NATIONAL INSITUTE FOR BASIC BIOLOGY  


National Institute for Basic Biology

DIVISION OF CELL MECHANISMS


Professor:
Mikio Nishimura
Associate Professor:
Ikuko Hara-Nishimura
Research Associates:
Makoto Hayashi
Tomoo Shimada
Post doctoral fellows:
Tetsu Kinoshita
Akira Kato (~ Aug. 30 )
Nagako Hiraiwa (~ March 30 )
Yasuko Ishimaru
Kanae Shirahama
Shoji Mano
Graduate Students:
Hiroshi Hayashi
Kenji Yamada
Naoto Mitsuhashi
Kazumasa Nito
Yuki Tachibe (from Hiroshima University)
Technical Staffs:
Maki Kondo
Katsushi Yamaguchi
JSPS Technical Staffs:
Miwa Kuroyanagi
Kanako Toriyama (~ Nov. 30 )
Miki Kinoshita (April .1 ~ )
Chizuru Ueda (April .1 ~ )
Chihiro Nakamori (April .1 ~ )
Visiting Scientists:
Roland R. Theimer (Bergische Univ. Germany )
Yasuko Koumoto


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. Regulation at the level of protein transport to microbodies during the microbody transition.

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 differentiate 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 microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Microbody enzymes function after their transport to microbodies. Since the enzyme compositions and functions of glyoxysomes and leaf peroxisomes differ from each other, it is likely that the two types of microbodies possess different machineries for protein import.
Microbody proteins are synthesized in the cytosol on free polysomes and are transported post-translationally into microbodies. Two types of targeting signals to microbodies have been reported. One type of targeting signal is a part of the mature protein. One such signal, the tripeptide Ser-Lys-Leu, occurs at the C-terminal. has been identified as a targeting signal. Ser-Lys-Leu and related amino acid sequences commonly function in mammals, insects, fungi, and plants.

T o characterize the microbody targeting signal in plants, we have examined the ability of 24 carboxy-terminal amino acid sequences to facilitate the transport of a bacterial protein, b-glucuronidase (GUS) into microbodies in green cotyledonary cells of transgenic Arabidopsis. Immunocytochemical analysis of the transgenic plants revealed that carboxy-terminal tripeptide sequences of the form [C/A/S/P]-[K/R]-[I/L/M] function as a microbody-targeting signal, while tripeptides with proline at the first amino acid position and isoleucine at the carboxyl terminus show weak targeting efficiencies. In contrast, another small group of microbody proteins are synthesized as precursors with N-terminal cleavable presequences; these include 3-ketoacyl-CoA thiolase, glyoxysomal citrate synthase (gCS), glyoxysomal malate dehydrogenase (gMDH) and long-chain acyl-CoA oxidase. These N-terminal sequences also function as a targeting signal to microbodies and are designated as PTS2. We generated a transgenic Arabidopsis plant that accumulated GUS-chimeric proteins with the N-terminal presequences of the pumpkin gCS or pumpkin gMDH. Immunocytochemical studies of the transgenic plant showed that the N-terminal sequences of gCS and gMDH function as targeting signals to plant microbodies. Site-directed mutagenesis studies of the chimeric proteins indicated that several amino acids in the consensus regions of the presequences were essential for targeting to microbodies or were necessary for the processing of the presequence. The fusion protein was transported into functionally different microbodies, such as glyoxysomes, leaf peroxisomes and unspecialized microbodies, and was subsequently processed. These observations indicated that the transport of gCS and gMDH is mediated by their amino-terminal presequences and that the transport system is functional in all plant microbodies. Therefore, it is unlikely that glyoxysomes and leaf peroxisomes posses different targeting machineries.



II. Microbody 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-dichlorophenoxybutyric acid (2,4-DB)-resistant mutants. It has been demonstrated previously that 2,4-dichlorophenoxybutyric 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. Twelve of the mutants survived; of these, four required sucrose for postgerminative growth (Figure 1, A B). This result suggests that these mutants have defects in peroxisomal fatty acid b-oxidation, because peroxisomal fatty acid b-oxidation plays an important role in producing sucrose from storage lipids during germination. 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 thiolase protein, an enzyme involved in fatty acid b-oxidation during germination and subsequent seedling growth, whereas the ped2 mutant has a defect in the intracellular transport of thiolase from the cytosol to glyoxysomes. Etiolated cotyledons of both ped1 (Figure 1D) and ped2 mutants have glyoxysomes with abnormal morphology.

Figure 1. A microbody-defective mutant of Arabidopsis.
Wild-type Arabidopsis (A) can germinate and grow after supplying only water. In contrast, the ped1 mutant (B), which has a defect in glyoxysomal fatty acid b-oxidation, requires sucrose for post-germinative growth. Glyoxysomes in wild-type plant (C; arrowhead) are surrounded by a single membrane with approximately 1 mm in diameter, whereas glyoxysomes in the ped1 mutant (D; arrowhead) become bigger, and contain tubular structures.



III. Transport of storage proteins to protein storage vacuoles is mediated by large PAC (precursor-accumulating) vesicles.

Novel vesicles that accumulate large amounts of proprotein precursors of storage proteins were purified from maturing pumpkin seeds. These vesicles were designated precursor-accumulating (PAC) vesicles and have diameters of 200 to 400 nm. We characterized them to answer the question of how seed protein precursors are accumulated in the vesicles to be delivered to protein storage vacuoles. They contain an electron-dense core of storage proteins surrounded by an electron-translucent layer, and some vesicles also contained small vesicle-like structures. An immunocytochemical analysis revealed numerous electron-dense aggregates of storage proteins within the endoplasmic reticulum (Figure 2). It is likely that these aggregates develop into the electron-dense cores of the PAC vesicles and then leave the endoplasmic reticulum. Immunocytochemical analysis also showed that complex glycans are associated with the peripheral region of PAC vesicles but not the electron-dense cores, indicating that Golgi-derived glycoproteins are incorporated into the PAC vesicles. These results suggest that the unique PAC vesicles might mediate a transport pathway for insoluble aggregates of storage proteins directly to protein storage vacuoles.

Figure 2. Electron microscopy of maturing pumpkin cotyledons showing numerous electron-dense aggregates within the ER.
(A) Electron-dense core aggregates (arrows) formed within the ER in cells at the middle stage of seed maturation. The electron-translucent space between the aggregate and the ER membrane is loaded with ribosomes. PAC vesicles (PV) are also visible. (B) Immunoelectron microscopy with 2S albumin-specific antibodies. The aggregates within the ER (arrows) are labeled with gold particles. PAC vesicles (PV) and a protein storage vacuole (V) are also visible. CW, cell wall; Lb, lipid body. Bars in (A) and (B) = 500 nm.



IV. Vacuolar processing enzymes in protein-storage vacuoles and lytic vacuoles.

Vacuolar processing enzymes (VPEs), which are responsible for maturation of various vacuolar proteins belong to a novel family of cysteine proteinases. Molecular characterization of castor bean VPE revealed that the latent precursor of VPE (proVPE) is converted into an active VPE by self-catalytic proteolysis. Thus, no other factor is necessary to produce active VPE, and VPE itself is a key enzyme in determining the final conformation of the vacuolar proteins. The VPE-mediated system is widely distributed in various plant organs. The temporal and spatial expression of the VPE system has been examined with three Arabidopsis VPEs, aVPE, bVPE and gVPE. The bVPE gene is expressed in seeds, suggesting that bVPE plays a role in maturation of seed proteins in protein storage vacuoles. On the other hand, both the aVPE and g VPE genes are expressed in senescent tissues and their expression patterns are correlated with programmed cell death. The vegetative VPEs might regulate the activation of senescence-associated hydrolytic enzymes in the lytic vacuoles of cells preparing for death.



V. 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. In addition to mitochondrial chaperonin10 homologues, we isolated a cDNA for chloroplastic chaperonin 10 homologues from Arabidopsis thaliana. The cDNA was 958 bp long and encoded a polypeptide of 253 amino acids. The deduced amino acid sequence showed that the protein contained an N-terminal chloroplast transit peptide and a mature region which was comprised of two distinct GroES-domains. The two halves of the Cpn20 show 42 % amino acid identity to each other. A Northern blot analysis revealed that the mRNA for the Cpn10 homologue was abundant in leaves and was increased by heat treatment. To examine the oligomeric structure of Cpn20, a histidine-tagged construct lacking the transit peptide was expressed in E. coli and purified by affinity chromatography. Gel filtration and cross-linking analyses showed that the expressed products form a tetramer. The expressed products can substitute for GroES to assist in the refolding of citrate synthase under non-permissive conditions. Further analysis on the subunit stoichiometry of the GroEL-Cpn20 complex revealed that the functional complex is composed of a GroEL tetradecamer and a Cpn20 tetramer.



Publication List:
Hara-Nishimura, I (1998) Asparaginyl endopeptidase. In Handbook of Proteolytic Enzymes, edited by A. J. Barrett, N. D. Rawlings and J. F. Woessner (Academic Press, London, UK, 1998) pp. 746-749.
Hara-Nishimura, I., Shimada, T., Hatano, K., Takeuchi, Y. and Nishimura, M. (1998) Transport of storage proteins to protein-storage vacuoles is mediated by large precursor-accumulating vesicles. Plant Cell 10, 825-836.
Hara-Nishimura, I., Kinoshita, T., Hiraiwa, N. and Nishimura, M. (1998) Vacuolar processing enzymes in protein-storage vacuoles and lytic vacuoles. J. Plant Physiol. 152, 668-674.
Hayashi, H., L. De Bellis, Yamaguchi, K., Kato, A., Hayashi, M. and Nishimura, M. (1998) Molecular characterization of a glyoxysomal long-chain acyl-CoA oxidase that is synthesized as a precursor of higher molecular mass in pumpkin. J. Biol. Chem. 273, 8301-8307.
Hayashi, M., Toriyama, K., Kondo, M. and Nishimura, M.(1998) 2,4-Dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects on glyoxysomal b-oxidation. Plant Cell 10, 183-195.
Hayashi, M., Toriyama, K., Kondo, M., Kato, A., Mano, S., L. De Bellis., Hayashi-Ishimaru, Y., Yamaguchi, K., Hayashi,.H. and Nishimura, M. (1998) Functional transformation of plant peroxisomes. Cell Biochem. Biophys. in press.
Kato, A., Hayashi, M., Kondo, M. and Nishimura, M.(1998) Transport of peroxisomal proteins that are synthesized as large precursors in plants. Cell Biochem. Biophys. in press.
Kato, A., Takeda-Yoshikawa, Y., Hayashi, M., Kondo, M., Hara-Nishimura, I. and Nishimura, M. (1998) Glyoxysomal malate dehydrogenase in pumpkin: cloning of a cDNA and functional analysis of its presequence. Plant Cell Physiol. 39, 186-195.
Mano, S., Hayashi, M. and Nishimura, M. (1998) A leaf-peroxisomal protein, hydroxypyruvate reductase, is produced by light-regulated alternative splicing. Cell Biochem. Biophys. in press.
Nishimura, M. (1998) Molecular chaperones and temperature stress. Stress Responses of Photosynthesis Organisms: Molecular Mechanisms and Molecular Regulations, Edited by K. Satoh and N. Murata, Elsevier. pp. 83-92.
Nishimura, M., Hayashi, M., Toriyama, K., Kato, A., Mano, S., Yamaguchi, K., Kondo, M. and Hayashi, H. (1998) Microbody defective mutants of Arabidopsis. J. Plant Res. 111, 329-332.


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Last Modified: 12:00, May 28, 1999