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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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Identification and antibody generation of sucrose binding protein1 in developing mung bean cotyledons. (A) Developing mung bean seeds were ground in grinding solution containing sucrose for organelle protection and then subjected to continuous sucrose gradient centrifugation. Proteins from fraction 8 (about 45% sucrose) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (lane 1). Individual protein bands were then isolated for trypsin digestion and subjected to MS/MS analysis for protein identification. The identified 64 kDa sucrose binding protein1 (VrSBP1, as indicated by a single asterisk) proteins were further purified and used as antigens to raise antibodies. CBB, Coomassie Brilliant Blue. MW, molecular weight marker in kDa. (B) Western blot analysis. Lanes 2 and 3 showed Western blot analysis of total proteins from developing mung bean seeds using the two newly generated anti-SBP1a or anti-SBP1b antibodies as indicated. VrSBP1, sucrose binding protein1 of mung bean. (C) MS/MS analysis and protein identification for protein bands 1–3. (D) N-terminal amino acid sequences and protein identification for protein band 1.
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fig1: Identification and antibody generation of sucrose binding protein1 in developing mung bean cotyledons. (A) Developing mung bean seeds were ground in grinding solution containing sucrose for organelle protection and then subjected to continuous sucrose gradient centrifugation. Proteins from fraction 8 (about 45% sucrose) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (lane 1). Individual protein bands were then isolated for trypsin digestion and subjected to MS/MS analysis for protein identification. The identified 64 kDa sucrose binding protein1 (VrSBP1, as indicated by a single asterisk) proteins were further purified and used as antigens to raise antibodies. CBB, Coomassie Brilliant Blue. MW, molecular weight marker in kDa. (B) Western blot analysis. Lanes 2 and 3 showed Western blot analysis of total proteins from developing mung bean seeds using the two newly generated anti-SBP1a or anti-SBP1b antibodies as indicated. VrSBP1, sucrose binding protein1 of mung bean. (C) MS/MS analysis and protein identification for protein bands 1–3. (D) N-terminal amino acid sequences and protein identification for protein band 1.

Mentions: We were interested in the identification of various proteins from various organelle fractions of developing mung bean seeds. As shown in Fig. 1, protein samples from a fraction representing the 45% sucrose gradient contained three major protein bands (Fig. 1A, as indicated by 1–3 in lane 1). Subsequent MS/MS analysis identified all these three protein bands as the soybean GmSBP2/S64 homologues with multiple peptides matching (Pedra et al., 2000; Elmer et al., 2003) sucrose binding proteins (SBPs) (Fig. 1C). To confirm the identity of these proteins as identified by MS/MS analysis further, the 64 kDa protein band was also cut out and used for N-terminal amino acid sequencing analysis by the Edman degradation method. The obtained amino acid sequences, KKETEVAADPELKTQKHQLL, match largely with the GmSBP2/SBP S64 of soybean (Fig. 1D; see Supplementary Fig. S1 at JXB online). Therefore, both MS/MS analysis with multiple peptides matching and the N-terminal amino acid analysis of the 64 kDa protein band of mung bean strongly suggest that it is a homologue of the soybean GmSBP2/S64. These three newly identified SBPs were therefore provisionally named VrSBP1, VrSBP2, and VrSBP3, respectively, as the possibility cannot be ruled out that VrSBP2 and VrSBP3 are the degraded products of VrSBP1. However, any uncertainty in this respect does not affect the findings of this study, which deliberately focused on an investigation of VrSBP1.


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)

Identification and antibody generation of sucrose binding protein1 in developing mung bean cotyledons. (A) Developing mung bean seeds were ground in grinding solution containing sucrose for organelle protection and then subjected to continuous sucrose gradient centrifugation. Proteins from fraction 8 (about 45% sucrose) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (lane 1). Individual protein bands were then isolated for trypsin digestion and subjected to MS/MS analysis for protein identification. The identified 64 kDa sucrose binding protein1 (VrSBP1, as indicated by a single asterisk) proteins were further purified and used as antigens to raise antibodies. CBB, Coomassie Brilliant Blue. MW, molecular weight marker in kDa. (B) Western blot analysis. Lanes 2 and 3 showed Western blot analysis of total proteins from developing mung bean seeds using the two newly generated anti-SBP1a or anti-SBP1b antibodies as indicated. VrSBP1, sucrose binding protein1 of mung bean. (C) MS/MS analysis and protein identification for protein bands 1–3. (D) N-terminal amino acid sequences and protein identification for protein band 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Identification and antibody generation of sucrose binding protein1 in developing mung bean cotyledons. (A) Developing mung bean seeds were ground in grinding solution containing sucrose for organelle protection and then subjected to continuous sucrose gradient centrifugation. Proteins from fraction 8 (about 45% sucrose) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (lane 1). Individual protein bands were then isolated for trypsin digestion and subjected to MS/MS analysis for protein identification. The identified 64 kDa sucrose binding protein1 (VrSBP1, as indicated by a single asterisk) proteins were further purified and used as antigens to raise antibodies. CBB, Coomassie Brilliant Blue. MW, molecular weight marker in kDa. (B) Western blot analysis. Lanes 2 and 3 showed Western blot analysis of total proteins from developing mung bean seeds using the two newly generated anti-SBP1a or anti-SBP1b antibodies as indicated. VrSBP1, sucrose binding protein1 of mung bean. (C) MS/MS analysis and protein identification for protein bands 1–3. (D) N-terminal amino acid sequences and protein identification for protein band 1.
Mentions: We were interested in the identification of various proteins from various organelle fractions of developing mung bean seeds. As shown in Fig. 1, protein samples from a fraction representing the 45% sucrose gradient contained three major protein bands (Fig. 1A, as indicated by 1–3 in lane 1). Subsequent MS/MS analysis identified all these three protein bands as the soybean GmSBP2/S64 homologues with multiple peptides matching (Pedra et al., 2000; Elmer et al., 2003) sucrose binding proteins (SBPs) (Fig. 1C). To confirm the identity of these proteins as identified by MS/MS analysis further, the 64 kDa protein band was also cut out and used for N-terminal amino acid sequencing analysis by the Edman degradation method. The obtained amino acid sequences, KKETEVAADPELKTQKHQLL, match largely with the GmSBP2/SBP S64 of soybean (Fig. 1D; see Supplementary Fig. S1 at JXB online). Therefore, both MS/MS analysis with multiple peptides matching and the N-terminal amino acid analysis of the 64 kDa protein band of mung bean strongly suggest that it is a homologue of the soybean GmSBP2/S64. These three newly identified SBPs were therefore provisionally named VrSBP1, VrSBP2, and VrSBP3, respectively, as the possibility cannot be ruled out that VrSBP2 and VrSBP3 are the degraded products of VrSBP1. However, any uncertainty in this respect does not affect the findings of this study, which deliberately focused on an investigation of VrSBP1.

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