Open AccessJournal Article
Multiple functional proteins are produced by cleaving asn-gln bonds of a single precursor by vacuolar processing enzyme
TLDR
The results suggested that VPE was responsible for cleaving Asn-Gln bonds of a single precursor, PV100, to produce multiple seed proteins, and it is likely that the Asn -Gln stretches not only provide cleavage sites for VPE but also produce aminopeptidase-resistant proteins.Abstract:
Precursor-accumulating vesicles mediate transport of the precursors of seed proteins to protein storage vacuoles in maturing pumpkin seeds. We isolated the precursor-accumulating vesicles and characterized a 100-kDa component (PV100) of the vesicles. Isolated cDNA for PV100 encoded a 97,310-Da protein that was composed of a hydrophobic signal peptide and the following three domains: an 11-kDa Cys-rich domain with four CXXXC motifs, a 34-kDa Arg/Glu-rich domain composed of six homologous repeats, and a 50-kDa vicilin-like domain. Both immunocytochemistry and immunoblots with anti-PV100 antibodies showed that <10-kDa proteins and the 50-kDa vicilin-like protein were accumulated in the vacuoles. To identify the mature proteins derived from PV100, soluble proteins of the vacuoles were separated, and their molecular structures were determined. Mass spectrometry and peptide sequencing showed that two Cys-rich peptides, three Arg/Glu-rich peptides, and the vicilin-like protein were produced by cleaving Asn-Gln bonds of PV100 and that all of these proteins had a pyroglutamate at their NH2 termini. To clarify the cleavage mechanism, in vitro processing of PV100 was performed with purified vacuolar processing enzyme (VPE). Taken together, these results suggested that VPE was responsible for cleaving Asn-Gln bonds of a single precursor, PV100, to produce multiple seed proteins. It is likely that the Asn-Gln stretches not only provide cleavage sites for VPE but also produce aminopeptidase-resistant proteins. We also found that the Cys-rich peptide functions as a trypsin inhibitor. Our findings suggested that PV100 is converted into different functional proteins, such as a proteinase inhibitor and a storage protein, in the vacuoles of seed cells.read more
Citations
More filters
Journal ArticleDOI
Subcellular targeting and biosynthesis of cyclotides in plant cells
TL;DR: The Vacuolar location of cyclotides supports the hypothesis that the vacuolar processing enzyme, asparaginyl endoproteinase, has a pivotal role in excision and cyclization from cyclotide precursors.
Journal ArticleDOI
Novel antifungal α-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and evolution
Anna A. Slavokhotova,Eugene A. Rogozhin,A. K. Musolyamov,Yaroslav A. Andreev,Peter B. Oparin,Antonina A. Berkut,Alexander A. Vassilevski,Tsezi A. Egorov,Eugene V. Grishin,Tatyana I. Odintsova +9 more
TL;DR: Sm-AMP-X is a new promising candidate for engineering disease resistance in plants and is assigned to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C1–C4 and C2–C3.
Journal ArticleDOI
A Novel Membrane Protein That Is Transported to Protein Storage Vacuoles via Precursor-Accumulating Vesicles
Naoto Mitsuhashi,Naoto Mitsuhashi,Yasuko Hayashi,Yasuko Koumoto,Tomoo Shimada,Tomoko Fukasawa-Akada,Mikio Nishimura,Ikuko Hara-Nishimura +7 more
TL;DR: Results suggest that PAC vesicles might mediate the delivery of not only storage proteins but also membrane proteins of the vacuoles.
Journal ArticleDOI
Seed storage albumins: biosynthesis, trafficking and structures.
TL;DR: This review will cover the subcellular movement, biochemical processing and mature structures of seed storage napins.
Journal ArticleDOI
Cloning and characterization of a novel peanut allergen Ara h 3 isoform displaying potentially decreased allergenicity
I.-H. Kang,M. Gallo +1 more
TL;DR: Results indicate that a new peanut arachin gene that encodes an expressed protein in peanut seed, Ara h 3-im, has potentially lower allergenic properties than previously characterized peanut allergens which may aid in the production of a hypoallergenic peanut.
References
More filters
Journal ArticleDOI
A simple method for displaying the hydropathic character of a protein
Jack Kyte,Russell F. Doolittle +1 more
TL;DR: A computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence has been devised and its simplicity and its graphic nature make it a very useful tool for the evaluation of protein structures.
Journal ArticleDOI
Improved tools for biological sequence comparison.
TL;DR: Three computer programs for comparisons of protein and DNA sequences can be used to search sequence data bases, evaluate similarity scores, and identify periodic structures based on local sequence similarity.
Journal ArticleDOI
Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
H. Schägger,G. von Jagow +1 more
TL;DR: A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for the separation of proteins in the range from 1 to 100 kDa is described, and the omission of glycine and urea prevents disturbances which might occur in the course of subsequent amino acid sequencing.
Journal ArticleDOI
A new method for predicting signal sequence cleavage sites.
TL;DR: A new method for identifying secretory signal sequences and for predicting the site of cleavage between a signal sequence and the mature exported protein is described.
Journal ArticleDOI
A plant vacuolar protease, VPE, mediates, virus-induced hypersensitive cell death
Noriyuki Hatsugai,Miwa Kuroyanagi,Kenji Yamada,Kenji Yamada,Tetsuo Meshi,Shinya Tsuda,Maki Kondo,Mikio Nishimura,Mikio Nishimura,Ikuko Hara-Nishimura +9 more
TL;DR: VPE deficiency prevented virus-induced hypersensitive cell death in tobacco plants and showed that plants have evolved a regulated cellular suicide strategy that, unlike PCD of animals, is mediated by VPE and the cellular vacuole.