NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

DIVISION OF CELL MECHANISMS

Professor:
Mikio Nishimura
Associate Professor:
Ikuko Hara-Nishimura
Associate Professor (adjunct):
Masayoshi Maeshima
Research Associates:
Makoto Hayashi
Tomoo Shimada
Post doctoral fellows:
Akira Kato 1
Tetsu Kinoshita 2
Nagako Hiraiwa 3
Yasuko Ishimaru 3
Kanae Shirahama 3
Graduate Students:
Shoji Mano
Hiroshi Hayashi
Kenji Yamada
Daigo Takemoto (Nagoya University)
Yuki Tachibe (Hiroshima University)
Technical Staffs:
Maki Kondo
Katsushi Yamaguchi
Visiting Scientists:
Luigi De Bellis (Pisa Univ. Italy)
Claus Schnarrenberger (from Free Univ. of Berlin, Germany)
Yasuko Koumoto
Miwa Kuroyanagi


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. The dynamic transformation of organellar function (differentiation of organelles) is responsible for flexibility of differentiation in higher plant cells. 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 the greening that occurs during 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 was 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 part of the mature protein. One such signal, the tripeptide Ser-Lys-Leu, occurs at the C-terminal end has been identified as a targeting signal. Ser-Lys-Leu and related amino acid sequences commonly function in mammals, insects, fungi, and plants. Glyoxysomal enzymes, such as malate synthase and isocitrate lyase, and leaf peroxisomal enzymes, such as glycolate oxidase and hydroxypyruvate reductase, contain the targeting signal at their C-terminal ends.

To characterize the targeting signal, 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, although tripeptides with proline at the first amino acid position and isoleucine at the carboxyl terminus show weak targeting efficiencies (Fig. 1). All known microbody enzymes that are synthesized in a form similar in size to the mature molecule, except catalase, contain one of these tripeptide sequences at their carboxyl terminus. These carboxyl tripeptides function as a targeting signal to the microbodies, not only to glyoxysomes but also to leaf peroxisomes. Therefore, it is unlikely that glyoxysomes and leaf peroxisomes possess different targeting machineries.

A
B
Substitutions of first amino acid Substitutions of second amino acid Substitutions of C-terminal amino acid
LRL - SIL - SRI +
FRL - SGL - SRV -
CRL ++ SSL - SRL ++
ARL ++ SHL - SRF -
GRL - SKL ++ SRM ++
SRL ++ SRL ++ SRS -
YRL - SRE -
PRL + SRK -
ERL -
KRL -

Fig. 1
Effects of Substitutions at the Carboxy-terminal Tripeptide on Subcellular Localization of Chimeric Proteins.
A) Subcellular localization of chimeric proteins in the cells of transgenic Arabidopsis was analyzed by immunoelectron-microscopy. Label on each photograph represents the carboxy-terminal tripeptide sequence of the chimeric protein. Arrowheads indicate microbodies. Bar = 1 µm.
B) Summary of the targeting efficiencies of carboxy-terminal tripeptides. Amino acid sequences of carboxy-terminal tripeptides are shown by single letter codes. Targeting efficiencies of proteins containing these tripeptides at the carboxy terminus were presented by ++ (efficient), + (detectable) and - (inefficient).


II. Light dependent alternative splicing in higher plants.

We previously showed that two different forms of hydroxypyruvate reductase which are localized in microbodies and in the cytosol, are produced by alternative splicing. In 1997, we found that stromal (sAPX) and thylakoid-bound ascorbate peroxidases (tAPX) are also produced by alternative splicing. cDNA for sAPX and tAPX were isolated and characterized. The cDNA for sAPX encodes a polypeptide with 372 amino acids and shares complete sequence identity with tAPX, except for the deletion of a putative membrane domain of tAPX. Southern blot hybridization and analysis of intron structure indicated that the mRNAs for sAPX and tAPX, whose suborganellar localizations in chloroplasts are different, are produced by alternative splicing. Immunoblot analysis showed that the accumulation of sAPX and tAPX was differently regulated and that the alternative splicing is regulated developmentally and by light.


III. A pumpkin 72-kDa membrane protein of precursor-accumulating vesicles has characteristics of a vacuolar sorting receptor.

Seed proteins are synthesized on the rough endoplasmic reticulum and are then delivered to the protein-storage vacuoles. Sorting and targeting them to the vacuoles requires the presence of a specific signal and a receptor. Previously we isolated transport vesicles that mediate the transport of the precursor of major storage proteins to protein-storage vacuoles in developing pumpkin cotyledons. We designated them precursor-accumulating (PAC) vesicles, as shown in Fig. 2A. The vesicles might contain a receptor protein for the storage protein. We characterized two homologous proteins from PAC vesicles, a 72-kDa protein (PV72) and an 82-kDa protein (PV82). PV72 and PV82 showed an ability to bind to peptides derived from both an internal propeptide and a C-terminal peptide of a proprotein precursor of 2S albumin, a major storage protein (Fig. 2B). PV72 was predicted to be a type I integral membrane protein with epidermal growth factor (EGF)-like motifs (Fig. 2A). These results suggest that PV72 and PV82 are potential sorting receptors for 2S albumin to protein-storage vacuoles. Previous studies have identified a potential receptor protein, BP-80, from the membranes of clathrin-coated vesicles of developing cotyledons of pea (Pisum sativum, Kirsch et al. 1994). Recently molecular structures of BP-80 (Paris et al. 1997) and the homolog from Arabidopsis (AtELP; Ahmed et al. 1997) were reported. They are membrane proteins that are homologous to PV72.

Fig. 2.
PV72 functions as a sorting receptor for intracellular transport of pro2S albumin.
A) A hypothetical scheme for the transport and processing of pro2S albumin. Pro2S albumin is synthesized on rER and transported to protein storage vacuoles via precursor-accumulating (PAC) vesicles. PV72 and pro2S albumin co-exist in the PAC vesicles. PV72 is predicted to be a type I integral membrane protein with three repeats of epidermal growth factor (EGF)-like motifs.
B) PV72 binds the peptides derived from pro2S albumin. PV72 binds to an affinity column with 2S-I peptide as a ligand and is specifically eluted with an excess amount of each peptide of 2S-I, 2S-C or PAP, but not 2S-N peptide. PAP is a peptide derived from a propeptide of barley aleurain, a thiol proteinase, that contained a vacuolar targeting signal. An NPIR (Asn-Pro-Ile-Arg) has been shown to be a consensus signal sequence for vacuolar targeting that is found in both propeptides of barley aleurain and sweet potato sporamin.


IV. Expression and activation of the vacuolar processing enzyme in Saccharomyces cerevisiae.

Vacuolar processing enzymes (VPE) are cysteine proteinases responsible for maturation of various vacuolar proteins in plants. A larger precursor to VPE synthesized on the rough endoplasmic reticulum is converted to an active enzyme in the vacuoles. We expressed a precursor to castor bean VPE in a pep4 strain of the yeast Saccharomyces cerevisiae to examine the mechanism of activation of VPE. Two VPE proteins with 59 kDa and 46 kDa were detected in the vacuoles of the transformant. They were glycosylated in the yeast cells, although VPE is not glycosylated in plant cells in spite of the presence of two N-linked glycosylation sites. During the growth of the transformant, the level of the 59-kDa VPE increased slightly until a rapid decrease occurred after 9 h. By contrast, the 46-kDa VPE appeared simultaneously with the disappearance of the 59-kDa VPE. Vacuolar processing activity increased with the accumulation of the 46-kDa VPE, but not an accumulation of the 59-kDa VPE. The specific activity of the 46-kDa VPE was similar to that of VPE in plant cells. The 46-kDa VPE mediated the conversion of procarboxypeptidase Y to the mature form instead of proteinase A. This indicated that proteinase A which is responsible for maturation of yeast vacuolar proteins, can be replaced functionally by plant VPE. These findings suggest that an inactive VPE precursor synthesized on the endoplasmic reticulum is transported to the vacuoles in the yeast cells and then processed to make an active VPE by self-catalytic proteolysis within the vacuoles.


V. An aspartic proteinase is involved in the maturation of storage proteins in concert with the vacuolar processing enzyme.

A 48-kDa aspartic proteinase was purified from protein bodies of dry seeds of castor bean. Immunocytochemical and cell fractionation analyses of the endosperm of maturing castor bean seed showed that the aspartic proteinase was selectively localized in the matrix of the protein storage vacuoles, where a variety of seed storage proteins were also present. To determine whether the aspartic proteinase is responsible for maturation of seed proteins, we prepared [35S]proproteins that were localized in the endoplasmic reticulum in pulse-labeled endosperm cells and used the authentic proproteins as substrate for in vitro processing experiments. The purified aspartic proteinase was unable to convert any of three endosperm proproteins into their mature forms, while the purified vacuolar processing enzyme could convert all three proproteins. We further examined the activity of aspartic proteinase on the cleavage of an internal propeptide of Arabidopsis pro2S albumin, that was post-translationally removed. The aspartic proteinase cleaved the propeptide at three sites at pH 3.0, but not at pH 5.5. These results suggest that aspartic proteinase cannot directly convert pro2S albumin into the mature form, but it may play a role in trimming the C-terminal propeptides from the subunits that are produced by the action of the vacuolar processing enzyme. However, the activity of the aspartic proteinase in the protein storage vacuoles might be limited.


VI. Role of molecular chaperones in organelle differentiation

Molecular chaperones are cellular proteins that function in the folding and assembly into oligomeric structures of certain other polypeptides 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 role in 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 the 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. A histidine-tagged construct lacking the transit peptide was expressed in E. coli and the functional and structural characterization is in progress.


Publication List:
Esaka, M., Yamada, N., Kitabayashi, M., Setoguchi, Y., Tsugeki, R., Kondo, M. and Nishimura, M. (1997) cDNA cloning and differential gene expression of three catalases in pumpkin. Plant Mol. Biol. 33: 141-155.
Fleurat-Lessard, P., Fragne, N., Maeshima, M., Ratajczak, R., Bonnemain, J. L. and Martinoia, E. (1997) Increased expression of vacuolar aquaporin and H+-ATPase related to motor cell function in Mimosa pudica L. Plant Physiol. 114: 827-834.
Hatano, K., Shimada, T., Hiraiwa, N., Nishimura, M. and Hara-Nishimura, I. (1997) A rapid increase in the level of binding protein (BiP) is accompanied by synthesis and degradation of storage proteins in pumpkin cotyledons. Plant Cell Physiol. 38: 344-351.
Hayashi, M., Aoki, M., Kondo, M. and Nishimura, M. (1997) Changes in targeting efficiencies of proteins to plant microbodies caused by amino acid substitution in the carboxy-terminal tripeptide. Plant Cell Physiol. 38: 759-768.
Hiraiwa, N., Kondo, M., Nishimura, M. and Hara-Nishimura, I. (1997) An aspartic proteinase is involved in the maturation of storage proteins in concert with the vacuolar processing enzyme. Eur. J. Biochem. 246: 133-141.
Hiraiwa, N., Nishimura, M. and Hara-Nishimura, I. (1997) Expression and activation of the vacuolar processing enzyme in Saccharomyces cerevisiae. Plant J. 12: 819-829.
Inoue, K., Wada, Y., Nishimura, M. and Hara-Nishimura, I. (1997) Heterologous expression and subcellular localization of pumpkin seed tonoplast intrinsic proteins (TIP) in yeast cells. Plant Cell Physiol. 38: 366-370.
Mano, S., Hayashi, M. Kondo, M. and Nishimura, M. (1997) Hydroxypyruvate reductase with a carboxy-terminal targeting signal to microbodies is expressed in Arabidopsis. Plant Cell Physiol. 38: 449-455.
Mano, S., Yamaguchi, K., Hayashi, M. and Nishimura, M. (1997) Stromal and thylakoid-bound ascorbate peroxidases are produced by alternative splicing in pumpkin. FEBS Lett. 413: 21-26.
Matsuoka, K., Higuchi, T., Maeshima, M. and Nakamura, K. (1997) A vacuolar H+-ATPase in a nonvacuolar organelle is required for the sorting of soluble vacuolar protein precursors in tobacco cells. Plant Cell 9: 533-546.
Minami, Y., Takao, H., Kanafugi, T., Miura, K., Kondo, M., Hara-Nishimura, I., Nishimura, M. and Matsubara, H. (1997) b-glucosidase in the Indigo plant: Intracellular localization and tissue specific expression in leaves. Plant Cell Physiol. 38: 1069-1074.
Nozue, M., Yamada, K., Nakamura, T., Kubo, H., Kondo, M. and Nishimura, M. (1997) Expression of a vacuolar protein (VP24) in anthocyanin-producing cells of sweet potato in suspension culture. Plant Physiol. 115: 1065-1072.
Odaira, M., Yoshida, S. and Maeshima, M. (1997) Accumulation of a glycoprotein homologous to seed storage protein in mung bean hypocotyls in the late stage of tissue elongation. Plant Cell Physiol. 38: 290-296.
Sato, M. H., Nakamura, M., Ohsumi, Y., Kouchi, H., Kondo, M., Hara-Nishimura, I., Nishimura, M. and Wada, Y. (1997) The AtVAM3 encodes a syntaxin-related molecule implicated in the vacuolar assembly in Arabidopsis thaliana. J. Biol. Chem. 272: 24530-24535.
Shimada, T., Kuroyanagi, M., Kondo, M., Nishimura, M. and Hara-Nishimura, I. (1997) A pumpkin 72-kDa membrane protein of precursor accumulating vesicles has characteristics of a vacuolar sorting receptor. Plant Cell Physiol. 38: 1414-1420.
Shiratake, K., Kanayama, Y., Maeshima, M. and Yamaki, S. (1997) Changes in H+-pumps and a tonoplast intrinsic protein of vacuolar membranes during the development of pear fruit. Plant Cell Physiol. 38: 1039-1045.
Takasu, A., Nakanishi, Y., Yamauchi, T. and Maeshima, M. (1997) Analysis of substrate binding site and carboxyl terminal region of vacuolar H+ translocating pyrophosphatase of mung bean with peptide antibodies. J. Biochem. 122: 883-889.

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Last Modified: 12:00, May 28, 1998