NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

Division of Cell Mechanisms

Professor:
Mikio Nishimura
Associate Professor (adjunct):
Masayoshi Maeshima
Research Associates:
Makoto Hayashi
Ikuko Hara-Nishimura
Tomoo Shimada
JSPS Postdoctoral Fellow:
Tetsu Kinoshita
NIBB Fellow:
Akira Kato
Graduate Students:
Nagako Hiraiwa
Shoji Mano
Hiroshi Hayashi
Kenji Yamada
Daigo Takemoto1)
Yuki Tachibe2)
Technical Staffs:
Maki Kondo
Katsushi Yamaguchi
Monbusho Foreign Scientist:
Luz Marina Melgarejo3)
Visiting Scientists:
Yasuko Koumoto
Miwa Kuroyanagi
1) from Nagoya University
2) from Hiroshima University
3) from Colombia National 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, while other organelles may degenerate. Because the dynamic transformation of organellar function (differentiation of organelles) is responsible for the flexibility of differentiation events in higher plant cells, the elucidation of regulatory mechanisms underlying organelle transformation are currently being 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 associated with 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 the targeting signal is part of the mature protein. One such signal, the tripeptide Ser-Lys-Leu 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, transgenic Arabidopsis plants that expressed a fusion protein composed of the C-terminal five amino acids of pumpkin malate synthase and a bacterial protein, b-glucuronidase (GUS) were generated. Immunocytochemical analysis of the transgenic plants revealed that the carboxy-terminal five amino acids of pumpkin malate synthase were sufficient for the transport of the fusion protein into glyoxysomes in etiolated cotyledons, into leaf peroxisomes in green cotyledons and in mature leaves, and into unspecialized microbodies in roots, although the fusion protein was no longer transported into microbodies when SRL at the carboxyl terminus was deleted. Transport of proteins into glyoxysomes and leaf peroxisomes was also observed when the carboxy-terminal amino acids of the fusion protein were changed from SRL to SKL, SRM, ARL or PRL.

A second type of targeting signal involved a cleavable N-terminal sequence. A small group of microbody proteins, such as 3-ketoacyl-coenzyme A (CoA) thiolase, malate dehydrogenase and glyoxysomal citrate synthase, (gCS) are synthesized as precursor proteins with larger molecular masses than those of the mature proteins. Each of these proteins has a cleavable presequence at its N-terminal end. Swinkels and colleagues showed that the N-terminal presequence of 3-ketoacyl-CoA thiolase from rat liver functions as a targeting signal. The N-terminal region of gCS is highly homologous to those of other microbody proteins that are synthesized as larger precursors. There are two conserved sequences in their N-terminal regions. One sequence is RL-X5-HL, first recognized by de Hoop and Ab. The other sequence, SXLXXAXCXA, is located at the cleavage site of the presequence. Transgenic Arabidopsis plants that expressed a fusion protein composed of the N-terminal region of gCS and GUS, were generated and their localization and processing were characterized by immunological and immunocytochemical analyses (Fig. 1). 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 is mediated by its amino-terminal presequence and that the transport system is functional in all plant microbodies. Therefore, it is unlikely that glyoxysomes and leaf peroxisomes possess different targeting machineries.

A similar analysis of transgenic Arabidopsis plants that expressed fusion proteins with substituted amino acid residues in the two consensus sequences by site-directed mutagenesis shows that RL-X5-HL functions in targeting to microbodies and SXLXXAXCXA plays a role in the processing of the N-terminal region.

Fig. 1
Targeting and processing of the chimeric proteins between N-terminal sequence of glyoxysomal citrate synthase (gCS) and b-glucuronidase (GUS). (A) Localization of the chimeric proteins in transgenic Arabidopsis plants analyzed by immunoelectron microscopy. 13-day-old cotyledons were fixed and embedded in LR-white resin. The thin sections were stained with anti-GUS antibodies. Gold particles indicated the localization of the GUS chimeric proteins in microbodies. Bar in panel DC42 indicates 1 mm. (B) Construction of the chimeric proteins. The yellow and green boxes show the consensus sequences which were found in the N-terminal region of the microbody proteins that contained the cleavable N-terminal presequences. Mutated amino acids are indicated by red letters. (C) Summary of the localization and processing of the chimeric proteins. Arg-16, Leu-17 and Leu-24 in gCS, which are located in the yellow box, function in the targeting to microbodies, whereas Cys-42 located in the green box functions in the processing of the presequence.


II. Two forms of hydroxypyruvate reductase might be produced by alternative splicing

Hydroxypyruvate reductase belongs to the leaf-peroxisome-specific enzymes and is induced and accumulated in microbodies during greening. Two different cDNAs encoding hydroxypyruvate reductase were isolated from a cDNA library of pumpkin cotyledons. One of the cDNAs, designated HPR1, encodes a polypeptide of 386 amino acids, while the other, HPR2, encodes a polypeptide of 381 amino acids. Although the nucleotide and deduced amino acid sequences of these cDNAs are almost identical, the deduced HPR1 protein contains Ser-Lys-Leu at its carboxy-terminal end, which is known as a microbody-targeting signal, while the deduced HPR2 protein does not. Analysis of genomic DNA strongly suggests that HPR1 and HPR2 are produced by alternative splicing. These findings show that two different hydroxypyruvate reductases, HPR1 and HPR2, are localized in microbodies and in the cytosol, respectively and accumulation of HPR1 in leaf peroxisomes is increased by light, suggesting that the microbody transition may be regulated by alternative splicing.


III. Membrane protein a-TIP of protein-storage vacuoles

The vacuole in the cells of plant seeds shows dramatic changes in its morphology and function during seed maturation through seed germination. To investigate this transition, we prepared protein-body membranes from dry seeds of pumpkin (Cucurbita sp.) and characterized their protein components. Five major membrane proteins, designated MP23, MP27, MP28, MP32 and MP73, are located in the protein body membrane of pumpkin seeds. Both MP28 and MP23 belong to the seed TIP (tonoplast intrinsic protein) subfamily. TIP is an integral membrane protein that is found in plant seeds and belongs to the MIP (major intrinsic protein) family. Both MP28 and the 29-kDa precursor to MP23 accumulate on vacuolar membranes before the deposition of storage proteins, and then the precursor is converted to the mature MP23 at the late stage. The two TIPs of pumpkin seeds, pMP23 and MP28, were expressed in yeast cells under control of the GAL1 promoter, and the subcellular localization of the proteins was analyzed. The pMP23 and MP28 proteins stably accumulated in the yeast vacuolar membrane when the proteins were expressed in the proteinase A-deficient strain (pep4), which lacks the activities of vacuolar proteases. However, pMP23 and MP28 did not accumulate in the wild-type strain; the expressed pMP23 and MP28 were degraded in a proteinase A-dependent manner. These results indicate that pMP23 and MP28 are transported to the vacuolar membrane when expressed in yeast. In vitro transport assays using the vacuolar membrane vesicles from the yeast transformants will allow us to further investigate the function(s) of pumpkin seed TIP.


IV. A rapid increase in the level of binding protein (BiP) is accompanied by synthesis and degradation of storage proteins in pumpkin cotyledons

The binding protein (BiP) is a member of the heat shock 70 protein (Hsp70) family that is localized to the endoplasmic reticulum (ER) of eukaryotic cells, where it functions as a chaperone and is believed to support proper protein folding and protein translocation into the ER lumen. To elucidate the involvement of BiP in the biosynthesis of vacuolar proteins, we have characterized the protein in pumpkin cotyledons during seed maturation and seedling growth. Isolated microsomes from maturing pumpkin cotyledons contained a significant amount of BiP, protein-disulfide isomerase and calreticulin. We have purified a 70-kDa protein; sequences of the N-terminus and internal fragments of this protein exhibited a high identity to the sequence of soybean BiP. Immunoblot analysis with specific antibodies raised against the purified BiP showed that the amount of BiP in cotyledons increased markedly at the middle stages and then decreased. The increase was accompanied by the synthesis of storage proteins and the development of the endoplasmic reticulum in the cotyledons at the middle stage of seed maturation. Most of these storage proteins degraded dramatically between 2 and 5 days after seed germination, and the degradation was also accompanied by a rapid increase in the level of BiP. Subcellular fractionation of the 4-day-old cotyledons showed a high accumulation of BiP in the endoplasmic reticulum. It is possible that BiP might be involved in the synthesis of seed storage proteins during maturation and in the synthesis of hydrolytic enzymes responsible for the degradation of the storage proteins during seed germination.


V. A vacuolar processing enzyme responsible for conversion of proprotein precursors into their mature forms

Processing enzymes responsible for the maturation of seed proteins belong to a novel group of cysteine proteinases with molecular masses of 37 to 39 kDa. However, the processing enzyme activity can be found not only in seeds but also in vegetative tissues such as hypocotyls, roots and mature leaves. Thus, we designated the enzyme as a vacuolar processing enzyme (VPE). Members of the VPE family can be separated into two subfamilies, one that is specific to seeds and another that is specific to vegetative organs. The molecular characterization of all the members of the VPE family in Arabidopsis is required if we are to elucidate the mechanisms of regulation of genes for VPE homologues and the physiological functions of these proteins in protein-storage vacuoles and vegetative vacuoles. We isolated the three genes of VPEs (a-VPE, b-VPE and g-VPE) from a genomic library of Arabidopsis. To demonstrate temporal and spatial expression of the promoters of the VPE genes, we transformed Arabidopsis plants with a reporter gene containing the promoter of the VPE genes and the coding region of b-glucuronidase (GUS). The b-VPE gene was expressed in seeds, but a-VPE and g-VPE were not. The GUS activity for g-VPE gene was predominatly expressed in the hydathode tissues of leaves (Fig. 2).

Fig. 2.
Histochemical localization of GUS activity in the hydathode of Arabidopsis transformed with the g-VPE promoter-gus fusion gene. Arabidopsis was transformed with a reporter gene composed of the promoter of the g-VPE gene and the coding region of b-glucuronidase (GUS). GUS activity was observed in the hydathodes at the ends of veins along with the leaf margins, as shown in (A). Hydathodes are structures for that discharge water from the interior of the leaf to its surface. A photograph of the hydathode with DIC optic of the enclosed region in A is shown in (B).


VI. 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 role in the translocation of proteins into chloroplasts. We isolated a cDNA for the chaperonin 10 homologues from Arabidopsis thaliana by functional complementation of the E. coli groES mutant. The cDNA was 647 bp long and encoded a polypeptide of 98 amino acids. The deduced amino acid sequence showed approximately 50% identity to mammalian mitochondrial Cpn10s and 30% identity to GroES. A. Northern blot analysis revealed that the mRNA for the Cpn10 homologue was expressed uniformly in various organs and was markedly induced by heat-shock treatment. The Cpn10 homologue was constitutively expressed in transgenic tobaccos. Immunogold and immunoblot analyses following the subcellular fractionation of leaves from transgenic tobaccos revealed that the Cpn10 homologue was localized in mitochondria and accumulated at a high level in transgenic tobaccos.


Publication List:
Hayashi, M., Aoki, M., Kato, A., Kondo, M. and Nishimura, M. (1996) Transport of chimeric proteins contain a carboxy-terminal targeting signal into plant microbodies. Plant J. 10: 225-234.
Hayashi, M., Tsugeki, R., Kondo, M., Mori, H. and Nishimura, M. (1996) Pumpkin hydroxypyruvate reductases with and without a putative C-terminal signal for targeting to microbodies may be produced by alternative splicing. Plant Mol. Biol. 30: 183-189.
Higuchi, T., Hisada, H. and Maeshima, M. (1996) Complete nucleotide sequence of a cDNA for an intrinsic protein in vacuolar membranes from radish roots (PGR 96-045). Plant Physiol., 111: 947.
Kato, A., Hayashi, M., Kondo, M. and Nishimura, M. (1996) Targeting and processing of a chimeric protein with the N-terminal presequence of the precursor to glyoxysomal citrate synthase. Plant Cell 8: 1601-1611.
Kato, A., Hayashi, M. Takeuchi, Y. and Nishimura, M. (1996) cDNA cloning and expression of a gene for 3-ketoacyl-CoA thiolase in pumpkin cotyledons. Plant Mol. Biol. 31: 843-852.
Koumoto, Y., Tsugeki, R., Shimada, T., Mori, H., Kondo, M., Hara-Nishimura, I. and Nishimura, M. (1996) Isolation and characterization of a cDNA encoding mitochondrial chaperonin 10 from Arabidopsis thaliana by functional complementation of an E. coli groES mutant. Plant J. 10: 1119-1125.
Maeshima, M., Nakanishi, Y., Matsuura-Endo, C. and Tanaka, Y. (1996) Proton pumps of vacuolar membrane in growing plant cell. J. Plant Res., 109: 119-125.
Matsumoto, H., Senoo, Y., Kasai, M. and Maeshima, M. (1996) Response of the plant root to aluminium stress: Analysis of the inhibition of the root elongation and changes in membrane function. J. Plant Res., 109: 99-105.
Mano, S., Hayashi, M., Kondo, M. and Nishimura, M. (1996) cDNA cloning and expression of a gene for isocitrate lyase in pumpkin cotyledons. Plant Cell Physiol. 37: 941-948.
Nishimura, M., Hayashi, M., Kato, A., Yamaguchi, K. and Mano, S. (1996) Functional Transformation of microbodies in higher plant cells. Cell Struct. Funct. 21: 387-393.
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.
Robinson, D.G., Haschke, H.-P. Hinz, G., Hoh, B., Maeshima, M. and Marty, F. (1996) Immunological detection of tonoplast polypeptides in the plasmamembrane of pea cotyledons. Planta, 198: 95-103.
Yamaguchi, K., Hayashi, M. and Nishimura, M. (1996) cDNA cloning for thylakoid-bound ascorbate peroxidase in pumpkin and its characterization. Plant Cell Physiol. 37: 405-409.

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Last Modified: 12:00, June 27, 1997