THE JOURNAL OFBIOLOGICAL CHE~~~ISTRY Vol.244, No. 16, Issueof August.25, PP. 4308-4315, 1969

Printed in U.S.A.

Biosynthesis of Serwtn Proteins and Ferritin by Free

and Attached Ribosomes of Rat Liver*

COLVIN M. REDMAN

(Received for publication, December 30, 1968)

From The New York Blood Center, New York, New York 10092

SUMMARY

Polysomes of rat liver appear to be either attached to the membrane of the endoplasmic reticulum or free in the cytoplasm. It has been postulated that attached ribosomes are engaged in the synthesis of secretory proteins while free ribosomes make nonexportable proteins. Experimental support for this hypothesis has been obtained by studying the site of synthesis of serum proteins, an example of secretory proteins, and of ferritin, an example of a non- exportable protein. Two to three minutes after an intra- venous injection of 3H-leucine into the rat there is equal and maximum incorporation, per mg of RNA, into trichloracetic acid-insoluble proteins bound to both free and attached ribosomes. The newly formed protein attached to the ribosomes was released by treatment with spermine and puromycin. About 80% of the newly formed serum protein recovered by this method was obtained from the attached ribosomes. The radioactive ferritin, however, was found to occur in the proteins released from the free ribosomes. Similar results were obtained by incubating free and attached polysomes (microsomes) in uifro with radioactive amino acids and under conditions for protein synthesis. Again, over 80% of the protein synthesized by attached ribosomes was found to be serum proteins, and the free ribosomes made 7 to 20 times more ferritin than did attached ribosomes. The radioactive proteins were detected and isolated by precipita- tion with specific antisera and the specificity of the methods checked by a combination of immunoelectrophoresis and autoradiography.

As seen in electron micrographs, the ribosomes of rat liver appear to be either attached to the membranes of the endoplas- mic reticulum or to be free in the cytoplasm (1, 2). Methods for the separation of these two classes of liver ribosomes have been developed and in the adult rat liver the attached ribosomes comprise about 60% of the ribosomal RNA (3, 4). It has been noted that cells which are actively engaged in protein synthesis

* This work was supported by Grant HE 09011 from the Na- tional Heart Institute and bv Grant T464 from the American Can- cer Society to A. Mazur. A part of this work was supported by Grant AM 13620 from the National Institute of Arthritis and Metabolic Diseases.

and secretion have larger numbers of attached ribosomes than cells which are engaged for the most part in making proteins for their own use. It was postulated that attached ribosomes make secretory proteins and free ribosomes make nonexport- able protein (5, 6).

Early biochemical support for this hypothesis was provided by studies on the pancreas. Siekevitz and Palade (5) showed that radioactive oc-chymotrypsinogen was bound to attached ribo- somes of pancreas rather than to free ribosomes, at 1 min after an intravenous injection of a radioactive amino acid into a guinea pig. Studies in viva of albumin synthesis in rat liver had indi- cated that the newly formed albumin first appeared in the deoxy- cholate-soluble extracts of the rough endoplasmic reticulum. This indicated that it was being made on attached ribosomes (7). Other studies in vivo of protein synthesis in rat liver showed that the attached ribosomes were more active in the incorporation of W-arginine into trichloracetic acid-insoluble protein, than were free ribosomes, but the synthetic products of these two classes of ribosomes were not identified (8). Later work has shown that both free and attached ribosomes of rat liver are equally active, in tivo, in incorporating radioactive leucine into trichloracetic acid-insoluble protein (9).

Microsomes (fragments of the endoplasmic reticulum) incu- bated in v&o synthesize albumin and other serum proteins (lO- 12). These earlier studies, however, have not considered whether naturally occurring free polysomes are also capable of synthesizing secretory proteins or whether the types of proteins made by free and attached ribosomes of rat liver differ. Earlier studies in vivo from this laboratory had shown that rat serum pro- teins are synthesized on attached rather than free ribosomes (13). Similar results were subsequently obtained by Takagi and Ogata using a system in vitro (14).

To test further the hypothesis that attached ribosomes synthe- size secretory proteins while free ribosomes make nonexportable proteins, I have compared the synthesis of serum proteins (secre- tory proteins) and ferritin (a nonexportable protein) by hepatic free and attached ribosomes both in vitro and in vivo.

EXPERIMENTAL PROCEDURE

Materials

L-Leucine-4,5-3H (5 Ci per mmole) and a mixture of I5 uni- formly labeled L-W-amino acids were purchased from New England Nuclear. Pyruvate kinase, was obtained from Sigma and sodium deoxycholate from Mann. Rabbit antisera to rat serum or to chicken serum were obtained from Hyland Labora-

4308

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Issue of August 25, 1969 C. M. Redman 4309

tories. Rabbit antiserum to cryst,alline horse ferritin, which has been shown to cross react with rat ferritin, was obtained from Dr. A. hlazur, and was prepared as previously reported (15). An RNase inhibitor, described by Blobel and Potter, was added to a stock solut,ion of 2.3 M sucrose in Buffer TKRI (50 mM Tris-EIcl, pH 7.5-25 rn3f KCl-5 rnM MgClJ to prepare the 1.35 M and 2.0 M sucrose in l3uffer TKM solutions that were used in the separation

of t,he ribosomal fractions (16).

A!! efh0d.S

Preparation oj Ribosomal Ikxtions--In the experiments in

viva, female albino rats weighing about 150 g were starved over- night and anaesthetized with Nembutal. L-Leucine-4, 5-3H (0.5 mCi) was injected into the femoral vein. The livers were removed at specified intervals of time after the injection and placed in cold 0.25 M sucrose in Buffer TKM. The washed livers were blotted, T\-eighed, and homogenized in 3 volumes of 0.25 M sucrose in Buffer TKM. The ribosomal fractions were isolated by the method of Loeb, Howell, and Tomkins (4) with some mod- ifications. A flow diagram of this procedure is presented in Fig. 1. The homogenate was filtered through a double layer of cheesecloth and then centrifuged at 700 x g for 20 min. The pellet was resuspended in 2.3 31 sucrose in Buffer TKXI and cen- trifuged at 54,450 x g for 1 hour to remove the nuclei. The material which packed at the top of the tube TTTas recovered with a spatula, resuspended in 0.25 111 sucrose in Buffer TKhI and

Livers from starved rats homogenized in 3 ~01s. of - _

sedimented at 6,590 x g for 15 min. The pellet was resus- pended in 0.25 M sucrose in Buffer TKM (Fraction I).

The second attached ribosomal fraction was prepared by cen- trifuging the 700 x g supernatant fluid at 20,000 x g for 20 min. This pellet \\Tas washed once with 0.25 M sucrose in Buffer TKM and resuspended in the same medium (Fraction II). The free polysomes were obtained from the 20,000 x g supernatant fluid (Fraction III). Fractions I and II were treated with 13% so- dium deoxycholate adding 0.5 ml for each gram, wet weight of liver. All three fractions were then layered over 3 ml of 2.0 M sucrose in Buffer TKM and 3 ml of 1.35 M sucrose in Buffer TKM, both containing RNase inhibitor, and centrifuged at 105,000 x g at 3” for 17 to 19 hours. The pellets which sedimented through the 2.0 M sucrose \I-ere resuspended in 0.25 M sucrose in Buffer TKM.

Studies in vitro were performed with free polysomes and micro- somes. These fractions were prepared as follows. Livers from starved rats were homogenized in 3 volumes of 0.25 M sucrose in Buffer TKM and centrifuged at 10,300 xg for 15 min to remove nuclei and mitochondria. The supernatant fluid was then lay- ered over a discontinuous gradient of 3 ml of 2.0 M sucrose and 3 ml of 1.35 M sucrose with Buffer TKM and RNase inhibitor and cent.rifuged in a Spinco No. 40 rotor for 17 to 19 hours at 105,000 x g. The pellet which sedimented through the 2.0 M sucrose was designat,ed as free polysomes. A band of material which cpllected above the 2.0 x sucrose layer was removed with a

0.25 M sucrose in TKM tiltered through cheesecloth spun 700 g x 10 min.

* pellet resuspended in 2.3 M

sucrose in TKM ~ f54>450giy

pellet top layer discarded resuspended

supernatant spun

f 0,000 g x 20 min.

pellet suspended in 0.25 M

supernatant

in 0.25 M sucrose

in TKM spun 6,590 g x

15 min.

resuspended in discarded 0.25 M sucrose

in TKM add 0.5 ml 13%

DT per g liver

Y pel et -4

supernatant resuspended in 0.25 M discarded

sucrose in TKM added 0.5 ml 13% DOC

per g. of liver layered on discontinuous

gradient over 1.35 M and 2.0 M sucrose spun 105,000 g x 17 hrs.

I

4 pellet

ATTACHED I

4 supernatant discarded

+ layered on discon- tinuous gradient over 1.35 M and 2.0 M sucrose spun 105,000 g x 17 hrs.

J pellet

ATTACKED II

supernatant

layered on discontinuous gradient over 1.35 M and 2.0 M sucrose spun 105,000 gx17 hrs.

\c pellet

FREE III

& supernatant discarded

FIG. 1. Procedure for the separation of attached (Fractions I and II) and free (Fraction III) rihosomes

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4310 Protein L3ynthesis by Free and Atlacked Ribosomes Vol. 244, No. 16

Pasteur pipette. It was diluted with Buffer TKM to give a final sucrose concentration of approximately 0.25 M and centrifuged for 1 hour at 105,000 x g. The pellet (microsomes) xas designated as attached ribosomes.

dssay of Nuscent Proteins from Free and Attached Ribosomes- In the experiments in vivo, the protein that was bound to the ribo- somes n-as assayed. Total protein synthesis xl-as calculated from the radioactivity of the hot trichloracetic acid-insoluble protein obtained from an aliquot of the ribosomal suspensions. When assaying for specific proteins, the ribosomes, suspended in 0.25 M

sucrose in Buffer TKRI (about 200 mg per ml), were incubated with 5 x IOP M puromycin and 2 X 1OP hf ITP for 10 min at 37” followed by addition of 5 x 1OF RI spermine and incubation for another 20 min. X large white precipitate was removed by centrifugation and the clear supernatant was used to dctcrmine the quantity of radioactivity which precipitated with either rabbit antiserum to rat serum or rabbit antiserum to horse ferritin. When determining the radioactivity of serum proteins, the super- natant was first cleared of nonspecific radioactivity by adding rabbit antiserum to chicken serum together n-ith some carrier chicken serum and incubating for 1 hour at 37”, and then 17 hours at 4” as suggested by Campbell, Greengard, and Kernot (10) and by Peters (7). Carrier rat serum and an excess of rabbit anti- serum to rat serum was added to the cleared supernatnnt and again the mixture was incubated for 1 hour at 37” and 17 hours at 4”. The resultant precipitate was washed 5 times xl-ith cold so- dium chloride solution and dissolved in 1 ml of Hyamine for counting. As a control, some of the tubes were treated a second time with chicken serum and antiserum to chicken serum. The amount of radioactivity from these control samples was sub- tracted from the values obtained with the rat serum precipitates.

In order to determine the radioactivity n-hich precipitated with rabbit antiserum to horse ferritin, the released proteins were incubated wit,h antiserum for 30 min at 37” and then mixed with carrier rat ferritin and incubated for another 30 min at 37”. n’or- rnal rabbit serum, instead of rabbit antiserum to horse ferritin, was added to control tubes and allowed t,o remain at 4” overnight. The controls with normal rabbit serum always contained some de- natured protein which precipitated on standing. The radioac- tivity obtained from this denatured protein was subtracted from the radioactivity of the ferritin which precipitat,ed with the anti- serum. In the experiments in vitro the reaction of incubated free and attached polysomes was stopped by freezing. The thawed material TT-as then souically disrupted for a total of 9 min with a Bronson Sonifier at, setting number 5 in a salt-ice water mixture to keep the temperature between O-5”. The incubation mixtures were centrifuged at 105,000 x g for 90 min and the su- pernatant fluids were used to determine the radioactivity which precipitated with antiserum to rat serurn or antiserum to ferritin, as described above. In some experiments the supernatant fluids were used for a partial purification of ferritin prior to treatment \J-ith the antiserum. In these cases carrier rat ferritin xas added to the supernatant fluid and t,hen it was heated Tvith stirring to 65” in a water bath. The mixture was cooled and the precipi- tated protein was removed by ccntrifugation. The ferritin was precipitated frorn the clear solution with 50% ammonium sulfate and then dialyzed overnight against cold sodium chloride solu- tion. The ~1-1 of the protein solution was then loIT-ered to 4.6 with acetic acid, and any protein which precipitated xx-as dis- carded. The pH of the solution mas then raised to 7.4 and the

ferritin in the solut.ion w-as recovered by precipitation n-ith its antiserum as described above.

Incubation in Vitro of Free and Attached Polysomes--Free poly- somes or microsomes from 1 to 2 g of rat liver were incubated with 0.1 ml of a 105,000 X g liver supernatant, fraction (0.5 g of liver per rnl) or with pH 5 enzyme from an equivalent amount of super- natant fluid. In addition, the incubation mixture contained 1 pmole of ATE’, 0.25 pmole of GTP, 5.5 prnoles of MgC12, 30 pmoles of KCl, 10 pmoles of sodium phosphoenolpyruvate, 50 pg of pyruvate kinase, 1 pmole of mercaptoethanol, and 50 pmoles of Tris-IICl, pH 7.6. Some expcrirnents were perforrned with 50 PCi of a uniformly labeled mixture of 14C-amiuo acids, and others with 10 &i of WL-leucine (5 Ci per pmole). These quantities are for a total volume of 1 ml. The incubations were performed at 37“ for 30 min and the volumes varied from 1 to 10 ml, depend- ing on the experiment. Some experiments were stopped by add- ing 10P4 M puromycin and incubating for another 15 min at 37”, and then freezing the reaction mixture.

Immunoelectrophoresis and 11 utoradiography-Bacto-Agar pur- chased from Difco was used. The gel n-as 1%; in 0.05 M sodium barbital buffer, pH 8.2, and the samples were run for 3 hours at 4 ma per microscope slide (75 x 25 mm). The antisera, was added to the troughs and allowed to react overnight in a moist atmos- phere at room temperature. The slides were then mashed with sodium chloride solution several times during a 24-hour period. The slides mere further soaked in distilled water for 8 hours with several changes of water and then dried overnight with bibulous paper. The dried slides Tvere stained with hmido s&n-nrz. Ko- dak Zjo-Screen X-ray filrn was used for autoradiography. Espo- sure times varied from a few days to 8 weeks. The antigen was prepared from the released proteins of the free and attached ribosomes. The clear supernatant fluids n-ere dialyzed overnight against cold 0.01 &I Tris, pH 7.5, lyophilized, and redissolved in water at & their original volume. Then, 15 ~1 of antigen were applied to the immunoplates and 0.1 ml of antiserum x-as placed in the troughs.

Other nnalyticul Methods-Protein radioactivity was deter- mined by the method of Siekevitz (17) after dissolving in 1 ml of H-amine. This mixture was added to 10 ml of toluene phosphor and counted in a Tri-Carb liquid scintillation counter. Radio- activity was corrected for quenchin, m by adding known amounts of W- or Wtoluene and recounting to calculate counting effi- ciency. Protein was determined by the method of Lowry et al. (18) using crystalline bovine serum albumin as standard. RSA \q-as determined by the orcinol method (19) u,\ing yeast Rx& hydrolyzed in 5% trichloracetic acid, as standard.

RESULTS

Use of Sodium Deoxycholate to Test Purity oj Ribosomal Frac- tions-To det,ermine the quantity of free ribosomes in the cell fractions obtained by differential centrifugation, the fractions were layered over 1.35 M and 2.0 h!r sucrose in Buffer TKM and centrifuged at 105,000 x g for 17 hours. The ribosomes, unat- tached to membranes, pelleted to the bott,orn of the tubes. When the fractions were treated with deoxycholat,e prior to cen- trifugation, the amount of ribosomes recovered is a measure of fret plus attached ribosomes. As reported by Loeb et al. (4) cell fractions obtained by differential centrifugation contained either free or attached ribosomes with only small cross contamina- tion (Table I). Attached Fraction II showed an 8 to 16% coil-

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TMILE I

EJect of deozycholale on amounts of libosomes obtained from various cell fractions

Cell fractions obtained as described under “Experimental Procedure” were layered over sucrose zones of 1.35 M and 2.0 JI sucrose with Buffer TKM aud centrifuged at, 105,000 X 9 for 17 hours. The llNA values were taken from the material which pelleted through the 2.0 w sucrose layer. Those samples treated with sodium deoxycholate received 65 mg per g of liver. The percentage of impurity was calculated from the total ribosomal RNA in the cell fraction. If there were no cross contaminations between free and attached ribosomes, the altached Fractions I and II would show no RNA which pelleted without dcoxycholate and also the amounts obtained from the free ribosomes (Fraction III), with and without deoxycholate. would have been equal.

Ribosomes pelleted through 2.0 M sucrose

Ribosomal fraction

Attached I

Attached II

Free III

Experiment

1

2 0.01

I 0.04 2 0.14

1 0.85 2 0.72

0.80 1

0.47 8 0.74 10

1.02 17 0.92 1S

Incorporation in vivo of 311-A-leucine into newly formecl jerritin, serum proteins, and trichloracetic o&-insoluble proteins of

free and attached ribosonzes

The values for each cxpcrimcnt are the averages of triplicate determinations =t S.D. The determination of ferritin and strum protein radioactivity is described under “Experimental Proce- dure.” Attached ribosomes (Fractions I and II) and fret ribo- somes (Fraction III) were prepared as described in Fig. 1. Rats were treated by intravenolls injections and their livers were re- moved 3 min after injection.

Radioactivity

Ribosomal fraction

Ferritin Serum proteins Trichloracetic acid- precipitable proteins

/

dflnt/g liver

Experimeni, 1 Attached I.. 341 f 30 3G,OOO z!z 2,400507,000 f 8,GOO At&shed II.. 734 i 164 23,500 f 2,200352,000 f 5,200 Free III... 9,756 & 1,736 4,480 & 480 430,000 + 3,GOO

Experiment 2 Attached I.. 328 XII 228 31,000 =!z 3, GO0 479,000 & 8,300

Attached II.. 132 & Gl 34,000 f- 2,200 439,000 f 10,200 Free III. 1,889 f 93 3,420 f 250 595,000 3~ 13,700

tamination with free ribosomes. The attached ribosomes in

Fraction III were not isolated in the normal experimental pro-

cedure, since this fraction is not treated with deoxycholate, and therefore, the 17 to 18% of attached ribosomes would normally

remain at the 1.35 M sucrose zone. Attached Fraction I had no free ribosomes, as indicated by the absence of ribosomes which

pelleted when the fraction was not treated with deoxycholate. Hence, the only contamination in these experiments is a possible 8 to 16% contamination of attached Fraction II by free ribo- somes. However, this does not take into account the probability t.hat some ribosomes, which may be attached in tioo, were re- moved from the endoplasmic reticulum during homogenization. Studies by Blobel and Potter (3), however, have shown that increasing the severity of homogenization did not increase the amount of free ribosomes recovered, but merely led to further fragmentation of the endoplasmic reticulum membrane.

Incorporation in VCJO of 3H-L-Leucine into Nascent Proteins on

Free and Attached Ribosomes-Three minutes after an intrave- nous injection of WL-leucine, the radioactivity, per g of liver, of the protein bound to the three ribosomal fractions \vas about the same. The two attached Fractions I and II were equally active

and the free ribosomes (Fraction III) were just as capable of syn-

thesizing trichloracetic acid-insoluble protein as were the at- tached ribosomes. The amounts of radioactive serum proteins found on attached and free ribosomes, however, were markedly different. Attached Fractions I and II had more nascent serum

,-0

E a 40-

ATTACHEDI&lI

L_;__L- I-LI

3 5 8 MINUTESAFTERINTRAVENOUS INJECTION

FIG. 2. Time course of incorporation of 3H-r-leucine into fer- ritin bound to free and attached ribosomes. The ferritin radioac- tivity was determined as described under “Experimental Pro- cedure.” A shows a single experiment representative of the time course of incorporation of 3H-L-leucine into ferritin. B shows the percentage of distribution of newly formed ferritin in free or at- tached ribosomes f S.D. In B the value at 1 min is the mean of three experiments while at 3, 5, and 8 min they are the mean of seven values f S.D.

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Protein Xynthesis by Free and Attached Ribosomes Vol. 244, No. 16

TABLE III Incubation in vitro of free and attached ribosomes and recovery of

released proteins with antisera to serum proteins and ferritin Free polysomes or attached ribosomes from 10 to 20 g of liver

were incubated with 1.0 ml of a 105,000 X g liver supernatant (0.5 g of liver per ml) or with pH 5 enzyme from an equivalent amount of supernatant. Also in the incubation mixture were: 10 pmoles of ATP, 2.5 pmoles of GTP, 52.5 pmoles of MgC12, 310 pmoles of KCl, 100 pmoles of sodium phosphoenolpyruvate, 500 pg of pyruvate kinase, 10 pmoles of mercaptoethanol, and 500 pmoles of Tris-HCl, pH 7.6. Experiment 1 had 50 pCi of a uni- formly labeled mixture of W-amino acids and Experiments 2 and 3 had 100 &i of 3H-n-leucine (5 Ci per pmole). Final volume, 10 ml, was incubated at 37” for 30 min and then IO-4 M puromycin was added and the reaction incubated for another 15 min. Serum protein and ferritin were assayed from the released proteins as described under “Experimental Procedure.” In Experiment 1, the ferritin in the released proteins was partially purified, as de- scribed under “Experimental Procedure,” before precipitation with its antiserum. The normal serum control values in Experi- ments 1,2, and 3 were 20,23, and 24a/., respectively, of the ferritin values in the free ribosomes.

Ribosomal fraction - Total

protein

Experiment 1 Free. . . . 80,500 Attached.. 136,400

Experiment 2 Free. 230,000 Attached. . 384,000

Experiment 3 Free. 377,000 Attached.. 882,000

- I -

Rsdioactivity

Released Serum protein protein

4Nmg RNA

- I -

34,600 4,440 1,395 13 26,800 20,250 202 75

37,000 4,440 1,110 12 24,500 18,620 122 76

150,000 4,800 3,000 226,000 117,200 226 I -

3 52

Ferritin

.-

Released radio- activity

Ser”F Ferritin protem

%

I -

4 0.1

3 0.5

2 0.1

proteins, per mg of RNA, or per g of liver, than did the free ri- bosomes (Table II). However, the radioactivity that was pre- cipitated by antiserum to ferritin was obtained mostly from free ribosomes. Table II illustrates two experiments in which 80 to 90% of the ferritin radioactivity on the ribosomes was associated with the free ribosomes.

The time course of 3H-n-leucine incorporation into the protein precipitated by antiserum to ferritin was similar to that of the trichloracetic acid-precipitable protein. Fig. 2A shows that maximum incorporation into ferritin on the ribosomes occurred at 3 min. At all time intervals most of the ferritin radioactivity was on the free ribosomes. The incorporation of 3H-L-leucine into ferritin varied considerably as can be seen in the two experi- ments shown in Table II. However, the percentage of total ribo- somal ferritin radioactivity which appeared on the free ribosomes was always more than that on the attached ribosomes, and this value was more constant then the total incorporation. There- fore, the percentage distribution between free (Fraction III) and attached ribosomes (Fractions I and II), with the standard devia- tion from the mean, is shown in Fig. 2B. One minute after intravenous injection there is overlap and this may be caused by the fact that the point represents the values from only three experiments whereas the other points represent the mean of seven

values. However, there was always more ferritin radioactivity found on the free ribosomes than on Fractions I and II combined.

Incorporation in Vitro of Radioactive Amino Acids into Serum Proteins and Ferritin-Both free and attached ribosomes (micro-

FIG. 3. Autoradiograms of immunoelectrophoresis plates. A, radioactive precipitin bands obtained with rabbit antiserum to rat serum with the nascent proteins released from free and at- tached ribosomes labeled in vivo with L-W-amino acids. The radioactive proteins were placed in the center well and electro- phoresis who performed as described under “Experimental Pro- cedure.” Each well contained about 400 dpm. The rabbit an- tiserum against rat serum was placed in troughs parallel to the direction of electrophoresis. Autoradiography was for 4 weeks. I and II are proteins derived from attached ribosomes and III is proteins derived from Fraction III or free ribosomes. B radioac- tive serum protein precipitin bands obtained from proteins syn- thesized by free and attached ribosomes (microsomes) incubated in vitro with n-‘%-amino acids. The released proteins from free and attached ribosomes were prepared as described under “Experi- mental Procedure”. The conditions for immunoelectrophoresis and autoradiography were as described in A. C, ferritin synthe- sized in vitro by free or attached ribosomes. The released radio- active proteins from either free or attached ribosomes (micro- somes) were heated to 60” in a boiling water bath. The denatured protein was removed by centrifugation and to the supernatant was added (NH4)sS04 to a final concentration of 50%. After re- maining at 0” for 2 hours, or 4” overnight, the precipitated pro- tein was collected by centrifugation and dissolved in a small vol- ume of sodium chloride solution. The precipitated protein was dialyzed overnight against cold NaCl solution and then reduced in a vacuum to about 0.01 of its original volume. Finally 15 ~1 of this solution containing about 1500 dpm was placed in the center well for immunoelectrophoresis. Rabbit antiserum against horse ferritin was used. Autoradiography was carried out for 8 weeks to obtain an image on the Kodak No-Screen X-ray film.

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somes) are active in protein synthesis in vitro. Under the condi- 100 tions used in these experiments the free polysomes were less '1 cn active (per mg of RNA) than the attached ribosomes, as meas- ,’ ured by the incorporation of radioactive amino acids into trichlor- acetic acid-precipitable protein. However, more of the protein I $ 8C

released by the free polysomes could be recovered since it was E g

released directly into the incubation medium. The protein g 6C

released by attached ribosomes (microsomes) was mostly trapped within the microsomal membrane (12,20,21). In order to meas-

$

ure this released protein, the microsomes were sonically treated t 4o to rupture the vesicles. This was only partially successful in $ releasing the entrapped proteins. Treatment with deoxycholate could not be used since it interfered with the antigen-antibody B ~ 2. reaction. Since the experiments were designed to evaluate the 5 relative amounts of secretory proteins (serum proteins) or of 2 proteins for internal use by the cell (ferritin) made by different ribosomal fractions, a quantitative recovery was not necessary and it is possible to compare the values for serum proteins and for

ferritin as a percentage of the released protein. This assumes that a mechanical method for disrupting the microsomal vesicle, such as sonic disruption, cannot selectively release some proteins

and not release others. Table III shows that attached ribosomes

FIG. 5. Precipitation of radioactive rat feiritin with rabbit an- tiserum to horse ferritin. Equal amounts of radioactive proteins synthesized in vitro by free ribosomes as described in the text were mixed with carrier rat ferritin. Varying athounts of rabbit anti- serum to horse ferritin were added and the immune precipitates collected and counted as described under “Experimental Proce- dures.” Control values were obtained by using normal rabbit serum instead of antiserum to ferritin. There was no iron de- tected in tli& control. The control tubes however did contain ra- dioactivity and this value was subtracted from the measurement obtained with the antiserum. Iro,ti in the precipitate was deter- mined by the method of Wong (22).

FIG. 4. Immunoelectrophoresis of rat ferritin mixed with ra- dioactive proteins synthesized in vitro by free and attached ribo- somes. The condition of immunoelectrophoresis and autoradiog- raphy were as described in Fig. 3C, except that 15 ~1 of carrier rat ferritin (1.2 mg per ml) were added to each of the radioactive so- lutions placed in the center well. A, immunoelectrophoresis plate stained with Amido schwarz. A is attached ribosomes and F is free ribosomes. B, autoradiograph of the above plate. The film was exposed to the plate for 8 weeks.

-RADIOACTIVITY

X---X IRON .

ANTI-SERUM ADDED (ml) 1.2

make about 6 times more serum proteins than do free ribosomes. Free polysomes, however, synthesize 6 to 20 times more ferritin than do attached ribosomes.

Specificity of Radioactive Assay for Serum Proteins and Ferritin -The nascent proteins obtained from ribosomes of Fractions I, II, III, labeled in vivo with 14C-amino acid mixture for 3 min after the intravenous injections, were used as antigens for immuno- electrophoresis with either rabbit antiserum to rat serum or to horse ferritin. Autoradiography of the immunoplates showed that only attached Fractions I and II have strong radioactive precipitin bands with the antiserum to rat serum while only a faint radioactive precipitin was seen in Fraction III (Fig. 3A). Fraction III showed a single protein precipitin band with anti- serum to horse ferritin on staining with Amido schwarz but no radioactive precipitin bands were seen on autoradiography with any of the three ribosomal fractions. A large amount of radio- active material ran toward the anode with Fraction III and was not washed from the plates. This was not, however, a precipitin reaction,

The radioactive proteins released by attached ribosomes, incu- bated in vitro, gave sharp dark autoradiographic images with antiserum to rat serum proteins while the free polysomes gave only little evidence of radioactive serum proteins (Fig. 3B). On the other hand only the proteins released by free polysomes, in- cubated in vitro, gave a radioactive image which coincided with the ferritin-antiserum precipitin band. This radioactive band was light, and the film had to be exposed for 8 weeks to obtain an image on Kodak No-Screen X-ray film. No radioactive ferritin band was observed with attached ribosomes whose products had been treated with antiserum to horse ferritin and which had also been exposed to film for the same length of time (Fig. 3C).

It is possible that the radioactive line observed may represent

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4314 Protein Synthesis by Free and Attached Ribosomes Vol. 244, No. 16

some nonspecific association of another radioactive protein with the immune precipitate. In order to rule out this possibility, nonradioactive rat ferritin was added to the radioactive proteins synthesized in vitro by both free and attached ribosomes. There- fore, on immunoelectrophoresis, immune precipitates were ob- tained from both samples when they were treated with antiserum to ferritin (Fig. 4A). However, on autoradiography, only one immune precipitate gave a radioactive image. The radioactivity was found only on the products from free ribosomes (Fig. 4B). I f there had been nonspecific association of radioactive proteins with the immune precipitate then both precipitin lines would have showed radioactivity.

Further evidence as to the reliability of the immunological test for ferritin was obtained by mixing a fixed amount of radioactive nascent protein, from free ribosomes incubated in vitro, with carrier rat ferritin. Different amounts of the antiserum to ferri- tin were then added to different tubes and the radioact,ivity pre- cipitated determined as previously described. This was com- pared to the amount of iron precipitated by the same amounts of antiserum. Normal rabbit serum was used as a control. There was a direct correlation between protein radioactivity precipi- tated and the amount of iron in the precipitate. (Fig. 5).

DISCUSSION

Other studies have shown that free and attached ribosomes from rat liver are active in protein synthesis (9) and that free and attached ribosomes make different proteins (13). The present studies show that both in viva and in vitro, attached ribosomes make about 7 times more secretory proteins than do free ribo- somes. Furthermore, free ribosomes are more active in the synthesis of ferritin, a nonexportable protein. This supports the hypothesis that attached ribosomes are sites of synthesis of secretory proteins and free ribosomes are engaged in the production of nonexportable proteins. The data on the pro- duction of serum proteins is clear. The liver cell is actively engaged in the synthesis of these proteins and their presence, either bound to attached ribosomes in vivo, or synthesized by at- tached ribosomes in vitro, was easily shown. Immunoelectro- phoresis, with subsequent autoradiography, showed that the assay was specific for these proteins. The synthesis of ferritin, however, was more difficult to follow since the hepatic cell makes much less of this protein as compared to the serum proteins. In the assay for radioactive ferritin, control samples were treated with normal rabbit serum instead of with rabbit antiserum to ferritin. During the 1 hour incubation at 37” and subsequent standing in the cold to precipitate the ferritin-antibody complex, other radioactive proteins became denatured and were sedi- mented during centrifugation. More of these denatured radio- active proteins were made by free than by attached ribosomes. The radioactivity of these contaminating proteins, determined from the control tubes, was subtracted from the radioactivity which precipitated with rabbit antiserum to ferritin. The con- trol values in the experiments in viva from free ribosomes usually represented about 50% of the radioactivity which precipitated with the ferritin antiserum. In the experiments in vitro the control values were 20 to 25% of the precipitated radioactivity. This correction was not minimized by partially purifying the ribosomal extract prior to treatment with the antiserum to ferri- tin (Table III). This large correction may have led to inac- curacies in the quantitative estimation of the ferritin. However,

a good correlation was obtained using these correction values, between the radioactivity precipitated by the antiserum and the amount of iron, from added ferritin, recovered in this precipitate (Fig. 5). Previous studies by Drysdale and lMunro had shown that the specific activity of W-labeled ferritin obtained by the immunological method bears a constant relationship to that ob- tained by isolating ferritin by chromatographic methods (23). These studies coupled with the experiments showing a single autoradiographic band from immunoelectrophoresis point to the validity of t.he ferritin measurements.

The failure to obtain an autoradiographic image of the precipi- tin band formed by ferritin and its antibody is caused by the small synthesis of ferritin, in &JO, as compared to serum proteins. In order to place enough ferritin radioactivity on the immunoelec- trophoresis plate it was overloaded with other protein thus ham- pering the separation during the immunoelectrophoresis. This problem could be avoided using experiments in vitro since under these conditions sufficient radioactivity is incorporated into fer- ritin. In these experiments it was possible to show that anti- serum to horse ferritin was not only chemically specific in precipitating only ferritin, but also was specific in that it only precipitated a single radioactive protein. The possibility that some other radioactive protein may be nonspecifically associated with the immune precipitate was ruled out by adding rat ferritin to the ribosomal extracts from both free and attached ribosomes. Both extracts therefore had precipitin lines with antiserum to ferritin but only the extract from the free ribosomes gave a radio- active image. This shows that the immune precipitate on the immunoplates does not absorb other radioactive material. The synthesis in vitro of ferritin has been previously reported by Saddi and von der Decken (24) and by Pearson (25).

These findings raise the question as to how polysomes that are programmed for making secretory proteins can recognize and be attached to the endoplasmic reticulum membrane while free polysomes which are making ferritin, or other nonexportable proteins, remain free in the cytoplasm. The mRNA on the polysomes determine what proteins are to be made. Therefore, it is probable that the mRNA also decides whether a polysome is to be bound to the membranes of the endoplasmic reticulum, or whether the polysomes are to be free in the cytoplasm. If the factor which determines whether a polysome is to be free or at- tached is the mRNA, then either (a) the mRNA for secretory proteins is on the membrane and therefore any ribosomes bound to it have to make secretory proteins, or (b) polysomes formed in the cytoplasm, containing mRNA for secretory proteins can recognize binding sites on the membranes, whereas polysomes making nonexportable proteins, cannot recognize these sites and remain free in the cytoplasm.

The second alternative implies that a polysome, scheduled to make secretory proteins, is preassembled in the cytoplasm before it is attached to the endoplasmic reticulum membranes. This polysome then proceeds to make secretory proteins before it is attached to the endoplasmic reticulum membrane but needs the membrane in order to complete the secretory proteins. This idea is not favored. I prefer the first suggestion which postulates that the mRNA for secretory proteins is attached to the mem- branes of the endoplasmic reticulum. This has many advan- tages. It has already been shown that membrane-bound ribo- somes are more stable in the presence of RNase (26) and also that the synthesis of albumin (which occurs on attached ribo-

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Issue of August 25, 1969 C. M. Redman

somes) is more refractory to the effects of starvation and actino- mycin D (27). The presence of mRNA for secretory proteins on the membrane also would assure that ribosomes bound to the membrane would make only secretory proteins, and it may also act by arranging the attached polysomes in the correct spatial configuration so that its protein product can be directed toward the cisternal space of the endoplasmic reticulum. It has been shown previously that liver ribosomcs are attached to the mem- brane in a specific manner and that protein synthesis plays a part in the binding of the ribosome to the membrane (28). There is, however, no direct proof of a stable mRNA within the endo- plasmic reticulum membrane although this idea has been sug- gested since nonribosomal RNA was found in the membranes (29-33).

-4chxowledgmenta-I wish to thank Dr. A. Mazur for his advice during the studies on fcrritin and to thank him also for helpful comments in the preparation of this manuscript. Thanks are also due to Miss Berry Burger for able technical assistance.

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Colvin M. Redmanof Rat Liver

Biosynthesis of Serum Proteins and Ferritin by Free and Attached Ribosomes

1969, 244:4308-4315.J. Biol. Chem. 

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