Ribosomal-membrane interaction: In vitro binding of ribosomes to microsomal membranes

J. Mol. Biol. (1974) 88, 559-580

Ribosomal-Membrane Interaction : In vitro Binding of Ribosomes to Microsomal Membranes NICA BORGESE

Center of Cytopharmacology of the Consiglio Nazionale delle Richerche Via Vanvitelli, 32, Milan, Italy WINNIE MOK, GERT KREIBICH AND DAVID

D. SABATINI

New Yorlc University School of Medicine Department of Cell Biology New York, N.Y. 10016, U.X.A.

(Received24 December 1973) Rat liver rough miorosomal membranes were stripped of bound ribosomes by treatment with puromycin and high concentrations of monovalent ions. Ribosomal subunits labeled in the RNA were detached from rough microsomes by the same procedure, recombined into monomers, and then incubated with stripped membranes in a medium of low ionic strength (25 mm-Kcl, 50 mM-Tris . HCI, 5 mM-Mgcls). These ribosomes readily attached to the stripped membranes, as determined by isopycnic flotation of the reconstituted microsomes. The binding reaction was complete after incubation for five minutes at 37”C, but also proceeded at O”C, at a lower rate. Seatchard plots showed a binding constant of NSX lo7 M-I and -5~ lo-* mol binding sites per gram of membrane protein. Native rough microsomes showed a much lower binding capacity at 0°C than stripped rough microsomes, but showed considerable uptake of ribosomes at 3’7’C. Smooth microsomes, treated for stripping and incubated at O”C, accepted less than half as many ribosomes as stripped rough microsomes. Erythrocyte ghosts were incapable of binding ribosomes. Microsomal binding sites were heat sensitive, were destroyed by a brief incubation with a mixture of trypsin and chymotrypsin in the cold, and were unaffected by incubation with phospholipase c. Ribosome binding was decreased by increasing the concentration of monovalent ions and was strongly inhibited by low4 Ma-aurintricarboxylic acid. Experiments with purified ribosomal subunits revealed that at concentrations of monovalent ions close to physiological concentrations (100 to 150 man-KCl), microsomal binding sites had a greater affinity for 60 S than for 40 S subunits. Stripped rough microsomes were also capable of accepting polysomes obtained from rough microsomes by detergent treatment. Although this binding presumably involves the correct membrane binding sites, polypeptides discharged from re-bound polymers were not transferred to the vesicular cavities, as in native microsomes. The released polypeptides remained firmly associated with the outer microsomal face, as shown by their accessibility to proteases.

1. Introduction The fate of proteins synthesized by cytoplasmic ribosomes appears to be related to the subcellular site of synthesis. Several reports indicate that in the rat liver, membrane-bound polysomes are engaged in the synthesis of secretory proteins, while 37

669

500

N.BORGESE

ETAL.

free polysomes are active in the synthesis of some proteins retained in the cell (Hit ks et al., 1969; Ganoza & Williams, 1969; Redman, 1969ab; Takagi et al., 1969,197O). However, the mechanism by which polysomes, operating in the translation of speci fit messengers, recognize the membrane, is not understood, and it appears clear that the recognization does not depend on structural or metabolic properties of the ribosomes themselves (Loeb et al., 1967; Talal $ Kaltreider, 1968; Tanaka et al., 1970). An analysis of the ribosome-membrane association, using rat liver microsomes (Adelman et aZ., 19733), has demonstrated that there are two main factors involved in maintaining ribosomes bound to microsomal membranes : ionic bonds, disruptable by high concentrations of monovalent ions, and a link provided by the nascent polypeptide ohain. In the presence of Mg2+ ions, ribosomes can be removed from the membranes through the combined action of puromycin and moderately high concentrations of monovalent ions (0.5 M-KCl). Undamaged ribosomal subunits and stripped membranes can be recovered separately. This provides a method for the non-destructive disassembly of rough miorosomes and a useful system in which to study the role of ribosomes and endoplasmic reticulum membranes on the assembly of rough microsomes, independently of nascent polypeptides and messenger RNA. Most studies on the afEinity of ribosomes for microsomal membranes have been carried out using polysomes, and rough microsomes freed of their ribosomes by methods which simultaneously lead to degradation or unfolding of the ribonucleoprotein particles (Siiss et al., 1966; James et d., 1969; Williams & Rabin, 1969; Khawaja $ Raina, 1970; Blyth et al., 1971,1972; Khawaja, 1971; Ragland et aZ., 1971; Roobol & Rabin, 1971; Scott-Burden 86 Hawtrey, 1971,1973; Shires et d., 197la,b,1973; Sunshine et al., 1971; Hochberg et al., 1972; Nolan & Munroe, 1972; Burke & Redman, 1973; Ekren et al., 1973; Jothy et al., 1973; Pitot & Shires, 1973; Shires $ Pitot, 1973). The system using ribosomes lacking nascent chains, and KCl/puromycin-stripped membranes (Borgese et al., 1972 ; Rolleston, 1972 ; Rolleston 82;Mak, 1973) presents the following advantages. (1) It is more appropriate for the quantitative measurement of ribosome binding sites, since in the case of messenger-containing polysomes, the binding of a single ribosome could lead to the loose association of the entire polysome with the membrane. (2) Any possible interference by the ribosome-associated nascent chain with the binding process is avoided. (3) Microsomes stripped by the KCl/puromycin procedure contain vectorially discharged peptidyl-puromycin molecules (Adelman et al., 19733 ; Kreibich & Sabatini, 1973), as opposed to nascent peptides, which presumably remain associated with membranes at the ribosome binding sites when ribosomes are removed by destructive procedures. In the present study, we have characterized the binding reaction of ribosomes to stripped rough microsomes, using the KCl/puromycin system. Our results indicate that there exist ribosome-acceptor sites characteristic of rough microsomal membranes and that, under certain conditions, there is a preference by these sites for the large over the small ribosomal subunit.

RIBOSOME-MEMBRANE

561

INTERACTION

2. Materials and Methods (a) General All solutions were prepared using double-distilled water, were Millipore-filtered (0.45 pm pore size for most, 1.2 pm for cone. sucrose stock sols) and were stored in the cold. The notation TriseHCl will be used for Tris*HCl, pH 7.6, at 20°C. Tris/K/Mg buffer is 50 mM-TriseHCl, 25 nx~-KCl, 5 mM-MgCl,. High salt buffer is 50 man-Tris*HCl, 500 mM-KCl, 5 mi%r-Mgcl,. All centrifugations were carried out in Spine0 ultracentrifuges (Beckman Instruments Inc., Palo Alto, Calif.), at 3”C, unless otherwise specified. (b) Fractio~xation

and labeling

of liver cells

(i) Microsomes and free polysomes These were prepared as described by Adelman et al. (1973a). The procedure allows the separation, by discontinuous sucrose-gradient centrifugation, of free polysomes, rough and smooth microsomes from a postmitochondrial supernatant solution containing more than 50% of the membrane-bound ribosomes of the tissue. Rough and smooth microsomes, collected separately from the discontinuous sucrose gradient, were sedimented (30,000 revs/mm, no.30 rotor; 20 min for rough microsomes, 1 h for smooth microsomes) after dilution with 6 vol. 0.25 M-sucrose-Tris/K/Mg buffer. The pellets of rough and smooth microsomes were resuspended in 0.25 ix-sucrose-Tris/K/Mg buffer (~5 mg protein/ml). Samples of the suspension (1 ml) were diluted with 2 vol. glycerol and stored at - 20°C for up to 2 months. Before use, the suspensions were diluted with 2 vol. Tris/K/ Mg buffer, and the microsomes recovered by centrifugation (15 min, 40,000 revs/mm no.40 rotor). (ii)

Preparation of stripped membrane fraction

Rough microsomes were stripped of the their ribosomes essentially as described by Adelman et al. (1973b). Freshly prepared rough or smooth microsomes were resuspended in 0.25 M-sucrose and a compensating buffer was added so that the final composition of the suspensions was 0.25 M-sucrose, 0.5 M-KCl, 0.05 M-Tris*HCl, 2.5 mm-MgCl,, 5 x lo-*

TABLE 1 Chemical

analysis

of rough and smooth microsomes before and after ribosomes by the KCljpuronzycin procedure Sample

Rough microsomes

Stripped

RM

Smooth microsomes

Stripped

SM

RNA/protein

RNA/PL

PL/protein

0.197

0.577 0.526

0.364 0.374

0.037 0.032

0.084 0.066

0.440 0.489

0.210

removal

of

0.098 O-046

0.110

0.416

0.008 0.0067

0.018 0.016

0.430 0.422

Rough microsomes (RM) and smooth microsomes (SM) were treated for ribosome stripping as described in the text. Appropriate samples were taken in duplicate for RNA, protein and phospholipid (PL) determinations. Each number represents an average value obtained from duplicate assays. These generally agree to within 5% for RNA and protein and to within 10% for PL assays run from separate extracts. The Table gives values from 2 different membrane preparations.

562

N.

BORGESE

ET AL.

M-puromycin. The final membrane concentration protein was ~5 mgjml. The suspensions were incubated for 1 h at O”C, followed by 10 min at 25”C, after which they were diluted B-fold with 0.25 M-sucrose-high salt buffer and centrifuged for 20 min at 30,000 revs/min in the no.30 rotor. The resulting microsomal pellets were resuspended in 0.25 M-sucrose-high salt buffer (~0.5 mg protein/ml) and sedimented again. The final stripped microsomal membrane preparations were resuspended in 0.25 M-sucrose-Tris/K/Mg buffer, stored with glycerol and recovered from the suspensions as described for rough and smooth microsomes. Table 1 shows the chemical composition of the different microsomal fractions used in this study?. (iii)

Bound ribosornes, ribosomal subunits and polysovnes

These were obtained either from rough microsomes prepared as described above or from a crude microsomal fraction obtained as follows: a rat liver homogenate, prepared in O-25 M-sucrose-Tris/K/Mg buffer (2 ml/g wet liver), was centrifuged at 10,000 revs/min for 10 min in the no.40 rotor. Microsomes were recovered from the resulting supernatant solution by centrifugation for 1 h at 40,000 revs/mm in the no.40 rotor. The microsomal pellets were washed once in 0.25 M-sucrose-Tris/K/Mg buffer. To detach bound ribosomes, the microsomal fraction was incubated in 0.25 M-SUCIOSe, 0.5 M-KCl, 0.050 M-Tris*HCl, 2.5 mu-MgCls, 5 x 10-4 M-puromycin at a concn of ~3 mg RNA/ml for 10 min at 37°C. After incubation, the microsomes were sedimented into a pellet by centrifugation for 12 min at 40,000 revs/min in the no.40 rotor. The supernatant solution, containing the released ribosomes, was decanted, diluted B-fold with Tris/K/Mg buffer and layered over 1 ml 1.6 ivr-sucrose-Tris/K/Mg buffer cushions, in centrifuge tubes of the no.40 rotor. The ribosomes were sedimented into pellets by centrifugation overnight at 40,000 revs/mm. Ribosomes prepared in this way exist in the form of monomers at low ionic strengths (25 to 150 mM-KCl) and dissociate into subunits at higher concentrations of monovalent ions (results not shown). To obtain ribosomal subunits, the microsomes were incubated as described above, and samples of the incubation mixture (0.6 ml) were layered directly on 11*5-ml linear sucrose gradients (10% to 25%)$, containing high salt buffer, which were centrifuged (2 h 45 min, 35,000 revs/min, SW41 rotor) at 20°C. The gradients were withdrawn from the top by an auto densiflow probe (Buchler Instruments, Fort Lee, N.J.) connected to a peristaltic pump, and passed through a U.V. analyzer (LKB Uvicord II, type 8303A; LKB Productor AB, Bromma, Sweden) attached to a recorder. The fractions corresponding to the 40 S and 60 S subunit peaks were collected separately, diluted 1: 1 with Tris/K/Mg buffer and sedimented by centrifugation overnight at 40,000 revs/min in the no.40 rotor. Analysis by sucrose-gradient centrifugation under conditions where formation of subunit aggregates does not occur, shows that subunits prepared in this way are satisfactorily pure (see Borgese et aZ., 1973). To obtain bound polysomes, the microsomes were resuspended in Tris/K/Mg buffer at ~3 mg RNA/ml and made O5o/o in deoxycholate. Samples (7 ml) of the suspension were layered over 2-ml 2.0 M-sucrose cushions, containing Tris/K/Mg buffer, and centrifuged overnight (40,000 revs/mm, no.40 rotor). Pellets of ribosomes, subunits and polysomes were stored at - 80°C for up to a month. (iv)

Radioactive labeling in vivo

Ribosomes labeled in the ribosomal RNA (referred to in this paper as 3H-ribosomes) ~200 &i of [5-3H]orotic acid intraperitowere prepared from rats that had received neally, 36 to 40 h before killing. The specific radioactivity in ribosomes varied between 0.5 x lo6 and 1.5 x 10” distints/min per mg RNA, according to the amounts of radioactive precursor injected per g of body weight. Polysomes containing labeled nascent chains were prepared from rats that had received 200 &i of L-[4,5-eH]leucine, injected into the portal vein under ether anesthesia. Two t ably tion $

The values differ somewhat from those reported previously (Adehnan et al., 1973b), presumbecause in the present study microsomal protein was measured without previous preoipitaby the addition of trichloroacetic acid. Percentage sucrose values are w/v.

RIBOSOME-MEMBRANE

563

INTERACTION

minutes after beginning the injection, the portal vein was cut and the liver excised. Specific radioactivity in the ribosomes was ~10~ distints/min per mg RNA. Microsomal membranes containing labeled phospholipids were obtained from rats that had received 200 pCi of choline [metIqZ-3H]chloride intraperitoneally 4 h before killing. When labeled microsomes prepared from these rats were extracted with chloroform/ methanol (Folch et al., 1957), approximately 98% of the radioactivity was recovered in the organic phase. (c) Preparation of erythrocyte ghosts These were prepared as described by Dodge et al. (1963) by hemolysis of whole human blood erythrocytes in hypotonic sodium phosphate buffer (pH 7.6). The erythrocyte ghosts were treated with high salt and puromyoin and washed with high salt buffer as described for the preparation of stripped miorosome fractions (see section (b) (ii), above). (d) Binding

assay

The incubation mixtures for ribosome binding contained, in 0.12 ml, membranes (0.1 to 0.4 mg protein) and “H-ribosomes, polysomes or subunits in quantities specified in the Figure legends. The ionic conditions are also described in the Figure legends. At the end of the incubation, 1.08 ml cold 2 x 2 M-sucrose, with the same ionic composition as the incubation mixture, were added to the samples so that the final sucrose concentration was ~2.0 JK. After thorough mixing, 0.8 ml of the mixture was underlayed, by means of prepared in centrifuge tubes of a syringe, below a 3&ml continuous sucrose gradient, the SW50.1 rotor. The gradients had the same ionic composition as the incubation mixtures, and the sucrose concentration range varied according to the membrane fraction used, so that the membranes would float to the upper part of the gradient (I.3 to 1.9 M1.0 to 1.9 M-sucrose for stripped smooth microsucrose for stripped rough microsomes, somes, 0.9 to 1.9 M-SUerOSe for erythrocyte ghosts, 1.55 to 1.9 M-sucrose for untreated rough microsomes). All gradients were overlayed with 0.4 ml Tris/K/Mg buffer to avoid exposure of any material to a liquid-air interface. The gradients were generally centrifuged for 45 min at 50,000 revs/min. They were then withdrawn from the top, as described above (see section (b), (iii)) and 3 fractions were collected: a top fraction (3.0 ml), an intermediate fraction (0.4 ml) and a bottom fraction (1.6 ml). Material in the pellet was resuspended in water and combined with the bottom fraction. After the addition of 1 mg bovine serum albumin as carrier, the fractions received an equal volume of ice-cold 10% trichloroacetic acid, and the acid-insoluble material was sedimented, dissolved in 0.5 ml Protosol (New England Nuclear, Boston, Mass), and counted in 10 ml toluene/liquifluor with an efficiency for 3H of -400/O. All values were corrected for background. Recovery of radioactivity in the gradient fractions ranged from 85 to 100%. The acidinsoluble radioactivity recovered in the top fraction was considered to be membranebound. In order to determine the time of centrifugation sufficient to float the membranes to the upper part of the gradient, preliminary experiments were run, using [3H]cholinelabeled membranes and cold ribosomes. As can be seen from Table 2, 45 min of centrifugation were sufficient to float w9Oo/o of the stripped rough microsomes and stripped smooth microsomes. However, non-treated rough microsomes required a much longer time to reach equilibrium. Therefore, in experiments designed to study the binding of ribosomes to untreated rough microsomes, centrifugation was for 5 h. In all other cases (e.g. erythrocyte ghosts, trypsin-chymotrypsin treated microsomes), 45 min were always sufficient to recover practically all membranes in the top fraction of the gradient. Table 2 also shows the distribution of radioactivity on a 1.9 M to 1.3 M-sucrose gradie:it containing Tris/K/Mg buffer, when 3H-ribosomes were analyzed in the absence of membranes. It can be seen that less than 1% of the total radioactivity was recovered in the top fraction. All values presented in the Results have been corrected for this small background. The results have not been corrected for recovery of membranes on the gradient, and thus represent a slight underestimation of the degree of binding actually obtained. Protein was assayed as standard.

(e) Analytical by the method of Lowry

methods et al. (1951), with

bovine

serum

albumin

45

300

(3) [3H]choline RM + cold ribosomes

(4) [3H]choline RM + cold ribosomes 35 (0.2%)

8495 (87%)

5061 (50.1%)

8077 (88.9%)

5067 (93.7%)

Radioactivity in top fraction (cts/min)

15 (0.1%)

621 (6.3%)

1617 (15.0%)

389 (4.2%)

126 (2.3%)

Radioactivity in intermediate fraction (cts/min)

14,127 (99.7%)

614 (6.7%)

3394 (33.9%)

616 (6.9%)

210 (4%)

Radioactivity in bottom fraction (cts/min)

14,177 (100%)

9730 (100%)

10,062 (100%)

9082 (100%)

5402 (100%)

Total radioactivity (cts/min)

tin 80 ~1, the samples contained: (1) 3.6 Azso units of cold ribosomes + 0.064 mg protein of stripped RM; (2) 3.6 A 260 units of cold ribosomes + 0.120 mg protein of stripped SM; (3) and (4) 3.6 A280 units of cold ribosomes + 0.140 mg protein of untreated RM; (5) 1.36 A 280 units of 3H-labeled ribosomes. Ah samples were in Tris/K/Mg buffer, and were incubated for 30 min at 0°C before dilution with heavy sucrose. Specific radioactivity of [3H]choline-labeled microsomes was -80,000 cts/min per mg protein. Specific radioactivity of 3H-labeled ribosomes was 12,500 cts/min per A,,, unit.

45

45

(2) [3H]choline stripped SM + cold ribosomes

(5) [3H]ribosomes

45

Time of centrifugation (min)

microsomes and [3H]orotic acid-labeled ribosomes on sucrose gradients after isopycnic Jtotation

(1) L3H]cholinestripped RM + cold ribosomes

Sample?

Distribution of [3H]chdi~e-labeled

TABLE 2

RIBOSOME-MEMBRANE

INTERACTION

565

RNA was determined by a modified Schmidt-Tannhauser procedure (Schmidt & Tannhauser, 1945; Fleck & Munro, 1962), using E,,I% = 313 at 260 nm (Munro & Fleck, 1966). Alternatively, ribosome concentrations were determined directly, using E:& = 135 at 260 nm (Tashiro & Siekevitz, 1965), and the RNA concentration was derived assuming that the ribosomes contain 52% RNA. The concentration of ribosomes was calculated assuming M, = 4.5~ lo6 (Hamilton et al., 1971). For phospholipid phosphorus determinations, lipids were extracted from membrane suspensions with 20 vol. chloroform/methanol (2: 1) and purified according to Folch et al. (1957). Appropriate fractions of the extracts were dried in a dessicator, and assayed for phosphorus as described by Ames (1966). Values for phospholipid phosphorus were converted to phospholipid using a factor of 25. ( f ) Materials Male albino rats (100 to 150 g) of the Sprague-Dawley strain, maintained on Purina Chow diet, were fasted for 15 to 20 h before killing. Whole human blood was obtained from the New York Blood Center (courtesy of Dr C. Redman). Chemicals were obtained from the following sources: puromycin dihydrochloride from Nutritional Biochemical Corp., Cleveland, Ohio; [5-3H]orotic acid (1 mCi/O*156 mg), choline [meth$-3H]chloride (1.00 mCi/O*253 mg), Protosol and Liquifluor from New England Nuclear, Boston, Mass. ; n-[4,5-3H]leucine (40 to 50 Ci/mmol) from Schwartz Bioresearch Inc., Orangeburg, N.Y. ; sodium deoxycholate from Matheson, Coleman and Bell, Cincinnati, Ohio; Triton Xl00 from Rohm and Haas, Philadelphia, Penn. ; aurintrocarboxylic acid (practical grade) from Eastman Kodak Co., Rochester, N. Y. ; 3 x crystallized bovine pancreatic trypsin and cr-chymotrypsin, and CZo&idizcm perfringens phospholipase C (86 units/mg) from Worthington Biochemical Corp., Freehold, N. J.

3. Results (a) Kinetics of ribosome binding It has been reported that, at low ionic strengths, polysomes can bind to rough microsomes “conditioned” to accept ribosomes, and that this binding can occur at 0°C (Siiss et al., 1966; Ragland et al., 1971; Shires et al., 1971a). As a first attempt to characterize the in vitro binding of ribosomes lacking nascent chains, we determined the time-course of ribosome attachment to different membrane fractions both at 0°C and at 37°C in Tris/K/Mg buffer. As can be seen from Figure l(a), binding of 3H-ribosomes to RMT and SM treated for stripping teached a maximum value after incubation for about five minutes, at 37°C. However, stripped RM also showed considerable binding capacity at 0°C even at early time points. After longer incubations (30 mm to 1 h) at 0°C the amount of ribosomes bound approached that reached at 37°C. Stripped SM showed a larger difference between binding capacities at 37°C and 0°C. The difference between binding at the two temperatures was much more marked for untreated RM (Fig. l(b)), which accepted essentially no ribosomes at early time points in the cold, but did bind considerable amounts after incubation at 37°C. Figure l(b) also shows the kinetics of ribosome binding to erythrocyte ghosts treated for stripping. It can be seen that these membranes were unable to accept ribosomes at both temperatures tested. Thus, it appears that the capacity to accept ribosomes rapidly at 0°C is characteristic for rough microsomal membranes after removal of bound polysomes. This capacity is lower in SM, very low in RM not treated for stripping, and tot&lly absent in erythrocyte ghosts. 7 Abbreviations

used : RM, rough miorosomes ; SM, smooth microsomes.

566

N.

BORGESE

ET AL.

80 -'--•\.,* S,605 ,+/------2 s I' ~ 40.7' ~-.-c---A

<. ---0 : .r./m’m

0

I

'0

I I I I I II ~ I-a 10 20 30 40 50 60 0 IO 20 30 40 50 6 I Time of incubation

(min )

FIG. 1. Time-course of binding of ribosomes to membrane fractions in Tris/K/Mg buffer. Samples contained, in 80 ~1: (a) 0.160 mg protein of stripped RM (-a--+and --O--O--) or stripped SM (--A--A-and --A--&--) and 0.725 AaeO unit’ of 3H-ribosomes (see Materials and Methods, section (b) (iv)); (b) native RM (0.176 mg of protein) (--m-m-and 0) or erythrocyte ghosts (0.210 mg of protein) (-+-+and --O--O--) and 0.957 A,,, unit of “H-ribosomes. The mixtures were incubated for the periods indicated at 37V (-•-•---, --m--m-, -+-+-, --A--A-) or 0% --O--O--, --A--&-, 0, --O--O--). The ordinate presents the radioactivity in the membrane fraction as the percentage of the total radioactivity recovered in the gradients.

Ribosomes

added ( pg )

FIG. 2. Saturation of membrane binding sites by ribosomes in Tris/K/Mg buffer. Samples contained, in 80 ~1: stripped (str) RM, 0.116 mg of protein (-•--a---); stripped SM, 0.184 mg of protein (-----A--); or native RM, 0.146 mg of protein (-*-m-.-m-.-), and the amounts of ribosomes indicated on the abscissa. Incubations were for 30 min at O’C.

SM

Stripped

fraction

0.025 0.067

0.106

0.268 0.225 0.254

0,125 0.104 0.118 0.045

mg RNA boundb mg PL

mg RNA bound” mg protein

O-618

0.123

0.343 0.300 0.329

xRNAO PL

1.2x 10-s

1.9 x lo- *

5.4x10-* 4-l x lo- s 4.9 x 10-s

m01 binding sites g protein,

buffer at 0°C

&OX

5.7

10’

X lo7

8.7 X lo7 6.2 x lo7 7.1 X lo’

M-l

M-l

M-l

M-l

M-l

a These values are computed from the radioactivity recovered in the membrane fraction after incubation with excess ribosomes (see Fig. 2), the specifio radioactivity of the added ribosomes, and the known amounts of membrane protein loaded onto the gradiants. b The values in this column are computed from the values in the first column of this Table and the average of the PL/protein ratios (PL, phospholipid) for each membrane fraction given in Table 1. c IRNA is the sum of RNA bound during the incubation and residual RNA of the stripped membranes before incubation for rebinding (see Table 1). d Caloulated from Scatchard plots by extrapolation to zero ordinate values (see Fig. 3). BAffinity aonstant calculated from the slope of Scatchard plots (see Fig. 3). f Values obtained with 3 different preparations of membranes.

RM

RM’

Stripped

Membrane

3

Binding of ribosomes to microsomal membranes in Tris/K/Mg

TABLE

568

N. BORGESE

ET

AL.

(b) Quantitative measurement of ribosome binding sites In order to measure quantitatively the amount of ribosomes that can bind to membranes, increasing quantities of 3H-ribosomes were added to a fixed amount of rough or smooth microsomes treated for stripping, or of untreated rough microsomes, and the membrane-associated radioactivity was determined after incubation for 30 minutes at 0°C. The results of these experiments are shown in Figure 2, where it can clearly be seen that both stripped RM and stripped SM have a finite number of binding sites and become saturated, when a sufficient amount of ribosomes is added. The RNA to phospholipid (PL) ratios in the reconstituted rough and smooth microsomes are shown in Table 3 (cRNA/PL). These have been calculated from the specific radioactivity of the added ribosomes, and the known RNA to phospholipid ratios of the stripped microsomal fractions (Table 1). A comparison of Tables 1 and 3 shows that the RNA to phospholipid ratio (average 0.326) in reconstituted RM was -60% of the value found in native RM (average O-545). On the other hand, the RNA to phospholipid ratio in SM, after ribosome binding, was close to that found in native SM. The data of Figure 2 were used for the Scatchard plots shown in Figure 3, from which the number of binding sites per gram membrane protein and the affinity constants of ribosomes for these sites have been calculated. It can be seen that stripped RM have more than twice as many binding sites as SM treated for stripping (Table 3). However, the two microsome fractions have similar affinity constants for ribosomes, which suggests that the same kind of binding sites are involved. Untreated RM, on the other hand, in addition to showing a much lower number of sites, have an affinity constant for ribosomes one order of magnitude lower than that of stripped microsomes.

FIG. 3. Scatchard plots of the data from the experiment of Fig. 2. The curves were fitted by least-squares linear fit using a computer programm (COLNRS) available on a GE Mark III timesharing service. The program also oomputed the 95% confidence limits, shown as bars in the Figure. R%m K = 8*7X lo7 M-l; SMstr; K = 5-7x 107 IX-~; RM, K = 6.5X 106 M-I.

RIBOSOME-MEMBRANE

569

INTERACTION

(c) Treatments affecting the binding reaction As can be seen from Table 4, the ribosome binding sites of stripped rough microsomes are heat labile, since preincubation of the membranes at 55°C for 15 minutes greatly reduced their capacity to accept ribosomes. Incubation of the membranes with phospholipase C had no significant effect on the binding reaction. At the concentration of phospholipase used, -92% of the choline was released from the membrane (results not shown). Thus, lecithin does not play an important role in ribosomemembrane interaction. It is of interest that the presence of 10e4 M-aurintricarboxylic acid almost totally eliminated ribosome binding. Pretreatment of the membranes TABLE 4 Effect of various treatments on the binding of ribosomes to stripped rough microsomes in TrislKlMglbuffer Expt

no.?

1

Cts/min associated with membranes

Condition

O/oinhibition of binding

4136

Control

82

Heat treatment$ 2

759

Control

1016 0

Phospholipase 3

C$

Control lo- 4 M-aurintricarboxylio acid]/

1058 4535 92 347

t In 80 ~1, samples contained: expt 1,0.184 mg protein of stripped RM and 0,620 A,,, unit of 3H-ribosomes (see Materials and Methods, section (b) (iv)) (spec. act., 9500 ots/min per Azso units) ; expt 2, O-2 mg protein of stripped RM and O-452 Az6,, unit of 3H-ribosomes (spec. act., 13,000 cts/min per AseO units); apt 3, 0.118 mg protein of stripped RM and 0.714 ASBO unit of 3Hribosomes (spec. act., 13,700 cts/min per A 260 unit). Incubations were for 10 min at 37°C (expt 1) or for 30 min at 0°C (expts 2 and 3). $ Stripped RM preincubated at 55°C for 15 min. $ Stripped RM were preincubated at 30°C for 15 min with or without phospholipese C (0.064 mgiml). The low binding obtained in the control samples is presumably due to this preincubation. jl lo- 4 M-aurintricarboxylic acid present in the incubation mixture.

with this dye and subsequent removal of the reagent did not affect the capacity of the membranes to accept ribosomes (results not shown). Thus, aurintricarboxylic acid presumably interacts with portions of the ribosome involved in the binding reaction. The effect of treatment with trypsin and chymotrypsin in the cold on stripped RM is shown in Figure 4. Proteolysis at 0°C rapidly eliminated binding tested either at 0°C .or 37°C. Binding was also reduced by prolonged storage of the stripped RM in the cold. We are currently correlating the observed loss of binding caused by proteolysis with changes in the protein complement of the membranes assessedby polyacrylamide gel electrophoresis.

570

N. BORGESE

E!P AL.

Time of proteolysis (h 1

4. Binding of ribosomes to stripped RM after treatment with trypsin-chymotrypsin. Samples containing stripped RM (-3 mg of protein/ml) were incubated with a mixture of trypsin and chymotrypsin (60 pg/ml each) in 50 mm-KCl, 60 mM-Tris.HCl (pH 7.6), MgC&. After proteolysis at 0°C for the times indicated, the samples were diluted by addition of 40 vol. high salt buffer, and the microsomes were sedimented by centrifugation for 20 min in the no. 30 rotor. The resulting pellets were rinsed with 0.26 M-Sucrose and then resuspended to a concentration of ~3 mg of protein/ml. These suspensions were used for incubation with “H-ribosomes (see Materials and Methods, section (b) (iv)) (0.76 A,,, unit per 80 ~1 of incubation mixture; spec. &et. or 30 13,400 cts/min per Azso unit). Binding was measured after 10 min at 37°C (-e--a-), min at 0°C (--A-A--). Control microsomes were kept in suspension at 0°C for 5 h, (0) end (A). The ordinate expresses pg bound per 0.1 mg microsomal membrane protein. FIG.

The effect of raising the concentration of monovalent ions was also tested (Fig. 5). There was an essentially linear decrease of ribosome binding from 25 mM to 200 mM-KCl. At concentrations of KC1 above 200 mu, the extent of binding was approximately 5% of that occurring in Tris/K/Mg buffer. Figure 5 also shows the binding obtained when stripped microsomes were incubated with ribosomes in Tris/K/Mg buffer and subsequently transferred to solutions containing varying concentrations of potassium ions. In this case the curve obtained was parallel to the one representing binding when incubations were carried out directly in the indicated salt solutions, suggesting that at the time point chosen for this experiment (incubation for 30 min at O’C), binding equilibrium was almost completed at each ion concentration. Thus, it is likely that the decreased binding, observed with increasing concentrations of monovalent ions, reflects a lower affinity constant. (d) Characterization of the binding capacity

of ribosonaul subunits

Since, in the rough endopbsmic reticulum, ribosomes are attached to membranes their large subunits (Sabatini et al., 1966), it was of interest to compare the capacity of 40 S and 60 S subunits to bind to stripped rough microsomes. This comparison was carried out at various ionic strengths, and at two different concentrations of membranes, after incubation for 30 minutes at 0°C. Figure 6 shows that between 100 and 150 m&r-KC& at the higher concentration of membranes (Fig. 6(a)),

via

RIBOSOME-MEMBRANE

KC1 concentration

INTERACTION

571

(mM 1

FIG. 5. Binding of ribosomes to stripped RM at different KC1 concns. All samples contained in 120 $:2-O A,,, units of 3H-ribosomes (see Materials and Methods, section (b) (iv)) (17,200 ct.s/min per A,,, unit) and stripped RM (0.192 mg of protein). One set of samples (-•-a---), conteining 50 ma%-Tris*HCl (pH 7.6), 5 mM-MgCl, and the indicated KC1 concns, was incubated for 30 min at O”C, after which 80 pl (~0.128 mg membrane protein) were assayed for ribosome binding as des-ribed in Materials and Methods. The other set of samples (--A--A--), containing Tris/ K/Mg buffer, was incubated for 30 min at O”C, after which 40 ~1 of a compensating buffer were added so as to obtain the indicated final KC1 concn. The samples were incubated again for 30 min at O”C, after which 80 pl were assayed for binding. The ordinate expresses pg ribosome bound per 0.128 mg of miorosomal membrane protein.

nearly four times as many large subunits were bound to the microsomes as small subunits. Moreover, a comparison of Figure 6(a) with Figure 5 shows that large subunit binding decreased with increasing potassium concentration in a manner similar to that shown by ribosomal monomers, while small subunit binding showed a more rapid decrease. However, when there was a lower membrane concentration, the difference between large and small subunit binding at 100 mM-KC1 was smaller (Fig. 6(b)), and in Tris/K/Mg buffer equimolar amounts of small and large subunits were bound. This effect may be due to binding of small subunit aggregates, which are easily formed at low ionic strengths (Nonomura et al., 1971). The results were not altered by lowering the magnesium concentration from 5 to 1 EIM (results not shown). The addition of unlabeled 60 S subunits to stripped RM increased the binding of 3H-labeled 40 S subunits (Table 5). This was shown by experiments in which membranes (at high concentrations) and labeled or unlabeled subunits were added sequentially to an incubation mixture containing 150 m&r-KCl, 50 m&r-TriseHCl, 5 m&t-Mgcl,. In the presence of unlabeled large subunits, the labeled small subunits bound as much as the labeled large subunits in the presence of unlabeled small subunits (compare experiments in E with experiments in B, and experiments in F with experiments in C, in Table 5). This result was achieved either when the subunits were first allowed to recombine into monomers and were then incubated with the membranes (experiments in E, Table 5) or when the small subunits were incubated with a preformed large subunit-membrane complex (experiments in F, Table 5).

572

N. BORGESE

ET

KC1 concentration

AL.

(mM)

Fm. 6. Binding of ribosomal subunits to stripped RM et different KC1 concns. Samples contained in 80 pl:stripped RM ((a) 0.181 mg protein; (b) 0.078 mg protein), 0.455 A,,, unit of large 50 m&r-Tris.HCl (pH subunits (-A-A-) or O-163 AzGO unit of small subunits (--a--+-), 7*6), 5 mm-MgCl, end the indicated KC1 concns. Incubation wes for 30 min at 0°C. Amounts of subunits bound are expressed in pmol, calculated on the basis that M, = 3.0~ lo6 for the large subunit and M, = 1.5~ 10” for the smell subunit (Hamilton et al., 1971).

TABLE

5

Binding of subunits to stripped rough microsomes at 150 m.&i-KCl, 50 mitt- Tris* HCt (pH 7.6), 5 mM-MgCl,t Expt

Order of additions

A 3H-labeled

large + stripped

no.

6.68 6.07 7.09

RM (30 min, 0°C)

3H-labeled

large + cold small (15 min, 0°C)

+ stripped

RM (15 mm, 0°C)

pmol bound

4.70

B Cold smsll + stripped

RM (15 min, O’C)

2

4.00

1

4.28

c + 3H-lebeled D 3H-labeled

large (15, min OW)

small + stripped

RM (30 min, O’C)

4.47 1.20 1.30 1.33

3H-labeled

+ cold large (15 min, 0°C)

3.02

+ stripped

RM (16 min, O°C)

3.08

E Cold lerge + stripped

RM (16 min, O’C)

4.38

F + 3H-labeled

small (15 min, 0°C)

4.39

t Incubation mixtures contained, in 80 ~1: stripped RM (0.78 mg protein) and, where indicated, &l*O67 Asso unit of labeled or unlabeled large subunits, and ~0.027 Asso unit of labeled or unlabeled small subunits.

RIBOSOME-MEMBRANE

573

INTERACTION

(e) Binding of polysomes to stripped rough microsomes: fate of the nascent chains It has been reported (Shires et al., 1973; Shires & Pitot, 1973) that reconstituted rough microsomes, obtained by incubation of “conditioned” membranes with detergent-prepared polysomes, are capable of translocating puromycin-released polypeptides to the interior of the membrane vesicles in a manner similar to that reported for native rough microsomes (Redman & Sabatini, 1966). This result has been interpreted as a proof of the functionality of the reconstituted polysomemembrane complex. On the other hand, Burke & Redman (1973) found that in reconstituted microsomes polypeptides are discharged by puromycin to the surrounding medium. We attempted to establish whether (i) nascent chains released by r-

a) “i

--c) 7;“I”

T

1

L

5 ml

FIG. 7. Fate of nascent chains discharged by puromycin from re-bound polysomes. The final samples contained in 1-O ml: 9.5 A 260 units of bound polysomes labeled in the nascent chains (speo. act. -400,000 cts/min per mg RNA), stripped RM (2.71 mg protein), 10m3 M-puromycin and 100 m&r-KCl, 50 mM-TriseHCl (pH 7.6), 5 mM-MgC1, (a), or high salt buffer ((b) and (c)). (a) Polysomes were first incubated with stripped RM for 10 min at 37°C in Tris/K/Mg buffer. The potassium concn was then raised to 100 mM by addition of a compensating buffer, puromyein was added, and the sample incubated for 20 mm at 26°C followed by 5 min at 37°C. (b) The sequence of events was the same as in (a), but the last incubation at 37°C was in high salt buffer. (c) Polysomes were first incubated for 30 min at 25% in 100 mM-KCl, 50 mM-TrisvHCl (pH 7.6), 6 m&r-MgCl,, and in the presence of lOA M-puromycin, without membranes. The salt concentration was then reduced, by dilution, to Tris/K/Mg buffer, stripped RM were added and the sample incubated for 10 min at 37°C. Incubation was then continued for 10 min at 37°C in high salt buffer. Portions (0.5 ml) of the samples were layered on 15% to 56% sucrose,gradients, containing the same salt concentrations as the flnal incubation mixtures. The gradients were centrifuged for 1.2 h at 40,000 revs/mm in the SB283 rotor of the IECBGO oentrifuge at 2O’C. The ordinate expresses cts/min determined by the method of Mans & Novelli (1961) on lOO-~1 samples of 0*6-ml fractions collected from the gradient. Sedimentation is from left to right and the large point to the right of each tracing represents radioactivity recovered in the pellet. M, ribosomal monomers; 8, small subunits; L, large subunits; Mb, membranes. ( ) Absorbance at 254 nm; (--e--e--), 3H radioactivity.

574

N. BORGESE

ET

AL.

puromycin from polysomes rebound to stripped RM would be recovered with the membranes, and (ii) if this were the case, whether the discharged chains would be protected by the microsomal membranes from attack by proteolytic enzymes. It has been shown previously (Kreibich & Sabatini, 1973) that in native RM, at least 50% of the nascent chains released by puromycin are protected from proteolysis by the limiting membrane, and become accessible to digestive enzymes only in the presence of detergents. Polysomes obtained from microsomes by treatment with detergent and labeled in the nascent chains with [3H]leucine, were allowed to bind to stripped rough microsomes in Tris/K/Mg buffer. After incubation for binding (10 min, 37”C), 10e3 M-puromycin was added and the KC1 concentration was raised to 100 mM. At this concentration the puromycin reaction occurs rapidly (Adelman et al., 1973a,b) but a substantial amount of ribosomes remains bound to microsomes (see Fig. 5). The sucrose gradient analysis of Figure 7(a) shows that a large proportion of the labeled nascent chains was recovered with the membrane band. By further raising the salt concentration after the puromycin reaction, ribosomal subunits were released from the membranes (Fig. 7(b)), with a concomitant shift of the isopycnic density of the microsomes to a lighter region of the gradient. However, the amount of labeled nascent chains recovered with the membranes was only slightly decreased. These results are similar to those of Shires et al. (1973), which were taken to suggest that the reconstituted rough microsomes were capable of carrying out vectorial discharge

Time of proteolysis

(hI

FIQ. 8. Proteolytic digestion of nascent chains disoharged from re-bound polysomes by puromycin. A %5-ml fraction from the sample used for the analysis of Fig. 7(b) was diluted 12-fold with high salt buffer, and the membranes were sedimented by centrifugation for 30 min at 30,000 revs/mm in the no.30 rotor. The microsomes were resuspended in 50 mu-KCI, 50 mM-Tris*HCl (pH 7.6), 5 min-MgCls, and incubated at 30°C with & mixture of trypsin-chymotrypsin (50 pg each/ml) in the presence (-a--•-) or absence (--A--A--) of 0.025% deoxycholate. After 6 h, Triton Xl00 was added to e concn of 2% to completely dissolve the membranes and inoubstion was continued for another hour. At the times indicated, IOO-(~1samples were withdrawn and trichloroecetic acid-precipitable radioactivity was determined by the method of Mans & Novelli (19Gl). The 100% value corresponds to -8500 cts/min.

RIBOSOME-MEMBRANE

INTERACTION

576

of the nascent polypeptides into the microsomal oavities. However, our further experiments demonstrated that this is not the case, because: (1) when the order of the reactions was inverted (i.e. puromycin first in the absence of membranes, then addition of membranes in Tris/Mg buffer, then high salt), a large portion of the nascent chains was still recovered with the miorosomal vesicles (Fig. 7(c)), suggesting that the association of nascent polypeptides with microsomal membranes does not require the formation of a polysome-membrane complex as an intermediate step; (2) when the membranes of the experiment in Figure 7(b) were subjected to proteolysis (Fig. S), the radioactive material was readily digested, and the presence of detergent had essentially no effect; (3) the nascent chains did not anchor polysomes to the membranes in reconstituted rough microsomes. In fact, the release of polysomes occurred in high salt buffer and did not require the addition of puromycin (results not shown). Thus, the results indicate that the nascent chains were deposited on the outside of the microsomal vesicles, and were not transferred across the membranes, as is the case with native rough microsomes.

4. Discussion The data reported in this paper demonstrate that ribosomes lacking nascent chains can attach to rough microsomal membranes stripped of ribosomes by the KCl/puromycin procedure. We have attempted to characterize both the membrane and the ribosome sites involved in the rebinding process. The binding of .ribosomes to stripped rough microsomes occurred readily at 0°C. This finding is in, agreement with previous observations in a system somewhat different from ours, consisting of detergent-prepared polysomes and “conditioned” rough microsomal membranes, which were prepared to accept ribosomes by treatment either with pyrophosphate and citric acid (Ragland et al., 1971) or with pancreatic ribonuclease and EDTA (Shires et cd., 1971a). We also found that, at O”C, untreated rough microsomes had a very low rib&some-accepting capacity, suggesting that, in the reconstitution reaction, ribosomes interact with the same sites that are normally occupied by polysomes in native rough microsomes. However, we did observe a considerable binding of ribosomes to untreated rough microsomes at 37°C. One possible explanation for this phenomenon could be the activation of additional, specific ribosome-acceptor sites at 37°C. This explanation is unlikely, since analysis by Scatchard plots showed that the “binding sites” of intact rough microsomes have a much lower affinity for ribosomes than those of stripped microsomes. It is more probable that binding to rough microsomes is due to non-specific interactions between ribosomes and membrane-bound polysomes. The possibility should be considered that a portion of the ribosome binding to stripped RM observed at 37°C is also due to non-specific interactions between ribosomes. We also investigated the binding of ribosomes to rat liver smooth microsomes and to erythrocyte ghosts, both treated by the same stripping procedure as the RM. In agreement with other workers (Burka & Schickliug, 1970), we found that erythrocyte ghosts were totally incapable of accepting ribosomes. Smooth microsomes, on the other hand, were capable of accepting ribosomes, but to much.lower levels than stripped RM. This binding can be accounted for partially by the presence of membrane-bound polysomes in the untreated SM fraction. The RNA content of the SM 38

576

N. BORGESE

ET

AL.

fraction before stripping (Table 1) is about 20 to 25% that of the rough fraction, which can account for approximately half of the observed binding. The remaining binding indicates that SM themselves do have vacant ribosome binding sites, although the total number of sites is less than in RM. It is of interest that these two membrane fractions, which are derived mainly from the endoplasmic reticulum and which are similar in oomposition and enzyme activities (Colbeau et al,, 1971; Fleischer & Fleischer, 1971; Kreibich & Sabatini, 1973), differ with respect to their capacity to accept ribosomes. Thus, in the cell, the absence of ribosomes in smooth portions of the endoplasmic reticulum probably does not only depend on a topological segregation of these membranes in areas where ribosomes are not available. There was somewhat more ribosome binding to SM at 37°C than at 0°C. This effect may be artificial and similar to the binding to untreated RM observed at this temperature. Other workers have reported a temperature-dependent binding of polysomes to untreated smooth microsomes, and interpreted the phenomenon as a heat-requiring activation of binding sites (Shires et al., 1971b). Rolleston & Mak (1973) studied the binding of polysomes to smooth microsomes only at 37°C and found that this reaction was more sensitive to potassium ions than the binding of polysomes to stripped RM. These authors suggested that polysome binding to SM is an artifact of the in vitro system. Although this is likely to be the case at 37”C, the Scatchard plot analysis suggests that binding to SM at 0°C is similar to that of stripped RM. It has also been reported that binding sites in SM become activated by steroid hormones (James et al., 1969; Williams & Rabin, 1969) and, moreover, that the action of the hormones in promoting the binding is sex-specific (Sunshine et al., 1971; Blyth et al., 1971). However, in these studies the ribosome-membrane complex was not isolated. Instead, the binding reaction was followed indirectly by measuring, with added substrates, the activity of the microsomal enzyme that catalyses disulfide exchange and which is thought to be masked by bound ribosomes (Williams et al., 1968). Our data further suggest that a heat-labile membrane protein(s) is involved in the ribosome-membrane interaction. In agreement with other authors (Shires et al., 1971a), we found that proteolytic digestion of stripped microsomes strikingly reduced their capacity to accept ribosomes. We also found that release of choline by treatment with phospholipase C had no effect on ribosome-membrane interaction. This finding is in agreement with the results of others (Jothy et al., 1973) on polysome-membrane interaction. Our results on heat denaturation of the binding sites at 55°C do not support the conclusions of Hochberg et al. (1972), who reported that preincubation of conditioned rough microsomes at 100°C for five minutes did not decrease the polysome binding reaction, which was taken to be non-specific. We attempted to characterize the portion of the ribosome involved in the binding reaction by carrying out experiments with purified subunits. Although small subunits could bind extensively to stripped rough microsomes at low ionic strengths (Tris/K/Mg buffer), at concentrations of potassium ions closer to physiological concentrations, we found that the membranes accepted large subunits better than small subunits. A preference for large subunits was also observed by Rolleston (1972) in a similar system derived from mouse liver. However, in contrast to the rebinding of rat liver subunits mouse liver subunits differed in their ability to bind to KCl/puromycinstripped rough microsomes throughout the whole range of monovalent ion

RIBOSOME-MEMBRANE

INTERACTION

577

concentrations tested (50 to 250 171~).Bar rat liver, Ekren et aE. (1973) have reported a large preferential in vitro binding of large subunits even at low ionic strengths (Tri.s/K/Mg buffer). This result, with membranes conditioned by pyrophosphate/ citrate treatment, differs from our results with KCl/puromycin-stripped membranes and those of others (Khawaja, 1971; Scott-Burden & Hawtrey, 1971) with membranes stripped by 2 M-LiCI. In the presence of cold 60 S subunits, the labeled 40 S subunits became associated with membranes as much as labeled 60 S subunits in the presence of unlabeled 40 S subunits, suggesting that the reconstitution reaction involving monomers and stripped rough microsomes occurs via the large ribosomal subunit. Although it is not clear why purified small subunits were bound at all to the microsomes (although in lesser amounts than 60 8 subunits), it must be emphasized that, in the cell, the proportion of free subunits is low (Joklik & Becker, 1965 ; Hogan & Korner, 1968 ; Kohler et al., 1968) and, moreover, that native small subunits are associated with a dissociation factor (Subramanian et d., 1968; Davis, 1971; Merrick et d., 1973). Thus, addition of a large amount of artificially prepared small subunits to stripped rough microsomes represents a very unnatural situation. Preliminary experiments suggest that a crude preparation of dissociation factor indeed decreases the aflinity of small subunits for microsomal membranes. The finding that aurintricarboxylic acid inhibits ribosome binding may also be useful to characterize further the portion of the ribosome involved in the binding reaction. Taken together, our data suggest that the reconstitution of rough microsomes from stripped membranes and ribosomes at 0°C is a phenomenon involving the recognition by large ribosomal subunits of specific binding sites, characteristic of rough microsomal membranes. However, in our attempt to reconstitute rough microsomes from polysomes and stripped membranes, we found that after reaction with puromycin the nascent chains were deposited on the outer surface of the microsomal vesicles, as opposed to the situation with native rough miorosomes. Thus, it appears that operation of the mechanism for vectorial transfer requires, in addition to binding of the ribosomes to the correct membrane sites, a recognition mechanism which ensures that assembly of a membrane-bound polysome proceeds in concert with the growth of a proper polypeptide chain. Since at ionic strengths presumed to approach those found under physiological conditions, the large ribosomal subunit had a considerable affinity for membrane sites (although to a much lesser degree than in Tris/K/Mg buffer), our results are compatible with a model for in vivo assembly of membrane-bound polysomes, in which the large ribosomal subunit can attach to membranes independently of protein synthesis. Such a mechanism has been suggested on the basis of in vivo (Baglioni et al., 1971) and in vitro (Rolleston, 1972; Borgese et nl., 1973) studies. Baglioni et al. (1971) followed the entry of newly synthesized subunits to a fraction of myeloma cells containing endoplasmic reticulum membranes, and found that the attachment of large subunits proceeded even after inhibition of protein synthesis, In an in vitro system it was found (Borgese et al., 1973) that large subunits remained associated with the microsomal membranes after natural termination or release of nascent chains by puromycin, while small subunits exchanged rapidly with added small subunits under the same conditions. Thus, it is possible that, in viva, large subunits do not necessarily detach from the membranes following completion of a round of protein synthesis.

578

N. BORGESE

E!f’

AL.

A type of model in which a variable proportion of inactive ribosomes can be bound io endoplasmic reticulum membranes is consistent with results of in oivo st,udies on changes in the distribution of free and membrane-bound ribosomes induced by a reduction in the level of the protein synthetic activity of the cell. Conditions such as starvation (Lee et al., 1971; Faiferman et al., 1971) and agents such as puromycin or NaP (Blobel & Potter, 1967; Rosbash & Penman, 1971; Bloiberg et al., 1972), which result in accumulation of inactive ribosomcs in the cell, have been reported t.o produce only a partial or no decrease in t.he amount of membrane-bound ribosomes found in microsomal f&actions. However, it must be pointed out that t.he distribut,ion of ribosomes bet,ween the free and bound states determined biochemically after cell fractionation may not represent the situation in the cell, since ribosomes may attach t,o or detach from membranes once the cells are rupt.ured and t.heir components are introduced into difIerent ionic conditions. If large subunits could attach to microsomal membranes independemly from protein synthesis, it would be unlikely that the nascent chain itself is the only factor involved in the recognition mechanism directing polysomes, engaged in the synthesis of defining products, to become ‘membrane-bound (Milstein et al., 1972; Blobel & Sabatini, 1971). An alternative mechanism could operate by which specific initiation complexes would recognize membrane-bound large subunits. The latter process could be mediated by modified initiation factors, capable of recognizing both messenger RNAs and membrane-bound large subunits. The existence of factors, which, by interacting with initiation factors, regulate the affinity of small ribosomal subunit.s for different messenger RNAs, has been reported both for bacterial (Groner et al., 1972; Lee-Huang & Ochoa, 1972) and reticulocyte (Nude1 et aE., 1973) cells. It remains to be explained why, in the cell, free polysomes do not bind to endoplasmic reticulum membranes aia their large subunits. To date, in vitro experiments have not answered this question, and contradictory results have appeared in the literature. Scott-Burden & Hawtrey (1971) reported that detergent-prepared bound polysomes at,tached to LiCl-stripped rough microsomes better than free polysomes, while Khawaja (1971) found that free polysomcs bound better than total polysomes prepa,red from a postmitochondrial supernate. Most workers, however? have detected no difference between the affinity of free and bound polysomes for microsoma,l membranes st.ripped by various procedures, both at low ionic strengths (Solan & Munro, 1972; Shires et al., 1973) and at higher concentrations of monovalent ions (Rollcston & Mak, 1973). Moreover, in t,he mouse liver system, Rolleston (1972) could detect no difference in the binding of large subunits obtained from free or bound polysomes to membranes, tested at a variet,y of ionic strengths. Alt,hough it is attractive to post,ulate that t.he ext,ra protein, which has been observed on free large subunits in various systems (Brown BEAbrams, 1970; Pridlender & Wett,stein, 1970; Borgese et al., 1973), represents a tightly bound factor that prevents free polysomes from binding t,o membranes, further experimental work is needed to clarify this problem. A preliminary report of part of the American Society of Cell WBS supported in part by U.S. at the Rockefeller University, We thank. Stelios Papadopoulos by the leaA-squares method.

of this work was presented at the 12th Annua.1 Meetillg Biology in St. Louis, 1972 (Borgme et ul., 1972). This work Public Hcmlth Service grant GM20277 and was initiated Now York. mltl Steven Hori for the fittin, (1 of the lirlcs in I’igurc 3

RIBOSOME-MEMBRANE

INTERACTION

579

REFERENCES Adelman, M. R., Blobel, G. & Sabatini, D. D. (1973a). J. CeZE Biol. 56, 191-205. Adelman, M. R., Sabatini, D. D. & Blobel, G. (19733)b J. Cell Biol. 56, 206229. Ames, B. N. (1966). In Methods in EnqmoZogy (Neufeld, 0. & Ginsburg 0. eds), val. 8, pp. 115-118, Academic Press, Inc., New York. Baglioni, C., Bleiberg, I. & Zaudcrer, M. (1971). Nature New Biol. 232, &12. Bleiberg, I., Zauderer, M. & Baglioni, C. (1972). Bioch~m. Biophys. Acta, 269, 453-464. Blobel, G. 6t Potter, V. R. (1967). J. Mol. BioZ. 26, 293-301. (Mason, L. A., ed.), vol. 2, pp. 193Blobel, G. & Sabatini, D. D. (1971). In Biomembranee 195, Plenum Publishing Corp., New York. Blyth, C. A., Freedman, R. B. & Rabin, B. R. (1971). Nature New BioZ. 230, 137-139. Blyth, C. A., Cooper, M. B., Roobol, A. & Rabin, B. R. (1972). Eur. J. Biochem. 29,293300. Borgese, N., Kreibich, G. & Sabatmi, D. D. (1972). J. Cell BioZ. 55, 24a. Borgese, N., Blobel, G. & Sabatini, D. D. (1973). J. Mol. BioZ. 74, 415-438. Brown, D. G. & Abrams, A. (1970). Biochim. Biophys. Acta, 200, 522-537. Burka, E. R. & Schickling, L. F. (1970). Biochemistry, 9, 459-463. Burke, G. T. & Redman, C. M. (1973). Biochim. Biophye. Acta, 299, 312-324. Colbeau, A., Nachbaur, J. & Vignais, P. M. (1971). Biochim. Biophys. Acta, 249, 262-292. Davis, B. D. (1971). iVatuve (Londolz), 231, 153-157. Dodge, J. T., Mitchell, C. & Hanahan, D. J. (1963). Arch. Biochem. Biophys. 100, 119-130. Ekren, T., Shires, T:& Pitot, H. C. (1973). Biochem. Biophys. Res. Commun. 54, 283-289. Faiferman, I., Cordunella, L. & Pogo, A. 0. (1971). Nature New BioZ. 233, 234-237. Fleck, A. & Munro, H. N. (1962). Biochim. Biophys. Acta, 55, 571-583. Fleischer, B. & Fleisoher, S, (1971). In Biomembranes (Mason, L. A., ed.), vol. 2, pp. 7594, Plenum Press, New York. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957). J. BioZ. Chem. 226, 497-509. Fridlender, B. R. & Wettstein, F. 0. (1970). Biochem. Biophye. Res. Commun. 39, 247-253. Ganoza, M. C. & Williams, C. A. (1969). Proc. Nat. Acad. Sk., U.S.A. 63, 1370-1376. Groner, Y., Pollack, Y., Berissi, H. & Revel, M. C. (1972). Nature New BioZ. 239, 16-19. Hamilton, H. G., Pavlovee, A. & Peterman, H. L. (1971). Biochemistry, 10, 3424-3427. Hicks, S. J., Drysdale, J. W. & Munro, H. N. (1969). Science, 164, 584-585. Hochberg, A. A., Stratman, F. W., Sahlten, R. N., Morris, H. P. & Lardy, H. A. (1972). Biochem. J. 129, 721-731. Hogan, B. L. M. & Korner, A. (1968). Biochim. Biophys. Acta, 169, 129-138. James, D. W., Rabin, B. R. & Williams, D. J. (1969). Nature (London), 224, 371-373. Joklik, W. K. & Becker, Y. (1965). J. Mol. BioZ. 13, 496-510. Jothy, S., Tay, S. & Simkins, H. (1973). Biochem. J. 132, 637-640. Khawaja, J. A. (1971). Biochim. Biophys. Acta, 254, 117-128. Khawaja, J. A. & Raina, A. (1970). Biochem. Biophys. Res. Commun. 41, 512-518. Kohler, R. E., Ron, E. Z. & Davis, B. D. (1968). J. Mol. BioZ. 36, 71-82. Kreibich, G. & Sabatini, D. D. (1973). Fed. Proc. Fed. Amer. Sot. Exp. BioZ. 32, 2133-2138. Loeb, J. N., Howell, R. R. & Tomkins, G. M. (1967). J. BioZ. Chem. 242, 2069-2074. Lee, S. Y., Krsmanovic, V. & Brawerman, G. (1971). J. Cell BioZ. 49, 683-691. Lee-Huang, S. & Ochoa, S. C. (1972). Biochem. Biophys. Res. Commun. 49, 371-376. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. BioZ. Chem. 193, 265275. Mans, R. J. & Novelli, G. D. (1961). Arch. Biochem. Biophys. 94, 48-53. Merrick, W. C., Lubsen, N. H. & Anderson, W. F. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 2220-2223. Milstein, C., Brownlee, G. G., Harrison, T. M. & Mathews, M. B. (1972). Nature New B&Z. 239, 117-122. Munro, H. N. & Fleck, A. (1966). Analyst, 91, 7&T-88. Nolan, R. D. & Munro, H. N. (1972). Biochim. Biophys. Acta, 272, 473-485. Nonomura, Y., Blobel, G. & Sabatini, D. D. (1971). J. Mol. BioZ. 60, 303-324. Nudel, U., Lebleu, B. & Revel, M. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 2139-2144. Pitot, H. C. & Shires, T. K. (1973). Fed. Proc. Fed. Amer. Exp. BioZ. 32, 76-79.

580

N. BORGESE

ET

AL.

Ragland, W. L., Shires, T. K. & Pitot, H. C. (1971). Biochem. J. 121, 271-278. Redman, C. M. (1969a). Fed. Proc. .Fed. Amer. Sot. Exp. Biol. 28, 2613a. Redman, C. M. (19696). J. Biol. Chem. 244, 4308-4315. Redman, C. M. & Sabatini, D. D. (1966). Proc. Nat. Acad. Sci., U.S.A. 56, 608-615. Rolleston, F. S. (1972). .&o&em. J. 129, 721-732. Rolleston, F. S. & Mak, D. (1973). Biochem. J. 131, 851-853. Roobol, A. & Rabin, B. R. (1971). FEBS Letters, 14, 165-169. Rosbach, M. & Penman, S. (1971). J. Mol. Biol. 59, 227-242. Sabatini, D. D., Tashiro, Y. & Palade, G. E. (1966). J. Mol. Biol. 19, 503-524. Schmidt, G. & Tannhauser, S. J. (1945). J. Biol. Chem. 161, 83-89. Scott-Burden, T. & Hawtrey, A. 0. (1971). Hoppe-Seyler’s 2. Physiol. Chem. 352, 575-5S2. Scott-Burden, T. & Hawtrey, A. 0. (1973). Biochem. Biophys. Res. Commun. 54, 12881295. Shires, T. K. & Pitot, H. C. (1973). Biochem. Biophys. Res. Commun. 50, 344-351. Shires, T. K., Narurkar, L. M. & Pitot, H. C. (197la). Biochem. J. 125, 67-79. Shires, T. K., Narurkar, L. M. & Pitot, H. C. (19715). Biochem. Biophys. Res. Commun. 45, 1212-1218. Shires, T. K., Ekren, T. E., Narurkar, L. M. & Pitot, H. C. (1973). Nature Nezo Biol. 242, 198-201. Subramanian, A. R., Ron, E. Z. & Davis, B. D. (1968). Proc. Nat. Acad. Sk., U.S.A. 61, 761-767. Sunshine, G. H., Williams, D. J. & Rabin, B. R. (1971). Nature New Biol. 230, 133-136. Siiss, R., Blobel, G. & Pitot, H. C. (1966). Biochem. Biophys. Res. Commun. 23, 299-304. Takagi, M., Tanaka, T. & Ogata, K. (1969). J. Biochem. 65, 651-653. Takagi, M., Tanaka, T. & Ogata, K. (1970). Biochim. Biophys. Acta, 217, 148-158. Talal, N. & Kaltreider, H. B. (1968). J. BioE. Chem. 243, 65046510. Tanaka, T., Takagi, M. & Ogata, K. (1970). Biochim. Biophys. Acta, 224, 507-517. Tashiro, Y. & Siekevitz, P. (1965). J. Mol. Biol. 11, 149-165. Williams, D. J. & Rabin, B. R. (1969). FEBS Letters, 4, 103-107. Williams, D. J., Gurari, D. & Rabin, B. R. (1968). E%BS Letters, 2, 133-135.