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Plasma membrane proteins ofDictyostelium: The spore coat proteins
DEVELOPMENTAL
BIOLOGY
71,
Plasma Membrane
297-307
(1979)
Proteins of Dictyostelium:
The Spore Coat Proteins
MICHAEL ORLOWSKI' AND WILLIAM F. LOOMIS Department
of Biology,
University
Received November
of California,
San Diego, La Jolla,
California
20, 1978; accepted in revised form March
8, 1979
92093
Spore coats were isolated following germination of Dictyostelium discoideum spores. They were found to contain five major proteins of apparent molecular weights of 60,000 (SP60),68,000 (SP68), 70,000 (SP70), 96,000 (SP96), and 209,000 (SP209). Of these SP68 and SP96 were found to be glycoproteins. Analysis of the tryptic and chymotryptic fingerprints indicated that the major spore coat proteins are independent gene products and not derived by proteolytic cleavage of common percursors. These spore coat proteins are the major newly synthesized proteins seen in membranes late in development and account for the majority of the changes seen in membranes during culmination. They can be observed in membranes isolated from spores, most likely while in transit to the spore coat. SP60, SP68, and SP70 appear to be on the outer face of the spore coat since they are sensitive to proteolysis by Pronase added to intact spores while SP96 and SP200 are protected until the spore case is broken during germination. The spore coat proteins are not found in stalk cells or in several mutant strains blocked in spore formation including one strain, HL-6, which undergoes many of the biochemical differentiations of culmination but forms only stalk cells, The spore coat proteins do accumulate in a spore shape mutant, HU26, which forms small round spores rather than ellipsoid ones. Characterization of the spore coat proteins further defines the changes in membrane proteins during development of Dictyostelium and the processes of terminal differentiation in spores.
the electron microscope the spore coat is Development of Dictyostelium discoi- 1000-1500 A thick and appears to be condeum leads the majority of cells from amoe- structed from an outer amorphous layer 200-300 A thick, a central fibrous layer of bas to encapsulated spores. Following the initiation of development the amoebas ag- cellulose 700-900 A thick, and an inner electron-dense layer 100-300 A thick gregate into groups of about lo5 cells which closely opposed to the plasma membrane become integrated into slug-shaped orga(Hohl and Hammamoto, 1969; Cotter et al., nisms. After about 18 hr of development, 1969; Loomis, 1975). It is likely that specific the anterior 20% of the cells construct a proteins are incorporated into the spore stalk perpendicular to the substratum coat along with the cellulose fibrils. which lifts the rest of the cells into the air. When the pattern of total cellular protein During this process of culmination, each of synthesis is monitored during development the cells in the posterior 80% of the slug by labeling proteins with [““Slmethionine constructs a thick extracellular coat around for 2 hr at various stages and then separatitself. Both the spore coats and the stalk ing them by electrophoresis on SDS-acrylwalls contain cellulose which makes up about 5% of the total dry weight and gives amide gels, it can be seen that the rates of them structural integrity (Rosness and synthesis of proteins banding at seven positions corresponding to molecular weights Wright, 1974; Freeze and Loomis, 1978). In of 40,000 to 100,000 increase dramatically during culmination (Siu et al., 1977). Four ’ Present address: Department of Microbiology, Louisiana State University, Baton Rouge, La. 70803. of these bands are found in purified plasma 297 INTRODUCTION
OO12-1606/79/080297-11$02.00 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
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DEVELOPMENTAL BIOLOGY VOLUME71, 1979
membranes and make up more than half of the newly synthesized membrane proteins after 16 hr of development as judged by the rate of incorporation of [35S]methionine (Siu et al., 1977). These proteins accumulate in the membranes during culmination and can be seen as intense bands when electrophoretically separated membrane proteins are stained with Coomassie brilliant blue. We have considered that these may be proteins destined for the spore coat which are seen in transit across the plasma membrane. When the spores germinate, they swell slightly and then split longitudinally. The amoebas crawl out of the slit leaving the split spore coats behind intact. By isolating these spore coats we have been able to analyze their protein composition. MATERIALS
AND METHODS
Strains and cultivation. Cells of the wildtype strain, NC-4, the stalky mutant, HL-6, and the round spore-forming strain, HU-26, were grown in association with Klebsiella aerogenes on nutrient agar plates until most of the bacteria were consumed and then collected for synchronous development on filters or allowed to proceed to terminal differentiation on the plates (Sussman, 1966). Strain HL-6 was isolated as a “stalky” mutant from a mutagenized population of strain NC-4; it differentiates quantitatively into stalk cells (Morrissey and Loomis, unpublished). Strain HU-26 carries the mutation sprA on linkage group I which results in the formation of small round spores rather than the larger wildtype ellipsoid spores (Katz and Sussman, 1972). Preparation of spore coats. Spores which had formed on agar growth plates were collected from the lids of petri dishes after they were sharply rapped on the bench. The spores were washed with distilled water several times by centrifugation at 27,000g for 3 min. The pelleted washed spores which were free of undifferentiated
amoebas and stalks were stored at -20°C until needed. Germination was induced by resuspending the spores at 2 x lo7 ml/in 20 mM potassium phosphate buffer, pH 6.5, containing 20% dimethylsulfoxide (DMSO) (Ennis and Sussman, 1975). After 30 min at 22”C, the activated spores were collected by centrifugation, washed once with buffer, and resuspended at 2 x 107/ml in 20 mJ4 potassium phosphate buffer, pH 6.5, containing penicillin and streptomycin to inhibit bacterial growth. The suspension was shaken for 6 hr at 22°C. After 3 hr, amoebas began to emerge from the spore coats and by 6 hr 80-90s of the spores had germinated leaving split coats behind. After 6 hr of germination the material was concentrated by centrifugation at 27,000g for 10 min and resuspended in 30 ml phosphate buffer. The spores and amoebas were pelleted by centrifugation at 500g for 3 min, and the supernatant containing the spore coats was collected. The pellet was reextracted four times by centrifugation at 500g for 3 min and the supernatants were pooled. Spore coats were pelleted from the combined supernatants by centrifugation at 27,000g for 3 min. Following resuspension in 20 ml of buffer, contaminating spores and amoebas were removed by centrifugation at 500g for 3 min. Small debris was removed from the supernatant by pelleting the spore coats by centrifugation at 3000g for 4 min. By phase-contrast microscopy the final preparation contained less than 0.1% contaminating whole spores or amoebas. Purified spore coats were stored at -20°C until used. Preparation of spores and stalks. Spores were collected from plates as described above and stored at -30°C. Stalks were collected from fruiting bodies which had developed on growth plates by vigorously washing the agar surface with distilled water. Stalks were collected on Nitex 200mesh filters which allowed spores, amoebas,
ORLOWSKI
AND
LOOMIS
Spore Coat Proteins in Dictyostelium
and bacteria to pass (Freeze and Loomis, 1978). The stalks were repeatedly resuspended, shaken, and filtered until free of contaminating cells. Purified stalks were stored at -20°C. Isolation of plasma membranes. Vegetative and developed cells were washed by repeated centrifugation from 20 mM potassium phosphate buffer, pH 6.5, at 270g for 3 min and broken with 50 strokes of a Dounce homogenizer. Washed spores were broken under liquid nitrogen by grinding for 3 min in a mortar and pestle. The plasma membranes were isolated by the method of Brunette and Till (Brunette and Till, 1971) using a two-phase system. The effectiveness of this technique for both vegetative and developed cells of Dictyostelium discoideum has been demonstrated previously (Siu et al., 1977). Polyacrylamide gel electrophoresis. Proteins were extracted by suspending the samples in 1% SDS, 2.5% P-mercaptoethano1 in 25 mM Tris buffer, pH 7.0, and heating to 100°C for 3 min. The samples, which contained 100 pg protein, were centrifuged for 3 min on an Eppendorf microfuge and the supernatants applied to gradient (8 to 17 or 5 to 15%) polyacrylamide slab gels prepared according to the method of Laemmli (1970). The following proteins were used as molecular weight markers: filamin (250,000); spectrin (240,000 and 220,000), myosin (200,000), phosphorylase a (96,000)) bovine serum albumin (68,000), immunoglobulin G y-chain (52,000), carboxypeptidase A (34,000), hemoglobin (15,500). Electrophoresis was performed at 24-mA constant current. Gels were fixed in 50% methanol and 10% acetic acid and stained either with 0.05% Coomassie brilliant blue or with the periodic acid Schiffs reagent (Fairbanks et al., 1975). After destaining the gels were scanned on a Joyce-Loebl microdensitometer and photographed with a Polaroid camera by transmitted light. Peptide analysis. The bands visualized by Coomassie blue staining of electropho-
299
retically separated spore coat proteins were cut from the gels with a razor blade and analyzed by the method of Elder et al. (1977a). Very briefly, the proteins in the slices were labeled with 125Iby chloramineT, washed and dried, and then digested with either trypsin (50 pg/ml) or chymotrypsin (50 pg/ml) overnight at 37°C. The eluted lz51-labeled peptides were then analyzed in two dimensions by autoradiography (Elder et al., 197713). These analyses were generously carried out by Dr. John Elder. Pronase treatment. Whole washed spores were suspended at lO’/ml in 20 mM potassium phosphate buffer, pH 6.5, containing 2 mg/ml Pronase CB for 75 min at 22°C. The spores were then extensively washed and activated in 20% DMSO. Activated spores were collected after a 30 min incubation in 20% DMSO, washed, and treated with 2 mg/ml Pronase for 75 min at 22°C before being extensively washed. The spore coats were collected after 6 hr of germination as described above. Germination efficiency was not affected by this treatment. Purified spore coats (2.5 mg/ml) were likewise treated with 2 mg/ml Pronase in 20 mM potassium phosphate buffer, pH 7.5, for 75 min at 22°C before being washed and extracted. Treatment with Pronase did not affect the appearance of the spore coats observed by phase contrast at 480X on a Zeiss compound microscope. Protein determination. The method of Lowry et al. (1951) was used to measure protein content using crystalline bovine serum albumin as the standard. RESULTS
Plasma membranes were isolated from cells and spores late in development and their proteins separated on SDS-acrylamide gels (Fig. 1). After 26 hr of development the cells had undergone terminal cytodifferentiation to spores and stalk cells. Membranes prepared from spores were free of
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DEVELOPMENTAL
MW x IO
BIOLOGY
VOLUME 71, 1979
-3
9668-
SP60
52 -
34 -
t5.5-
0
22 HOURS
26
28
32
OF DEVELOPMENT
FIG. 1. Electrophoretograms of plasma membrane proteins. Plasma membranes were isolated from vegetative cells (0 hr) and cells late in development (22 to 32 hr), solubilized in 1% SDS, reduced with 2.5% ,f3mercaptoethanol, and heated to 100°C for 3 min just before being applied to a slab gel containing a gradient (7 to 18%) of polyacrylamide. Following electrophoresis the slab was stained with Coomassie blue. Each sample contained 100 pg of protein. Proteins of known molecular weights (See Materials and Methods) were also run and their locations are indicated. Culmination was underway by 22 hr and most cells had differentiated into spores by 26 hr.
whole spores, spore coats, stalk material, or cells as judged microscopically. Since the general complement of proteins present in the membranes prepared from these cells was similar to that prepared at earlier stages and is not significantly more complex, the membrane purification technique appears applicable to fully differentiated
cells. While most of the bands extracted from membranes of vegetative cells can still be seen in membranes late in development, a prominent protein of apparent molecular weight 60,000 was found to accumulate after 22 hr of development and to make up the most prominent band in membranes isolated from mature spores. Other major
ORLOWSKI AND LOOMIS
301
Spore Coat Proteins in Dictyostelium
bands observed in membranes of developed cells can be seen at the positions of proteins of approximate molecular weights 68,000, 70,000, 96,000, and 200,900 (Fig. 1). The spore coats left behind by germinated spores can be separated from amoebas and ungerminated spores by differential centrifugation (see Materials and Meth-
ods). Protein accounts for 67% of the dry weight of the spore coats and can be quantitatively extracted by 1% SDS and 2.5% /?-mercaptoethanol. When separated on SDS-acrylamide gels by electrophoresis, most of the spore coat protein bands at positions of proteins of molecular weight 60,000, 70,000, 96,000 (Fig. 2b). There are
-
SP200
-
SF’96 SP70 SP60
-
6852-
a
b
c
d
FIG. 2. Spore coat proteins. Proteins were extracted from (b) purified spore coats, (c) whole spores, and (d) membranes of purified spores and analyzed by gradient (7 to 18%) acrylamide electrophoresis. The proteins were stained by Coomassie blue. (a) Marker proteins were co-electrophoresed. The spore coat proteins are indicated by their apparent molecular weight X IO-” prefaced by “SP.”
302
DEVELOPMENTAL BIOLOGY
also bands at the positions of proteins of molecular weights 68,000 and 200,000. In some electrophoretic analyses a band was seen at the position of molecular weight 94,000, but was not consistently found and may be a consequence of the fact that the 96,000 molecular weight protein is glycosylated (see Fig. 3). These proteins appear to be integral components of the coats since they are not extracted by a 10% solution of the nonionic detergent Triton X-100. We will refer to these spore coat proteins by their apparent molecular weights x 10m3 preceded by the letters “SP.” The spore coat proteins can be seen in extracts of whole spores but make up a smaller proportion of the total complement of proteins (Fig, 2~). The minor bands seen in extracts of spore coats are all found in whole spores and may result from cytoplasmic contaminants adhering to the coats throughout the purification procedure. Therefore, these minor bands are not considered specific to the spore coat. The apparent molecular weight of the spore coat proteins does not noticeably I
I
L
I
I
I
I
n
change during germination, since the major proteins extracted from the coats of germinated spores were found in ungerminated spores as well as in plasma membranes isolated from the mature spores (Fig. 2d). Thus, any major processing of these proteins would have to occur before they accumulate in the membranes and are transferred to the spore coat. Spore coat proteins separated on SDSacrylamide gels were stained for carbohydrate by the PAS technique. SP68 and SP96 are glycoproteins by staining criteria whereas SP60, SP70, and SP200 are not stained. A minor PAS-positive band was seen which migrated slightly more slowly than SP70 and may be a minor spore coat glycoprotein of approximately molecular weight 72,000. Densitometer traces of stained gels indicated that 70 f 5% of the total PAS-positive material was found in the SP96 band (Fig. 3). Densitometer tracings of the bands stained with Coomassie blue showed that SP60 is present at about twice the amount of the other major spore coat proteins and that together they make up more than half of the extracted material (Fig. 3). Peptide Analysis
ULl
SP60 I, SP96 SP70
I
VOLUME 71, 1979
2
3
4
5
6
Migration (mm) FIG. 3. Densitometer traces of separated spore coat proteins. Slab gels of 7 to 18% polyacrylamide stained either for glycoproteins with the PAS technique (upper trace) or for total proteins with Coomassie blue (lower trace) were analyzed with a microdensitometer. The distance toward the anode is indicated as are the positions of the major spore coat proteins.
Proteolytic processing has been observed to generate several membrane proteins from a common precursor during the early aggregation stage of D. discoideum (Bordier et al., 1978). To determine whether the major spore coat proteins were relat.ed, the bands were cut from SDS-acrylamide gels and the proteins marked with radioactive iodine (Elder et al., 1977a). Fingerprints of the tryptic as well as the chymotryptic peptides generated from SP60, SP68, SP70, SP96, or SP200 showed no similarity. It would appear that these membrane proteins are the products of independent genes. Location
of Spore Coat Proteins
Mature spores appear to be impermeable to exogenous proteases since they germi-
ORLOWSKI
AND
LOOMIS
Spore Coat Proteins
nate well after being incubated in the presence of Pronase. However, we found that several of the major spore coat proteins are cleaved by treatment of intact spores with Pronase (Fig. 4). While SP200 and SP96 remain intact in the spore coats isolated from Pronase-treated spores, SP60, SP68, and SP70 are removed (Fig. 4~). A new
in Dictyostelium
303
band of approximate molecular weight 62,000 occurs in the spore coats of proteasetreated spores which may be a cleavage product of either SP70 or SP68 but is likely to be a fragment of SP70 since it is in higher abundance than SP68. The fragments smaller than molecular weight 56,000 occur over a broad range. Treatment of spores
MWxlO"
68 52
15.5
a
b
c
d
FIG. 4. Sensitivity to Pronase. The spore coat proteins were analyzed along with standards by gradient (‘i to 18%) acrylamide gel electrophoresis. (a) Molecular weight standards; (b) spore coats of untreated spores; (c) spore coats of Pronase-treated spores; (d) spore coats treated with Pronase. 100 pg of protein from each sample was added to the slots.
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DEVELOPMENTAL BIOLOGY VOLUME71, 1979
with Pronase immediately after activation by DMSO likewise resulted in removal of SP60, SP68, and SP70 while SP200 and SP96 were unaffected. All of the major spore coat proteins are degraded when the coats of germinated spores are treated with Pronase (Fig. 4d). Thus, the spore coat proteins SP200 and SP96 are protected from proteolysis in intact spores and in spores activated by DMSO but are susceptible to Pronase following germination, most likely because the enzyme can enter through the germination slit and attack the inner face of the coats.
Another strain, HU26, carries a mutation on linkage group I, sprA, which gives rise to spores which are round rather than the wild-type ellipsoid (Katz and Sussman, 1972). The spore coat proteins do accumulate in this strain and are indistinguishable in size from those of wild-type strain NC-4 (Fig. 5). Thus, the defect in spore formation in the sprA mutant must involve structures other than the major spore coat proteins. DISCUSSION
Several of the major proteins synthesized late in development of Dictyostelium appear to be destined for the spore coat. PuSpore Coat Proteins in Mutant Cells rified spore coats contain five major proThere are many mutant strains of D. teins not found at high abundance in amoediscoideum affecting morphogenesis which bas or stalks. Further analysis by the twohave been isolated and partially character- dimensional (2-D) gel technique of Garrels ized. Not surprisingly strains which fail to and Gibson (1976) using isoelectric focusing aggregate and form neither spores nor stalk in the ranges of pH 5 to 7 and pH 6 to 8 has cells, such as strains WL3, WL4, and WL5, not revealed any further complexity of the do not accumulate the spore coat proteins proteins of approximate molecular weight (Siu et al., 1977). Of more interest are mu- 60,000, 68,000, 70,000, or 96,000 (MacLeod, tant strains which undergo culmination but unpublished). Thus, together with SP200 either fail to form spores or form aberrant which was not seen on these 2-D gels, these five proteins account for the majority of the spores. Recently a strain, HL-6, was isolated protein content of spore coats. As judged from strain NC-4 which undergoes all of by the rate of incorporation of [35S]methithe morphological differentiations up to the onine, the major spore coat proteins each culmination stage but fails to form spores comprise 5-10s of the total protein synthe(Morrissey and Loomis, in preparation). All sis during culmination and thus would be of the cells enter the stalk tube and thereby expected to be synthesized from high-abunconstruct an exceedingly tall stalk up to 2 dance messenger RNA (Siu et al., 1977). cm high. Cells of this strain accumulate all This is probably the consequence of both of the stage-specific enzymes which have the rapid demand for spore coats as well as been monitored including those associated the general decrease in synthetic activity as with culmination and appear to differen- the cells prepare for dormancy within tiate normally through most of develop- spores. The spore coat proteins account for ment (Morrissey and Loomis, in prepara- the majority of the newly synthesized protion). However, none of the spore coat pro- teins in the membranes late in developteins accumulate (Fig. 5). The protein pat- ment. Identification of their final positiontern seen in terminally differentiated cells ing and isolation of them from the bulk of of strain HL-6 is complex and appears iden- other membrane proteins has allowed us to tical to that seen in purified stalks of wild- further characterize them. Of the nine type strain NC-4 (Fig. 5). Cells of this strain membrane proteins whose synthesis was appear to be blocked in the biochemical noted to be stage specific by Siu et al. (1977), we have isolated and individually differentiations specific to spores.
ORLOWSKI AND LOOMIS
Spore Coat Proteins in Dictyostelium
MWxlOd
-
15.5,
e FIG. 5. Terminal differentiation patterns of proteins. Proteins were extracted by SDS and separated by SDS-acrylamide gel electrophoresis. In this experiment the gradient of polyacrylamide was 5 to 15%. (a) Molecular weight standards; (b) the proteins of fully differentiated cells of strain HL-6 which forms only stalk; (c) purified stalks of strain NC-4; (d) purified spore coats of strain HU26; (e) purified spore coats of strain NC4.
characterized four different proteins. One is present in a trimeric complex during aggregation (Bordier et al., 1978) and at least three others are spore coat proteins. The spore coat proteins are synthesized between 15 and 21 hr of development and accumu-
late in the membranes before being incorporated into the spore coat. Judging by the intensity of autoradiograms of the membrane proteins labeled with [““Sjmethionine from 16 to 18 hr of development the spore coat proteins make up to 50% of the
306
DEVELOPMENTAL BIOLOGY
newly synthesized membrane proteins, yet they accumulate to only about 10% of the total protein in the membranes as judged by intensity of Coomassie blue staining. Thus it appears that they are only in transit across the membranes to the spore coats. There appears to be little processing during the transit from the membrane to spore coats since distinct bands are seen in the membrane proteins at the positions of the spore coat proteins. However, there is relatively more of SP60 in the membranes than in the spore coats in proportion to the other proteins present in these structures. The spore coat proteins of molecular weights 68,000 and 96,000 are glycosylated. The major spore coat proteins are not significantly modified during germination since their electrophoretic mobility is the same in ungerminated spores as in the spore coats of germinated spores. The spore coat proteins may be extensively crosslinked by disulfide bonds since we found it was essential to have P-mercaptoethanol present during the extraction to solubilize most of the spore coat proteins. In the absence of @mercaptoethanol only the glycoprotein SP96 was extracted. The major spore coat proteins SP60, SP68, and SP70 are susceptible to attack by external protease and thus appear to be at least partially exposed on the surface of intact spores. Their integrity does not appear to be required for germination, since cleavage by added Pronase did not affect either the rate or extent (>90%) of spore germination. On the other hand, the heavily glycosylated protein SP96 as well as the minor spore coat protein SP200 was protected from proteolysis in intact spores as well as in DMSO-activated spores but SUSceptible in the opened spore coats. It is likely that they are positioned on the inner face of the spore coat opposed to the cellular plasma membrane. Although spores appear as smooth ellipsoidal bodies in the light microscope, in the scanning electron microscope the surface
VOLUME 71, 1979
can be seen to be covered by rough ridges (Loomis, 1975). It is unlikely that the overall shape is controlled by self-assembly of the spore coat proteins. Nevertheless, these specific proteins serve as a valuable molecular marker for terminal differentiation of spores and may lead us to a better understanding of the biochemical processes involved in encapsulation. We thank David Finney for technical assistance, John Elder for peptide analyses, and Carol MacLeod for two-dimensional gel analyses. This work was supported by Grant GB 28955 from the National Science Foundation and Grant GM 19543 from the Public Health Service. REFERENCES BORDIER,C., LOOMIS,W. F., ELDER, J., and LERNER, R. (1978). The major developmentally regulated protein complex in membranes of Dictyostelium. J. Biol. Chem. 253,5133-5138. BRUNETTE, D. M., and TILL, J. F. (1971). A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Membrane Biol. 5, 215-225. COTTER,D., MIURA-SANTA, L., and HOHL, H. (1969). Ultrastructural changes during germination of Dictyostelium discoideum spores. J. Bacterial. 100, 1020-1026. ELDER, J., JENSEN, F., BRYANT, M., and LERNER, R. (1977a). Polymorphism of the major envelope glycoprotein (gp70) of murine C-type viruses: Virion associated and differentiation antigens encoded by a multi-gene family. Nature (London) 267, 23-28. ELDER, J., PICKETT, R., HAMPTON, J., and LERNER, R. (1977b). Radioiodination of proteins in single polyacrylamide gel slices: Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein, J. Biol. Chem. 252,6510-6515. ENNIS, M., and SUSSMAN,M. (1975). Mutants of Dictyostelium discoideum defective in spore germination. J. Bacterial. 124, 62-64. FAIRBANKS, G., STECK, T., and WALLACH, D. (1971). Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10,2606-2609. FREEZE,H., and LOOMIS,W. F. (1978). Chemical analysis of stalk components of Dictyostelium. Biochim. Biophys. Acta 539,529-537. GARRELS, J., and GIBSON, W. (1976). Identification and characterization of multiple forms of actin. Cell 9, 793-805. HOHL, H., and HAMMAMOTO,S. (1969). Ultrastructure of spore differentiation in Dictyostelium: The pre-
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spore vacuole. J. Ultrastruct. Res. 26, 442-453. KATZ, E., and SUSSMAN, M. (1972). Parasexual recombination in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 69,495-498. LAEMMLI, V. K. (1970). Cleavage of structural proteins during assembly of the bacteriophage T4. Nature (London) 227,680-685. LOOMIS, W. F (1975). “Dictyostelium discoideum.” Academic Press, New York. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275.
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ROSNESS, P., and WRIGHT, B. (1974). In UCL’Ochanges of cellulose, trehalose, and glycogen during differentiation of Dictyostelium discoideum. Arch. Biothem. Biophys. 64,60-72. SIU, C. H., LERNER, R., and LOOMIS, W. F. (1977). Rapid accumulation and disappearance of plasma membrane proteins during development of wildtype and mutant strains of Dictyostelium. .I. Mol. Biol. 116,469-473. SUSSMAN, M. (1966). Biochemical and genetic methods in the study of cellular slime mold development. in “Methods in Cell Physiology” (D. Prescott, ed.), Vol. 2, pp. 397-410. Academic Press, New York.
BIOLOGY
71,
Plasma Membrane
297-307
(1979)
Proteins of Dictyostelium:
The Spore Coat Proteins
MICHAEL ORLOWSKI' AND WILLIAM F. LOOMIS Department
of Biology,
University
Received November
of California,
San Diego, La Jolla,
California
20, 1978; accepted in revised form March
8, 1979
92093
Spore coats were isolated following germination of Dictyostelium discoideum spores. They were found to contain five major proteins of apparent molecular weights of 60,000 (SP60),68,000 (SP68), 70,000 (SP70), 96,000 (SP96), and 209,000 (SP209). Of these SP68 and SP96 were found to be glycoproteins. Analysis of the tryptic and chymotryptic fingerprints indicated that the major spore coat proteins are independent gene products and not derived by proteolytic cleavage of common percursors. These spore coat proteins are the major newly synthesized proteins seen in membranes late in development and account for the majority of the changes seen in membranes during culmination. They can be observed in membranes isolated from spores, most likely while in transit to the spore coat. SP60, SP68, and SP70 appear to be on the outer face of the spore coat since they are sensitive to proteolysis by Pronase added to intact spores while SP96 and SP200 are protected until the spore case is broken during germination. The spore coat proteins are not found in stalk cells or in several mutant strains blocked in spore formation including one strain, HL-6, which undergoes many of the biochemical differentiations of culmination but forms only stalk cells, The spore coat proteins do accumulate in a spore shape mutant, HU26, which forms small round spores rather than ellipsoid ones. Characterization of the spore coat proteins further defines the changes in membrane proteins during development of Dictyostelium and the processes of terminal differentiation in spores.
the electron microscope the spore coat is Development of Dictyostelium discoi- 1000-1500 A thick and appears to be condeum leads the majority of cells from amoe- structed from an outer amorphous layer 200-300 A thick, a central fibrous layer of bas to encapsulated spores. Following the initiation of development the amoebas ag- cellulose 700-900 A thick, and an inner electron-dense layer 100-300 A thick gregate into groups of about lo5 cells which closely opposed to the plasma membrane become integrated into slug-shaped orga(Hohl and Hammamoto, 1969; Cotter et al., nisms. After about 18 hr of development, 1969; Loomis, 1975). It is likely that specific the anterior 20% of the cells construct a proteins are incorporated into the spore stalk perpendicular to the substratum coat along with the cellulose fibrils. which lifts the rest of the cells into the air. When the pattern of total cellular protein During this process of culmination, each of synthesis is monitored during development the cells in the posterior 80% of the slug by labeling proteins with [““Slmethionine constructs a thick extracellular coat around for 2 hr at various stages and then separatitself. Both the spore coats and the stalk ing them by electrophoresis on SDS-acrylwalls contain cellulose which makes up about 5% of the total dry weight and gives amide gels, it can be seen that the rates of them structural integrity (Rosness and synthesis of proteins banding at seven positions corresponding to molecular weights Wright, 1974; Freeze and Loomis, 1978). In of 40,000 to 100,000 increase dramatically during culmination (Siu et al., 1977). Four ’ Present address: Department of Microbiology, Louisiana State University, Baton Rouge, La. 70803. of these bands are found in purified plasma 297 INTRODUCTION
OO12-1606/79/080297-11$02.00 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
298
DEVELOPMENTAL BIOLOGY VOLUME71, 1979
membranes and make up more than half of the newly synthesized membrane proteins after 16 hr of development as judged by the rate of incorporation of [35S]methionine (Siu et al., 1977). These proteins accumulate in the membranes during culmination and can be seen as intense bands when electrophoretically separated membrane proteins are stained with Coomassie brilliant blue. We have considered that these may be proteins destined for the spore coat which are seen in transit across the plasma membrane. When the spores germinate, they swell slightly and then split longitudinally. The amoebas crawl out of the slit leaving the split spore coats behind intact. By isolating these spore coats we have been able to analyze their protein composition. MATERIALS
AND METHODS
Strains and cultivation. Cells of the wildtype strain, NC-4, the stalky mutant, HL-6, and the round spore-forming strain, HU-26, were grown in association with Klebsiella aerogenes on nutrient agar plates until most of the bacteria were consumed and then collected for synchronous development on filters or allowed to proceed to terminal differentiation on the plates (Sussman, 1966). Strain HL-6 was isolated as a “stalky” mutant from a mutagenized population of strain NC-4; it differentiates quantitatively into stalk cells (Morrissey and Loomis, unpublished). Strain HU-26 carries the mutation sprA on linkage group I which results in the formation of small round spores rather than the larger wildtype ellipsoid spores (Katz and Sussman, 1972). Preparation of spore coats. Spores which had formed on agar growth plates were collected from the lids of petri dishes after they were sharply rapped on the bench. The spores were washed with distilled water several times by centrifugation at 27,000g for 3 min. The pelleted washed spores which were free of undifferentiated
amoebas and stalks were stored at -20°C until needed. Germination was induced by resuspending the spores at 2 x lo7 ml/in 20 mM potassium phosphate buffer, pH 6.5, containing 20% dimethylsulfoxide (DMSO) (Ennis and Sussman, 1975). After 30 min at 22”C, the activated spores were collected by centrifugation, washed once with buffer, and resuspended at 2 x 107/ml in 20 mJ4 potassium phosphate buffer, pH 6.5, containing penicillin and streptomycin to inhibit bacterial growth. The suspension was shaken for 6 hr at 22°C. After 3 hr, amoebas began to emerge from the spore coats and by 6 hr 80-90s of the spores had germinated leaving split coats behind. After 6 hr of germination the material was concentrated by centrifugation at 27,000g for 10 min and resuspended in 30 ml phosphate buffer. The spores and amoebas were pelleted by centrifugation at 500g for 3 min, and the supernatant containing the spore coats was collected. The pellet was reextracted four times by centrifugation at 500g for 3 min and the supernatants were pooled. Spore coats were pelleted from the combined supernatants by centrifugation at 27,000g for 3 min. Following resuspension in 20 ml of buffer, contaminating spores and amoebas were removed by centrifugation at 500g for 3 min. Small debris was removed from the supernatant by pelleting the spore coats by centrifugation at 3000g for 4 min. By phase-contrast microscopy the final preparation contained less than 0.1% contaminating whole spores or amoebas. Purified spore coats were stored at -20°C until used. Preparation of spores and stalks. Spores were collected from plates as described above and stored at -30°C. Stalks were collected from fruiting bodies which had developed on growth plates by vigorously washing the agar surface with distilled water. Stalks were collected on Nitex 200mesh filters which allowed spores, amoebas,
ORLOWSKI
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Spore Coat Proteins in Dictyostelium
and bacteria to pass (Freeze and Loomis, 1978). The stalks were repeatedly resuspended, shaken, and filtered until free of contaminating cells. Purified stalks were stored at -20°C. Isolation of plasma membranes. Vegetative and developed cells were washed by repeated centrifugation from 20 mM potassium phosphate buffer, pH 6.5, at 270g for 3 min and broken with 50 strokes of a Dounce homogenizer. Washed spores were broken under liquid nitrogen by grinding for 3 min in a mortar and pestle. The plasma membranes were isolated by the method of Brunette and Till (Brunette and Till, 1971) using a two-phase system. The effectiveness of this technique for both vegetative and developed cells of Dictyostelium discoideum has been demonstrated previously (Siu et al., 1977). Polyacrylamide gel electrophoresis. Proteins were extracted by suspending the samples in 1% SDS, 2.5% P-mercaptoethano1 in 25 mM Tris buffer, pH 7.0, and heating to 100°C for 3 min. The samples, which contained 100 pg protein, were centrifuged for 3 min on an Eppendorf microfuge and the supernatants applied to gradient (8 to 17 or 5 to 15%) polyacrylamide slab gels prepared according to the method of Laemmli (1970). The following proteins were used as molecular weight markers: filamin (250,000); spectrin (240,000 and 220,000), myosin (200,000), phosphorylase a (96,000)) bovine serum albumin (68,000), immunoglobulin G y-chain (52,000), carboxypeptidase A (34,000), hemoglobin (15,500). Electrophoresis was performed at 24-mA constant current. Gels were fixed in 50% methanol and 10% acetic acid and stained either with 0.05% Coomassie brilliant blue or with the periodic acid Schiffs reagent (Fairbanks et al., 1975). After destaining the gels were scanned on a Joyce-Loebl microdensitometer and photographed with a Polaroid camera by transmitted light. Peptide analysis. The bands visualized by Coomassie blue staining of electropho-
299
retically separated spore coat proteins were cut from the gels with a razor blade and analyzed by the method of Elder et al. (1977a). Very briefly, the proteins in the slices were labeled with 125Iby chloramineT, washed and dried, and then digested with either trypsin (50 pg/ml) or chymotrypsin (50 pg/ml) overnight at 37°C. The eluted lz51-labeled peptides were then analyzed in two dimensions by autoradiography (Elder et al., 197713). These analyses were generously carried out by Dr. John Elder. Pronase treatment. Whole washed spores were suspended at lO’/ml in 20 mM potassium phosphate buffer, pH 6.5, containing 2 mg/ml Pronase CB for 75 min at 22°C. The spores were then extensively washed and activated in 20% DMSO. Activated spores were collected after a 30 min incubation in 20% DMSO, washed, and treated with 2 mg/ml Pronase for 75 min at 22°C before being extensively washed. The spore coats were collected after 6 hr of germination as described above. Germination efficiency was not affected by this treatment. Purified spore coats (2.5 mg/ml) were likewise treated with 2 mg/ml Pronase in 20 mM potassium phosphate buffer, pH 7.5, for 75 min at 22°C before being washed and extracted. Treatment with Pronase did not affect the appearance of the spore coats observed by phase contrast at 480X on a Zeiss compound microscope. Protein determination. The method of Lowry et al. (1951) was used to measure protein content using crystalline bovine serum albumin as the standard. RESULTS
Plasma membranes were isolated from cells and spores late in development and their proteins separated on SDS-acrylamide gels (Fig. 1). After 26 hr of development the cells had undergone terminal cytodifferentiation to spores and stalk cells. Membranes prepared from spores were free of
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MW x IO
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VOLUME 71, 1979
-3
9668-
SP60
52 -
34 -
t5.5-
0
22 HOURS
26
28
32
OF DEVELOPMENT
FIG. 1. Electrophoretograms of plasma membrane proteins. Plasma membranes were isolated from vegetative cells (0 hr) and cells late in development (22 to 32 hr), solubilized in 1% SDS, reduced with 2.5% ,f3mercaptoethanol, and heated to 100°C for 3 min just before being applied to a slab gel containing a gradient (7 to 18%) of polyacrylamide. Following electrophoresis the slab was stained with Coomassie blue. Each sample contained 100 pg of protein. Proteins of known molecular weights (See Materials and Methods) were also run and their locations are indicated. Culmination was underway by 22 hr and most cells had differentiated into spores by 26 hr.
whole spores, spore coats, stalk material, or cells as judged microscopically. Since the general complement of proteins present in the membranes prepared from these cells was similar to that prepared at earlier stages and is not significantly more complex, the membrane purification technique appears applicable to fully differentiated
cells. While most of the bands extracted from membranes of vegetative cells can still be seen in membranes late in development, a prominent protein of apparent molecular weight 60,000 was found to accumulate after 22 hr of development and to make up the most prominent band in membranes isolated from mature spores. Other major
ORLOWSKI AND LOOMIS
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Spore Coat Proteins in Dictyostelium
bands observed in membranes of developed cells can be seen at the positions of proteins of approximate molecular weights 68,000, 70,000, 96,000, and 200,900 (Fig. 1). The spore coats left behind by germinated spores can be separated from amoebas and ungerminated spores by differential centrifugation (see Materials and Meth-
ods). Protein accounts for 67% of the dry weight of the spore coats and can be quantitatively extracted by 1% SDS and 2.5% /?-mercaptoethanol. When separated on SDS-acrylamide gels by electrophoresis, most of the spore coat protein bands at positions of proteins of molecular weight 60,000, 70,000, 96,000 (Fig. 2b). There are
-
SP200
-
SF’96 SP70 SP60
-
6852-
a
b
c
d
FIG. 2. Spore coat proteins. Proteins were extracted from (b) purified spore coats, (c) whole spores, and (d) membranes of purified spores and analyzed by gradient (7 to 18%) acrylamide electrophoresis. The proteins were stained by Coomassie blue. (a) Marker proteins were co-electrophoresed. The spore coat proteins are indicated by their apparent molecular weight X IO-” prefaced by “SP.”
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also bands at the positions of proteins of molecular weights 68,000 and 200,000. In some electrophoretic analyses a band was seen at the position of molecular weight 94,000, but was not consistently found and may be a consequence of the fact that the 96,000 molecular weight protein is glycosylated (see Fig. 3). These proteins appear to be integral components of the coats since they are not extracted by a 10% solution of the nonionic detergent Triton X-100. We will refer to these spore coat proteins by their apparent molecular weights x 10m3 preceded by the letters “SP.” The spore coat proteins can be seen in extracts of whole spores but make up a smaller proportion of the total complement of proteins (Fig, 2~). The minor bands seen in extracts of spore coats are all found in whole spores and may result from cytoplasmic contaminants adhering to the coats throughout the purification procedure. Therefore, these minor bands are not considered specific to the spore coat. The apparent molecular weight of the spore coat proteins does not noticeably I
I
L
I
I
I
I
n
change during germination, since the major proteins extracted from the coats of germinated spores were found in ungerminated spores as well as in plasma membranes isolated from the mature spores (Fig. 2d). Thus, any major processing of these proteins would have to occur before they accumulate in the membranes and are transferred to the spore coat. Spore coat proteins separated on SDSacrylamide gels were stained for carbohydrate by the PAS technique. SP68 and SP96 are glycoproteins by staining criteria whereas SP60, SP70, and SP200 are not stained. A minor PAS-positive band was seen which migrated slightly more slowly than SP70 and may be a minor spore coat glycoprotein of approximately molecular weight 72,000. Densitometer traces of stained gels indicated that 70 f 5% of the total PAS-positive material was found in the SP96 band (Fig. 3). Densitometer tracings of the bands stained with Coomassie blue showed that SP60 is present at about twice the amount of the other major spore coat proteins and that together they make up more than half of the extracted material (Fig. 3). Peptide Analysis
ULl
SP60 I, SP96 SP70
I
VOLUME 71, 1979
2
3
4
5
6
Migration (mm) FIG. 3. Densitometer traces of separated spore coat proteins. Slab gels of 7 to 18% polyacrylamide stained either for glycoproteins with the PAS technique (upper trace) or for total proteins with Coomassie blue (lower trace) were analyzed with a microdensitometer. The distance toward the anode is indicated as are the positions of the major spore coat proteins.
Proteolytic processing has been observed to generate several membrane proteins from a common precursor during the early aggregation stage of D. discoideum (Bordier et al., 1978). To determine whether the major spore coat proteins were relat.ed, the bands were cut from SDS-acrylamide gels and the proteins marked with radioactive iodine (Elder et al., 1977a). Fingerprints of the tryptic as well as the chymotryptic peptides generated from SP60, SP68, SP70, SP96, or SP200 showed no similarity. It would appear that these membrane proteins are the products of independent genes. Location
of Spore Coat Proteins
Mature spores appear to be impermeable to exogenous proteases since they germi-
ORLOWSKI
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Spore Coat Proteins
nate well after being incubated in the presence of Pronase. However, we found that several of the major spore coat proteins are cleaved by treatment of intact spores with Pronase (Fig. 4). While SP200 and SP96 remain intact in the spore coats isolated from Pronase-treated spores, SP60, SP68, and SP70 are removed (Fig. 4~). A new
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band of approximate molecular weight 62,000 occurs in the spore coats of proteasetreated spores which may be a cleavage product of either SP70 or SP68 but is likely to be a fragment of SP70 since it is in higher abundance than SP68. The fragments smaller than molecular weight 56,000 occur over a broad range. Treatment of spores
MWxlO"
68 52
15.5
a
b
c
d
FIG. 4. Sensitivity to Pronase. The spore coat proteins were analyzed along with standards by gradient (‘i to 18%) acrylamide gel electrophoresis. (a) Molecular weight standards; (b) spore coats of untreated spores; (c) spore coats of Pronase-treated spores; (d) spore coats treated with Pronase. 100 pg of protein from each sample was added to the slots.
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with Pronase immediately after activation by DMSO likewise resulted in removal of SP60, SP68, and SP70 while SP200 and SP96 were unaffected. All of the major spore coat proteins are degraded when the coats of germinated spores are treated with Pronase (Fig. 4d). Thus, the spore coat proteins SP200 and SP96 are protected from proteolysis in intact spores and in spores activated by DMSO but are susceptible to Pronase following germination, most likely because the enzyme can enter through the germination slit and attack the inner face of the coats.
Another strain, HU26, carries a mutation on linkage group I, sprA, which gives rise to spores which are round rather than the wild-type ellipsoid (Katz and Sussman, 1972). The spore coat proteins do accumulate in this strain and are indistinguishable in size from those of wild-type strain NC-4 (Fig. 5). Thus, the defect in spore formation in the sprA mutant must involve structures other than the major spore coat proteins. DISCUSSION
Several of the major proteins synthesized late in development of Dictyostelium appear to be destined for the spore coat. PuSpore Coat Proteins in Mutant Cells rified spore coats contain five major proThere are many mutant strains of D. teins not found at high abundance in amoediscoideum affecting morphogenesis which bas or stalks. Further analysis by the twohave been isolated and partially character- dimensional (2-D) gel technique of Garrels ized. Not surprisingly strains which fail to and Gibson (1976) using isoelectric focusing aggregate and form neither spores nor stalk in the ranges of pH 5 to 7 and pH 6 to 8 has cells, such as strains WL3, WL4, and WL5, not revealed any further complexity of the do not accumulate the spore coat proteins proteins of approximate molecular weight (Siu et al., 1977). Of more interest are mu- 60,000, 68,000, 70,000, or 96,000 (MacLeod, tant strains which undergo culmination but unpublished). Thus, together with SP200 either fail to form spores or form aberrant which was not seen on these 2-D gels, these five proteins account for the majority of the spores. Recently a strain, HL-6, was isolated protein content of spore coats. As judged from strain NC-4 which undergoes all of by the rate of incorporation of [35S]methithe morphological differentiations up to the onine, the major spore coat proteins each culmination stage but fails to form spores comprise 5-10s of the total protein synthe(Morrissey and Loomis, in preparation). All sis during culmination and thus would be of the cells enter the stalk tube and thereby expected to be synthesized from high-abunconstruct an exceedingly tall stalk up to 2 dance messenger RNA (Siu et al., 1977). cm high. Cells of this strain accumulate all This is probably the consequence of both of the stage-specific enzymes which have the rapid demand for spore coats as well as been monitored including those associated the general decrease in synthetic activity as with culmination and appear to differen- the cells prepare for dormancy within tiate normally through most of develop- spores. The spore coat proteins account for ment (Morrissey and Loomis, in prepara- the majority of the newly synthesized protion). However, none of the spore coat pro- teins in the membranes late in developteins accumulate (Fig. 5). The protein pat- ment. Identification of their final positiontern seen in terminally differentiated cells ing and isolation of them from the bulk of of strain HL-6 is complex and appears iden- other membrane proteins has allowed us to tical to that seen in purified stalks of wild- further characterize them. Of the nine type strain NC-4 (Fig. 5). Cells of this strain membrane proteins whose synthesis was appear to be blocked in the biochemical noted to be stage specific by Siu et al. (1977), we have isolated and individually differentiations specific to spores.
ORLOWSKI AND LOOMIS
Spore Coat Proteins in Dictyostelium
MWxlOd
-
15.5,
e FIG. 5. Terminal differentiation patterns of proteins. Proteins were extracted by SDS and separated by SDS-acrylamide gel electrophoresis. In this experiment the gradient of polyacrylamide was 5 to 15%. (a) Molecular weight standards; (b) the proteins of fully differentiated cells of strain HL-6 which forms only stalk; (c) purified stalks of strain NC-4; (d) purified spore coats of strain HU26; (e) purified spore coats of strain NC4.
characterized four different proteins. One is present in a trimeric complex during aggregation (Bordier et al., 1978) and at least three others are spore coat proteins. The spore coat proteins are synthesized between 15 and 21 hr of development and accumu-
late in the membranes before being incorporated into the spore coat. Judging by the intensity of autoradiograms of the membrane proteins labeled with [““Sjmethionine from 16 to 18 hr of development the spore coat proteins make up to 50% of the
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newly synthesized membrane proteins, yet they accumulate to only about 10% of the total protein in the membranes as judged by intensity of Coomassie blue staining. Thus it appears that they are only in transit across the membranes to the spore coats. There appears to be little processing during the transit from the membrane to spore coats since distinct bands are seen in the membrane proteins at the positions of the spore coat proteins. However, there is relatively more of SP60 in the membranes than in the spore coats in proportion to the other proteins present in these structures. The spore coat proteins of molecular weights 68,000 and 96,000 are glycosylated. The major spore coat proteins are not significantly modified during germination since their electrophoretic mobility is the same in ungerminated spores as in the spore coats of germinated spores. The spore coat proteins may be extensively crosslinked by disulfide bonds since we found it was essential to have P-mercaptoethanol present during the extraction to solubilize most of the spore coat proteins. In the absence of @mercaptoethanol only the glycoprotein SP96 was extracted. The major spore coat proteins SP60, SP68, and SP70 are susceptible to attack by external protease and thus appear to be at least partially exposed on the surface of intact spores. Their integrity does not appear to be required for germination, since cleavage by added Pronase did not affect either the rate or extent (>90%) of spore germination. On the other hand, the heavily glycosylated protein SP96 as well as the minor spore coat protein SP200 was protected from proteolysis in intact spores as well as in DMSO-activated spores but SUSceptible in the opened spore coats. It is likely that they are positioned on the inner face of the spore coat opposed to the cellular plasma membrane. Although spores appear as smooth ellipsoidal bodies in the light microscope, in the scanning electron microscope the surface
VOLUME 71, 1979
can be seen to be covered by rough ridges (Loomis, 1975). It is unlikely that the overall shape is controlled by self-assembly of the spore coat proteins. Nevertheless, these specific proteins serve as a valuable molecular marker for terminal differentiation of spores and may lead us to a better understanding of the biochemical processes involved in encapsulation. We thank David Finney for technical assistance, John Elder for peptide analyses, and Carol MacLeod for two-dimensional gel analyses. This work was supported by Grant GB 28955 from the National Science Foundation and Grant GM 19543 from the Public Health Service. REFERENCES BORDIER,C., LOOMIS,W. F., ELDER, J., and LERNER, R. (1978). The major developmentally regulated protein complex in membranes of Dictyostelium. J. Biol. Chem. 253,5133-5138. BRUNETTE, D. M., and TILL, J. F. (1971). A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Membrane Biol. 5, 215-225. COTTER,D., MIURA-SANTA, L., and HOHL, H. (1969). Ultrastructural changes during germination of Dictyostelium discoideum spores. J. Bacterial. 100, 1020-1026. ELDER, J., JENSEN, F., BRYANT, M., and LERNER, R. (1977a). Polymorphism of the major envelope glycoprotein (gp70) of murine C-type viruses: Virion associated and differentiation antigens encoded by a multi-gene family. Nature (London) 267, 23-28. ELDER, J., PICKETT, R., HAMPTON, J., and LERNER, R. (1977b). Radioiodination of proteins in single polyacrylamide gel slices: Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein, J. Biol. Chem. 252,6510-6515. ENNIS, M., and SUSSMAN,M. (1975). Mutants of Dictyostelium discoideum defective in spore germination. J. Bacterial. 124, 62-64. FAIRBANKS, G., STECK, T., and WALLACH, D. (1971). Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10,2606-2609. FREEZE,H., and LOOMIS,W. F. (1978). Chemical analysis of stalk components of Dictyostelium. Biochim. Biophys. Acta 539,529-537. GARRELS, J., and GIBSON, W. (1976). Identification and characterization of multiple forms of actin. Cell 9, 793-805. HOHL, H., and HAMMAMOTO,S. (1969). Ultrastructure of spore differentiation in Dictyostelium: The pre-
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spore vacuole. J. Ultrastruct. Res. 26, 442-453. KATZ, E., and SUSSMAN, M. (1972). Parasexual recombination in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 69,495-498. LAEMMLI, V. K. (1970). Cleavage of structural proteins during assembly of the bacteriophage T4. Nature (London) 227,680-685. LOOMIS, W. F (1975). “Dictyostelium discoideum.” Academic Press, New York. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275.
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ROSNESS, P., and WRIGHT, B. (1974). In UCL’Ochanges of cellulose, trehalose, and glycogen during differentiation of Dictyostelium discoideum. Arch. Biothem. Biophys. 64,60-72. SIU, C. H., LERNER, R., and LOOMIS, W. F. (1977). Rapid accumulation and disappearance of plasma membrane proteins during development of wildtype and mutant strains of Dictyostelium. .I. Mol. Biol. 116,469-473. SUSSMAN, M. (1966). Biochemical and genetic methods in the study of cellular slime mold development. in “Methods in Cell Physiology” (D. Prescott, ed.), Vol. 2, pp. 397-410. Academic Press, New York.