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Transformation-associated changes in nuclear-coded mitochondrial proteins in 3T3 cells and SV40-transformed 3T3 cells
Biochimica et Biophysica Acta, 804 (1984) 285-290
285
Elsevier BBA 11294
TRANSFORMATION-ASSOCIATED CHANGES IN NUCLEAR-CODED MITOCHONDRIAL PROTEINS IN 3"1"3CELLS AND SV40-TRANSFORMED 3T3 CELLS STEVEN H. Z U C K E R M A N *, STIG LINDER ** and JEROME M. EISENSTADT ***
Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510 (U.S.A.) (Received December 27th, 1983)
Key words: Transformation," Protein composition," Mitochondrial protein; (3T3 cell)
Comparative two-dimensional gel electrophoretic studies were performed on mitochondrial proteins in nontransformed mouse 3T3 cells and in SV40-transformed 3T3 cells, SV-T2. Two polypeptides, of 58 and 40 kDa, were present in increased mounts in SV40-transformed cells. These polypeptides were demonstrated to be nuclear-ceded mitochondrial proteins by their absence in mitochondrial preparations, when labeling was performed in the presence of a mitochondrial-specific inhibitor, Rhodamine 6G. Temperature-sensitive mutants for transformation were derived from 3T3 cells by transfection with cloned SV40 DNA containing the ts A58 mutation. Increased amounts of the 58 kDa protein were apparent in these cells at the permissive temperature (33°C) compared to the restrictive temperature (39.5°C).
Introduction Warburg's hypothesis that cancer cells have an elevated rate of glycolysis due to an impaired respiratory capacity [1] and the finding that mitochondria are the principal site for oxidative phosphorylation and ATP generation in animal cells [2] resulted in extensive investigations of mitochondria from neoplastic cells. Differences in mitochondrial structure and numbers, as well as in mitochondrial enzyme content, membrane lipids, transport mechanisms and substrate specificities, have been reported [3,4]. However, these differences are variable depending on the tumor source, mitochondrial isolation procedures and the * Present address: Department of Immunology, Lilly Research Laboratories, 307 East McCarty Street, Indianapolis, IN 46285, U.S.A. ** Present address: Department of Medical Genetics, The Biomedical Centre, Uppsala University, 75123 Uppsala, Sweden.
*** To whom correspondence should be addressed. Abbreviation: R6G, rhodamine 6G. 0167-4889/84/$03.00 © 1984 Elsevier Science Publishers B.V.
in vivo and in vitro growth properties of the tumors. Accordingly, it has been difficult to define which changes are associated with neoplastic transformation. In a different approach, sarcomavirus-transformed mink fibroblasts were reported to have a decreased mitt~hondrial membrane potential compared to control, nontransformed mink fibroblasts [5]. These differences in membrane potential were based on the decreased uptake and retention of the mitochondrial-specific cationic dye .Rhodamine 123 and were reversible following addition of the ionophore nigericin. Further studies on the retention of Rhodamine 123 in various tumors indicated that many transitional cell carcinomas, adenocarcinomas and chemically transformed epithelial cell lines retained Rhodamine 123 for extended periods, while oat cell carcinomas did not [6]. These results demonstrate that neoplastic transformation is associated with discrete mitochondrial changes. In the present study, we have performed comparative studies on nuclear-coded mitochondrial proteins in nontransformed 3T3 cells and in
286
Materials and Methods
Charles Cole, Dartmouth College, Hanover, NH). The temperature-sensitive mutation has been mapped within the large T antigen [8]. Calcium phosphate coprecipitates of ts A58 and carrier DNA were incubated with 3T3 cells [9]. Following incubation, cells were seeded in soft agar and incubated at 33°C. Four soft agar clones (3.4, 4.4, 5.1 and 6.1) were isolated, which grew in soft agar at 33°C, but not at 39.5°C.
Cell culture Balb/c 3T3 cells clone A31 and SV40-transformed 3T3 cells, SV-T2, were obtained from the American Type Culture Collection. These cell lines were maintained at low density in Dulbecco's modified Eagle's media supplemented with 5% calf sera [7]. Temperature-sensitive transformation mutants of 3T3 were obtained using the ts A58 SV40 mutant inserted in pBR322 (provided by Dr.
Mitochondria preparation and gel electrophoresis 2.106 cells were seeded in 25 cm2 flasks (Falcon) and labeled for 90 min (short pulse) or 18 h (long pulse) with 200 and 40 #Ci/ml, respectively, of [35S]methionine (Amersham International) in methionine-free media (GIBCO, Grand Island, NY). In parallel cultures, Rhodamine 6G (Kodak) was present at 2 #g/ml during the overnight labeling [10]. Following labeling, cultures were washed
SV40-transformed 3T3 cells by two-dimensional gel electrophoresis. Increased quantities of two nuclear-coded mitochondrial proteins, of 58 and 40 kDa, were detected in SV40-transformed 3T3 cells. Increased synthesis of the 58 kDa protein was also detected in 3T3 cells transformed with the SV40 ts A58 mutant.
Fig. 1. R6G-sensitivity of nuclear-coded mitochondrial proteins in SV-T2. SV-T2 was labeled for 18 h with 40 ~ C i / m l of [35S]methionine in the absence (a) or presence (b) of 2 ~ g / m l R6G. Mitochondria were then isolated, and nuclear-coded mitochondrial polypeptides were resolved by two-dimensional gel electrophoresis. Approx. 30 polypeptides were absent or significantly reduced, including the predominant polypeptides 1, 2 and 3. The position of these polypeptides in R6G-treated SV-T2 is indicated by circles. Two R6G-sensitive polypeptides (arrows) are present in greater quantities in SV-T2 relative to non-transformed 3T3 cells (see Fig. 2). The contaminating cytoskeletal proteins actin (Ac) tubulin (T) and vimentin (vt) are not sensitive to R6G. A pH gradient between 4.0 and 6.5 was generated in the isoelectric-focusing gels. The basic region of the iscelectric-focusing gel is to the left. Molecular weights are indicated in thousands.
287
and mitochondria were prepared using the procedure of Bogenhagen and Clayton [11]. Unlabeled mitochondria were added as carrier prior to centrifugation in a 1.0-1.5 M sucrose step gradient. The mitochondria were washed and lysed in O'Farrell lysis buffer [12]. Mitochondrial lysates were electrophoresed on two-dimensional gels with isoelectric focusing in the first and SDS-10.5% polyacrylamide gels in the second dimension [12]. The isolation of an inner nutochondnal membrane-enriched preparation was performed by digitonin solubilization of the outer membrane and Lubrol WX fractionation of the inner membrane and matrix [13]. In additional experiments, mitochondria were lysed with 0.5% NP-40 and incubated for 30 min at 37 °C with 10 U of bovine alkaline phosphatase (Sigma, St. Louis, MO) at pH 8.5 in the presence of 2 mM phenylmethylsulfonyl fluoride. Alkaline phosphatase-treated lysates were then subjected to two-dimensional gel electrophoresis. All gels were dried and exposed to Kodak X-Omat film. •
#
.
Results
Mitochondria prepared from [35S]methioninelabeled 3T3 cells and 3T3 cells transformed with SV40 were lysed and resolved using two-dimensional gel electrophoresis. Preliminary experiments indicated that polypeptides comigrating with cytoskeletal components were abundant in sucrose gradient purified mitochondrial preparations. It was thus necessary to distinguish between mitochondrial specific polypeptides and contaminating cytoplasmic polypeptides. This was achieved by labeling parallel cultures in the presence or absence of 2 # g / m l of R6G, a mitochondrial-specific dye which inhibits oxidative phosphorylation [10]. R6G-treated cells are unable to transport nuclearcold mitochondrial proteins into and across the inner mitochondrial membrane. Therefore, only polypeptides that were sensitive to R6G were considered for analysis. Fig. 1 shows the polypeptide patterns of SV-T2 cells labeled in the absence (Fig. la) or presence (Fig. lb) of R6G. Approx. 30 polypeptides were absent or significantly diminished in mitochondrial preparations from R6G-treated cells. These polypeptides represent nuclear-coded mitochondrial proteins and differ in
their relative abundance, molecular weights and isoelectric points. A number of polypeptides are common to both preparations, notably actin, tubulins and vimentin. The identity of these polypeptides were confirmed by tryptic peptide analysis (not shown). The predominant R6G-sensitive polypeptides 1-3 correspond to the Mitcon 1-3 polypeptides described by Anderson [14]. 3T3 and SV-T2 mitochondrial polypeptides were then analyzed by two-dimensional gel electrophoresis in cells labeled for 90 min with [35S]methionine (Fig. 2). Two R6G-sensitive polypeptides, of 58 and 40 kDa, were present in approx. 6- and 3-fold greater quantities respectively in SV-T2 (Fig. 2b) compared to 3T3 cells (Fig. 2a). The 40 kDa polypeptide remained associated with the inner membrane during mitochondrial fractionation with digitonin and Lubrol WX (Fig. 2c). The 58 kDa polypeptide could not be unequivocally assigned to matrix or membrane fractions• Protein 1 (Mitcon 1) was enriched in the inner membrane fraction and was found to react with an anti-F1 ATPase serum in immunoblot experiments [20]. Mitcon 1 was identified as the fl-subunit of the F1 ATPase complex, based on its molecular weight and isoelectric point [15,16]• Proteins 2 and 3 were enriched in the matrix fraction• Incubation of detergent solubilized mitochondria with alkaline phosphatase resulted in the altered electrophoretic mobility of several polypeptides, presumably due to phosphate removal (Fig. 2d and e). This procedure did not alter the mobility of the 40 kDa polypeptide, suggesting that this transformationrelated polypeptide is not phosphorylated. The 58 kDa polypeptide appeared to be more labile and could not be detected after incubating mitochondrial lysates at 37 °C, with or without phosphatase. In further experiments, neither the 40 nor the 58 kDa protein were labeled with [32p]orthophosphate. In a different series of experiments, four SV40 ts A58-transformed clones of 3T3 were isolated which were capable of growth in soft agar at the permissive temperature of both 33°C and 39.5 °C. One of these lines, ts 6.1, was labeled with [35S]methionine for 18 h at 33°C, but not at 39.5 o C. Two-dimensional gel analysis (Fig. 3) indicated that at 33°C (Fig. 3b) the ts 6.1 line had 5-6-fold greater amounts of the 58 kDa protein
288
3T3
SV-T 2
-68
-45
-25
.....
i~i,
:oii!!!iii!!i
~ii ¸ ~
©
rig. z. t ranstormaUon-related changes in nuclear-coded mitochondrial polypeptides. 3T3 cells (a) or the SV40-transformed SV-T2 (b) were labeled for 90 rain with 200 pCi/ml of [35S]methionine. Mitochondria Were then isolated and nuclear-coded mitochondrial polypeptides were resolved by two-dimensional gel electrophoresis. Two R6G-sensitive polypeptides (arrows) of 58 and 40 kDa, were present in greater quantities in SV-T2 compared to 3T3 cells. (c) The 40 kDa polypeptide, actin, and polypeptide 1 fractionate with an inner mitochondrial membrane preparation, while polypeptides 2 and 3 are lost and represent matrix proteins. [ 35S]Methionine-labeled mitochondrial lysates were incubated in the absence (d) or presence (e) of bovine alkaline phosphatase. The 40 kDa polypeptide (arrow) is not altered by alkaline phosphatase treatment, while the mobility of two other polypeptides (open arrowheads) was altered.
289
Fig. 3. Temperature-dependentchanges in the 58 kDa protein in the ts 6.1 cell line. 3T3 cells were transformed with the ts A58 mutant of SV40, and the 6.1 cell line was isolated. The 6.1 line was labeled with 40 #Ci/ml of [35S]methioninefor 18 h at the restrictivetemperatureof 39.5o C (a) or permissivetemperature of 33° C (b) for transformation.Note increased amounts of the 58 kDa protein at the permissivetemperature(b).
compared to the same line at 39.5 °C (Fig. 3a). Similar results were observed with the three other temperature-sensitive transformed lines. None of these lines revealed any significant differences in the expression of the 40 kDa protein at the two temperatures.
Discussion The present experiments suggest that transformation of 3T3 cells with SV40 results in quantitative changes in nuclear-coded mitochondrial proteins. This was evident in both SV-T2 and in the ts A58-transformed mutants at the permissive temperature for transformation. Two proteins, of 58 and 40 kDa, were found in increased quantities in the SV-T2 cells. These proteins were identified as nuclear-coded mitochondrial proteins by their sensitivity to R6G. Increased synthesis of the 58 kDa protein, but not the 40 kDa protein, was also detected in the temperature-sensitive 3T3 transformants. Whether this reflects the variability inherent in different transformed lines is not known. In additional studies, 3T3 and SV-T2 were labeled with [32p]orthophosphate, and the mitochondrial polypeptides were resolved on twodimensional gels. A similar pattern of phosphoproteins was observed on two-dimensional gels of 3T3 and SV-T2 cells (data not shown). None of the approx. 50 phosphoproteins that we resolved were R6G-sensitive. This observation could be ex-
plained by the phosphorylation of proteins already inside the mitochondria. We were therefore unable to distinguish between nuclear-coded mitochondrial phosphoproteins and cytoplasmic phosphoprotein contaminants. Biochemical and structural differences have been reported in mitochondria from tumor vs. control cells [1-4]. However, although two-dimensional gel electrophoretic patterns have been reported for mitochondria preparations [14,16-18], we are unaware of any comparative two-dimensional gel studies of mitochondrial proteins between closely related normal and transformed cells. This may in part be related to the problem of cytoplasmic contamination of mitochondrial preparations. The use of the mitochondrial inhibitor rhodamine 6G enabled us to identify nuclear-coded mitochondrial-specific proteins on our two-dimensional gels. The potassium ionophore nonactin has been used in a similar approach to identify nuclear-coded mitochondrial proteins in whole-cell lysates [14]. A nuclear-coded mitochondrial protein (Mitcon 5) was lost in human lymphocytes, following treatment with phorbol ester tumor promoters. Furthermore, this transformation-sensitive protein was not detected in lymphoblastoid cell lines. Further studies are required to identify other transformation-related changes in nuclear-coded mitochondrial proteins. Recently, Gay and Walker [19] have reported that a portion of p21, the product of the bladder carcinoma oncogene, exhibits significant sequence homology to the nucleotide-binding region of the fl-subunit of the nuclear-coded mitochondrial A T P synthase. Clearly, it is necessary to define how transformation-related qhanges in nuclear-coded mitochondrial proteins affect the function and regulation of the mitochondrial genome.
Acknowledgements This work was supported by N I H grant G M 21873 awarded to J.M.E.S.H.Z. was supported by a Swebilius Cancer Research Award. S.L. was supported by a James Hudson Brown Postdoctoral fellowship. S.H.Z. would like to thank Dr. Sandra Zuckerman for her stimulating discussions.
290 References 1 Warburg, O. (1956) Science 123, 309-314 2 Kennedy, E.P. and Lehninger, A.L. (1948) J. Biol. Chem. 172, 847-848 3 Pederson, P.L. (1978) Prog. Exp. Tumor Res. 22, 190-274 4 Spitsberg, V.L. (1979) Arch. Biochem. Biophys. 195, 136-144 5 Johnson, L.V., Summerhayes, I.C. and Chen, L.B. (1982) Cell 28, 7-14 6 Summerhayes, I.C., Lampidis, T.J., Bernal, S.D., Nadakavukaren, J.J., Nadakavukaren, K.K., Shepherd, E.L and Chen, L.B. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5292-5296 7 Aaronson, S.A. and Todaro, G.J. (1968) J. Cell. Physiol. 72, 141-148 8 Lai, C.-J. and Nathans, D. (1975) J. Virol. 66, 70-81 9 Abrahams, P.J. and Van der Eb, A.J. (1975) J. Virol. 16, 206-209
10 Gear, A.R.L. (1974) J. Biol. Chem. 249, 3628-3637 11 Bogenhagen, D. and Clayton, D.A. (1974) J. Biol. Chem. 249, 7991-7995 12 O'Farrell, P.H, (1975) J. Biol. Chem. 250, 4007-4021 13 Greenawalt, J.W. (1979) Methods Enzymol. 31,310-323 14 Anderson, L. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2407-2411 15 Penefsky, H.S. (1979) Adv. Enzymol. 49, 223-280 16 Henslee, J.G. and Srere, P.A. (1979) J. Biol. Chem. 254, 5488-5497 17 Burnett, K.G. and Scheffler, I.E. (1982) J. Cell Biol. 90, 108-115 18 Hare, J.F. and Hodges, R. (1982) J. Biol. Chem. 257, 3375-3580 19 Gay, N.J. and Walker, J.E. (1983) Nature 301,262-264 20 Ludwig, B. and Capaldi, R.A. (1979) Bioehem. Biophys. Res. Commun. 87, 1159-1167.
285
Elsevier BBA 11294
TRANSFORMATION-ASSOCIATED CHANGES IN NUCLEAR-CODED MITOCHONDRIAL PROTEINS IN 3"1"3CELLS AND SV40-TRANSFORMED 3T3 CELLS STEVEN H. Z U C K E R M A N *, STIG LINDER ** and JEROME M. EISENSTADT ***
Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510 (U.S.A.) (Received December 27th, 1983)
Key words: Transformation," Protein composition," Mitochondrial protein; (3T3 cell)
Comparative two-dimensional gel electrophoretic studies were performed on mitochondrial proteins in nontransformed mouse 3T3 cells and in SV40-transformed 3T3 cells, SV-T2. Two polypeptides, of 58 and 40 kDa, were present in increased mounts in SV40-transformed cells. These polypeptides were demonstrated to be nuclear-ceded mitochondrial proteins by their absence in mitochondrial preparations, when labeling was performed in the presence of a mitochondrial-specific inhibitor, Rhodamine 6G. Temperature-sensitive mutants for transformation were derived from 3T3 cells by transfection with cloned SV40 DNA containing the ts A58 mutation. Increased amounts of the 58 kDa protein were apparent in these cells at the permissive temperature (33°C) compared to the restrictive temperature (39.5°C).
Introduction Warburg's hypothesis that cancer cells have an elevated rate of glycolysis due to an impaired respiratory capacity [1] and the finding that mitochondria are the principal site for oxidative phosphorylation and ATP generation in animal cells [2] resulted in extensive investigations of mitochondria from neoplastic cells. Differences in mitochondrial structure and numbers, as well as in mitochondrial enzyme content, membrane lipids, transport mechanisms and substrate specificities, have been reported [3,4]. However, these differences are variable depending on the tumor source, mitochondrial isolation procedures and the * Present address: Department of Immunology, Lilly Research Laboratories, 307 East McCarty Street, Indianapolis, IN 46285, U.S.A. ** Present address: Department of Medical Genetics, The Biomedical Centre, Uppsala University, 75123 Uppsala, Sweden.
*** To whom correspondence should be addressed. Abbreviation: R6G, rhodamine 6G. 0167-4889/84/$03.00 © 1984 Elsevier Science Publishers B.V.
in vivo and in vitro growth properties of the tumors. Accordingly, it has been difficult to define which changes are associated with neoplastic transformation. In a different approach, sarcomavirus-transformed mink fibroblasts were reported to have a decreased mitt~hondrial membrane potential compared to control, nontransformed mink fibroblasts [5]. These differences in membrane potential were based on the decreased uptake and retention of the mitochondrial-specific cationic dye .Rhodamine 123 and were reversible following addition of the ionophore nigericin. Further studies on the retention of Rhodamine 123 in various tumors indicated that many transitional cell carcinomas, adenocarcinomas and chemically transformed epithelial cell lines retained Rhodamine 123 for extended periods, while oat cell carcinomas did not [6]. These results demonstrate that neoplastic transformation is associated with discrete mitochondrial changes. In the present study, we have performed comparative studies on nuclear-coded mitochondrial proteins in nontransformed 3T3 cells and in
286
Materials and Methods
Charles Cole, Dartmouth College, Hanover, NH). The temperature-sensitive mutation has been mapped within the large T antigen [8]. Calcium phosphate coprecipitates of ts A58 and carrier DNA were incubated with 3T3 cells [9]. Following incubation, cells were seeded in soft agar and incubated at 33°C. Four soft agar clones (3.4, 4.4, 5.1 and 6.1) were isolated, which grew in soft agar at 33°C, but not at 39.5°C.
Cell culture Balb/c 3T3 cells clone A31 and SV40-transformed 3T3 cells, SV-T2, were obtained from the American Type Culture Collection. These cell lines were maintained at low density in Dulbecco's modified Eagle's media supplemented with 5% calf sera [7]. Temperature-sensitive transformation mutants of 3T3 were obtained using the ts A58 SV40 mutant inserted in pBR322 (provided by Dr.
Mitochondria preparation and gel electrophoresis 2.106 cells were seeded in 25 cm2 flasks (Falcon) and labeled for 90 min (short pulse) or 18 h (long pulse) with 200 and 40 #Ci/ml, respectively, of [35S]methionine (Amersham International) in methionine-free media (GIBCO, Grand Island, NY). In parallel cultures, Rhodamine 6G (Kodak) was present at 2 #g/ml during the overnight labeling [10]. Following labeling, cultures were washed
SV40-transformed 3T3 cells by two-dimensional gel electrophoresis. Increased quantities of two nuclear-coded mitochondrial proteins, of 58 and 40 kDa, were detected in SV40-transformed 3T3 cells. Increased synthesis of the 58 kDa protein was also detected in 3T3 cells transformed with the SV40 ts A58 mutant.
Fig. 1. R6G-sensitivity of nuclear-coded mitochondrial proteins in SV-T2. SV-T2 was labeled for 18 h with 40 ~ C i / m l of [35S]methionine in the absence (a) or presence (b) of 2 ~ g / m l R6G. Mitochondria were then isolated, and nuclear-coded mitochondrial polypeptides were resolved by two-dimensional gel electrophoresis. Approx. 30 polypeptides were absent or significantly reduced, including the predominant polypeptides 1, 2 and 3. The position of these polypeptides in R6G-treated SV-T2 is indicated by circles. Two R6G-sensitive polypeptides (arrows) are present in greater quantities in SV-T2 relative to non-transformed 3T3 cells (see Fig. 2). The contaminating cytoskeletal proteins actin (Ac) tubulin (T) and vimentin (vt) are not sensitive to R6G. A pH gradient between 4.0 and 6.5 was generated in the isoelectric-focusing gels. The basic region of the iscelectric-focusing gel is to the left. Molecular weights are indicated in thousands.
287
and mitochondria were prepared using the procedure of Bogenhagen and Clayton [11]. Unlabeled mitochondria were added as carrier prior to centrifugation in a 1.0-1.5 M sucrose step gradient. The mitochondria were washed and lysed in O'Farrell lysis buffer [12]. Mitochondrial lysates were electrophoresed on two-dimensional gels with isoelectric focusing in the first and SDS-10.5% polyacrylamide gels in the second dimension [12]. The isolation of an inner nutochondnal membrane-enriched preparation was performed by digitonin solubilization of the outer membrane and Lubrol WX fractionation of the inner membrane and matrix [13]. In additional experiments, mitochondria were lysed with 0.5% NP-40 and incubated for 30 min at 37 °C with 10 U of bovine alkaline phosphatase (Sigma, St. Louis, MO) at pH 8.5 in the presence of 2 mM phenylmethylsulfonyl fluoride. Alkaline phosphatase-treated lysates were then subjected to two-dimensional gel electrophoresis. All gels were dried and exposed to Kodak X-Omat film. •
#
.
Results
Mitochondria prepared from [35S]methioninelabeled 3T3 cells and 3T3 cells transformed with SV40 were lysed and resolved using two-dimensional gel electrophoresis. Preliminary experiments indicated that polypeptides comigrating with cytoskeletal components were abundant in sucrose gradient purified mitochondrial preparations. It was thus necessary to distinguish between mitochondrial specific polypeptides and contaminating cytoplasmic polypeptides. This was achieved by labeling parallel cultures in the presence or absence of 2 # g / m l of R6G, a mitochondrial-specific dye which inhibits oxidative phosphorylation [10]. R6G-treated cells are unable to transport nuclearcold mitochondrial proteins into and across the inner mitochondrial membrane. Therefore, only polypeptides that were sensitive to R6G were considered for analysis. Fig. 1 shows the polypeptide patterns of SV-T2 cells labeled in the absence (Fig. la) or presence (Fig. lb) of R6G. Approx. 30 polypeptides were absent or significantly diminished in mitochondrial preparations from R6G-treated cells. These polypeptides represent nuclear-coded mitochondrial proteins and differ in
their relative abundance, molecular weights and isoelectric points. A number of polypeptides are common to both preparations, notably actin, tubulins and vimentin. The identity of these polypeptides were confirmed by tryptic peptide analysis (not shown). The predominant R6G-sensitive polypeptides 1-3 correspond to the Mitcon 1-3 polypeptides described by Anderson [14]. 3T3 and SV-T2 mitochondrial polypeptides were then analyzed by two-dimensional gel electrophoresis in cells labeled for 90 min with [35S]methionine (Fig. 2). Two R6G-sensitive polypeptides, of 58 and 40 kDa, were present in approx. 6- and 3-fold greater quantities respectively in SV-T2 (Fig. 2b) compared to 3T3 cells (Fig. 2a). The 40 kDa polypeptide remained associated with the inner membrane during mitochondrial fractionation with digitonin and Lubrol WX (Fig. 2c). The 58 kDa polypeptide could not be unequivocally assigned to matrix or membrane fractions• Protein 1 (Mitcon 1) was enriched in the inner membrane fraction and was found to react with an anti-F1 ATPase serum in immunoblot experiments [20]. Mitcon 1 was identified as the fl-subunit of the F1 ATPase complex, based on its molecular weight and isoelectric point [15,16]• Proteins 2 and 3 were enriched in the matrix fraction• Incubation of detergent solubilized mitochondria with alkaline phosphatase resulted in the altered electrophoretic mobility of several polypeptides, presumably due to phosphate removal (Fig. 2d and e). This procedure did not alter the mobility of the 40 kDa polypeptide, suggesting that this transformationrelated polypeptide is not phosphorylated. The 58 kDa polypeptide appeared to be more labile and could not be detected after incubating mitochondrial lysates at 37 °C, with or without phosphatase. In further experiments, neither the 40 nor the 58 kDa protein were labeled with [32p]orthophosphate. In a different series of experiments, four SV40 ts A58-transformed clones of 3T3 were isolated which were capable of growth in soft agar at the permissive temperature of both 33°C and 39.5 °C. One of these lines, ts 6.1, was labeled with [35S]methionine for 18 h at 33°C, but not at 39.5 o C. Two-dimensional gel analysis (Fig. 3) indicated that at 33°C (Fig. 3b) the ts 6.1 line had 5-6-fold greater amounts of the 58 kDa protein
288
3T3
SV-T 2
-68
-45
-25
.....
i~i,
:oii!!!iii!!i
~ii ¸ ~
©
rig. z. t ranstormaUon-related changes in nuclear-coded mitochondrial polypeptides. 3T3 cells (a) or the SV40-transformed SV-T2 (b) were labeled for 90 rain with 200 pCi/ml of [35S]methionine. Mitochondria Were then isolated and nuclear-coded mitochondrial polypeptides were resolved by two-dimensional gel electrophoresis. Two R6G-sensitive polypeptides (arrows) of 58 and 40 kDa, were present in greater quantities in SV-T2 compared to 3T3 cells. (c) The 40 kDa polypeptide, actin, and polypeptide 1 fractionate with an inner mitochondrial membrane preparation, while polypeptides 2 and 3 are lost and represent matrix proteins. [ 35S]Methionine-labeled mitochondrial lysates were incubated in the absence (d) or presence (e) of bovine alkaline phosphatase. The 40 kDa polypeptide (arrow) is not altered by alkaline phosphatase treatment, while the mobility of two other polypeptides (open arrowheads) was altered.
289
Fig. 3. Temperature-dependentchanges in the 58 kDa protein in the ts 6.1 cell line. 3T3 cells were transformed with the ts A58 mutant of SV40, and the 6.1 cell line was isolated. The 6.1 line was labeled with 40 #Ci/ml of [35S]methioninefor 18 h at the restrictivetemperatureof 39.5o C (a) or permissivetemperature of 33° C (b) for transformation.Note increased amounts of the 58 kDa protein at the permissivetemperature(b).
compared to the same line at 39.5 °C (Fig. 3a). Similar results were observed with the three other temperature-sensitive transformed lines. None of these lines revealed any significant differences in the expression of the 40 kDa protein at the two temperatures.
Discussion The present experiments suggest that transformation of 3T3 cells with SV40 results in quantitative changes in nuclear-coded mitochondrial proteins. This was evident in both SV-T2 and in the ts A58-transformed mutants at the permissive temperature for transformation. Two proteins, of 58 and 40 kDa, were found in increased quantities in the SV-T2 cells. These proteins were identified as nuclear-coded mitochondrial proteins by their sensitivity to R6G. Increased synthesis of the 58 kDa protein, but not the 40 kDa protein, was also detected in the temperature-sensitive 3T3 transformants. Whether this reflects the variability inherent in different transformed lines is not known. In additional studies, 3T3 and SV-T2 were labeled with [32p]orthophosphate, and the mitochondrial polypeptides were resolved on twodimensional gels. A similar pattern of phosphoproteins was observed on two-dimensional gels of 3T3 and SV-T2 cells (data not shown). None of the approx. 50 phosphoproteins that we resolved were R6G-sensitive. This observation could be ex-
plained by the phosphorylation of proteins already inside the mitochondria. We were therefore unable to distinguish between nuclear-coded mitochondrial phosphoproteins and cytoplasmic phosphoprotein contaminants. Biochemical and structural differences have been reported in mitochondria from tumor vs. control cells [1-4]. However, although two-dimensional gel electrophoretic patterns have been reported for mitochondria preparations [14,16-18], we are unaware of any comparative two-dimensional gel studies of mitochondrial proteins between closely related normal and transformed cells. This may in part be related to the problem of cytoplasmic contamination of mitochondrial preparations. The use of the mitochondrial inhibitor rhodamine 6G enabled us to identify nuclear-coded mitochondrial-specific proteins on our two-dimensional gels. The potassium ionophore nonactin has been used in a similar approach to identify nuclear-coded mitochondrial proteins in whole-cell lysates [14]. A nuclear-coded mitochondrial protein (Mitcon 5) was lost in human lymphocytes, following treatment with phorbol ester tumor promoters. Furthermore, this transformation-sensitive protein was not detected in lymphoblastoid cell lines. Further studies are required to identify other transformation-related changes in nuclear-coded mitochondrial proteins. Recently, Gay and Walker [19] have reported that a portion of p21, the product of the bladder carcinoma oncogene, exhibits significant sequence homology to the nucleotide-binding region of the fl-subunit of the nuclear-coded mitochondrial A T P synthase. Clearly, it is necessary to define how transformation-related qhanges in nuclear-coded mitochondrial proteins affect the function and regulation of the mitochondrial genome.
Acknowledgements This work was supported by N I H grant G M 21873 awarded to J.M.E.S.H.Z. was supported by a Swebilius Cancer Research Award. S.L. was supported by a James Hudson Brown Postdoctoral fellowship. S.H.Z. would like to thank Dr. Sandra Zuckerman for her stimulating discussions.
290 References 1 Warburg, O. (1956) Science 123, 309-314 2 Kennedy, E.P. and Lehninger, A.L. (1948) J. Biol. Chem. 172, 847-848 3 Pederson, P.L. (1978) Prog. Exp. Tumor Res. 22, 190-274 4 Spitsberg, V.L. (1979) Arch. Biochem. Biophys. 195, 136-144 5 Johnson, L.V., Summerhayes, I.C. and Chen, L.B. (1982) Cell 28, 7-14 6 Summerhayes, I.C., Lampidis, T.J., Bernal, S.D., Nadakavukaren, J.J., Nadakavukaren, K.K., Shepherd, E.L and Chen, L.B. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5292-5296 7 Aaronson, S.A. and Todaro, G.J. (1968) J. Cell. Physiol. 72, 141-148 8 Lai, C.-J. and Nathans, D. (1975) J. Virol. 66, 70-81 9 Abrahams, P.J. and Van der Eb, A.J. (1975) J. Virol. 16, 206-209
10 Gear, A.R.L. (1974) J. Biol. Chem. 249, 3628-3637 11 Bogenhagen, D. and Clayton, D.A. (1974) J. Biol. Chem. 249, 7991-7995 12 O'Farrell, P.H, (1975) J. Biol. Chem. 250, 4007-4021 13 Greenawalt, J.W. (1979) Methods Enzymol. 31,310-323 14 Anderson, L. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2407-2411 15 Penefsky, H.S. (1979) Adv. Enzymol. 49, 223-280 16 Henslee, J.G. and Srere, P.A. (1979) J. Biol. Chem. 254, 5488-5497 17 Burnett, K.G. and Scheffler, I.E. (1982) J. Cell Biol. 90, 108-115 18 Hare, J.F. and Hodges, R. (1982) J. Biol. Chem. 257, 3375-3580 19 Gay, N.J. and Walker, J.E. (1983) Nature 301,262-264 20 Ludwig, B. and Capaldi, R.A. (1979) Bioehem. Biophys. Res. Commun. 87, 1159-1167.