EFFECT OF CELLULAR LOCATION ON THE FUNCTION OF FERROCHELATASE

Ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is a nuclear encoded protein that is synthesized in the cytoplasm in a precursor form and then is translocated to the matrix side of the inn er mitochondrial membrane, Since the product of the enzymatic reaction , protoheme IX, is utilized almost exclusively in the cytoplasmic comp artment or on the cytoplasmic side of the inner mitochondrial membrane , it was of interest to determine if the intracellular location of fer rochelatase is crucial for its effective functioning, In a ferrochelat ase-deficient strain of the yeast Saccharomyces cerevisiae vectors tha t coded for full-length ferrochelatase and a truncated form of the enz yme that lacked the mitochondrial targeting sequence were expressed, B oth of these transformed cells produce approximately equal total amoun ts of ferrochelatase, as determined by enzyme assays and Western blot analysis, but only with the full-length construct was ferrochelatase p roperly localized. In cells containing the truncated construct, ferroc helatase activity was found in all membrane fractions but was not loca ted on the matrix side of the inner mitochondrial membrane, Cells cont aining either construct produced heme, although the amount of heme syn thesized by cells with the truncated construct was significantly less, Interestingly in cells with improperly localized ferrochelatase the a mount of b-type cytochrome decreased by 80% as opposed to c- and a-typ e cytochromes where the decreases were only 60 and 40%, respectively.