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I Hara-Nishimura

Publications and source records attributed to I Hara-Nishimura.

At least 37 records · Page 2Linked to original sources
PubMed1997

The AtVAM3 encodes a syntaxin-related molecule implicated in the vacuolar assembly in Arabidopsis thaliana.

The vacuole constitutes a large compartment in plant and fungal cells. The VAM3 gene of Saccharomyces cerevisiae encodes a syntaxin-related protein required for vacuolar assembly. An Arabidopsis thaliana cDNA library, designed for expression in S. cerevisiae, was screened for cDNAs able to complement defective vacuolar assembly of the Deltavam3 mutation. One cDNA, encoding a 33-kDa protein with structural similarities to the other syntaxins, was identified. The product of AtVAM3 (AtVam3p) was expressed in various tissues including roots, leaves, inflorescence stems, flower buds, and young siliques. The AtVAM3 transcripts were abundant in undifferentiated cells in the meristematic region. AtVam3p fractionated predominantly to an 8,000 x g pellet fraction where a vacuolar membrane protein H+-translocating inorganic pyrophosphatase (H+-PPase) also fractionated. Immunoelectron microscopy showed that AtVam3p was localized to restricted regions on the vacuolar membranes. We propose that AtVam3p provides the t-SNARE function in the vacuolar assembly in A. thaliana.

Amino Acid Sequence↗
PubMed1997

An aspartic endopeptidase is involved in the breakdown of propeptides of storage proteins in protein-storage vacuoles of plants.

To understand the mechanism of the maturation of various proteins in protein-storage vacuoles, we purified a 48-kDa aspartic endopeptidase composed of 32-kDa and 16-kDa subunits from castor bean. Immunocytochemical and cell fractionation analyses of the endosperm of maturing castor bean seed showed that the aspartic endopeptidase was localized in the matrix of the protein-storage vacuoles, where a variety of seed storage proteins were also present. The amount of the aspartic endopeptidase increased at the mid-maturation stage of the seeds before accumulation of the storage proteins. To determine how the aspartic endopeptidase is responsible for maturation of seed proteins in concert with the vacuolar processing enzyme, we prepared 35S-labeled proproteins of seed proteins from the endoplasmic reticulum fraction of pulse-labeled maturing endosperm and used the authentic proproteins as substrates for in vitro processing experiments. The purified aspartic endopeptidase was unable to convert any of three endosperm proproteins, pro2S albumin, proglobulin, and proricin, into their mature sizes, while the purified vacuolar processing enzyme could convert all three proproteins. We further examined the activity of aspartic endopeptidase on the cleavage of an internal propeptide of Arabidopsis pro2S albumin, which is known to be removed post-translationally. The aspartic endopeptidase cleaved the propeptide at three sites under acidic conditions. These results suggest that aspartic endopeptidase 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.

2S Albumins, Plant↗
PubMed1997

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

Vacuolar processing enzymes (VPEs) are cysteine proteinases responsible for maturation of various vacuolar proteins in plants. A larger precursor to VPE synthesized on rough endoplasmic reticulum is converted to an active enzyme in the vacuoles. In this study, a precursor to castor bean VPE was expressed in a pep4 strain of the yeast Saccharomyces cerevisiae to examine the mechanism of activation of VPE. Two VPE proteins of 59 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 of the 59 kDa VPE. The specific activity of the 46 kDa VPE was at a similar level to that of VPE in plant cells. The 46 kDa VPE instead of proteinase A mediated the conversion of procarboxypeptidase Y to the mature form. This indicates that proteinase A 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.

Amino Acid Substitution↗
PubMed1997

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

Precursor-accumulating (PAC) vesicles were previously shown to mediate the transport of the precursor of a major storage protein (pro2S albumin) to protein-storage vacuoles in developing pumpkin cotyledons. In this study, 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 pro2S albumin. PV72 was predicted to be a type I integral membrane protein with epidermal growth factor (EGF)-like motifs. These results suggest that PV72 and PV82 are potential sorting receptors for 2S albumin to protein-storage vacuoles.

2S Albumins, Plant↗
PubMed1997

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) has been implicated in cotranslational folding of nascent polypeptides, and in the recognition and disposal of aberrant polypeptides. 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. Immunoblot analysis with specific antibodies raised against the purified BiP showed that the amount of BiP in a cotyledon 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.

Amino Acid Sequence↗
PubMed1997

Heterologous expression and subcellular localization of pumpkin seed tonoplast intrinsic proteins (TIP) in yeast cells.

Two tonoplast intrinsic proteins (TIP) 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 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.

Cloning, Molecular↗
PubMed1997

beta-Glucosidase in the indigo plant: intracellular localization and tissue specific expression in leaves.

beta-Glucosidase of indigo plant (Polygonum tinctorium) has a high substrate specificity for indican (indoxyl beta-D-glucoside). To examine the localization of this beta-glucosidase, we fractionated the cells of the leaves and analysed them immunocytochemically. Immunoelectron micrographs with specific antibodies against the beta-glucosidase clearly showed that the beta-glucosidase was localized in the stroma of the chloroplasts in mesophyll cells, but not in the thylakoid membrane. Chloroplasts were isolated from the crude homogenate of the fresh leaves by Percoll density gradient centrifugation and then subjected to suborganellar fractionation. beta-Glucosidase activity was specifically detected in the stromal fraction, but not in the thylakoid membrane. This was also supported by the result of an immunoblot of the fraction with anti-beta-glucosidase antibodies. The beta-glucosidase was immunocytochemically localized in the chloroplasts of mesophyll cells, but not in any chloroplasts in marginal cells of the vascular bundle or epidermal cells; ribulose 1,5-bisphosphate carboxylase (Rubisco), a typical stromal protein, was observed in all chloroplasts in these cells. These results suggest that beta-glucosidase is tissue specific in its expression in the leaves of the indigo plant.

Plant Leaves↗
PubMed1996

Isolation and characterization of a cDNA encoding mitochondrial chaperonin 10 from Arabidopsis thaliana by functional complementation of an Escherichia coli groES mutant.

Chaperonin (Cpn) is one of the molecular chaperones. Cpn10 is a co-factor of Cpn60, which regulates Cpn60-mediated protein folding. It is known that Cpn10 is located in mitochondria and chloroplasts in plant cells. The Escherichia coli homologue of Cpn10 is called GroES. A cDNA for the Cpn10 homologue was isolated 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 Cpn 10 homologue was localized in mitochondria and accumulated at a high level in transgenic tobaccos.

Amino Acid Sequence↗
PubMed1995

Changes in physical properties of vacuolar membrane during transformation of protein bodies into vacuoles in germinating pumpkin seeds.

Changes in membrane molecular dynamics associated with the transformation of protein body membranes into vacuolar membranes during pumpkin seed germination, were monitored by EPR-spin probe technique. Using highly purified membrane preparations as well as 5-SASL and 16-SASL spin labels, parameters like general membrane lipid fluidity, order parameter, semicone angle, rotational correlation times tau 2B and tau 2C, ratio of immobilized to mobile lipids were determined and the activation energy for rotational diffusion of 16-SASL was calculated. Analysis of these parameters at different temperatures indicated a more rigid nature of protein body membrane comparing to vacuolar membrane, as a result of a more restricted motional freedom of lipids. These differences are discussed in terms of protein composition and various functional specialization of both types of membranes.

Cyclic N-Oxides↗
PubMed1995

Homologues of a vacuolar processing enzyme that are expressed in different organs in Arabidopsis thaliana.

Vacuolar processing enzymes (VPEs) are responsible for the maturation of seed proteins. These processing enzymes belong to a novel group of cysteine proteinases with molecular masses of 37 to 39 kDa. We isolated two genes of VPEs from a genomic library of Arabidopsis. The gene products were designated alpha-VPE and beta-VPE, and they were 56% identical in terms of amino acid sequence. The amino acid sequences of alpha-VPE and beta-VPE were also 55% and 67% identical to that of castor bean VPE, respectively. The gene for alpha-VPE had 7 introns, while that of beta-VPE had 8 introns. Northern blot analysis revealed that alpha-VPE is expressed in rosette leaves, cauline leaves and stems of Arabidopsis, while beta-VPE is predominantly expressed in the flowers and buds. Neither alpha-VPE nor beta-VPE is expressed in the siliques. This result strongly suggests that the isolated genes encode isozymes of VPE that are specific to vegetative organs.

Amino Acid Sequence↗
PubMed1995

Characterization of two integral membrane proteins located in the protein bodies of pumpkin seeds.

Two integral membrane proteins, MP28 and MP23, were found in protein bodies isolated from pumpkin (Cucurbita sp.) seeds. Molecular characterization revealed that both MP28 and MP23 belong to the seed TIP (tonoplast intrinsic protein) subfamily. The predicted 29 kDa precursor to includes six putative membrane-spanning domains, and the loop between the first and second transmembrane domains is larger than that of MP28. The N-terminal sequence of the mature MP23 starts from residue 66 in the first loop, indicating that an N-terminal 7 kDa fragment that contains one transmembrane domain is post-translationally removed. During maturation of pumpkin seeds, mRNAs for MP28 and MP23 became detectable in cotyledons at the early stage, and their levels increased slightly until a rapid decrease occurred at the late stage. This is consistent with the accumulation of the 29 kDa precursor and MP28 in the cotyledons at the early stage. By contrast, MP23 appeared at the late stage simultaneously with the disappearance of the 29 kDa precursor. Thus, it seems possible that the conversion of the 29 kDa precursor to the mature MP23 might occur in the vacuoles after the middle stage of seed maturation. Both proteins were localized immunocytochemically on the membranes of the vacuoles at the middle stage and the protein bodies at the late stage. These results suggest that both MP28 and the 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. These two TIPs might have a specific function during the maturation of pumpkin seeds.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of proteins in protein-body membrane that disappear most rapidly during transformation of protein bodies into vacuoles.

During the post-germination growth of seeds, protein bodies fuse with one another and are converted to a central vacuole. To investigate this transition, protein-body membranes from dry seeds of pumpkin (Cucurbita sp.) were prepared and their protein components characterized. Five major proteins (designated MP23, MP27, MP28, MP32 and MP73) were detected in the protein-body membranes. A cDNA clone encoding both MP27 and MP32 has been isolated. The deduced precursor polypeptide was composed of a hydrophobic signal sequence, MP27 and MP32, in that order. A putative site of cleavage between MP27 and MP32 was located on the COOH-terminal side of asparagine 278, an indication that the post-translational cleavage may occur by the action of a vacuolar processing enzyme that converts proprotein precursors of seed proteins into the mature forms. Immunoelectron microscopic analysis showed that MP27 and MP32 were associated with protein-body membrane of dry pumpkin seeds. Among the five membrane proteins, MP27 and MP32 disappeared most rapidly during seedling growth. The degradation of MP27 and MP32 starts just after seed germination and proceeds in parallel with the transformation of the protein bodies into a vacuole.

Amino Acid Sequence↗
PubMed1995

The sequence and expression of the gamma-VPE gene, one member of a family of three genes for vacuolar processing enzymes in Arabidopsis thaliana.

Vacuolar processing enzymes (VPEs) are responsible for the maturation of seed proteins. Southern blot analysis showed that a family of genes for VPEs in Arabidopsis thaliana was composed of three genes, for alpha-VPE, beta-VPE and gamma-VPE, respectively. The gene for gamma-VPE was isolated from a genomic library. It was composed of nine exons and eight introns. The positions of the introns were fully conserved among the three genes, with the exception that the alpha-VPE gene was missing the fifth intron found in the beta-VPE and gamma-VPE genes. The predicted gamma-VPE protein was 80% and 57% identical in terms of amino acid sequence to the alpha-VPE protein and beta-VPE protein, respectively. Northern blot analysis demonstrated that the gamma-VPE gene was expressed predominantly in the stems, with a lower level of expression in rosette and cauline leaves. However, the expression was not detected in roots, flowers plus buds, or green siliques, in contrast to the high-level expression of the beta-VPE gene in the flowers plus buds. Thus, gamma-VPE seems to be an isoform that is specific to vegetative organs. 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, such as leaves and stems. The members of the seed subfamily might function in the protein-storage vacuoles of seeds, while those of the vegetable subfamily might function in the lytic vacuoles of non-storage organs.

Amino Acid Sequence↗
PubMed1994

Molecular characterization and functional expression of squid retinal-binding protein. A novel species of hydrophobic ligand-binding protein.

The primary structure of squid retinal-binding protein (RALBP) was determined by cDNA and protein sequencing. Squid RALBP contains 342 amino acid residues in a single N-terminal-blocked chain with a molecular weight of 39,111. The N alpha-blocking group was identified as an acetyl moiety by mass spectrometry. The amino acid sequence revealed that the protein is highly hydrophilic and acidic, but it has several hydrophobic regions that are located mainly in the middle part of the polypeptide chain. It is also predicted that these hydrophobic regions form beta-sheet structures. The primary structure of RALBP is, however, quite distinct from those of other retinoid-binding proteins, showing that squid RALBP is a novel hydrophobic ligand-binding protein that functions in intracellular retinoid transport. Using the cloned cDNA, squid RALBP was expressed in vitro. By carrying out the translation at 20 degrees C in reticulocyte lysates, the protein having retinol binding activity was produced.

Amino Acid Sequence↗