collected in glass conical tubes in O-6-ml fractions. The pellets in the gradient tubes were resuspended in water and quantitatively transferred to glass conical tubes. All fractions, cooled to O”C, received 1 mg of bovine serum albumin as carrier and 2 ml of ice-cold 10% trichloroacetic acid. After 15 min, the resulting precipitate was sedimented by low-speed centrifugation. Supernatants were withdrawn and the acid-insoluble material was dissolved in 0*5 ml of NCS (see section (f), below) solubilizer. Samples were transferred to glass sointillation vials and counted with 8 ml of toluene/Liquifluor as scintillator. Efficiency for t o/o Sucrose will be used for o/o sucrose w/v.

418

D. BORGESE.

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AND

D. D. SABATINI

tritium was 30% to 35%. All values were corrected for background. Recovery of T&Oactivity in the gradient fractions ranged from 85% to 95%. In all sedimentation profiles the top of the gradient is on the left, and the large point at the right represents radioactivity recovered in the pellet. For qua&it&ion of the distribution of U.V. absorbing material, areas under the optical density tracings were cut out and weighed. RNA was determined by a modified Schmidt-Tannhauser procedure (Fleck & Munro, 1962), using E:& = 312 at 260 nm (Munro & Fleck, 1966). In most cases, however, ribosome concentmtions were determined directly, using Ef& = 135 at 260 nm (Tashiro & Siekevitz, 1965) and correcting for ferritin (Jackson et al., 1964). To estimate the ribosomal content of microsomal suspensions, fractions were made 0.5% in deoxycholate and read at 260 nm against a 0*5% deoxycholate blank. Estimates thus obtained agreed well with those from RNA determinittions. (f ) Mate&& Male albino rats (100 to 160 g) of the Sprague-Dawley strain, maintained on a Purina Chow diet, were fasted for 15 to 20 h before sacrifice. Chemicals were obtained from the following sources: dithiothreitol, puromycin dihydrochloride and cycloheximide (actidione) from Nutritional Bioohemioals Corp., Cleveland, Ohio; [5-3H]orotic acid (1 mCi/O*156 mg) and Liquifluor from New England Nuclear, Boston, Mass. ; equine muscle ATP (disodium salt), GTP type IIS, (trisodium salt) and sodium dodecyl sulfate from Sigma Chemical Co., St. Louis, MO.; phosphoenolpyruvate (trisodium salt) and pyruvate kinsse (2230 international units/ml) from Calbiochem., San Diego, C&f. ; amino acids, n-[4,5-3H]leucine (40 to 50 Ci/mmol) and L-(14C]leucine (316 mCi/mmol) from Schwarz Bioresearch Inc., Orengeburg, N.Y. ; sodium deoxycholate from Matheson, Coleman and Bell, Cincinnati, Ohio; Sephadex G50 medium from Pharmaoia, Piscataway, N.J. ; NCS solubilizer from Amersham/Searle, Arlington Heights, Ill. ; Coomassie brilliant blue from Schwarz/Mann, Orangeburg, N.Y. ; acrylamide, N,N,N’,N’tetramethylethylenediamine and N,N’-methylenebisacrylamide from Eastman Kodak Co., Rochester, N.Y.

3. Results (a) Exchange betweenfree and microsomal small subunits Ribosomes squire the ability to exchange their subunits only after the release of their nascent chains. In the in vitro subunit exchange experiments with eukaryotic ribosomes reported in the literature, chain release has been achieved either naturally following read-out of mRNA (Falvey & Staehlin, 1970), or artificially after the action of puromycin (Kaempfer & Meselson, 1969). In the present study for release have been used. In rough microsomes, however, natural

both procedures release was pre-

viously found to be ineficient, comprising only ~20% of the nascent chains (Sabatini & Blobel, 1970), whereas puromycin-induced release was found to be almost complete under the ionic conditions generally used for amino acid incorporation (Adelman et al., 1973b). Furthermore, it has been shown that neither natural (Redman & Sabatini, 1966; Blobel & Potter, 1967b) nor puromycin-induced chain release carried out at moderate ionic strength (Adelman et al., 19736) lead to a concomitant release of ribosomes from membranes. When membrane-bound ribosomes were allowed to release their nascent chains in the presence of added small subunits derived from free polysomes (ratio of added small subunits to miorosomal small subunits ~1 :I) in an exchange-in assay (see Materials and Methods), the results shown in Figure 1 and Table 1 were obtained. After only partial chain release, resulting from natural termination in the amino

EXCHANGE

BETWEEN

FREE

AND

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419

RIBOSOMES

M

RM

t

1

/5000

4000

3000

-2 \2 i? 1 .g

i : 2000 d

IO00

0 Volume (ml) (b)

(ci

1. Exchange of added SH-labeled smell subunits with the smell subunits of membrenebound ribosomes in an amino acid incorporation medium. Incubation mixtures contained in 1 ml; 11.2 O.D .ses units rough microsomes and 2.2 o.D.~~,, units 3H-lebeled smell subunits (spec. ect., 22,400 cts/min/o.n.,,, unit). After the incubetion, O&ml samples were layered onto IS% to 60% sucrose gradients, containing 150 mu-NH,Cl, 20 mnn-Tris*HCl and 5 miid-MgCls, which were centrifuged 2 h 36 min, 40,000 revs/min, SB283 rotor. S, small ribosomal subunit; M, ribosomal monomer; RM, rough microsomes. -. ) (e) No inhibitors; (b) plus 10e3 M-pU.rOmyCin; (c) plus 10-e M-cycloheximide. ( Optical density; (--a--@--) 3H radioaotivity. FIU.

acid incorporation medium, -20% of the added labeled small subunits were shifted to the rough microsomes (Fig. l(a), Table 1). However, a much larger shift occurred after the more complete puromyoin-induced chain release (Fig. l(b), Table 1). Furthermore, the optical density peak of small subunits was not reduced after puromycin addition, that is, the small subunits (labeled) which were transferred to rough microsomes were replaced by an equivalent amount of small subunits (not labeled) released from rough microsomes. Therefore, we concluded that exchange between free and microsomal small subunits had occurred. It should be noted (Fig. 1) that some ribosomal monomers were detached from rough miorosomes in the incubation medium. In the presence of puromycin (Fig. l(b)) this detachment was somewhat increased, with a resulting shift in the isopycnic position of rough microsomes to a lighter region of the gradient. The presence of radioactivity in the monomer peak (Fig. l(b), Table 1) indicates the participation of the free monomers as well as of the membrane-bound ribosomes in the small subunit exchange. When natural chain release was reduced, following an 80% inhibition of amino acid incorporation by cyoloheximide (data not shown), there was a -2Oo/o inhibition of the shift of labeled small subunits to rough microsomes with respect to the sample incubated without added drugs (Fig. l(c) versus l(a), Table 1). This apparently low

420

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SABATINI

TAESLE1 Distribution of radioactivity (ctslmin) in .sucroSegradients after incubafiolz of 3H-labeled small subunits with rough microsomes in amino acid incorporation medium Region

of gradient

Top fractions 40 s Monomers Miorosomes Pellet Total ots/min

No inhibitors 1028 (5%) 12,421 (61%) 356 (4%) 3966 (20%) 2053 (10%) 20,315 (100%)

+Puromycin

(10m3 M)

+ Cyoloheximide

1209 (6%) 8677 (41%) 2470 (12%) 7420 (35%) 1802 (7%) 21,278 (100%)

See legend to Fig. 1 for experimental

( 10m2 M)

1279 (7%) 12,549 (65%) 961 (5%) 3108 (16%) 1428 (7%) 19,225 (100%) details.

inhibition of the exchange by cycloheximide could be explained by a high background binding of small subunits to microsomal membranes, which would decrease the sensitivity of the exchange-in assay. Background binding, however, should not interfere with the exchange-out assay, which follows the release of labeled microsomal subunits caused by addition of non-labeled competing subunits. The results of an exchange-out experiment are summarized in Table 2. The first column shows the amounts of labeled small subunits released from rough microsomes into the 40 S region in the absence of added small subunits (“basal ” release). The second column of the Table shows the amounts of small subunits released when added competing subunits (in a 1 :l ratio) were present. The difference between the second and the 6rst column (A cts/min) represents the amount of small subunits released specifically by addition of competing subunits and is taken as a measure of exchange. It can be seen that the exchange occurring upon natural termination (1st row) was inhibited by cycloheximide by about 60% (3rd row). Puromycin, on the other hand (2nd row), in addition to causing a larger basal release of microsomal small subunits, produced an exchange TABLET Eflect of added non-labeled small subunits on the release of 3H-.?ubeledsubunits from rough microsomes in amino acid incorporation medium

Condition

3H (ots/min) -Non-labeled S

of incubation

No drug +h.rOmyOiIt

+Cyoloheximide

(lom3

M)

(lob2

M)

1298 3786 1124

in 40 S region +Non-labeled 2826 7946 1750

S

d ots/min 1528 4160 626

Incubation mixtures contained in 1 ml; 8.0 0.D .26,, units of 3H-labeled rough miorosomes unit), with or without the addition of 3.3 0.D.260 units of un(speo. act. 22,200 ots/min/o.n.,,, labeled small subunits. Composition of the sucrose gradients and conditions of oentrifugation were the came as described in Fig. 1. The Table shows the radioactivity in fractions corresponding to the 40 S region. Total radioactivity recovered in each gradient was ~75,000 ots/min. S is small ribosomal subunit.

EXCHANGE

of more than drugs. Thus, Furthermore, the inhibition

BETWEEN

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421

twice as many subunits as in the samples incubated without added these results confirm those obtained from the exchange-in assay. since background binding does not interfere in an exchange-out assay, of the exchange by cycloheximide was now clearly deteded. (b) Requirements for the puromycin-induced

exchange

The previous results indicate that, under conditions optimal for amino acid incorporation, puromycin promotes small subunit exchange between bound ribosomes and added subunits. To elucidate the mechanism of the puromycin-induced exchange, we attempted to establish its minimum requirements by omitting components of the amino acid incorporation medium. The results are given in Table 3. The difference (A cts/min) in the ambunt of radioactivity in the presence and absence of puromycin found in the region comprising rough microsomes and monomers is taken as a measure of the extent of the exchange. TABLE 3 Requirements jar puromycin induced exchange of 3H-lubeled small subunits with rough microsomes Experiment n0.t

lb) (b) 2(a) (b) (cl 3(a)

(b) 4(a)

(b) 5(a)

(bj Ccl

idi 6W (b) (cl

Condition Complete system -&&$ --&a ---aa-PEP-PK --aa,-PEP-PK 1.5 miaMgCla as 2(c) a,s 2(c) but no G50 fraction ---a& ---&a + heat-treated G50 fraction§ as 2(c) as 2((tj but no ATP as 210) but no GTP as 2icj but no ATP no GTP as B(b) as 5(b) but no GTP as 5(b) but no GTP and 1.0 mM-Mgcl,

3H (ots/min) in monomer-microsome region -Puromycin +Puromycin

A cts/min

2313 2822 1556 2539 2513

4910 6081 3261 2836 4175

2597 2259 1705 297 1662

3047 3652

5754 4167

2707

3081 2115

5144 2527

2063 412

4568 3862 3364 3323

7598 7826 6671 5550

3012 3964 3309 2227

4472 4338 4636

9513 7644 7755

5041 3307

615

3119

Samples were analyzed on sucrose gradients as described in the legend to Fig. 1. Radioactivity (cts/min) was measured in the pooled fractions from the monomer-microsome regions of the gradients. For each row, d ots/min indicates the difference between radioactivities in the presence and absence of puromycin. t Spec. act. of small subunits was: exp. 1, 17,700 cts/min/o.n.,,, unit; exp. 2, 11,045 cts/min/ o.D.~~,, unit; exp. 3, 23,500 cts/min/o.r, .260 unit: exp. 4, 19,800 ots/min/o.n.,,, unit; exps. 5 and 6, 22,500 cts/mm/o.n.zso unit. Total radioactivity recovered on gradients was: exp. 1, -10,800 cts,/min; exp. 2, -8400 cts/min; exp. 3(a), ~10,800 cts/min; exp. 3(b), ~8750 cts/min; exp. 4(a), ~11,000 cts/min; exp. 4(b), ~8100 cts/min; exp. 5, ~14,800 cts/min; exp. 6, ~16,100 cts/min. Inputs of rough microsomes were: exps. 1 and 2, 4.0 0.D.2e0 units; exp. 3, 4.5 o.D.~~~ units; exps. 4 and 6, 3.0 o.D.~~~ units; exp. 6, 5.5 O.D.~~~ units. $ aa, amino acids: PEP, phosphoenolpyruvate; PK, pyruvate kinase. 5 55"C, 15 min.

422

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Experiment 1 shows that the presence of amino acids is not required for the puremycin-induced exchange. Omission of phosphoenolpyruvate and pyruvate kinase, on the other hand, resulted in a large inhibition of small subunit exchange (exp. 2). Phosphoenolpyruvate, however, is a chelating agent; therefore, its function in promoting subunit exchange at 5 nmr-Mg2 + may be due to a decrease in the effective Mg2 + concentration rather than to a supply of chemical energy. For this reason we examined whether exchange might be restored in the absence of phosphoenolpyruvate and pyruvate kinase by lowering the Mg2+ concentration. A series of Mg2+ concentrations was tested, and it was found that between 1.0 and 2-O m&r-MgCl, the exchange did indeed occur in the absence of phosphoenolpyruvate and pyruvate kinase. The results for 15 mM-MgCl, are shown in Table 3 (exp. Z(c)). Omission of the G50 fraction of the high speed supernatant greatly decreased the puromycin-induced exchange (Table 3, exp. 3). The stimulatory activity in this fraction is thermolabile (exp. 4), since preincubation of the G50 fraction at 55°C for 15 minutes reduced its ability to promote the exchange. The presence of either ATP or GTP (Table 3, exps 5 and 6) stimulated the exchange, but the effects of the nucleoside triphosphates were not additive. It is, therefore, unlikely that they act simply by chelating Mg2 + . Moreover, lowering the Mg2 + concentration to 1.0 mM in the absence of ATP and GTP did not restore the exchange to the control value (exp. 6). Because of these observations, 0.5 m&r-GTP was kept as a component of the minimal exchange medium. Other parameters, not shown in Table 3, were also investigated. It was found that no further exchange occurred when incubation was extended for more than five minutes. Incubation for 30 minutes at 0°C produced only 15% of the exchange which occurred at 37°C. The exchange was not affected by increasing the amount of G50 supernatant fraction. Raising the monovalent ion concentration (200 m&r-NH&l instead of 150 mM) produced a larger detachment of monomers from membranes and thus led to a proportional increase of small subunit exchange into the pool of free monomers. The total extent of the exchange, however, was not altered. From the results described above, a minimal exchange medium for the puromycininduced exchange was designed. It contained 0.5 mM-GTP, 150 mM-NH&l, 20 mMTris-HCl, I.5 mM-MgCl,, 0.25 M-sucrose, and 0.15 ml-G50 supernatant fraction per ml incubation medium. The requirement for the G.50 fraction deserves further analysis. A comparison of Figure 2(b) and (d) demonstrates the large stimulatory effect of the G50 fraction on small subunit exchange. However, from Figure 2(c) and (d) it can be seen that the sedimentation properties of small subunits were altered in the absence of the 650 fraction. About half of the subunits sedimented at a faster rate, indicated by a second peak in the sedimentation profile, which may correspond to dimers of the 40 S particles. Moreover, the supernatant fraction served to protect small subunits from degradation during incubation, since there was more radioactivity at the top of the gradient, and the recovery of radioactivity was consistently lower (~20%) when the G50 supernatant fraction was omitted. For these reasons it could not be decided if the role of the G50 fraction in promoting exchange was due entirely to its effect iu protecting the integrity of the small subunits and preventing their aggregation, or if an additional exchange-promoting macromolecular component exists in this fraction. From Figure 2(a) and Table 3 it can be seen that in minimal exchange medium, even in the absence of puromyoin, there was membrane-associated radioactivity.

EXCHANGE

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423

S M RM II

1

(0) .-

I

1000

3000

zoo0

loo0

; \ 2 0 02 .? rj .-z 73 E

S M II

S M RM

RM

2

Ill

1

(cl

(dl 4000

3000

ti ’ : 3I-5

2000

IO00

0

Volume (ml1

2. Effect of G50 supernatant fraction on the exchange of small subunits induced by puromycin in minimal exchange medium. Incubation mixtures contained in 1 ml; 9.0 O.D .260 units of rough microsomes and 1.1 o.D.~S,, units of 3H-labeled small subunits (spec. act. 23,400 Ci%/min/0.D.2So unit). After incubation, O&ml samples were layered onto sucrose gradients. Composition of the gradients and conditions of oentrifugation were as described in the legend to Fig. 1. (a) and (c) no puromycin; (b) and (d) 10e3 M-puromycin; (a) and (b) with G50 supernatant fraction; (c) and (d) no G50 supernatant fraction. S, M and RM as in legend to Fig. 1. ( ) Optical density; (--e--e--) 3H radioactivity. FIG.

424

D. BORGESE,

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SABATINI

Since in minimal exchange medium the protein synthetic apparatus is non-operative, this membrane-bound radioactivity was most probably due to background binding and not to exchange. To investigate the nature of the membrane-bound material, a “product analysis ” of rough microsomes after incubation for exchange-in was carried out as follows. After incubation in minimal exchange medium with 311-labeled small subunits in the presence or absence of puromycin, rough microsomes were separated from free subunits and monomers and incubated in high-salt buffer with puromycin to detach membrane-bound ribosomes as 40 S and 60 S subunits (Adelman et al., 1970). The results of this experiment are shown in Figure 3. It can be seen that labeled small subunits bound to microsomes in the absence of puromycin (Fig. 3(b)) were degraded, aggregated or could not be released from microsomal membranes, because after high-salt buffer/puromycin treatment the radioactivity was recovered mainly in the upper fractions of the gradient or in the pellet. Therefore, we concluded that the membrane-associated radioactivity found after incubation in minimal exchange

Control

+ 40s

subunlis

+ 405 subunirs + puromycin

zoo0

7t

.L t

e 1500

IO00

: >r .z .> t x23 I 10

500

0 Volume (ml) (b)

(cl

3. KCl/puromycin analysis of rough microsomes recovered after incubation in minimal exchange medium with 3H-labeled small subunits. Incubation mixtures contained in 1 ml; 9.5 0.D . 260 units of rough miorosomes. Samples (b) and (c) contained in 1 ml; 2.0 0.DsZ6,, units of 3H-labeled small subunits (spec. act. 23,500 cts/min/ o.~.~s~ unit). Sample (c) contained low3 M-pUrOmyCin. Volumes of the incubation mixtures were 5 ml. After incubation eaoh sample was layered onto two 106ml sucrose grrtdients containing ranges were: (a) and 150 mM-i%B,Cl, 20 mnn-Tris*HCl, 5 rnM-MgCl,. The sucrose concentration (b) 15% to 60%; (c) 15% to 55%. After centrifugation (1 h, 40,000 revs/mm, SB283 rotor) the membrane bands from the three samples were collected from the lower third of the gradient, diluted 1: 1 with TKM buffer and sedimented into pellets by centrifugation (15 min, 59,000 revs/min, A321 rotor) at 3°C. The microsome pellets were resuspended in water and a compensating buffer was added so that the final composition of the microsome suspensions was high-salt buffer with 10e3 M-puromyoin. The samples were then incubated for 10 min at 37”C, after which O&ml samples were layered onto the final snslytical 10% to 30% sucrose gradients in high-salt buffer. Centrifugation (3.5 h, at 40,000 revs/min, SB283 rotor at ZOW). L, large ribosomal subunit; ) Optical density; (--m--e--) SH radioactivity. S, small ribosomal subunit. ( FIG.

EXCHANGE

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RIBOSOMES

426

medium in the absence of puromycin was due to non-specific adsorption of altered small subunits and not to exchange. On the other hand, the presence of puromycin in minimal exchange medium increased the amount of membrane-associated radioactivity, and the additional labeled small subunits were recovered mainly as intact 40 S particles after high-salt buffer/puromycin treatment (Fig. 3(c)). From the specific activities of the 40 S particles in Figure 3(c) and of the initially added labeled small subunits it can be computed that in this experiment (in which the ratio of added to microsomal small subunits was ~1: 1) labeled added small subunits represented, after exchange, -20% of all the membrane-associated small subunits. Since the ratio of large to small membrane-associated subunits was unchanged with respect to a control (incubated in minimal exchange medium in the absence of puromycin and without added subunits (Fig. 3(a)), we concluded that the replacement of released small subunits by undegraded added small subunits was stoichiometric. (c) Quantitation of the exchange The extent to which the small subunits of membrane-bound ribosomes are exchangeable in minimal exchange medium was determined in an exchange-out assay. A constant amount of 3H-labeled rough microsomes was incubated with increasing amounts of free small subunits. It can be seen from Figure 4 that, in the presence of puromycin, the percentage of microsomal small subunits released was enhanced by increasing the ratio of competing to microsomal small subunits. In the absence of puromycin, on the other hand, addition of unlabeled small subunits to the system had no effect on the release of microsomal small subunits. These results, therefore corroborate the conclusion reached from the product analysis experiment (Fig. 3), that is, that in minimal exchange medium small subunit exchange is entirely dependent on the presence of puromycin.

0

2

4

Added

4OS/

6 ‘H-lob&d

8

10 405

FIG. 4. Effect of addition of cold subunits and puromycin on the release of sH-labeled small subunits from rough microsomes in minimal exchange medium. Incubation mixtures contained in 1 ml; 7.5 O.D .260 units of 3H-labeled rough miorosomes (spec. act. 11,000 cts/min/o.n.,,, unit). Small subunits were added in the amounts indioated. The ratios of added subunits to miorosomal small subunits, as well as the extent of microsomal subunit release, were calculated by assuming a molecular weight ratio of 2.6 of 28 S to 18 S RNA (Loening, 1968). After incubation for 10 min at 37”C, 0.5-ml samples were layered onto 15% to 30% sucrose gradients containing 160 mM-NH,Cl, 20 m&r-Tris*HCl, 5 mnn-MgCla, which were centrifuged (2 h, 45 min, 40,000 revs/min, SB283 rotor). The ordinate represents radioactivity recovered in pooled fractions corresponding to the small subunit optical density peak, as percentage of total radioactivity reoovered on the gradient multiplied by 3.6. -@--aWith lo-” M-pUr0Iqk.n; --A--A--no puromycm.

426

D. BORGESE,

G. BLOBEL

AND

D. D. SABATINI

From Figure 4 it can also be seen that, in the presence of puromycin, with increasing amounts of added competing subunits, the release of microsomal small subunits approached a limit value of 60% of the total microsomal small subunits. Roughly 60% of the expected equilibration was in fact attained with all ratios of added to microsomal small subunits. l?or example, with a ratio of added to microsomal small subunits of 1: 1, 30% of the microsomal small subunits were released (50% would have been released if complete equilibration between small subunits had been attained). It is likely that the exchange does not reach completion because of rapid inactivation of the system during incubation at 37°C. The occurrence of ribosome degradation is in fact shown in Figure 3. (d) Experiments with kzrge subunits Since ribosomes are attached to membranes via the large subunits (Sabatini et al., 1966), it was of special interest to investigate whether the bonds between ribosomes and membranes would impose any restriction on the exchangeability of large subunits. Exchange-in and exchange-out experiments were therefore carried out as in the case of small subunits. The results of an exchange-in experiment (added subunits: microsomal subunits =1 : 1) are shown in Figure 5, in which profiles from experiments with added labeled small subunits are also included for comparison. l?rom an examination of Figure 5(c) and (d) it can be seen that addition of puromycin to the system containing added large subunits derived from free polysomes had the following effects.

(1) Reduction in both the optical density and radioactivity

of the 60 S peak (L in Figure 5(c) versus (d)), indicating a large decrease in the amount of added large subunits which remained as free 60 S particles. This is in marked contrast to the results obtained with small subunits where a slight increase in the optical density of the 40 S peak occurred upon addition of puromycin (Fig. 5(a) versus (b), see also Fig. 1). The 60 S subunits which remained free after incubation had approximately the same or only slightly reduced specific activity as the initially added subunits. Therefore the incubation with puromycin led to an association of some of the added large subunits with other components of the system with little or no replacement by microsomal large subunits.

(2) A large increase in the amount of monomers, as indicated by the optical density profiles in the 80 S region. This increase was considerably larger (at least twice) than the one observed in the presence of puromyoin and small subunits (Fig. 5(d) versus (b)). As indicated by the radioactivity peak in the 80 S region (peak M in Fig. 5(d)) the free monomers contained labeled, added large subunits which must have associated with unlabeled small subunits released from the rough miorosomes. This transfer of small subunits of membrane-bound ribosomes to a monomer pool should be expected from their exchangeability, demonstrated in section (a), and from the fact that ribosomes are attached to the membranes via large subunits only. (3) A shift of labeled large subunits to rough miorosomes, which was only approximately half of that observed for small subunits if the data are compared on a molar basis (i.e. taking into account the factor of 2.6 for the molar ratio of 28 S to 18 S RNA (Loening, 1968)). Since the shift was small and not accompanied

LM

RM I (c)

II

LM II

RM I (d

Volume (mil

FIG. 5. Puromycin-induced exchange of 3H-labeled small and large subunits with membranebound ribosomes in minimal exchange medium. Incubation mixtures contained in 1 ml; 10.2 O.D .260 units of rough microsomes. Samples (a) and (b) contained in 1 ml; 1.9 0.D.2e0 units of 3H-labeled small subunits; samples (c) and (d) contained in 1 ml; 3.76 o.D.~~~ units of 3H-labeled large subunits obtained from free polysomes. Spec. act. of the subunits w&s 23,700 cts/min/o.o .seOunit. After incubation, O.&ml samples were layered onto sucrose gradients containing 150 mu-NH,Cl, 20 mnir-TriseHCl, 5 m-MgCl,, which were centrifuged (2 h, 36 miu, 40,000 revs/min, KS283 rotor). The sucrose concentration ranges of the gradients were: (a) and (c) 15% to 60%; (b) 15% to 55%; (d) 15% to 60%. S, M, L and RM &8 in legend to Fig. 3. (a) and (c) No puromycin; (b) and (d) 10m3 M-puromyoin. ) Optical density; (--O--O--) 3H radioactivity. ( In order to facilitate the comparison of the data for small and large subunits, the radioactivity scale for (a) and (b) has been expanded by a factor of 2.6 with respeot to the scale for (c) and (d).

428

D. BORGESE,

G. BLOBEL

AND

D. D. SABATINI

by an obvious decrease in the specific activity of free large subunits (see above), it was dif&uilt to decide whether a significantly large subunit exchange had occurred. In order to obtain quantitative data on large subunit exchange, an exchange-out assay was carried out as follows, 3H-labeled rough microsomes were incubated in minimal exchange medium with puromycin, either in the absence or in the presence of a sixfold excess of free large subunits. As can be seen in Figure 6(b), even in the presence of this large excess of competing large subunits only 9% of the microsomal large subunits were recovered in the 60 S region of the gradient. This should be contrasted with the 50 to 60% of microsomal small subunits which can be released by adding excess free small subunits (Fig. 4). From a comparison of Figure 6(a) and (b) it can also be seen that addition of large subunits to the microsome-puromycin system led to the formation of free monomers (peak &I). These monomers were hybrids of added unlabeled large subunits and labeled microsomal small subunits released from the membranes, as is apparent from the low specific activity of the monomers in Figure 6(b), compared to that of the pure labeled monomers in Figure 6(a).

3 11 IE

,o-

\ 1.5-

9

I

o-

FIG. 6. Effect of added unlabeled large subunits on the release of 3H-labeled subunits from rough microsomes in minimal exchange medium. Incubation mixtures contained in 1 ml; 4.75 o.D.~~~ units of “H-labeled rough microsomes (spec. act. 6200 cts/min/o.n.zso unit). Sample (b) contained in 1 ml; 208 o.D.~~,, units of large subunits. After incubation, the samples were diluted by addition of an equal volume of 150 nn~NH,Cl, 20 mM-Tris*HCl, 1.5 mM-MgCl,, and 0.4-ml samples were layered onto 5% to 20% sucrose gradients containing 150 mm-N&Cl, 20 mM-Tris.HCI, 5 mivr-Mgcl,, which were centrifuged (10 h, 18,000 revs/min, SB283 rotor). Centrifugation at this low speed was chosen to avoid a pressure-induced dissociation of inactive monomers (Infante & Baierlein, 1971; Inf&nte & Erauss, 1971; Infante & Graves, 1971), which we observed at higher centrifugal fields. ( ) Optical density; (--e--m--) 3H-radioactivity. S, L & M as in legend to Fig. 3.

EXCHANGE

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FREE AND BOUND RIBOSOMES

429

Examination of Figure 6(a) shows that in the absence of added subunits, there was a preferential release of small subunits over large subumts from rough microsomes. It can be calculated that 40% of the miorosomal small subunits, but only 7% of the microsomal large subunits, were released. The release of microsomal small subunits was larger than in other experiments (see Fig. 4), presumably because in this experiment the microsomes were less concentrated than previously (see legend to Fig. 6). Because in association-dissociation equilibria, dilution causes a shift in the equilibrium in favor of the dissociation product, the release of small subunits from diluted microsomes treated with puromycin provides an independent confirmation of the results on the dissociation-association reaction occurring between subunits of membrane-bound ribosomes (sections (a), (lo) and (c) above). The much smaller amount of 60 S particles released (more than fivefold smaller) indicates that the binding constant of large subunits to the membrane is considerably higher than that of small subunits to membrane-bound large subunits. The results of this experiment indicate that in the in vitro system, added large subunits can remove small subunits from rough microsomes, but that an exchange of free and bound large subunits comparable to the exchange of small subunits does not occur. The large subunit exchange appears to be at most one-fifth of the small subunit exchange. This is a maximal estimate, since the labeled large subunits released into the 60 S region could also be accounted for by exchange of the added subunits with the labeled monomers detached from rough microsomes (peak M in Fig. 6(a)). The possibility that the added large subunits derived from free polysomes do not compete with microsomal large subunits for membrane binding sites as efficiently as would large subunits derived from membrane-bound polysomes was ruled out by an exchange-in experiment (not shown), which demonstrated that both types of large subunit had identical behavior in the exchange system. It should also be noted that in experiments carried out in the complete incorporation medium (not shown) the behavior of the large subunits was similar to their behavior in minimal exchange medium. In order to corroborate the results described above, a product analysis of rough microsomes after incubation for exchange-out was performed. “H-labeled rough microsomes were incubated in minimal exchange medium with puromycin and a fivefold excess of unlabeled small or large subunits. The control sample consisted of labeled rough mierosomes incubated with puromycin and no added subunits. The rough microsomes were recovered from the incubation medium and re-incubated in high-salt buffer/puromycin. This treatment, however, resulted in release of only 50% to 60% of the microsomal RNA into the 40 S and 60 S regions of the gradient, as opposed to the 85 to 90% which can be released from untreated rough microsomes (Adelman et al., 1970). The distribution of radioactivity in the subunits is plotted in Figure 7, in which the data have been corrected for equal inputs of microsomal membranes on the gradients (see legend). It can be seen that added small subunits and large subunits were equally effective in removing bound small subunits from microsomes (-50% with respect to the control without added subunits). The effect of added small and large subunits on removal of large subunits (22% and 35% of control, respectively) was considerably weaker than their effect on small subunits. Most likely the decrease of large subunits after incubation with either subunit was unspecific, and the difference between the effect of each subunit was not significant since the puromycin/KCl procedure was not equally effective in each case.

430

D. BORGESE,

G. BLOBEL

5

IO Fraction

AND

15

20

D. D.

SABATINI

Pellet

no.

FIG. 7. KCl/puromycin analysis of 3H-labeled rough microsomes recovered after incubation in minimal exchange medium. Incubation mixtures contained in 1 ml; 6.7 O.D .e6e units of 3H-labeled rough microsomes (speo. act. 12,800 cts/min/o.n.,e, unit) and 10e3 M-puromyoin. The total volumes of the incubation mixtures were 4.0 ml. After inoubetion, in the presence or absence of added unlabeled subunits, 0.26-ml portions of each sample were layered onto 15% to 30% sucrose grsdients containing 150 m&r-NH,Cl, 20 mnn-Tris.HCl, 5 mi%r-Mgcl, for direct antlysis of the incubation mixtures. From this analysis it was learned that, after incubation with small or large subunits, the amount of radioactivity which remained membrane-associated was 72% of the radioactivity which remained membrane-bound in the control, incubated with puromycin but without subunits. The rem&ing 3.75 ml of the incubation mixtures were layered onto ll&ml 15% to 55% sucrose gmdients oontaining 150 mu-NH,Cl, 20 mm-Tris.HCl, 6 mM-MgCl,, to recover the microsomes, as described in the legend to Fig. 3. The microsomal pellets were resuspended in O-6 ml water and incubated in high-salt buffer/puromycin for detachment of ribosomes, as described in the legend to Fig. 3. Samples (0.6 ml) of the high-salt incubation mixtures were layered onto the final amlytical sucrose gradients. Composition of the gradients and conditions of centrifugation were as described in the legend to Fig. 3. --a--+No added subunits in minimal exchange medium; --A--A--, 11.5 O.D.ZB~ units of small subunits/ml in minimal exchange medium; * * * n * . . n * * a, 26.5 O.D.,,, units of large subunits/ml in minimal exchange medium. The radioactivity value of each experimental point within the gmdient and in the pellet (A, n ) hrts been multiplied by a normalizing factor which makes the total radioactivity in each case correspond to 72% of the control (0). In this manner small differences have been compensated for in the recovery of miorosomes after incubation for exchange and the data are directly comparable on the basis of equal input of microsomal membranes in the final analytical gradient. The inset shows optical density profiles ( ) corresponding to --A--A-(a), and . . . n . . . n * 9 . (b).

Optical density profiles corresponding to radioactivity profiles have been plotted as an inset in Figure 7 for the samples incubated with added subunits. Table 4 compares the radioactivity and optical density ratios of large to small subunits from data in this Figure. It is clear that only in the case of pre-incubation with small subunits do these ratios differ significantly from each other. This difference indicates that subunit exchange occurred, since competing non-labeled small subunits replaced

EXCHANGE

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431

RIBOSOMES

microsomal labeled small subunits. This resulted in an unaltered optical density ratio but in an altered radioactivity ratio with respect to the control. In the case of preinoubation with large subunits, on the other hand, both ratios were changed with respect to the control sample, but did not differ significantly from each other. Therefore, in this case released labeled subunits were not replaced by competing unlabeled subunits, with a resulting excess of large over small subunits on the membrane. TABLET Radioactivity and [email protected] density ratios oj’ large to small subunits releasd from rough microsome recovered after incubation for exch4mge Sample

Control “H-labeled rough microsomes 3H-labeled rough microsomes + small subunits 3H-labeled rough microsomes + large subunits

Optical

Large/small density Radioactivity 2.6 2.3 3.2

2.4 3.4 3.0

The radioactivity ratios were calculated from the d&a plotted in Fig. 7. The optical ratios were estimated from the corresponding areas in the optical density profiles.

density

Although in this experiment only 50 to 60% of the membrane-associated subunits could be analyzed-most likely because of the deleterious effect of pre-incubationthe results are consistent with the inability of large subunits to exchange and with the interpretation that the main effect of added large subunits is to cause the net removal of microsomal small subunits into a newly formed monomer pool. (e) Exchange of small and large subunits with free polysomes The previous results demonstrate that upon natural or puromycin-induced release of peptide chains, membrane-bound ribosomes can undergo extensive exchange of small subunits with added subunits derived from free polysomes. However, a comparable exchange was not observed for large subunits. To establish if this difference is specifically due to the fact that in bound ribosomes large subunits are attached to the membranes, or rather to a general incompetence of our preparations of large subunits, we investigated whether small and large subunits are equally efhcient in exchanging with a system of free polysomes. An exchange of mouse small subunits with rat liver f
(cl

(b)

s

M

I

I

d)

L

M

1

i

IO00

0 Volume

(ml)

FIG. 8. Exchange of small and large subunits with free polysomes in the amino acid incorporation medium. Free polysomes, resuspended in 150 m~-NH,cl, 20 mM-Tris.HCI, 5 IIIM-&$&., were re-purified by centrifugation at 3°C (5 min, 10,000 revs/m& A321 rotor) to eliminate heavy aggregates. The resulting supernatant was layered over a 4-ml cushion of 2 M-SUCrOSe containing 150 mnoNH&l, 20 mnx-Tris.HCl, 5 mnn-MgCl, and centrifuged (2 h, 59,000 revs/min, A321 rotor) at 3°C. The polysomes in the resulting pellet were used for the exchange experiment. Each incubation mixture contained in 1 ml: 2.6 O.D.%m units purified polysomes. Samples (a), (b) and (c) contained in 1 ml; 1.32 O.D.26o units 3H-labeled small subunits; samples (d), (e) and (f) contained in 1 ml, 2.95 o.D.~~,, 3H-labeled large subunits. Spec. act. of the subunits w&s 15,000 cts/min/o.n.,,, unit. After incubation, 0.4-ml samples were layered onto 5% to 20% sucrose gradients, containing 150 mu-NH,Cl, 20 mm-Tris*HCI, 5 mivr-M&l,, whiah were centrifuged (10 h, 18,000 revs/min, SB283 rotor). (c) and (f) 10e2 M-oyoloheximide. (a) and (d) no inhibitors; (b) and (e) 10e3 M-puromycin; S, L and M as in legend to Fig. 3. ) Opt&l density; (--@--a--) 3H radioactivity. (

PLATE I. Electrophoresis of subunits obtained from free and bound ribosomes on 12.5% polyacrylamide, sodium dodecyl sulfate gels. Free polysomes and rough microsomes were washed once in high-salt buffer before high salt/ o.D.~~~ units bound small subunits; puromycin treatment to obtain subunits. Slot (a), -0.4 slot (c), -1.25 O.D.Z~~ units bound large subunits; slot (b), NO.5 0.D.260 units free small subunits; o.D.~~~ units free large subunits. slot (d), -1.5

f.faeing

p. 43.’

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433

monomers was essentially complete. A comparison of the radioactivity patterns in Figure 8 showed an increase in the amount of labeled added subunits shifted to the monomer peak, which paralleled the increase in monomers generated during incubation. It can be computed from the optical density and radioactivity profiles that when puromycin was added, the specific activity of subunits within monomers was 85% of that expected if total equilibration had occurred. The degree of equilibration reached with the puromycin-induced monomers was similar for both subunits, as indicated by the ratio of the radioactivities in monomers obtained after large and small subunit exchange, which was 2.7 (expected theoretical value: 2.6). (f) Electrophoresis of ribosoml proteins To further investigate if subunits of free and bound ribosomes can be functionally equivalent, we compared their protein composition by sodium dodecyl sulfate, polyacrylamide gel electrophoresis. Plate I(a) and (b) show the electrophoretic patterns for free and bound small subunits, respectively. Although as many as 17 protein bands are resolved on these gels, no difference between the two types of subunits is apparent. However, complete resolution of all proteins would be required to conclude that free and bound small subunits have identical protein complements. The electrophoretic patterns obtained from free and bound large subunits, respectively, are shown in Plate I(c) and (d). In this case there is also a close correspondence between both sets of protein bands, except for a prominent protein band (mol. wt ~50,000) in the ease of the free large subunit (arrow, Plate I). A band at the corresponding position in the bound large subunit pattern is in most cases absent or very weak. Control experiments demonstrated that the extra protein band of free large subunits is not due to contamination of free ribosomes with ferritin. A similar extra protein band was previously observed in free undissooiated chick embryo polysomes (Fridlender & Wettstein, 1970) and in large subunits from Streptococcus fecalis free ribosomes (Brown & Abrams, 1970).

4. Discussion Our results show that, upon release of polypeptide chains in vitro, small subunits of membrane-bound ribosomes are capable of undergoing extensive exchange with added small subunits derived from free ribosomes. This exchange is depicted in Figure 9, which also presents possible effects resulting from the addition of large subunits. The small subunit exchange was inferred from the transfer of added labeled small subunits to microsomal membranes, or vice versa, from the release of labeled microsomal small subunits upon addition of cold small subunits. Considering that small subunits become exchangeable upon release of polypeptide chains, two alternative situations were expected for the behaviour of large subunits which would indicate whether exchange occurred or not (Fig. 9(b) and (c)). Large subunit exchange-in would have resulted in a dilution of the specific activity of the added subunits, whereas large subunit exchange-out would have resulted in the release of radioactivity from the microsomes (Fig. 9(b)). Since we were unable to demonstrate either of these effects, which were clear in the case of small subunits, our results are better interpreted by the scheme of Figure 9(c), showing no in vitro large subunit exchange. The Figure also includes our finding that the main effect of adding large subunits and puromycin to rough microsomes was a large net removal of small subunits from

434

D. BORGESE,

G. BLOBEL

AND

D. D.

SABATINI

Exchange of small subunits

Exchange of large subunits

No exchange of large subunits

(a)

(b)

(cl

FIO. 9. Schematic bound ribosomes.

representation

of exchange

of small and large subunits

with

membrane-

bound ribosomes (Fig. 9(c)), due to the trapping of small subunits into a newly formed monomer pool. This effect is consistent with the observations that small subunits become exchangeable once polypeptide chains have been released and with previous reports (Sabatini et al., 1966) that ribosomes are attached to membranes via large subunits only. We also demonstrated that at sufficiently low concentrations of Mg2+ ions, the in vitro exchange of small subunits requires only the addition of a macromolecular fraction from the high-speed supernatant and possibly a nucleoside triphosphate. However, the effect of the high-speed supernatant fraction in promoting exchange is as yet difficult to interpret, since the sedimentation properties of the small subunits are changed during incubation with miorosomes in the absence of the high-speed supernatant fraction. Although it may be that the only function of the G50 fraction is to protect small subunits, the possibility remains open that a factor is contributed by the supernatant which is directly involved in promoting the exchange. An initiation factor with a ribosome dissociation activity is generally found in tight association with native small ribosomal subunits and is currently thought to regulate the subunit association-dissociation cycle in free polysomes (Subramanian et al., 1968; Subramanian et al., 1969; Subramanian & Davis, 19’70,197l; Davis, 1971; Lawford et al., 1971; Lubsen & Davis, 1972; Kaempfer, 1971). However, details of the mechanism by which the dissociation factor regulates subunit exchange are still a subject of controversy in the literature. While Subramanian and co-workers (Subramania.n et al., 1969; Subramanian & Davis, 1971) have concluded that the dissociation factor can reversibly dissociate inactive monomers, Kaempfer (197OJ971) has reported that the dissociation factor acts only by preventing the reassociation of ribosomal subunits produced at polypeptide chain termination and is incapable of dissociating inactive monomers. We have shown that the subunit exchange induced by puromycin occurs in a minimal medium where the protein synthetic apparatus is not operative. It is clear that under these conditions the exchange is a net result of nascent polypeptide chain release and does not depend on recycling of a dissociation factor due to initiation. However, we have not yet established whether the exchange occurs only concomitantly with the puromyoin-induced polypeptide

EXCHANGE

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435

chain release, or whether it occurs subsequent to release, involving the cyclic dissociation-association of inactive monomers generated by puromycin. If the latter were the case: even a small amount of dissociation factor present in the supernatant, acting reversibly, could account for a major exchange. The effect of GTP or ATP in increasing the exchange could, in this case, be explained by a stimulatory activity of the nucleoside triphosphates on the dissociation factor. A stimulatory activity of ATP or GTP (Subramanian et al., 1969), as well as of GTP specifically (Gonzales et al., 1969; Garcia-Patrone et al., 1971), on the bacterial dissociation factor has in fact been reported. However, some authors now attribute this effect to chelation of Mg2 + by the nucleoside triphosphates (Subramanian & Davis, 1970). Other possible functions of the supernatant could be to increase the eflicieney of the puromycin reaction through the action of a translocation factor (Skogerson & Moldave, 1968a,b), or to contribute a factor required for ribosome run-off from mRNA after polypeptide chain release (Hirashima I%Kaji, 1972). Evidence accumulated in recent years indicates that membrane-bound polysomes are responsible for translation of specific messengers directing the synthesis of secretory proteins (Campbell et al., 1960; Siekevitz & Palade, 1960; Takagi & Ogata, 1968; Redman, 1968; Takagi et al., 1969; Ganoza & Williams, 1969). It has also been suggested that membrane-bound ribosomes are involved in the synthesis of membrane proteins (Dallner et al., 1966a,b; Omura & Kuriyama, 1971), while some cell-sap proteins have been shown to be synthesized in free polysomes (Redman, 1969a,b; Hicks et al., 1969; Takagi et al., 1970). Thus, free and membrane-bound ribosomes appear to fulill different functions in the cell. Therefore, the possibility that subunits of membrane-bound ribosomes participate in a common ribosome cycle with free polysomes, is of particular interest in understanding the process by which specific proteins are distributed intracellularly. Our observations indicate that a subunit associationdissociation cycle similar to that described for free polysomes (Kaempfer, 1968,1969; Falvey & Staehlin, 1970) occurs in bound ribosomes and suggest that in viva, because of the operation of this cycle, at least small subunits can change from the free to the bound condition. Possible effects of chain release on the relationship of the large subunit to the membrane should be discussed, in view of the fate of nascent polypeptides contained in large subunits of bound ribosomes and their role in the ribosome-membrane association. In secretory cells, like the pancreatic exocrine cell and the hepatocyte, these nascent peptide chains are upon termination vectorially discharged into the cisternal cavities of the endoplasmic reticulum (Redman et al., 1966; Redman & Sabatini, 1966 ; Redman, 1967). In liver microsomes the amino terminal segment of a sufficiently long nascent polypeptide, which emerges from a membrane-bound ribosome, has been found to be iu close association with the underlying microsomal membrane and is thereby protected from the attack of added proteases (Sabatini & Blobel, 1970) which readily digest a similar segment in free polysomes (Blobel $ Sabatini, 1970). The relationship of the nascent polypeptide with the membrane is an important factor in maintaining the binding of ribosomes to membranes. At sufficiently high ionic strength the polypeptide is the only link between the membrane and the ribosome, since in vitro release of nascent chains by puromycin in high-salt buffer specifically causes an extensive release of both ribosomal subunits from liver rough microsomes (Adelman e.t al., 1970; Sabatini et al., 1971; Adelman et al., 19733). At more nearly physiological ionic strengths, however, in addition to the link mediated

436

D. BORGESE,

G. BLOBEL

AND

D. D. SABATINI

by the chain, there are also direct bonds sufficiently strong to retain inactive ribosomes attached to membranes after puromycin has caused an extensive release of polypeptide chains (Adelman et al., 1973b). Our experiments indicate that under these conditions release of polypeptide chains does not involve the detachment from and/or the re-attachment of large subunits to the membranes during the time-interval studied, although it does cause a dissociation-association reaction between subunits of membrane-bound ribosomes. A lack of large subunit exchange in vitro may reflect the presence in the cell of a stable large subunit-membrane complex, which could exist independently of protein synthesis. Such a complex could participate in the assembly of membrane-bound polysomes in a manner similar to that proposed by Baglioni et al. (1971). However, it should be emphasized that the results irz vitro may simply reflect the absence of factors necessary for detachment of large sibosomal subunits upon chain release, or for the subsequent re-attachment of added subunits to sites made available on the miorosomal membranes. The possibility should also be considered that added large subunits lost their capacity to bind the membranes during the in vitro treatment but that there was a dissociation-association between miorosomal large subunits and membrane-binding sites. This however is unlikely, since added large subunits were capable of exchanging with free polysomes and of combining with the small subunits released from miorosomes. Moreover, large subunits remained bound to membranes at dilutions of microsomal suspensions sufficient to produce a puromyoin-induced detachment of 40% of the small subunits. In spite of the results suggesting no in vitro exchange of large subunits, structural and metabolic similarities between free and membrane-bound ribosomes (Loeb et al., 1967; Talal & Kaltreider, 1968; Tanaka et al., 1970) suggest that, according to the physiological state of the cell, large subunits are capable of changing from the free to the bound condition during their biological lifetime. Moreover, physiological (Lee et al., 1971) and experimental (Bleiberg et al., personal communication) conditions have been reported to change (through effects on protein synthesis) the intracellular distribution of free and bound ribosomes in times shorter than the half-life of ribosomal RNA. As mentioned before, the process of in vivo exchange of bound large subunits may be regulated by factors absent in the in vitro system. In this context it is of interest to note that we found a more intense protein band in the electrophoretic pattern of large subunits from free polysomes than from bound sibosomes, which could be one possible factor involved in regulating the normal process of attachment. This work was supported in part by Public Health Service grant GM16583 We thank Dr George E. Palade for helpful

discussions and suggestions on the manuscript.

REFERENCES Adehnan, M. R., Blobel, G. & Sabatini, D. D. (1970). J. Cell Biol. 47, 4a. Adehnan, M. R., Blobel, G. & Sabatini, D. D. (1973a). J. Cell BioZ. 56, 191. Adehnan, M. R., Sabatini, D. D. & Blobel, 0. (19736). J. cell BioZ. 56, 206. Baglioni, C., Bleiberg, I. & Zauderer, M. (1971). Natwre New BioZ. 232, 8. Blobel, G. & Potter, V. R. (1967a). J. Mol. BioZ. 26, 279. Blobel, G. & Potter, V. R. (19673). J. Mol. BioZ. 26, 293. Blob& G. & Sabatimi, D. D. (1970). J. CeZE BioZ. 45, 130. Blobel, G. & Sabatini, D. D. (1971). Proc. Nat. Acud. SC<., Wash. 68, 390.

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