Interspecific variations in proteins synthesized by mammalian mitochondria1 ~ N D A L L W.

ATS SCOFF,^ LUKEAUJUME, A N D KARLB . FREE MAN^

Department of Biochemistry, McMaster University, Hamilton, Ont., Canuda L8S 4J9

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SAMUEL GOLDSTEIN Depurtnlents of Biochemistry and Medicine, McMaster University, Hamilton, On?., Canada L8S 4J9 Received December 6 , 1977 Revised April 3, 1978 Yatscoff, R. W., Aujame, L., Freeman, K. B. & Goldstein, S. (1978) Interspecific variations in proteins synthesized by mammalian mitochondria. Can. J . Biochem. 56,939-942 The products of mitochondria1 protein synthesis in established cell lines of various mammalian species were labelled with [3sS]methionine and their number and apparent molecular weights determined by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis and fluorography. Proteins synthesized by isolated rat liver mitochondria were labelled with [3H]valine and similarly characterized. Each species had a distinctive pattern of from 10 to 13 mitochondrially synthesized proteins with apparent molecular weights between 10 000 and 50000. No differences were detected in the number or electrophoretic mobility of the mitochondrially synthesized proteins of SV-40-transformed and nontransformed WI-38 cells. Yatscoff, R. W., Aujame, L., Freeman, K. B. & Goldstein, S. (1978) Interspecific variations in proteins synthesized by mammalian mitochondria. Can. J . Biochem. 56,939-942 Nous avons marque, avec la f3"]methionine, les produits de la synthese proteique mitochondriale dans des lignees cellulaires etablies de diverses especes de mammifkres et nous avons determine leur nombre et leur poids moleculaire apparent par electrophorese sur des tranches de gel de polyacrylamide avec dodecylsulfate de sodium et par fluorographie. Les proteines synthetisees par les mitochondries isolees du foie de rat sont marquees avec la ['H]valine et nous les avons caracterisees de la m2me faqon. Chaque e s g c e montre un profil different des 10 h 13 proteines synthetisees par les mitochondries, avec des psids molCculaires apparents allant de 10008 2 50000. Nous n'avons dicele aucune difference dans le nombre ou dans la mobilite electrophoretique des proteines synthetisees par les mitochondries des cellules SV-40 transformees et des cellules WI-38 non transformees. [Traduit par le journal]

Introduction Materials and Methods The sources of all chemicals are described elsewhere (1) exRelatively simple electrophoretic profiles have been reported for the proteins synthesized in mammalian cept for L-[2,3-3H]valinewhich was obtained from New England mitochondria (1 and references contained within). Jef- Nuclear, Boston, MA. Chinese hamster CHO TK-, bovine EBTR, mouse L, and rat freys and Craig (2) compared the proteins synthesized in HTC cell lines were obtained a s previously described (1, 3. 4). mitochondria of various animal species by sodium do- Human WI-38 cells originally derived from embryonic lung tisdecyl sulfate slab gel electrophoresis and fluorography. sue (5) were a gift of Dr. L. Hayflick (Stanford University, Seven to 1I bands were found for each species but back- Stanford, CA). The VA-13A cell line, a SV-40 transformant of ground radioactivity prevented a more accurate estima- WI-38 (61, was a gift of Dr. V. CristofaIo (Wistar Institute, tion. In the present work, improvements in methodology, Philadelphia, PA); Syrian hamster BHK-21 cells, originaily dewhich give better resolution of bands and a more readily rived from the kidneys of baby hamsters (7), were obtained from determined and constant base line, have enabled mork Dr. W. E. Rawls (Department of Pathology, McMaster Univerprecise enumeration of the proteins synthesized in situ in sity, Hamilton, Ont.). All cell lines were grown as previously mitochondria of various mammalian cell lines as well as described (1. 3, 4) except for the BHK-21, WI-38, and VA-l3A cell lines which were grown in monolayer in Eagle's minimal those synthesized by isolated rat liver mitochondria. essential medium supplemented with 15% fetal calf serum. ABBREVIATIONS: CHC) TK-, Chinese hamster ovary (thymidine kinase minus); EBTR, embryonic bovine trachea; HTC, hepatoma tissue cell. 'Supported by the Medical Research Council of Canada (grants No. MT-1940 and MT-3513). =Recipientof a Medical Research Council of Canada studentship, 'Author to whom all correspondence should be addressed,

The products of mitochondria1 protein synthesis in whole cells were labelled and mitochondria isolated as previously described (1). WI-38 cells were labelled about halfway through their limited replicative life-span (5). Rat liver mitochondria were isolated as described by Fukamachi et al. (8). Protein synthesis by isolated mitochondria was carried out in the medium described by Wallace et al. (9) in the presence of 22.5 gCi (1 Ci = 37 GBq) L-[2,3-3H]valine (17.7 Cilmmol) per millilitre. Electrophoresis in sodium dodecyl sulfate - 12.5%

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The number of distinct electrophoretic bands obtained for each species may not accurately reflect the number of proteins. Partial aggregation may have occurred which would increase the number of bands observed, or two genetically distinct proteins could comigrate by chance so as to reduce the number of detectable electrophoretic components (1). For example, in the densitornetric patResults and Discussion terns obtained for all species, the peak having an apFigure 1 shows the fluorographic patterns of the whole parent molecular weight of 48000 is the most intensely cell products of mitochondrial protein synthesis of labelled and has the greatest width. Thus, as considered human VA-13A cells (A) rat HTC cells (B), mouse L cells elsewhere (1-3), this peak could consist of two comigrat(D), and CHO TK- cells (E), along with the products of ing proteins. Attempts were made to improve resolution protein synthesis of isolated rat liver mitochondria (C). by two-dimensional (12) and exponential gradient gel Densitornetric tracings of the fluorographic patterns for electrophoresis (13) but the former has not been successthe CHO, L, and HTC cell lines and for isolated rat liver ful as yet and the latter did not yield better separations. It mitochondria are shown on the left side of Fig. 2 and is also possible that the synthesis of some proteins in those of the proteins synthesized in mitochondria of mitochondria would not be seen with a labelling time of BHK, EBTR, WI-38, and VA-13A cell lines are shown 1 h but previous results with CHO TK- ( I ) and HeLa on the right side. As shown in these figures, the number of cells (14, 15) suggest that the number does not sigbands varied from 10 to 13. At least one mitochondrially nificantly vary with time of labelling. The apparent synthesized protein of the CHQ TK- cell line is not molecular weights obtained for the mitochondrially labelled with methionine (I) so that the minimum number synthesized proteins of the VA-13A cell line (Fig. 1) are (numbered in the reverse order previously (1)) in this cell approximately 10% less than those obtained for these line is probably 11. At least 12 proteins were synthesized same proteins when they were run on another gel (shown in mitochondria of L, WI-38, VA-13A. and EBTR cells, in Fig. 2). Apparent molecular weights for the proteins while13 were synthesized in mitochondria of HTC and synthesized in the mitochondria of the CHO TK- cell line BHK cells. in the gel shown in Fig. 1are also approximately 10% less These are true components synthesized in mitochon- than previously reported (1). These lower molecular dria as shown by the inhibition of their synthesis by weights were only obtained with one particular lot of Tevenel, a specific inhibitor of mitochondrial protein acrylamide; with three other lots of acrylamide, the synthesis (I), in the case of CHO TK- cells (I), EBTW higher molecuar weight values were obtained. cells (3), HTC, and and VA-13A cells and rat liver It can be seen in Fig. 2 that the proteins synthesized in mitochondria (results not shown). The low backgrounds the mitochondria of different species differ not only in with BHK and L cells indicate that there is no contamina- electrophoretic mobility but also in relative amounts. tion with proteins synthesized on cytosolic ribosomes. However, it is not possible to state which band for each The proteins synthesized in mitochondria are in the species corresponds to bands of another species. Some molecular weight range of between 10888 and 50000. bands have been reported to be subunits of cytochrome c Approximately 5 x 18"altons of mitochondrial DNA oxidase (EC 1.9.3.1). The number suggested has been would be required to code for these 18-13 proteins. If the one (16), two (components 2 and 6 of bovine EBTR cells portion of the genome coding for mitochondrial rRNAs in Fig. 2) (3), or three (17). In any case, the patterns and tRNAs is included (ID), this accounts for about 80% obtained for the mouse L, CHO, and rat HTC cells were of the coding capacity of animal mitochondrial DNA if more similar to each other than those obtained for Syrian genes do not overlap and this DNA codes for these pro- hamster BHK-21, human WI-38, and VA-13A or bovine teins. EBTR cells. If these proteins are coded by mitochondrial Previous studies by Jeffreys and Craig (2) had found DNA (lo), then the variation may reflect known differthat 7 to 11 proteins were synthesized in mitochondria of ences in the mitochondrial DNA from species to species a number of animal cell lines. In particular, they ob- (18-21). Densitometric patterns obtained for proteins syntheserved eight predominant and three possible minor components synthesized in HeLa cell mitochondria, and sized by isolated rat liver mitochondria and mitochondria since one of these was thought to consist of two compo- of cat HTC cells were almost identical. The only excepnents. this brings to 12 the number of proteins synthe- tions were the possible absence of component'$ and the sized in mitochondria of human cells. This contrasts with presence of a few additional lower molecular weight the 10 reported by Lederman and Attardi (11). The components in the rat liver mitochondria1profile. In most slightay lower numbers seen by the other groups (2, 11) previous studies usually involving poorer resolution of probably reflect higher and ill-defined base lines and (or) bands (1, 11, 22, 23), similar although not identical patpoorer resolution. An additional component running be- terns were also seen suggestingthat although some minor tween bands 6 and 7 was detected in the fluorographic dgferences of unknown origin are present isolated mitopattern obtained for the VA-13A cell line in Fig. 1 and as a chondria synthesize the same proteins as mitochondria sf minor band in Fig. 2. In later preparations of the VA-13A whole cells. Previously, Birkmayer and Balda (24) found a differcell line and other human cell lines, this component was ence in the number and electrophoretic mobilities of the not observed and is probably not a true component.

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polyacrylamide slab gels and fluorography were by methods described previously (1, 3). Fluorograrms were scanned with a Joyce-Loebl scanning densitometer (I). Except for bands with very low or very high radioactivity, exposure was proportional to radioactivity. The radioactivity of the majority of bands was within the linear range.

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YATSCOFF ET AL.

FIG. I. Fluorogmphic patterns of mitochondria1 proteins of various mammalian cell lines labelled in the presence of cycloheximide. Mitochondria] proteins of each cell line were labelled with 20pCi [35S]methionineper millilitre in the presence of 308 pg cyclohexirnide per millilitre. Mitochondria were isolated and proteins subjected to sodium dodecyl sulfate polyacrylarnide slab gel electrophoresis as described in the Materials and Methods. Proteins synthesized by isolated iyatBiver mitochondria were labelled with [Wlvaline in the presence of 308pg cycloheximide per millilitre. Apparent molecular weights marked to the right of the gel were determined as previously described (1). (A) VA-138, (B) HTC, (C) isolated rat liver mitochondria with different amounts of protein run in each channel, (Dl L, (E) CHO TK- (F) electrophoretic front, (TI top of gel.

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labelling of this component between VA-13A and WI-38 ceils (Fig. 2) reflects this variation. In other preparations of VA-B3A cells, this protein was labelled as intensely as it was in the WI-38 cells. The results suggest that transformation, at least by SV-40 virus, does not alter the number o r electrophoretic mobility of proteins synthesized in mitochondria. Acknowledgements We thank Ms. Hansa Patel and Mrs. Elena Moerman for skilled technical assistance.

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FIG. 2. Densitometric tracings of fluorograms of proteins synthesized in mitochondria s f various mammalian species and those synthesized by isolated rat liver mitochondria. Proteins were labelled as described in Fig. 1 . All scans on the left were obtained from the gel shown in Fig. I. All scans on the right were obtained from another gel, except for EBTW cells, in which a gel was used where the marker proteins had run the same distance. Numbers above each peak represent distinct reprsducible components identified consecutive!y from the front for a given species. Numbers are not meant to be comparable from one cell type to another except for rat liver mitochondria which follow that of HTC cells. F, electrophoretic front.

proteins synthesized by isolated mitochondria from rat liver and hamster meianoma ceils and suggested that this reflected a difference between normal and tumor cells. From our present results, this difference probably reflects an interspecific variation. In contrast, comparison of a normal human cell line (WI-38) and its SV-40 transformant (VA-13A) revealed no difference in the number or molecular weights of proteins synthesized in mitochondria (Fig. I). The intensity of labelling of protein 3 varied in different preparations of VA-13A and other human cell lines, and the difference in intensity in

1 . Yatscoff, R. W. & Freeman, K. B. (1977)Can. J. Bhockem. 55, 1064- 1074 2. Jeffreys. A. J. & Craig, I. W. 4 1976) Nat~tre(London),259, 698-692 3. Yatscsff, R. W., Freeman, K. B. & Vail, W. J . (1977)FEBS Lett. 81,7-9 4. Mitra, R. S., Bartoov. B., Monahan, J. & Freeman, K. B. (1972)Biochena. J . 18,1833-1041 5. Hayflick, L, (1961)Exp. Cefl.Res. 25,585-621 6- Girardi, A. J., Weinstein, D. & Moorhead, B. S. (1966)Ann. Med. Exp. Biol. Fenn. 44,242-254 7 . Macpherson, J. A. B Stoker, M. G. P. (1962) Viro10g.y 16, 147-153 8. Fukamachi, S.. Bartoov, B., Mitra, R. S. & Freeman, K . B. (1972)Biochern. Biophys. Res- C0mm64n. 40,852-857 9. Wallace, R. B., Williams, T. M. & Freeman, K. B. (1975) Ertr. J . Biochenz. 59, 167- 173 10. Borst, P. (1977) Trends Biochenz. Sci. 2,31-34 1 1 . Lederrnan, M. & Attardi, 6 . (1973) J. Mol. Biol. 78, 275-283 12. Ames, 6 . F. L. & Nikaids, K. (1976) Bs'uchernisbry 15, 616-623 13. Douglas, M. G. & Butow, R . A. (1976) Proc. Natl. Acati. Sci. L1.S.A. 73. 1083-1086 14. Costantino, P. $t Attardi, G. (1975) J . Mul. BioE. 96, 297-306 15. Jeffreys, A. J . & Craig, 1. W. (1976) Eur. J . Biochena. 68, 301-311 16. JeffPeys, A. J. &Craig, I. W. (1977)FEBSLett.77, 151-154 17. Bernstein, J. D., Bucher, J. R. & Penniall, R. (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 15 13 78. Dawid, I . B. (1972)Dev.Biol. 29, 139-151 19. Coon, H. G., Horak. H. & Bawid, 1. B. (1973)J. Mol. Biol. 51,285-298 20. Jacovic, S., Carey, J. & Wabinowitz, M. (1975)Biockemisars; 14,2037-2043 21. Potter, S . S., Newbold, 9. E . , Hutchisen, C. A. & Edgell, M. H. (1975)Proc. Ncrtl. Acad. Sci L1.S.A. 7,3,44%-450 22. Ibrahim, N . G., Burke, J. P. & Beattie, D. J. (1972) FEBS Left.29,73-76 23. Coote, 9. L. & Work, T. S. (1971) Eur. J . Biuchen~.23, 564-574 24. Birkmayer, G. D. & Balda, B. 8. (1971) FEBS Lett. 15, 156- 168

Interspecific variations in proteins synthesized by mammalian mitochondria.

Interspecific variations in proteins synthesized by mammalian mitochondria1 ~ N D A L L W. ATS SCOFF,^ LUKEAUJUME, A N D KARLB . FREE MAN^ Departmen...
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