Isolation of a cDNA encoding chloroplast ferrochelatase from Arabidopsis thaliana by functional complementation of a yeast mutant.
Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme. It is located in the mitochondria in all eukaryotes and is also found in plastids in plants. Although it has been purified from animals and microorganisms, and genes for it isolated and characterized, very little is known about plant ferrochelatases. We have isolated a cDNA for ferrochelatase from the higher plant Arabidopsis thaliana by functional complementation of a mutant of Saccharomyces cerevisiae defective in this enzyme. The cDNA encodes a protein of 52 kDa, which has 25-35% sequence similarity to ferrochelatases from other organisms. There is an N-terminal extension of about 65 residues, which is almost certainly the chloroplast transit peptide, since the precursor protein, transcribed and translated in vitro, is efficiently imported and processed to the mature size by isolated pea chloroplasts. In contrast, the precursor was not processed by mitochondrial processing peptidase activity, nor could import into isolated yeast mitochondria be demonstrated conclusively, although, presumably, in the rescued yeast mutant, at least some of the Arabidopsis ferrochelatase must be present in the mitochondria. A single transcript the same size as the cDNA was detected in both Arabidopsis leaves and roots, although the amount of message was greater in the photosynthetic tissue. Southern analysis suggests that there is a single gene for chloroplast ferrochelatase in Arabidopsis.
Smith
A G
AG
Department of Plant Sciences, University of Cambridge, United Kingdom.
Santana
M A
MA
Wallace-Cook
A D
AD
Roper
J M
JM
Labbe-Bois
R
R
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002736:Chloroplasts / Q000201:enzymology*; D018076:DNA, Complementary; D005294:Ferrochelatase / Q000235:genetics*; D005816:Genetic Complementation Test; D006801:Humans; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology; D014170:Transformation, Genetic
D018076:DNA, Complementary; D005294:Ferrochelatase