Molecular characterization and subcellular localization of protoporphyrinogen oxidase in spinach chloroplasts.
Protoporphyrinogen oxidase (Protox) is the last common enzyme in the biosynthesis of chlorophylls and heme. In plants, there are two isoenzymes of Protox, one located in plastids and other in the mitochondria. We cloned the cDNA of spinach (Spinacia oleracea) plastidal Protox and purified plastidal Protox protein from spinach chloroplasts. Sequence analysis of the cDNA indicated that the plastid Protox of spinach is composed of 562 amino acids containing the glycine-rich motif GxGxxG previously proposed to be a dinucleotide binding site of many flavin-containing proteins. The cDNA of plastidal Protox complemented a Protox mutation in Escherichia coli. N-terminal sequence analysis of the purified enzyme revealed that the plastidal Protox precursor is processed at the N-terminal site of serine-49. The predicted transit peptide (methionine-1 to cysteine-48) was sufficient for the transport of precursors into the plastid because green fluorescent protein fused with the predicted transit peptide was transported to the chloroplast. Immunocytochemical analysis using electron microscopy showed that plastidal Protox is preferentially associated with the stromal side of the thylakoid membrane, and a small portion of the enzyme is located on the stromal side of the chloroplast inner envelope membrane.
Che
F S
FS
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama Ikoma, Nara 630-0101, Japan. fsche@bs.aist-nara.ac.jp
Watanabe
N
N
Iwano
M
M
Inokuchi
H
H
Takayama
S
S
Yoshida
S
S
Isogai
A
A
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D015388:Organelles / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure
D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase