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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 membrane-associated. The P100 vesicle fraction from developing mung bean cotyledons were first resuspended in solutions containing one of the three chemicals (0.1 M Na2CO3, 1% Triton X-100, and 1% SDS), as indicated, and incubated for 1 h at 4 °C, followed by centrifugation at 100 000 g for 1 h, resulting in pellet (P) and supernatant (S). The pellet (P) was resolubilized in the same volume of extraction solution containing 1.5% SDS and 150 mM NaCl. Equal volumes of protein samples were separated by SDS-PAGE, followed by Western blot analysis using anti-VrSBPa (A), anti-VSRat-1 (B), and anti-α-TIP (C), as indicated. Asterisks indicate the position of the detected proteins. The signals of the detected protein bands were further quantified with the software Quantity One (Bio-Rad) to determine the percentages of protein distribution between the pellet (P) and supernatant (S) in each treatment. MW, molecular weight marker in kDa.
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fig9: VrSBP1 is membrane-associated. The P100 vesicle fraction from developing mung bean cotyledons were first resuspended in solutions containing one of the three chemicals (0.1 M Na2CO3, 1% Triton X-100, and 1% SDS), as indicated, and incubated for 1 h at 4 °C, followed by centrifugation at 100 000 g for 1 h, resulting in pellet (P) and supernatant (S). The pellet (P) was resolubilized in the same volume of extraction solution containing 1.5% SDS and 150 mM NaCl. Equal volumes of protein samples were separated by SDS-PAGE, followed by Western blot analysis using anti-VrSBPa (A), anti-VSRat-1 (B), and anti-α-TIP (C), as indicated. Asterisks indicate the position of the detected proteins. The signals of the detected protein bands were further quantified with the software Quantity One (Bio-Rad) to determine the percentages of protein distribution between the pellet (P) and supernatant (S) in each treatment. MW, molecular weight marker in kDa.

Mentions: As shown in Fig. 9, after the P100 fraction was treated with 0.1 M Na2CO3 more than 87% of the VrSBP1 proteins were released from membrane association (Fig. 9A, lane 2) into the soluble fraction (Fig. 9A, lanes 3 versus 4), indicating that Na2CO3 was effective in disassociating VrSBP1 from the membrane. A similar result was obtained when the P100 fraction was treated with 1% Triton X-100 (Fig. 9A, lanes 5 and 6), whereas treatment of 1% SDS caused the nearly complete release of VrSBP1 into the soluble fraction (Fig. 9A, lanes 7 and 8). By contrast, when the same P100 fraction was treated with 0.1 M Na2CO3, followed by detection using either VSR or α-TIP antibodies, neither VSR nor α-TIP protein was released from the membrane because no protein was detected in the soluble fraction after 0.1 M Na2CO3 treatment (Fig. 9B, C, lane 3). However, both 1% Triton X-100 and 1% SDS treatments caused VSR or α-TIP to be released from the membrane (Figs 9B and 9C, lanes 5–8). These results strongly indicate that VrSBP1 proteins in mung bean are membrane-associated and are different from the integral membrane proteins of VSR or α-TIP.


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 membrane-associated. The P100 vesicle fraction from developing mung bean cotyledons were first resuspended in solutions containing one of the three chemicals (0.1 M Na2CO3, 1% Triton X-100, and 1% SDS), as indicated, and incubated for 1 h at 4 °C, followed by centrifugation at 100 000 g for 1 h, resulting in pellet (P) and supernatant (S). The pellet (P) was resolubilized in the same volume of extraction solution containing 1.5% SDS and 150 mM NaCl. Equal volumes of protein samples were separated by SDS-PAGE, followed by Western blot analysis using anti-VrSBPa (A), anti-VSRat-1 (B), and anti-α-TIP (C), as indicated. Asterisks indicate the position of the detected proteins. The signals of the detected protein bands were further quantified with the software Quantity One (Bio-Rad) to determine the percentages of protein distribution between the pellet (P) and supernatant (S) in each treatment. MW, molecular weight marker in kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: VrSBP1 is membrane-associated. The P100 vesicle fraction from developing mung bean cotyledons were first resuspended in solutions containing one of the three chemicals (0.1 M Na2CO3, 1% Triton X-100, and 1% SDS), as indicated, and incubated for 1 h at 4 °C, followed by centrifugation at 100 000 g for 1 h, resulting in pellet (P) and supernatant (S). The pellet (P) was resolubilized in the same volume of extraction solution containing 1.5% SDS and 150 mM NaCl. Equal volumes of protein samples were separated by SDS-PAGE, followed by Western blot analysis using anti-VrSBPa (A), anti-VSRat-1 (B), and anti-α-TIP (C), as indicated. Asterisks indicate the position of the detected proteins. The signals of the detected protein bands were further quantified with the software Quantity One (Bio-Rad) to determine the percentages of protein distribution between the pellet (P) and supernatant (S) in each treatment. MW, molecular weight marker in kDa.
Mentions: As shown in Fig. 9, after the P100 fraction was treated with 0.1 M Na2CO3 more than 87% of the VrSBP1 proteins were released from membrane association (Fig. 9A, lane 2) into the soluble fraction (Fig. 9A, lanes 3 versus 4), indicating that Na2CO3 was effective in disassociating VrSBP1 from the membrane. A similar result was obtained when the P100 fraction was treated with 1% Triton X-100 (Fig. 9A, lanes 5 and 6), whereas treatment of 1% SDS caused the nearly complete release of VrSBP1 into the soluble fraction (Fig. 9A, lanes 7 and 8). By contrast, when the same P100 fraction was treated with 0.1 M Na2CO3, followed by detection using either VSR or α-TIP antibodies, neither VSR nor α-TIP protein was released from the membrane because no protein was detected in the soluble fraction after 0.1 M Na2CO3 treatment (Fig. 9B, C, lane 3). However, both 1% Triton X-100 and 1% SDS treatments caused VSR or α-TIP to be released from the membrane (Figs 9B and 9C, lanes 5–8). These results strongly indicate that VrSBP1 proteins in mung bean are membrane-associated and are different from the integral membrane proteins of VSR or α-TIP.

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