Annual Report 2001
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DIVISION OF CELL MECHANISMS

Professor: NISHIMURA, Mikio
Associate Professor: HAYASHI, Makoto
Research Associate: MANO, Shoji
Technical Staff: KONDO, Maki
NIBB Research Fellow: SHIRAHAMA, Kanae
Post doctoral Fellows: HAYASHI, Yasuko (-March 15)

HAYASHI, Hiroshi (-March 31)

MITSUHASHI, Naoto (-March 31)
Graduate Students: NITO, Kazumasa

WATANABE, Etsuko

FUKAO, Youichiro

KAMADA, Tomoe (April1-)

HATSUGAI, Noriyuki (April 1-)
Technical Assistants: KUROYANAGI, Miwa (-March 31)

NAKAMORI, Chihiro

TAKEI, Rie (-April 30)

YAGI, Mina (March 1-)

SecretariesUEDA, Chizuru

KOMORI, Akiko


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, while other organelles may degenerate. Because the dynamic transformation of organellar function (differentiation of organelles) is responsible for flexibility of differentiation events in higher plant cells, the elucidation of regulatory mechanisms underlying organelle transformation are currently studied in this division.

I. Reversible transformation of plant peroxisomes

Dramatic metabolic changes which 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 diffe-rentiate into chloroplasts and mitochondria acquire the ability to oxidize glycine. Glyoxysomes which 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 transition of leaf peroxisomes to glyoxysomes occurs during senescence. The functional transformation between glyoxysomes and leaf peroxisomes is controlled by gene expression, alternative splicing, protein translocation and protein degradation.

To investigate the roles of peroxisomal membrane proteins in the reversible conversion of glyoxysomes to leaf peroxisomes, we characterized several membrance proteins of glyoxysomes. One of them (PMP38) was identified a putative ATP/ADP carrier protein. Cell fractionation and immunocytochemical analysis using pumpkin cotyledons revealed that PMP38 is localized on peroxisomal membranes as an integral membrane protein. The amount of PMP38 in pumpkin cotyledons increased and reached the maximum protein level after 6 d in the dark but decreased thereafter. Illumination of the seedlings caused a significant decrease in the amount of the protein. These results clearly showed that the membrane protein, PMP38 in glyoxysomes changes dramatically during transformation of glyoxysomes to leaf peroxisomes, as do the other glyoxysomal enzymes, especially enzymes of the fatty acid b-oxidation cycle, that are localized in the matrix of glyoxysomes. An ascorbate peroxidase (pAPX) was also identified as one of glyoxysomal membrane proteins. Its cDNA was isolated by immunoscreening. The deduced amino acid sequence encoded by the cDNA insert does not have a peroxisomal targeting signal (PTS), suggesting that pAPX is imported by one or more PTS-independent pathways. Subcellular fractionation of 3- and 5-d-old cotyledons of pumpkin revealed that pAPX was localized not only in the glyoxysomal fraction, but also in the ER fraction. A magnesium shift experiment showed that the density of pAPX in the ER fraction did not increase in the presence of Mg2+ , indicating that pAPX is not localized in the rough ER. Immunocytochemical analysis using a transgenic Arabidopsis which expressed pumpkin pAPX showed that pAPX was localized on peroxisomal membranes, and also on a unknown membranous structure in green cotyledons. The overall results suggested that pAPX is transported to glyoxysomal membranes via this unknown membranous structure.

II. Peroxisomes defective mutant of Arabidopsis.

It has been suggested that the functional conversion between glyoxysomes and leaf peroxisomes is controlled by gene expression, protein translocation, and protein degradation. A genetic approach is an effective strategy toward understanding the regulatory mechanism(s) of peroxisomal function at the level of gene expression, protein translocation, and protein degradation. We isolated and characterized 2,4-dichloro-phenoxybutyric acid (2,4-DB)-resistant mutants. It has been demonstrated previously that 2,4-dichlorophe-noxybutyric acid (2,4-DB) is metabolized to produce a herbicide, 2,4-D, by the action of peroxisomal fatty acid b-oxidation in higher plants. To isolate mutants that have defects in peroxisomal fatty acid b-oxidation, we screened mutant lines of Arabidopsis seedlings for growth in the presence of toxic levels of 2,4-DB. Genetic analysis revealed that these mutants can be classified as carrying alleles at three independent loci, which we designated ped1, ped2, and ped3, (where ped stands for peroxisome defective). The ped1 mutant lacks the 3-ketoacyl CoA thiolase, an enzyme involved in fatty acid b-oxidation during germination and subsequent seedling growth, while AtPex14p, the PED2 gene product, is a peroxisomal membrane protein that determines the peroxisomal protein targeting. PED3 gene was recently identified by positional cloning. The phenotype of the ped3 mutant indicated that the mutation in the PED3 gene inhibits the activity of fatty acid b-oxidation. Ped3p, the PED3 gene product, is a 149-kD protein that exists in peroxisomal membranes. The amino acid sequence of Ped3p had a typical characteristic for "full-size" ATP-binding cassette (ABC) transporter consisting of two transmembrane regions and two ATP-binding regions. This protein was divided into two parts, that had 32% identical amino acid sequences. Each domain showed a significant sequence similarity with peroxisomal "half" ABC transporters so far identified in mammals and yeast. Ped3p may contribute to the transport of fatty acids and their derivatives across the peroxisomal membrane. ped1/ped3 double mutant showed severe defects on leaves and inflorescences, and was sterile (Fig. 1). The phenotype may tell us unidentified function(s) of plant peroxisomes.

pict

Figure 1. Phenotype of ped1/ped3 double mutant.

ped1/ped3 double mutant showed vegetative and reproductive phenotypes. It had wavy leaves with irregular shapes (Fig. 1A). Influorescence of the double mutant was difficult to develop, but it occasionally had dwarf inflorescences with abnormal structure (Fig. 1B). Although the inflorescences had some flowers, it was sterile. These phenotypes were not found in the parents, ped1 and ped2.

 

III. ER derived organelles for transport of proteins to vacuoles.

Novel vesicles designated precursor-accumulating (PAC) vesicles that accumulate large amounts of proprotein precursors of storage proteins were purified and characterized from maturing pumpkin seeds. These vesicles had diameters of 300 to 400 nm and contained an electron-dense core of storage proteins surrounded by an electron-translucent layer and were shown that the PAC vesicles mediate a transport pathway for insoluble aggregates of storage proteins directly to protein storage vacuoles. We found a novel membrane protein with molecular mass of 73 kDa, MP73, on the membrane of protein storage vacuoles of pumpkin seeds. MP73 appeared during seed maturation and disappeared rapidly after seed germination, in association with the morphological changes of the protein storage vacuoles. Immunocytochemistry and an immunoblot analysis showed the PAC vesicles accumulated proMP73, but not MP73, on the membranes. Subcellular fractionation of the pulse-labeled maturing seeds demonstrated that the proMP73 form with N-linked oligosaccharides was synthesized on the ER and then transported to protein storage vacuoles via PAC vesicles. Tunicamycin-treatment of the seeds resulted in the efficient deposition of proMP73 lacking the oligosaccharides into the PAC vesicles, but no accumulation of MP73 in vacuoles. After arrival at protein storage vacuoles, proMP73 was cleaved by the action of a vacuolar enzyme to form a 100-kD complex on the vacuolar membranes. These results show that PAC vesicles mediate delivery of not only storage proteins but also membrane proteins of protein storage vacuoles. In order to investigate the mechanism of the PAC vesicle formation, we constructed chimeric genes that encode fusion proteins consisting to both various lengths of polypeptides derived from pumpkin 2S albumin and a selectable marker enzyme, phosphinothricin acetyltransferase and expressed in Arabidopsis. A fusion protein expressed by one of the chimeric genes is accumulated as a proprotein-precursor form, and localized in novel vesicles of vegetative cells, that show distinct features that well much to the PAC vesicles. Arabidopsis mutants that defect vesicular transport of the fusion protein are now screened and characterized by using the transgenic plants.

Plants degrade cellular materials during senescence and under various stresses. The precursors of two stress-inducible cysteine proteinases, RD21 and a vacuolar processing enzyme (VPE), were specifically accumulated in ~0.5 µm diameter x ~5 µm long bodies in Arabidopsis thaliana. Such bodies have previously been observed in Arabidopsis but their function was not known. Because these bodies contain precursors of lytic enzymes, we propose to call them ER bodies. They are surrounded with ribosomes and thus are assumed to be directly derived from the endoplasmic reticulum. ER bodies develop specifically in the epidermal cells of healthy seedlings. These cells are easily wounded and stressed by the external environment. When the seedlings are stressed with a concentrated salt solution, leading to death of the epidermal cells, the ER bodies start to fuse with each other and with the vacuoles, thereby mediating the delivery of the precursors directly to the vacuoles. This regulated, direct pathway differs from the usual case in which proteinases are transported constitutively from the endoplasmic reticulum to the Golgi complex and then to vacuoles, with intervention of vesicle-transport machinery, such as a vacuolar-sorting receptor or a syntaxin of the SNARE family. Thus, the ER bodies appear to be a novel proteinase-storing system that assists in cell death of the vegetative organs of higher plants.

IV. Role of molecular chaperones in organelle differentiation.

Molecular chaperones are cellular proteins that function in the folding and assembly of certain other polypeptides into oligomeric structures but that are not, themselves, components of the final oligomeric structure. To clarify the roles of molecular chaperones on organelle differentiation, we have purified and characterized chaperonin and Hsp70s and analyzed their roles in the translocation of proteins into chloroplasts.

Previously, we characterized a mitochondrial co-chaperonin (Cpn10) and a chloroplast co-chaperonin (Cpn20) from Arabidopsis thaliana. In 2001, we characterized a third co-chaperonin. The cDNA was 603 base pairs long, encoding a protein of 139 amino acids. From a sequence analysis, the protein was predicted to have one Cpn10 domain with an amino-terminal extension that might work as a chloroplast transit peptide. This novel Cpn10 was confirmed to be localized in chloroplasts, and we refer to it as chloroplast Cpn10 (chl-Cpn10). The phylogenic tree that was generated with amino acid sequences of other co-chaperonins indicates that chl-Cpn10 is highly divergent from the others. In the GroEL-assisted protein folding assay, about 30% of the substrates were refolded with chl-Cpn10, indicating that chl-Cpn10 works as a cochaperonin. A Northern blot analysis revealed that mRNA for chl-Cpn10 is accumulated in the leaves and stems, but not in the roots. In germinating cotyledons, the accumulation of chl-Cpn10 was similar to that of chloroplastic proteins and accelerated by light. It was proposed that two kinds of co-chaperonins, Cpn20 and chl-Cpn10, work independently in the chloroplast.

Publication List:

Fukao, Y., Y. Hayashi, S. Mano, M. Hayashi and M. Nishimura (2001) Developmental analysis of a putative ATP/ADP carrier protein localized on glyoxysomal membranes during the peroxisome transition in pumpkin. Plant Cell Physiol.42: 835-841

Hayashi, M. and M. Nishimura (2002) Genetic approaches to understand plant peroxisomes. in Plant peroxisomes, edited by A. Baker and I. Graham, Kluwer Acad. Pub. in press

Hayashi, M., K. Nito, R. Takei-Hoshi, M. Yagi, M. Kondo, A. Suenaga, T. Yamaya and M. Nishimura (2002) Ped 3p is a peroxisomal ATP-binding cassete transporter that might supply substrates for fatty acid b-oxidation. Plant Cell Physiol. 43:1-11

Hayashi, Y., M. Hayashi, H. Hayashi, I. Hara-Nishimura and M. Nishimura (2001) Direct interaction between glyoxysomes and lipid bodies in etiolated cotyledons of Arabidopsis thaliana ped1mutant . Protoplasma218: 83-94

Hayashi, Y., K. Yamada, T. Shimada, R. Matsushima, N. K. Nishizawa, M. Nishimura and I. Hara-Nishimura (2001) A proteinase-storing body that prepares for cell death or stresses in the epidermal cells of Arabidopsis. Plant Cell Physiol.42: 894-899

Kimura, Y., S. Matsuno, S. Tsurusaki, M. Kimura, I. Hara-Nishimura and M. Nishimura (2002) Subcellular localization of endo-b-N-acetylglucosaminidase and high-mannose type free N-glycans in plant cell. Biochim. Biophys. Acta in press

Koumoto, K., T. Shimada, M. Kondo, I. Hara-Nishimura and M. Nishimura. (2001) Chloroplasts have a novel Cpn10 in addition to Cpn20 as co-chaperonins in Arabidopsis thaliana. J. Biol. Chem. 276: 29688-29694

Mano, S., C. Nakamori, M. Hayashi, A, Kato, M. Kondo and M. Nishimura (2002) Distribution and characterization of peroxisomes in Arabidopsis by visualization with GFP. Plant Cell Physiol. in press

Minamikawa, T., K. Toyooka, T. Okamoto, I. Hara- Nishimura and M. Nishimura (2001) Degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase by vacuolar enzymes of senescing French bean leaves: Immunocytochemical and ultrastructural observations. Protoplasma218: 144-153

Mitsuhashi, N., Y. Hayashi, Y. Koumoto, T. Shimada, T. Fukasawa-Akada, M. Nishimura and I. Hara-Nishimura (2001) A novel membrane protein of protein bodies that is transported to protein-storage vacuoles via precursor-accumulating vesicles. Plant Cell13: 2361-2372

Nito, K., K. Yamaguchi, M. Kondo, M. Hayashi and M. Nishimura (2001) Pumpkin peroxisomal ascorbate peroxidase is localized on peroxisomal membranes and unknown membranous structures. Plant Cell Physiol.42: 20-27

Tanaka, H., H. Onouchi, M. Kondo, I. Hara-Nishimura, M. Nishimura, C. Machida and Y. Machida (2001) A subtilisin-like serine protease is required for epidermal surface formation in Arabidopsis embryos and juvenile plants. Development128: 4681-4689

Watanabe, E., T. Shimada, M. Kuroyanagi, M. Nishimura and I. Hara-Nishimura (2002) Calcium-mediated association of a putative vacuolar sorting receptor PV72 with a propeptide of 2S albumin. J. Biol. Chem.in press

Xu, W., K. Morita, K. Yamada, M. Kondo, M. Nishimura, H. Shioiri, M. Kojima and M. Nozue (2001) Expression and localization of a 36-kDa vacuolar protein (VP24) precursor in anthocyanin-producing sweet potato cells in suspension culture. Plant Biotechnol.18: 203-208

Yamada, K., R. Matsushima, M. Nishimura and I. Hara-Nishimura (2001) A unique cysteine protease with a granulin domain that slowly matures in the vacuoles of senescing Arabidopsis leaves. Plant Physiol.127: 1626-1634


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