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The maternal inheritance of proteins synthesized in mammalian mitochondria
Preliminary 11. Pokomy, K S, Ph.D. Thesis, Columbia Univ (1971). Received June 6, 1980 Revised version received September 24, 1980 Accepted September 29, 1980
Copyright @ 1981 hy Academic Press. Inc. All rights of reproduction in any form reserved 0014.4827/8l/OZO417-03$02.00/O
The maternal inheritance of proteins synthesized in mammalian mitochondria RANDALL W. YATSCOFF,’ JEREMY R. MASON,’ LARRY W. BELBECKZ and KARL B. FREEMAN,’ ‘Department of Biochemistry and $Department of Pathology, McMaster ton, Ont L.8N 325, Canada
University,
Hamil-
Interspecific variations between the proteins synthesized in the mitochondria of two closely related-mammalian species were demonstrated to be matemallv inherited bv examination of their hybrid progeny. -This, in accbrdance with the documented &parental inheritance of mitochondrial DNA (mtDNA) would indicate that the latter codes for the products of mitochondrial translation.
Summary.
The mitochondrial genome of lower eukaryotes has been shown to code for a limited number of RNA and protein species [ 1, 21. These cytoplasmic genes have been identified by reciprocal crosses in which they are transmitted only from the female parent [3]. In contrast, in higher eukaryotes, although the mitochondrial genome has been found to code for poly A-containing RNA species, it has not been directly ascertained whether this RNA is translated into mitochondrial protein [4] and uniparental inheritance of mitochondrial DNA (mtDNA) has only been demonstrated by means of molecular techniques [5-91. For example, Hutchison et al. [6] utilized difference between the restriction fragment patterns of the mtDNAs of the horse, Equus caballus, and the donkey, E. asinus, to show that reciprocal hybrids (mules and hinnies) had no (less than 5 %) paternal mtDNA. Printed
in Sweden
notes
417
Previously we and others have shown that between 10 and 13 mitochondrial translation products are synthesized by whole mammalian cells [l&13]. Interspecific variation in the number and electrophoretic mobilities of these proteins has been demonstrated, which could reflect known difference in the mtDNA from species to species [4, 14, 151.Alternatively, if the mRNA is coded in the nucleus but transported into and translated within mitochondria, this variation could reflect the divergence of the nuclear genome amongst species. Utilizing the known maternal inheritance of mtDNA and electrophoretic difference in the proteins synthesized in mitochondria of zebra (E. zebra) and donkey (E. asinus), the genes coding for these proteins were shown to be in mtDNA by characterization of the mitochondrial proteins synthesized by their hybrid, the zeedonk. Materials
and Methods
Biopsy samples were taken from the neck of a female donkey, from a male zebra and from their hybrid, a male zeedonk. The samples were explanted in Petri dishes and then crown as adherent cultures at 37°C in a-minimal es&tial medium (MEM) [16] supplemented with 15% (v/v) fetal calf serum (FCS). Approx. 3 months after explantation, lo8 cells from each suecies were harvested and labelled with PSLmethionine (1000 Ci mmol-I) as previously de&ibid [lo]. This was carried out in the presence of either 300 pg cycloheximide ml-l to specifically inhibit protein synthesis on cytosolic ribosomes or cycloheximide plus 150 pg Tevenel ml-’ to additionally inhibit mitochondrial translation. The mitochondria were isolated from these labelled cells [lo] and their polypeptides analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis incorporating 12.5% acrylamide monomer and utilizing the buffer system of Laemmli [17]. Fluorography was performed on the resulting gels [18].
Results
Fig. la shows a fluorogram of mitochondrial proteins from donkey cells labelled in the presence of cycloheximide, to permit mitochondrial translation only. Thirteen components were identified as products of Exp CellRrs 131 (1981)
418
Preliminary
notes
the mitochondrial protein synthesis, with apparent molecular weights ranging from 6500 to 45 000. Minor bands of molecular weight greater than component 2 represent residual cytosolic protein synthesis, except for the probable presence of component 1, deduced in comparison with earlier work [lo, 111. Cells labelled in the presence of Tevenel and cycloheximide gave rise to no detectable incorporation of radiolabel, in the region of bands 2-13 confirming that the electrophoretic bands shown in fig. 1a represent the products of mitochondrial translation. From fig. 1b it may be seen that there is a distinct species variation with respect to bands 5 and 6. This is more apparent in the densitometric scan of the region from bands 5 to 8 (fig. lc). In the donkey profile, band 5 runs behind and band 6 runs ahead of those in the zebra profile. The profile obtained for the zeedonk is identical with that of the maternal parent, the donkey. These differences were found with three separate preparations. Discussion
The results suggest that at least two proteins synthesized in mitochondria are maternally inherited and this is probably true for all 13. Since it has been shown that mtDNA is maternally inherited, unlike the nuclear genome which shows mendelian Fig. I. (a) Fluorographic pattern obtained for proteins svnthesized in the mitochondria of donkey cells. Mitochondrial proteins were Iabelled in whoie cells with 20 uCi PSlmethionine ml-’ in the nresence of 300 pg cycloheximide ml-’ and mitochondria isolated by differential centrifugation as described in the text. Proteins were subjected to electrophoresis in SDS12.5% polyacrylamide slab gels. (b) Fhrorogmphic patterns of proteins synthesized in mitochondtia of (A) donkey, (E) zeedonk and (C) zebra cells. Apparent molecular weights, the top of the gel and the electrophoretic front, are indicated. (c) Densitometric scans of region extending from band 5 to band 8 in (b). Due to limited incorporation of YS band 7 was not detectable. Exp Cell RPS 131 (1981)
Preliminary notes
419
segregation, this indicates that these pro- 20. Barrell, B G, Bankier, A T & Drouin, J, Nature 282 (1979) 189. teins are coded by the mitochondrial ge- 21. Steffens, G J & Buse, G, Hoppe-Seyler’s Z physiol nome. Previously, sequence analysis of them 360 (1979) 613. R W,‘Freeman, K B & Vail, W J, FEBS human mtDNA [20] and bovine subunit II 22. Yatscoff, lett 81 (1977) 7. of cytochrome oxidase [21] indicated that June 11, 1980 mtDNA codes for this protein which is syn- Received Revised version received September 26, 1980 thesized in bovine mitochondria [22]. If one Accepted October 9, 1980 takes into account the number and molecular weights of all proteins synthesized in mitochondria, approx. 7x lo6 D of mtDNA Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved would be required for their coding. If the 0014.4827/81/020419-05~Z.M)/0 portion of the genome which codes for mtrRNAs and tRNAs is included [4], this Receptors for hyaluronate on the surface accounts for approx. 80% of the coding of parent and virus-transformed cell lines Binding and aggregation studies capacity of mammalian mtDNA. We wish to thank Mr M. Homer of the African Lion Safari Park, Rockton, Ontario and Mr W. Clark of Story Book Gardens, London, Ontario for permission to biopsy the animals, and Mrs E. Moerman for technical assistance. This investigation was supported by the MRC of Canada, grant MT-1940.
References 1. Perlman, P S, Douglas, M G, Stausberg, R L & Butow, R A, J mol biol 115 (1977) 675. 2. Borst, P & Grivell, L A, Cell 15 (1978) 705. 3. Birky, C W, Jr, Ann rev genet 12 (1978) 471. 4. Borst, P, Trends biochem sci 2 (1977) 31. 5. Dawid, I B & Blackler, A W, Dev biol 29 (1972) 152. 6. Hutchison, C A, III, Newbold, J E, Potter, S S & Edaell. M N. Nature 251 (1974) 536. 7. B&o; K, Fouts, D L & Wolstenholme, D R, Proc natl acad sci US 75 (1978) 909. 8. Kroon, A M, deVos, W M & Bakker, H, Biochim biophys acta 519 (1978) 269. 9. Brown, G G & Simpson, M W, J supramol biol suppl. 3 (1979) 157. 10. Yatscoff, R W & Freeman, K B, Can j biochem 55 (1977) 1064. 11. Yatscoff, R W, Aujame, L, Freeman, K B & Goldstein, S, Can j biochem 56 (1978) 939. 12. Constantino, P & Attardi, G, J molec biol96 (1975) 291. 13. Jeffreys, A J & Craig, I W, Nature 259 (1976) 690. 14. Dawid, I B, Dev bio129 (1972) 139. 15. Potter, S S, Newbold, J E, Hutchison, C A & Edge& M H. Proc natl acad sci US 72 (1975)4496. 16. Stamters, C P, Eliceiri, C L & Green, H, ‘Nature new biol 230 (1971) 52. 17. Laemmli, U K, Nature 227 (1970) 680. 18. Bonner, W M & Laskey, R A, Eur j biochem 46 (1974) 83. 19. Yatscoff, R W, Goldstein, S & Freeman, K B, Som cell genet 4 (1978) 633. Printed
in Sweden
CHARLES B. UNDERHILL’, * and BRYAN P. TOOLE,’ Developmental Biology Laboratory, Medical Service, Massachusetts General Hospital and Department of Medicine and Anatomy, Harvard Medical School, Boston, MA 02114, USA Summary. Two sets of parent and virus-transformed cell lines (3T3 vs SV-3T3; BHK vs PY-BHK) were compared with respect to the extent of divalentcation independent aggregation which previously has been shown to depend upon the interaction of endogenous hyaluronate with specific receptors on the cell surface. When measured under conditions of physiological ionic strength, a significant amount of hvahuonidase-inhibitable aaareaation was found in the v&us-transformed cell linesBVL3T3 and PY-BHK) but not in their parent counterparts (3T3 and BHK). However, when the same experiment was performed in a high ionic strength solution (0.5 M NaCl), the hyaluronidase inhibitable aggregation was detected in all of the cell lines. The differences in the aggregation between the various cell lines was also reflected in the binding of r3Hlhvahtronate. In uhvsioloaic~ saline. the viru&&for%d cells bound greater -amounts of hyahrronate (higher B,,,) with a greater affinity (lower &) than did their untransformed counterparts. Increasing the ionic strength to 0.5 M NaCl increased the binding of [3H]hyaIuronate by each cell line; however, the relative differences between the cell lines remained. These results indicate that variations in the ability of the cells to bind hyahtronate can partially account for the differences between the parent and the virus-transformed cells with respect to their ability to aggregate.
In previous studies we have shown that there are hyaluronate-binding receptors on i Present address: Department of Anatomy, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA. * To whom offprint requests should be addressed. Exp Cell Res 131 (1981)
Copyright @ 1981 hy Academic Press. Inc. All rights of reproduction in any form reserved 0014.4827/8l/OZO417-03$02.00/O
The maternal inheritance of proteins synthesized in mammalian mitochondria RANDALL W. YATSCOFF,’ JEREMY R. MASON,’ LARRY W. BELBECKZ and KARL B. FREEMAN,’ ‘Department of Biochemistry and $Department of Pathology, McMaster ton, Ont L.8N 325, Canada
University,
Hamil-
Interspecific variations between the proteins synthesized in the mitochondria of two closely related-mammalian species were demonstrated to be matemallv inherited bv examination of their hybrid progeny. -This, in accbrdance with the documented &parental inheritance of mitochondrial DNA (mtDNA) would indicate that the latter codes for the products of mitochondrial translation.
Summary.
The mitochondrial genome of lower eukaryotes has been shown to code for a limited number of RNA and protein species [ 1, 21. These cytoplasmic genes have been identified by reciprocal crosses in which they are transmitted only from the female parent [3]. In contrast, in higher eukaryotes, although the mitochondrial genome has been found to code for poly A-containing RNA species, it has not been directly ascertained whether this RNA is translated into mitochondrial protein [4] and uniparental inheritance of mitochondrial DNA (mtDNA) has only been demonstrated by means of molecular techniques [5-91. For example, Hutchison et al. [6] utilized difference between the restriction fragment patterns of the mtDNAs of the horse, Equus caballus, and the donkey, E. asinus, to show that reciprocal hybrids (mules and hinnies) had no (less than 5 %) paternal mtDNA. Printed
in Sweden
notes
417
Previously we and others have shown that between 10 and 13 mitochondrial translation products are synthesized by whole mammalian cells [l&13]. Interspecific variation in the number and electrophoretic mobilities of these proteins has been demonstrated, which could reflect known difference in the mtDNA from species to species [4, 14, 151.Alternatively, if the mRNA is coded in the nucleus but transported into and translated within mitochondria, this variation could reflect the divergence of the nuclear genome amongst species. Utilizing the known maternal inheritance of mtDNA and electrophoretic difference in the proteins synthesized in mitochondria of zebra (E. zebra) and donkey (E. asinus), the genes coding for these proteins were shown to be in mtDNA by characterization of the mitochondrial proteins synthesized by their hybrid, the zeedonk. Materials
and Methods
Biopsy samples were taken from the neck of a female donkey, from a male zebra and from their hybrid, a male zeedonk. The samples were explanted in Petri dishes and then crown as adherent cultures at 37°C in a-minimal es&tial medium (MEM) [16] supplemented with 15% (v/v) fetal calf serum (FCS). Approx. 3 months after explantation, lo8 cells from each suecies were harvested and labelled with PSLmethionine (1000 Ci mmol-I) as previously de&ibid [lo]. This was carried out in the presence of either 300 pg cycloheximide ml-l to specifically inhibit protein synthesis on cytosolic ribosomes or cycloheximide plus 150 pg Tevenel ml-’ to additionally inhibit mitochondrial translation. The mitochondria were isolated from these labelled cells [lo] and their polypeptides analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis incorporating 12.5% acrylamide monomer and utilizing the buffer system of Laemmli [17]. Fluorography was performed on the resulting gels [18].
Results
Fig. la shows a fluorogram of mitochondrial proteins from donkey cells labelled in the presence of cycloheximide, to permit mitochondrial translation only. Thirteen components were identified as products of Exp CellRrs 131 (1981)
418
Preliminary
notes
the mitochondrial protein synthesis, with apparent molecular weights ranging from 6500 to 45 000. Minor bands of molecular weight greater than component 2 represent residual cytosolic protein synthesis, except for the probable presence of component 1, deduced in comparison with earlier work [lo, 111. Cells labelled in the presence of Tevenel and cycloheximide gave rise to no detectable incorporation of radiolabel, in the region of bands 2-13 confirming that the electrophoretic bands shown in fig. 1a represent the products of mitochondrial translation. From fig. 1b it may be seen that there is a distinct species variation with respect to bands 5 and 6. This is more apparent in the densitometric scan of the region from bands 5 to 8 (fig. lc). In the donkey profile, band 5 runs behind and band 6 runs ahead of those in the zebra profile. The profile obtained for the zeedonk is identical with that of the maternal parent, the donkey. These differences were found with three separate preparations. Discussion
The results suggest that at least two proteins synthesized in mitochondria are maternally inherited and this is probably true for all 13. Since it has been shown that mtDNA is maternally inherited, unlike the nuclear genome which shows mendelian Fig. I. (a) Fluorographic pattern obtained for proteins svnthesized in the mitochondria of donkey cells. Mitochondrial proteins were Iabelled in whoie cells with 20 uCi PSlmethionine ml-’ in the nresence of 300 pg cycloheximide ml-’ and mitochondria isolated by differential centrifugation as described in the text. Proteins were subjected to electrophoresis in SDS12.5% polyacrylamide slab gels. (b) Fhrorogmphic patterns of proteins synthesized in mitochondtia of (A) donkey, (E) zeedonk and (C) zebra cells. Apparent molecular weights, the top of the gel and the electrophoretic front, are indicated. (c) Densitometric scans of region extending from band 5 to band 8 in (b). Due to limited incorporation of YS band 7 was not detectable. Exp Cell RPS 131 (1981)
Preliminary notes
419
segregation, this indicates that these pro- 20. Barrell, B G, Bankier, A T & Drouin, J, Nature 282 (1979) 189. teins are coded by the mitochondrial ge- 21. Steffens, G J & Buse, G, Hoppe-Seyler’s Z physiol nome. Previously, sequence analysis of them 360 (1979) 613. R W,‘Freeman, K B & Vail, W J, FEBS human mtDNA [20] and bovine subunit II 22. Yatscoff, lett 81 (1977) 7. of cytochrome oxidase [21] indicated that June 11, 1980 mtDNA codes for this protein which is syn- Received Revised version received September 26, 1980 thesized in bovine mitochondria [22]. If one Accepted October 9, 1980 takes into account the number and molecular weights of all proteins synthesized in mitochondria, approx. 7x lo6 D of mtDNA Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved would be required for their coding. If the 0014.4827/81/020419-05~Z.M)/0 portion of the genome which codes for mtrRNAs and tRNAs is included [4], this Receptors for hyaluronate on the surface accounts for approx. 80% of the coding of parent and virus-transformed cell lines Binding and aggregation studies capacity of mammalian mtDNA. We wish to thank Mr M. Homer of the African Lion Safari Park, Rockton, Ontario and Mr W. Clark of Story Book Gardens, London, Ontario for permission to biopsy the animals, and Mrs E. Moerman for technical assistance. This investigation was supported by the MRC of Canada, grant MT-1940.
References 1. Perlman, P S, Douglas, M G, Stausberg, R L & Butow, R A, J mol biol 115 (1977) 675. 2. Borst, P & Grivell, L A, Cell 15 (1978) 705. 3. Birky, C W, Jr, Ann rev genet 12 (1978) 471. 4. Borst, P, Trends biochem sci 2 (1977) 31. 5. Dawid, I B & Blackler, A W, Dev biol 29 (1972) 152. 6. Hutchison, C A, III, Newbold, J E, Potter, S S & Edaell. M N. Nature 251 (1974) 536. 7. B&o; K, Fouts, D L & Wolstenholme, D R, Proc natl acad sci US 75 (1978) 909. 8. Kroon, A M, deVos, W M & Bakker, H, Biochim biophys acta 519 (1978) 269. 9. Brown, G G & Simpson, M W, J supramol biol suppl. 3 (1979) 157. 10. Yatscoff, R W & Freeman, K B, Can j biochem 55 (1977) 1064. 11. Yatscoff, R W, Aujame, L, Freeman, K B & Goldstein, S, Can j biochem 56 (1978) 939. 12. Constantino, P & Attardi, G, J molec biol96 (1975) 291. 13. Jeffreys, A J & Craig, I W, Nature 259 (1976) 690. 14. Dawid, I B, Dev bio129 (1972) 139. 15. Potter, S S, Newbold, J E, Hutchison, C A & Edge& M H. Proc natl acad sci US 72 (1975)4496. 16. Stamters, C P, Eliceiri, C L & Green, H, ‘Nature new biol 230 (1971) 52. 17. Laemmli, U K, Nature 227 (1970) 680. 18. Bonner, W M & Laskey, R A, Eur j biochem 46 (1974) 83. 19. Yatscoff, R W, Goldstein, S & Freeman, K B, Som cell genet 4 (1978) 633. Printed
in Sweden
CHARLES B. UNDERHILL’, * and BRYAN P. TOOLE,’ Developmental Biology Laboratory, Medical Service, Massachusetts General Hospital and Department of Medicine and Anatomy, Harvard Medical School, Boston, MA 02114, USA Summary. Two sets of parent and virus-transformed cell lines (3T3 vs SV-3T3; BHK vs PY-BHK) were compared with respect to the extent of divalentcation independent aggregation which previously has been shown to depend upon the interaction of endogenous hyaluronate with specific receptors on the cell surface. When measured under conditions of physiological ionic strength, a significant amount of hvahuonidase-inhibitable aaareaation was found in the v&us-transformed cell linesBVL3T3 and PY-BHK) but not in their parent counterparts (3T3 and BHK). However, when the same experiment was performed in a high ionic strength solution (0.5 M NaCl), the hyaluronidase inhibitable aggregation was detected in all of the cell lines. The differences in the aggregation between the various cell lines was also reflected in the binding of r3Hlhvahtronate. In uhvsioloaic~ saline. the viru&&for%d cells bound greater -amounts of hyahrronate (higher B,,,) with a greater affinity (lower &) than did their untransformed counterparts. Increasing the ionic strength to 0.5 M NaCl increased the binding of [3H]hyaIuronate by each cell line; however, the relative differences between the cell lines remained. These results indicate that variations in the ability of the cells to bind hyahtronate can partially account for the differences between the parent and the virus-transformed cells with respect to their ability to aggregate.
In previous studies we have shown that there are hyaluronate-binding receptors on i Present address: Department of Anatomy, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA. * To whom offprint requests should be addressed. Exp Cell Res 131 (1981)