VIROLOGY
65,226237
(1975)
Alterations
in Membrane
Polypeptides
Induced
by Transformation
Fibroblasts
Sarcoma TOMOE Faculty
ISAKA,
of Pharmaceutical
of Chick Embryo with Avian
Viruses
MITSUAKI
YOSHIDA
Sciences, University
of Tokyo, Hongo, Japan
AND
MASATOSHI Department
OWADA,
of Tumor Viruses, Research Institute
KUMAO
AND
for Microbial Japan
Accepted January
TOYOSHIMA
Diseases, Osaka Uniuersity,
Suita City, Osaka,
29, 1975
Membrane proteins of chick embryo fibroblasts (CEF) transformed with various strains of avian sarcoma viruses were analyzed by electrophoresis in SDS-polyacrylamide gels and compared with those of untransformed cells. The following differences were consistently detected in CEF transformed with B77, the Prague strain of Rous sarcoma virus (PR-RSV) or the Schmidt-Ruppin strain of RSV (SR-RSV): (1) The appearance of a polypeptide band with an apparent molecular weight of 90,000, (2) increase in amount of a polypeptide of 79,000 daltons, (3) significant decrease in amount of a polypeptide of 50,000 daltons and (4) marked decrease in amount of a protein of 200,000 daltons. CEF infected with the temperature-sensitive (ts) mutants of these strains, LA334 (of B77), LA31 (of PR-RSV) or OS122 (of SR-RSV) showed similar changes at 36”, but at 41”, except for alteration (4), the profiles of the membrane proteins were similar to those of uninfected cells. Changes (1) and (3) were reversible and clearly observable within a few hours after a temperature shift of CEF infected with ts mutants. Fusiform transformation induced by a variant of B77 was also shown to induce alterations (1) and (3). From these and other results, the appearance of the polypeptide band of 90,000 daltons, which could not be detected in untransformed cells, and the marked decrease in amount of a protein of 50,090 daltons in cell membranes were concluded to be closely correlated with transformation of CEF. INTRODUCTION
Alterations in membrane components have been reported in RSV-transformed cells; namely alterations in glycoprotein (Warren et al., 1972, 1973), glycolipids (Hakomori et al., 1971), carbohydrates of the cell surface (Hartmann et al., 1972; Perdue et al., 1972), and polypeptides (Hynes, 1973). Bussell and Robinson (1973) demonstrated that the amount of a major protein component of uninfected cell membranes, with a molecular weight of 142,000, decreased greatly on transformation of cells with the Bryan high-titer strain of RSV. Wickus and Robbins (1973)
Rous sarcoma virus (RSV) induces alterations of cell morphology (Manaker and Group& 1956), loss of contact inhibition of cell movement (Stoker and Rubin, 1967), increased activity of sugar transport (Hatanaka and Hanafusa, 1970), increased agglutinability by plant lectins (Burger and Martin, 1972), and reduced activity of membrane-bound adenylate cyclase (Anderson et al., 1973a,b; Yoshida et al., 1975). These changes suggest that transformation by RSV is associated with alterations in cell membranes. 226 Copyright All rights
0 1975 by Academic Press. of reproduction in any form
Inc. reserved.
MEMBRANE
PROTEINS
OF RSV-TRANSFORMED
also reported that another protein component with a molecular weight of 45,000 decreased when cells were transformed with the Schmidt-Ruppin strain of RSV (SR-RSV). Very recently, Stone et al. (1974) reported a remarkable increase in amount of a polypeptide of 73,000 daltons, a small increase in amount of a polypeptide of 95,000 daltons, and decreases in the amounts of proteins of 250,000 and 39,000 daltons in cells transformed by avian sarcoma viruses. This paper describes the appearance of a membrane polypeptide band with apparent molecular weight of 90,000 in polyacrylamide-gel electrophoresis, a remarkable increase in the amount of a 79,000-dalton polypeptide and decrease in amount of a protein of 50,000 daltons when CEF were transformed by avian sarcoma viruses. A protein with a molecular weight of 90,000 was detected as one of the major components in transformed cell membranes but was not detectable in uninfected cells. Furthermore, these changes were shown to be rapidly reversible when cells infected with temperature-sensitive (ts) mutants were shifted between the permissive (36”) and nonpermissive (41”) temperature. Our findings are similar to those of Stone et al. (1974), except that they observed only a small increase in the amount of protein of 95,000 daltons, which was a main component in uninfected cell membranes. This band seems to correspond to our proteins of 91,000 and 90,000 daltons. We found that the protein of 91,000 daltons was a main component of uninfected and transformed cells, while the protein of 90,000 daltons was only visible in transformed cells. MATERIALS
AND
METHODS
Cells and viruses. Chick embryo fibroblast (CEF) cultures were prepared from 11-day-old C/E chick embryos, as described by Vogt (1969), and grown in loo-mm plastic dishes in complete medium, consisting of Eagle’s minimal essential medium (MEM; Nissui), 10% tryptose phosphate broth, 5% calf serum, 0.5% chick serum, and 0.056% sodium bicarbonate. Infected CEF were usually transferred once before analysis to induce extensive trans-
CELLS
227
formation of cultured cells. The permissive temperature for ts mutants of avian sarcoma viruses in their transforming capacity was 36”, and the nonpermissive temperature was 41”. After formation of monolayers, 1% dimethylsulfoxide was added to culture media. LA31, a ts mutant of the Prague strain of RSV, PR-RSV-A, was a generous gift from Dr. P. K. Vogt and was characterized previously (Wyke and Linial, 1973). LA334 and OS122, the ts mutants of B77 and SR-RSV-D, have been reported previously (Toyoshima and Vogt, 1969; Owada and Toyoshima, 1973; Toyoshima et al., 1973). QV-2, a subgroup E variant of B77 which will be described elsewhere, induced fusiform transformation when C/E CEF were infected with the RAV-1 pseudotype of QV-2. Labeling of cultures. Normal and transformed cells were labeled with L[35S]methionine (145 Ci/mmole, Radiochemical Centre, Amersham, England) at a concentration of 3.5 j&i/ml in complete medium for about 20 hr. In the experiment on the temperature shift of cultures, L[35S]methionine was added at a concentration of 7 &X/ml when the temperature was shifted, and, 6-7 hr later, the cells were used for preparation of membranes. Preparation of the membrane fraction. A crude membrane fraction was prepared essentially by the method described by Atkinson and Summers (1971), as modified by Bussell and Robinson (1973). In most experiments, one loo-mm dish of a secondary or tertiary culture was used for preparation of membranes. The culture medium was decanted, and the cells were rinsed twice with phosphate-buffered saline (PBS). Then they were transferred to about 5 ml of PBS using a rubber policeman and washed twice by centrifugation at 250 g for 6 min. The pellet was resuspended in the homogenizing medium (15 mM iodoacetate, 10 mM NaN,, 10 mM Tris-HCl, pH 8.0) and kept at room temperature for 5-8 min. Then the cells were homogenized with four strokes at 1,000 rpm of a loose fitting Potter homogenizer. The homogenate was layered over a discontinuous sucrose density gradient consisting of 1.5 ml of 45% (w/w) sucrose overlaid with 2.5 ml of
228
ISAKA
30% (w/w) sucrose, both containing 10 mM Tris-HCl (pH 8.0) and 10 mM NaN,, and centrifuged at 10,000 rpm and 4” for 20 min in a Hitachi RPS 50 rotor. The band of membranes at the interphase between the 45 and 30% sucrose layers was used directly as the membrane preparation for electrophoresis. The distribution of enzyme markers for plasma cell membranes, Na+, K+stimulated ATPase (Perdue and &eider, 1970) and adenylate cyclase (Pohl et al., 1971), were measured, and more than 90% of both the enzyme activities were found in the interphase fractions. However, about half of the glucose-6-phosphatase activity, used as marker enzyme for endoplasmic reticulum (Hiibscher and West, 1965), was also found in the interphase fraction. These observations indicated that the membrane fraction used here contained not only plasma cell membrane but also internal membranous structures. The top fraction in the 30% sucrose layer was recentrifuged at 200,000 g for 2 hr in a Hitachi RPS 50 rotor to obtain the 200,000 g supernatant fraction. SDS-polyacrylamide slab-gel electrophoresis. Slab-gel (180 x 150 x 1 mm) electrophoresis was performed as described by Laemmli (1970) with minor modifications. Samples containing up to 50-70 pg of protein, 0.5% sodium dodecyl sulfate (SDS), 2.5% 2-mercaptoethanol, 5% glycerol, 0.001% bromophenol blue, and 0.025 M Tris-HCl, pH 6.8, were boiled for 3 min before electrophoresis. Electrophoresis was carried out for 4 hr at 20 mA. Gels were fixed in 50% trichloroacetic acid (TCA) for 2 hr, stained for 20 min with 0.1% Coomassie brilliant blue in 50% TCA, and destained by diffusion in 7% acetic acidmethanol. After washing overnight, gels were dried on filter paper in vacua and exposed to X-ray film (Kodax RP Royal XOmat) for 2-3 weeks. Optical scanning of stained gels and autoradiograms were carried out with a densitometer (Fujiriken, Model FD-A IV). Cell surface labeling. The cell surface was labeled with carrier-free lz51 by the lactoperoxidase method described by Vitetta et al. (1971). The cells in a 50-mm
ET AL.
petri dish were rinsed with PBS and iodinated for 10 min in 2 ml of PBS containing 100 PCi of carrier-free 1251and 50 ~1 of 0.03% H,O,. The reaction was initiated by addition of an adequate amount of lactoperoxidase and stopped by addition of 10 ml of 5 mM cysteine hydrochloride. During the reaction, two additional volumes of 50 ~1 of 0.03% H,O, were added to the reaction mixture. After the reaction, either the cells were rinsed five times with PBS, scraped off with a rubber policeman and used to prepare the membrane fraction as described above or the whole cells were disrupted with 0.1% SDS solution and used directly as the sample for electrophoresis. Preparation of nuclei. Nuclei were isolated as described by Ohtsuki and Amano (1971) in the presence of detergent to minimize the contamination by cytoplasmic materials. Cells were washed with PBS and then transferred with a rubber policeman into 2 ml of 0.25 M sucrose containing 3 mM CaCl,. Nonidet P-40 was added to the cell suspension to a final concentration of 0.5%. The mixture was allowed to stand for 20 min at 4” and then centrifuged at 750 g for 15 min. The pellet was resuspended in 1 ml of 0.25 M sucrose containing 3 mM CaCl, and 0.2% Nonidet P-40. After standing for 5 min at 4”, the mixture was centrifuged again at 750 g for 15 min. The pellet was resuspended in a small amount of the same solution, layered over 4 ml of 2 M sucrose containing 3 mM CaCl, and 0.2% Nonidet P-40, and centrifuged at 50,000 g for 60 min. The pellet was washed with 0.25 M sucrose-3 mM CaCl, by centrifugation at 750 g for 15 min and used as the nuclear fraction for electrophoresis. RESULTS
Polypeptide Constitution of the Membrane Fraction from CEF Transformed by B77 Chick embryo fibroblasts transformed by avian sarcoma viruses and uninfected CEF were labeled with L- [35S]methionine for 18-24 hr, and a fraction rich in large fragments of cell membrane was isolated by a method similar to that established by Atkinson and Summers (1971) and modi-
MEMBRANE
PROTEINS
OF RSV-TRANSFORMED
fied by Bussell and Robinson (1973). In most experiments, treatment of cells with EDTA was avoided. Figure 1A shows autoradiograms of 6.5% polyacrylamide-SDS gels after electrophoresis of polypeptides labeled with L-
229
[35S]methionine in the membrane fraction isolated from uninfected CEF and CEF transformed with B77. The polypeptide composition of membranes from transformed cells consistently showed the following differences from that of uninfected B
A
(a)
CELLS
(b)
(a)
(b)
FIG. 1. (A) Autoradiograms of 6.5% polyacrylamide-SDS slab-gels after electrophoresis of L-[35S]methionine-labeled proteins of membrane fractions from CEF transformed with B77 (a) and uninfected cells (b). (B) Electropherograms, stained with Coomassie brilliant blue, of membrane proteins from cells transformed by B77 (a) and uninfected cells (b). The numbers refer to the molecular weights in thousands of polypeptide components.
230
ISAKA
cells: (1) The appearance or remarkable increase in amount of polypeptide of 90,000 daltons, (2) significant increase in the amount of a polypeptide of 79,000 daltons, and (3) marked decreases in amounts of polypeptides with molecular weights of 200,000 and 50,000 daltons. The polypeptide of 90,000 daltons could not be detected as a clear visible band in uninfected cells, but it was easily detectable as a new clear band in transformed cells, and its appearance was the most pronounced change in
ET AL.
membrane components. These changes were clearly visible in stained gels (Fig. 1B) and were clearer when the stained gels or autoradiograms were scanned with an optical densitometer and compared semiquantitatively (Fig. 2a). When total cell homogenates were analyzed on 6.5% polyacrylamide-SDS gels, the profiles were obviously more complex. However, a clearly visible band was detected in transformed cells at a position corresponding to a molecular weight of
FIG. 2. Comparison of membrane constituent proteins from uninfected CEF (-----) and from CEF transformed by (a), B77; (b), SR-RSV-A; and (c), PR-RSV-C (-). The gels were stained with Coomassie brilliant blue, and the direction of electrophoresis was from left to right.
MEMBRANE
PROTEINS
OF RSV-TRANSFORMED
231
CELLS
FIG. 3. COXnDariSOn of the protein components of total cell homogenate from CEF transformed by B77 (-) and uninfected CEF (-----). The gels were stained with Coomassie brilliant blue and the direction electrophoresis was from left to right.
90,000, while no clear band was observed in this position in uninfected cells (Fig. 3). These results strongly suggest that the existence of this polypeptide in the membranes was not a reflection of a different distribution of this polypeptide in uninfected and transformed cells but that the polypeptide was a product of new synthesis or proteolytic hydrolysis of high molecular weight protein, which was increased by cell transformation. The amount of the polypeptide of about 200,000 daltons in membranes from uninfected cells was variable: When EDTA was included in the buffer used for washing the cells, its content was especially small. On the other hand, CEF transformed with B77 consistently contained a small amount of this polypeptide. Polypeptide Constitution of the Membrane Fraction from CEF Transformed by SR-RSV and PR-RSV Alterations in polypeptide composition similar to those described above were observed in membrane preparations from CEF transformed with SR-RSV of subgroups A and D or the Prague strain of RSV (PR-RSV) of subgroups A and C (Fig. 2). Again the change in the polypeptide of 90,000 daltons was remarkable. From these results, these changes in membrane proteins appear to be common characteristics
of CEF transformed viruses.
with
of
avian sarcoma
Alterations in Membrane Polypeptides CEF Infected with ts Mutants
of
When CEF are infected with the ts mutants of avian sarcoma viruses, morphological transformation of the cells is reversibly controlled by the temperature of cell cultivation (Toyoshima and Vogt, 1969; Martin, 1970; Kawai and Hanafusa, 1971). To demonstrate that the changes in membrane polypeptides described above are correlated with cell transformation, CEF were infected with the ts mutants, LA334 (of B77), LA31 (of PR-RSV-A) or OS122 (of SR-RSV-D), and grown at 36”. Pairs of cultures were set up and one of each was shifted and maintained at 41’ for more than 25 or 48 hr. Then the cells were harvested and their membrane fractions were prepared. As shown in Fig. 4, when the cells were transformed by LA334 at 36”, all the changes of the membrane polypeptides described above were observed, but when the cells were maintained at 41” for 25 hr, the profile was very similar to that of uninfected cells, except for a decrease in the amount of protein of about 200,000 daltons. The same effects of temperature were observed with infected cells with LA31 or OS122 (Fig. 5). In uninfected cells,
232
ISAKA
ET AL.
B
FIG. 4. Comparison of polypeptide components of membranes from uninfected cells (-----) and cells infected with LA334 (-1. (A), Profiles of membrane components of cells maintained at 41” for more than 24 hr; (B), profiles of membrane components of cells maintained and transformed at 36”. Autoradiograms of membrane polypeptides labeled with L- [3sS]methionine were scanned as described in Materials and Methods.
the profiles of membrane polypeptides were virtually identical at 36 and 41”. The observations that the amount of the protein of 200,000 daltons decreased even at 41” in ts mutant-infected cells indicates that this protein has no direct correlation with regulation of the temperature-sensitive step of these ts mutants. These results suggest that, among the changes in the membrane polypeptides, the appearance of the polypeptide band of 90,006 daltons, increase in the amount of polypeptide of 79,000 daltons and reduction in the amount of protein of 50,000 daltons are related to cell transformation by avian sarcoma viruses but not to viral replication, since LA31 and OS122 can replicate at 41” as efficiently as at 36”.
Effects of Temperature Shift on Changes in Membrane Polypeptides of CEF Infected with LA334 To examine the relationship of these changes to morphological transformation, the time of these changes in membrane polypeptides after the temperature shift 11as studied. When cells at 41” infected with LA334 were shifted to 36”, the morphology of most of the cells was observed to change within a few hours. Seven hours after the shift, the membrane fraction from LA334-infected cells showed a distinct band with a molecular weight of 90,000 (Fig. 6a), which could not be detected at 41” (Fig. 6d), though this band was less than that in cells maintained at 36” (Fig.
MEMBRANE
(a)
(b)
PROTEINS
OF RSV-TRANSFORMED
(a)
CELLS
233
(b)
FIG. 5. Electropheroprams of6.5% polyacrylamide-SDS slab-gels stained with Coomassie brilliant blue. (A) CEF infected with OS122 of SR-RSV-D and maintained at 41” (a) and at 36” (b), for more than 24 hr. (B) CEF infected with LA31 of PR-RSV-A and maintained at 41” (a) and at 36” (b) for more than 24 hr.
234
ISAKA
6b). Decrease in the amount of protein of 50,000 daltons was also noticeable. Upon the reverse shift, the morphology of the transformed cells reverted to that of uninfected cells within a few hours and the
(a)
(b)
(4
(d
-
91 90
-
79
-
50
FIG. 6. Autoradiograms of polya6.5% crylamide-SDS slab-gels after electrophoresis of polypeptides labeled with L- [35S]methionine in membranes from CEF infected with LA334. LA334infected CEF 7 hr after shifting down to 36” from 41” (a) or after shifting up to 41” from 36” (c) and maintained for more than 24 hours at 36” (b) or at 41° (d).
ET AL.
band of polypeptide of 90,000 daltons became significantly less distinct (Fig. 6~). Meanwhile the amount of polypeptide of 50,000 daltons increased to almost the level of that in cells maintained at 41”. Change in amount of the polypeptide with a molecular weight of 79,000 on temperature shift up or down was less than those of the two proteins described above. Thus, the time of appearance of the protein band of 90,000 daltons and marked decrease in amount of protein of 50,000 daltons coincided precisely with the time of morphological transformation. Addition of cycloheximide (5 pg/ml) at the time of the down shift of LA334infected cells inhibited both the appearance of the polypeptide band of 90,000 daltons and transformation. On shift up did not from 36 to 41”, cycloheximide prevent the disappearance of the protein of 90,000 daltons. Alterations in Polypeptide Composition the Membranes of CEF Induced Fusiform Transformation
of by
Uninfected CEF are long, fibroblastictype cells, while cells transformed by B77 or RSV are round. QV-2, a variant of B77, induces fusiform transformation of CEF. It seemed interesting to see whether the changes of membrane polypeptides of CEF induced by fusi- and round-form transformation were the same. As shown in Fig. 7, the profile of membrane polypeptides from QV-2-transformed CEF was very similar to that from B77-transformed cells, that is, both showed a band of 90,000 daltons, increase in the amount of polypeptide of 79,000 daltons and decrease in the amount of 50,000-dalton polypeptide. These results provide evidence for the correlation between the appearance of the polypeptide of 90,000 daltons and decrease in the amount of polypeptide of 50,000 daltons on viral transformation, irrespective of the type of morphology on the transformed cells. Localization of Polypeptide of 90,000 Daltons in Transformed Cells As described above, a protein band corresponding to a molecular weight of 90,000 was a characteristic of transformed cells
MEMBRANE
FIG. 7. Comparison by QV-‘2 (RAV-1) (-).
PROTEINS
OF RSV-TRANSFORMED
of the profiles of membrane proteins from uninfected
and was found in the membrane fraction. After sucrose gradient centrifugation, the top fraction in 30% sucrose contained a protein band with this molecular weight. But this was not detected in the supernatant fluid after centrifugation at 200,000 g for 2 hr. A partially purified nuclear fraction did not show a similar band. These observations suggest that the polypeptide of 90,000 daltons is mainly bound to a membranous structure. This polypeptide of 90,000 daltons was not iodinated by lactoperoxidase-catalyzed iodination (Vitetta et al., 1971) of the cell surface and was not removed from the cells by washing with 1 A4 urea-l mM EDTA, which removed some high molecular weight proteins from the cell surface (Weston and Hendricks, 1972). These results can be explained by supposing that this polypeptide was not exposed on the surface of the cells. DISCUSSION
This paper describes characteristic changes of polypeptides in CEF membranes associated with transformation of cells by avian sarcoma viruses. These changes are (1) the appearance of a polypeptide band with an apparent molecular weight of 90,000 daltons, (2) increase in amount of polypeptide of 79,000 daltons, and (3) marked decrease in amounts of proteins of 50,000 and 200,000 daltons.
CELLS
235
CEF (-----) and cells transformed
These changes were all relatively large so that they could be detected on stained gels, and they were found in CEF transformed by either B77, SR-RSV, or PR-RSV. Among these changes, the appearance of the polypeptide band of 90,000 daltons was especially noticeable, since it was not detected as a clear visible band in gels from uninfected or untransformed cells. These observations suggest that this protein of 90,000 daltons was a membrane polypeptide induced by viral transformation, although the possibility of the presence of an undetectable amount of this polypeptide in uninfected cells is not excluded. When CEF infected with LA334 and maintained at 41” were shifted to 36”, the appearance of the band of 90,000 daltons and marked decrease in the amount of polypeptide of 50,000 daltons were clearly detected within 7 hr, and at this time most of the infected cells were observed to be morphologically transformed. The reverse temperature shift induced changes in the opposite direction within a few hours. LA31, the ts mutant of PR-RSV, and OS122, that of SR-RSV, also showed temperature-dependent induction of changes in membrane polypeptides. These results indicate that these two changes of membrane polypeptides at least are closely correlated with morphological transformation of cultured cells but not with viral replication. This conclusion is also sup-
236
ISAKA
ported by the finding that cycloheximide, an inhibitor of protein synthesis which suppresses cell transformation (Kawai and Hanafusa;l971), also inhibited the appearance of the polypeptide of 90,000 daltons. This polypeptide also appeared in fusiform cells transformed with QV-2, which suggests that it is correlated with viral transformation, irrespective of the type of morphology of the transformed cells. The polypeptide band of 90,000 daltons which was detected only in transformed cells may be mainly in a membrane-bound state since it was not found in either the supernatant fluid after centrifugation at 200,000 g or the nuclear fraction. These results also suggest that the appearance of this polypeptide band is not simply due to a different distribution of this polypeptide in transformed and uninfected cells. The possibility that the polypeptide is formed by proteolytic modification of high molecular weight protein in the membrane cannot be excluded, but it seems likely that morphological transformation of cells induces new synthesis of the polypeptide of 90,000 daltons and that this protein is exclusively incorporated into membrane structures. Other explanations are also possible such as incomplete or abnormal glycosylation of a component which may result in the appearance of the 90,000-dalton protein. The presence of a similar polypeptide with the molecular size of 91,000 daltons in transformed and normal cell membranes may suggest a structural relationship between proteins of 90,000 and 91,000 daltons. However, the observations that the amount of 91,000-dalton protein was not affected by the increasing amount of 90,000-dalton protein leads to the speculation that these two proteins are not related. Attempts to isolate and characterize these proteins are in progress. The appearance of the polypeptide band of 90,000 daltons and increase in the amount of polypeptide of 79,000 daltons seem to correspond to the increase in amounts of proteins of 95,000 and 73,000 daltons, respectively, described by Stone et al. (1974) on transformation by PR-RSV-A and B77. However, they observed only a small increase in the amount of protein of
ET AL.
95,000 daltons, which was the main component in uninfected cells. This discrepancy between their results and ours is due to the better separation achieved under our conditions of gel electrophoresis rather than to the difference in the methods used for membrane preparation, since the protein of 90,000 daltons moved just in front of a main band of 91,000 daltons, which was also a main band in uninfected cells. The results of Stone et al. (1974) on the effects of a temperature shift of cells infected with a ts mutant on these polypeptide changes also differ from ours. We found that more than half the maximum amount of polypeptide of 90,000 daltons appeared within 7 hr of the temperature shift, while they reported that it was detected only after 24 hr. This might be due to a difference in the character of the ts mutant or the conditions used for cell cultures. The significant decrease in the amount of polypeptide of 50,000 daltons induced by transformation probably corresponds to the decrease in the amount of protein of 45,000 daltons in SR-RSV-transformed cells reported by Wickus and Robbins (1973). This change was not observed by Stone et al. (1974), possibly due to marked differences in the methods used to isolate membranes. The method used in this work was separation by centrifugation on a sucrose gradient and was in principle similar to that of Wickus and Robbins (1973), while Stone et al. (1974) used a completely different system, namely a two-phase polymer procedure. The big differences in the profiles they observed in the region of molecular weight higher than 200,000 daltons might also be a result of the different method used to isolate membranes. REFERENCES W. B., JOHNSON, G. S., and PASTAN, I. (1973a). Transformation of chick-embryo fibroblasts by wild-type and temperature-sensitive Rous sarcoma virus alters adenylate cyclase activity. Proc. Nat. Acad. Sci. USA 70, 1055-1059. ANDERSON, W. B., LOVELACE, E., and PASTAN, I. (1973b). Adenylate cyclase activity is decreased in chick embryo fibroblasts transformed by wild type and temperature sensitive Schmidt-Ruppin Rous sarcoma virus. Biochem. Bioph,ys. Res. Commun. 52,1293-1299. ANDERSON,
MEMBRANE
PROTEINS
OF RSV-TRANSFORMED
ATKINSON, P. H., and SUMMERS, D. F. (1971). Purification and properties of HeLa cell plasma membranes. J. Biol. &hem. 246, 5162-5175. BURGER, M. M., and MARTIN, G. S. (1972). Agglutination of cells transformed by Rous sarcoma virus by wheat germ agglutinin and concanavalin A. Nature New Biol. 237, 9-12. BUSSELL, R. H., and ROBINSON, W. S. (1973). Membrane proteins of uninfected and Rous sarcoma virus-transformed avian cells. J. Virol. 12,320-327. FRIIS, R. R., TOYOSHIMA, K., and VOGT, P. K. (1971). Conditional lethal mutants of avian sarcoma viruses. I. Physiology of ts 75 and ts 149. Virology 43, 375-389. HAKOMORI, S.-I., SAITO, T., and VOGT, P. K. (1971). Transformation by Rous sarcoma virus: Effects on cellular glycolipids. Virology 44, 609-621. HARTMANN, J. F., BUCK, C. A., PEFENDI, V., GLICK, M. L. (1972). The carbohydrate C., and WARREN, content of control and virus-transformed cells. J. Cell Physiol. 80, 159-165. HATANAKA, M., and HANAFUSA, H. (1970). Analysis of a functional change in membrane in the process of cell transformation by Rous sarcoma virus: Alteration in the characteristics of sugar transport. Virology 41, 647-652. HOBSCHER, G., and WEST, G. R. (1965). Specific assay of some phosphatases in subcellular fractions of smal. intestinal mucosa. Nature (London) 205, 799800. HYNES, R. 0. (1973). Alteration of cell-surface proteins by viral transformation and by proteolysis. Proc. Nat. Acad. Sci. USA 70, 3170-3174. KAWAI, S., and HANAFUSA, H. (1971). The effects of reciprocal changes on the transformed state of cells infected with a Rous sarcoma virus mutant. Virol0g.v 46, 470-479. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. MANAKER, R. A., and GROUP& V. (1956). Discrete foci altered chicken embryo cells associated with Rous sarcoma virus in tissue culture. Virology 2.838-840. MARTIN, G. S. (1970). Rous sarcoma virus: A function required for the maintenance of the transformed state. Nature (London) 227, 1021-1023. OHTXKI, H., and AMANO, M. (1971). Isolation of nuclei from ascites hepatoma AH-130 cells of rat by a detergent. Gann 62, 427-430. OWADA, M., ~~~TOYOSHIMA, K. (1973). Analysis of the reproducing and cell-transforming capacities of a temperature sensitive mutant (ts 334) of avian sarcoma virus B77. Virology 54, 170-178. PEROUE, J. F., KLETZIEN, R., and WRAY, V. L. (1972). The isolation and characterization of plasma membrane from cultured cells. IV. The carbohydrate composition of membranes isolated from oncogenic RNA virus-converted chick embryo fibroblasts. Bi-
CELLS
237
ochim. Biophys. Acta. 266, 505-510. PERDUE, J. F., and SNEIDER, J. (1970). The isolation and characterization of the plasma membrane from chick embryo fibroblasts. Biochim. Biophys. Acta 196, 125-140. POHL, S. L., BIRNBAUMER, L., and RODBELI., M. (1971). The glucagon-sensitive adenyl cyclase system in plasma membrane of rat liver. I. Properties. J. Biol. Chem. 246, 1849-1856. STOKER, M. G. P., and RUBIN, H. (1967). Density dependent inhibition of cell growth in culture. Nature (London) 215, 171-172. STONE, K. R., SMITH, R. E., and JOKLIK, W. K. (1974). Changes in membrane polypeptides that occur when chick embryo fibroblasts and NRK cells are transformed with avian sarcoma viruses. Virologv 58, 86-100. TOYOSHIMA, K., and VOCT, P. K. (1969). Temperature sensitive mutants of avian sarcoma virus. Virolog,% 39, 930-931. TOYOSHIMA, K., OWADA, M., and KOZAI, Y. (1973). Tumor producing capacity of temperature sensitive mutants of avian sarcoma viruses in chicks. B&en J. 16, 103-110. VITETTA, E. S., BAUR, S., and UHR, J. W. (1971). Cell surface immunoglobulin. II. Isolation and characterization of immunoglobulin from mouse splenic lymphocytes. J. Exp. Med. 134, 242-264. VOGT, P. K. (1969). Focus assay of Rous sarcoma virus. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 198-211. Academic Press, New York. WARREN, L., CURTCHLEY, D., and MACPHERSON, I. (1972). Surface glycoproteins and glycolipids of chicken embryo cells transformed by a temperature sensitive mutant of Rous sarcoma virus. Nature (London) 235, 275-277. WARREN, L., FUHRER, L. P., and BUCK, C. A. (1973). Surface glycoproteins of cells before and after transformation by oncogenic viruses. Fed. Proc. 32, 80-85. WESTON, J. A., and HENDRICKS, K. L. (1972). Reversible transformation by urea of contact-inhibited fibroblasts. Proc. Nat. Acad. Sci. USA 69, 3727-3731. WICKUS, G. G., and ROBBINS, P. W. (1973). Plasma membrane proteins of normal and Rous sarcoma virus transformed chick embryo fibroblasts. Nature Neu! Biol. 245, 65-67. WYKE, L. A., and LINIAL, M. (1973). Temperature sensitive avian sarcoma viruses: A physiological comparison of twenty mutants. Virologv 53, 152-161. YOSHIDA, M.. OWADA, M., and TOYOSHIMA, K. (1975). Strain specificity of changes in adenylate cyclase activity in cells transformed by avian sarcoma viruses. Virolog.y 63, 68-77.
65,226237
(1975)
Alterations
in Membrane
Polypeptides
Induced
by Transformation
Fibroblasts
Sarcoma TOMOE Faculty
ISAKA,
of Pharmaceutical
of Chick Embryo with Avian
Viruses
MITSUAKI
YOSHIDA
Sciences, University
of Tokyo, Hongo, Japan
AND
MASATOSHI Department
OWADA,
of Tumor Viruses, Research Institute
KUMAO
AND
for Microbial Japan
Accepted January
TOYOSHIMA
Diseases, Osaka Uniuersity,
Suita City, Osaka,
29, 1975
Membrane proteins of chick embryo fibroblasts (CEF) transformed with various strains of avian sarcoma viruses were analyzed by electrophoresis in SDS-polyacrylamide gels and compared with those of untransformed cells. The following differences were consistently detected in CEF transformed with B77, the Prague strain of Rous sarcoma virus (PR-RSV) or the Schmidt-Ruppin strain of RSV (SR-RSV): (1) The appearance of a polypeptide band with an apparent molecular weight of 90,000, (2) increase in amount of a polypeptide of 79,000 daltons, (3) significant decrease in amount of a polypeptide of 50,000 daltons and (4) marked decrease in amount of a protein of 200,000 daltons. CEF infected with the temperature-sensitive (ts) mutants of these strains, LA334 (of B77), LA31 (of PR-RSV) or OS122 (of SR-RSV) showed similar changes at 36”, but at 41”, except for alteration (4), the profiles of the membrane proteins were similar to those of uninfected cells. Changes (1) and (3) were reversible and clearly observable within a few hours after a temperature shift of CEF infected with ts mutants. Fusiform transformation induced by a variant of B77 was also shown to induce alterations (1) and (3). From these and other results, the appearance of the polypeptide band of 90,000 daltons, which could not be detected in untransformed cells, and the marked decrease in amount of a protein of 50,090 daltons in cell membranes were concluded to be closely correlated with transformation of CEF. INTRODUCTION
Alterations in membrane components have been reported in RSV-transformed cells; namely alterations in glycoprotein (Warren et al., 1972, 1973), glycolipids (Hakomori et al., 1971), carbohydrates of the cell surface (Hartmann et al., 1972; Perdue et al., 1972), and polypeptides (Hynes, 1973). Bussell and Robinson (1973) demonstrated that the amount of a major protein component of uninfected cell membranes, with a molecular weight of 142,000, decreased greatly on transformation of cells with the Bryan high-titer strain of RSV. Wickus and Robbins (1973)
Rous sarcoma virus (RSV) induces alterations of cell morphology (Manaker and Group& 1956), loss of contact inhibition of cell movement (Stoker and Rubin, 1967), increased activity of sugar transport (Hatanaka and Hanafusa, 1970), increased agglutinability by plant lectins (Burger and Martin, 1972), and reduced activity of membrane-bound adenylate cyclase (Anderson et al., 1973a,b; Yoshida et al., 1975). These changes suggest that transformation by RSV is associated with alterations in cell membranes. 226 Copyright All rights
0 1975 by Academic Press. of reproduction in any form
Inc. reserved.
MEMBRANE
PROTEINS
OF RSV-TRANSFORMED
also reported that another protein component with a molecular weight of 45,000 decreased when cells were transformed with the Schmidt-Ruppin strain of RSV (SR-RSV). Very recently, Stone et al. (1974) reported a remarkable increase in amount of a polypeptide of 73,000 daltons, a small increase in amount of a polypeptide of 95,000 daltons, and decreases in the amounts of proteins of 250,000 and 39,000 daltons in cells transformed by avian sarcoma viruses. This paper describes the appearance of a membrane polypeptide band with apparent molecular weight of 90,000 in polyacrylamide-gel electrophoresis, a remarkable increase in the amount of a 79,000-dalton polypeptide and decrease in amount of a protein of 50,000 daltons when CEF were transformed by avian sarcoma viruses. A protein with a molecular weight of 90,000 was detected as one of the major components in transformed cell membranes but was not detectable in uninfected cells. Furthermore, these changes were shown to be rapidly reversible when cells infected with temperature-sensitive (ts) mutants were shifted between the permissive (36”) and nonpermissive (41”) temperature. Our findings are similar to those of Stone et al. (1974), except that they observed only a small increase in the amount of protein of 95,000 daltons, which was a main component in uninfected cell membranes. This band seems to correspond to our proteins of 91,000 and 90,000 daltons. We found that the protein of 91,000 daltons was a main component of uninfected and transformed cells, while the protein of 90,000 daltons was only visible in transformed cells. MATERIALS
AND
METHODS
Cells and viruses. Chick embryo fibroblast (CEF) cultures were prepared from 11-day-old C/E chick embryos, as described by Vogt (1969), and grown in loo-mm plastic dishes in complete medium, consisting of Eagle’s minimal essential medium (MEM; Nissui), 10% tryptose phosphate broth, 5% calf serum, 0.5% chick serum, and 0.056% sodium bicarbonate. Infected CEF were usually transferred once before analysis to induce extensive trans-
CELLS
227
formation of cultured cells. The permissive temperature for ts mutants of avian sarcoma viruses in their transforming capacity was 36”, and the nonpermissive temperature was 41”. After formation of monolayers, 1% dimethylsulfoxide was added to culture media. LA31, a ts mutant of the Prague strain of RSV, PR-RSV-A, was a generous gift from Dr. P. K. Vogt and was characterized previously (Wyke and Linial, 1973). LA334 and OS122, the ts mutants of B77 and SR-RSV-D, have been reported previously (Toyoshima and Vogt, 1969; Owada and Toyoshima, 1973; Toyoshima et al., 1973). QV-2, a subgroup E variant of B77 which will be described elsewhere, induced fusiform transformation when C/E CEF were infected with the RAV-1 pseudotype of QV-2. Labeling of cultures. Normal and transformed cells were labeled with L[35S]methionine (145 Ci/mmole, Radiochemical Centre, Amersham, England) at a concentration of 3.5 j&i/ml in complete medium for about 20 hr. In the experiment on the temperature shift of cultures, L[35S]methionine was added at a concentration of 7 &X/ml when the temperature was shifted, and, 6-7 hr later, the cells were used for preparation of membranes. Preparation of the membrane fraction. A crude membrane fraction was prepared essentially by the method described by Atkinson and Summers (1971), as modified by Bussell and Robinson (1973). In most experiments, one loo-mm dish of a secondary or tertiary culture was used for preparation of membranes. The culture medium was decanted, and the cells were rinsed twice with phosphate-buffered saline (PBS). Then they were transferred to about 5 ml of PBS using a rubber policeman and washed twice by centrifugation at 250 g for 6 min. The pellet was resuspended in the homogenizing medium (15 mM iodoacetate, 10 mM NaN,, 10 mM Tris-HCl, pH 8.0) and kept at room temperature for 5-8 min. Then the cells were homogenized with four strokes at 1,000 rpm of a loose fitting Potter homogenizer. The homogenate was layered over a discontinuous sucrose density gradient consisting of 1.5 ml of 45% (w/w) sucrose overlaid with 2.5 ml of
228
ISAKA
30% (w/w) sucrose, both containing 10 mM Tris-HCl (pH 8.0) and 10 mM NaN,, and centrifuged at 10,000 rpm and 4” for 20 min in a Hitachi RPS 50 rotor. The band of membranes at the interphase between the 45 and 30% sucrose layers was used directly as the membrane preparation for electrophoresis. The distribution of enzyme markers for plasma cell membranes, Na+, K+stimulated ATPase (Perdue and &eider, 1970) and adenylate cyclase (Pohl et al., 1971), were measured, and more than 90% of both the enzyme activities were found in the interphase fractions. However, about half of the glucose-6-phosphatase activity, used as marker enzyme for endoplasmic reticulum (Hiibscher and West, 1965), was also found in the interphase fraction. These observations indicated that the membrane fraction used here contained not only plasma cell membrane but also internal membranous structures. The top fraction in the 30% sucrose layer was recentrifuged at 200,000 g for 2 hr in a Hitachi RPS 50 rotor to obtain the 200,000 g supernatant fraction. SDS-polyacrylamide slab-gel electrophoresis. Slab-gel (180 x 150 x 1 mm) electrophoresis was performed as described by Laemmli (1970) with minor modifications. Samples containing up to 50-70 pg of protein, 0.5% sodium dodecyl sulfate (SDS), 2.5% 2-mercaptoethanol, 5% glycerol, 0.001% bromophenol blue, and 0.025 M Tris-HCl, pH 6.8, were boiled for 3 min before electrophoresis. Electrophoresis was carried out for 4 hr at 20 mA. Gels were fixed in 50% trichloroacetic acid (TCA) for 2 hr, stained for 20 min with 0.1% Coomassie brilliant blue in 50% TCA, and destained by diffusion in 7% acetic acidmethanol. After washing overnight, gels were dried on filter paper in vacua and exposed to X-ray film (Kodax RP Royal XOmat) for 2-3 weeks. Optical scanning of stained gels and autoradiograms were carried out with a densitometer (Fujiriken, Model FD-A IV). Cell surface labeling. The cell surface was labeled with carrier-free lz51 by the lactoperoxidase method described by Vitetta et al. (1971). The cells in a 50-mm
ET AL.
petri dish were rinsed with PBS and iodinated for 10 min in 2 ml of PBS containing 100 PCi of carrier-free 1251and 50 ~1 of 0.03% H,O,. The reaction was initiated by addition of an adequate amount of lactoperoxidase and stopped by addition of 10 ml of 5 mM cysteine hydrochloride. During the reaction, two additional volumes of 50 ~1 of 0.03% H,O, were added to the reaction mixture. After the reaction, either the cells were rinsed five times with PBS, scraped off with a rubber policeman and used to prepare the membrane fraction as described above or the whole cells were disrupted with 0.1% SDS solution and used directly as the sample for electrophoresis. Preparation of nuclei. Nuclei were isolated as described by Ohtsuki and Amano (1971) in the presence of detergent to minimize the contamination by cytoplasmic materials. Cells were washed with PBS and then transferred with a rubber policeman into 2 ml of 0.25 M sucrose containing 3 mM CaCl,. Nonidet P-40 was added to the cell suspension to a final concentration of 0.5%. The mixture was allowed to stand for 20 min at 4” and then centrifuged at 750 g for 15 min. The pellet was resuspended in 1 ml of 0.25 M sucrose containing 3 mM CaCl, and 0.2% Nonidet P-40. After standing for 5 min at 4”, the mixture was centrifuged again at 750 g for 15 min. The pellet was resuspended in a small amount of the same solution, layered over 4 ml of 2 M sucrose containing 3 mM CaCl, and 0.2% Nonidet P-40, and centrifuged at 50,000 g for 60 min. The pellet was washed with 0.25 M sucrose-3 mM CaCl, by centrifugation at 750 g for 15 min and used as the nuclear fraction for electrophoresis. RESULTS
Polypeptide Constitution of the Membrane Fraction from CEF Transformed by B77 Chick embryo fibroblasts transformed by avian sarcoma viruses and uninfected CEF were labeled with L- [35S]methionine for 18-24 hr, and a fraction rich in large fragments of cell membrane was isolated by a method similar to that established by Atkinson and Summers (1971) and modi-
MEMBRANE
PROTEINS
OF RSV-TRANSFORMED
fied by Bussell and Robinson (1973). In most experiments, treatment of cells with EDTA was avoided. Figure 1A shows autoradiograms of 6.5% polyacrylamide-SDS gels after electrophoresis of polypeptides labeled with L-
229
[35S]methionine in the membrane fraction isolated from uninfected CEF and CEF transformed with B77. The polypeptide composition of membranes from transformed cells consistently showed the following differences from that of uninfected B
A
(a)
CELLS
(b)
(a)
(b)
FIG. 1. (A) Autoradiograms of 6.5% polyacrylamide-SDS slab-gels after electrophoresis of L-[35S]methionine-labeled proteins of membrane fractions from CEF transformed with B77 (a) and uninfected cells (b). (B) Electropherograms, stained with Coomassie brilliant blue, of membrane proteins from cells transformed by B77 (a) and uninfected cells (b). The numbers refer to the molecular weights in thousands of polypeptide components.
230
ISAKA
cells: (1) The appearance or remarkable increase in amount of polypeptide of 90,000 daltons, (2) significant increase in the amount of a polypeptide of 79,000 daltons, and (3) marked decreases in amounts of polypeptides with molecular weights of 200,000 and 50,000 daltons. The polypeptide of 90,000 daltons could not be detected as a clear visible band in uninfected cells, but it was easily detectable as a new clear band in transformed cells, and its appearance was the most pronounced change in
ET AL.
membrane components. These changes were clearly visible in stained gels (Fig. 1B) and were clearer when the stained gels or autoradiograms were scanned with an optical densitometer and compared semiquantitatively (Fig. 2a). When total cell homogenates were analyzed on 6.5% polyacrylamide-SDS gels, the profiles were obviously more complex. However, a clearly visible band was detected in transformed cells at a position corresponding to a molecular weight of
FIG. 2. Comparison of membrane constituent proteins from uninfected CEF (-----) and from CEF transformed by (a), B77; (b), SR-RSV-A; and (c), PR-RSV-C (-). The gels were stained with Coomassie brilliant blue, and the direction of electrophoresis was from left to right.
MEMBRANE
PROTEINS
OF RSV-TRANSFORMED
231
CELLS
FIG. 3. COXnDariSOn of the protein components of total cell homogenate from CEF transformed by B77 (-) and uninfected CEF (-----). The gels were stained with Coomassie brilliant blue and the direction electrophoresis was from left to right.
90,000, while no clear band was observed in this position in uninfected cells (Fig. 3). These results strongly suggest that the existence of this polypeptide in the membranes was not a reflection of a different distribution of this polypeptide in uninfected and transformed cells but that the polypeptide was a product of new synthesis or proteolytic hydrolysis of high molecular weight protein, which was increased by cell transformation. The amount of the polypeptide of about 200,000 daltons in membranes from uninfected cells was variable: When EDTA was included in the buffer used for washing the cells, its content was especially small. On the other hand, CEF transformed with B77 consistently contained a small amount of this polypeptide. Polypeptide Constitution of the Membrane Fraction from CEF Transformed by SR-RSV and PR-RSV Alterations in polypeptide composition similar to those described above were observed in membrane preparations from CEF transformed with SR-RSV of subgroups A and D or the Prague strain of RSV (PR-RSV) of subgroups A and C (Fig. 2). Again the change in the polypeptide of 90,000 daltons was remarkable. From these results, these changes in membrane proteins appear to be common characteristics
of CEF transformed viruses.
with
of
avian sarcoma
Alterations in Membrane Polypeptides CEF Infected with ts Mutants
of
When CEF are infected with the ts mutants of avian sarcoma viruses, morphological transformation of the cells is reversibly controlled by the temperature of cell cultivation (Toyoshima and Vogt, 1969; Martin, 1970; Kawai and Hanafusa, 1971). To demonstrate that the changes in membrane polypeptides described above are correlated with cell transformation, CEF were infected with the ts mutants, LA334 (of B77), LA31 (of PR-RSV-A) or OS122 (of SR-RSV-D), and grown at 36”. Pairs of cultures were set up and one of each was shifted and maintained at 41’ for more than 25 or 48 hr. Then the cells were harvested and their membrane fractions were prepared. As shown in Fig. 4, when the cells were transformed by LA334 at 36”, all the changes of the membrane polypeptides described above were observed, but when the cells were maintained at 41” for 25 hr, the profile was very similar to that of uninfected cells, except for a decrease in the amount of protein of about 200,000 daltons. The same effects of temperature were observed with infected cells with LA31 or OS122 (Fig. 5). In uninfected cells,
232
ISAKA
ET AL.
B
FIG. 4. Comparison of polypeptide components of membranes from uninfected cells (-----) and cells infected with LA334 (-1. (A), Profiles of membrane components of cells maintained at 41” for more than 24 hr; (B), profiles of membrane components of cells maintained and transformed at 36”. Autoradiograms of membrane polypeptides labeled with L- [3sS]methionine were scanned as described in Materials and Methods.
the profiles of membrane polypeptides were virtually identical at 36 and 41”. The observations that the amount of the protein of 200,000 daltons decreased even at 41” in ts mutant-infected cells indicates that this protein has no direct correlation with regulation of the temperature-sensitive step of these ts mutants. These results suggest that, among the changes in the membrane polypeptides, the appearance of the polypeptide band of 90,006 daltons, increase in the amount of polypeptide of 79,000 daltons and reduction in the amount of protein of 50,000 daltons are related to cell transformation by avian sarcoma viruses but not to viral replication, since LA31 and OS122 can replicate at 41” as efficiently as at 36”.
Effects of Temperature Shift on Changes in Membrane Polypeptides of CEF Infected with LA334 To examine the relationship of these changes to morphological transformation, the time of these changes in membrane polypeptides after the temperature shift 11as studied. When cells at 41” infected with LA334 were shifted to 36”, the morphology of most of the cells was observed to change within a few hours. Seven hours after the shift, the membrane fraction from LA334-infected cells showed a distinct band with a molecular weight of 90,000 (Fig. 6a), which could not be detected at 41” (Fig. 6d), though this band was less than that in cells maintained at 36” (Fig.
MEMBRANE
(a)
(b)
PROTEINS
OF RSV-TRANSFORMED
(a)
CELLS
233
(b)
FIG. 5. Electropheroprams of6.5% polyacrylamide-SDS slab-gels stained with Coomassie brilliant blue. (A) CEF infected with OS122 of SR-RSV-D and maintained at 41” (a) and at 36” (b), for more than 24 hr. (B) CEF infected with LA31 of PR-RSV-A and maintained at 41” (a) and at 36” (b) for more than 24 hr.
234
ISAKA
6b). Decrease in the amount of protein of 50,000 daltons was also noticeable. Upon the reverse shift, the morphology of the transformed cells reverted to that of uninfected cells within a few hours and the
(a)
(b)
(4
(d
-
91 90
-
79
-
50
FIG. 6. Autoradiograms of polya6.5% crylamide-SDS slab-gels after electrophoresis of polypeptides labeled with L- [35S]methionine in membranes from CEF infected with LA334. LA334infected CEF 7 hr after shifting down to 36” from 41” (a) or after shifting up to 41” from 36” (c) and maintained for more than 24 hours at 36” (b) or at 41° (d).
ET AL.
band of polypeptide of 90,000 daltons became significantly less distinct (Fig. 6~). Meanwhile the amount of polypeptide of 50,000 daltons increased to almost the level of that in cells maintained at 41”. Change in amount of the polypeptide with a molecular weight of 79,000 on temperature shift up or down was less than those of the two proteins described above. Thus, the time of appearance of the protein band of 90,000 daltons and marked decrease in amount of protein of 50,000 daltons coincided precisely with the time of morphological transformation. Addition of cycloheximide (5 pg/ml) at the time of the down shift of LA334infected cells inhibited both the appearance of the polypeptide band of 90,000 daltons and transformation. On shift up did not from 36 to 41”, cycloheximide prevent the disappearance of the protein of 90,000 daltons. Alterations in Polypeptide Composition the Membranes of CEF Induced Fusiform Transformation
of by
Uninfected CEF are long, fibroblastictype cells, while cells transformed by B77 or RSV are round. QV-2, a variant of B77, induces fusiform transformation of CEF. It seemed interesting to see whether the changes of membrane polypeptides of CEF induced by fusi- and round-form transformation were the same. As shown in Fig. 7, the profile of membrane polypeptides from QV-2-transformed CEF was very similar to that from B77-transformed cells, that is, both showed a band of 90,000 daltons, increase in the amount of polypeptide of 79,000 daltons and decrease in the amount of 50,000-dalton polypeptide. These results provide evidence for the correlation between the appearance of the polypeptide of 90,000 daltons and decrease in the amount of polypeptide of 50,000 daltons on viral transformation, irrespective of the type of morphology on the transformed cells. Localization of Polypeptide of 90,000 Daltons in Transformed Cells As described above, a protein band corresponding to a molecular weight of 90,000 was a characteristic of transformed cells
MEMBRANE
FIG. 7. Comparison by QV-‘2 (RAV-1) (-).
PROTEINS
OF RSV-TRANSFORMED
of the profiles of membrane proteins from uninfected
and was found in the membrane fraction. After sucrose gradient centrifugation, the top fraction in 30% sucrose contained a protein band with this molecular weight. But this was not detected in the supernatant fluid after centrifugation at 200,000 g for 2 hr. A partially purified nuclear fraction did not show a similar band. These observations suggest that the polypeptide of 90,000 daltons is mainly bound to a membranous structure. This polypeptide of 90,000 daltons was not iodinated by lactoperoxidase-catalyzed iodination (Vitetta et al., 1971) of the cell surface and was not removed from the cells by washing with 1 A4 urea-l mM EDTA, which removed some high molecular weight proteins from the cell surface (Weston and Hendricks, 1972). These results can be explained by supposing that this polypeptide was not exposed on the surface of the cells. DISCUSSION
This paper describes characteristic changes of polypeptides in CEF membranes associated with transformation of cells by avian sarcoma viruses. These changes are (1) the appearance of a polypeptide band with an apparent molecular weight of 90,000 daltons, (2) increase in amount of polypeptide of 79,000 daltons, and (3) marked decrease in amounts of proteins of 50,000 and 200,000 daltons.
CELLS
235
CEF (-----) and cells transformed
These changes were all relatively large so that they could be detected on stained gels, and they were found in CEF transformed by either B77, SR-RSV, or PR-RSV. Among these changes, the appearance of the polypeptide band of 90,000 daltons was especially noticeable, since it was not detected as a clear visible band in gels from uninfected or untransformed cells. These observations suggest that this protein of 90,000 daltons was a membrane polypeptide induced by viral transformation, although the possibility of the presence of an undetectable amount of this polypeptide in uninfected cells is not excluded. When CEF infected with LA334 and maintained at 41” were shifted to 36”, the appearance of the band of 90,000 daltons and marked decrease in the amount of polypeptide of 50,000 daltons were clearly detected within 7 hr, and at this time most of the infected cells were observed to be morphologically transformed. The reverse temperature shift induced changes in the opposite direction within a few hours. LA31, the ts mutant of PR-RSV, and OS122, that of SR-RSV, also showed temperature-dependent induction of changes in membrane polypeptides. These results indicate that these two changes of membrane polypeptides at least are closely correlated with morphological transformation of cultured cells but not with viral replication. This conclusion is also sup-
236
ISAKA
ported by the finding that cycloheximide, an inhibitor of protein synthesis which suppresses cell transformation (Kawai and Hanafusa;l971), also inhibited the appearance of the polypeptide of 90,000 daltons. This polypeptide also appeared in fusiform cells transformed with QV-2, which suggests that it is correlated with viral transformation, irrespective of the type of morphology of the transformed cells. The polypeptide band of 90,000 daltons which was detected only in transformed cells may be mainly in a membrane-bound state since it was not found in either the supernatant fluid after centrifugation at 200,000 g or the nuclear fraction. These results also suggest that the appearance of this polypeptide band is not simply due to a different distribution of this polypeptide in transformed and uninfected cells. The possibility that the polypeptide is formed by proteolytic modification of high molecular weight protein in the membrane cannot be excluded, but it seems likely that morphological transformation of cells induces new synthesis of the polypeptide of 90,000 daltons and that this protein is exclusively incorporated into membrane structures. Other explanations are also possible such as incomplete or abnormal glycosylation of a component which may result in the appearance of the 90,000-dalton protein. The presence of a similar polypeptide with the molecular size of 91,000 daltons in transformed and normal cell membranes may suggest a structural relationship between proteins of 90,000 and 91,000 daltons. However, the observations that the amount of 91,000-dalton protein was not affected by the increasing amount of 90,000-dalton protein leads to the speculation that these two proteins are not related. Attempts to isolate and characterize these proteins are in progress. The appearance of the polypeptide band of 90,000 daltons and increase in the amount of polypeptide of 79,000 daltons seem to correspond to the increase in amounts of proteins of 95,000 and 73,000 daltons, respectively, described by Stone et al. (1974) on transformation by PR-RSV-A and B77. However, they observed only a small increase in the amount of protein of
ET AL.
95,000 daltons, which was the main component in uninfected cells. This discrepancy between their results and ours is due to the better separation achieved under our conditions of gel electrophoresis rather than to the difference in the methods used for membrane preparation, since the protein of 90,000 daltons moved just in front of a main band of 91,000 daltons, which was also a main band in uninfected cells. The results of Stone et al. (1974) on the effects of a temperature shift of cells infected with a ts mutant on these polypeptide changes also differ from ours. We found that more than half the maximum amount of polypeptide of 90,000 daltons appeared within 7 hr of the temperature shift, while they reported that it was detected only after 24 hr. This might be due to a difference in the character of the ts mutant or the conditions used for cell cultures. The significant decrease in the amount of polypeptide of 50,000 daltons induced by transformation probably corresponds to the decrease in the amount of protein of 45,000 daltons in SR-RSV-transformed cells reported by Wickus and Robbins (1973). This change was not observed by Stone et al. (1974), possibly due to marked differences in the methods used to isolate membranes. The method used in this work was separation by centrifugation on a sucrose gradient and was in principle similar to that of Wickus and Robbins (1973), while Stone et al. (1974) used a completely different system, namely a two-phase polymer procedure. The big differences in the profiles they observed in the region of molecular weight higher than 200,000 daltons might also be a result of the different method used to isolate membranes. REFERENCES W. B., JOHNSON, G. S., and PASTAN, I. (1973a). Transformation of chick-embryo fibroblasts by wild-type and temperature-sensitive Rous sarcoma virus alters adenylate cyclase activity. Proc. Nat. Acad. Sci. USA 70, 1055-1059. ANDERSON, W. B., LOVELACE, E., and PASTAN, I. (1973b). Adenylate cyclase activity is decreased in chick embryo fibroblasts transformed by wild type and temperature sensitive Schmidt-Ruppin Rous sarcoma virus. Biochem. Bioph,ys. Res. Commun. 52,1293-1299. ANDERSON,
MEMBRANE
PROTEINS
OF RSV-TRANSFORMED
ATKINSON, P. H., and SUMMERS, D. F. (1971). Purification and properties of HeLa cell plasma membranes. J. Biol. &hem. 246, 5162-5175. BURGER, M. M., and MARTIN, G. S. (1972). Agglutination of cells transformed by Rous sarcoma virus by wheat germ agglutinin and concanavalin A. Nature New Biol. 237, 9-12. BUSSELL, R. H., and ROBINSON, W. S. (1973). Membrane proteins of uninfected and Rous sarcoma virus-transformed avian cells. J. Virol. 12,320-327. FRIIS, R. R., TOYOSHIMA, K., and VOGT, P. K. (1971). Conditional lethal mutants of avian sarcoma viruses. I. Physiology of ts 75 and ts 149. Virology 43, 375-389. HAKOMORI, S.-I., SAITO, T., and VOGT, P. K. (1971). Transformation by Rous sarcoma virus: Effects on cellular glycolipids. Virology 44, 609-621. HARTMANN, J. F., BUCK, C. A., PEFENDI, V., GLICK, M. L. (1972). The carbohydrate C., and WARREN, content of control and virus-transformed cells. J. Cell Physiol. 80, 159-165. HATANAKA, M., and HANAFUSA, H. (1970). Analysis of a functional change in membrane in the process of cell transformation by Rous sarcoma virus: Alteration in the characteristics of sugar transport. Virology 41, 647-652. HOBSCHER, G., and WEST, G. R. (1965). Specific assay of some phosphatases in subcellular fractions of smal. intestinal mucosa. Nature (London) 205, 799800. HYNES, R. 0. (1973). Alteration of cell-surface proteins by viral transformation and by proteolysis. Proc. Nat. Acad. Sci. USA 70, 3170-3174. KAWAI, S., and HANAFUSA, H. (1971). The effects of reciprocal changes on the transformed state of cells infected with a Rous sarcoma virus mutant. Virol0g.v 46, 470-479. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. MANAKER, R. A., and GROUP& V. (1956). Discrete foci altered chicken embryo cells associated with Rous sarcoma virus in tissue culture. Virology 2.838-840. MARTIN, G. S. (1970). Rous sarcoma virus: A function required for the maintenance of the transformed state. Nature (London) 227, 1021-1023. OHTXKI, H., and AMANO, M. (1971). Isolation of nuclei from ascites hepatoma AH-130 cells of rat by a detergent. Gann 62, 427-430. OWADA, M., ~~~TOYOSHIMA, K. (1973). Analysis of the reproducing and cell-transforming capacities of a temperature sensitive mutant (ts 334) of avian sarcoma virus B77. Virology 54, 170-178. PEROUE, J. F., KLETZIEN, R., and WRAY, V. L. (1972). The isolation and characterization of plasma membrane from cultured cells. IV. The carbohydrate composition of membranes isolated from oncogenic RNA virus-converted chick embryo fibroblasts. Bi-
CELLS
237
ochim. Biophys. Acta. 266, 505-510. PERDUE, J. F., and SNEIDER, J. (1970). The isolation and characterization of the plasma membrane from chick embryo fibroblasts. Biochim. Biophys. Acta 196, 125-140. POHL, S. L., BIRNBAUMER, L., and RODBELI., M. (1971). The glucagon-sensitive adenyl cyclase system in plasma membrane of rat liver. I. Properties. J. Biol. Chem. 246, 1849-1856. STOKER, M. G. P., and RUBIN, H. (1967). Density dependent inhibition of cell growth in culture. Nature (London) 215, 171-172. STONE, K. R., SMITH, R. E., and JOKLIK, W. K. (1974). Changes in membrane polypeptides that occur when chick embryo fibroblasts and NRK cells are transformed with avian sarcoma viruses. Virologv 58, 86-100. TOYOSHIMA, K., and VOCT, P. K. (1969). Temperature sensitive mutants of avian sarcoma virus. Virolog,% 39, 930-931. TOYOSHIMA, K., OWADA, M., and KOZAI, Y. (1973). Tumor producing capacity of temperature sensitive mutants of avian sarcoma viruses in chicks. B&en J. 16, 103-110. VITETTA, E. S., BAUR, S., and UHR, J. W. (1971). Cell surface immunoglobulin. II. Isolation and characterization of immunoglobulin from mouse splenic lymphocytes. J. Exp. Med. 134, 242-264. VOGT, P. K. (1969). Focus assay of Rous sarcoma virus. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 198-211. Academic Press, New York. WARREN, L., CURTCHLEY, D., and MACPHERSON, I. (1972). Surface glycoproteins and glycolipids of chicken embryo cells transformed by a temperature sensitive mutant of Rous sarcoma virus. Nature (London) 235, 275-277. WARREN, L., FUHRER, L. P., and BUCK, C. A. (1973). Surface glycoproteins of cells before and after transformation by oncogenic viruses. Fed. Proc. 32, 80-85. WESTON, J. A., and HENDRICKS, K. L. (1972). Reversible transformation by urea of contact-inhibited fibroblasts. Proc. Nat. Acad. Sci. USA 69, 3727-3731. WICKUS, G. G., and ROBBINS, P. W. (1973). Plasma membrane proteins of normal and Rous sarcoma virus transformed chick embryo fibroblasts. Nature Neu! Biol. 245, 65-67. WYKE, L. A., and LINIAL, M. (1973). Temperature sensitive avian sarcoma viruses: A physiological comparison of twenty mutants. Virologv 53, 152-161. YOSHIDA, M.. OWADA, M., and TOYOSHIMA, K. (1975). Strain specificity of changes in adenylate cyclase activity in cells transformed by avian sarcoma viruses. Virolog.y 63, 68-77.
