title: Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble Chl-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera).
abstract: A water-soluble Chl-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera), hereafter termed BoWSCP, is categorized into the Class II WSCPs (non-photoconvertible WSCPs). Previous studies on BoWSCP have focused mainly on its biochemical characterization. In this study, we cloned the cDNA encoding BoWSCP. Sequence analysis revealed that the BoWSCP gene was composed of a single exon corresponding to 654 bp of an open reading frame encoding 218 amino acid residues, including 19 residues of a deduced signal peptide targeted to the endoplasmic reticulum (ER). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of native BoWSCP revealed that the molecular mass of the subunit was 19,008.523 Da, corresponding to a mature protein of 178 amino acids, indicating the removal of 21 residues in the C-terminal region. Functional BoWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (BoWSCP-His). When BoWSCP-His was mixed with thylakoid membranes in aqueous solution, BoWSCP-His was able to remove Chls from the thylakoid membranes. The absorption spectrum of the reconstituted BoWSCP-His was identical to that of the native BoWSCP. Chl binding analyses of BoWSCP-His revealed that the BoWSCP-His bound both Chl a and Chl b with almost the same affinity in 40% methanol solution, although the native BoWSCP had a higher content of Chl a. To reveal the intracellular localization of BoWSCP, we constructed a transgenic plant expressing the fluorescent protein fused with the N-terminal deduced signal peptide of BoWSCP. The fluorescence emitted from the chimeric protein was detected in the ER body, an ER-derived compartment observed only in Brassicaceae plants.
authors.affiliation: Department of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
authors.forename: Shigekazu
authors.initials: S
authors.lastname: Takahashi
authors.forename: Haruna
authors.initials: H
authors.lastname: Yanai
authors.forename: Yurino
authors.initials: Y
authors.lastname: Nakamaru
authors.forename: Akira
authors.initials: A
authors.lastname: Uchida
authors.forename: Katsumi
authors.initials: K
authors.lastname: Nakayama
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Satoh
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001937:Brassica / Q000235:genetics*; D002734:Chlorophyll / Q000378:metabolism; D061146:Chlorophyll Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism; D017343:Genes, Plant / Q000235:genetics; D042541:Intracellular Space / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013057:Spectrum Analysis; D013347:Subcellular Fractions / Q000378:metabolism; D020524:Thylakoids / Q000378:metabolism; D014867:Water / Q000378:metabolism*
chemical_list: D061146:Chlorophyll Binding Proteins; D018076:DNA, Complementary; D014867:Water; D002734:Chlorophyll