protein composition of plasma membranes from embryonic chick erythroid cells at various stages of maturation. Significant

Proc. NatI. Acad. Sci. USA Vol. 74, No. 3, pp. 1062-1066, March 1977 Cell Biology Changes in the composition of plasma membrane proteins during differentiation of embryonic chick erythroid cell (red blood cell/development/membrane isolation and characterization/sodium dodecyl sulfate-polyacrylamide gels) LEE-NIEN'L. CHAN Physiology Department, University of Connecticut Health Center, Farmington, Conn. 06032 Communicated by George E. Palade, November 29,1976 ABSTRACT Erythroid cells which are homogeneous with regard to sta e of maturation are naturally available from the circulation of chick embryos at various times of development. This provides a convenient system for examining the changes in plasma membrane protein composition during red cell maturation, Plasma membranes are isolated from chick embryonic erythroid cells at various stages of maturation. Extensive characterization of the isolated membranes show that they are pure and their roteins undegraded. Analyses by sodium dodecyl sulfate/polyacrylamide gel electrophoresis show that both qualitative and quantitative changes occur in membrane protein composition during the early stage of erythroid differentiation. Specific proteins of red cell membrane such as "spectrin" and band three proteins are present in low levels in early erythroblasts but increase in their relative amounts with maturation. A steady-state membrane protein composition seems to be established by the late polychromatophilic erythroblast stage. The erythrocyte membrane is a most useful system for the study of cell membrane structure and membrane-associated functions. The protein composition of erythrocyte plasma membranes has been extensively studied (see ref. 1). In general, the major erythrocyte membrane polypeptides, as resolved by sodium dodecyl sulfate (NaDodSO4/polyacrylamide gel electrophoresis), are common in all mammalian species as well as in certain avian species (1-5). Since the morphology and cellular functions of erythroid cells change dramatically during differentiation, it is of importance to know the specific developmental changes that occur in the plasma membrane. However, very little is known about the plasma membrane of immature erythroid cells (6). This is presumably due to the technical difficulties in obtaining erythroid cells at early stages of maturation in sufficient quantity and homogeneity. Erythropoiesis in the chick embryo, however, provides an exceptionally suitable system for such developmental studies. The series of primitive chick erythroid cells naturally develop as a synchronized cohort in the embryonic circulation, such that erythroid cells taken from embryos between 2 and 6 days of incubation are at each of the stages of maturation between basophilic erythroblasts and reticulocytes, respectively (7). The definitive erythroid cell series, which predominate in the circulation after 7 days of incubation, is relatively more heterogeneous because they are stem-cell derived. However, because progressively more mature cells are released into the circulation, the majority of the definitive cells are also at a similar state of maturation at any one embryonic age (7). Thus, homogeneous populations of erythroid cells at each stage of maturation between basophilic erythroblasts and mature erythrocytes can be easily obtained by harvesting erythroid cells from the circulation of embryos at the appropriate ages of development. The present paper describes a systematic examination of the Abbreviations: NaDodSO4, sodium dodecyl sulfate; Con A, concanavalin A. 1062 protein composition of plasma membranes from embryonic chick erythroid cells at various stages of maturation. Significant changes both in quality and in quantity of membrane proteins occur during embryonic erythroid differentiation. MATERIALS AND METHODS Materials. Fertilized White Leghorn eggs are obtained from the Spafas Co., Norwich, Conn. The eggs are incubated in a Humidaire Incubator (model no. 50) at 380 (dry bulb) and 290 (wet bulb). Red blood cells are collected from the circulation of the embryos after various times of incubation by cutting open the main blood vessels and allowing the blood cells to be pumped or drained out. The cells are washed extensively with cold Howard Ringer's saline before use (7). Contamination by other cell types is negligible in red cell samples collected in this manner. Furthermore, the white blood cell counts in the embryonic circulation is insignificant until hatching (8). The stages of maturation of erythroid cells from embryos at various days of incubation are as follows: basophilic erythroblast, 2.5 days; midpolychromatophilic erythroblast, 3.5 days; late polychromatophilic erythroblast, 4.5 days; reticulocyte, 6 days; proerythrocyte and erythrocyte, 8 days; erythrocyte, 18 days. 125I-Labeled concanavalin A (Con A) is the generous gift of M. Sheetz, Physiology Department, University of Connecticut Health Center, Farmington, Conn. Nonidet P-40 is obtained from Imperial Shell. Sodium tetrathionate is from Pfaltz and Bauer, and NaDodSO4 is from BDH. All other chemicals are from Sigma or Baker. Isolation of Plasma Membranes. All operations are carried out at 40. The red blood cells are suspended in a hypotonic buffer [10 mm Tris-HCl at ph 7.5, 10mM KCl, 1.5 mm MgCl2, with 5 mm Na2S406 present as a proteolytic enzyme inhibitor (4)] and then homogenized in a tight-fitting Dounce homogenizer. An appropriate volume of 2 M sucrose is added immediately to the homogenate to restore isotonicity. The homogenate is then layered over a sucrose step gradient [3 volumes of 28% sucrose (wt/vol) over 1 volume of 50% sucrose (wt/vol) in a buffer containing 5 mm Tris-HCl at ph 7.4, 2.4 mm Mg(OAc)2, 0.14 M NaCl, and 5 mm Na2S406)] and centrifuged in swinging buckets (Beckman SW 27 rotor) at 117,000 X g for 40-45 min. The membrane fraction at the 28% and 50% sucrose interphase is collected, resuspended in 10 ml 0.02 M Tris-HCl at ph 7.4 and spun at 15,000 X g for 10 min. The pellet is washed once more before it is taken up in a buffer containing 10mM Tris.HCI at ph 8,1 mm EDTA, and 2% NaDodSO4. The protein concentration is determined and then adjusted to 1 mg/ml in Fairbanks loading buffer (3) before storage at -20. Electron Microscopy. The isolated membranes are fixed chemically in 3% glutaraldehyde buffered with 0.1 M sodium phosphate at ph 7.3, postfixed with 2% osmium tetroxide

Cell Biology: Chan buffered with 0.1 M S-collidine (ph 7.3), stained intact in a 2% uranyl acetate solution at 60, dehydrated in a graded series of ethanol dilutions, and embedded in an epoxy resin. Thin sections of the membrane pellets are stained with uranyl acetate and lead citrate and examined with a Philips EM 300 electron microscope. Characterization of Membrane Preparation. For protein determinations, the method of Lowry et al. (9) is used. The RNA and DNA contents of the membrane preparations are measured by means of the orcinol and the diphenylamine techniques, respectively (10). The specific activity of cytochrome oxidase in the whole cell homogenate, all fractions of the sucrose step gradient, and the isolated membranes is assayed by the method of Cooperstein and Lazarow (11). The amount of membrane protein per ghost is determined by using '25I-Con A binding as an indirect measure of the number of membrane ghosts per preparation. Packed cells (0.1 ml) are incubated at 4 in 1 ml of Howard Ringer's saline containing a specific amount of '25I-Con A (6 X 105 cpm/ml is routinely used). After 30 min, the cells are washed extensively with ice-cold saline and aliquots are removed to determine cell number as well as the amount of 125I-Con A bound per cell. Membranes are then prepared from the remaining cells and the amount of '25I-Con A bound per,ug of membrane protein is determined. From these values, the amount of membrane protein per ghost is estimated. NaDodSO4/Polyacrylamide Gel Electrophoresis. The isolated membranes are solubilized in buffer containing 2% NaDodSO4 and are analyzed on NaDodSO4/polyacrylamide slab gels using a modified version of the system of Fairbanks et al. (3). These slab gels contain an exponential gradient of 4% acrylamide at the top and 10% acrylamide at the bottom and provide good resolution of the membrane polypeptides. The gels are stained with Coomassie brilliant blue and the stained protein patterns are scanned with a spectrodensitometer (Schoeffel Instruments Corp.) at 550 nm. The relative amounts of proteins per band are estimated by measuring the area under each peak. To check the possibility of losing certain protein components from the membrane during isolation, we analyzed proteins in the supernatant fractions from the sucrose step gradients, after concentration by vacuum dialysis, on NaDodSO4 polyacrylamide gels. Specific Elution of Membrane Protein Components. The techniques for eluting membrane protein components from isolated ghosts by means of nonionic detergents (Triton X-100 and Nonidet P-40) and low ionic strength medium are essentially those of Yu et al. (12) and Steck and Yu (13). RESULTS Purity of Isolated Chick Embryonic Erythroid Cell Membranes. The isolation technique involves the hypotonic swelling and then Dounce homogenization of the red blood cells. This procedure is highly effective in releasing the plasma membrane from the nucleus and other cytoplasmic contents. Electron microscopic examination of the membranes thus prepared shows that the plasma membranes are essentially entire membrane ghosts or very large pieces of membrane (Fig. 1). The recovery of '25I-Con A in the isolated membranes is between 40% to 60% of total cell surface bound 125I-Con A. The amount of enrichement in 125I-Con A specific activity (cpm/mg of protein) of the isolated membranes is between 30- and 50- fold that of the intact cells. X. Proc. Natl. Acad. Sci. USA 74 (1977) 1063 FIG. 1. Electron micrographs of a typical plasma membrane preparation. This particular sample was prepared from 7-day-old erythroid cells. Magnification X10,790; bar, 1 gsm. Inset magnification X166,000; bar, 100 A. Contamination by membranes from three nonplasma membrane sources are of concern; namely, nuclear membrane, endoplasmic reticulum, and mitochondrial membrane. The breakage of nuclei during the isolation procedure is minimized by the presence of magnesium in the buffers used and by the speed of the process. Also, the immediate addition of sucrose to restore isotonicity to the homogenate helps in keeping the nuclei intact. Electron micrographs of typical membrane samples show virtually no detectable contamination by nuclei, chromatin, mitochondria, or any other subcellular components (Fig. 1). Furthermore, almost negligible amounts of DNA and RNA are detected in the membrane samples: the percent of DNA to membrane proteins (wt/wt) from erythroid cells of 2.5, 3, 7, and 17 day-old embryos as well as adult erythrocytes is 4, 0, 8,3, and 3%, respectively, and the percent (wt/wt) of RNA to membrane proteins is 2.5, 5, 2, 4, and 4%, respectively. The presence of mitochondria in the whole cell homogenate, the isolated membranes, as well as all fractions of the sucrose step-gradient are monitored by assaying for a mitochondriaspecific enzyme, cytochrome oxidase. The results show that in cells of every embryonic age tested (5,7,9, 13, 15, and 18 days) less than 1% of specific cytochrome oxidase activity present in the whole cell homogenate remained in the membranes after the isolation procedure. Essentially all of the cytochrome oxidase activity is recovered in the pellet of the sucrose step-gradient, with between 1 and 2% present in the 50% sucrose fraction. Other subcellular components of lower density, if present in the interphase membrane fraction, are very likely eliminated during the moderate speed washes of the membranes. Changes intprotein Composition of Erythroid Cell Membranes during Development. The protein patterns of plasma membranes of red blood cells from chick embryos at various stages of development as analyzed blue by NaDodSO4/pory-

1064 Cell Biology: Chan Proc. Natl. Acad. Sci. USA 74 (1977) M, X-13 I8 8 6 4.5 3 5 25 -lq 250 1-_ - -.M 230 2- I 220 2 1- w %at=# - 42 100 3- # 3 --.0~ 80 4-43 -_4 2 42 5-5 -6-7 -g E 0 2 U, U,) w 0 a~~.ys: FIG. 2. NaDodSO4/polyacrylamide slab gel stained with Coomassie brilliant blue showing the protein composition of plasma membranes isolated from chick erythroid cells at various stages of development. H, human erythrocyte membrane proteins. The numbers 2.5, 3.5, 4.5, 6, 8, and 18 are membrane proteins from embryonic chick erythroid cells at those days of incubation. g, globin; Mr, molecular weight. Approximately 15 Aig of protein was loaded per track. acrylamide gel electrophoresis and stained with Coomassie brilliant blue are shown in Fig. 2. The electrophoretic pattern of proteins from human erythrocyte membranes is also shown. Fig. 3 shows the densitometer tracings of the stained proteins patterns. As previously observed, the chicken erythrocyte membrane proteins show a pattern similar to that of human erythrocyte membranes (2, 4, 5) on NaDodSO4 gels. For the sake of consistency, the chick membrane protein components are given a similar nomenclature as that commonly used for human erythrocyte membrane polypeptides (3). The approximate molecular weights of the chick membrane polypeptides are shown in Fig. 3 and Table 1. These estimates are derived from Table 1. Relative amounts of erythroid membrane polypeptides at various stages of embryonic development Age (days) Coin- Mr X ponents 103 2.5 3.5 4.5 6 8 18 Adult 1 250 2 5 8 9 10 10 15 2 230 1 3 6 8 10 9 12 2.1 220 1 2 3 4 6 6 6 120 15 6 7 4 0 0 0 105 8 4 4 4 3 3 4 3 100 2 7 15 16 17 16 14 3.1 92 2 8 21 26 29 27 27 4 80 15 8 8 7 5 5 4 45 15 8 1 0 0 0 0 5 42 3 10 11 8 6 8 4 29 21 15 2 1 0 0 0 Globin 16 1 1 < 1 0 0 0 0 Data are the average of three determinations expressed as percent of total stained protein (wt/wt). Mr, molecular weight. CO) 0 2 4 6 8 10 1 2 14 o) MIGRATION (CM) 0 FIG. 3. Densitometric scans of a slab gel stained with Coomassie brilliant blue showing the membrane protein components of erythroid cells at various stages of development. Approximately 15,ug of protein was loaded per track. Mr, molecular weight. molecular weight standards: myosin, 220,000; (3-galactosidase, 130,000; phosphorylase a, 100,000; bovine serum albumin, 68,000; actin, 43,000; globin, 16,000. Table 1 also shows the relative amounts of each component expressed as percent of total membrane protein (wt/wt). Several points of interest can be noted. The most obvious differences, both in types of protein components present as well as in the relative amounts of each species, are apparent in membranes of the very early cells, those from 2.5-4.5 days. The membrane protein patterns seem to stabilize by 6-8 days, and the pattern of late embryonic red cell membranes, aside from some minor quantitative differences, are essentially the same as that of adult erythrocytes. There are three components of 120,000, 45,000, and 29,000 molecular weight that are present as major components in the most immature cells, but gradually decrease in relative amounts until they are no longer present by 6-8 days. The possibility that these components may be precursors for other membrane

Cell Biology: Chan proteins is unlikely because data from pulse-chase studies indicate that the specific activities of these components d-not decrease significantly during the chase period (L-N. L. Chan, unpublished results). Note the polypeptide which is of molecular weight slightly larger than component 3 and is present in relatively high amounts at 2.5 days, but decreases rapidly and is seen only as a small shoulder of constant relative amount by 4.5 days and thereafter. These observations suggest that component 3 may be composed of more than one protein species, some of which are present at high amounts in immature cells but gradually decrease with age and others which develop in the opposite direction. Thus, in the chick component 3 seems to be composed of at least two distinct protein species. Conversely, several other components increase in relative amounts with developmental age. Components 1 and 2.1 increase by about 5- to 6-fold between 2.5 days and 18 days. More dramatically, component 2 and 3 increase 8- to 10-fold, whereas component 3.1 increases by about 14-fold in relative amounts during this time. In the more mature embryonic cells, the two most prominent membrane protein species are components 3 and 3.1, and there are equal but lesser amounts of 1 and 2. In the adult, component 3.1 is the single most abundant specie and there are about equal amounts of components 1, 2, and 3. The relative amounts of components 1 and 2 change with respect to each other; at 2.5 days there is about two times the amount of component 1 to component 2, but this ratio changes until there is almost equal amounts of the two by 6 days. The relative amounts of components 3 and 3.1 also change with time; there are about the same amounts of the two components at 2.5 days, but after 4.5 days, there is about 1.5 times more component 3.1 than 3. The amount of contamination by hemoglobin is extremely low, ranging from 1% of total membrane protein to undetectably low levels. This further attests to the purity of the membrane preparations. The amount of total protein present per membrane ghost of erythroid cells at different stages of development is shown in Table 2. The decrease in the amount of total protein per membrane ghost is accompanied by a decrease in cell surface area which is estimated from known cell size and volume measurements (8). From the values above, the "density" of membrane proteins (amount of protein per unit membrane area) can be estimated. The estimated densities increase slightly during the course of development (Table 2, last column). To estimate the numbers of molecules of each membrane protein component per cell, it is necessary to take into account the decrease in amount of total membrane protein per cell with developmental age. The numbers of molecules of each component per ghost at different stages of development has been estimated. From these data it can be approximated that, on a per ghost basis, there are about one third as many molecules of components 1, 2, and 2.1 at 2.5 days as there are at 4.5 days and older. Similarily, the number of molecules of components 3 and 3.1 per cell at 2.5 days is about 17-20% of the number at later times. The possibility that certain proteins, such as components 1 and 2, are more easily lost from the membranes of younger cells than older cells during the isolation procedure, and therefore are seen to be at relatively lower levels in the immature cells, is examined by NaDodSO4/polyacrylamide gel electrophoresis analyses of supernatant fractions of the sucrose-step gradients. The results (not shown) indicate that membrane proteins are not lost into the soluble fraction during the isolation procedure for membranes from cells at any stage of maturation. Results from experiments with Triton X-100 and Nonidet Proc. Natl. Acad. Sci. USA 74 (1977) 1065 Table 2. Cell volume, cell surface area, and amount of protein per erythroid membrane ghost at various stages of development Amount of Cell Amount of protein per Cell surface protein per unit surface Age volume* areat ghostt (mg area (mg X (days) (gim3) (Am2) x 10-10) 10-'0/Am2) 2.5 700 518 15.5 30 4.5 465 312 10.0 32 8 200 222 6.9 31 13 150 179 6.1 34 18 150 179 6.0 34 * Values from ref. 8. t Estimated by assuming cell shape to be flat discs. I Average of at least three determinations. P-40 indicate that components 3 and 3.1 are preferentially extracted by these nonionic detergents. At all of the detergent concentrations tested (0.5%, 1%, 5%, and 10% Triton X-100, 5% Nonidet P-40) about 50% of the two components are eluted after 30 min of incubation. Successive elutions do not result in complete removal of these components from the pellet. Treatment of isolated membranes with low ionic strength medium at ph 12 results in the preferential elution of components 1, 2, and 2.1, as well as components 4 and 5. Successive extractions continue to elute components 1, 2, and 2.1, but at decreasing concentrations. The amount of components 1, 2, and 2.1 that remain in the pellet after the final extraction is about 70% of control. DISCUSSION The purity of the plasma membrane preparation is of crucial importance in a comparative study such as the present report. Thus, a great deal of effort was placed on minimizing the levels of contamination from nonplasma membrane sources. From all of the different methods of analyses used, including electron microscopic examination and biochemical assays, there appears to be negligible amounts of nuclear, endoplasmic reticular, and mitochondrial contamination of our plasma membrane preparations. Furthermore, from the NaDodSO4/polyacrylamide gel profiles, those polypeptides which are specific to nuclear membranes (2) are virtually absent in the plasma membranes isolated by our method. Proteolytic degradation of the membrane proteins is also of concern. It is evident, however, that the presence of 5 mm sodium tetrathionate in the buffers used in membrane isolation is very effective against protease activity. The degradation products (molecular weight 175,000, 150,000, and 140,000) typically seen in chick plasma membranes prepared without protease inhibitor protection (2, 4, 5) are not seen in any of our preparations. Moreover, small degradation products (molecular weight <16,000) are also absent from our membrane samples (Fig. 2). Thus, we are fairly confident that the differences seen in the NaDodSO4 gel profiles reflect actual changes in membrane protein composition. In this report, the total protein compositions, as analyzed by NaDodSO4/polyacrylamide gel electrophoresis, of plasma membranes of a complete maturation series of erythroid cells are shown. The most striking changes take place during the early stages of maturation not only in types of membrane proteins but also in their relative amounts. Of special interest are components 3, 3.1, and their closely associated neighbors since

1066 Cell Biology: Chan these components are in many respects very similar to their counterpart(s) (band 3) in human erythrocytes (4, 14) and are very likely involved in certain membrane-associated functions (15-17). By using the chick system, it is now possible to correlate in a systematic fashion the changes in membrane protein composition with differences in membrane-associated functions (18). The rapid increases and decreases in the relative amounts of the protein components during the early phases of erythroid maturation indicate that the rates of synthesis and degradation of these components must also change during development. After 6 days of incubation the protein profiles have stabilized and, therefore, the rates of turnover of each component most likely have reached steady state. Because these immature erythroid cells of the chick embryo are actively synthesizing plasma membrane proteins, they constitute a most useful system for studies of plasma membrane biosynthesis (19). I thank Dr. Michael J. Weise for stimulating and helpful discussions and Dr. Peter Cooke for preparing the electron micrographs. The excellent technical assistance of Ms. Barbara Hyer and Ms. Katherine Mahoney is gratefully acknowledged. This work was supported by a Grant-in-Aid from the American Heart Association, a Basil O'Connor Starter Grant from the National Foundation-March of Dimes and National Institutes of Health Grant HL 19068. 1. Steck, T. L. (1974) J. Cell Biol. 62, 1-19. 2. Blanchet, J. B. (1974) Exp. Cell Res. 84, 159-166. Proc. Natl. Acad. Sci. USA 74 (1977) 3. Fairbanks, G., Steck, T. L. & Wallach, D. F. (1971) Biochemistry 10,2606-2617. 4. Jackson, R. C. (1975) J. Biol. Chem. 250,617-622. 5. Weise, M. J. (1975) Ph.D. Dissertation, Massachusetts Institute of Technology. 6. Koch, P. A., Gardner, F. H. & Carter, J. R., Jr. (1973) Biochem. Biophys. Res. Commun. 54,1296-1299. 7. Bruns, G. A. & Ingram, V. M. (1973) Philos. Trans. R. Soc. London Ser. B 226,225-305. 8. Romanoff, A. L. (1969) The Avian Embry (MacMillan Co., New York), pp. 571-605. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951)J. Biol. Chem. 193,265-275. 10. Shatkin, A. J. (1969) Fundamental Techniques in Virology, eds. Habel, K. & Salzman, N. P. (Academic Press, New York), pp. 231-237. 11. Cooperstein, S. J. & Lazarow, A. (1951) J. Biol. Chem. 189, 665-670. 12. Yu, J., Fischman, D. A. & Steck, T. L. (1973) J. Supramol. Struct. 1,233-248. 13. Steck, T. L. & Yu, J. (1973) J. Supramol. Struct. 1, 220-232. 14. Weise, M. J. & Ingram, V. M. (1976) J. Biol. Chem. 251, 6667-6673. 15. Brown, P. A., Feinstein, M. B. & Sha'afi, R. I. (1975) Nature 254, 523-524. 16. Capantchik, F. I. & Rothstein, A. (1974) J. Membr. Biol. 15, 207-226. 17. Knauf, P. A., Proverbio, F. & Hoffman, J. F. (1974) J. Gen. Physiol. 63, 305-323. 18. Chan, L.-N., Wacholtz, M. & Sha'afi, R. I. (1977) Memb. Biochem., in press. 19. Weise, M. J. & Chan, L.-N. (1976) Fed. Proc. 35,475.