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 inner mitochondrial membrane. Since the product of the enzymatic reaction, protoheme IX, is utilized almost exclusively in the cytoplasmic compartment or on the cytoplasmic side of the inner mitochondrial membrane, it was of interest to determine if the intracellular location of ferrochelatase-deficient strain of the yeast Saccharomyces cerevisiae vectors that coded for full-length ferrochelatase and a truncated form of the enzyme that lacked the mitochondrial targeting sequence were expressed. Both of these transformed cells produce approximately equal total amounts of ferrochelatase, as determined by enzyme assays and Western blot analysis, but only with the full-length construct was ferrochelatase properly localized. In cells containing the truncated construct, ferrochelatase activity was found in all membrane fractions but was not located on the matrix side of the inner mitochondrial membrane. Cells containing either construct produced heme, although the amount of heme synthesized by cells with the truncated construct was significantly less. Interestingly in cells with improperly localized ferrochelatase the amount of b-type cytochrome decreased by 80% as opposed to c- and a-type cytochromes where the decreases were only 60 and 40%, respectively.
Prasad
A R
AR
Department of Microbiology, University of Georgia, Athens 30602-2605, USA.
Dailey
H A
HA
D001483:Base Sequence; D001692:Biological Transport; D015415:Biomarkers; D002451:Cell Compartmentation; D002458:Cell Fractionation; D017936:Cytochrome a Group / Q000032:analysis; D003573:Cytochrome b Group / Q000032:analysis; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D005294:Ferrochelatase / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000096:biosynthesis*; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013267:Stilbenes / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013451:Sulfonic Acids / Q000494:pharmacology
C094955:4,4'-maleimidylstilbene-2,2'-disulfonic acid; D015415:Biomarkers; D017936:Cytochrome a Group; D003573:Cytochrome b Group; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D013267:Stilbenes; D013451:Sulfonic Acids; D006418:Heme; D005294:Ferrochelatase