NATIONAL INSTITUTE FOR BASIC BIOLOGY

DIVISION OF CELL MECHANISMS

Professor:

Mikio Nishimura

Research Associates:

Kazuo Ogawa
Makoto Hayashi
lkuko Hara-Nishimura
Hitoshi Mori

JSPS-Post-doctoral Fellow:

Ryuji Tsugeki

Graduate Students:

Kaori Inoue
Akira Kato
Tomoo Shimada
Tetsu Kinoshita
Nagako Hiraiwa (1)
Masahiro Aoki (2)

Technical Staff:

Maki Kondo
Katsushi Yamaguchi

(1) from Aichi University of Education
(2) from Shinshu University

I. Transformation of leaf peroxisomes to glyoxysomes in senescing pumpkin cotyledons.

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.

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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 confirmed.

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.

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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 (Fig. 2). 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 tissues.

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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.

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V. Role of molecular chaperones on translocation of proteins into chloroplasts.

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 progress.

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Publication List:

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. 29, 395-402.

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.