Printed in Sweden Copyright © 1977 by Academic' Press, Inc. All rights of reproduction in any Jbrrn reserved I$SN 0014-4827

Experimental Cell Research 108 (1977) 321-330

INTERACTION BETWEEN MEMBRANE PROPERTIES AND PROTEIN SYNTHESIS

In vitro Synthesis of Membrane Proteins during Erythropoiesis D. WRESCHNER, H E L E N MISHAN-DAHAN, M. RAAB and M. HERZBERG

Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel

SUMMARY Membrane protein synthesis was investigated by incubating rabbit reticulocytes, in vitro, with radioactive amino acids. The kinetics of membrane protein synthesis showed linear incorporation for approx. 15 min, after which there was only a slight increase in incorporation. On the other hand, intracellular protein synthesis was linear for an incubation period of 60 min. Membranes isolated from such rabbit reticulocytes were analysed on sodium dodecyl sulfate (SDS)polyacrylamide gels. Two major radioactive bands were found in the 50-60000 D region, whilst another labelled band had a molecular weight of 43 000 D. This latter band had an electrophoretic mobility identical with rabbit muscle actin (and chick brain actin), when run on one-dimensional SDS polyacrylamide gels. Absolute identity between rabbit brain actin and a newly synthesized reticulocyte membrane protein was shown by comigration on a two-dimensional (first dimension isoelectric focusing and second dimension SDS gel) electrophoresis system. Another band that was radioactively labelled was found to have a molecular weight of approx. 32 000 D. Separation of reticulocytes into different age groups showed that young reticulocytes synthesized a membrane protein species that was not radioactively labelled in the old reticulocyte population.

The rabbit reticulocyte has been exten"sively used as a convenient system for the study of protein synthesis and its regulatory mechanisms. As a rapidly differentiating cell that does not contain a nucleus, control of protein synthesis occurs at the !evel of translation. Mechanisms controlling protein synthesis include a hemin-regulated repressor [1] as well as possible membrane involvement [2]. Globln synthesis constitutes almost 95 % 9f the total protein synthesis in the reticuloCyte and thus has been the subject of ex!ensive research. Several recent reports have investigated the synthesis of nonglobin proteins by the reticulocyte. Harris i

.

,

,

& Johnson [3] first described a puromycin sensitive incorporation of [14C]glucosamine into reticulocyte membrane proteins, whilst following work most notably by Lodish and his co-workers [4, 5] detected the synthesis of two major and two to three minor membrane proteins by the intact rabbit reticulocyte. Koch et al. [6] have shown by working in vivo with anemic rabbits that the first membrane proteins to be synthesized after 8 h of administration of radioactive leucine to rabbits, were of the low molecular weight species (lower than 90 000 D) whilst larger membrane proteins were found to be labelled after 24 h. These results indicate a programmed Exp Cell Res 108 (1977)

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translation of messenger RNA (mRNA), in which circulating reticulocytes synthesize a certain class of membrane proteins whilst reticulocytes situated in bone marrow synthesize different larger species of membrane proteins. Indeed the biological significance of the membrane proteins synthesized by the circulating rabbit reticulocyte is unknown. The mature erythrocyte must have a flexible membrane that can undergo distortion in order to pass through capillaries in the circulatory system. In this regard, it has been shown by Tilney I7] that the three spectrin polypeptides (2 in the 200000 D region and one of 43 000 D) associate to form a network beneath the erythrocyte membrane. In this work we studied the membrane proteins synthesized in vitro by reticulocytes in suspension with a special emphasis on: (a) the kinetics of synthesis; (b) trying to elucidate the nature of the proteins synthesized; (c) observing the membrane synthesis at different stages of maturation.

MATERIALS AND METHODS Anemic blood was obtained by subcutaneously injecting rabbits with a 2.5 % solution of phenylhydrazine. The blood was collected from the ear vein into heparinized isotonic buffer solution (154 mM NaCI, 10 mM Tris-HCl, pH 7.4, 5 mM MgCI~) and immediately cooled to 4oc. A sample of the blood was stained with brilliant cresyl blue for reticulocyte percentage determination. The reticulocytes were washed three times (3 500 rpm, 5 rain, Sorvall rotor SS34) with isotonic buffer solution and then resuspended (10% packed cells v/v) in a solution that had the following constituents: 127 mM NaC1; 5 mM KCI; 2 mM MgSO,; 2 mg/ml glucose; 30 p.g/ml FeSO4; 1 mM CaCI2; 20 mM pH 7.4 Na + phosphate buffer; 19 cold amino acids 0.25 raM, [asS]methionine to give 70 t~Ci/ml. In certain instances [14C]protein hydrolysate was used instead of [3~S]methionine. Incubation of the cell suspension was carried out at the temperature and for the times indicated in the legends. Radioactivity incorporated into cell protein was determined by placing a sample on a glass filter (GF/C) and placing this into cold 10% (w/v) trichloroacetic acid (TCA) for 15 min. This was followed by Exp Cell Res 108 (1977)

5% TCA at 90"C for 10 rain and then two washes with cold 5 % TCA, two washes with ethanol-ether 1 : 1 and finally one wash with ether. The filters were dried and counted in a scintillation liquid in a Packard Scintillation Counter. Incorporation of radioactivity into reticulocyte cel~ membranes was checked by isolating cell membranes by hypotonic shock in 10 mM NaC1, 5 mM MgCl~ 1 mM Tris.HC1, pH 7.4, return to isotonicity with 0.1 vol of 1.5 M NaC1 and cantrifugation at 7000 rpnt for 5 rain (Sorvall rotor SS34). This process was r¢~ peated until "hemoglobin poor" membranes were obtained. These membranes were washed with cold 5 % TCA and the membranes then solubilized overnight in 0.5 cm ~ Packard's soluene 300. The solubilized membranes were then added to a dioxane scintil. lation mix and counted in a Packard Scintillation Counter. Membranes were prepared for electrophoresis by lysing the reticulocytes with 10-20 vol of 5 mM Na + phosphate at pH 8.0. The membranes were sediment¢.d at 7 000 rpm for 5 rain (Sorvall rotor SS34) and the procedure repeated 3--4 times. The final membrane pellet obtained was yellow-white in colour. The membranes were prepared and run on SDS-acrylamide gels according to Laemmli [8]. Routinely; the samples were separated using a 3 % stacking p~ with a 10 % separating gel. For the determination of radioactivity in the gels;, the gel was sliced using a Miles-Yeda gel cutter and the gel slices placed in vials containing 0.5 em 8 of soluene 300. The vials were incubated for 3 h at 50°C, after which scintillation fluid was added and the radioactivity checked. In certain cases, the gel was autoradiographed by drying the 1.5 mm thick gel in a lyophilizer, for 6--7 h. The dried gel was then exposed for 5--6 days to Ilg ford Red Seal 100FW X-ray film safety base and the film developed in D-19 developer. Actin was prepared either from rabbit brain or rabbit muscle by washing the tissue three times in ace, tone (10 ml/l g wet weight) at room temperature in a mortar. The acetone was decanted and the extracted tissue lyophilized. The tissue was ground in buffer | (5 ml/l g wet weight) containing 2 mM Tris-HCl, pH 8.0, 0.5 mM mercaptoethanol, 0.5 mM CaCls, 0.2 ATP and left for 30 rain at 0*C. The ground extract was centrifuged at 10000 rpm (Sorvall rotor SS34) for 10 rain and the supernatant spun for 90 rain al 48 000 rpm in a Spinco Ti-50 rotor. Four M KC1 w u added to the supernatant to obtain a final concentration of 0.6 M, and 1 M MgCls added to obtain a final concentration of 2 mM. The solution was left overnight at 0*C, and then layered on 6 ml of buffer I con, taining 0.6 M KC1, 2 mM MgCI2 and 20% glycerol, This was centrifuged at 4"C for 90 rain at 48 000 rpm in a Spineo Ti-50 rotor. The pellet obtained is F-aetin. The two-dimensional electrophoresis system was performed exactly according to O'Farelrs procedure [17]. The reticulocyte membranes were isolated by lysing the reticulocytes with 10-20 vol of 5 mM N,a÷ phosphate at pH 8.0 and then washed several tim®sl. These membranes were then solubilized according t o the method of Ames [18]. Isolated rabbit brain actin (prepared as above) was also subjected to the same solubilization procedure. These samples were then rula

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Table 1. Cycloheximide inhibition of membrane and intracellular protein synthesis in rabbit reticulocytes

the radioactivity associated with the membranes of control reticulocytes. Cycloheximide inhibited the synthesis of membrane proteins by 88% whilst globin synthesis +50/~g/ml CH was almost totally inhibited by 99% (taMin A B A B ble 1). It was necessary to verify that the obMembrane.bound radioactivity served membrane protein synthesis indeed 10 4 397 4 576 484 584 60 16 165 17 043 1 922 2 066 reflected the de novo synthesis of mem(100%) (100%) (12%) (12%) brane proteins, and was not due to difIntracellular protein synthesis ferential contamination of the membrane 10 22 050 21 800 0 1 050 fraction by cytoplasmic proteins. In order 60 113 I00 113 700 850 2 350 (100%) (100%) (0,75%) (2,1%) to test this possibility reticulocytes that had been incubated with [ssS]methionine were Fifty/~l of packed reticulocytes were added to 450 ~1 of medium minus methionine, Fifteen /~Ci of [~sS]- lysed and the membranes isolated as demethionine was added and the suspension incubated scribed in Methods. The supernatant (retiat 34°C. At the indicated times 50/~1 aliquots were removed and placed in cold phosphate buffer (5 raM, culocyte cytoplasmic lysate fraction) was pH 8.0). The tubes were mixed and then centrifuged also removed and recentrifuged at 100 000 g at 6 000 rpm for 5 rain. An aliquot of the supernatant was removed and placed on a GF/C filter to deter- in a Spinco ultracentrifuge for 3 h in order mine TCA-precipitable radioactivity (equivalent to in- to pellet the ribosomal fraction. The 100 000 tracellular protein synthesis), The pellet was washed with phosphate buffer, twice with cold 10% TCA g supernatant fraction was taken and run and then solubilized overnight with 0.5 cm8 soluene. Scintillation fluid was then added. Values for radioac- alongside the solubilized reticulocyte memtivity have been corrected such that the membrane branes, on an SDS-polyacrylamide gel. protein synthesis and intracellular synthesis refer to Five times more radioactivity was present the same volume of cell suspension, in the 100000 g supernatant fraction than in the reticulocyte membrane sample than were loaded onto the SDS gel. on the first dimension isoelectric focusing cylindrical Fig. I shows the autoradiogram obtained gels, The gels were then equilibrated [17] and layered and run on an SDS acrylamide gel composed of 3 % from these two samples when run on the stacking and 10% separating gels. The radioactivity associated with the actin protein of the reticulocyte SDS gel. It can be clearly seen that the membrane was detected by slicing the gel into squares, cytoplasmic fraction contains only two solubilizing these squares with soluene (as above), radioactive peptides, corresponding to the adding scintillation fluid and then counting the radioactivity in a Packard Scintillation counter, globin region at the lower portion of the gel and a peptide in the 20-30 000 D region. On the other hand, the reticulocyte membrane RESULTS fraction shows distinctly radioactive pepA de novo synthesis of membrane proteins tides at positions in the gel described below by the rabbit reticulocyte was demonstrated where there are absolutely no newly synby addition of cycloheximide to the cells thesized peptides in the cytoplasmic fracsuspended in a medium allowing protein tion. synthesis. Table 1 shows that the cell susFig. 2 shows the kinetics of both intrapension incubated in the presence of cyclo- cellular and membrane protein synthesis. heximide gave a minimal labelling of the This result shows that intracellular synmembrane components as compared with thesis proceeds at a linear rate until at least Exp Cell Res 108 (1977)

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Fig. 1. Cytoplasmic and membrane proteins synthesized by rabbit reticulocytes. Packed, washed reticulocytes were added to the medium in the ratio of 1 : 9. Twenty/.tl of [85S]methionine (4.75 mCi/ml) was added to the cell suspension (total vol 1 ml) and incubation was carried out at 34°C for 40 min. Cells were then washed and lysed with 1 vol of 0.005 M sodium phosphate buffer at pH 8. The lysed cells were then centrifugated and the supernatant recentrifuged in a Spinco ultracentrifuge at 100000 g for 3 h. The reticulocyte membranes were prepared as in Materials and Methods and SDS gel electrophoresis then performed on both the solubilized reticulocyte membranes and the 100000 g supernatant fraction. 250000 cpm of the supernatant and 50 000 cpm of the reticulocyte membranes were loaded on the gel. The gel was then dried and exposed to X-ray film. Autoradiogram of (a) the 100000 g supernatant fraction; (b) reticulocyte membrane sample.

35 min of incubation. On the other hand, membrane protein synthesis was linear until approx. 15-20 min of incubation after which further synthesis ceased or even showed a Exp Cell Res 108 (1977)

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Fig. 2. Abscissa: time of incubation (min); ordinate: radioactivity (cpm). ---, whole blood; - - , packed reticulocytes resuspended in medium. Packed reticulocytes were added to the medium (minus leucine) in the proportion 1 : 9 [14C]leucine was then added and the cell suspension incubated at 37°C. When whole blood was used, only [14C]leucine was added to the incorporation test tube that contained untreated and unwashed anemic blood. Sampling procedures for the determination of membrane and intracellular protein synthesis were as in table 1. Kinetics of (A) intracellular protein synthesis, and 03) of membrane protein synthesis.

slight decrease. The kinetics of membrane protein synthesis also showed a vigorous initial burst in the first 5 min in contrast to that shown by intracellular synthesis. These results were obtained using an artificial medium. When whole blood was in-

Reticulocyte membrane protein synthesis

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Iobm.

20

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act n hke

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7000t 5000F 5000~2000'ti2

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Fig. 3. Abscissa: migration along gel (from left to right); ordinate: (left) OD560nm; (right) radioactivity in ~ n t s per 10 r a i n . . . . , Spectrophotometric scan at ~ nm; - - , radioactivity profile. Gel electrophoretic analysis of reticulocyte membrane proteins and radioactively labelled proteins after incubation with [14C]protein hydrolysate. Washed reticulocytes were obtained as in Materials and Methods ~ 50 ~1 of packed reticulocytes were resuspended in 450 NI of medium. Fifteen ~1 of [14C]protein hydrolysate was added (50 ~Ci/ml) and incubation continued at 37°C for 40 min. At the end of the incubation period, membranes were prepared by repeated hypotonic lysis of the reticulocytes. The membranes were solubilized as in Materials and Methods and then electrophoresed on a 3-7-12 % acrylamide$DS gel. Only the 12% region of the gel is presented. The gel was stained with Coomassie Blue and scanned at 560 nm in a Gilford spectrophotometer. Radioactivity was determined on the gel slices as in Mal~erials and Methods.

cubated with radioactive leucine, the kinetics of globin synthesis, compared with that Of membrane protein synthesis, was almost identical. Incorporation of radioactive leueine into membrane proteins was linear until at least 35 min, as was the synthesis of intracellular proteins. The extent of ineorporation was lower due to the dilution

40

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Fig. 4. Abscissa: slice no.; ordinate: radioactivity in cpm. Radioactively labelled proteins after incubation of rabbit reticulocytes with [asS]methionine. Cells were prepared as noted in fig. 2 except that 10/.d of [a~S]methionine (4.75 mCi/ml) was added to 600 /zl of a 10% (v/v) reticulocyte cell suspension. Incubation was carried out at 34°C for 40 rain. Preparation of cell membranes, gel electrophoresis and determination of radioactivity were performed as in Materials and Methods.

of the radioactive amino acid with endogenous amino acids present in the plasma. An electrophoretic gel analysis superimposed on the radioactivity profile of membranes isolated from reticulocytes that had been incubated with [14C]protein hydrolysate is shown in fig. 3. From the radioactivity profile it can be seen that there are five major peaks of radioactivity associated with the gel analysis of the membrane components. Three of these major peaks appear in the region of 50000--60000 D; the following fourth peak appears at a location corresponding to a molecular weight of 43 000 D, whilst the last radioactive component occurs immediately behind the "broad peak" of the reticulocyte membrane. As the above experiment gave a membrane preparation with a relatively low specific activity (as witnessed by the low valExp Cell Res 108 (1977)

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Wreschner et al. A

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actln-llke (5)

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b r o a d peek (9)

rocytes run alongside purified chick brai~ actin. It is evident from these gels that bot h the reticulocyte and erythrocyte membrane samples contain a band of similar if not identical electrophoretic mobility to that of purified actin. In order to further characterize this protein band, the region corresponding to the actin-like protein found in the reticulocyte was cut out and the protein eluted from the gel. This eluted protein was then rerun on a polyacrylamide next to purified rabbit muscle actin and chick brain actin. Fig. 6 shows that both the rabbit muscl~ actin and chick brain actin had electropho/

Fig. J. Coelectrophoresis of reticulocyte and erythrocyte membrane samples alongside purified chick brain actin. Reticulocytes and their membranes were prepared as in Materials and Methods, Erythrocyte membranes were obtained from normal, untreated rabbit blood. Actin had been previously prepared as noted in Methods from chick brain, The three samples were solubilizedand run on a 3-7-12 % SDS polyacrylamide gel. Only the 12% region of the gel is shown in this figure. (A) Reticulocyte membrane sample; (B) purified actin; ((7)erythrocyte membrane sample.

A

B

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D

ues of the counts on the gel) it was imperative to verify the results using an amino acid of high specific activity. The experiment was repeated using [asS]methionine as a radioactive amino acid in the medium. Fig. 4 shows the gel analysis and the radioactive profile of the membranes isolated from incubated reticulocytes. Corre6. Characterization of the actin.like band in the spondence with fig. 3 is almost complete Fig, reticulocyte membrane by coelectrophoresis with with all the major peaks being synthesized purified actins, Solubilized reticulocyte membranU were run on SDS acrylamide gels and the region corat identical positions. responding to actin was excised and the proteins elutsd The radioactively labelled band corre- by macerating the gel in 5 mM sodium phosphate bufat pH 8.0. The eluate was concentrated by diasponding to the 40 000--45 000 D region was fer lysis against polyvinyl pyrillidone (PVP). Actin wits of special interest due to the recent report purified from newly.born rabbit muscle and from chick as in Methods. The reticulocyte membrmm [7] of an actin-like protein situated in the brain, actin-like protein, rabbit muscle and chick brain actln erythrocyte membrane. Fig. 5 shows the gel were then coelectrophoresed on a 3-10 % SDS pc4p gel. A, Chick brain actin; B, C, rabi~ pattern obtained with membrane samples acrylamide muscle actin; D, reticulocyte membrane actin.like ~ from rabbit reticulocytes and rabbit eryth- tein. Exp CellRe$108 (1977)

Reticulocyte membrane protein synthesis

Fig. 7. Two-dimensional gel electrophoresis of actin

and reticulocyte membranes. Washed reticulocytes were washed and incubated with [ssS]methionine and the membranes isolated as in Materials and Methods. Rabbit brain actin was purified by one cycle of polymerization. Both the ret~culocyte membranes and purified actin were solubilized according to Ames [18] and then immediately run on isoelectric focusing acrylamide gels. These gels were then run in the second dimension on 3 % stacking, 10% separating SDS acrylamide gels. The gels were then stained with Coomassic blue. (A) Isolated reticulocyte membranes (200 g,g protein); (B) isolated reticulocyte membranes (70/zg protein)+purified aetin (25/.~g); (C) purified actin (25 /~g). Radioactivity was counted on slices of a radioactivity labelled reticulocyte membrane sample and we found that only these two gel squares (see Materials and Methods) which corresponded to the isolated active spot showed radioactivity (380 and 205 cpm) as compared with spectrin (30-35 and 32 cpm). Some other fractions corresponding to other peptides were also labelled.

retie mobilities identical with that of the protein eluted from the gel. The above results show that a radioactively labelled reticulocyte membrane protein comigrates on one-dimensional SDS gels with actin. The possibility exists

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though that this newly synthesized membrane protein is simply a very minor membrane protein that fortuitously co-migrates with actin. Thus, in order to further characterize this radioactively labelled membrane protein as actin, reticulocyte membranes were subjected to two-dimensional electrophoresis (first dimension isoelectric focusing followed by SDS gel electrophoresis). Fig. 7A shows a Coomassie blue stain of the two-dimensional gel of isolated reticulocyte membranes. The position of purified actin in the reticulocyte membrane pattern was determined by electrophoresizing a mixed sample comprised of a dilute concentration of solubilized reticulocyte membranes to which purified rabbit actin had been added. The reticulocyte membrane peptides x and y (fig, 7A) were found on fig. 7B, and thus the peptide indicated by the arrow (fig. 7B) was concluded to be actin. This was further verified by running only the purified rabbit brain actin sample on the two-dimensional gel system (fig. 7C) and indeed this reinforced our previous conclusion as to the position of the actin protein. Due to slight shrinkage of the gel during drying (and thus position distortion of the separated peptides), the two-dimensional gel of the radioactively labelled reticulocyte membrane sample was sliced into a grid shape at the region of the putative actin, and these acrylamide sections were then solubilized and counted. We found that the two gel squares (see Materials and Methods) which corresponded to the isolated actin spot showed radioactivity (380 and 205 cpm) as compared to slices in the spectrin region (3035 cpm). Some other fractions corresponding to other peptides were also labelled. In order to determine whether membrane protein synthesis was identical in different reticulocyte age groups, the cells were separated by differential floatation [9] after Exp Cell Res 108 (1977)

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Fig. 8. Membrane protein synthesis by reticulocytes of different ages. One hundred /zl washed reticulocytes were resuspended in medium and 10 /~1 of [asS]methionine (5 mCi/ml) was added. The cells were then incubated at 34°C for 40 min, after which they were washed in isotonic saline and then resuspended in autologous plasma. The cell suspension was then left to equilibrate to room temperature, and were separated on phthalate ester oils according to Danon & Marikovsky [9]. Membranes of old and young reticulocyte age groups were isolated as in Methods, solubilized and run on a 3-10% acrylamide gel. The 1.5 mm thick gel was dried for 8 h in a perspex press placed in a lyophilizer and then exposed to X-ray film for 6 days. The film was developed in a Kodak D-19 Developer Radioautogram of (A) old; (B) young reticulocyte membranes. The band labelled (1) is the most extensively synthesized membrane protein (tool. w. 53 000 D) in both the young and old reticulocyte. The band labelled (2), having a molecular weight of 42 000 D, is only synthesized in the young reticulocyte whilst band (3), presumably actin, is synthesized to a greater extent in the young reticulocyte than in the old reticulocyte.

they had been incubated in artificial medium supplemented with [aSS]methionine. Fig. 8 shows the autoradiogram obtained of membrane samples prepared from young and old cells. The major membrane protein synthesized by both the old and young retiExp Cell Res 108 (1977)

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Fig. 9. Abscissa: mol. wt (D x 10-a); ordinate: 100 kg Rf (where Ry is the relative mobility in relation to the dye). Mol. wt calibration curve of a 10% SDS-acrylamide gel. The following purified proteins obtained from commercial sources were run on a 3-10% SDSacrylamide gel (approx. mol. wt in D of the protein monomer appears in brackets)--rabbit albumin (+) (68000), creatine phosphokinase (©) (40500), horseradish peroxidase (A) (40 000), DNAse I (@) (31 000) trypsin ( n ) (24 K). The arrows on the curve represent the positions of the major membrane proteins synthesized by the young rabbit reticulocyte.

culocyte has a molecular weight of 53 000 D. In addition, the young reticulocyte synthesizes membrane proteins of 49K, 46K, 43K, 40K, 38K, 29K and 25K D (fig. 9). On the other hand, the old reticulocyte does not synthesize the membrane protein species at 42K and the 40K protein, presumably actin, is synthesized to a lesser extent (fig. 8). DISCUSSION In our work--as in the work of Koch et al. [10J--one can show a differential inhibitory action of cycloheximide versus synthesis of intracellular and membrane proteins. A possible explanation would be that, as was suggested by Bulova & Burka [11], membrane proteins are synthesized by membrane-bound ribosomes. In this case the

Reticulocyte membrane protein synthesis membrane environment could protect the polysomes against the action of the drug. Another possibility is that cycloheximide, being an inhibitor both of initiation [12, 13] and of translocation, i t a c t s differentially for different mRNAs requiring specific initiation factors. The reticulocyte membranes used in this study were not purified by centrifugation through a sucrose gradient, and thus the possibility existed that the observed membrane protein synthesis was simply a result of contamination by cytoplasmic proteins. The experiment devised and shown in fig. 1 completely ruled out this possibility; if the membranes were contaminated with cytoplasmic proteins then these proteins should appear also in the autoradiographed lysate supernatant fraction. Not one of the radioactive membrane proteins was found in the cytoplasmic fraction. Moreover, the rabbit reticulocyte is composed solely of a cell membrane, intracellular ribosomal network and very few, if any, degenerating mitochondria. Thus, contamination by cellular organelles of the membrane fraction is highly unlikely. This was verified by observing sections of the membranal pellet in the electron microscope (data not shown), which only showed reticulocyte membranes and some tightly membrane-bound ribosomes. However, in this work we do not distinguish between integral and peripheral reticulocyte membrane proteins and it may indeed be that the membrane~proteins synthesized are mostly peripheral. As far as the nature of the proteins which are synthesized at the reticulocyte stage of differentiation, it is clear from our work and from Lodish's work [4, 14], that only some of the membrane proteins are synthesized. Particularly one could note that the high molecular weight proteins, spectrins and

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band 3 protein, are not synthesized. On the contrary, the most actively synthesized proteins are below 55 000 D. It should be pointed out here that not all of the medium molecular weight proteins are synthesized. This leads us to the question regarding the fate or, more precisely, the differential fate of various mRNA during erythropoietic differentiation. Indeed, up to now there is no clear explanation altogether regarding the degradation of mRNA in a cell which does not contain classical RNAase. Indeed a measure of its nucleotide monophosphate content resulting from degradation of R N A was recently found to be extremely low [151. Together with this differential fate of mRNA it is interesting to notice in our work the differential kinetics of synthesis of different proteins. When incubating cells at 37°C we always found a burst of membrane protein synthesis for the first 10-15 min after the onset o f incubation, whilst intracellular protein synthesis is linear for at least 40 min under the same conditions. Lodish [4], on the other hand, found that at 30°C synthesis of both intracellular and membrane proteins was linear for 40 min. This difference in conditions could also explain why these authors found a precursor to band B which could not be found under our conditions. Another interesting point is the striking difference observed between cells which were incubated in "natural" conditions, i.e., whole blood and cells which were first washed and then resuspended in artificial medium. The latter synthesized much more membrane proteins while the former showed an identical pattern of synthesis for both membrane and intracellular components. However, in the two cases the nature of the synthesized proteins was the same (data not shown). Exp Cell Res 108 (1977)

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One of the most interesting components which is synthesized corresponds to band 5 which has a molecular weight ranging between 40000 and 45 000 D. This band seems to correspond quite exactly to actin as we demonstrated here and as was shown in human erythrocytes by Tilney & Detmers [7]. This component was described to be a part of a complex in conjunction with bands 1 and 2 of spectrin [16]. We further verified the identity of the putative actin protein by showing co-migration on two-dimensional gels of a purified actin sample with a radioactively labelled reticulocyte membrane protein. This twodimensional system used was that of O'Farell [17], where the first dimension is based on isoelectric focusing and the second dimension separation on molecular weight. The chances that two different proteins comigrate to the same position on these gels are very slight (for discussion see O'Far~',ll [17]). As reported in this paper, high molecular spectrins are not synthesized. The putative complex comprising bands 1,2 and 5 should then be enriched in band 5 during differentiation. If band 5 is indeed actin, this could explain the added flexibility of the cells in the erythrocyte phase. That there are proteins which are synthesized at different stages of differentiation is further established by the fact that we have shown

Exp Cell Res 108 (1977)

that even reticulocytes of different age groups have a different pattern of membrane protein synthesis. This research was conducted with the aid of a grant from D616gation O6n(.~ralea la Recherche Scientifique et Technique, and a Jane Coffin Childs Foundation grant. We wish to thank Mr Uri Caro and Mr Uri Yoran for graphical illustrations.

REFERENCES 1. Adamson, S C, Mo-Ping Yau, P, Herbert, E & Zucker, W V, J mol biol 63 (1972) 247. 2. Wreschner, D & Herzberg, M, Eur j biochem 64 (1976) 399. 3. Harris, E D & Johnson, C A, Biochemistry 8 (1970) 512. 4. Lodish, H F & Small, B, J cell bio165 (1975) 51. 5. Lodish, H F, Proc natl acad sci US 70 (1973) 1526. 6. Koch, P A, Gartrell Jr, J E, Gardner, F M & Carter, J R, Biochim biophys acta 389 (1975) 162. 7. Tilney, L G & Detmers, P, J cell bio166 (1975) 508. 8. Laemmli, V K, Nature 227 (1970) 680. 9. Danon, D & Marikovsky, Y, J lab clin med 64 (1964) 668. 10. Koch, P A, Gardner, F H, Gartrell Jr, J E & Carter, J R, Biochim biophys acta 389 (1975) 177. 11. Bulova, S I & Burka, E R, J biol chem 245 (1970) 4907. 12. Lin, S Y, Mostewlar, R D & Hardesty, B, J mol bio121 (1966) 51. 13. Obrig, T G, Culp, W J, McKeehan, W L & Hardesty, B, J biol them 246 (1971) 174. 14. Wreschner, D, Foglizzo, R & Herzberg, M, FEBS lett 52 (1975) 255. 15. Bartlett, G R, Biochem biophys res commun 70 (1976) 1055. 16. Fairbanks, G, Steck, T L & Wallach, D F H, Biochemistry 10 (1971) 2606. 17. O'Farell, P H, J biol them 250 (1975) 4007. 18. Ames, G F & Nikaido, K, Biochemistry 15 (1976) 616. Received March 16, 1977 Accepted April 13, 1977

Interaction between membrane properties and protein synthesis. In vitro synthesis of membrane proteins during erythropoiesis.

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