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Cell surface proteins in a cultured cell line of Drosophila melanogaster
Insect Biochem. Vol. 14, No. l, pp. 87 95, 1984 Printed in Great Britain. All fights reserved
0020-1790/84$3.00+ 0.00 Copyright © 1984PergamonPress Ltd
C E L L S U R F A C E P R O T E I N S IN A C U L T U R E D C E L L L I N E OF DROSOPHILA M E L A N O G A S T E R T, K. JOHNSON, L. A. BROWN and R. E. DENELL Division of Biology, Kansas State University, Manhattan, KS 66506, U.S.A.
(Received 14 February 1983) Abstrac~Cell surface proteins and glycoproteins of a cultured Drosophila melanogaster cell line were studied by two-dimensional electrophoresis. Proteins radiolabelled by the lactoperoxidase method were examined for hydrophilic or hydrophobic properties using a phase separation technique, as well as for trypsin sensitivity. The populations of proteins labelled by lactoperoxidase catalyzed radio-iodination were compared to the populations of glyeoproteins labelled by either metabolic incorporation of tritiated glucosamine or by binding of radiolabelled concanavalin A. The general distribution and characteristics of the proteins labelled by the lactoperoxidase method are also discussed. Key Word Index." Radio-iodination, cell surface proteins, glycoproteins, insect cells, Drosophila melanogaster
INTRODUCTION Proteins associated with the external surface of cell membranes and integral membrane proteins with exposed residues are thought to play significant roles in important biological processes such as cell-cell recognition and communication, growth regulations during differentiation and carcinogenesis. Our knowledge concerning the characterization and biological activity of cell-surface proteins is derived primarily from studies of cultured vertebrate cells. The results of these studies have provided important insights into the complex nature of cell membranes and the diversity of functions mediated by membrane proteins. In contrast, only a limited amount of information is available concerning the biochemical composition and characteristics of invertebrate cell membranes. However, invertebrates have been extensively characterized at the developmental and genetic levels. The synthesis of this information with molecular approaches, particular in Drosophila, provides attractive model systems for studying a wide variety of biological phenomena, including the structure and function of the cell surface. Thus a number of laboratories have begun to utilize invertebrates, and particularly insect in vitro cell cultures, to study plasma membrane-associated macromolecules. It has been observed by a number of workers that a variety of insect tissues maintained in organ culture, as well as cells from primary cultures and established cell lines, exhibit alterations in both behaviour and morphology in response to the insect moulting hormone, 20-hydroxyecdysone, and its analogues (Courgeon, 1972; Judy and Marks, 1971; Lanir and Cohen, 1978 ; Oberlander et al., 1981). Such observations suggest that the plasma membrane of these cells may also be affected. Recent work by a number of laboratories using cells derived from Drosophila melanogaster, has shown that fundamental changes in the membrane occur in response to the hormone and its
analogues as assayed by cell adhesion (Cherbas et al., 1980), lectin agglutination (Muckenthaler and FaustoSterling, 1981), and analysis of cell-surface proteins (Dennis and Haustein, 1982; Johnson et al., 1983). The goal of these studies was to further our understanding of the role played by the cell surface in sensing and responding to changes in its environment. The present study describes the use of lactoperoxidase (LPO) catalyzed radio-iodination of proteins and their separation by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to detect and analyze the population of proteins present on external cell membranes of cultured cells of Drosophila melanogaster. The radiolabelled proteins were characterized further on the basis of sensitivity to tryptic digestion and solubility in a nonionic detergent. The population of glycoproteins present was also examined by metabolic labelling with glucosamine and by binding of the radiolabelled lectin, concanavalin A. MATERIALS AND METHODS Chemicals for these experiments were purchased from the following sources: ampholines (LKB) ; urea (Schwart~ Mann); SDS, NP-40 (BDH); DTE, acrylamide, BIS, TEMED, ammonium persulphate (Bio-Rad); enzymes, 20-hydroxyecdysone, lectins and other biochemical reagents (Sigma); 125I(Amersham, sp. act. 17 Ci/mg); D-[1,63H(N)]glucosamine hydrochloride (New England Nuclear, sp. act. 32.5 Ci/mM); and agarose without EEO (Serva). Our studies were initiated with a Kc-H cell line (kindly provided by Dr L. Cherbas of Harvard University) which was derived from the original Kc embryonic cell line of Echalier and Ohanessian (1969). When we received this line, it grew predominantly in suspension. We subsequently isolated by selection, a subline in which most cells are attached to the culture vessel, and this derived subline was utilized for all experiments reported here. Cells were cultured in D22 medium + 10~ofoetal calf serum (Echalier, 1976) in plastic tissue culture flasks (25 cm2, Coming) at 24°C. For all experiments, cells from several culture flasks 87
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T.K. JOHNSON et al.
were pooled, and then used to seed new flasks with an initial 0.005°~{~citric acid-0.005°~o formalin until background colour starts to appear (approx. 10 min); (8) stop development by density of 2 × 106 cells/ml. replacing the developer with 15~/o methanol-5~/o acetic Cell surface proteins were labelled by the radioiodination of exposed tyrosine residues using a modification acid. The above steps are carried out with slow shaking at a of the method of Hynes (1973). After 5 days of culture, cells rate sufficient to maintain gel movement. Stained gels were were harvested by placing them into suspension with a photographed and then dried for autoradiography if they rubber policeman, and then collected and washed three contained radio-iodinated proteins. 12Si.labelled con-A was used to localize glycoproteins which bind this lectin. times in cold Drosophila saline (Ephrussi and Beadle, Concanavalin A labelled by the method of Greenwood et al. 1936) followed by centrifugation for 2 rain at 800 g at 4°C. (1963) (100ltCi/mg) was kindly provided by Drs D. Cells from each flask were resuspended in 0.25 ml of a Groswald and P. Kelly (Division of Biology, Kansas State solution containing 12Sl (600pCi/ml), glucose oxidase University). Gels containing 2D separated proteins from (0.2 units/ml), and lactoperoxidase (LPO) (40 #g/ml) in Gehring's salt solution (Poodry and Bryant, 1971) for 15 unlabelled cells were placed in 50% methanol-10~ acetic rain. The labelling reaction was terminated by collecting acid overnight (12 18 hr) to fix the proteins. The next day the gels were washed five times in distilled water, 10 min each, the cells by centrifugation at 800g for 2 rain at 4:'C and washing the cells three times in Drosophila saline in which and three times in buffer (150 mM NaCI-1 mM CAC12-20 mM Tris-HC1, pH 7.0), 30 min each. Following washing, NaCI had been replaced by Nal. As a control, cells were the gels were equilibrated in 150 ml/gel of buffer containing treated in the same manner except that the enzymes, LPO 50 itCi of labelled con-A for 3 hr, washed six times in buffer, and glucose oxidase, were omitted from the reaction mixture. 30 min each, and dried on filter paper prior to autoTotal cellular glycoproteins were labelled by adding 1 pCi radiography. of tritiated glucosamine (32.5 Ci/mM)/ml of culture to Proteins radiolabelled with 125I or binding 125I-labelled cultures 72 hr after initiation. Forty-eight hours thereafter con-A were detected by autoradiography using DuPont the cells were harvested using a rubber policeman, collected Lighting-Plus intensifying screens. Gels containing 3Hby centrifugation at 800g for 2 min at 4 C and washed labelled proteins were impregnated with a scintillator for three times in Drosophila saline with recentrifugation. For experiments which did not involve the incorporation of autofluorography following the procedure of Bonnet and Lasky (1974). Both autoradiography and autofluorography radioisotopes into cell proteins, unlabelled cells were were performed at 7 0 C using Kodak X-Omat film. collected and washed in the same manner. The washed The film was processed following the manufacturer's cells were solubilized in 40 Ill of 1~i; (w/v) SDS-20 mM recommendation using Kodak GBX developer• DTE, treated with 40 pl of micrococcal nuclease (50/g/ml), The nature of the proteins labelled by the LPO method was and lypholized. Samples were resolubilized in isoelectric investigated by examining the consequences of trypsin focusing buffer (9.5 M urea, 100mM DTE, 0.1 mM treatment on the pattern of labelled proteins and the phenylthiourea, 5 mM phenylmethylsulphonyl fluoride, 0.8,°/,, pH 4 6 ampholine, 0.8°~i pH 5-7 ampholine, 0.4~i~ solubility of these' proteins in a nonionic detergent. To assess the effect of trypsin on proteins labelled by the LPO 3.5-10 ampholine and 4°~, NP-40) and analyzed using a method, cells were treated with trypsin either before or modification of the two-dimensional electrophoretic proafter radio-iodination. Cells were suspended in 0.5 ml of cedure of O'Farrell (1975). Proteins were run on 13 cm Drosophila saline containing various concentrations of long, 2.5 mm internal diameter isoelectric focusing gels Difco Bacto-Trypsin, and incubated on ice for 15 rain. The (5% acrylamide, 9.5°~, urea, 0.8°~, p H 4 - 6 ampholine, cells were then washed three times in Drosophila saline and 0.8% pH 5-7 ampholine, 0.4~, pH 3•5-10 ampholine and analyzed by the methods already described. The partition 2 ~ NP-40) for 15 hr using a power supply set for constant of amphiphilic membrane proteins from hydrophilic propower with a starting voltage of 400 V and programmed for teins during the phase separation of the nonionic detergent a maximum voltage of 750 V. The gels were then focused for 2 h r at 1125 V, extruded into equilibration buffer (10°/O Triton X-114 was recently demonstrated by Bordier (1981) and using that technique a phase separation of radio(v/v) glycerol, 5 0 p M DTE, 2.5~o (w/v) SDS and 0.125 M iodinated Drosophila proteins was performed. The resulting Tris HC1, pH 6.8), frozen immediately and stored at aqueous and detergent phases were recovered, washed and 70~'C until run in the second dimension. The isoelectric lypholized. The lypholized samples were resolubilized in focusing gel was affÉxed with a 1};, (w/v) agarose solution in isoelectric focusing sample buffer and analyzed by 2Dequilibration buffer without DTE to the top of a 10~, PAGE as described above. Prior to autoradiography the separating gel 1.5 mm thick and 15 cm long with a 5 mm pattern of total proteins in the gels was examined by silver high stacking gel prepared according to the method of staining as described above• Laemmli (1970), and run at 30 mA per gel until the phenol red tracking dye reached the bottom. The patterns of proteins separated by 2D-PAGE were RESULTS detected using several methods. Gels containing radiolabelled proteins in which total proteins were not localized The LPO technique is a well established m e t h o d for by post treatment of the gel were dried immediately after •the selective incorporation of radioisotope into proteins electrophoresis. Total protein profiles were examined in located on the outer surfaces of vertebrate cell memsome gels using a modifiication of the silver staining techbranes with little or no incorporation into other nique of Poehling and Neuhoff (1981). Gels to be silver stained were placed in 125 ml/gel of 50},; (v/v) methanol: 10~0 proteins. We felt that the identity of the population of proteins labelled by this technique on Drosophila cells (v/v) acetic acid immediately after electrophoresis and maintained in solution for 48 hr or longer, with a change of under the conditions used in the present study required solution after 24hr, to remove compounds (such as confirmation. T o this end, experiments were carried o u t glycine and DTE) which interfere with staining and to fix the to assess the a m o u n t o f radiolabel i n c o r p o r a t e d as a proteins. The gels were stained using the following protocol : direct consequence of the action o f lactoperoxidase a n d (1) wash in 15~o (v/v) methanol three times, 20 min each; the location of proteins labelled by LPO catalyzed (2) soak in fresh 10?/O (w/v) glutaraldehyde, 30min; (3) radio-iodination. In control experiments, cells inwash in 15'?~,(v/v) methanol three times, 30 rain each; (4) cubated under n o r m a l labelling conditions in the wash in distilled water, 10 rain; (5) incubate for 15 min in absence of lactoperoxidase h a d less than 0.1% of the silver stain, 125 ml/gel (made by adding 0.25 g of AgNO3 to a m o u n t o f 1251 associated with L P O labelled cells. A 125 ml of 0.08°~i NaOH-0,6~o NHaOH); (6) wash in distilled water, 5 min and transfer to clean dish ; (7) develop in comparison of the patterns of proteins labelled by the
I soeleetric f o c u s i n g
03 r~ (13
Fig. 1. The effect of trypsin treatment on the pattern of radiolabelled proteins recovered. (A) cells untreated. (B) cells treated with 1.0~/otrypsin following radio-iodination. (C) cells treated with 1.0~ trypsin prior to radiolabelling. (D) cells treated with 0.25~,, trypsin prior to radiolabelling.
89
Fig. 2. Phase separation of cell proteins. (A) total proteins profile (silver stained) from unfractionated cells. (B) autoradiograph of gel A. (C) total protein in aqueous fraction. (D) autoradiograph of gel C. (E) total protein in Triton X-114 fraction. (F) autoradiograph of gel E. (G) total protein in residue fraction. (H) autoradiograph of gel G.
A
B [] lo
1
A
f|
4
|
Fig. 3. Cell surface proteins and glycoproteins. (A) autoradiograph of proteins radiolabelled by the lactoperoxidase method. (B) composite representation of proteins labelled by lactoperoxidase catalyzed radio-iodination, glucosamine incorporation and/or lectin binding. Spots enclosed in a square ([]) are proteins labelled by all three methods, spots indicated by the symbol A are proteins labelled by LPO radioiodination and by glucosamine incorporation, spots indicated by the symbol • are proteins labelled by LPO radio-iodination and by lectin binding, and unlabelled spots are those proteins which are labelled by glucosamine incorporation and by lectin binding. (C) autofluorograph of labelled proteins from cells incubated for 48 hr in tritiated glucosamine. (D) autoradiograph of 12Si.labelled concanavalin A binding to separated proteins from unlabelled cells.
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Drosophila cell surface proteins
LPO method with the pattern of silver stained total cellular proteins indicated that only a small subset of total proteins is radiolabelled (data not shown). The physical location of the labelled proteins was assayed in a series of experiments examining the release of label from radio-iodinated cells by mild proteo|ysis. Cells labelled by the LPO method were treated for 15 min with various concentrations (0.0012.5~o) of trypsin prior to solubilization and analysis by 2D-PAGE and autoradiography. Cell morphology as assayed by phase contrast microscopy did not appear to be altered by exposure to trypsin, although concentrations of 1.0~o or greater resulted in agglutination of the cells. When compared with 2D gel patterns from cells not subjected to proteolysis, cells treated with trypsin concentrations of 0.001 or 0.005~ display a decrease in the amount of incorporated label recovered in resolvable protein species and concentrations of 0.02~ or greater result in the loss of almost all labelled proteins. A comparison of autoradiographs of proteins from control and treated cells (Fig. 1A and B) clearly shows that most if not all of the 160 labelled species present on control cells are reduced below the limits of detection by exposure to trypsin. The release of incorporated label by concentrations as low as 0.02~o is significant in light of the fact that typsin concentrations of 0.02 to 0.2~o are routinely used during subculturing of various insect cell lines (Hinks, 1979 ; Schneider and Blumenthal, 1978). In addition, the effect of exposure to the same range of trypsin concentrations (0.001-2.5~o) prior to labelling was also examined. Treatment with increasing concentrations in the range of 0.001 to 0.2~ results in the progressive modification of the pattern of labelled proteins. At concentrations of 0.2~ or greater the population of iodinatable proteins is reduced to a total of 71 protein species identifiable in autoradiographs of 2D gels (compared to the 160 species recovered from untreated control cells) (Fig. 1A, C and D). These 71 species consist of 55 species which appear to correspond to proteins resolved in control gels and 16 species without obvious counterparts in control cells. We also investigated whether the labelled proteins are associated with the external surface of the cell membrane (peripheral proteins) or are components of the membrane per se with hydrophobic domains inserted into the lipid bilayer (integral membrane proteins). Bordier (1981) has recently shown that the phase separation of solutions containing the nonionic detergent Triton X-114 is an effective method for the partition ofhydrophilicand amphiphilic (hydrophobic) proteins. This technique takes advantage of the formation of micelles by proteins with hydrophobic domains in association with detergents, whereas hydrophilic proteins do not require association with the detergent to maintain their solubility. Thus, when detergent solutions undergo a phase separation, proteins containing hydrophobic domains (integral membrane proteins) are partitioned into the detergent phase while hydrophilic proteins remain associated with the aqueous phase. When labelled material is fractionated by this method the distribution of radioactivity recovered was nonrandom; 27~o aqueous phase, 40~ detergent phase and 30~o in residue (material not solubilized during the detergent extraction). Figure 2 shows 2D gel patterns
of radiolabelled proteins recovered in the aqueous, detergent and residue fractions; gel patterns of unfractionated labelled proteins and total proteins of each fraction are included for comparison. For reasons to be discussed later, a total of only 72 proteins were radiolabelled by the LPO method in this experiment. Of the 72 labelled species present, a total of 56 were recovered in sufficient quantity to be detected following fractionation. These proteins are distributed approximately equally between the aqueous (28) and detergent (21) phases, with the remainder (7) being associated with the unsolubilized material. We were also interested in assessing the presence of glycosylated proteins in the cells being studied. Thus, cells were grown in the presence of tritiated glucosamine for 48 hr and proteins separated by 2D-PAGE. An autofluorograph of the resulting gel pattern is presented in Fig. 3C. A total of 36 glycoproteins were resolved by a four month exposure and other less abundant species could presumably be detected with longer exposures. When compared to a 2D gel pattern of radio-iodinated proteins (Fig. 3A), 12 of the tritiated glycoproteins appear to correspond to protein species labelled by the LPO method. A subset of the population of total cellular glycoproteins was also identified by lectin binding to proteins separated by 2D-PAGE. A total of 73 proteins bound sufficient ~zSI-conjugated con-A to be resolved by autoradiography (Fig. 3D). A comparison of the profile of radio-iodinated (Fig. 3A) and con-A binding (Fig. 3D) proteins reveals 11 species which appear to be labelled by both methods. A comparison of the autoradiographs of 2D gel patterns of proteins labelled by these three techniques reveals a group of 9 proteins which appear to be labelled by both methods used to identify glycoproteins as well as by the LPO technique.
DISCUSSION
Several recent reports (Goldstein and McIntosh, 1980; Butters and Hughes, 1980; Dennis and Haustein, 1981; Johnson et al., 1983) have shown that LPO catalyzed radio-iodination effectively labels a large number of presumptive external cell surface proteins in various cultured insect cell lines. The present studies have focused on the population of proteins radioiodinated by the method of Hynes (1973) and resolved by 2D-PAGE (O'Farrell, 1975). Work using vertebrate cells has demonstrated that the incorporation of label is a result of the action of the exogenous LPO and that this relatively large macromolecule (mol. wt 84,000) is effectively excluded from the interior of the cells, such that only exposed external cell surface proteins are accessible for radio-iodination. Several kinds of evidence provide confirmation that similar groups of proteins are labelled on D. melanogaster cells under the conditions utilized here. Firstly, the failure to incorporate significant amounts of label in the absence of exogenous LPO indicates that there is no significant level of endogenous peroxidase activity capable of mediating the iodination reaction. Secondly, radiolabelled proteins represent a small subset of total cellular proteins as resolved by silver staining (Fig. 2A, B and additional data not shown). Thirdly, incorporated label is sensitive to release by mild proteolysis
94
T.K. JOHNSONet al.
under conditions which maintain cellular integrity (Fig. 1). Thus, we can conclude that the LPO method specificially detects the cell surface proteins of these insect cells. We are presently able to identify over 160 species in autoradiographs of 2D gels of proteins labelled by the LPO technique. However, we are examining only limited pH and molecular weight ranges during 2D-Page and an unknown number of proteins would be expected to fall outside the ranges examined. Similarly, the number of cell surface proteins present but not labelled by the LPO method as a consequence of conformation, glycosylation, etc., is not known. Thus, in the absence of data from an independent approach, it is not possible to determine what proportion of total cell surface proteins are resolved by this method. Many vertebrate cell surface proteins are known to be glycosylated. Several studies have shown that insect cells contain glycosylated proteins (Metakovski et al., 1977; Rizki et al., 1977; Goldstein and McIntosh, 1980). However, the one dimensional SDS-PAGE analysis employed in these studies allows only limited insight into the actual number and distribution of these glycoproteins. Using metabolic labelling and lectin binding to 2D separated proteins we are able to identify a total of 94 cellular glycoproteins. Comparisons of 2D gel patterns of glycosylated proteins with the pattern of LPO radiolabelled proteins indicate that at least 14 of the glycoproteins are located on the cell surface. Since these methods identify neither all cell surface proteins nor all glycoproteins, a more definitive conclusion concerning the proportion of external cell surface proteins which are glycosylated is not possible at present. Finally, two experiments reflect on the organization of the cell surface of this insect material. Mild proteolysis of cells before the cell surface proteins were labelled by the LPO method results in marked changes in the 2D pattern resolved when compared to patterns from untreated control cells or cells treated with trypsin after iodination. Treatment of cells with dilute solutions of trypsin (0.02~) results in the disappearance of approximately one-half of the proteins detected in 2D gels of proteins from control cells. A total of 55 of the 71 proteins detected on pretreated cells appear to be shared with control cells, with the remaining 16 representing proteins without obvious counterparts on control cells. This observation is significant in several respects. Firstly, the reduction in the number of proteins available for LPO catalyzed labelling as a result of exposure to dilute trypsin solutions provides confirmatory evidence for the peripheral location of the proteins labelled by the LPO technique. Secondly, the failure to radio-iodinate cytoplasmic proteins under any of the conditions examined shows the inability of this method to label internal proteins even in severely trypsinized cells. Thirdly, only 16 "new" polypeptides are detected in 2D gels ofpretreated cells. The available data do not allow us to state with any certainty whether these polypeptides represent degradation products of proteins detected on untreated control cells or whether they represent proteins normally present but inaccessible to labelling by the LPO method in untreated cells. Based on the small number of "new" peptides observed, the maximum number of polypeptide species
not resolved by LPO labelling because they are masked by other proteins must be relatively low. In addition, the solubility and thereby the presumptive location of the radiolabelled proteins was also examined. Using the phase separation technique of Bordier (1981), we fractionated the population of labelled proteins on the basis of their hydrophilic or hydrophobic properties. Our results indicated that within the population of labelled proteins, hydrophilic (peripheral) proteins are slightly more prevalent than amphiphilic (hydrophobic, integral membrane) proteins. Two important caveats concerning this partition experiment must be noted. Firstly, the total number of proteins labelled was less than the number of proteins labelled in other experiments (72 vs 1604 ). Distinct qualitative changes in the profile of proteins labelled in a Drosophila cell line have been noted by at least one other laboratory (Dennis and Haustein, 1981), although the basis of such changes has not been identified. Secondly, since there is some dilution of sample during the fractionation procedure some minor species are not recovered in sufficient quantities to be detected in our system. Although our conclusions are limited to those proteins resolved following fractionation, our results clearly indicate that a significant proportion of the proteins identified represent integral membrane proteins having exposed polypeptides on the external cell surface, with the remainder representing membrane associated peripheral proteins. Acknowledgements--We would like to express our appreciation to Dr W. A. Ramoska of the Department of Entomology, Kansas State University for his advice and instruction concerning insect tissue culture. This work was supported by grant GM-27195 from the National Institutes of Health.
REFERENCES
Bonner W. M. and Lasky R. A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88. Bordier C. (1981) Phase separation of integral membrane proteins in Triton X-114 solution. J. biol. Chem. 256, 1604 1607. Butters T. D. and Hughes R. C. (1980) Isolation and characterization of mosquito cell membrane glycoproteins. Biochim. biophys. Acta 640, 655-671. Cherbas L., Yonge C. D., Cherbas P. and Williams C. M. (1980) The morphological response of Kc-H cells to ecdysteroids: hormonal specificity. Wilhelm Roux's Archs 189, 1 15.
Courgeon A. M. (1972) Action of insect hormones at the cellular level. Morphological changes of a diploid cell line of Drosophila melanogaster treated with ecdysone and several analogues in vitro. Expl Cell Res. 74, 327 336. Dennis R. and Haustein D. (1981) Radioiodination of cell surface proteins in a Drosophila cell line. Insect Biochem. It, 699 705. Dennis R. and Haustein D. (1982) Ecdysteroid-related changes in cell-surface properties of a Drosophila cell line. Insect Biochem. 12, 83 89. Echalier G. (1976) In vitro established lines of Drosophila cells and applications in physiological genetics. In Invertebrate Tissue Culture, p. 131. Academic Press, New York. Echalier G. and Ohanessian A. (1969) lsolement, en cultures
Drosophila cell surface proteins in vitro, de lignees cellulaires diploides de Drosophila melanogaster. C.r. Acad. Sci. 268, 1771 1773. Ephrussi B. and Beadle G. W. (1936) A technique of transplantation in Drosophila. Am. Nat. 70, 218 225. Goldstein N. I. and McIntosh A. H. (1980) The proteins and glycoproteins of insect cells in culture. Insect Biochem. 10, 419~27, Greenwood F. C., Hunter W. M. and Glover J. S. (1963) The preparation of ~3~I-labelled human growth hormone of high specific radioactivity, Bioehem. J. 89, 114--123. Hinks W. F. (1979) Cell lines from invertebrates. Meth. Enzym. 58, 450~,66. Hynes R. O. (1973) Alteration of cell-surface proteins by viral transformation and by proteolysis. Proc. nam. Acad. Sci., U.S.A. 70, 3170~3174. Johnson T. K., Brown L. A. and Denell R. E. (1983) Changes in cell surface proteins of cultured Drosophila cells exposed to 20-hydroxyecdysone. Wilhelm Roux's Archs 192, 103-107. Judy K. J. and Marks E. P. (1971) Effects of ecdysone in vitro on hindgut and hemocytes of Manduca sexta (Lepidoptera). Gen. comp. Endocr. 17, 351-359. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lanir N. and Cohen E. (1978) Studies on the effect of the
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moulting hormone in a mosquito cell line. J. Insect Physiol. 24, 613-621. Metakovski E. V., Cherdanteseva E. M. and Gvozdev V. A. (1977) Action of ecdysterone on surface membrane glycoproteins of Drosophila melanogaster cells in culture. Molec. Biol. 11, 158-170. Muckenthaler F. A. and Fausto-Sterling, A. (1981) Normal and lectin-mediated agglutination in a Drosophila cell line. Wilhelm Roux's Archs 190, 118-122. O'Farrell P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. biol. Chem. 250, 4007 4021. Oberlander H., Leach C. E. and Lynn D. E. (1981) Effects of cyclohexamide on cell elongation in a Manduca sexta cell line. Wilhelm Roux's Arehs 190, 61-66. Poehling H. M. and Neuhoff V. (198l) Visualization of proteins with a silver "stain": A critical analysis. EleCtrophoresis 2, 141 147. Poodry C. and Bryant P. J. (1971) Intercellular adhesivity and pupal morphogenesis in Drosophila melanogaster. Wilhelm Roux's Archs 168, 1 9. Rizki T. M., Rizki R. M. and Andrews C. A. (1977) The surface features of Drosophila embryonic cell lines. Devl Growth Differ. 19, 354 356. Schneider I. and Blumenthal A. B. (1978) Drosophila cell and tissue culture. In The Genetics and Biology gf Drosophila, Vol. 2a, p. 265. Academic Press. New York.
0020-1790/84$3.00+ 0.00 Copyright © 1984PergamonPress Ltd
C E L L S U R F A C E P R O T E I N S IN A C U L T U R E D C E L L L I N E OF DROSOPHILA M E L A N O G A S T E R T, K. JOHNSON, L. A. BROWN and R. E. DENELL Division of Biology, Kansas State University, Manhattan, KS 66506, U.S.A.
(Received 14 February 1983) Abstrac~Cell surface proteins and glycoproteins of a cultured Drosophila melanogaster cell line were studied by two-dimensional electrophoresis. Proteins radiolabelled by the lactoperoxidase method were examined for hydrophilic or hydrophobic properties using a phase separation technique, as well as for trypsin sensitivity. The populations of proteins labelled by lactoperoxidase catalyzed radio-iodination were compared to the populations of glyeoproteins labelled by either metabolic incorporation of tritiated glucosamine or by binding of radiolabelled concanavalin A. The general distribution and characteristics of the proteins labelled by the lactoperoxidase method are also discussed. Key Word Index." Radio-iodination, cell surface proteins, glycoproteins, insect cells, Drosophila melanogaster
INTRODUCTION Proteins associated with the external surface of cell membranes and integral membrane proteins with exposed residues are thought to play significant roles in important biological processes such as cell-cell recognition and communication, growth regulations during differentiation and carcinogenesis. Our knowledge concerning the characterization and biological activity of cell-surface proteins is derived primarily from studies of cultured vertebrate cells. The results of these studies have provided important insights into the complex nature of cell membranes and the diversity of functions mediated by membrane proteins. In contrast, only a limited amount of information is available concerning the biochemical composition and characteristics of invertebrate cell membranes. However, invertebrates have been extensively characterized at the developmental and genetic levels. The synthesis of this information with molecular approaches, particular in Drosophila, provides attractive model systems for studying a wide variety of biological phenomena, including the structure and function of the cell surface. Thus a number of laboratories have begun to utilize invertebrates, and particularly insect in vitro cell cultures, to study plasma membrane-associated macromolecules. It has been observed by a number of workers that a variety of insect tissues maintained in organ culture, as well as cells from primary cultures and established cell lines, exhibit alterations in both behaviour and morphology in response to the insect moulting hormone, 20-hydroxyecdysone, and its analogues (Courgeon, 1972; Judy and Marks, 1971; Lanir and Cohen, 1978 ; Oberlander et al., 1981). Such observations suggest that the plasma membrane of these cells may also be affected. Recent work by a number of laboratories using cells derived from Drosophila melanogaster, has shown that fundamental changes in the membrane occur in response to the hormone and its
analogues as assayed by cell adhesion (Cherbas et al., 1980), lectin agglutination (Muckenthaler and FaustoSterling, 1981), and analysis of cell-surface proteins (Dennis and Haustein, 1982; Johnson et al., 1983). The goal of these studies was to further our understanding of the role played by the cell surface in sensing and responding to changes in its environment. The present study describes the use of lactoperoxidase (LPO) catalyzed radio-iodination of proteins and their separation by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to detect and analyze the population of proteins present on external cell membranes of cultured cells of Drosophila melanogaster. The radiolabelled proteins were characterized further on the basis of sensitivity to tryptic digestion and solubility in a nonionic detergent. The population of glycoproteins present was also examined by metabolic labelling with glucosamine and by binding of the radiolabelled lectin, concanavalin A. MATERIALS AND METHODS Chemicals for these experiments were purchased from the following sources: ampholines (LKB) ; urea (Schwart~ Mann); SDS, NP-40 (BDH); DTE, acrylamide, BIS, TEMED, ammonium persulphate (Bio-Rad); enzymes, 20-hydroxyecdysone, lectins and other biochemical reagents (Sigma); 125I(Amersham, sp. act. 17 Ci/mg); D-[1,63H(N)]glucosamine hydrochloride (New England Nuclear, sp. act. 32.5 Ci/mM); and agarose without EEO (Serva). Our studies were initiated with a Kc-H cell line (kindly provided by Dr L. Cherbas of Harvard University) which was derived from the original Kc embryonic cell line of Echalier and Ohanessian (1969). When we received this line, it grew predominantly in suspension. We subsequently isolated by selection, a subline in which most cells are attached to the culture vessel, and this derived subline was utilized for all experiments reported here. Cells were cultured in D22 medium + 10~ofoetal calf serum (Echalier, 1976) in plastic tissue culture flasks (25 cm2, Coming) at 24°C. For all experiments, cells from several culture flasks 87
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T.K. JOHNSON et al.
were pooled, and then used to seed new flasks with an initial 0.005°~{~citric acid-0.005°~o formalin until background colour starts to appear (approx. 10 min); (8) stop development by density of 2 × 106 cells/ml. replacing the developer with 15~/o methanol-5~/o acetic Cell surface proteins were labelled by the radioiodination of exposed tyrosine residues using a modification acid. The above steps are carried out with slow shaking at a of the method of Hynes (1973). After 5 days of culture, cells rate sufficient to maintain gel movement. Stained gels were were harvested by placing them into suspension with a photographed and then dried for autoradiography if they rubber policeman, and then collected and washed three contained radio-iodinated proteins. 12Si.labelled con-A was used to localize glycoproteins which bind this lectin. times in cold Drosophila saline (Ephrussi and Beadle, Concanavalin A labelled by the method of Greenwood et al. 1936) followed by centrifugation for 2 rain at 800 g at 4°C. (1963) (100ltCi/mg) was kindly provided by Drs D. Cells from each flask were resuspended in 0.25 ml of a Groswald and P. Kelly (Division of Biology, Kansas State solution containing 12Sl (600pCi/ml), glucose oxidase University). Gels containing 2D separated proteins from (0.2 units/ml), and lactoperoxidase (LPO) (40 #g/ml) in Gehring's salt solution (Poodry and Bryant, 1971) for 15 unlabelled cells were placed in 50% methanol-10~ acetic rain. The labelling reaction was terminated by collecting acid overnight (12 18 hr) to fix the proteins. The next day the gels were washed five times in distilled water, 10 min each, the cells by centrifugation at 800g for 2 rain at 4:'C and washing the cells three times in Drosophila saline in which and three times in buffer (150 mM NaCI-1 mM CAC12-20 mM Tris-HC1, pH 7.0), 30 min each. Following washing, NaCI had been replaced by Nal. As a control, cells were the gels were equilibrated in 150 ml/gel of buffer containing treated in the same manner except that the enzymes, LPO 50 itCi of labelled con-A for 3 hr, washed six times in buffer, and glucose oxidase, were omitted from the reaction mixture. 30 min each, and dried on filter paper prior to autoTotal cellular glycoproteins were labelled by adding 1 pCi radiography. of tritiated glucosamine (32.5 Ci/mM)/ml of culture to Proteins radiolabelled with 125I or binding 125I-labelled cultures 72 hr after initiation. Forty-eight hours thereafter con-A were detected by autoradiography using DuPont the cells were harvested using a rubber policeman, collected Lighting-Plus intensifying screens. Gels containing 3Hby centrifugation at 800g for 2 min at 4 C and washed labelled proteins were impregnated with a scintillator for three times in Drosophila saline with recentrifugation. For experiments which did not involve the incorporation of autofluorography following the procedure of Bonnet and Lasky (1974). Both autoradiography and autofluorography radioisotopes into cell proteins, unlabelled cells were were performed at 7 0 C using Kodak X-Omat film. collected and washed in the same manner. The washed The film was processed following the manufacturer's cells were solubilized in 40 Ill of 1~i; (w/v) SDS-20 mM recommendation using Kodak GBX developer• DTE, treated with 40 pl of micrococcal nuclease (50/g/ml), The nature of the proteins labelled by the LPO method was and lypholized. Samples were resolubilized in isoelectric investigated by examining the consequences of trypsin focusing buffer (9.5 M urea, 100mM DTE, 0.1 mM treatment on the pattern of labelled proteins and the phenylthiourea, 5 mM phenylmethylsulphonyl fluoride, 0.8,°/,, pH 4 6 ampholine, 0.8°~i pH 5-7 ampholine, 0.4~i~ solubility of these' proteins in a nonionic detergent. To assess the effect of trypsin on proteins labelled by the LPO 3.5-10 ampholine and 4°~, NP-40) and analyzed using a method, cells were treated with trypsin either before or modification of the two-dimensional electrophoretic proafter radio-iodination. Cells were suspended in 0.5 ml of cedure of O'Farrell (1975). Proteins were run on 13 cm Drosophila saline containing various concentrations of long, 2.5 mm internal diameter isoelectric focusing gels Difco Bacto-Trypsin, and incubated on ice for 15 rain. The (5% acrylamide, 9.5°~, urea, 0.8°~, p H 4 - 6 ampholine, cells were then washed three times in Drosophila saline and 0.8% pH 5-7 ampholine, 0.4~, pH 3•5-10 ampholine and analyzed by the methods already described. The partition 2 ~ NP-40) for 15 hr using a power supply set for constant of amphiphilic membrane proteins from hydrophilic propower with a starting voltage of 400 V and programmed for teins during the phase separation of the nonionic detergent a maximum voltage of 750 V. The gels were then focused for 2 h r at 1125 V, extruded into equilibration buffer (10°/O Triton X-114 was recently demonstrated by Bordier (1981) and using that technique a phase separation of radio(v/v) glycerol, 5 0 p M DTE, 2.5~o (w/v) SDS and 0.125 M iodinated Drosophila proteins was performed. The resulting Tris HC1, pH 6.8), frozen immediately and stored at aqueous and detergent phases were recovered, washed and 70~'C until run in the second dimension. The isoelectric lypholized. The lypholized samples were resolubilized in focusing gel was affÉxed with a 1};, (w/v) agarose solution in isoelectric focusing sample buffer and analyzed by 2Dequilibration buffer without DTE to the top of a 10~, PAGE as described above. Prior to autoradiography the separating gel 1.5 mm thick and 15 cm long with a 5 mm pattern of total proteins in the gels was examined by silver high stacking gel prepared according to the method of staining as described above• Laemmli (1970), and run at 30 mA per gel until the phenol red tracking dye reached the bottom. The patterns of proteins separated by 2D-PAGE were RESULTS detected using several methods. Gels containing radiolabelled proteins in which total proteins were not localized The LPO technique is a well established m e t h o d for by post treatment of the gel were dried immediately after •the selective incorporation of radioisotope into proteins electrophoresis. Total protein profiles were examined in located on the outer surfaces of vertebrate cell memsome gels using a modifiication of the silver staining techbranes with little or no incorporation into other nique of Poehling and Neuhoff (1981). Gels to be silver stained were placed in 125 ml/gel of 50},; (v/v) methanol: 10~0 proteins. We felt that the identity of the population of proteins labelled by this technique on Drosophila cells (v/v) acetic acid immediately after electrophoresis and maintained in solution for 48 hr or longer, with a change of under the conditions used in the present study required solution after 24hr, to remove compounds (such as confirmation. T o this end, experiments were carried o u t glycine and DTE) which interfere with staining and to fix the to assess the a m o u n t o f radiolabel i n c o r p o r a t e d as a proteins. The gels were stained using the following protocol : direct consequence of the action o f lactoperoxidase a n d (1) wash in 15~o (v/v) methanol three times, 20 min each; the location of proteins labelled by LPO catalyzed (2) soak in fresh 10?/O (w/v) glutaraldehyde, 30min; (3) radio-iodination. In control experiments, cells inwash in 15'?~,(v/v) methanol three times, 30 rain each; (4) cubated under n o r m a l labelling conditions in the wash in distilled water, 10 rain; (5) incubate for 15 min in absence of lactoperoxidase h a d less than 0.1% of the silver stain, 125 ml/gel (made by adding 0.25 g of AgNO3 to a m o u n t o f 1251 associated with L P O labelled cells. A 125 ml of 0.08°~i NaOH-0,6~o NHaOH); (6) wash in distilled water, 5 min and transfer to clean dish ; (7) develop in comparison of the patterns of proteins labelled by the
I soeleetric f o c u s i n g
03 r~ (13
Fig. 1. The effect of trypsin treatment on the pattern of radiolabelled proteins recovered. (A) cells untreated. (B) cells treated with 1.0~/otrypsin following radio-iodination. (C) cells treated with 1.0~ trypsin prior to radiolabelling. (D) cells treated with 0.25~,, trypsin prior to radiolabelling.
89
Fig. 2. Phase separation of cell proteins. (A) total proteins profile (silver stained) from unfractionated cells. (B) autoradiograph of gel A. (C) total protein in aqueous fraction. (D) autoradiograph of gel C. (E) total protein in Triton X-114 fraction. (F) autoradiograph of gel E. (G) total protein in residue fraction. (H) autoradiograph of gel G.
A
B [] lo
1
A
f|
4
|
Fig. 3. Cell surface proteins and glycoproteins. (A) autoradiograph of proteins radiolabelled by the lactoperoxidase method. (B) composite representation of proteins labelled by lactoperoxidase catalyzed radio-iodination, glucosamine incorporation and/or lectin binding. Spots enclosed in a square ([]) are proteins labelled by all three methods, spots indicated by the symbol A are proteins labelled by LPO radioiodination and by glucosamine incorporation, spots indicated by the symbol • are proteins labelled by LPO radio-iodination and by lectin binding, and unlabelled spots are those proteins which are labelled by glucosamine incorporation and by lectin binding. (C) autofluorograph of labelled proteins from cells incubated for 48 hr in tritiated glucosamine. (D) autoradiograph of 12Si.labelled concanavalin A binding to separated proteins from unlabelled cells.
91
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Drosophila cell surface proteins
LPO method with the pattern of silver stained total cellular proteins indicated that only a small subset of total proteins is radiolabelled (data not shown). The physical location of the labelled proteins was assayed in a series of experiments examining the release of label from radio-iodinated cells by mild proteo|ysis. Cells labelled by the LPO method were treated for 15 min with various concentrations (0.0012.5~o) of trypsin prior to solubilization and analysis by 2D-PAGE and autoradiography. Cell morphology as assayed by phase contrast microscopy did not appear to be altered by exposure to trypsin, although concentrations of 1.0~o or greater resulted in agglutination of the cells. When compared with 2D gel patterns from cells not subjected to proteolysis, cells treated with trypsin concentrations of 0.001 or 0.005~ display a decrease in the amount of incorporated label recovered in resolvable protein species and concentrations of 0.02~ or greater result in the loss of almost all labelled proteins. A comparison of autoradiographs of proteins from control and treated cells (Fig. 1A and B) clearly shows that most if not all of the 160 labelled species present on control cells are reduced below the limits of detection by exposure to trypsin. The release of incorporated label by concentrations as low as 0.02~o is significant in light of the fact that typsin concentrations of 0.02 to 0.2~o are routinely used during subculturing of various insect cell lines (Hinks, 1979 ; Schneider and Blumenthal, 1978). In addition, the effect of exposure to the same range of trypsin concentrations (0.001-2.5~o) prior to labelling was also examined. Treatment with increasing concentrations in the range of 0.001 to 0.2~ results in the progressive modification of the pattern of labelled proteins. At concentrations of 0.2~ or greater the population of iodinatable proteins is reduced to a total of 71 protein species identifiable in autoradiographs of 2D gels (compared to the 160 species recovered from untreated control cells) (Fig. 1A, C and D). These 71 species consist of 55 species which appear to correspond to proteins resolved in control gels and 16 species without obvious counterparts in control cells. We also investigated whether the labelled proteins are associated with the external surface of the cell membrane (peripheral proteins) or are components of the membrane per se with hydrophobic domains inserted into the lipid bilayer (integral membrane proteins). Bordier (1981) has recently shown that the phase separation of solutions containing the nonionic detergent Triton X-114 is an effective method for the partition ofhydrophilicand amphiphilic (hydrophobic) proteins. This technique takes advantage of the formation of micelles by proteins with hydrophobic domains in association with detergents, whereas hydrophilic proteins do not require association with the detergent to maintain their solubility. Thus, when detergent solutions undergo a phase separation, proteins containing hydrophobic domains (integral membrane proteins) are partitioned into the detergent phase while hydrophilic proteins remain associated with the aqueous phase. When labelled material is fractionated by this method the distribution of radioactivity recovered was nonrandom; 27~o aqueous phase, 40~ detergent phase and 30~o in residue (material not solubilized during the detergent extraction). Figure 2 shows 2D gel patterns
of radiolabelled proteins recovered in the aqueous, detergent and residue fractions; gel patterns of unfractionated labelled proteins and total proteins of each fraction are included for comparison. For reasons to be discussed later, a total of only 72 proteins were radiolabelled by the LPO method in this experiment. Of the 72 labelled species present, a total of 56 were recovered in sufficient quantity to be detected following fractionation. These proteins are distributed approximately equally between the aqueous (28) and detergent (21) phases, with the remainder (7) being associated with the unsolubilized material. We were also interested in assessing the presence of glycosylated proteins in the cells being studied. Thus, cells were grown in the presence of tritiated glucosamine for 48 hr and proteins separated by 2D-PAGE. An autofluorograph of the resulting gel pattern is presented in Fig. 3C. A total of 36 glycoproteins were resolved by a four month exposure and other less abundant species could presumably be detected with longer exposures. When compared to a 2D gel pattern of radio-iodinated proteins (Fig. 3A), 12 of the tritiated glycoproteins appear to correspond to protein species labelled by the LPO method. A subset of the population of total cellular glycoproteins was also identified by lectin binding to proteins separated by 2D-PAGE. A total of 73 proteins bound sufficient ~zSI-conjugated con-A to be resolved by autoradiography (Fig. 3D). A comparison of the profile of radio-iodinated (Fig. 3A) and con-A binding (Fig. 3D) proteins reveals 11 species which appear to be labelled by both methods. A comparison of the autoradiographs of 2D gel patterns of proteins labelled by these three techniques reveals a group of 9 proteins which appear to be labelled by both methods used to identify glycoproteins as well as by the LPO technique.
DISCUSSION
Several recent reports (Goldstein and McIntosh, 1980; Butters and Hughes, 1980; Dennis and Haustein, 1981; Johnson et al., 1983) have shown that LPO catalyzed radio-iodination effectively labels a large number of presumptive external cell surface proteins in various cultured insect cell lines. The present studies have focused on the population of proteins radioiodinated by the method of Hynes (1973) and resolved by 2D-PAGE (O'Farrell, 1975). Work using vertebrate cells has demonstrated that the incorporation of label is a result of the action of the exogenous LPO and that this relatively large macromolecule (mol. wt 84,000) is effectively excluded from the interior of the cells, such that only exposed external cell surface proteins are accessible for radio-iodination. Several kinds of evidence provide confirmation that similar groups of proteins are labelled on D. melanogaster cells under the conditions utilized here. Firstly, the failure to incorporate significant amounts of label in the absence of exogenous LPO indicates that there is no significant level of endogenous peroxidase activity capable of mediating the iodination reaction. Secondly, radiolabelled proteins represent a small subset of total cellular proteins as resolved by silver staining (Fig. 2A, B and additional data not shown). Thirdly, incorporated label is sensitive to release by mild proteolysis
94
T.K. JOHNSONet al.
under conditions which maintain cellular integrity (Fig. 1). Thus, we can conclude that the LPO method specificially detects the cell surface proteins of these insect cells. We are presently able to identify over 160 species in autoradiographs of 2D gels of proteins labelled by the LPO technique. However, we are examining only limited pH and molecular weight ranges during 2D-Page and an unknown number of proteins would be expected to fall outside the ranges examined. Similarly, the number of cell surface proteins present but not labelled by the LPO method as a consequence of conformation, glycosylation, etc., is not known. Thus, in the absence of data from an independent approach, it is not possible to determine what proportion of total cell surface proteins are resolved by this method. Many vertebrate cell surface proteins are known to be glycosylated. Several studies have shown that insect cells contain glycosylated proteins (Metakovski et al., 1977; Rizki et al., 1977; Goldstein and McIntosh, 1980). However, the one dimensional SDS-PAGE analysis employed in these studies allows only limited insight into the actual number and distribution of these glycoproteins. Using metabolic labelling and lectin binding to 2D separated proteins we are able to identify a total of 94 cellular glycoproteins. Comparisons of 2D gel patterns of glycosylated proteins with the pattern of LPO radiolabelled proteins indicate that at least 14 of the glycoproteins are located on the cell surface. Since these methods identify neither all cell surface proteins nor all glycoproteins, a more definitive conclusion concerning the proportion of external cell surface proteins which are glycosylated is not possible at present. Finally, two experiments reflect on the organization of the cell surface of this insect material. Mild proteolysis of cells before the cell surface proteins were labelled by the LPO method results in marked changes in the 2D pattern resolved when compared to patterns from untreated control cells or cells treated with trypsin after iodination. Treatment of cells with dilute solutions of trypsin (0.02~) results in the disappearance of approximately one-half of the proteins detected in 2D gels of proteins from control cells. A total of 55 of the 71 proteins detected on pretreated cells appear to be shared with control cells, with the remaining 16 representing proteins without obvious counterparts on control cells. This observation is significant in several respects. Firstly, the reduction in the number of proteins available for LPO catalyzed labelling as a result of exposure to dilute trypsin solutions provides confirmatory evidence for the peripheral location of the proteins labelled by the LPO technique. Secondly, the failure to radio-iodinate cytoplasmic proteins under any of the conditions examined shows the inability of this method to label internal proteins even in severely trypsinized cells. Thirdly, only 16 "new" polypeptides are detected in 2D gels ofpretreated cells. The available data do not allow us to state with any certainty whether these polypeptides represent degradation products of proteins detected on untreated control cells or whether they represent proteins normally present but inaccessible to labelling by the LPO method in untreated cells. Based on the small number of "new" peptides observed, the maximum number of polypeptide species
not resolved by LPO labelling because they are masked by other proteins must be relatively low. In addition, the solubility and thereby the presumptive location of the radiolabelled proteins was also examined. Using the phase separation technique of Bordier (1981), we fractionated the population of labelled proteins on the basis of their hydrophilic or hydrophobic properties. Our results indicated that within the population of labelled proteins, hydrophilic (peripheral) proteins are slightly more prevalent than amphiphilic (hydrophobic, integral membrane) proteins. Two important caveats concerning this partition experiment must be noted. Firstly, the total number of proteins labelled was less than the number of proteins labelled in other experiments (72 vs 1604 ). Distinct qualitative changes in the profile of proteins labelled in a Drosophila cell line have been noted by at least one other laboratory (Dennis and Haustein, 1981), although the basis of such changes has not been identified. Secondly, since there is some dilution of sample during the fractionation procedure some minor species are not recovered in sufficient quantities to be detected in our system. Although our conclusions are limited to those proteins resolved following fractionation, our results clearly indicate that a significant proportion of the proteins identified represent integral membrane proteins having exposed polypeptides on the external cell surface, with the remainder representing membrane associated peripheral proteins. Acknowledgements--We would like to express our appreciation to Dr W. A. Ramoska of the Department of Entomology, Kansas State University for his advice and instruction concerning insect tissue culture. This work was supported by grant GM-27195 from the National Institutes of Health.
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moulting hormone in a mosquito cell line. J. Insect Physiol. 24, 613-621. Metakovski E. V., Cherdanteseva E. M. and Gvozdev V. A. (1977) Action of ecdysterone on surface membrane glycoproteins of Drosophila melanogaster cells in culture. Molec. Biol. 11, 158-170. Muckenthaler F. A. and Fausto-Sterling, A. (1981) Normal and lectin-mediated agglutination in a Drosophila cell line. Wilhelm Roux's Archs 190, 118-122. O'Farrell P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. biol. Chem. 250, 4007 4021. Oberlander H., Leach C. E. and Lynn D. E. (1981) Effects of cyclohexamide on cell elongation in a Manduca sexta cell line. Wilhelm Roux's Arehs 190, 61-66. Poehling H. M. and Neuhoff V. (198l) Visualization of proteins with a silver "stain": A critical analysis. EleCtrophoresis 2, 141 147. Poodry C. and Bryant P. J. (1971) Intercellular adhesivity and pupal morphogenesis in Drosophila melanogaster. Wilhelm Roux's Archs 168, 1 9. Rizki T. M., Rizki R. M. and Andrews C. A. (1977) The surface features of Drosophila embryonic cell lines. Devl Growth Differ. 19, 354 356. Schneider I. and Blumenthal A. B. (1978) Drosophila cell and tissue culture. In The Genetics and Biology gf Drosophila, Vol. 2a, p. 265. Academic Press. New York.