THE S E P A R A T I O N OF RAT L I V E R ENDOPLASMIC R E T I C U L U M M E M B R A N E P R O T E I N S BY TWO D I M E N S I O N A L P O L Y A C R Y L A M I D E GEL E L E C T R O PHORESIS

HENRYK H. CZOSNEK and ABRAHAM A. HOCHBERG

Dept. of Biological Chemistry, The Hebrew University, Jerusalem, Israel

(Received 3 December, 1974) Abstract. The separation of rat liver endoplasmic reticulum membrane proteins by two dimensional polyacrylamide gel electrophoresis is described. By this method, the proteins of the rough membrane ribosomes could be separated from the other rough membrane proteins. Both rough and smooth membrane fractions contain at least 30 defined membranal proteins. The electrophoretic patterns of rough and smooth membrane proteins are clearly different. I. iNTRODUCTION Electron microscopic pictures of liver cells clearly reveal the existence of an interlacing network of membranes, some of them studded with dense ribosomal particles (the rough membrane), and some lacking ribosomes (the smooth membrane) [ 1, 2]. Smooth and rough membranes can be separated in vitro by discontinuous sucrose gradient centrifugation, and then studied separately [3, 4]. The relationship between the rough and the smooth membrane fractions has been investigated but not completely clarified [5, 6]. Erikson studied the chemical composition and the enzymatic activities of the smooth and rough membrane fractions, and clearly showed that they differ both in their chemical composition and pattern of enzymatic activities [7]. The presence of structural proteins in the microsomal fraction was claimed by Criddle et al. [8], and Ward and Pollack [9]. The protein composition of rough and smooth membranes was investigated by SDS-polyacrylamide gel electrophoresis by Schnaitman, who found that the two microsomal membrane fractions contained a number of similar proteins, however similar proteins were not present in the same amounts in both microsomal membrane fractions, and some proteins were observed that were unique to each fraction [ 10]. Burka and Bulova reported on the difference in the ribosomal proteins of membrane-bound and free ribosomes [ 11 ], difference which could have been due to contamination of the bound ribosomes by membrane proteins [ 12]. Hanna et al. reported that free and membrane-bound ribosomes released by deoxycholate treatment have identical protein composition [ 13]. We decided to compare the protein composition of polyribosomes, smooth and rough membrane fractions by two dimensional polyacrylamide gel electrophoresis, in order to gain more information on the protein pattern of these different fractions. 19 Molecular Biology Reports 2 (1975) 19-25. All Rights reserved. Copyright 9 1975 by D. Reidel Publishing Company, Dordrecht-Holland.

II. MATERIALS AND METHODS 1. Preparation o f Rat Liver Polyribosomes, Rough and Smooth Membranes Male albino rats, 150-200 g, were starved overnight before sacrifice. The livers were homogenized (1:2 w/v) in 0.25 M sucrose TKM (Tris HC1, pH = 7.5, 50 mM; KC1 25 mM; MgC12 10 mM). The homogenate was centrifuged for 20 rain at 19 000 • g. The free polyribosomes, rough and smooth membranes were prepared by combining the methods described by Bloemendal et al. [3] and Sunshine et al. [4]. The postmitochondrial supernatant was layered on a discontinuous sucrose gradient consisting of 8 ml of 2.0 M sucrose TKM overlayered with 8 ml of 1.35 M sucrose TKM, and spun for 3.5 hr at 78 000 • g. The smooth membrane and the rough membrane were aspired, diluted with 0.25 M sucrose TKM (1:1) and layered on a discontinuous gradient consisting of 5 ml of 2.0 M sucrose TKM overlayered with 8 ml of 1.45 M sucrose TKM, and spun for 1.5 hr at 78 000 • g. This procedure was repeated twice. The smooth and rough membranes were pelleted by centrifugation for 1.5 hr at 78 000 X g and homogenized in 1 M sucrose TKM. Free polyribosomes were suspended in 0.25 M sucrose TKM and stored in liquid nitrogen. The rough and smooth membrane fractions so obtained had RNA/protein ratio of 0.6 and 0.2, respectively. Electron microscopic pictures of the rough and smooth membrane fractions were very similar to those obtained by Bloemendal et al. [3]. The free polyribosomes, the rough and the smooth membrane fractions were tested for their in vitro amino acid incorporation activity. The free polyribosomes and the rough membrane fractions were found to be active in amino acid incorporation; in comparison the smooth membrane fraction had a very low amino acid incorporation activity. RNA was measured according to Bloemendal et al. [3] and protein according to Lowry et al. [ 14]. 2. Two Dimensional Polyacrylamide Gel Electrophoresis The solubilization of membrane proteins for the electrophoresis was done as follows: the pelleted membranes were washed six times with 20 volumes of ethanol-ether (1 : 1, v/v) to extract lipids. The extracted membrane fractions were pelleted each time by centrifugation at 1000 • g for 10 min. After the last ethanol-ether extraction, the pellet was extracted with ether and dried at 37 ~ The dry pellet was suspended in a minimal volume of Tris HC1 pH-7.6, 10 mM containing Mg acetate 100 mM. The proteins were solubilized by the addition of two volumes of glacial acetic acid and extracted three times essentially as described by Sherton and Wool [15]. By this method 100% of the proteins were solubilized after the third extraction. The combined acetic acid extracts were dialysed for 12 hr against the first dimensional electrode buffer used by Kaltschmidt and Wittman [ 16]. After the dialysis, the dialysis bag was placed into polyethyleneglycol 6000, until the protein concentration in the dialysis bag reached approx. 4 mg ml- 1. Each fraction was subjected to electrophoresis in the first dimension on two separated gels; one was run from cathode to anode and the second from anode to cathode. Both gels were composed of a separation gel (13.5 • 0.6 cm) overlaid with spacer gel (0.3 • 0.6 cm); preparation of the gels was done according to Kaltschmidt and Wittman [ 16]. Four mg of protein were applied on the top of each gel and the electrophoresis was carried out essentially as described [ 16]. pH adaptation from the first dimension electrophoresis to the second was done according to Avital and Elson [ 17]. After the first dimensional separation, each gel was applied on a different plate for 20

Fig. la. Figs. la-c. Two dimensional electrophoretograms of liver ribosomal (a) rough membrane (b) smooth membrane (c) proteins. Condition for electrophoresis is described in material and methods. The anode was at the left in plates (A) and (B) in the fflrst dimension, at the top in the second. 4 mg of ribosomal, rough membrane or smooth membrane proteins were subjected to electrophoresis from anode to cathode and 3 mg from cathode to anode. The photographs were developed to give maximum contrast.

Fig. lb. 21

Fig. lc.

separation in the second dimension [ 16, 18]. Staining and destaining were performed as described [ 16]. III. RESULTS AND DISCUSSION The two dimensional polyacrylamide gel electrophorogram of the free ribosomal proteins from rat liver is shown in Figure la and the pattern obtained is portrayed schematically in Figure 2a. The pattern seen is essentially the same as described by Sherton and Wool [ 15] and Hanna e t al. [ 13]. The numbers given to some of the spots in Figures 2a, b are the same as those given to the corresponding spots in the electrophorogram shown by Sherton and Wool [ 15]. In the gel which is shown in Figure la, three spots of protein which had migrated from cathode to anode ('acidic' proteins, Figure la-A), and 55 spots of protein which had migrated from anode to cathode ('basic' proteins, Figure la-B) were dearly visible. Since we were interested in comparing the protein patterns of ribosomes, rough membrane and smooth membrane, the ribosomes were not treated with high KC1 Concentration, because treatment of rough membranes with high salt concentration is known to strip partly the membrane from its attached ribosomes [ 19], and might affect the protein composition of the rough membrane [20]. The electrophorogram of the rough membrane proteins is shown in Figure lb and portrayed schematically in Figure 2b. Corresponding spots to all the ribosomal proteins present in free ribosomes (Figure 1a-B) appear in the protein pattern of the rough membrane (Figure 1b-B). The electrophorogram reveals in addition 8 'basic' proteins which are absent from the free ribosomal protein pattern; these additional proteins (Figure lb-B) might be 'basic' membranal proteins, or 22

ribosomal proteins which are associated with bound ribosomes and are not present in free ribosomes. In the electrophorogram of the rough membrane proteins (Figure lb-A), as many as 37 deemed 'acidic' protein spots are seen; at least 34 o f them do not appear in the free ribosomal protein pattern. These 'acidic' protein spots appear with the same intensity in the electrophorogram o f the rough membrane after stripping it from its ribosomes with KC1 and puromycin [ 19] and therefore are very likely genuine membranal proteins. The electrophorogram of the smooth membrane proteins is shown in Figure lc and portrayed schematically in Figure 2c. At least 20 'basic' protein spots are seen faintly (Figure 1c-B); all or some of these proteins might represent ribosomal proteins due to contamination with rough membrane, or proteins which are part o f the smooth membrane [20]. However, two 'basic' protein spots are common to rough and smooth membrane (Figures 2b-B and 2c-B) and do not appear in the ribosomal protein pattern (Figure 2a-B). In Figure 1c-A, as many as 29 'acidic' protein spots are seen. In comparing the 'acidic' protein pattern of rough (Figures lb-A and 2b-A) and smooth membrane (Figures lc-A and 2c-A), it can be seen that there is at least one spot unique to smooth membrane (spot number 11, Figure 2c-A) and not less than 6 protein

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spots unique to rough membrane (spots number 13-16, 22, 33, Figure 2b-A). The rest of the protein spots are common to both membrane fractions. From the results described, it can be seen that, by introducing some modifications in the method used by Kaltschmidt and Wittman to separate ribosomal proteins [ 16], one can obtain good separation of smooth and rough membrane proteins. All the ribosomal proteins are seen in the rough membrane protein pattern. Most of the membranal proteins are more acidic than the ribosomal proteins and therefore the technique described allows a complete separation of the ribosomal proteins from the membranal proteins, both present in the rough membrane fraction.

REFERENCES 1. Palade, G. E. and Siekevitz, P.,J. Biophys. Bioche~ Cytol. 2, 171 (1956). 2. Blobel, G. and Potter, V. R.,J. Mol. Biol. 28, 539 (1967). 3. Bloemendal, H., Bont, W. S., de Vries, M., and Benedetti, E. L., Biochem. J. 103, 177 (1967). 4. Sunshine, G. H., Williams, D. J., and Rabin, B. R., Nature NewBiol. 230, 133 (1971). 5. DaUner, G., Siekevitz, P., and Palade, G. E., J. Cell Biol. 30, 73 (1966). 6. DaUner, G., Siekevitz, P., and Palade, G. E.,J. CellBiol. 30, 97 (1966). 7. Erikson, L. C., Acta Pathologica etMicrobiologica Scandinavica, Section A 1973, supplement 239. 8. Criddle, R. S., Bock, R. M., Green, D. E., and Tisdale, H. D.,Biochemistry 1,827 (1962). 9. Ward, K. A. and Pollack, J. K., Biochem. J. 114, 41 (1969). 10. Schnaitman, C. A.,Proc. Nat. Acad. Sci. U.S.A. 63,412 (1969). 11. Burka, E. R. and Bulova, S. I.,Biochem. Biophys. Res. Commun. 42, 801 (1971). 12. Sherton, C. C. and Wool, I. G.,J. Biol. Che~ 249, 2258 (1974). 13. Hanna, N., Bellemare, G., and Godin, C., Biochem. Biophys. Acta 331,141 (1973). 14. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 254 (1951). 15. Sherton, C. C. and Wool, I. G.,J. Biol. Chem. 247, 4460 (1972). 16. Kaltschmidt, E. and Wittman, H. G., Anal. Biochem. 36, 401 (1970). 17. Avital, S, and Elson, D., Anal. Bioehem. 57,287 (1974). 18. Jones, B. L., Nagabhnshan, N., Gulyas, A., and Zalik, S., FEBS Letters 23, 167 (1972). 19. Adelman, M. R., Sabatini, D. D., and Blobel, G.,J. CellBiol. 56, 206 (1973). 20. Czosnek, H. H. and Hochberg, A. A., to be published.

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The separation of rat liver endoplasmic reticulum membrane proteins by two dimensional polyacrylamide gel electrophoresis.

The separation of rat liver endoplasmic reticulum membrane proteins by two dimensional polyacrylamide gel electrophoresis is described. By this method...
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