title: Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble chlorophyll-binding protein (WSCP) from Virginia pepperweed (Lepidium virginicum), a unique WSCP that preferentially binds chlorophyll b in vitro.
abstract: Various plants possess non-photosynthetic, hydrophilic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins (WSCPs). WSCPs are categorized into two classes; Class I (photoconvertible type) and Class II (non-photoconvertible type). Among Class II WSCPs, only Lepidium virginicum WSCP (LvWSCP) exhibits a low Chl a/b ratio compared with that found in the leaf. Although the physicochemical properties of LvWSCP have been characterized, its molecular properties have not yet been documented. Here, we report the characteristics of the LvWSCP gene, the biochemical properties of a recombinant LvWSCP, and the intracellular localization of LvWSCP. The cloned LvWSCP gene possesses a 669-bp open reading frame. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis revealed that the precursor of LvWSCP contains both N- and C-terminal extension peptides. RT-PCR analysis revealed that LvWSCP was transcribed in various tissues, with the levels being higher in developing tissues. A recombinant LvWSCP and hexa-histidine fusion protein (LvWSCP-His) could remove Chls from the thylakoid in aqueous solution and showed an absorption spectrum identical to that of native LvWSCP. Although LvWSCP-His could bind both Chl a and Chl b, it bound almost exclusively to Chl b when reconstituted in 40 % methanol. To clarify the intracellular targeting functions of the N- and C-terminal extension peptides, we constructed transgenic Arabidopsis thaliana lines expressing the Venus protein fused with the LvWSCP N- and/or C-terminal peptides, as well as Venus fused at the C-terminus of LvWSCP. The results showed that the N-terminal peptide functioned in ER body targeting, while the C-terminal sequence did not act as a trailer peptide.
authors.affiliation: Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan.
authors.forename: Shigekazu
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan.
authors.forename: Haruna
authors.initials: H
authors.lastname: Yanai
authors.affiliation: Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Oka-Takayama
authors.affiliation: Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Zanma-Sohtome
authors.affiliation: Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan.
authors.forename: Kosaku
authors.initials: K
authors.lastname: Fujiyama
authors.affiliation: Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Uchida
authors.affiliation: Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Nakayama
authors.affiliation: Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan. hsatoh@biomol.sci.toho-u.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Satoh
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002734:Chlorophyll / Q000378:metabolism; D000077194:Chlorophyll A; D061146:Chlorophyll Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017343:Genes, Plant; D029686:Lepidium / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D030821:Plants, Genetically Modified; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility
chemical_list: D061146:Chlorophyll Binding Proteins; D018744:DNA, Plant; D011993:Recombinant Fusion Proteins; D002734:Chlorophyll; C037184:chlorophyll b; D000077194:Chlorophyll A
keywords: Chlorophyll scavenger; Class II WSCP; Endoplasmic reticulum body (ER body); Lepidium; Water-soluble chlorophyll-binding protein (WSCP)