Activity and Cellular Location inSaccharomyces cerevisiaeof Chimeric Mouse/Yeast andBacillus subtilis/Yeast Ferrochelatases

We have constructed a series of chimeric yeast/mouse and yeast/Bacillus subtilisferrochelatase genes in order to investigate domains of the ferrochelatase that are important for activity and/or association with the membrane. These genes were expressed in aSaccharomyces cerevisiaemutant in which the endogenous ferrochelatase gene (HEM15) had been deleted, and the phenotypes of the transformants were characterized. Exchanging the ∼40-amino-acid C-terminus between the yeast and mouse ferrochelatases caused a total loss of activity and the hybrid proteins were unstable when overproduced inEscherichia coli.The water-soluble ferrochelatase ofB. subtilisdid not complement the yeast mutant, although a large amount of active protein accumulated in the cytosol. Addition of the N-terminal leader sequence of yeast ferrochelatase to theB. subtilisenzyme targeted the fusion protein to mitochondria, but both the precursor and the mature forms of the enzyme were inactivein vivoand had residual activity when measuredin vitro.An internal ∼45-amino-acid segment located at the N-terminus of yeast ferrochelatase was identified, which, when replaced with the corresponding 30-amino-acid segment of theB. subtilisenzyme, caused the yeast enzyme to be located in the mitochondrial matrix as a soluble protein. The fusion protein was inactivein vivoand had residual activityin vitro.We speculate that this segment, which shows the greatest variability between species, is responsible for the association of the enzyme with the membrane.