Developmentally regulated proteins of the plasma membrane of Dictyostelium discoideum. The carbohydrate-binding protein

J. Mol. Biol. (1976) 100, 157-178

Developmentally Regulated Proteins of the Plasma Membrane of Dictyostelium discoideum. The Carbohydrate-binding Protein CHI-I-IuI~IGSIu, RIC~rARDA. LERNER

Department of Immunopathology Scripps Clinic and Research Foundation La Jolla, Calif. 92037, U.S.A. GLORIA M_A, RICHARD A. ~-tIRTEL AND WILLIAM F. LOOMIS

Department of Biology University of California San Diego La Jolla, Calif. 92037, U.S.A. (Received 6 August 1975) Proteins of the p l a s m a m e m b r a n e of Dictyostelium discoideum were characterized. A carbohydrate-binding protein was found to be associated with the external surface of the p l a s m a membrane. The appearance of the CBP~f on the cell surface is a developmentally regulated event. I t was detected in the soluble protein fraction at four hours and on the membrane a t five hours after the initiation of development. Studies with inhibitors showed t h a t both R N A and protein synthesis were required for the expression of CBP. Synthesis of messenger R N A coding for the CBP was initiated between one and two hours of developm0nt, and synthesis of the protein was detected a t three to four hours. Q u a n t i t a t i v e studies showed t h a t CBP was not detectable at zero a n d two hours of development a n d it represented only 0"1 ~/o of the soluble cellular proteins a t four hours. Between four a n d ten hours, a r a p i d accumulation of the CBP occurred until it represented a b o u t 1% of the soluble proteins. The concentration of CBP in the cell began to level off a t ten hours of development and remained constant for at least the n e x t six hours. The time of expression of CBP corresponds closely to the period when cells begin to acquire intercellular cohesion. Three non-cohesive mutants, WL3, W L 4 and WLS, were examined for the presence of CBP. W L 3 and W L 4 did not have a n y detectable a m o u n t of CBP on the cell surface or in the cytoplasm, while CBP was present in reduced amounts on the surface of W L 5 cells. These d a t a suggest t h a t CBP m a y have a role in cellular cohesion.

1. Introduction P l a s m a m e m b r a n e c o m p o n e n t s m a y p l a y a n essential role in t h e i n t e r a c t i o n o f cells d u r i n g e a r l y e m b r y o g e n e s i s a n d d i f f e r e n t i a t i o n ( G a r b e r & Moscona, 1972; H e n k a r t et al., 1973). I n t h e cellular slime m o l d Dictyostelium discoideum, t h e free-living amoebae respond to the depletion of nutrients by aggregating and increasing their m u t u a l cohesiveness. This results in t h e f o r m a t i o n o f a n a g g r e g a t e w h i c h u n d e r g o e s f u r t h e r d e v e l o p m e n t as a m u l t i c e l l u l a r unit. A v a r i e t y o f b i o c h e m i c a l d i f f e r e n t i a t i o n s such as t h e a c c u m u l a t i o n o f d e v e l o p m e n t a l e n z y m e s occur a t specific stages, ultim a t e l y r e s u l t i n g in t h e f o r m a t i o n o f e i t h e r s t a l k cells or spores (for a r e v i e w see Abbreviations used: CBP, carbohydrate-binding protein; IgG, immunoglobulin G; IgM, immunoglobulin M. 157

158

C.-H. SIU E T A L .

Loomis, 1975). H o w e v e r , if t h e cells a r e d i s p e r s e d f r o m p s e u d o p l a s m o d i a a n d k e p t from reassociating, t h e a c c u m u l a t i o n o f c e r t a i n late-stage-specific e n z y m e s ceases (Newell et al., 1971). Thus, i t a p p e a r s t h a t i n t i m a t e cell c o n t a c t is r e q u i r e d for d e v e l o p m e n t in this s y s t e m . A t t h e t i m e t h a t cohesiveness increases a m o n g cells o f D. discoideum, a b o u t six hours a f t e r t h e i n i t i a t i o n o f d e v e l o p m e n t , n e w a n t i g e n i c d e t e r m i n a n t s a p p e a r on "Ghe cell surface (Sonneborn et al., 1964; Beug et al., 1970,1973). Changes in s e v e r a l p l a s m a m e m b r a n e p r o t e i n s h a v e also b e e n f o u n d to occur a t t h i s s t a g e ( S m a r t & H y n e s , 1974; Siu et al., 1975). R o s e n et al. (1973) h a v e i s o l a t e d from solubilized a m o e b a e a specific p r o t e i n b y v i r t u e of its affinity t o galactose. This has lectin-like p r o p e r t i e s a n d c a n a g g l u t i n a t e sheep e r y t h i ' o c y t e s . B y using a n t i s e r a p r e p a r e d a g a i n s t a purified p r e p a r a t i o n o f this C B P t we h a v e b e e n a b l e to show t h a t a f r a c t i o n o f this p r o t e i n is l o c a t e d on t h e e x t e r n a l surface o f t h e p l a s m a m e m b r a n e a n d t h a t its a p p e a r a n c e a t t h i s site is a d e v e l o p m e n t a l l y r e g u l a t e d e v e n t w h i c h requires R N A a n d p r o t e i n s y n t h e s i s a f t e r t h e i n i t i a t i o n o f d e v e l o p m e n t . T h e close c o r r e l a t i o n of t h e a p p e a r a n c e o f this p r o t e i n a n d a l t e r e d cohesiveness t o g e t h e r w i t h t h e finding t h a t C B P is a b s e n t in several non-cohesive m u t a n t s suggest t h a t it m a y p l a y a role in t h e d e v e l o p m e n t o f cohesiveness in t h i s s y s t e m . 2. M a t e r i a l s a n d M e t h o d s (a) Cell strain and growth conditions D. discoideum strain A3 was used in all experiments except where noted. Amoebae were grown on S1K agar (Sussman, 1966) in association with Klebsiella aerogenes. Cells were grown until the bacterial lawn was p a r t i a l l y cleared giving a yield of a b o u t 1 • l0 s cells per 9-em diam. plate, and then harvested in cold distilled water. Bacteria were removed b y repeated differential eentrifugation. Cells were deposited for development on W h a t m a n no. 50 filters s u p p o r t e d on absorbent pads s a t u r a t e d with a buffered salt solution containing KC1 (1-5 g/l), MgC12 (0-5 g/l), s t r e p t o m y c i n sulfate (0.5 g/l), a n d 0-01 M-potassium phosphate buffer (pH 6.5) (Sussman, 1966). Cells were also groom axenieally in HL-5 medium (Coeucei & Sussman, 1970) containing per l: 10 g proteose peptone, 10 g glucose, 5 g yeast extract, 0.25 g Na2HPO4, and 0-4 g KH2PO4 (adjusted to p H 6.5). (b) Isolation of carbohydrate-binding protein CBP was isolated from cells with a procedure modified from t h a t of Simpson et al. (1974). Cells of either strain A3 or strain G4 (a m u t a n t of A3 lacking fl-glucosidase) (Diam o n d & Loomis, 1975) were grown in axenic culture a n d collected a t a cell density of 6 • l0 s to 7 • 106 cells/m]. Cells were washed a n d resuspended in T r i s / E D T A buffered saline solution containing 1 mM-EDTA, 75 m_~-KC1, 75 m~-NaC1 and 15 m_~-Tris (pH 7.3). Cells were disrupted b y sonieation and the homogenate was centrifuged a t 100,000 g for 60 min in a Beckman ultracentrifuge. The soluble protein fraction was loaded on a 2-1 reverse-flow Sepharose 4B column a t 4~ The u n b o u n d m a t e r i a l was washed out b y 3 to 4 column volumes of T r i s / E D T A buffered saline and the adsorbed protein was eluted with the same buffered saline containing 0-4 M-galactose. (c) Radioiodination of protein Purified protein preparations were labeled with 12sI using the chloramine T m e t h o d (McConahey & Dixon, 1966) or the solid-state lactoperoxidase m e t h o d (David & Reisfeld, 1974). I n general, 100 ~g of protein was labeled with 2 mCi of carrier-free Na12SI (New England Nuclear) to give a spec. act. of 2 • 106 to 4 X 106 cts/min per ~g protein. t See footnote on page 157.

MEMBRANE PROTEIN IN D I C T Y O S T E L I U M

159

(d) Radioiodination of cell surface The cell surface proteins were radioiodinated using a modification of the lactoperoxidase method previously described b y Kennel & Lerner (1973). Cells were harvested a n d washed in 17 m ~ - K / N a 2 phosphate buffer (pH 6"5) and resuspended a t a cell concentration of 5 • 107 cells/ml for vegetative cells and 108 cells/ml for developing cells in a labeling m e d i u m containing 17 m_~-phosphate buffer a n d 10 -8 M-KI. Lacteperoxidase (a gift of Drs S. J. K e n n e l and R. L. Levy) was added to a final concentration of 20 ~g/ml and carrier-free Na12SI was added to give 1 to 2 mCi/ml. The reaction was started b y adding H202 to a final concentration of 17 ~M and allowed to proceed for 2 min. Cells were then washed free of the enzyme and unreacted iodide.

(e) Preparation of antisera Antisera to the purified CBP were prepared in rabbits or goats b y i m m u n i z a t i o n with complete F r e u n d ' s a d j u v a n t . Goat a n t i - r a b b i t IgG serum was a gift of D r S. J. Kennel. The specificity of the antisera was tested b y double diffusion in 1% agarose or i m m u n o electrophoresis. (f) Cell solubilization Cells labeled with 12sI were resuspended in phosphate buffered saline (pH 7.2) at 5 • 107 cells/ml and solubilized in 0.5% NP40 (Shell Chemical, London, England). The lysate was further dispersed b y ultrasonication in a 20 k t t z Branson sonifier S125 equipped with 0.125 in probe for small volumes. Samples were sonicated at a m a x i m u m o u t p u t of 10 A (d.c.) for two 15-s intervals in a n ice bath. The homogenate was centrifuged at 100,000 g for 60 mh~ in a Beckman ultracentrifuge. The s u p e r n a t a n t solution was collected a n d used for immunologic studies. (g) Immunologic precipitation of the carbohydrate-binding protein To minimize loss due to adsorption of proteins to glass, tubes were coated with 50 ~I normal goat serum a n d 50 ~1 5~/o NP40. Labeled ceils were solubilized as described a n d 100-~1 samples of the soluble protein fraction were added to pre-coated tubes and chal: lenged with 2-5 ~1 of rabbit anti-CBP serum or normal r a b b i t serum as a control. Phosphate buffered saline was added to make a final volume of 0"5 rnl and the mixture was incubated at 37~ for 15 rain. Then goat anti-rabbit IgG serum was added to precipitate the r a b b i t I g G - C B P complex. The i m m u n e precipitate was collected b y centrffugation at 2000 g for 15 rain at 4~ a n d washed once with 0.5% iNP40 in phosphate buffered saline a n d finally with cold distilled water. The precipitates were stored dry i n v a c u u m desiccators. W h e n goat anti-CBP serum was used in the experiment, the same procedure was followed, except t h a t r a b b i t anti-goat IgG was used as the second antibody. (h) Polyacrylamide gel electrophoresis Sodium dodecyl sulfate/polyacrylamide gels were prepared according to Weber & Osborn (1969). I n most cases, a final concentration of 6% (w/v) acrylamide was used. The ~, 7, K/h subtmits of h u m a n myeloma immunoglobulin proteins were iodinated with 131I a n d included as markers in every gel. Immunoprecipitates were solubflized a n d reduced in 100 ~1 of 1~ sodium dodeeyl sulfate, 8 M-urea, a n d 2% ~-mercaptoethanol b y heating at 90~ for 3 to 5 min. The marker proteins am completely reduced b y this procedure. Samples were layered on 6 m m • 100 m m gels a n d r u n at 15 mA/gel for 2"5 to 3 h. Frozen gels were sectioned into l-ram slices using a 5oyce-Loebl automatic gel slicer a n d radioactivity was determined in a Nuclear Chicago automatic g a m m a radiation counter. D a t a were corrected for crossover and efficiency of isotope detection. Acrylamide gradient slab gels were prepared according to the method of Laemmli {1970). Gels with a linear gradient of 5~/o to 17~/o acrylamide a n d of 1.5 m m thickness were polymerized between 14 cm • 17 em glass plates. The stacking gel was 3~o acrylamide. Protein samples were solubilized a n d reduced as described above. Electrophoresis was performed with a constant current of 20 m A for 3"5 to 4 h. Gels were fixed a n d stained in a solution of 25% isopropanol, 10% acetic acid, 0.05% Coomassie brillant blue for 6 to 12 h

160

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a n d then destained in 10~/o methanol, 7~o acetic acid. For autoradiography, gels were dried under a heat lamp and placed in direct contact with Kodak Royal X p a n film in light-proof boxes for 1 to 7 days. (i) Amino acid analysis Protein samples were reduced and separated on an acrylamide gradient slab gel. Strips of stained protein bands were cut a n d proteins were extracted as described previously (Houston, 1971). Protein samples were hydrolyzed for 24 h in 6 N-HC1 at 100~ in v a c u u m tubes. Amino acid composition was analyzed in a Beckman amino acid analyzer using the chromatographic system of Spackman et al. (1958).

( j ) Radiaimmunoassay Radioimmunoassays were performed according to the method described by Leruer et al. (1972) with slight modifications. To estimate the a m o u n t of antigen in a test solution, the ability of the solution to inhibit the binding of 12SI-labeled antigen to a k n o w n a m o u n t of antibody is determined. The a m o u n t of antigen in this solution can be estimated by comparison with a standard inhibition curve generated with unlabeled antigen of known concentrations. To construct the standard curve, unlabeled antigen was diluted to a wide range of concentrations with 0.1 l~-borate buffer (pH 8.4) containing 2 mg bovine serum albumin/ml. A fixed a m o u n t of rabbit antiserum to CBP (25 ~1 of a 1 : 1000 dilution in 10% normal rabbit serum) was added to 100 ~1 of each dilution of the unlabeled CBP, mixed, a n d incubated at 4~ for 60 rain. The a m o u n t of antiserum used was determined empirically to precipitate 50~o of 5 ng 12~I-labeled CBP. Then, 5 ng 125I-labeled CBP were added and incubated at 4~ for another 60 rain. Goat antiserum against rabbit IgG was added to precipitate the rabbit a n t i b o d y - C B P complex. 13~I-labeled normal r a b b i t IgG a n d 221~a+ were generally added to the reaction mixture to monitor the extent of precipitation of the first antibody and the extent of contamination of the precipitate with the unreacted radioactive antigen. To q u a n t i t a t e the a m o u n t of cytoplasmic CBP during development, cells of strain NC4 were allowed to develop, and then collected every 2 h for assay. Cells were lysed by sonication in 1 ml phosphate buffered saline containing 0.3 M-galactose. The homogenate was centrifuged at 100,000 g for 60 m i n and the s u p e r n a t a n t solution was collected for subsequent determination of CBP content. The protein concentration of each sample was determined b y the method of Lowry et al. (1951). Radioimmunoassay was performed by incubating 100 ~1 of the soluble protein fraction with 25 ~1 rabbit anti-CBP serum (1 : 100 dilution in 10~/o normal serum) at 4~ for 60 min. For each time point, the assay was performed on six 5-fold dilutions of the protein sample and each dilution was done in triplicate. After 1 h, 25 ~1 of a mixture containing 5 ng 12aI-labeled CBP (about 20,000 cts/min), lalI-labeled r a b b i t normal IgG (about 20,000 ors/rain), and 221~a+ (about 20,000 cts/min) was added, a n d the sample was incubated for another 60 rain at 4~ Then 100 ~l goat anti-rabbit IgG serum was added to precipitate the rabbit I g G - C B P complex. The immune precipitate was collected by centrifugation at 14,000 r p m for 5 rain in a Beckman 152 microcentrifuge. The radioactivity of the three isotopes in the precipitate was measured in a Nuclear Chicago automatic g a m m a counter. A computer program was designed for correcting the crossover, non-specific trapping (22Na+), background, and for calculating the percentage of inhibition by the soluble protein sample. The a m o u n t of CBP in each dilution was estimated from the standard blocking curve constructed simultaneously and expressed as the averaged a m o u n t of antigen per ~g of soluble protein. 3. R e s u l t s (a) Lactoperoxidase radioiodination of surface proteins (i) Labeling conditions I n i t i a l studies were carried o u t to d e t e r m i n e t h e o p t i m a l c o n d i t i o n s for l a b e l i n g

D. discoideum b y t h e lactoperoxidase r a d i o i o d i n a t i o n m e t h o d . W e h a v e e x a m i n e d t h e effect of various c o n c e n t r a t i o n s of laetoperoxidase a n d h y d r o g e n peroxide o n t h e

MEMBRANE

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161

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incorporation of 12sI into membrane proteins and on cell viability. The results are summarized in Tables 1 and 2. The optimal peroxide concentration was about 17 ~M under our standard labeling conditions: l0 s cells/ml, 10 -s M-KI and 3.3 • 10 -~ Mlaetoperoxidase. Concentrations of peroxide above 70 ~M caused a significant amount of cell death. The reaction is completely dependent on the presence of exogenous laetoperoxidase (Table 2). This eliminates the possibility of protein iodination b y non-enzymic mechanisms. On the other hand, in the absence of exogenous H202, incorporation of iodine was about 200/o compared with the case of the complete reaction mixture, suggesting that the cells m a y be able to generate a small amount of endogenous peroxide. The kinetics of cell labeling are shown in Figure 1. Using the above conditions, the reaction was over in one minute. Cells were thus generally labeled for one to two minutes followed b y rapid processing to remove the peroxide and the enzyme. To demonstrate that only cell surface proteins were selectively labeled b y this method, electron microscope autoradiography was done. Grains on 150 cell sections were counted and the results showed that 80% of the grains were found within 0-5 ~m of the plasma membrane. The rest of the grains (20~ were found inside the cytoplasm

TABLE 1

Effects of hydrogen peroxide on labeling and cell viability H202 concentration (#.M)

Radioactivity (ets/min per l0 S cells)

Dead cells (%)

0 3.5 17-5 35 70 175

1320 3088 5931 5034 5332 5135

O.1 0.3 O.3 1.2 8.9 59.4

A3 cells (10s eells]ml) were labeled in 17 mM-phosphate buffer (pH 6.5) containing 10 -5 MKI, 2 mCi Nat2SI/ml, 3.3 • 10- v M-lactoperoxidase. Cells were lysed in 2% sodium dodecyl sulfate and incorporation of i2sI was determined by trichloroacetie acid-precipitated radioactivity. Cell viability was estimated by Trypan blue (0.05%) exclusion. TABLE 2

Effects of lactoperoxidase concentration on labeling Enzyme concentration (• 10 .7 ~)

Radioactivity (cts/min per 103 cells)

0 0.33 0.66 1.65 3-3 6-6

28 2152 2902 3866 5493 5319

Iodination (%) 0.5 39.2 52.8 70.4 100.0 96-8

A3 ceils (108 cells/ml) were labeled in 17 mM-phosphate buffer (pH 6.5) containing 10 -5 M-KI, 2 mCi Naxa6I/ml, 17 pM-H202.

162

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Fro. 1. Kinetics of surface labeling of vegetative A3 cells. Cells were labeled at a concentration of 5 • l0 T eells]ml in phosphate buffer {pit 6.5) containing 10 .5 ~ - K I (2 mCi l~SI]ml), 17 tLMH202, 3"3 • 10-7 M-lactoperoxidase. The enzyme action was stopped by adding NaN3 to a final concentration of 5 mM at various times. Cells were lysed with 2~o sodium dodecyl sulfate and precipitated with 10~o trichloroacetie acid. Precipitates were washed twice with cold 5~o trichloroaeetie acid before counting.

and this was possibly due to the influx of radioactive iodide during the reaction. To demonstrate whether these internal grains represent labeled soluble proteins, cells were disrupted using a Dounce homogenizer and then centrifuged at 100,000 g for one hour. About 92% (average of five determinations) of the trichloroacetic acidprecipitable radioactivity was found in the pellet, thus indicating that the soluble proteins were not labeled to a significant extent. (ii) Cell surface proteins To identify proteins associated with the external surface of the plasma membrane, surface proteins of vegetative cells were selectively radioiodinated with the lactoperoxidase method. Plasma membranes were isolated and purified by the Brunette & Till (1971) two-phase aqueous polymer system. It was found that membrane proteins had an approximate tenfold increase in 125I specific activity over the whole-cell homogenate, thus indicating that the membrane fraction was enriched in plasma membranes. Membrane proteins were solubilized by the addition of sodium dodeeyI sulfate and separated by electrophoresis on an SDS/acrylamide gradient slab gel. The electrophoretogram is shown in Figure 2. The Coomassie brilliant blue-stained gel profile showed about 30 distinct bands with a molecular size distribution between a range of 200,000 to 15,000 (Fig. 2(b)). The plot of log molecular weight versus migration distance was linear between 68,000 (albumin) and 25,000 (ehymotrypsinogen) (Fig. 2(a)). Proteins were labeled with a number preceded by p, designating its molecular weight • 10-3. Those that fell between the linear range were determined from the protein markers, while others were designated by an approximate molecular weight estimation. An autoradiogram of the same gel is shown in Figure 2(c). Only a fraction of the stained bands were labeled with the lactoperoxidase system and thus are considered surface proteins. The p59 and p90 bands were present as major stained bands and were also highly labeled. By contrast, the other labeled bands appeared s be

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Fro. 2. Electrophoretograms of plasma membrane proteins. Plasma membranes of surface iodinated vegetative A3 cells (grown on K. aerogenes) were isolated with the two-phase aqueous polymer system as described by Brunette & Till (1971). Membrane proteins were solubilized and reduced for gradient slab gel (5% to 17%) eleetrophoresis at 20 mA for 4 h. (a) Protein markers stained with Coomassie brilliant, blue, molecular weight is indicated; (b) Coomassie-sgained pattern of plasma membrane proteins from vegetative cells; and (c) autoradiogram of the surface labeled material, p = molecular weight • 10 -3. m i n o r c o m p o n e n t s o f t h e m e m b r a n e b y m a s s as j u d g e d b y t h e i r s t a i n i n g i n t e n s i t y w i t h Coomassie b r i l l i a n t blue. A n u m b e r o f t h e s e l a b e l e d surface p r o t e i n s h a v e b e e n f o u n d t o c h a n g e d u r i n g d e v e l o p m e n t (Siu et al., 1975). (b) Association of the carbohydrate-binding protein with the external surface of the

plasma membrane To a c q u i r e a n u n d e r s t a n d i n g o f t h e e x p r e s s i o n o f p l a s m a m e m b r a n e p r o t e i n s d u r i n g t h e d e v e l o p m e n t a l cycle o f Dictyostelium, we h a v e chosen t o s t u d y t h e C B P d e s c r i b e d b y R o s e n et al. (1973), b e c a u s e i t c a n be easily purified a n d has i n t e r e s t i n g biological i m p l i c a t i o n s . This p r o t e i n h a s a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 100,000 12

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and consists of four subunits with a molecular size of 26,0009 The CBP was isolated on a Sepharose 4B column as described b y Simpson et al. (1974). On reduction and separation in an acrylamide gradient slab gel, two Coomassie blue-stained bands with molecular sizes of approximately 26,000 and 24,000 were present in all preparations of the purified protein (Fig. 3). These two proteins had similar amino acid compositions (Table 3) and no N-terminus could be detected (data not shown). R a b b i t and goat antisera to a preparation of CBP in which the 26,000 molecular weight species represented 90 to 95% of the protein were prepared and used as an immunologic probe of the cell surface. Since conclusions reached from such experiments depend on the specificity of the antisera, studies were carried out to (a) determine the number of immunogenic proteins present in our purified CBP preparation and (b) to insure t h a t the material precipitated b y antisera has the same biochemical properties as the purified protein. As can be seen from the gel diffusion studies in Figure 4, our antisera detected one major protein in the purified CBP preparation. When CBP was radioiodinated and then precipitated b y our antisera, the protein detected had a molecular size identical to the CBP subunit, showing t h a t our antisera reacted with this protein (Fig. 5). *'_

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Fie. 3. Eleetrophoretogram of the CBP subunit (8 gg) in a 5% to 17~/o gradient slab gel. The protein was purified by adsorption on a Sepharose 4B column and eluted in 0.3 ~-galactose (Simpson et al., 1974). The subunit protein had a molecular size of about 26,000. A minor band with a molecular size of about 24,000 was also evident.

MEMBRANE P R O T E I N I1~ D I C T Y O S T E L I U M

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TABLE 3

Amino acid composition of Dictyostelium carbohydrate-binding protein

Lys His Arg Asp Thr Ser Glu Pro Gly Ala Val Met Ilo Leu Tyr Pho

Main band t protein

Minor bandJ~ protein

54"5 21 "3 48.3 138.8 84"0 75.8 107.1 44.0 88.4 77.5 72.7 3.9 41.4 54.0 38.3 35.3

63"0 33"5 48"9 147.4 52"9 82-4 87.1 56.3 79-O 54-9 84.4 6.0 47.6 61-6 36.2 38.9

Values are expressed as residues/1000 residues and represent the average of three independent determinations. "~Mr = 26,000. :~M r = 24,000.

To determine if the CBP was associated with the plasma membrane, the surface proteins of cells at the aggregation stage (12 h after the initiation of development) were selectively radioiodinated. The cells were solubilized in NP40 and the CBP was precipitated with a rabbit anti-CBP serum, which was in turn precipitated b y a goat anti-rabbit IgG serum. The presence of 125I radioactivity in the precipitate would indicate the association of the CBP with the external surface of the plasma membrane. When such a precipitate was solubilized and reduced for acrylamide gel eleetrophoresis, two predominant protein species with molecular sizes of approximately 56,000 and 26,000 were evident (Fig. 6). Neither protein was present in the precipitate where normal rabbit serum was substituted for antibody against CBP, and thus both proteins were present in the precipitate b y virtue of specific antigen-antibody union with at least one of the proteins. The 26,000 molecular weight protein has a molecular size expected for the subunit of the CBP, but the 56,000 molecular weight protein was unexpected. As will be described below, the appearance or disappearance of the 56,000 molecular weight protein is always co-ordinate with the 26,000 molecular weight protein.

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FIG. 5. Gel profiles of immunoprecipitates of laSI-labeled CBP. Purified CBP radioiodinated with the chloramine T method and precipitated with rabbit anti-CBP serum. The immunopreeipitare was solubilized and reduced by heating for 3 to 5 rain at 90~ in 1% sodium dodecyl sulfate, 8 ~i-urea, and 2o/o ~-mercaptoethanol, and analyzed by sodium dodecyl sulfate/polyacrylamide (6%) gel eleetrophoresis for 2.5 h at 15 mA/gel. 131I-labeled human IgG and IgM were added to each gel as protein markers. (a) Control immunoprecipitate using normal rabbit serum ( - - { } - - 0 - - ) and the reduced protein markers ( - - O - - O - - ) ; (b) Immunoprecipitate of the CBP ( - - O - - O - - ) and 125I-labeled CBP used for the immunoprecipitation studies ( - - C ) - - O - - ) . (c) Appearance of the carbohydrate-binding protein on the plasma membrane surface

during development To d e t e r m i n e t h e t i m e w h e n t h e C B P a p p e a r s on t h e p l a s m a m e m b r a n e d u r i n g d e v e l o p m e n t , cells g r o w n on K. aerogenes were h a r v e s t e d d u r i n g t h e e x p o n e n t i a l g r o w t h phase, w a s h e d free o f b a c t e r i a , a n d t h e n t r a n s f e r r e d t o filters to i n i t i a t e d e v e l o p m e n t . A t 0, 3, 5, 7, 9, 12 a n d 18 hours o f d e v e l o p m e n t , cells were collected a n d t h e surface r a d i o i o d i n a t e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . R a d i o a c t i v e l y l a b e l e d surface p r o t e i n s were a n a l y z e d for t h e presence o f t h e C B P b y i m m u n e p r e c i p i t a t i o n a n d c h a r a c t e r i z a t i o n o f t h e solubilized p r e c i p i t a t e s in a c r y l a m i d e gels. As sho~m in F i g u r e 7, b o t h t h e 26,000 m o l e c u l a r w e i g h t s u b u n i t o f t h e C B P a n d t h e 56,000 m o l e c u l a r w e i g h t p r o t e i n a p p e a r e d c o - o r d i n a t e l y a f t e r 5 hours o f d e v e l o p m e n t . T h e s e t w o p r o t e i n s r e m a i n e d a s s o c i a t e d w i t h t h e cell surface t h r o u g h t h e c u l m i n a t i o n s t a g e a t 20 hours of d e v e l o p m e n t . S i m i l a r results h a v e also b e e n o b t a i n e d w i t h o t h e r

168

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so

(rain)

FIG. 6. Gel profile of an anti-CBP immunoprecipitate of A3 cells. Cells grown on K. aerogenes collected for development for 12 h, and then surface radioiodinated with the lactoperoxidase method. Cells were lysed in 0-5% 1WP40and the 100,000 g supernatant solution was precipitat6d with antibodies to the CBP. Sodium dodecyl sulfate/acrylamide gel electrophoresis was carried out as described in Fig. 5. (a) Marker proteins (reduced samples of lalI-labeled human IgG and IgM) ( - - O - - O--), and the non-specific trapping control using normal rabbit serum (-- 9 9 (b) anti-CBP precipitate ( - - 9 1 4 9

strains, such as NC4. I n repeated attempts, neither protein was detectable on the surfaces of cells growing on bacteria or during the first three hours of development. To find out if C B P was present in the c y t o p l a s m of vegetative cells, soluble proteins prepared from a zero-time cell sonicate were radioiodinated. Using the i m m u n e precipitation method, no radioactive C B P was detectable in this preparation. Thus, the appearance of b o t h proteins on the external surface of the plasma m e m b r a n e appears to be a developmentally regulated event and the d a t a suggest t h a t the C B P is n o t synthesized until the first few hours of the developmental cycle. (d) Presence of the carbohydrate-binding protein on the plasma membrane of

axenically grown cells Since the C B P has been found in the soluble protein fraction of vegetative cells g r o ~ axenically in liquid medium, we have a t t e m p t e d to determine if this protein is also expressed on the cell surface of these cells. A3 cells were harvested from H L - 5 m e d i u m at two different cell titers, 3>< l0 s cells/ml a n d 5 • l0 s eells/ml. Surface

MEMBRANE PROTEIN IN DICTYO,.~TELIUM 56

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80

Fro. 7. Gel profiles of anti-CBP precipitates of 12SI-labeled surface proteins from developing cells. Cells were grown on K . aerogenes and then harvested for development on Whatman no. 50 filters saturated with buffered salt solution (see Materials and Methods). Developing cells were collected at different times and their surfaces were radioiodinated. Soluble proteins from zero time were also radioiodinated for immunoprecipitation. Anti-CBP precipitate of 125I-labeled surface proteins from (a) 0-h cells ( - - 0 - - 0 - - ) ; (b) 3-h cells; (e) 5-h cells; (d) 7-h cells and (e) 9-h cells. 131I-labeled human IgG and IgM were included in each gel as protein markers (-- 9 Gel profiles of precipitates prepared from 12, 18 and 20-h cells were similar to that of the 9-h sample. About 3 to 5~o of the initial triehloroaeetic acid-insoluble input radioactivity was precipitated by the antibody for samples prepared between 9 and 20 h of development.

proteins of these cells were labeled with 125I a n d t h e C B P was p r e c i p i t a t e d w i t h a n t i body. T h e results show t h a t , as for developing cells, b o t h t h e C B P a n d t h e 56,000 molecular weight p r o t e i n were p r e s e n t on the surface of axenic cells e v e n d u r i n g t h e e x p o n e n t i a l g r o w t h phase (Fig. 8). These two p r o t e i n s c o n t i n u e to be associated with t h e cell surface t h r o u g h o u t t h e d e v e l o p m e n t a l cycle. T h e presence of t h e C B P o n t h e surface of a x e n i c a l l y growing cells is c o n s i s t e n t w i t h t h e n o t i o n t h a t cells g r o w n i n this w a y are p a r t i a l l y starved, a n d t h u s some of t h e e a r l y d e v e l o p m e n t a l e v e n t s h a v e a l r e a d y occurred.

170

C.-H. SIU E T AL. 7O

g-

56

23

9-

6-

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Distance migrated (mm)

FIG. 8. Presence of CBP on the cell surface of axenically grown vegetative cells. Cells grown in HL-5 medium were harvested at a density of 3 • 105 cells/ml and surface proteins were radioiodinated. Anti-CBP precipitate was prepared from the cell lysate and analyzed with sodium dodecyl sulfate/acrylamide gel electrophoresis as described in Fig. 5 legend.

(e) Effect of actinomycin D and cycloheximide on the expression of the carbohydratebinding protein To determine ff R N A a nd protein synthesis are necessary after the initiation of development for the expression of the CBP on the cell surface, developing cells were treated with actinomycin D and daunomycin, and cycloheximide at various stages during the developmental cycle. I t has been found t h a t a combination of aetinomycin D (125 Fg/ml) and daunomycin (250 Fg/ml) is more effective in inhibiting the synthesis of m R N A t h a n actinomycin D alone (Firte] et al., 1973). Cells deposited on filters for development were transferred to pads saturated with actinomycin D and daunomycin at 0, 1, 2 and 3.5 hours. Cells were collected at 6 hours and plasma membranes were analyzed for the presence of the CBP. For comparative purposes, surface m e m b r a n e proteins were iodinated to yield similar specific activities or the same amount of triehloroaeetie acid-preeipitable 125I counts were used for the immunoprecipitation studies. As shown in Figure 9, when cells were treated at either 0 hour or 1 hour with actinomycin D and daunomyein, no CBP was detectable on the surface of the cells 6 hours after the initiation of development, while the CBP was present in reduced amounts when cells were treated at 2 hours. T r e a t m e n t with the drugs after 3.5 hours of development had little or no effect on the appearance of the CBP on the slime mold cell surface at 6 hours. To determine if de nero protein synthesis is required for the expression of the CBP on cell surface, cells at 3 hours and 4 hours of development were treated with the inhibitor cyeloheximide (500 Fg/ml). Cells were collected at 6 hours and analyzed for the presence of the CBP on the plasma membrane. Cycloheximide was effective in preventing the expression of the CBP in both cases (Fig. 10(a)). Since the cell size after cycloheximide t r e a t m e n t was reduced to about half oi t h a t before treatment, there might be rapid turnover or other changes for m e m b r a n e

MEMBRANE

PROTEIN

IN DIOTYOSTELIUM

171

(a) I0

70

56

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8

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Distance migrated (mrn) F I a . 9. Effect of actinomycin D and daunomyein on the expression of CBP on the cell surface. W h a t m a n no. 50 filters with developing ceils were transferred to Millipore pads saturated w i t h buffered salt solution (see Materials and Methods) containing actinomycin D (125 ~g/ml) s n d daunomycin (250 ~g/ml) at different development times. Cells w e r e h a r v e s t e d at 6 h and surface proteins were radioiodinated with lactoperoxidase. Immunoprecipitates were prepared with NP40-soluble materials and sodium dodeeyl sulfate/acrylamide gel electrophoresis was performed as described in Fig. 5. Precipitate of developing cells t r e a t e d w i t h drugs at 1 h (b), 2 h (c), and 3.5 h (d). T h e c o n t r o l is s h o w n in (a).

proteins. Therefore, to investigate the effect of cycloheximide on the stability of CBP already present on the cell surface, developing cells were treated with cycloheximide at 7 hours and the presence of CBP on the cell surface was determined at 9 hours (Fig. 10(b)). The presence of CBP on the plasma membrane appeared to be stable. When cells were allowed to recover after treatment with cycloheximide between 4 hours and 6 hours, the CBP appeared on the cell surface only 3 to 3.5 hours after the removal of the inhibitor (Fig. 10(c)). CBP was detected at 9.5 hours even when cells were treated with a combination of actinomycin D and daunomyein from 6 to 9"5

C . - H . S I U E T AL.

172 (o)

70

56

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6-

3-

(b) A N

b

129-

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9-

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0

20

40

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migrated

60

80

(re,r,)

Fzo. I0. Effect of cycloheximide on the expression of CBP on the cell surface. (a) Gel profile of anti-CBP precipitate prepared from a lysate of cells treated with cycloheximide (500 #g/ml) at 4 h and collected at 6 h of development. (b) Gel profile of anti-CBP precipitate of cells treated with cycloheximide at 7 h and collected at 9 h of development. (c) Gel profile of anti-CBP precipitate of cells treated with cycloheximide between 4 and 6 h of development and then allowed to recover for 3 h after transfer to a new pad of buffered salt solution (see Materials and Methods) alone. h o u r s a f t e r t h e r e m o v a l of c y c l o h e x i m i d e , suggesting t h a t such a r e c o v e r y d i d n o t r e q u i r e c o n c o m i t a n t m R N A synthesis. (f) Absence of the carbohydrate-binding protein from the surface of non-cohesive

mutants To assess t h e role of t h e C B P in d e v e l o p m e n t , a n u m b e r o f m u t a n t s w i t h a l t e r a t i o n s in t h e d e v e l o p m e n t a l p r o g r a m were s t u d i e d . T h r e e o f t h e s e m u t a n t s t h a t failed to e x h i b i t i n t e r c e l l u l a r cohesion also l a c k e d t h e a b i l i t y t o a g g l u t i n a t e sheep e r y t h r o c y t e s , a n in vitro a s s a y for t h e C B P ( R o s e n etal., 1973). S o m e o f t h e p r o p e r t i e s o f t h e s e m u t a n t s arc s u m m a r i z e d in T a b l e 4. W h e n t h e p l a s m a m e m b r a n e s o f m u t a n t s W L 3 , W L 4 a n d W L 5 were s t u d i e d for t h e presence o f t h e CBP, n o n e was d e t e c t e d in W L 3 a n d W L 4 , while in W L 5 i t was p r e s e n t in r e d u c e d a m o u n t s (Fig. 11). B o t h t h e 56,000 m o l e c u l a r w e i g h t p r o t e i n a n d t h e 26,000 m o l e c u l a r w e i g h t s u b u n i t o f t h e C B P were c o - o r d i n a t e l y lost in W L 3 a n d W I A .

MEMBRANE P R O T E I N I N D I C T Y O S T E L I U M

173

TABLE 4

Characterization of three non-cohesive mutantst of D. discoideum Strain A3 WL3 WL4 WL5

Aggregation:~ + . . --

Cell cohesion w + . .

. . --

Agglutination of82 sheep erythrocytes

Presence of CBP [[ on cell surface

-[. . --

+ :h

t WL3, WL4 and WL5 are new non-cohesive mutants and were isolated from strain A3 following mutagenesis with N'-nltro-N-methyl-N-nitrosoguanidine. Morphological mutants were isolated by plating the cells at a titer of 30/plate and colonies that failed to aggregate were selected. $ Cell aggregation was determined by morphological observation 12 h after depositing on filters. wCohesiveness of 12-h developed cells was quantitated by the roller-tube culturing technique according to Geriseh (1961) and Rosen et al. (1973). 82The crythrocyte agglutination assay was performed and scored according to Rosen el at. (1973). II Presence of the 26,000 molecular weight CBP subunit was detected by 125I laetoperoxidase surface labeling and immunopreeipitation of cells 18 h after ~he initiation of development. To test whether the defect in these mutants was in the synthesis of the CBP or in its integration into the membrane, the soluble cytoplasmic proteins from WL3, WL4 and A3 cells at 18 hours of development were labeled with a2~I and assayed for the presence of CBP by immunoprecipitation. The CBP was detected in the cytoplasm of A3 cells, but not in the cytoplasm of the non-cohesive m u t a n t strains (Fig. 12), and thus the defect appears to be in the synthesis of the protein. This is further suggested b y the fact t h a t CBP was not detectable b y radioimmunoassay in the wholecell homogenate of WL3 and WL4 as discussed in section (g), below. (g) Quantitation of total carbohydrate-binding protein during development The above results show that CBP does not appear on the cell surface until 5 hours of development and the inhibitor studies suggest t h a t the protein is synthesized around 4 hours. I n order to estimate the amount of CBP present in each cell, we have employed radioimmunoassay techniques, which can give sensitive and accurate quantitation of a n y antigen. With this technique, we have determined the time when CBP began to appear in the cytoplasm and its concentration in the cell at different developmental stages. As shown in Figure 13, the soluble protein fractions from vegetative and 2-hour cells grown on bacteria did not have a n y detectable CBP and their inhibitory effect on the rabbit antiserum against CBP was negligible. The antigen was present in a very low concentration in 4-hour cells, about 0.1~/o of the soluble protein fraction. I t began to accumulate rapidly and linearly between 4 hours and 10 hours of development for an approximate tenfold increase. At 10 hours, the concentration of CBP in the soluble protein fraction appeared to level off and remained constant for at least the next six hours. At its maximum concentration, CBP was present at about 10 ng/~g (1 ~ ) of soluble protein. The average amount of soluble protein per ceil at the 10-hour stage was determined to be 2 • 10 -2 ng, and so there was 2 • 10 -9 ng CBP per cell. Since the molecular weight of the native CBP molecule is 100,000 (Simpson et al., 1974), there were approximately 1.2 • 106 CBP molecules per cell at 10 hours of development.

174

C.-H. S I U E T (a

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c

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I

.

.

I

.

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0

WL5

20

40 Distance

migrated

60

80

( rnrn )

FIG. ll. Anti-OBP precipitates of NP40-soluble material from three nonieohesive mutants. Surface proteins were labeled with 12sI 18 h after the initiation of development and anti-CBP

immunoprecipitates were prepared for elecbrophoresis. (a) A3 cells; (b) WL3; (c) WL4; and (d) WL5.

As a control for specificity of the radioimmunoassay, the non-cohesive m u t a n t WL3 was incubated for development and then collected at different time-points to assay for the presence of CBP. None of these protein samples exhibited any significant inhibiting effect on the antiserum (Fig. 13), indicating that CBP was absent from these cells. Similar observation was also made with m u t a n t strain WL4.

4. D i s c u s s i o n

The laetoperoxidase method of radioiodination provides a useful tool to identify the membrane proteins located on the external cell surface. Control experiments have shown that the reaction is completely dependent on the addition of exogenous lactoperoxidase and that proteins t h a t become iodinated are exclusively associated with the external surface of the plasma membrane not only in Dictyostelium but also other cell types (Kennel & Lerner, 1973; Gates et al., 1974; Graham et al., 1975).

MEMBRANE

(a)

I0

IN DIGTYOSTELIUM

PROTEIN

70

56

25

{

{

{

175

A5

b

6

~4 x

.E -~ 4 - ( b ) -

I

l

I

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/

2

-

0

( C )

WL4

20

40

60

80

Distance migrated (rnm)

FIG. 12. Gel profiles of anti-CBP immunoprecipitates of 12SI-labeled cytoplasmic proteins from A3 cells and the non-cohesive mutants. Cells at 18 h of development were lysed b y sonication. The cell homogenate was centrifuged a t 100,000 g for 1 h and the soluble protein fraction was labeled with 1251 by the solid-state lactoperoxidase method. Immunoprecipitates prepared with the labeled soluble proteins and the rabbit anti-CBP serum were studied with sodium dodecyl sulfate/polyaerylamide gel electrophoresis. (a) A3 cells; (b) WL3; and (e) WL4.

B y combining this methodology with the use of immunologic probes, we can study various aspects of the expression of a specific protein, such as the CBP. As discussed previously (Kennel & Lerner, 1973), this method of radioiodination works on the premise that only "available" tyrosine residues at the cell surface are derivatized. Thus, uniform labeling is usually not achieved and consequently quantitation is difficult. So, for comparative purposes, we have labeled surface proteins to similar overall specific activity or used the same amount of trichloroaeetie acidinsoluble radioactive material for immunologic studies. One can, therefore, determine the availability of a protein to lactoperoxidase and this approach gives a reasonable first approximation as to which molecules are likely to be "seen" b y other cells, and thus m a y be important in cell-cell interaction. The plasma membrane of D. discoideum consists of about 30 Coomassie-stained bands after acrylamide gel eleetrophoresis, of which about haft a dozen are associated with the external surface, since they are radioiodinated by lactoperoxidase. However, an accurate estimation of the number of proteins exposed to the external surface is difficult, because proteins of similar molecular sizes may band at the same region while some peripheral proteins can be lost during the membrane purification procedures. During the development of D. discoideum, it has been found that new proteins are inserted into the plasma membrane, while others disappear after a particular stage

176

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SIU

ET

AL.

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Time (h)

Fro. 13. Quantitation of total CBP with radioimmunoassay. NC4 cells were harvested from bacteria growth plates for development. Cells were collected at 2-h intervals and the soluble protein fractious were used for radioimmunoassays as described in the Materials and Methods. The non-cohesive mutant WL3, which was shown to lack CBP both on the surface and in the cytoplasm, was used as the control (A). The results of two experiments using NC4 were plotted together (O, O) and each experimental point represents the average concentration of CBP in the soluble protein fraction calculated from the values obtained with different dilutions of the protein sample. Cells at 0 h and 2 h had no inhibitory effect on the antibody even when 250 ~g soluble proteins per sample were used.

(Smart & Hynes, 1974; Sin et al., 1975). Thus, the plasma membrane must be constantly undergoing rearrangement during the course of development. The CBP is found to be one of those proteins associated with the plasma membrane on its external surface. This protein is absent from both the cell surface and the cytoplasm when cells are grown on a bacterial lawn. However, it begins to appear on the cell surface about five hours after the initiation of development. The data suggest an increasing amount of CBP is transported to the external cell surface between 5 and 9 hours. Quantitative studies using radioimmunoassay show that CBP was not detectable in zero-time and 2-hour cells in spite of the sensitivity of our technique, which can detect as little as 1 ng of antigen in 10 Fg of soluble proteins. CBP represents only 0.1% of the soluble proteins at 4 hours of development. This is followed by a rapid accumulation of the protein and an approximate tenfold increase in the CBP concentration between 4 hours and 10 hours. The data thus suggest that the gene coding for CBP was not activated until a time between 1 and 2 hours after the initiation of development and that CBP is one of those proteins synthesized early in the developmental cycle. I t probably reaches its maximum rate of synthesis between 6 hours and 10 hours. This view is supported b y our preliminary results on the rate of CBP synthesis during development. Studies with inhibitors of m R N A synthesis, such as actinomycin D and daunomycin, show t h a t m R N A synthesis is required for the expression of CBP. Since treating the cells with a combination of these two drugs at 2 hours of development does not completely eliminate the appearance of CBP on the cell surface at 6 hours, this suggests t h a t synthesis of the m R N A coding for the CBP is probably initiated

MEMBRANE P R O T E I N I N D I C T Y O S T E L I U M

177

between 1 hour and 2 hours. Studies with cycloheximide and the quantitative analysis of CBP in the cytoplasm indicate t h a t synthesis of CBP is initiated around 3 to 4 hours. Thus, a lag period appears to occur between the initiation of m R N A synthesis and translation. Contrary to some other proteins which are present in the plasma m e m b r a n e for only a specific duration in the developmental cycle (Sin et al., 1975), the CBP remains exposed on the external cell surface from the pre-aggregation stage through the culmination stage. Although there is an apparent accumulation of the CBP on the cell surface during the initial period of its appearance, it is not known at present whether the concentration and distribution of the CBP remain unchanged throughout the course of development. The appearance of CBP on the cell surface corresponds closely to the time when cells begin to acquire intercellular cohesion (Beug et al., 1970; Rosen et al., 1973). Indeed, the cohesive property is retained b y cells until the end of the developmental cycle, and so is the CBP on the cell surface. This, together with the observation t h a t the CBP is either missing or present only in reduced amounts in the non-cohesive m u t a n t s tend to suggest t h a t the CBP m a y have a role in cell cohesion. Plasma membrane-associated, carbohydrate-binding proteins could theoretically have a wide range of specificities or affinities for other molecules such as glycoproteins. B y altering the rate of synthesis and/or the availability of either the lectin or its ligand, differential interactions could be controlled. This mechanism m a y be of general importance, since carbohydrate groups or carbohydrate-binding proteins have been reported to be involved in cell adhesion in different experimental systems (Turner & Burger, 1973; Rosen et al., 1974; Balsamo & Lilien, 1975). I n the case of D. discoideum, the 56,000 molecular weight protein t h a t coprecipitated with the CBP in immunoprecipitates appears to be a likely candidate for the CBP receptor. Purification and characterization of this protein are currently being undertaken. Cultures in the early exponential growth phase in HL-5 medium have the CBP on the cell surface. I t is intriguing to note t h a t axenically grown cells also acquire intercellular cohesiveness, even at low cell titers (about 5 • 105 cells/ml). Ashworth & Quance (1972) have also reported t h a t cells grown in axenic medium accumulate certain enzymes which normally appear only during the early phase of development. Therefore, cells, when grown axenically, m a y acquire some of the characteristics of developing cells. This is publication nmnber 1013 from the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, Cal. 92037, U.S.A. and publication no. 3 from the Consortium Program in Developmental Aspects of Membrane Biology, La Jolla, Cal. 92037, U.S.A. The authors thank Dr F. J. Dixon and Ms P. J. McConahey for advice and providing space and equipment for the radioimmunoassay work; Dr S. J. Kennel for advice on radioiodination by the lactoperoxidase system ; Dr T. Hugli for performing the amino acid composition analyses and A. Willard for cohesion tests on the mutants. The excellent technical assistance of H. P. Burstyn and S. M. Clark is acknowledged. We also thank L. M. Beligotti for preparation of the manuscript. This research was supported by grants from the American Cancer Society (NP 168), the Research Corporation, Cancer Research co-ordinating committee of the University of California to one of us (R. A. F.), and National Science Foundation (GB 28959) to one of us (W. F. L.). One of us (R. A. L.) is a recipient of a National Institutes of l=Iealth career development award (no. AI-46372).

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