Functional expression of rat cytochrome c in Saccharomyces cerevisiae. |
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| Authors: | R C Scarpulla and S H Nye |
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| Abstract: | To determine whether a mammalian cytochrome c could efficiently replace iso-1-cytochrome c, which is encoded by the yeast CYC1 gene, the coding sequence of RC9 (a nondefective processed gene from rat) was cloned in both single- and multiple-copy expression vectors under the direction of the yeast alcohol dehydrogenase 1 (ADC1) promoter. Upon transformation of a CYC1 deletion strain, the multiple-copy construct restored a wild-type growth rate on lactate medium; such growth normally requires derepressed amounts of iso-1-cytochrome c. These transformants expressed a level of hybrid ADC1/RC9 mRNA approximately 5- to 10-fold greater than the amount of message from the endogenous ADC1 gene and produced a steady-state level of rat cytochrome c equivalent to that of the wild-type yeast protein. A requirement for the vector was evidenced by its absence in all transformants that lost the lactate growth phenotype after propagation in nonselective medium. In contrast, the level of vector-specific message in single copy was equivalent to that of the endogenous ADC1 mRNA, but transformants exhibited no significant growth on lactate. Constructions having a small deletion or a mammalian intron within the rat cytochrome c coding region failed to support lactate-dependent growth, indicating that complementation depends upon proper translation of the correct rat coding sequence. Therefore, the rat polypeptide, when expressed at normal physiological levels, is recognized by the yeast machinery involved in the multiple steps required for the processing and transport of an active cytochrome c as well as its functional interaction with the respiratory apparatus. |
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