NATIONAL INSTITUTE FOR BASIC BIOLOGY
DIVISION OF CELL MECHANISMS
- Mikio Nishimura
- Research Associates:
- Kazuo Ogawa
- JSPS-Post-doctoral Fellow:
- Ryuji Tsugeki
- Graduate Students:
- Kaori Inoue
Nagako Hiraiwa (1)
Masahiro Aoki (2)
- Technical Staff:
- Maki Kondo
- (1) from Aichi University of Education
(2) from Shinshu University
Higher plant cells contain several distinct organelles that play vital roles
in cellular physiology. During proliferation and differentiation of the cells,
the organelles often undergo dynamic changes. The biogenesis of new organelles
may occur; existing organelles may undergo a transformation of function, which
other organelles may degenerate. In this division, the regulatory mechanisms
underlying organelle transformation are currently studied to examine the
dynamic transformation of organellar functions (differentiation of organelles)
responsible for differentiation events in higher plant cells.
I. Transformation of leaf peroxisomes to glyoxysomes in senescing
- Dramatic metabolic changes that underlie the shift from heterotrophic to
autotrophic growth occur in greening of seed germination. Accompanying
these metabolic changes, many constitutive organelles are functionally
transformed. For example, etioplasts differentiate into chloroplasts and
mitochondria acquire the ability to oxidize glycine. Glyoxysomes that
are microbodies engaged in the degradation of reserve oil via
B-oxidation and the glyoxylate cycle, are transformed into leaf
peroxisomes that function in several crucial steps of photorespiration.
- After the functional transition of glyoxysomes to leaf peroxisomes
during the greening of pumpkin cotyledons, the reverse microbody
transition of leaf peroxisomes to glyoxysomes occurs during senescence.
Immunocytochemical labeling with protein A-gold was performed to analyze
the reverse microbody transition using antibodies against a
leaf-peroxisomal enzyme, glycolate oxidase, and against two glyoxysomal
enzymes, namely, malate synthase and isocitrate lyase. The intensity of
labeling for glycolate oxidase decreased in the microbodies during
senescence whereas in the case of malate synthase and isocitrate lyase
intensities increased strikingly. Double labeling experiments with
protein A-gold particles of different sizes showed that the
leaf-peroxisomal enzymes and the glyoxysomal enzymes coexist in the
microbodies of senescing pumpkin cotyledons
(Fig. 1) ,
indicating that leaf peroxisomes are directly transformed to glyoxysomes
during senescence. These findings indicate that transformation of
microbodies is a reversible process and glyoxysomes and that leaf
peroxisomes are directly transformed to other microbodies in greening
and senescing cotyledons, respectively.
II. Characterization of aconitase respon-sible for glyoxylate cycle.
- As a step to understand the regulatory mechanisms that operate during
microbody transition in pumpkin cotyledons (Cucurbita sp., Amakuri
Nankin), nine microbody enzymes, i.e., three glyoxysomal enzymes, three
leaf peroxisomal enzymes and three enzymes present in both microbodies
have been purified and characterized. The developmental changes in the
level of mRNA and protein have been analyzed. This year, we will start
analyzing aconitase which catalyzes the reversible interconversion of
citrate, isocitrate, and cis-aconitate. Aconitase is involved in the
Krebs cycle, localized in mitochondria, and in the glyoxylate cycle in
glyoxysomes, but the enzyme has also been reported to be localized in
the cytosol. Aconitase has been detected in glyoxysomes, but only less
than 0.50/0 of the total aconitase activity was recovered in glyoxysomal
fractions after sucrose gradient centrifugation. Whether mitochondria
and glyoxysomes contain specific isoenzymes of aconitase remains to be
- Three isoforms of aconitase (AcoI, AcoII, AcoIII) were found in
etiolated cotyledons of pumpkin and two of them (AcoI, AcoII) were
purified. The specific antibody raised against AcoI crossreacted with
all isoforms of aconitase. Sub-cellular fractionation and immunoblot
analyses showed that the activity of aconitase and the immunopositive
polypeptide were not detected in glyoxysomes. These findings indicate
that aconitase is not localized in glyoxysomes although the enzyme is a
member of the glyoxylate cycle. cDNA cloning and immunocytochemical
analysis are in progress.
III. Vacuolar processing enzyme responsible for conversion of
proprotein precursors into mature forms.
- Proprotein precursors of vacuolar components are transported from the
endoplasmic reticulum to the dense vesicles, and then targeted to the
vacuoles. In the vacuoles, the proproteins are processed proteolyiically
to their mature forms by a unique vacuolar processing enzyme. However,
the processing mechanism in plant vacuoles is very obscure. We isolated
a processing enzyme, a 37-kD cysteine proteinase, from castor bean
endosperm (Hara-Nishimura et al., 1991, FEBS Lett. 294, 89-93). Our
findings show that a single vacuolar processing enzyme is capable of
converting several proprotein precursors with a large variability of
molecular structure into their mature forms.
i) Molecular characterization and localization of a vacuolar
processing enzyme. Immunocytochemical localization of a vacuolar processing enzyme
in the endosperm of maturing castor bean (Ricinus communis) seeds
places the enzyme selectively in the dense vesicles as well as in
the vacuolar matrix, where a variety of proproteins is also
present. However, endogenous processing of the proproteins was
not observed in the isolated dense vesicles. This suggests that
the vacuolar processing enzyme is a latent form in the vesicles
To characterize the molecular structure of vacuolar processing
enzyme, we isolated a cDNA for the enzyme. The deduced primary
structure of a 55-kD precursor is 330/0 identical to a putative
cysteine proteinase of the human parasite Schistosoma mansoni.
The precursor is composed of a signal peptide, a 37-kD active
processing enzyme domain, and a propeptide fragment. Although the
precursor expressed in Escherichia coli has no vacuolar
processing activity, a 36-kD immunopositive protein expressed in
E. coli. is active. These activation of findings suggest that
the vacuolar processing enzyme requires proteolytic cleavage of
the 14-kD C-terminal propeptide fragment of the precursor.
ii) Developmental changes of a vacuolar processing enzyme in
maturing and germinating seeds.
- During seed maturation of castor bean, an increase in the
activity of the vacuolar processing enzyme in the endosperm
precedes the increase in the amount of total protein. The
enzymatic activity reaches a maximum at the late stage of seed
maturation and then decreases during seed germination
concomitantly with the degradation of seed storage proteins. We
examined the distribution of the enzyme in different tissues of
various plants. The processing enzyme was found in cotyledons of
castor bean, pumpkin and soybean, as well as in endosperm, and a
slight processing activity was also detected in roots, hypocotyls
and leaves of castor bean, pumpkin, soybean, mung bean and
spinach. These findings suggest that the proprotein-processing
machinery is widely distributed in vacuoles of various plant
IV. Mechanism for biosynthesis and vacuolar processing of 2S
albumin, a major seed protein of pumpkin.
- Cell fractionation of pulse-chaselabeled developing pumpkin cotyledons
demonstrated that the proprotein precursor to 2S albumin is transported
from the endoplasmic reticulum to dense vesicles and then to the
vacuoles, in which pr02S albumin is processed to the mature 2S albumin.
Immunocytochemical analysis showed that dense vesicles of about 300 nm in
diameter mediate the transport of pr02S albumin to the vacuoles. The
primary structure of the precursor to 2S albumin has been deduced from
the nucleotide sequence of an isolated cDNA insert. The presence of a
hydrophobic signal peptide at the N-terminus indicates that the precursor
is a preproprotein that is converted into pr02S albumin after cleavage of
the signal peptide. N-terminal sequencing of the pr02S albumin in the
isolated vesicles revealed that the signal peptide is cleaved off
co-translationally on the C-terminal side of alanine residue 22 of
prepro2S albumin. By contrast, posttranslational cleavage occurs on the
C-terminal side of both asparagine residues 35 and 74, which are
conserved among precursors to 2S albumin from different plants.
Hydropathy analysis revealed that the two asparagine residues are located
in the hydrophilic regions of pr02S albumin. These findings suggest that
a vacuolar processing enzyme can recognize exposed asparagine residues on
the molecular surface of pr02S albumin and cleave the peptide bond on the
C-terminal side of each asparagine residue to produce mature 2S albumin
in the vacuoles.
V. Role of molecular chaperones on translocation of proteins into
- Molecular chaperones are a class of cellular proteins that function in
the folding and assembly into oligomeric structures of certain other
polypeptides but that are not components of the final oligomeric
structure. To investigate the role(s) of molecular chaperones in the
chloroplasts, we examined whether the homologues of the 70-kDa heat-shock
protein (Hsp70) and chaperonin 60 (Cpn60) interact with newly imported
proteins to assist in their maturation. Ferredoxin NADP+ reductase (FNR)
imported into chloroplasts in vitro could be immunoprecipitated with
antisera raised against the homologue of Hsp70 from pumpkin chloroplasts
and against GroEL from Escherichia coli, which is a bacterial homologue
of chaperonin 60 (Cpn60), in an ATP-dependent manner, an indication that
newly imported FNR interacts physically with homologues of Hsp70 and
Cpn60 in chloroplasts. Timecourse analysis of the import of FNR showed
that imported FNR interacts transiently with the homologue of Hsp70 and
that the association of FNR with the homologue of Hsp70 precedes that
with the homologue of Cpn60. These findings suggest that homologues of
Hsp70 and Cpn60 in chloroplasts sequentially assist in the maturation of
newly imported FNR in an ATP-dependent manner. cDNA for chloroplast Cpn10
has been isolated and characterized. Further investigation on the
function of Cpn10 in the transport of protein into chloroplasts is in
- De Bellis, L., Tsugeki, R., Alpi, A. and Nishimura, M. (1993)
Purification and characterization of aconitase isoforms from etiolated
pumpkin cotyledons. Physiof Plant. 88, 485-492.
- Hara-Nishimura, I., Kondo, M., Nishimura, M., Hara, R. and Hara, T.
(1993) Cloning and nucleotide sequence of cDNA for rhodopsin of the squid
Todarodes pacificus. FEBS Letters 317, 5-11.
- Hara-Nishimura, I., Kondo, M., Nishimura, M., Hara, R. and Hara, T. (1993)
Amino acid sequence surrounding the retinal-binding site in retinochrome
of the squid, Todarodes pacificus. FEBS Letters 335, 94-98.
- Hara-Nishimura, I., Takeuchi, Y., Inoue, K. and Nishimura, M. (1993)
Vesicle transport and processing of the precursor to 2S albumin in
pumpkin. Plant Journal 4, 793-800.
- Hara-Nishimura, I., Takeuchi, Y. and Nishimura, M. (1993) Molecular
characterization of a vacuolar processing enzyme related to a putative
cysteine proteinase of Schistosoma mansoni. Plant Cell 5, 1651-1659.
- Hiraiwa, N., Takeuchi, Y., Nishimura, M. and Hara-Nishimura, I. (1993)
A vacuolar processing enzyme in maturing and germinating seeds: Its
distribution and associated changes during development. Plant Cell
PhysioL 34, 1197-1204.
- Nishimura, M., Takeuchi, Y., De Bellis, L. and Hara-Nishimura, I. (1993)
Leaf peroxisomes are directly transformed to glyoxysomes during
senescence of pumpkin cotyledons. Protoplasma 175, 131-137.
- Nozue, M., Kubo, H., Nishimura, M., Kato, A., Hattori, C., Usuda, N.,
Nagata, T. and Yasuda, H. (1993) Characterization of intravacuolar
pigmented struqtures in anthocyanin-containing cells of sweet potato
suspension cultures. Plant Cell Physiot 34, 803-808.
- Ogawa, K. and Shimizu, T. (1993) cDNA sequence for mouse caltractin.
Biochim. Biophys. Acta 1216, 126-128.Tezuka, K., Hayashi, M., Ishihara,
H., Onozaki, K., Nishimura, M. and Takahashi, N.(1993) Occurrence of
heterogeneity of N-Iinked oligosaccharides attached to sycamore (Acer
pseudoplatanus L.) Iaccase after excretion. Biochem. MoL Biot Internat.
- Tezuka, K., Hayashi, M., Ishihara, H., Nishimura, M., Onozaki, K. and
Takahashi, N. (1993) Purification and substrate specificity of
B-xylosidase from sycamore cell (Acer pseudoplatanus L.): application for
structural analysis of xylose-containing N-Iinked oligosaccharides.
Anal. Biochem. 211, 205 209.
- Tsugeki, R., Hara-Nishimura, I., Mori, H. and Nishimura, M. (1993)
Cloning and sequencing of cDNA for glycolate oxidase from pumpkin
cotyledons and northern blot analysis. Plant Cell PhysioL 34, 51 57.
- Tsugeki, R. and Nishimura, M. (1993) Interaction of homologues of Hsp70
and Cpn60 with ferredoxin-NADP+ reductase upon its import into
chloroplasts. FEBS Letters 320, 198-202.