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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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Related in: MedlinePlus

Protein profiles of sucrose binding protein1 during mung bean seed development and germination. Total proteins were isolated from various stages of developing (A) or germinating (B) mung bean seeds as indicated, followed by protein separation via SDS-PAGE and Western blot analysis with anti-VrSBP1a, anti-VrSBP1b, and anti-8S globulin antibodies, as indicated. DAF, days after flowering; M, mature dry seed; D, days after imbibitions.
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fig2: Protein profiles of sucrose binding protein1 during mung bean seed development and germination. Total proteins were isolated from various stages of developing (A) or germinating (B) mung bean seeds as indicated, followed by protein separation via SDS-PAGE and Western blot analysis with anti-VrSBP1a, anti-VrSBP1b, and anti-8S globulin antibodies, as indicated. DAF, days after flowering; M, mature dry seed; D, days after imbibitions.

Mentions: To determine whether the VrSBP1 proteins would display characteristics of storage proteins during seed development and germination, Western blot analysis was performed next with VrSBP1 antibodies on total proteins extracted at various stages from developing mung bean seeds or at various times from germinating seeds. As a control, antibodies for the mung bean major storage protein 8S globulin were also used. As shown in Fig. 2A, little VrSBP1 protein was detected in very young seeds at 8 d after flowering, but the amounts of VrSBP1 proteins as detected by either VrSBP1a or VrSBP1b antibodies gradually increased between 10–16 d after flowering and levelled off thereafter at amounts comparable to those found in mature seeds. Similar patterns of protein accumulation were also observed for the 8S globulin storage proteins during mung bean seed development (Fig. 2A). By contrast, the levels of both VrSBP1 and 8S globulin proteins gradually decreased upon seed germination from day 0 to day 3 after seed imbibitions (Fig. 2B). At day 4 after seed germination, the detectable levels of VrSBP1 or 8S globulin proteins had fallen dramatically (Fig. 2B).


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)

Protein profiles of sucrose binding protein1 during mung bean seed development and germination. Total proteins were isolated from various stages of developing (A) or germinating (B) mung bean seeds as indicated, followed by protein separation via SDS-PAGE and Western blot analysis with anti-VrSBP1a, anti-VrSBP1b, and anti-8S globulin antibodies, as indicated. DAF, days after flowering; M, mature dry seed; D, days after imbibitions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Protein profiles of sucrose binding protein1 during mung bean seed development and germination. Total proteins were isolated from various stages of developing (A) or germinating (B) mung bean seeds as indicated, followed by protein separation via SDS-PAGE and Western blot analysis with anti-VrSBP1a, anti-VrSBP1b, and anti-8S globulin antibodies, as indicated. DAF, days after flowering; M, mature dry seed; D, days after imbibitions.
Mentions: To determine whether the VrSBP1 proteins would display characteristics of storage proteins during seed development and germination, Western blot analysis was performed next with VrSBP1 antibodies on total proteins extracted at various stages from developing mung bean seeds or at various times from germinating seeds. As a control, antibodies for the mung bean major storage protein 8S globulin were also used. As shown in Fig. 2A, little VrSBP1 protein was detected in very young seeds at 8 d after flowering, but the amounts of VrSBP1 proteins as detected by either VrSBP1a or VrSBP1b antibodies gradually increased between 10–16 d after flowering and levelled off thereafter at amounts comparable to those found in mature seeds. Similar patterns of protein accumulation were also observed for the 8S globulin storage proteins during mung bean seed development (Fig. 2A). By contrast, the levels of both VrSBP1 and 8S globulin proteins gradually decreased upon seed germination from day 0 to day 3 after seed imbibitions (Fig. 2B). At day 4 after seed germination, the detectable levels of VrSBP1 or 8S globulin proteins had fallen dramatically (Fig. 2B).

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
Related in: MedlinePlus