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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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Sucrose binding proteins localized to the PSV tonoplasts of developing mung bean. Ultrathin sections prepared from high-pressure frozen/freeze-substituted developing mung bean seeds were immunogold-labelled with either SBP1a antibodies (A) or SBP1b antibodies (B), as indicated. Arrows indicate examples of gold particles on the tonoplast of protein storage vacuole (PSV). Scale bar=200 nm.
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fig3: Sucrose binding proteins localized to the PSV tonoplasts of developing mung bean. Ultrathin sections prepared from high-pressure frozen/freeze-substituted developing mung bean seeds were immunogold-labelled with either SBP1a antibodies (A) or SBP1b antibodies (B), as indicated. Arrows indicate examples of gold particles on the tonoplast of protein storage vacuole (PSV). Scale bar=200 nm.

Mentions: Immunogold EM studies were carried out next to determine the subcellular localization of VrSBP1 in developing mung bean seeds. As shown in Fig. 3, the gold particle labelling with either VrSBP1a or VrSBP1b antibodies was mainly found on the limiting membrane (tonoplast) of the PSVs (Fig. 3A, B, indicated by arrows as examples), and there was very little background labelling. Similarly, as a positive control, α-TIP antibodies also mainly labelled PSV tonoplasts in similar ultra-thin sections (Fig. 4A, examples indicated by arrows). By contrast, gold particle labelling for the 8S globulin storage proteins was found to be evenly distributed inside the PSVs (Fig. 4B, examples indicated by arrowheads).


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)

Sucrose binding proteins localized to the PSV tonoplasts of developing mung bean. Ultrathin sections prepared from high-pressure frozen/freeze-substituted developing mung bean seeds were immunogold-labelled with either SBP1a antibodies (A) or SBP1b antibodies (B), as indicated. Arrows indicate examples of gold particles on the tonoplast of protein storage vacuole (PSV). Scale bar=200 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Sucrose binding proteins localized to the PSV tonoplasts of developing mung bean. Ultrathin sections prepared from high-pressure frozen/freeze-substituted developing mung bean seeds were immunogold-labelled with either SBP1a antibodies (A) or SBP1b antibodies (B), as indicated. Arrows indicate examples of gold particles on the tonoplast of protein storage vacuole (PSV). Scale bar=200 nm.
Mentions: Immunogold EM studies were carried out next to determine the subcellular localization of VrSBP1 in developing mung bean seeds. As shown in Fig. 3, the gold particle labelling with either VrSBP1a or VrSBP1b antibodies was mainly found on the limiting membrane (tonoplast) of the PSVs (Fig. 3A, B, indicated by arrows as examples), and there was very little background labelling. Similarly, as a positive control, α-TIP antibodies also mainly labelled PSV tonoplasts in similar ultra-thin sections (Fig. 4A, examples indicated by arrows). By contrast, gold particle labelling for the 8S globulin storage proteins was found to be evenly distributed inside the PSVs (Fig. 4B, examples indicated by arrowheads).

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