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 protein composition of plasma membranes from embryonic regard to sta e of maturation are naturally available from the chick erythroid cells at various stages of maturation. Significant circulation of chick embryos at various times of development. This provides a convenient system for examining the changes changes both in quality and in quantity of membrane proteins in plasma membrane protein composition during red cell mat- occur during embryonic erythroid differentiation. uration, Plasma membranes are isolated from chick embryonic erythroid cells at various stages of maturation. Extensive char- MATERIALS AND METHODS acterization of the isolated membranes show that they are pure Materials. Fertilized White Leghorn eggs are obtained from and their roteins undegraded. Analyses by sodium dodecyl sulfate/polyacrylamide gel electrophoresis show that both the Spafas Co., Norwich, Conn. The eggs are incubated in a qualitative and quantitative changes occur in membrane protein Humidaire Incubator (model no. 50) at 380 (dry bulb) and 290 composition during the early stage of erythroid differentiation. (wet bulb). Red blood cells are collected from the circulation Specific proteins of red cell membrane such as "spectrin" and of the embryos after various times of incubation by cutting open band three proteins are present in low levels in early erythro- the main blood vessels and allowing the blood cells to be blasts but increase in their relative amounts with maturation. A pumped or drained out. The cells are washed extensively with steady-state membrane protein composition seems to be es- cold Howard Ringer's saline before use (7). Contamination by tablished by the late polychromatophilic erythroblast stage. other cell types is negligible in red cell samples collected in this The erythrocyte membrane is a most useful system for the study manner. Furthermore, the white blood cell counts in the em- of cell membrane structure and membrane-associated func- bryonic circulation is insignificant until hatching (8). The stages tions. The protein composition of erythrocyte plasma mem- of maturation of erythroid cells from embryos at various days branes has been extensively studied (see ref. 1). In general, the of incubation are as follows: basophilic erythroblast, 2.5 days; major erythrocyte membrane polypeptides, as resolved by so- midpolychromatophilic erythroblast, 3.5 days; late poly- dium dodecyl sulfate (NaDodSO4/polyacrylamide gel elec- chromatophilic erythroblast, 4.5 days; reticulocyte, 6 days; trophoresis), are common in all mammalian species as well as proerythrocyte and erythrocyte, 8 days; erythrocyte, 18 in certain avian species (1-5). days. Since the morphology and cellular functions of erythroid cells 125I-Labeled concanavalin A (Con A) is the generous gift of change dramatically during differentiation, it is of importance M. Sheetz, Physiology Department, University of Connecticut to know the specific developmental changes that occur in the Health Center, Farmington, Conn. Nonidet P-40 is obtained plasma membrane. However, very little is known about the from Imperial Shell. Sodium tetrathionate is from Pfaltz and plasma membrane of immature erythroid cells (6). This is Bauer, and NaDodSO4 is from BDH. All other chemicals are presumably due to the technical difficulties in obtaining from Sigma or Baker. erythroid cells at early stages of maturation in sufficient Isolation of Plasma Membranes. All operations are carried quantity and homogeneity. Erythropoiesis in the chick embryo, out at 40. The red blood cells are suspended in a hypotonic however, provides an exceptionally suitable system for such buffer [10mM Tris-HCl at pH 7.5, 10mM KCl, 1.5mM MgCl2, developmental studies. The series of primitive chick erythroid with 5 mM Na2S406 present as a proteolytic enzyme inhibitor cells naturally develop as a synchronized cohort in the em- (4)] and then homogenized in a tight-fitting Dounce homoge- bryonic circulation, such that erythroid cells taken from em- nizer. An appropriate volume of 2 M sucrose is added imme- bryos between 2 and 6 days of incubation are at each of the diately to the homogenate to restore isotonicity. The homoge- stages of maturation between basophilic erythroblasts and re- nate is then layered over a sucrose step gradient [3 volumes of ticulocytes, respectively (7). The definitive erythroid cell series, 28% sucrose (wt/vol) over 1 volume of 50% sucrose (wt/vol) in which predominate in the circulation after 7 days of incubation, a buffer containing 5 mM Tris-HCl at pH 7.4, 2.4 mM is relatively more heterogeneous because they are stem-cell Mg(OAc)2, 0.14 M NaCl, and 5 mM Na2S406)] and centrifuged derived. However, because progressively more mature cells are in swinging buckets (Beckman SW 27 rotor) at 117,000 X g for released into the circulation, the majority of the definitive cells 40-45 min. The membrane fraction at the 28% and 50% sucrose are also at a similar state of maturation at any one embryonic interphase is collected, resuspended in 10 ml 0.02 M Tris-HCl age (7). Thus, homogeneous populations of erythroid cells at at pH 7.4 and spun at 15,000 X g for 10 min. The pellet is each stage of maturation between basophilic erythroblasts and washed once more before it is taken up in a buffer containing mature erythrocytes can be easily obtained by harvesting 10mM Tris.HCI at pH 8,1 mM EDTA, and 2% NaDodSO4. The erythroid cells from the circulation of embryos at the appro- protein concentration is determined and then adjusted to 1 priate ages of development. mg/ml in Fairbanks loading buffer (3) before storage at The present paper describes a systematic examination of the -20°. Electron Microscopy. The isolated membranes are fixed Abbreviations: NaDodSO4, sodium dodecyl sulfate; Con A, concana- chemically in 3% glutaraldehyde buffered with 0.1 M sodium valin A. phosphate at pH 7.3, postfixed with 2% osmium tetroxide 1062 Downloaded by guest on September 27, 2021 Cell Biology: Chan Proc. Natl. Acad. Sci. USA 74 (1977) 1063 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. X. 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 FIG. 1. Electron micrographs of a typical plasma membrane isolated membranes are solubilized in buffer containing 2% preparation. This particular sample was prepared from 7-day-old NaDodSO4 and are analyzed on NaDodSO4/polyacrylamide erythroid cells. Magnification X10,790; bar, 1 gsm. Inset magnification slab gels using a modified version of the system of Fairbanks X166,000; bar, 100 A. et al. (3). These slab gels contain an exponential gradient of 4% acrylamide at the top and 10% acrylamide at the bottom and Contamination by membranes from three nonplasma provide good resolution of the membrane polypeptides. The membrane sources are of concern; namely, nuclear membrane, gels are stained with Coomassie brilliant blue and the stained endoplasmic reticulum, and mitochondrial membrane. The protein patterns are scanned with a spectrodensitometer breakage of nuclei during the isolation procedure is minimized (Schoeffel Instruments Corp.) at 550 nm. The relative amounts by the presence of magnesium in the buffers used and by the of proteins per band are estimated by measuring the area under speed of the process. Also, the immediate addition of sucrose each peak. to restore isotonicity to the homogenate helps in keeping the To check the possibility of losing certain protein components nuclei intact. Electron micrographs of typical membrane from the membrane during isolation, we analyzed proteins in samples show virtually no detectable contamination by nuclei, the supernatant fractions from the sucrose step gradients, after chromatin, mitochondria, or any other subcellular components concentration by vacuum dialysis, on NaDodSO4 polyacryl- (Fig. 1). Furthermore, almost negligible amounts of DNA and amide gels. RNA are detected in the membrane samples: the percent of Specific Elution of Membrane Protein Components. The DNA to membrane proteins (wt/wt) from erythroid cells of 2.5, techniques for eluting membrane protein components from 3,7, and 17 day-old embryos as well as adult erythrocytes is 4, isolated ghosts by means of nonionic detergents (Triton X-100 0, 8,3, and 3%, respectively, and the percent (wt/wt) of RNA and Nonidet P-40) and low ionic strength medium are essen- to membrane proteins is 2.5, 5, 2, 4, and 4%, respectively. tially those of Yu et al. (12) and Steck and Yu (13). The presence of mitochondria in the whole cell homogenate, the isolated membranes, as well as all fractions of the sucrose RESULTS step-gradient are monitored by assaying for a mitochondria- Purity of Isolated Chick Embryonic Erythroid Cell specific enzyme, cytochrome oxidase. The results show that in Membranes. The isolation technique involves the hypotonic cells of every embryonic age tested (5,7,9, 13, 15, and 18 days) swelling and then Dounce homogenization of the red blood less than 1% of specific cytochrome oxidase activity present in cells. This procedure is highly effective in releasing the plasma the whole cell homogenate remained in the membranes after membrane from the nucleus and other cytoplasmic contents. the isolation procedure. Essentially all of the cytochrome oxi- Electron microscopic examination of the membranes thus dase activity is recovered in the pellet of the sucrose step-gra- prepared shows that the plasma membranes are essentially dient, with between 1 and 2% present in the 50% sucrose frac- entire membrane ghosts or very large pieces of membrane (Fig. tion. Other subcellular components of lower density, if present 1). in the interphase membrane fraction, are very likely eliminated The recovery of '25I-Con A in the isolated membranes is during the moderate speed washes of the membranes. between 40% to 60% of total cell surface bound 125I-Con A. The Changes intProtein Composition of Erythroid Cell Mem- amount of enrichement in 125I-Con A specific activity (cpm/mg branes during Development. The protein patterns of plasma of protein) of the isolated membranes is between 30- and 50- membranes of red blood cells from chick embryos at various fold that of the intact cells. stages of development as analyzed blue by NaDodSO4/pory- Downloaded by guest on September 27, 2021 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 42 %at=#

100 3- # 3 --.0~ 80 4-__ 43 -_4 2

42 5 -5 -6 -7

E a~~.ys: 0 2 -g U, U,) w 0

FIG. 2. NaDodSO4/polyacrylamide slab gel stained with Coom- CO) assie 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 num- bers 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, mo- lecular 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 con- sistency, the chick membrane protein components are given a similar nomenclature as that commonly used for human 0 2 4 6 8 10 12 14 erythrocyte membrane polypeptides (3). The approximate o) molecular weights of the chick membrane polypeptides are MIGRATION (CM) 0 FIG. 3. Densitometric scans of a slab gel stained with Coomassie shown in Fig. 3 and Table 1. These estimates are derived from brilliant blue showing the membrane protein components of erythroid cells at various stages of development. Approximately 15,ug of protein Table 1. Relative amounts of erythroid membrane was loaded per track. Mr, molecular weight. polypeptides at various stages of embryonic development molecular weight standards: myosin, 220,000; (3-galactosidase, Age (days) 130,000; phosphorylase a, 100,000; bovine serum albumin, Coin- Mr X 68,000; actin, 43,000; globin, 16,000. Table 1 also shows the ponents 103 2.5 3.5 4.5 6 8 18 Adult relative amounts of each component expressed as percent of 1 250 2 5 8 9 10 10 15 total membrane protein (wt/wt). 2 230 1 3 6 8 10 9 12 Several points of interest can be noted. The most obvious 2.1 220 1 2 3 4 6 6 6 differences, both in types of protein components present as well 120 15 6 7 4 0 0 0 as in the relative amounts of each species, are apparent in 105 8 4 4 4 3 3 4 membranes of the very early cells, those from 2.5- 4.5 days. The 3 100 2 7 15 16 17 16 14 membrane protein patterns seem to stabilize by 6-8 days, and 3.1 92 2 8 21 26 29 27 27 the pattern of late embryonic red cell membranes, aside from 4 80 15 8 8 7 5 5 4 some minor quantitative differences, are essentially the same 45 15 8 1 0 0 0 0 as that of adult erythrocytes. 5 42 3 10 11 8 6 8 4 There are three components of 120,000, 45,000, and 29,000 29 21 15 2 1 0 0 0 molecular weight that are present as major components in the Globin 16 1 1 < 1 0 0 0 0 most immature cells, but gradually decrease in relative amounts Data are the average of three determinations expressed as percent until they are no longer present by 6-8 days. The possibility that of total stained protein (wt/wt). Mr, molecular weight. these components may be precursors for other membrane Downloaded by guest on September 27, 2021 Cell Biology: Chan Proc. Natl. Acad. Sci. USA 74 (1977) 1065

proteins is unlikely because data from pulse-chase studies in- Table 2. Cell volume, cell surface area, and amount of dicate that the specific activities of these components d-not protein per erythroid membrane ghost at various stages decrease significantly during the chase period (L-N. L. Chan, of development unpublished results). Note the polypeptide which is of molec- Amount of ular weight slightly larger than component 3 and is present in relatively high amounts at 2.5 days, but decreases rapidly and Cell Amount of protein per Cell surface protein per unit surface is seen as a constant amount only small shoulder of relative by Age volume* areat ghostt (mg area (mg X 4.5 days and thereafter. These observations suggest that com- (days) (giM3) (Am2) x 10-10) 10-'0/Am2) ponent 3 may be composed of more than one protein species, some of which are present at high amounts in immature cells 2.5 700 518 15.5 30 but gradually decrease with age and others which develop in 4.5 465 312 10.0 32 the opposite direction. Thus, in the chick component 3 seems 8 200 222 6.9 31 to be composed of at least two distinct protein species. 13 150 179 6.1 34 Conversely, several other components increase in relative 18 150 179 6.0 34 amounts with developmental age. Components 1 and 2.1 in- crease by about 5- to 6-fold between 2.5 days and 18 days. More * Values from ref. 8. t Estimated by assuming cell shape to be flat discs. dramatically, component 2 and 3 increase 8- to 10-fold, whereas I component 3.1 increases by about 14-fold in relative amounts Average of at least three determinations. during this time. In the more mature embryonic cells, the two most prominent membrane protein species are components 3 P-40 indicate that components 3 and 3.1 are preferentially nonionic At and 3.1, and there are equal but lesser amounts of 1 and 2. In extracted by these detergents. all of the detergent the adult, component 3.1 is the single most abundant specie and concentrations tested (0.5%, 1%, 5%, and 10% Triton X-100, 5% there are about equal amounts of components 1, 2, and 3. Nonidet P-40) about 50% of the two components are eluted not in The relative amounts of components 1 and 2 change with after 30 min of incubation. Successive elutions do result respect to each other; at 2.5 days there is about two times the complete removal of these components from the pellet. amount of component 1 to component 2, but this ratio changes Treatment of isolated membranes with low ionic strength until there is almost equal amounts of the two by 6 days. The medium at pH 12 results in the preferential elution of compo- 4 relative amounts of components 3 and 3.1 also change with nents 1, 2, and 2.1, as well as components and 5. Successive time; there are about the same amounts of the two components extractions continue to elute components 1, 2, and 2.1, but at at 2.5 days, but after 4.5 days, there is about 1.5 times more decreasing concentrations. The amount of components 1, 2, and component 3.1 than 3. 2.1 that remain in the pellet after the final extraction is about The amount of contamination by hemoglobin is extremely 70% of control. low, ranging from 1% of total membrane protein to undetec- tably low levels. This further attests to the purity of the mem- DISCUSSION brane preparations. The purity of the plasma membrane preparation is of crucial The amount of total protein present per membrane ghost of importance in a comparative study such as the present report. erythroid cells at different stages of development is shown in Thus, a great deal of effort was placed on minimizing the levels Table 2. The decrease in the amount of total protein per of contamination from nonplasma membrane sources. From membrane ghost is accompanied by a decrease in cell surface all of the different methods of analyses used, including electron area which is estimated from known cell size and volume microscopic examination and biochemical assays, there appears measurements (8). From the values above, the "density" of to be negligible amounts of nuclear, endoplasmic reticular, and membrane proteins (amount of protein per unit membrane mitochondrial contamination of our plasma membrane prep- area) can be estimated. The estimated densities increase slightly arations. Furthermore, from the NaDodSO4/polyacrylamide during the course of development (Table 2, last column). gel profiles, those polypeptides which are specific to nuclear To estimate the numbers of molecules of each membrane membranes (2) are virtually absent in the plasma membranes protein component per cell, it is necessary to take into account isolated by our method. the decrease in amount of total membrane protein per cell with Proteolytic degradation of the membrane proteins is also of developmental age. The numbers of molecules of each com- concern. It is evident, however, that the presence of 5 mM so- ponent per ghost at different stages of development has been dium tetrathionate in the buffers used in membrane isolation estimated. From these data it can be approximated that, on a is very effective against protease activity. The degradation per ghost basis, there are about one third as many molecules of products (molecular weight 175,000, 150,000, and 140,000) components 1, 2, and 2.1 at 2.5 days as there are at 4.5 days and typically seen in chick plasma membranes prepared without older. Similarily, the number of molecules of components 3 and protease inhibitor protection (2, 4, 5) are not seen in any of our 3.1 per cell at 2.5 days is about 17-20% of the number at later preparations. Moreover, small degradation products (molecular times. weight <16,000) are also absent from our membrane samples The possibility that certain proteins, such as components 1 (Fig. 2). Thus, we are fairly confident that the differences seen and 2, are more easily lost from the membranes of younger cells in the NaDodSO4 gel profiles reflect actual changes in mem- than older cells during the isolation procedure, and therefore brane protein composition. are seen to be at relatively lower levels in the immature cells, In this report, the total protein compositions, as analyzed by is examined by NaDodSO4/polyacrylamide gel electrophoresis NaDodSO4/polyacrylamide gel electrophoresis, of plasma analyses of supernatant fractions of the sucrose-step gradients. membranes of a complete maturation series of erythroid cells The results (not shown) indicate that membrane proteins are are shown. The most striking changes take place during the not lost into the soluble fraction during the isolation procedure early stages of maturation not only in types of membrane pro- for membranes from cells at any stage of maturation. teins but also in their relative amounts. Of special interest are Results from experiments with Triton X-100 and Nonidet components 3, 3.1, and their closely associated neighbors since Downloaded by guest on September 27, 2021 1066 Cell Biology: Chan Proc. Natl. Acad. Sci. USA 74 (1977) these components are in many respects very similar to their 3. Fairbanks, G., Steck, T. L. & Wallach, D. F. (1971) Biochemistry counterpart(s) (band 3) in human erythrocytes (4, 14) and are 10,2606-2617. very likely involved in certain membrane-associated functions 4. Jackson, R. C. (1975) J. Biol. Chem. 250,617-622. (15-17). By using the chick system, it is now possible to correlate 5. Weise, M. J. (1975) Ph.D. Dissertation, Massachusetts Institute the changes in membrane protein of Technology. in a systematic fashion 6. Koch, P. A., Gardner, F. H. & Carter, J. R., Jr. (1973) Biochem. composition with differences in membrane-associated functions Biophys. Res. Commun. 54,1296-1299. (18). 7. Bruns, G. A. & Ingram, V. M. (1973) Philos. Trans. R. Soc. The rapid increases and decreases in the relative amounts of London Ser. B 226,225-305. the protein components during the early phases of erythroid 8. Romanoff, A. L. (1969) The Avian Embry (MacMillan Co., New maturation indicate that the rates of synthesis and degradation York), pp. 571-605. of these components must also change during development. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. After 6 days of incubation the protein profiles have stabilized (1951)J. Biol. Chem. 193,265-275. and, therefore, the rates of turnover of each component most 10. Shatkin, A. J. (1969) Fundamental Techniques in Virology, eds. likely have reached steady state. Because these immature Habel, K. & Salzman, N. P. (Academic Press, New York), pp. 231-237. erythroid cells of the chick embryo are actively synthesizing 11. Cooperstein, S. J. & Lazarow, A. (1951) J. Biol. Chem. 189, plasma membrane proteins, they constitute a most useful system 665-670. for studies of plasma membrane biosynthesis (19). 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. I thank Dr. Michael J. Weise for stimulating and helpful discussions 14. Weise, M. J. & Ingram, V. M. (1976) J. Biol. Chem. 251, and Dr. Peter Cooke for preparing the electron micrographs. The ex- 6667-6673. cellent technical assistance of Ms. Barbara Hyer and Ms. Katherine 15. Brown, P. A., Feinstein, M. B. & Sha'afi, R. I. (1975) Nature 254, Mahoney is gratefully acknowledged. This work was supported by a 523-524. Grant-in-Aid from the American Heart Association, a Basil O'Connor 16. Capantchik, F. I. & Rothstein, A. (1974) J. Membr. Biol. 15, Starter Grant from the National Foundation-March of Dimes and 207-226. National Institutes of Health Grant HL 19068. 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. Bio- 1. Steck, T. L. (1974) J. Cell Biol. 62, 1-19. chem., in press. 2. Blanchet, J. B. (1974) Exp. Cell Res. 84, 159-166. 19. Weise, M. J. & Chan, L.-N. (1976) Fed. Proc. 35,475. Downloaded by guest on September 27, 2021