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Plant Physiol, September 2000, Vol. 124, pp. 59-70
Molecular Characterization and Subcellular Localization of
Protoporphyrinogen Oxidase in Spinach
Chloroplasts1
Fang-Sik
Che,2*
Naohide
Watanabe,2
Megumi
Iwano,
Hachiro
Inokuchi,
Seiji
Takayama,
Shigeo
Yoshida, and
Akira
Isogai
Graduate School of Biological Sciences, Nara Institute of Science
and Technology, 8916-5, Takayama Ikoma, Nara 630-0101, Japan (F.S.C.,
N.W., M.I., S.T., A.I.); Department of Biophysics, Faculty of Science,
Kyoto University, Sakyoku, Kyoto, 606-8502, Japan (H.I.); and The
Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1,
Wako-shi, Saitama, 351-0198, Japan (S.Y.)
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.
1
This work was supported in part by a
Grant-in-Aid for Encouragement of Young Scientists from the Ministry of
Education, Science, Sports and Culture of Japan (grant no. 09760304).
2
These authors contributed equally to the paper.
*
Corresponding author; e-mail fsche{at}bs.aist-nara.ac.jp; fax
81- 743-72-5459.
© 2000 American Society of Plant Physiologists
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