On the attachment of ribosomes to microsomal membranes

J. Mol. Biol. (1966) 19, 503-524

On the Attachment of Ribosomes to Microsomal Membranes D. D.

SABATINI,

Y.

TASHIROt AND

G. E .

PALADE

The Rockefeller University , New York, N.Y. 10021, U.S.A. (Received 14 F ebruary 1966, and in revised form 20 May 1966) Guinea pig hepatic micr osom es were treated with increasing conc entrations of EDTA to d issociate into subunits t he ir atta ched ribosom es. The dis sociation process was followed by zon e centrifugation a n d its products characterized by analytical centrifugation. At 20 pmoles EDTA p er 0·5 g tissue equivalent of microsomes, only small subunits ("" 32 s) were released to the sucrose density-gradients in amounts which , a ccording to RNA determinations, accounted for practically all 32 s subunit s originally presen t in the mi crosom al preparat ion. At hi gher EDTA concentrations (20 to 200 p.molesjO·5 g tissue equivalent ), t he relea sed 32 s subunits were degraded, and increasing amounts of large su bunits ("" 47 s) were detached from the microsomal membranes. After in vivo labeling with (3H]leucin e, the detached large subunits cont aine d newly synt hesized pro te ins. Ul tracentrifugal analysis confirmed the preferential release of small over large subunit s at EDTA concentrations below 40 p.M. A fra ction comprising,..., 30% of the mi crosomal RNA remained bound to mi crosomal membranes even after treatment with 500 p.moles EDTAjO'5 g tissue equiva lent . Successive treatment with EDTA (500 p.moles) a nd deo xy chola te (0'5 %) led to the release of this residual R NA which, by analytical centrifugation, was found to be ass ociate d with particles, primarily ,..., 50 s parti cles, presumably large subunits. After labeling in vivo with [3H]leu cine, the particles resistant to detachment by EDTA eontained vv 70 % of the n ewly synthesized protein: presumably the large su bunit s of a ctive ribosomes are more firmly atta ched to the microsomal m embrane. The initial release of small subunits sug gest ed that ribosomes are attached to the microsomal membrane by their large subunit s. This ass umpt ion is supported by elect ron microscopy findings on isolated, negat ively stained microsomes. In favorable specimens, the gro ov e that separa tes the ribo somal subunits could be recognized and was found to be ori ented generally parallel to the en doplas mic reticulum or mi crosomal m embrane. Th e functional implications of these findings are discussed in relation to the processes of synthesis of secretory proteins on attached ribosom es and their subsequent release into the cisternal sp ace.

1. Introduction In cells synthesizing protein for secretion, the majority of ribosom es is attached to the membranes of the endoplasmic reticulum (Palade, 1956,1958; Porter, 1961). Upon cell fractionation, this organelle is fragm ented into vesicles which are isolated as the microsomal fraction (Palade & Siekevitz, 1956). The association of ribo somes and endoplasmic reticulum membranes, which is preserved in the mlcro somes, has t Present address: Department of Physiology, Kansa i Medic al School, Fumizonocho, Morigu chi, Osaka, Japan. 503

504

D. D. SABATINI, Y. TA SHIRO AND G. E. PALADE

special functional significance . On one hand, microsomes constitute in oioo, on an RNA basis, a more efficient protein- synthesizing system than free ribo somes (Siekevitz & P alade, 1960). A higher efficiency of isolated microsomes over free ribo somes was also shown in vitro. Furtherm ore, the addition of synthetic polynucleotide is required for high in corporation rates by free ribosomes, but is not necessary for microsomes, which ar e in fact less st imulated (Henshaw, Bojarski & Hi att, 19tm). On the other hand, combined morphological and biochemical work (Siekevitz & Palade, 1960; R edman , Siekevitz & P alade, 1966) points to the transfer of newly synt hesized protein, from its site of synthesis on the attached ribosomes to the interior of the ciste rnae of the endoplasmic reti culum as another fun ctional correlate of the attachme nt . This would be the first step in a postul ated cycle of protein secret ion which involves several cell organelles (Palade, Siekevitz & Caro , 1962). If, as it appears from recent data (Redman et al., 1966; Redman & Sabatini, 1966), secret ory proteins pa ss directly from the attached ribosom es into the cisternal (i.e. microsomal) cavities across the endoplasmic reticulum (i.e. microsomal) membrane, then the passage route should lie close to, or be provided by, the ribosomemembrane junction. Further elucidat ion of the proces s of protein transport requires a mor e detailed investigation of the relationship between attached ribosomes and ER (microsomal) membranes. That the ribo some-membrane junction may involve specific sites, at least on the rib osomal side, was suggested t o us by the fact that liver ribosomes (Tashiro & Siekevitz, 1965a; Ta shiro & Yphantis, 1965), like Escherichia coli ribo somes (Tiasieres, Watson, Schlessinger & H ollingworth, 1959), are asymmetric particles with definite subunits . In the case of the liver, the large (47 s) and t he small subunits (32 s ) ar e obtained from purified ribosomes dissociated by ehelating Mg 2 + with EDTA. Fo r the ribo somal subunits, evidence of surface specialization concernin g the binding of newly made polypeptide (Gilbert, 1963; Ta shiro & Siekevitz, 1965b), sR NA (Cannon, Krug & Gilbert, 1963; but cf. Suzuka, K aji & K aji , 1965) and synthetic polynucleotide (Takan ami & Okamoto, 1963) ha ve recently been provided . To determine by which subuni t liver ribo somes are attached to microsomal membranes, whether by the large or t he small subunit or by both subunits, we decided to investi gate the possibilit y that by adding EDTA t o intact micro somes one could dissociate the ribosomes while t hey were still attached to microsomal membranes. If this could be accomplished, sediment at ion analysis of preparations thus treated should show which kind of subunit, if any, had been released and which had sedimented away bound to the membranes. It is known from previous work (Palade & Siekevitz, 1956; Sachs, 1958b) that EDTA can release large amounts (50 to 60%) of RNA fr om the microsomes, and it has been shown (Sachs, 1958a) t ha t treatment with another chelating agent, pyrophosphate, can release in particulate form about 80% of ribonucleopr otein from the . microsomes t ogether wit h a fraction of newly synthesized protein. TIlls work also indicate d that microsomes treated wit h pyrophosphate are rendered inactive in protein synthesis in vitro. As material for our work, we have chosen t he guinea pig liver, since ribo somes and rib osomal subunit s of t his organ have been recently charac terized ph ysicochemically (Tashiro & Siekevi tz , 1965a; Tashiro & Yphantis, 1965). In guinea pig liver, the attached form of ribo somes is predominant. Although undoubtedly the liver cell must synt hesize a variety of proteins to match the complexity of it s many

ATTACHMENT OF RIBOSOMES

505

functions, the bulk of the proteins made in the liver of the adult animal appears to he seru m albumin which is exported (Peters, 1959).

2. Materials and Methods R eagents. Tris ("Sigma 7.9") , fr om Sigma Chemical Company, St. Louis, Mo.; was used for t h e pH 7·6 Tris-H'Cl buffer. EDTA was a cert.ified reagent from the Fisher Scientific Com pany , Fairlawn, N.J. A 5 % solut ion of DOCt was prepared by neutralizing with KaOH deoxycholic a cid fr om t he Mann Research Laboratories, Inc., New York, K.Y. D,L.[4,5. 3H]l eu cine (4 m c /m-mole] was ob tained from the New England Nuclear Corp., Boston, Maes. A O'IM solution of p -(diisobutyl.cresoxyethox yethyl)dimethylbenzylammonium hydroxide in methanol (hydroxide of hyarnine 10·X from the Packard Instrument Company, Tnc., La Grange, Ill.) wa s us ed t o d issol ve protein for counting radioactivity. Tho liquid scintillator was "Liquiftuor" (Pil ot Chemicals, Inc., Watertown, Masa.), diluted 25 times with t oluene. Solution A: 0·25 M-sucrose, 0·005 M-MgCI 2 , 0·025 M.KCI, 0·050 M-Tris-HCI buffer (pH 7'6). Solution D: 0·002 M·MgCI2 , 0·050 M-RCI, O·OOIM·Tris -HCI buffer (pH 7·6). Solution E: 0·050 M-RCI, 0·001 1\1.Tris-HCl buffer (pH 7,6). (a) Pr eparation of microsomal fraction s Male albino guinea pigs weighing 300 to 350 g, and fasted for 24 hr were killed by d ecapitation after a blow on the head. In labeling experiments, I me of [3H]leucine was inj ected intravenously in the saphena and the animals were killed at chosen intervals from 1 to 10 min after the end of the injection. Following removal of the gall bladder, the livers were collected in an ice -cooled beaker, and weighed and homogenized in 5 vol. of solution A, with a glass Potter-Elvejehm homogenizer provided with a Teflon pestle. Homogonizat.ion and all subsequent operations in t he isolation procedure were performed at 4°C. Nuclei and mitochondria were discarded as a common p ellet after centrifugation for 10 min at 12,000 tev Jtnu: in the no. 40 rotor of the Spinco L centrifuge. The supernatant solut ion was spun 30 min at 40,000 xev.hnu: in the same rotor and the ensuing mi cro some pellet resuspended in solution A (5 ml. j I g tissue equivalent). The final mi cro. somal preparation was obtained from the former after spinning it at 40,000 rev./min for 30 min and resuspending the pellet in solution D (4 ml./l g tissue equivalen t ). Micro somal pellets were resuspended either by hand, in a small glass homogenizer, or mechanically using a motor-driven Teflon pestle fitting the plastic tubes of the centrifuge. Microsomal preparations contained more free ribosomes after the second, than after the flrst resuspension procedure.

(b) Purification of microsomal fract ione When microsomes, resuspended mechanically with a T eflon pestle, were used to study the attachment site of ribosomes, they were further purified by sedimenting through a 5 to 20% sucrose density-gradient containing the same ions as solution D. Magnesium chloride was omitted from the sucrose density-gradient experiments designed to study the effect of EDTA on microsomes, AIl sucrose gradients were made in tubes of the SW25 rotor of the Spinco L centrifuge, with a device similar to that described by Britten & Roberts (1960). The concentration gradients, with a total volume of 30·5 mI., were linear as t ested by measuring spectrophotometrically the distribution of the dye Orange G introduced into the sucrose solution in one of the chambers of the mixing device. The sedimentation coefficients of t he peaks were calculated a ccording t o Nomura, Hall & Spiegelman (1960) and Martin & Am es (1961). EDTA was added to the samples from 0'1 or 0·41\1 stock solutions adjusted to pH 7,6, and the mi crosomes thus treated were kept in the cold for 10 min before centrifugations were started . The samples were carefully layered on top of the gradients in a volu me of approximately 2 mI. which usually contained microsomos from 0·5 g of liver. The S\V25 rotors wcr e st opped without braking. A hole was punctured at the bottom of the plastic tubes, avoiding the pellets, and fractions of 13 drops each ('" 30 fractions from each tube) were collected in the cold room with an automatic fraction collector. t Abbreviations used: DOC. deoxycholate; PTA, phosphotungstic ac id .

506

D . D . SABATINI, Y. T A SHIRO AND G. E. PALADE (c) Electron microscopy

Material for elec t ro n m icroscop y was fixed in t he cold (4°0) in dilute sus pe nsions b y adding glutaralde hyde n eutralized with 1 N-NaOH and d iluted t o a final concentration of 6'5 % (Sabatini, Bensch & Barrnet t , 1963). Durin g t he first 10 m in of fixat ion , drops of the sa mples were lai d on carbon-coated grids for negative stain ing , and after was hing with a dr op of water were stained with a 1 % solution of phosphot u ngstic ~cid neut ralized to pH 7·0 (Brenner & H orne, 1959). Ob servations wer e made in a Siemens Elmiskop electron mi cr oscope, at 40,000 and 80,00 0 elect ro n op t ica l magnificat ion . . (d) Oentrifugal analysis

The analytica l cen trifuges were Sp inco E, equippe d with ul traviolet and sch liere n optics. The runs were don e in t he cold at 2 to 9°C. P hotographs were taken every 4 min after arriv ing at t he maximum spe ed of 44,770 tev. lta in , The ul t ra violet p ictures wer e scanned with a Spinco model R An a ly trol with a m icroden sit omet ry attachment. Initial conce nt rations were ca lculated from dens it ometry aft er correc t ion for radial dilutions, an d are sh own in the Figures with standard de v iat ions . (e) Ohemical procedures Ultraviolet absorption m easurements were done at 260 mIL in a Beckman DU spec t ro phot omet er. RNA was measured b y t he orc in ol method (Mejbaum, 1939) after extract ion by the Schneide r procedure (Schneid er, 194 5). Radi oa cti v it y in prot ein -polypeptides was counted in a Tricarb scintillation cou n ter. F or t his purpose, t he residual pellet s of the Schneider procedure were washed in alcoho l-ethe r (3: 1) and ethe r, d issolved in H yamine or in 88 % formic a cid, and t he n t ransferre d with 5 ml. methanol t o v ia ls contain ing 15 ml , of liquid scintillator .

3. Results (a) Purification of microsomes

For our experiments on the effect of EDTA on attached ribosomes, we needed .a preparation of micros omes completely, or nearly completely, freed of free ribosomes. If present, free ribo somes would be expect ed to dissociate in the normal manner upon addition of EDTA, and the resulting subunits would appear in the gradient and ma sk the effect of EDTA on the microsomes proper. F or purification and analysis, microsomes derived from 0·5 g of liver and resuspended mechanically in 2 ml. of solution D, were passed t hrough a sucrose densitygradient as describ ed under Materi als and Methods. Fi gure l(a) shows a typical result. The micro somes sedimente d to the bottom of the tube afte r two hours centrifugation in a SW25 rotor at 25,000 rev. ltuu: and were not visible in the gradient . A peak of monomers, amounting to from 10 to 15% of the RNA of the sample in different experiments, remained within the gradient , sedimenting at a velocity of approximately 78 s. RNA in the gradient was estimated from ultraviolet absorption, t aking 135 as t he extinction coefficient at 260 mfL for a 1% ribosome solut ion (Tashiro & Siekevit z, 1965a), or from t he difference in the results of the chemical det erminations made on the sample and the pellet. Th e mi crosomes obtained in this ste p as a pellet in the SW25 tube were free of contaminat ing or easily detachabl e ribo somes. F igure l (b) shows t hat release of more ribo somes did not occur when , afte r resuspension in a solution without sucrose, the microsomes were centrifuged again t hrough a similar gradient. In thi s case, all the ribo somes, probably bound t o membranes, have sedimented to the bottom of the tube. Microsomes purified by this method, and containing approximately 0·750 to 1·0 mg of RNA, were resuspended in a volume of 2 ml, and used for the experiments in which the effect of EDTA was st udied . It was

ATTACHMENT OF RIBOSOMES

507

later found, as mentioned under Materials and Methods, that purification through a density gradient was not necessary if the original microsomal pellets were resuspended gently by hand, with a glass homogenizer. In density-gradient analysis, most of these preparations gave patterns similar to that of Fig. l(b) and, in the analytical centrifuge, showed a small percentage of monomer ribosomes (less than 10%, Fig. 9). In most experiments, we used microsomes resuspended by hand, since this procedure yielded pure preparations faster. In these cases, control density gradients were run in parallel to establish the absence of free ribosomes. Experiments with both kinds of microsome preparations gave similar results.

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FIG. 1. Purification of guinea pig liver microsomes. (a) Washed microsomes were resuspended in solution D (0,5 g tissue equivalentf2 ml.), layered on top of a sucrose density-gradient and centrifuged for 2 hr at 25,000 rev.fmin in the SW25 rotor of the Spinco L centrifuge. (b) The pellet from (a) was resuspended and centrifuged again through a sucrose densitygradient as in (a).

(b) Dissociation of attached ribosomes by EDTA

No ultraviolet-absorbing material was released from 0·5 g tissue equivalent of microsomes resuspended in 1 ml. of solution Dj', by the addition of up to 20 p.moles of EDTA. In these cases, the density-gradient analysis appeared similar to the untreated controls (Fig. l(b)). However, such small amounts of EDTA had an unexpected effect on some preparations in which small amounts of free ribosomes were present as contaminants. The effect could be detected either by control densitygradient analysis of miorosomes, or by analytical centrifugation. The addition of less

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508

D. D. SABATINI, Y. TASHIRO AND G. E. PALADE

than 20 fLmoles of EDTA to such preparations produced the disappearance of the free ribosomes from the gradients, all the material in the samples sedimenting with the microsomes to the bottom of the tube. A similar aggregating effect has been observed with pancreas ribosomes (Madison & Dickman, 1963). Upon addition of 20 fLmoles of EDTA (Figs 2 and 3) to 0·5 g tissue equivalent of purified microsomes, a particulate material sedimenting as a normal 32 s subunit (with which it can be compared in the parallel control, Fig. 2) was released from the microsomes. With this amount of EDTA, no material corresponding to the 47 s component of dissociated ribosomes appeared in the gradients. The sedimentation coefficient of the particles first released was confirmed in the analytical centrifuge (Fig. 10) (see Results (i) under section (e)). HNA determinations in a portion of the sample and in the pellet showed that 20 to 30% of the HNA in the sample was released from the microsomes at this EDTA concentration.

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FlO. 2. Comparison between sedimentation patterns of EDTA-treated ribosomes (a) and microsomes (b). After treatment with EDTA, the samples were kept 10 min in the cold (4°C) before loading the donsity gradients. The latter were centrifuged for 8 hr at 25,000 rev.fmin in the SW25 rotor of the Spinco L centrifuge. (a) 10 mg of purified guinea pig liver ribosomes prepared according to Tashiro & Siekevitz (1965a) and treated with 25 JLmolesofEDTA. The dissociated ribosomal subunits serve as markers in the density gradients. (b) 0·5 g tissue equivalent of purified microsomes resuspended in 2 ml. of solution D and treated with 20 JLmolos of EDTA.

ATTACHMENT OF RIBOSOMES

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Equal portions of microsomes to which 30 Ikmoles of EDTA were added (Fig. 3) revealed a second component in the density gradients, sedimenting with the velocity of 47 s. At this EDTA concentration, the ratio of 32 s to 47 s material was 2 : 1. At 40 Ikmoles of EDTA, the amount under the 47 s peak increased, and the area under the 32 s peak decreased while a slower heterogeneous component appeared (Fig. 3). It is assumed that this lighter material results from the degradation of the 32 s

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component at this high concentration of EDTA, and this interpretation is supported .by a similar effect of high concentrations of ED'fA on isolated ribosomes split into subunits (Tashiro & Siekevitz, 1965a) and maintained in the cold. Finally, at

50 Ikmoles or higher amounts of EDTA, a notable peak of the 47 s component was found in the gradient, while that corresponding to 32 s was reduced to a shoulder on a high peak of lighter material assumed to be the 32 s degradation product (Fig. 3). The 47 s peak increased with the EDTA added until it remained almost unchanged beyond 100 fLmoles, at which concentration the 32 s peak disappeared or was masked under the lighter heterogeneous component. RNA determinations in the samples and in the pellets obtained in these experiments indicated that, as EDTA concentration was increased, microsomal RNA was progressively released from the pellet to the gradient until the I~KA released reached a limiting value of 70% of the original RNA content at 100 fLmoles or higher amounts of EDTA. The curve of RNA release is shown in Fig. 4. It can be seen from it that

D. D. SABATINI, Y. TASHIRO AKD G. E. PALADE

510

at 20 fLmoles of EDTA, when only 32 s material (small subunits) were found in the gradient, 20 to 30% of the RNA was released. Since 85 to 90% of the RNA of the original preparation (cf. Table I) could be recovered in ribosomes when these were prepared by conventional DOC treatment, and since there is evidence indicating that the 32 s ribosomal subunit contains slightly less than one-third of the ribosomal RNA (Tashiro & Morimoto, results to be published), it follo~s that the RKA released at 2U fLmoles of EDTA as 32 s particles should represent the' large majority or almost all of the small subunits in the microsomal preparation.

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TABLE

1

Distribution of insoluble radioactive material in microsomest Radioactivity (ctsjrnin]

Microsomes from 0·5 g Ribosomcsf from 0·5 g

9934 (100%) 8443 (85%)

Specific activity (cts/min/mg RNA)

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Animals were injected intravenously with 1 me DI"-[4,5-3H]leucine 1 min before killing. Obtained as a pellet after treating the microsomos with 0'5% DOC and centrifuging for 2 hr at 105,000 g.

(c) EDTA-dissociation of attached ribosomes labeled in vivo with [3H]leucine Liver microsomes were isolated from guinea pigs injected intravenously with 1 me of [3H]leucine, and killed at various times thereafter. The microsomes were subjected to similar EDTA treatment followed by gradient analysis as described above. After reading the ultraviolet absorption at 260 uu», the material from each gradient fraction was precipitated with cold trichloroacetic acid added to a final concentration of 5%. In

ATTA CHMEXT OF RIBOSO;\IES

some cases, 200 fLg of bovin e ser um albumin were added to each fraction as a carrier. The protein was extrac ted and the radioactivity counte d as described under Materials and Methods. In agr eement with previous results, no material sediment ing as eit her 32 or 47 s was released fr om mierosomes deriv ed from 0·5 g of liver by amounts of EDTA smaller than 20 fLmoles, but such amounts were found to release a peak of sol uble radioactive material. Most of this soluble radi oacti ve material , whi ch in creased at higher EDTA concentrations, is assumed to be newly synt hesized protein whi ch has alrea dy been discharged from the ribo somes and which, because of the action of EDTA, either leaks from the ciste rnae or is released from the microsomal membranes. H owever, it is known (Tashiro & Siekevitz, I965b) that EDTA is able to detach some soluble radioacti ve material from isolated labeled rib osomes, hence part of the soluble radioactive material released from the microsomes may be of direct rib osomal origin. At 20 fLmoles of EDTA, tho 32 s component released from the miorosomes was not a ccompanied by radioactive protein (Fig. 5). At 25 and 30 fLmoles of EDTA, a small peak sedimenting as 47 s appeared in the gradient, a ccompanied by radioactivity (Fig. 5), whereas the 32 s material was not radioactive. At 50 fLmoles of EDTA (Fig. 5) the non-radioactive 32 s material started to be degraded to lighter, heterogeneous components, resulting in the appearance of a shoulder of the 32 s peak rising continuo usly toward the meniscus side of t he gradient . At 50 (Fig. 5), 100 and 150 fLmolcs of EDTA, the radioacti ve 47 s material increased in am ount, but beyond 100 fLmoles EDTA the appearance of 47 s components in the gradient leveled off and the increase of material in the gradients produced by 150 fLmoles EDTA over t ha t produced by 100 fLmoles EDTA was only small. A peak at 32 s was not recogniz abl e at these EDTA concent rat ions. Taken to gether, t he results of the la st two experim ents indi cate t hat : (a) there is no radioactivity accompanying the 32 s peak which app ears first after addition of EDTA, and (b) part of the newly synt hesized protein in the rib osomes is detached while it is still bound to t he 47 s particles. Th ese results suppo rt t he conclusion that the 47 s p ar ti cle released from the microsomes at higher EDTA concentra tions is in fact iden tical with the large subunit obtained fr om dissociation of isolated ribo somes, which is known to contain t he newly synthesized protein (Tashiro & Siekevitz, 1965b). (d} Label distribution among attached ribosomes An additional finding of the labeling experiments called for a more careful analysis. Wh cn the distributions of total radioactivity and RNA in the pellets and in the gradient fractions were compared, it became apparent that even at the highest EDTA concentration used (150 fLmoles per microsomes derived from 0·5 g of liver), 70% of the radioactivity in the microsomes remained in the pellet s although their RNA content was redu ced to less than 40 %. Thi s discrepancy between the proportions of RNA and radioactivity released by EDTA from the microsomes was confirmed and put on a mor e stringent quantitati ve basis in experiments utilizing differential centrifugation. Th e processing times were thus shor tened and t he pro cedure simplified because the measurement s of R NA and radioa ctivity were performed only in t he pellet s and in portions of the original samples. As is shown below, by shortening the time of lab eling in vivo it was possible to reduce t he amount of non-ribosomal radioactivity in t he miorosomes, whi ch appears as a slow-moving peak in the gradients (sec abo ve). Thi s radioactivity (which is not

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FIG. 5. Sucrose density-gradient analysis of material released from labeled microsomes upon treatment with increasing amounts of EDTA. The animals were killed 2 min after the intravenous injection of 1 me of [3H]leucine. Centrifugation for 3 hr at 25,000 rev./min in the SW25 rotor of the Spinco L. Optical density at 260 m,..; - - 0 - - 0 - -, radioactivity in protein (ctsjmin],

-e-e-,

associated with ribosomal subunits) is presumed to be initially contained in the microsomal cavities and to be released therefrom at low EDTA concentration. (i) Effect of EDTA treatment of labeled microsomes studied by differential centrifugation

(Tables 1 to 3)

The microsomes for these experiments were obtained from guinea pigs injected with 1 me of [3H]leucine and killed one minute after the end of the injection. To portions of these microsomes, DOC was added to 0·5% final concentration and ribosomes were obtained by differential centrifugation (40,000 rev.fmin for two hours in the no. 40 rotor) of the treated suspensions. In agreement with previous reports in

ATTACHMEN T O F RIBOS OMES TABLE

513

2

Release of RNA and insoluble radioactive material by addition of EDTA to microsomest EDTA added to mi crosomes from 0'5g liver

None 50 fLmoles 100 fLmoles 200 fLmoles 500 fLmoles

pgRNA in pellett

Cta/min in p ellett

9525 7434 7952 7468 7382

890 429 365 318 310

(100 %) (78 %) (83-4 %) (78'4 %) (77,5 %)

Specific activity (cts/min/mg RNA)

(100 %) (48 ,2 %) (41,0%) (35,7 %) (34-8 %)

10,702 17,328 21,786 23,484 23,812

t

Animals were inj ect ed intravenously with 1 me DL.[4,5-3H]leucine 1 min be fore kill ing. t Obtained by cent rifuging at 105,000 g for 30 min. TABLE

3

DistribuJion of RNA and radioactivity after applying DOC to EDTA-treated microsomest

EDTA (fLmoles)

Percentage radioactive material insoluble in D OC

P erc entage RNA insoluble in DOCt

100 200 500

72·0 69· 3 73·2

79·4 65·7 72·9

t 0·2 % DOC was applied t o labeled mi cro somes sedimented after addition of various amount s of E D TA. t Pellets obtained by centrifuging for 10 hr 40 m in at 114,000 g. the literature (Palade & Siekevitz, 1956), it was found that 85 to 90% of the RNA in the microsomes (Table 1) was ribosomal. As mentioned ab ove, at t his early time of labeling it was also found that 85 to 90% of the radioactivity of the microsomal protein extract ed by the Schneider procedure was also recovered in the ribosomes obtained after DOO treatment. Graded amounts of the chelating agent, up to 500 ftmoles , were added to 0·5 g tissue equivalent of these lab eled microsomes resusp end ed in 2 ml. of solut ion D . After t en minutes in the cold (4°0) , the volume of each sample was made up to 10 ml. with solution E, and the treated microsomes spun in the no . 40 rotor of the Spinco L centrifuge for 30 minutes at 40,000 rev .jmin, Portions of the original preparations and the ensuing pellets were submitted to the Schneid er procedure and the trichloroacetic acid -insoluble (polypeptide protein) radioactive material determined (Table 2). The result s in Table 2 confirm ed the preliminary

+

514

D. D. SABATINI, Y . TASHIRO

A~D

G . B . PAL ADE

observations in the density-gradi ent exp eri ments, that is, the discrepancy between the release of RNA and the release of radioactive material from EDTA -treat~d miorosomes. Even t he highest am ount of EDTA (500 fLmoles) added to miorosomes derived fr om 0·5 g of liver , which det aches approximat ely 65% of the R NA and consequently at least 57% of the ribosomal RNA of the sample, releases only ",23 % of its radioactive material. The course of release of RNA and of radioacti ve material from mierosomes is depicted in F ig. 6. It is seen that beyond 200 fLmoles, the effect of EDTA is not pronounced. In t he range of 200 and 500 fLmoles EDTA, the rati o in the pellet of radioactive pr otein a nd polyp eptides to RNA ha s doubled with respect to t he non-treated control. 100 \

90 80

\

\

\

\

b~'

,,0.......

...........0 - -

·

70

...

~ ua. 60 .~

'"en

50

'"

40

0

-:= ~

~

30

-,

----._--------.

20 10

o

50 100

200

300

4 00

500

flmoles EDTA/microsomes from 0 '5 9

F IG. 6. Releas e of RXA a nd rad ioac t ive prot ein from labeled microsomos treate d with increasing amounts of EDl'A. The anim al s were inj ect ed intra ve nous ly with I me of ['H]lou cine ] m in boforo killin g. The mic rosomes were r esu sp ended in solution D (0'5 g tissue equivalcnt/2 ml.) t o wh ich EDTA was added in t he amounts indicated. Aftor ] 0 min in the cold (4°C), tho sam ples wero diluted to 10 m l. with solution E and centrifuged for 30 min at 105,000 g. RNA a nd r a di oactivity were measured in portions of the original mi cr osomes and in the pellets obtained fr om the EDl'A-treated samples. RNA; - - 0 - - 0 - -, r adioactivity (cts/rnin).

-e-e-,

(ii) Effects of successive treatment with EDTA and DOG To determine what proportion of t he ribosomal material still bound to the microsomes after EDTA treatment could be recovered in a particulate form , the following experiment was performed . P ellets recovered from microsomes t reated with EDTA as above were first resuspended in 5 ml, of solution E and subsequently treated with DOC to a. final concentration of 0'2 %. This concentrat ion of DOC is lower than customarily used, but the ratio of DOC to protein was ade quate.'] Th e DOC-treat ed suspen sion was spun in the rotor no. 40.3 of the Spineo L for 10 hours and 40

t Micro somes from 0·5 g wer e resu spended in 5 ml. and the suspen sion was cla rified with DO C. 0·2 % DO C was us ed in a n a tt em p t to inc rease t ho y ield of R XA sed imont.a ble b y subsequent centrifugat ion. However, simila r r esults woro obtained by t reating with 0·5 % DO C m icr osomos resuspended in solution D .

ATTACHMEXT OF RIBOSOMES

515

minutes at 40,000 xer.huin, This centrifugation is expected to spin down ribosomal subunits as a pellet. The results of the experiment (Table 3) show that 70% of the RNA left in microsomes after EDTA treatment can be recovered as particles after spinning as mentioned. A similar proportion of the insoluble radioactive material (protein + polypeptides) left in microsomcs upon EDTA treatment sediments with the ribonucleoprotein after DOC treatment. As a result, the specific activity of the pellets (cts/min/mg of RNA) is similar to the specific activity in the suspension before the addition of DOC. Ultracentrifugal analysis of suspensions treated with 0'5% DOC after EDTA (see below) showed the presence in this material of particles sedimenting as 47 S, as well as of heterogeneous components, some sedimcnting very rapidly. (iii) Effect of DOG concentration on the release of RNA and radioactivity from microsomes Experiments were performed to determine whether different concentrations of DOC have a discriminating effect on the release of radioactive material and RNA from microsomes. 2"6r------r----.------r-r------r----.-----~,...,1300

1200

4

-

2·2

1100 1000

~ ,I

\·8

900

,I"

I

I

,\

I I I

.,,

"

IIV·

1'4

I I

1·2

I

I

\·0

~

.4

1·8

,\..f ..... f

1-6

............

.'

0'4 0'2

:

o

'~~

~./\/i ~ .J

/I

oP-q,

POe! 0\ / I 1/ ./0

\/

500

I

I

400 300

• I / /

200

/

100

1"\ I o """'"0

10

20

30

10

20

30

Tube no.

FIG. 7.

FIG. 8. FIG. 7. Ribosomes released from labeled microsomes by treatment with DOC. One minute before killing, the animals received I me of [3HJleucine intravenously. The microsomes were resuspended in solution D (0,5 g tissue equivalcnt/2 ml.), treated with 0·125% DOC and loaded on density gradients. Specific activity = 0·467 cts/min/c.D. These were centrifuged for 8 hr at 25,000 rev./min in the SW25 rotor of the Spinco L centrifuge. FIG. 8. Sarno as Fig. 7, except that the final DOC concentration was 0'5%. 33

·zu

"

.Q

\.........• IJ

'J

?;.:;:

"U

rI

I

\,od

0

I I

I I

/7

.~

0

600

I I I

r

I

I

.'

•

ti

\

'lI\\

I

I

1/ I

700

o

0:::

516

D. D. SABATINI, Y. TASHIRO AND G. E. PALADE

Preparations of microsomes, labeled as described above, were submitted to the action of DOC at two different concentrations, 0'125% and 0'5 %. Figure 7 shows that at 0'125% DOC, the material detached from the microsomes as ribosome monomers has a specific activity of 467 cts/min/o.D. unit. At this DOC concentration, the monomer peak represents 76% of the total RNA of the sample. At 0·5% DOC (Fig. 8), when practically all of the ribosomes were detached by DOC (96% of the RNA), the specific activity of the monomers was practically unchanged: 469 cts/ min/o.D. unit. Thus a difference in the mode of action of DOC and EDTA on microsomes was reflected in the radioactive content of the detached particles. (e) UUracentrifugal analysis

The effect of EDTA on microsomes was studied also with the analytical centrifuge. The previous results were confirmed and a more exact determination of the sedimentation coefficients was obtained. The figures provided additional support for identifying as ribosomal subunits the particles released in density gradients. (i) Detachment of particles by EDTA treatment

Microsomal pellets were suspended in solution D to give a tissue equivalent of 0·5 g wet weight liver/ml. This suspension was used as stock preparation for EDTA experiments. After addition of EDTA (5 to 40 ",moles/ml.), the microsomes were kept for ten minutes in the cold (O°C) and subsequently diluted with solution E. The dilution was adjusted to bring den sitometry tracings of photographs within the region of linear response. Usually a dilution of 20 times or more was necessary for a preparation treated with EDTA at the concentration of 20 ",moles/ml. of microsomal suspension. At concentrations of less than 20 /Lmoles, best results were obtained with a five or ten times dilution. Each series of experiments was carried out starting with the highest EDTA concentration. At the end of the series, a portion of the stock microsome suspension, to which no EDTA was added, was diluted five or ten times with solution D and used as control to estimate the amount of free ribosomes which could have been liberated from purified miorosomes by resuspension and/or storage in the cold for several hours. The controls contained a small amount of a rapidly sedimenting component (76 ± 2 s), which most probably corresponded to ribosome monomers. Since the controls were run at the end of a series of experiments, we assumed that this monomer represents the maximum amount of ribosomes released from the microsomcs during the cold storage. Figure 9 combines data from different series of experiments which were carried out in the linear region of the densitometry. The abscissa gives the amount of EDTA in /.Lmoles added to I ml. of the original microsomal suspension, and the ordinate shows the analytical reading, which is proportional to optical density at 260 m", and therefore to the concentration of ribosomes. As will be seen, the results are in good general agreement with those obtained by sucrose density-gradient centrifugation. In the analytical centrifuge, however, release of small amounts of large subunits was observed even at 20 /.Lmoles of EDTA, and less degradation of the small subunits occurred. /) ",moles of EDTA. Almost no monomers were present in microsomal suspensions treated with this small amount of EDTA. Although no discrete boundaries were observed after the treatment, a very faint deflection was detected with a sedimentation

(a)

(b)

PLATE I. Sed imentation patterns from m icrosom es treated with EDTA (a) and with DOC after EDTA (b) . The photographs were taken with the ultraviolet absorption system eve ry 4 m in at a sp eed of 44,770 rev ./ m in. (a) A suspension of m icrosomes (0, 5 g tissue eq uivalentjrnl.) was treated w ith 20llmoles E DTA/mi. After 10 min in the cold (O°C), t he treated m iorosornes were di luted 20 t imes with so lu tion E and analyzed in t he Spinco E u lt racen t rifu ge at 3·3 °C. T h e S20 .w of the main componen t , which cons t it u t ed 73% of t h e to tal material released, was 33 s, that of t he minor compon ent was 49 s . (b) Mierosomes treated w ith 20 llmo les E D T A /m i. as in (a ) wer e sedimented in the Spi n co L centrifuge (105,000 g for 30 m in) . T he pe lle t s were r esu sp en d ed in solution D (0'5 g t issue eq uivalentjm l.) and then treated wi t h 0'5% DO C. The treat ed mi cr osom es we re di luted 15 t imes with solution D and analyzed in t h e Sp ineo E cent r ifuge at 3·10C . T h e S20 .w of t h e m ajor component was 49 s. I n ad dition to t his component, h et erogen eou s ~ 80 t o 90 s and ~ l Os materia ls a re present.. A comparison with (a) shows that t he re is n o boundary correspond ing to t he small subu nit.

[fO C;llg 1). !'d 6

/

( b) PLATE II. (a) Microsomal vesicle (M) negatively stained with sodium phosphotungstate. Attached ribosomes are visible on the face and along the edge of the flattened vesicle. Those at the periphery (between arrows) show indications of subunits separated by a groove which lies approximately parallel to the microsomal surface. Some free ribosomes are also visible in the background. Guinea pig liver microsomes isolated by differential centrifugation from a 1 : 5 homogenate prepared in 0·14M-KCI, 0·005M-MgCl z, O'OlOM-Tris-HCI (pH 7,6) and fixed in suspension with 6·5% neutral glutaraldehyde. X 200,000. (b) Liver ribosomes attached to the membrane (m) of a negatively stained microsome (M). Some ribosomes (arrows) have a partitioned appearance. In these cases, only one of the subunits appears to be in contact with the microsomal membrane. Microsomes prepared and fixed as indicated for (a). X 200,000. (c) Part of a microsomal vesicle (M) in which only one attached ribosome is visible. The groove (arrows) separating the subunits appears as a dense line parallel to the microsomal membrane (m). Microsomes prepared as indicated for (a). X 400,000. (d) Face view of a negatively stained microsomal vesicle. Only half of the vesicle is shown. At its periphery, the groove (arrow) between the subunits of an attached ribosome appears as a dense line parallel to the microsomal surface. Preparation of microsomes as for (a). X 240,000.

517

ATTACHMENT OF RIBOSOMES

30

T

t

Small

subunit

o

~

W

30

~

pmoies EDTA/ml.

FIG. 9. Effect of EDTA on tho release of ribosomal particles from microsomes. P ortions of a. suspension of microsomes in solution D (0'5 g tissue equivalent/ml.) wero treated with various amounts of EDTA (5 to 40 /Lmoles/ml.). Aftor 10 min in tho cold (O°C), tho portions were diluted with solution E and analyzed in the Spinco E ultracontrifuge using the ultraviolet ab sorption system. The dilution was adjusted to bring the den sitometry tracing of the photo. graphs wit h in tho region of linear response. Tho ordinate shows the analytical reading, which is proportional to the amount of material rel eased.

coefficient of ap proximate ly 39 s. The ab sence of the small amount of free (monomer) ribosomes usually present as contaminants in the microsome preparation was surprising, because we expecte d the appearance of two peaks corresponding to the large and small subuni ts derived from free monomers. It seems likely that these small concentrations of EDTA resulted in tho transient aggregation of the few monom ers present or in their unstable binding to the accompanying microsom es; as pointed out previously, any of these circumstances could lead to their rapid removal from the suspension. At 10 umoles EDTA, a component with a sedim entation coefficient of approximately 37 s was clearly seen at the proper dilution. The av erage value was 37 ± 2 s, which probably corresponds to t hat of the small subunit under the conditions used. At 15 psnoles EDTA, definitely two sedim entation boundaries were observed. Tho major one was a remarkably sharply defined 33 s component (33 ± 2 s), probably identical with the small ribosomal subunit. The second boundary corresponded to a 49 s component (49 ± 2 s), probably the large ribosomal subunit. Tho ratio of the amount of ultraviolet-absorbing material present as a 4-9 s component to that present as a 33 s component was only about 0,3, in remarkable contrast with the value of 2·0 ± 0·1 (determined by schlieren optical system) for the weight ratio of large to small subunits isolated from purified ribosomes dissociated with EDTA (Tashiro & Siekevitz, 1965a). At 20 p.moles EDTA (Plate I(a», the patterns were similar to that at 15 p.mo!cs, except that both the 33 s (33 ± 1 s) and 49 s (49 ± 1 s) components increased in amount. At 30 pmoles and 40 psnoles EDTA, the amount of large subunits relea sed COIltinued to increase with EDTA concentration, while the amount of small subunits detached from the microsomes remained at a constant level after 20 p.molesof EDTA. The ratio of the two materials approaches one at 40 p.moles of EDTA. At 100 psnoles EDTA, the S20,W of the main component was 48 s, which most

518

D. D. SABATDH, Y. TASHIRO AND G. E. PALADE

probably corresponds to the large subunit. The boundary, however, was quite broad, suggesting extensive heterogeneity. In some runs, with samples diluted 20 times in solution E, a component with a sedimentation coefficient of approximately 26 s, possibly corresponding to the small subunit, was also detected. This boundary was not clearly separable from the degraded ultraviolet-absorbing materials, and the relative amount of this component was less than 10% of the total, the rest being composed of heterogeneous material, degraded and slower as well as aggregated and faster. When microsomcs treated with 100 fLmoles of EDTA were diluted with solution D (containing 0·002 M-MgCI 2 instead of solution E containing no MgCI2 ) no boundary was observed. Probably all the ribosomal material aggregated under these conditions. 60,---,---,------,-----,

50

~.

--:. o

o

o

20 10

o

10

20

30

40

!"moles EDTA/ml. FIG. 10. S20. w of the large (0) and small (L) particles released from microsomos by treatment with various amounts of EDTA. Samples were analyzed as indicated in Fig. 9, and the S20.w values were plotted against the amount of EDTA added to I m!' of the original microsomal suspension. Each point represents one independent determination, except the filled circle (.) and filled triangle ( ... ) at 20 I'moles of EDTA/m!., which are averages of eight determinations with 48·5 ± 1·2 and 32·6 ± 0·7 s, respectively.

Sedimentation coefficients of ribosomal particles released from microsomes at various concentrations of EDTA. The present experiments show that the sedimentation coefficient of the smaller particle released from microsomes increases at lower EDTA concentrations: the s-value (Fig. 10) at 40 fLmolcs of EDTAjml. and 10 or 20 times dilution is r-..J 32 s, but at 15 fLmoles EDTAjml. or below this concentration it increases considerably, arriving at r-..J 40 s. The sedimentation coefficient of the larger particles released from microsomes by 15 to 40 fLmoles EDTAjml. was r-..J 50 s after 10 or 20 times dilution. At lower EDTA concentrations, the s-value of the larger particles could not be determined, because they remain attached to the microsomal membranes. The sedimentation coefficients of tho particles released from microsomes and the increase in the s-values of the smaller particles at low EDTA concentrations closely agree with the results obtained by Tashiro & Siekevitz (19600) on subunits from purified ribosomes. Hence we can conclude that the small and large particles released from micro somes in our experiments arc the small and large ribosomal subunits respectively.

ATTA CHMEKT OF RIBO SOME S

51!)

(ii) Ultracentrif ugal analysis of the particles detached by deoxycholate treatment from

EDTA-treated microsomes P ellets of washed microsomes, each deriv ed from 0·5 g wct weight of liver, were resuspended in 1 ml. of solut ion D. To this suspension 20/-Lmoles or 100/-Lmoles of EDTA at pH 7·6 were added , and the mixture was cent rifuged at 40,000 rev .jmin in the no. 40 rotor for 30 minutes. The pellets obtained were again resuspended either in 1 ml. of solut ion D or in 1 ml. of solution :E (see below), and then treated with 5% DOC (pH 7,6), to a final concentration of 0·5% . The DOC-treated mi crosomes were diluted 5 to 20 times just before the run in the analytical centrifuge. All these proc edures were carried out in the cold (0 t o 5°C). Twenty /-Lmoles/ml. of EDTA were added becau se the amount of 32 s component released from the microsomes r eached an almost const ant level at this concentration, while the amount of 47 s component was st ill quite sma ll (see Fig. 9 above). The ultraviolet absorption of the DOC, present in the final samples after dilution for the run at concentrations of 0·2 to 0,04%, was neglected because the E 2 6 0 mu of 1% DOC was only 0·042/cm. Sedimentation analysis in solution D (Plate l(b». In samples treated in succession with 20/-Lmoles EDTA and 5% DOC, the presence of components sediment ing as 80 to 90 s, 50 s and v--. 10 s was observed. The relative proportions of these components, immediately after the addition of DOC, was 36, 45 and 19%, respectively. Th e 80 to 90 s material was heterogeneous and probably contained several components, bu t tho po or resolution of the ultraviolet optical system prevented attempts to separ ate t hem . The component with v-> 50 s was mu ch more homogeneous, and the S20.W was apparently ind ependent of time up to 4·5 hours. The aver age value from four runs was 48 ± 2 s. The slowest component was also heterogeneous and did not make a clear boundary, sprea ding from i--. 15 s to 0 s. No boundary corresponding approximately to the small subunit, i.e. 32 s, was detected at any of the dilutions tested . When 100 /-Lmoles of EDTA were used, the sedimentation pattern s were essent ially similar, except for an increase in t he amount of degraded material s (......, 10 s). Thi s result suggests that the maj or component detached by DOC (48 + 2 s) is the large subunit left still attached to the microsomal membrane by the previou s EDTA treatment. Wc cann ot , however, ignore the possibility that this particle of '"'" 50 s is a mixture of large subunits and dim ers of small subunit s (Tashiro & Siekevitz, 1965a), because aggregation of ribosomes and/or their subunit s is probable in the presence of 0·002 lI-I-MgCI2 • This is the reason why a similar sedimentation analy sis was carried out in the absence of Mg 2 + , i.e. in solution E. Sedimentation analysis in soluti on E. In this series of experiments, pellets from microsomes previously treated with 20 or 100 /-Lmoles of EDTA/ml. were resuspended in solut ion E, solubilized with DOC (0,5% final DOC concent ra tion) and then diluted 5 to 20 times with the same soluti on just before sedimentation analysis. The sedimentation patt erns in the absence of Mg 2 + were similar t o t hose reported above in the presence of this cation. That is, a heterogeneous 80 t o 90 s material was observed to gether with a 50 s and a heterogeneous 10 s component . Th e 50 s was less homogeneous than in solution D, and in some instances the sedimentation coefficient was considerably less than 50 s. Plots of the S 20 .w again st time after addition of DOC revealed the gradual decrease of sedime ntat ion coefficient , with a value of 47 ± 2 s determined by extrapolat ion to zero t ime. Thi s value is similar to that determined with the schlieren opti cal syste m for the .s~o of the larg e subuni t

520

D. D. SABATINI, Y. TASHIRO AND G. E. PALADB

of purified DOC-prepared ribosomes (Tashiro & Siekevitz, 19600). The "" 80 sand 10 s components were quite heterogeneous. The relative proportion of the 10 s to the f""oo/47 s component was different from experiment to experiment, changing from 1 : 2 to almost 2 : 1. Presumably the 47 s component is less stable in the absence of Mg 2 + . As in the presence of Mg 2 + , no component of 30 s was detectable when this cation was omitted from the solution. When microsomes were treated with 100 fLmoles EDTA, the sedimentation patterns were essentially similar to those described above, but the proportion of degraded and aggregated material was larger. f""oo/

f""oo/

f""oo/

4. Discussion and Electron Microscopy Taken together our results indicate that: (a) At low concentrations of EDTA ("" 20 fLmoles EDTA per 0·5 g tissue equivalent of microsomes), particles with a sedimentation coefficient similar to that of the small ribosomal subunits are preferentially released from microsomes. If these particles arc small subunits (as is here assumed), they amount to all or nearly all small subunits expected to be present on the basis of their RNA content and of the total ribosomal RNA content of the microsomes. (b) The release of particles with sedimentation coefficients close to that of the large subunit (47 s) requires higher concentrations of EDTA. Moreover, it does not occur in the weight ratio of 2 : 1 with respect to small subunits, as would be expected if whole ribosomes were to become detached and then dissociate, or if the two processes were simultaneous. This discrepancy is clearly shown by the distribution of material in the density gradients and by the results obtained with the analytical centrifuge. At any given concentration of EDTA below 40 fLmoles, when both subunits were present and the amount of degraded material was small, an excess of small subunits was clearly demonstrated by the weight ratio of released subunits. The identification of the two classes of particles released from microsomes with the corresponding classes of subunits that result from the dissociation of purified ribosomes relied initially only on the close similarity of the corresponding sedimentation coefficients. The basis of this identification was broadened later by the finding that newly synthesized protein is associated with the larger of the subunits released from the microsomes. That the large subunits derived from purified ribosomes contain the nascent polypeptide has been shown in the case of guinea pig liver ribosomes (Tashiro & Siekevitz, 1965b) and E. coli ribosomes (Gilbert, 1963) by experiments involving the incorporation of radioactive amino acids into proteins. (c) Even at the highest concentrations of EDTA (500 fLmolesfO'5 g tissue equivalent of miorosomes), 35% of the RNA (and consequently a similar proportion of the ribosomal material) remains undetached from the microsomes. The assumption that this RNA corresponds mainly to 47 s subunits unaffected by EDTA and possibly to some "stuck monomers" resistant to dissociation (Tashiro & Siekevitz, 1965b; Gilbert, 1963) is supported by the results obtained in the study of the particulate material released by DOC from EDTA-treated microsomes, Though this material is heterogeneous, and aggregation and degradation always occur, the two main boundaries after 20 fLmoles of EDTA and 0'5% DOC correspond to 80 sand 50 s. No small subunits were observed, although if present they would not be expected to be totally degraded at this EDTA concentration. Another indication comes from the sedimentation analysis of microsomal suspensions treated in f""oo/

f""oo/

ATTACHMENT OF RIBOSOMES

521

succession with 100 fLmoles of EDTA and 0·5% DOC. When the runs were performed under conditions of Mg 2 + protection against subunit instability (solution D), only 47 s particles were observed. The presence of small subunits dimerized to 50 s (Tashiro & Siekevitz, 19600) among the particles released under these conditions, is not excluded; but their number is expected to be small or negligible since most small subunits are detached at low EDTA concentrations, and since the material released by DOC after EDTA contains the nascent polypeptide. As was mentioned before, this is a property of the large subunit. (d) After a short period oflabeling, most ofthe newly synthesized protein, although still bound to ribosomes (as shown by DOC treatment of intact labeled microsomes) is not released therefrom by the action of EDTA. In fact, even after treating microsomes derived from 0·5 g of liver with the highest EDTA concentration (Table 2), 77 % of the radioactive protein remained in the microsomes, Part of this radioactive protein could be released from the microsomes in a form still in association with ribonucleoprotein particles by subsequent treatment with DOC. We have indicated above that these particles are most likely large subunits and possibly "stuck monomers". This property of EDTA in distinguishing between ribosomes containing newly synthesized proteins and those "inactive" at the time in protein synthesis, contrasts with the effect of DOC on labeled microsomes. When two different concentrations of DOC were used, different proportions of ribosomes were released from the microsomes (Figs 7 and 8), but their specific radioactivity (expressed as counts per minute found in the protein-polypeptide of the Schneider procedure per unit of optical density at 260 mfL) did not vary with detergent concentration. It follows that in detaching the ribosomes, presumably by affecting the membranes (Palade & Siekevitz, 1956), DOC failed to discriminate between labeled ("active") and unlabeled ("inactive") ribosomes. Different mechanisms and/or a different point of attack at the ribosome-membrane junction are thus suggested for EDTA and DOC. Discarding the unlikely possibility of rearrangement of the ribosomes on the membranes under our conditions, points (a) and (b) of the above enumeration show that of the three possible arrangements of the ribosomes on the membranes depicted in Fig. 11, the one crossed out (adhesion through the small subunits) can be eliminated. The systematic way in which the subunits are detached sequentially by EDTA also eliminates the additional possibility that the ribosomes are attached to the membrane by anyone of these subunits at random. With the evidence just presented, it is not possible to decide between possibilities at the left and right of Fig. 11, i.e. (i) that only the large subunit is attached to the ('oJ

FIG. 11. Three possible ways of ribosome attachment to a microsomal membrane. The crossed-out possibility (attachment through the small ribosomal subunit) is eliminated because, upon addition of EDTA. a. ~ 32 s particle is released first from microsomes. A choice between the two other possibilities is made on the basis of electron microscopy (see Plate U(a) ,to (d)).

522

D. D. SABATINI, Y. TASHIRO AND G. E. PALADE

membrane, or (ii) that both subunits are attached but the forces binding the large subunit to the membrane are stronger or more resistant to EDTA than those acting on the small subunit which, for this reason, is released first. A choice between these two possibilities could be made by electron microscopy, provided that the partitioned structure of the ribosomes could be visualized on attached particles. If the arrange. ment at the left (Fig. 11) holds, the groove separating the subunits should be parallel to the microsomal membrane. If the arrangement at the right applies (Fig. 11), the groove should be perpendicular to the membrane. (a) Electron microscopy of isolated mierosomes

We have found (Sabatini, Tashiro & Palade, unpublished results) that the treatment of guinea pig liver and pancreas ribosomes with sucrose- or deoxycholate-containing solutions tends to mask the partitioned appearance of the ribosomes in the electron microscope. Thus, to be able to recognize both subunits and the groove separating them within a ribosome, microsomes were prepared in salt-containing media free of sucrose. Under these conditions, the surface configuration of ribosomes is more favorable for the visualization of the subunits by negative staining with sodium phosphotungstate. (b) Electron microscopy

Microsomes were prepared from guinea pig liver and pancreas homogenized in O·14M.KCI, O'005M-MgCI 2 , O'OlOM-Tris (pH 7,6), and fixed in suspension with 6·5% glutaraldehyde. Preparations negatively stained with sodium phosphotungstate showed in the electron microscope the presence of microsomal vesicles varying widely in size and surrounded by the contrast mass. Ribosomes were easily recognizable on the surface of many vesicles, but they could be studied in more detail at the periphery of the vesicles. Here they lay embedded within a thin film of phosphotungstate, and presented the plane separating the subunits parallel to the electron beam axis. In these unpurified preparations, free ribosomes were also visible, as well as numerous smooth vesicles and mitochondrial fragments. Plate II shows a microsomal vesicle; six ribosomes are indicated on the upper part of it. All of these ribosomes show a suggestion of a groove separating the subunits. This groove is in each case approximately parallel to the membrane surface and only one ribosomal subunit makes contact with this surface. Grooves with a similar orientation appear more clearly in the two ribosomes attached to the bottom of the same microsome (Plate II(a». Another example where the ribosomes are less compact but indications of the grooves can still be seen is shown in Plate II(b). In Plate II(c) only one ribosome is visible on the membrane of a small microsomal vesicle; in this ribosome a thin dense line of phosphotungstate marks the position of the groove which, as in the previous examples, runs parallel to the membrane. Grooves parallel to the membranes can also be seen in microsomes appearing in the micrographs in face view. These microsomes are almost completely embedded within the PTA film and for this reason the contrast metal provides no optical section of the microsomal vesicle. The plane between the subunits of a ribosome located at the periphery of one vesicle completely embedded in PTA is marked in Plate II(d). Here the dense line is thicker, presumably because the plane between the subunits is slightly inclined with respect to the beam axis. An additional electron microscopic observation worth mentioning can be made on Plate II(a) and (b). The ribosomes on these microsomes appear at close distances of

ATTACHMENT OF RIBOSOMES

523

each other, forming an almost continuous layer of particles on the membrane. This arrangement differs from the one seen in sections of osmium tetroxide-fixed, plasticembedded microsomes and endoplasmic reticulum clements. In the latter case, in between ribosomes there are stretches of particle-free membrane. Several reasons can be given for the discrepancy between the results given by the two techniques. (a) Since the negatively stained vesicles are presented to the beam in toto, not in sections, several rows of ribosomes along the edge of the flattened vesicles, not necessarily close neighbors, can be projected in the same image plane. (b) In the case of ribosomes on the face of these vesicles, apparent crowding of particles could also result from the superposition of two images, one for each face (top and bottom) of the flattened vesicle. Against these possibilities one should consider that, in general, the PTA film is thin enough to provide an optical section of the vesicle, and overlap of ribosome images rarely occurs. Other observations to be taken into account concern the diameter of the ribosomes, which in sections of Os04-fixed, plastic-embedded material usually measures 150 to 200 A, while in negatively stained preparations it reaches 250 to 300 A. Taken together, these findings suggest that micrographs of Os04-fixed, plastic-embedded specimens give a reduced ribosome diameter because of lack of sufficient contrast at the periphery of the particles, or because of ribosome shrinkage during dehydration, or finallyin the case of isolated microsomes and ribosomes-because of ribonucleoprotein degradation at the periphery ofthe particles (cf. Huxley & Zubay, 1960). Reduction ofspacings among ribosomes can also result, however, from shrinkage or "contraction" of microsomal membranes. Available information suggests that the first factor (incomplete visualization) is more important than the second (membrane contraction), but further work is needed to determine the extent.to which each ofthem is involved. As was indicated in points (a), (b) and (c) of the enumeration at the beginning of this discussion, our results showed that a fraction of the microsome-bound ribosomes is resistant to detachment by EDTA. The results obtained by treating miorosomes in succession with EDTA and DOC indicate that most of the particles left on the membranes by EDTA are probably 47 s subunits, and contain newly synthesized proteins and/or nascent polypeptides. The recovery of RNA still in particulate but partially degraded form after DOC treatment of EDTA-treated microsomes shows that these particles suffer some degradation during the prolonged procedures required for their isolation. However, the recovery of radioactive material parallels that of the RNA and the specific radioactivity remains constant. The coincidence of strong attachment of, and presence of recently synthesized polypeptide on, large subunits suggests that these two conditions are functionally correlated. The finding has particular significance in connection with current views on the role taken by the endoplasmic reticulum of protein-exporting cells in the first step of the secretory cycle. According to these views, which are supported by cell fractionation (Siekevitz & Palade, 1960; Redman et al., 1966) and autoradiographic work (Caro & Palade, 1964) and both (Jamieson & Palade, 1966), the cycle starts with the transfer of protein from the ribosome to the interior of the cisternae of the endoplasmic reticulum. As such, it seems plausible to consider the possibility that strong attachment of "active ribosomes" is related to passage of the protein through the membrane. However, our work does not indicate which of the following possibilities holds. (a) The ribosomes which are strongly attached are, because of this situation, more active in protein synthesis and therefore become labeled in vivo. (b) The presence

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D. D. SABATINI, Y. TASHIRO AND G. E. PALADE

of the product of protein synthesis on the ribosomes is what makes them stick to the membrane. In any event, it can be said that if magnesium ions are involved in the attachment of ribosomes affected by EDTA, an extra factor, not affected by the chelating agent, is present in active undetached ribosomes. This work was partially supported by grant HE 5648 from the National Institutes Health to one of us (G. E. P.) and grant AM 01635 from the same institution to Dr Siekevitz. We are also grateful to Dr Siekevitz for discussions related to the subject the paper. A preliminary report has been presented to the 6th International Congress Biochemistry (Sabatini & Tashiro, 1964, Abstracts, p. 65).

of P. of of

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