Abstract
A new temperature-sensitive nuclear mutant affecting the biogenesis of functional mitochondria has been identified. This pet mutant was formerly characterized by a complete block of mitochondrial translation at the restrictive temperature. The analysis of mitochondrial transcripts demonstrates the accumulation of precursors for the small ribosomal RNA. Transformation of the mutant with plasmids from gene banks identified a chromosomal DNA fragment which can restore growth at the restrictive temperature. A reading frame of 2034 base pairs was found to be responsible for complementation of the mutant phenotype. Sequence analysis identified this gene as the γ-glutamylcysteine synthetase of yeast. This enzyme catalyses the first reaction in the γ-glutamyl cycle for the synthesis of glutathione. Disruption of yeast γ-glutamylcysteine synthetase causes a drastic reduction of growth on glucose medium. The insertion mutants were not able to grow on plates with glycerol as the sole carbon source indicating the special dependence of mitochondria on this substance. Crosses between the pet-ts mutant and the disruption mutant produced diploid cells with a complementation of all their genetic defects indicating that the pet-ts mutation and the insertion mutation are located in different genes. This finding demonstrates that the cloned yeast gene acts as an extragenic suppressor when present on a high-copy-number plasmid inside the pet mutant.
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References
Attardi G, Schatz G (1988) Annu Rev Cell Biol 4:289–333
Auld AM, Meister A (1991) Proc Natl Acad Sci USA 88:1913–1917
Bennetzen JL, Hall BD (1982) J Biol Chem 257:3026–3031
Biggin MD, Gilson TJ, Hong GF (1983) Proc Natl Acad Sci USA 80:3963–3965
Birnboim HC, Doly JD (1979) Nucleic Acids Res 7:1513–1523
Bousquet I, Dujardin G, Slonimski PP (1991) EMBO J 10:2023–2031
Broach JR, Strathern JN, Hicks JB (1979) Gene 8:121–133
Clayton DA (1984) Annu Rev Biochem 53:573–594
Colowick SP, Kaplan NO (eds) (1985) Methods in enzymology, vol 113. Academic Press, New York
Grivell LA (1989) Eur J Biochem 182:477–493
Hanahan D (1983) J Mol Biol 166:557–580
Hartl F-U, Pfanner N, Donald WN, Neupert W (1989) Biochim Biophys Acta 988:1–45
Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986) Yeast 2:163–167
Ito H, Fukuda Y, Murata K, Kimura A (1983) J Bacteriol 153:163–168
Kistler M, Summer KH, Eckhardt F (1986) Mutat Res 173:117–120
Kistler M, Maier K, Eckhardt-Schupp F (1990) Mutagenesis 5:39–44
Langford CJ, Gallwitz D (1983) Cell 33:519–527
Lisowsky T (1984) Thesis (Diploma), University of Bielefeld
Lisowsky T (1992) Mol Gen Genet 214:218–223
Lisowsky T, Michaelis G (1989) Mol Gen Genet 219:125–128
Martinsson J, Lai JCK, Meister A (1991) Proc Natl Acad Sci USA 87:7185–7189
Meister A (1988a) J Biol Chem 263:17205–17208
Meister A (1988b) Trends Biochem Sci 13:185–188
Michaelis G, Mannhaupt G, Pratje E, Fischer E, Naggert J, Schweizer E (1982) Mitochondrial translation products in nuclear respiratory-deficient pet mutants of Saccharomyces cerevisiae. In: Slonimski P, Borst P, Attardi G (eds) Mitochondrial genes. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 311–321
Mooz ED (1979) Biochem Biophys Res Commun 90:1221–1228
Mooz ED, Wigglesworth L (1976) Biochem Biophys Res Commun 68:1066–1072
Morimoto R, Locker J, Synenki RM, Rabinowitz M (1979) J Biol Chem 254:12461–12470
Ohtake Y, Yabuuchi S (1991) Yeast 953–961
Osinga KA, Evers RF, Van der Laan JC, Tabak HF (1981) Nucleic Acids Res 9:1351–1364
Pearson WR, Lipman DJ (1988) Proc Natl Acad Sci USA 85:2444–2448
Rothstein RJ (1983) Methods Enzymol 101:202–211
Russel M, Holmgren A (1988) Proc Natl Acad Sci USA 85:990–994
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) Proc Natl Acad Sci USA 74:5463–5467
Schmelzer C, Schweyen RJ (1982) Nucleic Acids Res 10:513–523
Seelig GF, Meister A (1985) Methods Enzymol 113:379–390
Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 76:1035–1039
Tzagoloff A (1982) Mitochondria. In: Siekevitz P (ed) Cellular organelles. Plenum Press, New York, pp 235–264
Tzagoloff A, Dieckmann CL (1990) Microbiol Rev 54:211–225
Verner K, Schatz G (1988) Science 241:1307–1313
Wiesenberger G, Link TA, von Ahsen U, Waldherr M, Schweyen RJ (1991) J Mol Biol 217:23–38
Yanisch-Perron C, Vieira J, Messing J (1985) Gene 33:103–119
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Lisowsky, T. A high copy number of yeast γ-glutamylcysteine synthetase suppresses a nuclear mutation affecting mitochondrial translation. Curr Genet 23, 408–413 (1993). https://doi.org/10.1007/BF00312627
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DOI: https://doi.org/10.1007/BF00312627