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+ transformants were selected at 25°C and screened for growth at 25° and 37°C on minimal glucose media containing 5-fluoro-orotic acid and the appropriate growth supplements.
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3 (25) and resistance to proteinase K in mitoplasts (26).
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23
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14444284674
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note
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For cross-linking, in vitro import was for 10 min at 20°C, and nonimported precursor was digested with proteinase K (30 μg/ml). Mitochondria were washed, resuspended at 1 mg/ml in import buffer, and incubated with 1 mM dithiobis(succinimidylpropionate) (DSP) for 30 min at 4°C followed by a quench with 100 mM tris-base, pH 8.0. For immunoprecipitation, solubilized mitochondria were incubated with monospecific polyclonal antibodies coupled to protein A-Sepharose (27).
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24
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5 transformants in strain tim10-1, 46 transformants grew at 37°C; all of them contained only TIM10 as the "suppressor" gene.
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25
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We thank N. Pfanner for antiserum against yeast AAC and phosphate carrier and for plasmid pGEM4-PIC for in vitro transcription; F. Palmieri for antiserum against yeast dicarboxylate carrier; S. Helliwell and M. Hall for the yeast genomic library; and N. Kralli, S. Merchant, and members of the Schatz and Schweyen laboratories for helpful discussions. This study was supported by grants from the Swiss National Science Foundation (G.S.), the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (R.J.S.), the Human Capital and Mobility Program of the European Economic Community (G.S. and R.J.S.), and the Louis-Jeantet Foundation (G.S.), and by fellowships from the Damon Runyan-Walter Winchell Cancer Research Foundation (C.M.K.), the National Science Foundation (C.M.K), the European Molecular Biology Organization (E.J.), the Human Frontiers Science Program Organization (K.T.), and the Roche Research Foundation (K.T.).
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