  National Insitute for Basic Biology

DIVISION OF CELL MECHANISMS

Professor:: Mikio Nishimura

Research Associates:: Kazuo Ogawa; Makoto Hayashi; Ikuko Hara-Nishimura

Graduate Students:: Kaori Inoue; Akira Kato; Tomoo Shimada; Tetsu Kinoshita; Nagako Hiraiwa; Masahiro Aokil ¹⁾

Technical Staff:

Maki Kondo

Katsushi Yamaguchi

Yasuko Koumoto

Visiting Scientists:: Kyoko Hatano ²⁾; Gerhard Bytof ³⁾

( ¹⁾ from Shinshu University)
( ²⁾ from Kyoto University)
( ³⁾ from Technischen Universitat Braunschweig, Germany)

The cells of higher plants cells contain several distinct organelles that play vital roles in cellular physiology. During the proliferation and differentiation of these cells, the organelles often undergo dynamic changes. The biogenesis of new organelles may occur, existing organelles may undergo a change in function, which other organelles may degenerate. The dynamic transformation of organellar functions (differentiation of organelles) is responsible for the flexibility of differentiation events in higher plant cells. Therefore, the efforts of this division are focussed on the elucidation of regulatory mechanisms that underlie such transformation.

I. Development of microbody membrane proteins during the microbody transition.

Dramatic metabolic changes that underlie the shift from heterotrophic to autotrophic growth occur during the greening associated with the germination of seeds. As these metabolic changes take place, 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 the leaf peroxisomes that function at several crucial steps in 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. As part of our attempts to clarify the molecular mechanisms that underlie the microbody transition, that changes with development in microbody membrane proteins during the transformation of glyoxysomes to leaf peroxisomes were characterized. Two proteins in glyoxysome membranes, with molecular masses of 31 kDa and 28 kDa respectively, were purified and polyclonal antibodies were raised against the each protein. Analysis of these membrane proteins during development revealed that the amounts of these proteins decreased during the microbody transition. The larger one was retained in leaf peroxisomes, whereas the small one could not be found in leaf peroxisomes after completion of the microbody transition. The results clearly showed that membrane proteins in glyoxysomes change dramatically during the microbody transition, as do the enzymes in the matrix.

II. Characterization of the aconitase that participates in the glyoxylate cycle.

Aconitase catalyzes the reversible interconversion of citrate, isocitrate and cis-aconitate. This enzyme is thought to function as a component of the glyoxylate cycle in glyoxysomes, as well as the Krebs cycle in mitochondria. It was reported, however, that less than 0.5% of the total aconitase activity could be recovered in glyoxysomal fractions after sucrose gradient centrifugation, with most of the activity being found in mitochondria and the cytosol. It remained to be confirmed whether mitochondria and glyoxysomes contain different and specific isoenzymes of aconitase.

Three isoforms of aconitase (AcoI, AcoII, AcoIII) are found in etiolated cotyledons of pumpkin, and we purified two of them (AcoI, AcoII). Antibodies raised against AcoI allowed us to isolate a cDNA for aconitase. The amino acid sequence deduced from the cDNA was very similar to those of mammalian iron responsive element-binding proteins that are also known as cytosolic aconitases. Antibodies were raised against a fusion protein that consisted of glutathione-S-transferase and the partial sequence ( 162 amino acids) of the cloned aconitase. Not only the antibodies against purified AcoI but also the antibodies against the fusion protein cross-reacted with all three isoforms of aconitase. Further analysis by subcellular fractionation, immunoblotting and immunofluorescene microscopy, revealed that AcoI and AcoIII were localized in the cytosol, while AcoII was localized exclusively in mitochondria (Fig. 1). No aconitase was found in glyoxysomes. AcoI was specifically found in etiolated pumpkin cotyledons during the early stage of seedling growth, while AcoII and AcoIII were present in all tissues of pumpkin plants. The pattern of expression of AcoI was similar to those of other enzymes of the glyoxylate cycle. These data suggest that cytosolic AcoI functions as a component of the glyoxylate cycle despite the fact that all other enzymes of the glyoxylate cycle appear to be localized in glyoxysomes.

Fig. 1.
Localization of aconitase and isocitrate lyase within a cell from a pumpkin cotyledon, as analyzed by double immunofluorescence staining. Pumpkin seedlings were grown for 5 days in darkness. A thin section of a cotyledon was double-stained first with aconitase-specific antibodies which were visualized by use of FITC-conjugated second antibody, and then with isocitrate lyase-specific antibodies that were visualized by use of rhodamine-conjugated second antibody. (a) Immunofluorescent image due to FITC. (b) Immunofluorescent images in a and c are superimposed. (c) Immunofluorescent image due to rhodamine of the same field as in (a). Bar in panel c indicates 10痠. Magnification in a, b and c is the same.

III. Mechanisms for vesicular transport of seed proteins.

i) Dense vesicles responsible for the transport of precursors to seed proteins.

Most vacuolar proteins are synthesized on the rough endoplasmic reticulum and are then delivered to vacuoles via vesicle-mediated transport systems. Developing seeds, in which large amounts of seed proteins are synthesized and transported to protein-storage vacuoles, are particularly useful materials with which to explore the cellular machinery involved in the sorting of vacuolar proteins in plant cells. Proprotein precursors to seed proteins are delivered to the vacuoles via unique vesicles, known as dense vesicles. Electron microscopic studies of developing seeds of pumpkin ( Cucurbita sp.) and castor bean (Ricinus communis) have shown that the dense vesicles have a highly electron-dense core; they are about 300 nm in diameter and are different from Golgi vesicles. Such dense vesicles were isolated from developing pumpkin cotyledons and were shown to contain large amounts of the precursors to various seed proteins. This result was supported by results of immunocytochemical analysis with specific antisera against two seed proteins, namely, 2S albumin and 11S globulin. The dense vesicles are directly targeted to vacuoles and fuse to the vacuolar membranes. Biochemical analysis of the isolated dense vesicles should provide insight into the specific targeting and fusion of vesicles to vacuolar membranes.

ii) Isolation and characterization of the dense vesicles, association of small GTP-binding proteins on the membrane of dense vesicles.

To date, a vesicle-mediated transport system through secretory pathway has been investigated exclusively in yeast. Distinct members of the ras superfamily of small GTP-binding proteins facilitate the targeting of vesicles with the appropriate membranes in a GTP-dependent manner to regulate specific steps in the secretion pathway in yeast. However, the mechanism of the vesicular transport that is targeted to vacuoles is still obscure.

Isolation of these vesicles allows us to analyze the components responsible for the specific targeting of vesicles to vacuoles and for the fusion between vesicles and vacuoles. To explore the vesicle-mediated transport system that is targeted to vacuoles in plant cells, we isolated dense vesicles and examined them for the presence of guanine nucleotide-binding proteins. GTP-binding proteins of 25 kDa and 27 kDa were detected on the dense vesicles isolated from developing pumpkin cotyledons. The two different, small GTP-binding proteins might function in the targeting and/or fusion of the vesicles to the vacuolar membranes or in the budding of the vesicles (Fig. 2).

Fig. 2.
Dense vesicles mediate the final step in the delivery of seed proteins to vacuoles in developing pumpkin ( Cucurbita sp.) cotyledons. Two different, small GTP-binding proteins of 25 kDa and 27 kDa might function in the targeting and/or fusion of the vesicles to the vacuolar membranes or in the budding of the vesicles.

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

Proprotein precursors of vacuolar components are transported from the endoplasmic reticulum to the dense vesicles and then they are targeted to the vacuoles. In the vacuoles, the proproteins are processed proteolytically to their mature forms by a unique vacuolar processing enzyme. A vacuolar process-ing enzyme responsible for the maturation of seed proteins was isolated from castor bean and soybean. The processing enzyme is a novel cysteine proteinase with a molecular mass of 37 kDa (castor bean) or 39 kDa (soybean). The enzyme splits a peptide bond on the C-terminal side of an exposed asparagine residue of a proprotein precursor to produce a mature seed protein such as 11S globulin and 2S albumin. The immunocytochemical localization of the enzyme in the vacuolar matrix of the maturing endosperm of castor bean indicated that the maturation of the seed proteins occurs in the vacuoles. Molecular characterization revealed that the enzyme is synthesized as an inactive precursor with a larger molecular mass. The results of immunoelectron microscopy suggested that the precursor is transported to vacuoles via dense vesicles together with the pro-protein forms of other seed proteins. Upon arrival in the vacuole, the inactive precursor is converted to the active enzyme. This result suggests that processing of proprotein for the maturation of seed proteins involves a cascade of reactions. The activity of the vacuolar processing enzyme was found in various plant tissues and several cDNAs corresponding to homologues of the enzyme were isolated from different plants. Thus, a similar processing enzyme ap-pears to be widely distributed in plant tissues and to play a crucial role in the maturation of a variety of proteins in plant vacuoles.

V. Role of molecular chaperones in the translocation of proteins into chloroplasts.

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 chaperonin (Cpn) and Hsp 70 in the translocation of chloroplast proteins, we examined the interaction of Cpn and Hsp 70 with chloroplast proteins during their import into chloroplasts. Ferredoxin NADP⁺ reductase (FNR), imported into pea chloroplasts in vitro, was immunoprecipitated not only with antisera raised against Hsp 70, but also with against Cpn 60 in an ATP-depend- ent manner, indicating that newly imported FNR interacts physically with Hsp 70 and Cpn 60 in chloroplasts. Time-course and temperature-shift analysis of these interactions revealed that imported FNR binds transiently to Hsp 70 and that the association of FNR with Hsp 70 precedes that with Cpn 60. These results suggest that Hsp 70 and Cpn 60 in chloroplasts might sequentially assist in the maturation of newly imported FNR in an ATP-dependent matter.

Immunoblot analysis revealed that, in germinating pumpkin seedlings, not only chloroplasts but also etioplasts accumulate heat shock-induced Hsp 70 proteins. Moreover, cDNAs for homologues of Cpn60 and Cpn10 have been isolated and characterized. Further investigations on the function of Cpn10 in the trans-port of proteins into chloroplasts are in progress.

Publication List:

De Bellis, L., M. Hayashi, I. Hara-Nishimura, A. Alpi and M. Nishimura (1994) Immunological analysis of aconitase in pumpkin cotyledons: The absence of aconitase in glyoxysomes. Physiol. Plant. 90, 757-762.

Kinoshita, T., I. Hara-Nishimura, H. Shiraishi, K. Okada, Y. Shimura and M. Nishimura (1994) Nucleotide sequence of a transmembrane protein (TMP-C) cDNA in Arabidopsis thaliana. Plant Physiol. 105, 1441-1442.

Maeshima, M., I. Hara-Nishimura, Y. Takeuchi and M. Nishimura (1994) Accumulation of vacuolar H⁺ -pyrophosphatase and H⁺-ATPase during reformation of the central vacuole in germinating pumpkin seeds. Plant Physiol. 106, 61-69.

Ozaki, K., A. Terakita, M. Ozaki, R. Hara, T. Hara, I. Hara-Nishimura, H. Mori and M. Nishimura (1994) Molecular characterization and functional expression of squid retinal-binding protein: A novel species of hydro-phobic ligand-binding protein. J. Biol. Chem. 269, 3838-3845.

Shigemori, Y., J. Inagaki, H. Mori, M. Nishimura, S. Takahashi and Y. Yamamoto (1994) The presence of the precursor to the nucleus-encoded 30-kDa protein of photosystem 11 in Euglena gracilis Z includes two hydro-phobic domains. Plant Mol. Biol. 24, 209 215.

Shimada, T., N. Hiraiwa, M. Nishimura and I. Hara-Nishimura (1994) Vacuolar processing enzyme of soybean that converts proproteins to the corresponding mature forms. Plant Cell Physiol. 35, 713 718.

Shimada, T., M. Nishimura and I. Hara-Nishimura (1994) Small GTP-binding proteins are associated with the vesicles targeting to vacuoles in developing pumpkin cotyledons. Plant Cell Physiol. 35, 995 1001.

Strzalka, K., R. Tsugeki and M. Nishimura ( 1994) Heat shock induces synthesis of plastid-associated hsp 70 in etiolated and greening pumpkin cotyledons. Folia Histochem. Cyto-biol. 32, 45-49.