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A 64 kDa sucrose binding protein is membrane-associated and tonoplast-localized in developing mung bean seeds.

Wang J, Suen PK, Xu ZF, Jiang L - J. Exp. Bot. (2009)

Bottom Line: Immunogold electron microscope (EM) studies on ultra-thin sections of high-pressure freezing/frozen substituted developing mung bean cotyledons demonstrated that VrSBP1 was localized specifically to the tonoplast of the protein storage vacuole and to the limiting membrane of a novel putative prevacuolar compartment.Biochemical and subcellular fractionation studies further demonstrated that VrSBP1 proteins were membrane-associated in developing mung beans, consistent with their tonoplast localization.This study thus shows convincing evidence of tonoplast-localization of a plant SBP for its future functional characterization and provides a model of studying non-integral membrane proteins associated with the tonoplasts in plant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Centre for Cell and Development Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.

ABSTRACT
Sucrose binding proteins (SBPs) were predicted to be membrane-associated, but have been shown to localize in the lumen of protein storage vacuoles of various seeds. In this study, a new 64 kDa SBP has been identified from developing mung bean (Vigna radiata) seeds (here termed VrSBP1) via MS/MS analysis and N-terminal amino acid sequencing analysis and specific antibodies were generated using purified VrSBP1 proteins. Western blot analysis with the new VrSBP1 antibodies showed that, similar to most seed storage proteins, VrSBP1 proteins accumulated during seed development and were subsequently mobilized once the mung bean seeds germinated. Immunogold electron microscope (EM) studies on ultra-thin sections of high-pressure freezing/frozen substituted developing mung bean cotyledons demonstrated that VrSBP1 was localized specifically to the tonoplast of the protein storage vacuole and to the limiting membrane of a novel putative prevacuolar compartment. Biochemical and subcellular fractionation studies further demonstrated that VrSBP1 proteins were membrane-associated in developing mung beans, consistent with their tonoplast localization. This study thus shows convincing evidence of tonoplast-localization of a plant SBP for its future functional characterization and provides a model of studying non-integral membrane proteins associated with the tonoplasts in plant cells.

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VrSBP1 is not a major protein in developing mung bean cotyledons. Total proteins from developing mungbean cotyledons were extracted and separated by SDS-PAGE, followed by either Coomassie Brilliant Blue staining for protein visulization (lane 1) or Western blot analysis using anti-VrSBP1a (lane 2) and anti-8S-globulin antibodies (lane 3), respectively. A single asterisk and a double asterisk indicate the positions of VrSBP1 and 8S-globulin, respectively.
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fig11: VrSBP1 is not a major protein in developing mung bean cotyledons. Total proteins from developing mungbean cotyledons were extracted and separated by SDS-PAGE, followed by either Coomassie Brilliant Blue staining for protein visulization (lane 1) or Western blot analysis using anti-VrSBP1a (lane 2) and anti-8S-globulin antibodies (lane 3), respectively. A single asterisk and a double asterisk indicate the positions of VrSBP1 and 8S-globulin, respectively.

Mentions: To determine the relative protein abundance of VrSBP1, total proteins were also extracted from developing mung bean cotyledons for analysis. As shown in Fig. 11, the amount of VrSBP1 proteins, as detected and quantified in a CCB-stained protein gel (Fig. 11, lane 1, as indicated by single asterisk), only represents about 1.98% of the total seed protein, while the major storage protein 8S globulin accounts more than 70% of the total proteins in developing mung bean cotyledons (Fig. 11, lane 1, as indicated by double asterisks). Thus, VrSBP1 is unlikely to be a storage protein in developing mung bean.


A 64 kDa sucrose binding protein is membrane-associated and tonoplast-localized in developing mung bean seeds.

Wang J, Suen PK, Xu ZF, Jiang L - J. Exp. Bot. (2009)

VrSBP1 is not a major protein in developing mung bean cotyledons. Total proteins from developing mungbean cotyledons were extracted and separated by SDS-PAGE, followed by either Coomassie Brilliant Blue staining for protein visulization (lane 1) or Western blot analysis using anti-VrSBP1a (lane 2) and anti-8S-globulin antibodies (lane 3), respectively. A single asterisk and a double asterisk indicate the positions of VrSBP1 and 8S-globulin, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2651462&req=5

fig11: VrSBP1 is not a major protein in developing mung bean cotyledons. Total proteins from developing mungbean cotyledons were extracted and separated by SDS-PAGE, followed by either Coomassie Brilliant Blue staining for protein visulization (lane 1) or Western blot analysis using anti-VrSBP1a (lane 2) and anti-8S-globulin antibodies (lane 3), respectively. A single asterisk and a double asterisk indicate the positions of VrSBP1 and 8S-globulin, respectively.
Mentions: To determine the relative protein abundance of VrSBP1, total proteins were also extracted from developing mung bean cotyledons for analysis. As shown in Fig. 11, the amount of VrSBP1 proteins, as detected and quantified in a CCB-stained protein gel (Fig. 11, lane 1, as indicated by single asterisk), only represents about 1.98% of the total seed protein, while the major storage protein 8S globulin accounts more than 70% of the total proteins in developing mung bean cotyledons (Fig. 11, lane 1, as indicated by double asterisks). Thus, VrSBP1 is unlikely to be a storage protein in developing mung bean.

Bottom Line: Immunogold electron microscope (EM) studies on ultra-thin sections of high-pressure freezing/frozen substituted developing mung bean cotyledons demonstrated that VrSBP1 was localized specifically to the tonoplast of the protein storage vacuole and to the limiting membrane of a novel putative prevacuolar compartment.Biochemical and subcellular fractionation studies further demonstrated that VrSBP1 proteins were membrane-associated in developing mung beans, consistent with their tonoplast localization.This study thus shows convincing evidence of tonoplast-localization of a plant SBP for its future functional characterization and provides a model of studying non-integral membrane proteins associated with the tonoplasts in plant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Centre for Cell and Development Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.

ABSTRACT
Sucrose binding proteins (SBPs) were predicted to be membrane-associated, but have been shown to localize in the lumen of protein storage vacuoles of various seeds. In this study, a new 64 kDa SBP has been identified from developing mung bean (Vigna radiata) seeds (here termed VrSBP1) via MS/MS analysis and N-terminal amino acid sequencing analysis and specific antibodies were generated using purified VrSBP1 proteins. Western blot analysis with the new VrSBP1 antibodies showed that, similar to most seed storage proteins, VrSBP1 proteins accumulated during seed development and were subsequently mobilized once the mung bean seeds germinated. Immunogold electron microscope (EM) studies on ultra-thin sections of high-pressure freezing/frozen substituted developing mung bean cotyledons demonstrated that VrSBP1 was localized specifically to the tonoplast of the protein storage vacuole and to the limiting membrane of a novel putative prevacuolar compartment. Biochemical and subcellular fractionation studies further demonstrated that VrSBP1 proteins were membrane-associated in developing mung beans, consistent with their tonoplast localization. This study thus shows convincing evidence of tonoplast-localization of a plant SBP for its future functional characterization and provides a model of studying non-integral membrane proteins associated with the tonoplasts in plant cells.

Show MeSH