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Keywords:

  • dense vesicles;
  • Golgi apparatus;
  • legumin;
  • pea;
  • protein storage vacuole;
  • protein targeting;
  • sucrose-binding-protein

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

Developing pea seeds contain two functionally distinct vacuoles – lytic vacuoles and protein storage vacuoles (PSV). The Golgi apparatus of these cells has to discriminate between proteins destined for these vacuolar compartments. Whereas it is known that sorting into the lytic vacuole is performed via the conserved clathrin-coated vesicle pathway, sorting of proteins into the protein storage vacuole remains enigmatic. In developing pea cotyledons, the major storage proteins are sorted via ‘dense vesicles’. In this report we examined the sorting of a minor protein of the protein storage vacuole, the sucrose-binding-protein homolog (SBP), along the secretory pathway employing immunoelectron microscopy on cryosectioned pea cotyledons. SBP follows the same vesicular route into the PSV as the main storage proteins legumin and vicilin, via the dense-vesicles. Furthermore, legumin and SBP are sorted together into the same dense vesicle population at the stack. Although soluble cargo proteins of the dense vesicles, they show a stratified distribution in the lumen of the dense vesicles. Whereas the legumin label is equally distributed across the lumen, the SBP label is concentrated at the membrane of the vesicle. This observation is discussed with respect to a putative receptor-mediated sorting of the proteins into the dense vesicles.

Plant cells may contain functionally distinct vacuoles in the same cell at some time during their life cycle (1). Because most vacuolar proteins are sorted via the Golgi apparatus, the Golgi apparatus of these cells has to discriminate between the different types of vacuolar cargo vesicles in order to avoid mistargeting. Accordingly, two different transport pathways that sort vacuolar proteins in plant cells have so far been described. One, the clathrin-coated vesicle (CCV)-dependent pathway, is common to all eukaryotic cells and transports vacuolar acid hydrolases into the lytic vacuole (2). Both the vacuolar sorting signals in the cargo proteins and the vacuolar sorting receptors for this pathway have been identified and characterized (3,4). The second, the so-called dense vesicle (DV)-mediated pathway, seems to be unique to plants and is responsible for the transport of storage proteins into the protein storage vacuole in seeds (5).

Despite the importance of storage protein deposition for human nutrition, the mechanism underlying the formation of DV is only poorly understood. DV are clearly distinguishable from CCV. Mature DV do not have a clathrin coat (6), nor do they contain the vacuolar sorting receptor for vacuolar acid hydrolases (7). Sorting of unglycosylated storage proteins, like legumin, the major storage protein in pea seeds, into DV starts at the cis- and not at the trans-Golgi cisternae (8). Several lines of evidence indicate that this is an ongoing process, and that the DV remain attached to the cisternae as they mature. Firstly, DV show an increasing degree of electron opacity in the cis to the trans direction, reflecting a gradual filling with osmiophilic protein cargo (9). Secondly, DV at the trans-Golgi network (TGN) label with antibodies against complex glycosylated proteins, whereby the transfer of xylose and fucose residues is known to be a property of the median and trans cisternae of the Golgi stack (10). Thirdly, although sorting of legumin already occurs in the cis cisternae of the Golgi stack, legumin-containing DV are present at all Golgi cisternae (6). Finally, CCV form at the surface of DV prior to their release from the TGN, suggesting either removal of mistargeted proteins or the recycling of sorting receptors (6,7,9).

Until now, only the sorting of the two major storage globulins of pea seeds, vicilin and legumin, into DV has been studied. In order to understand the formation of DV in more detail, the sorting of minor constituents of the protein storage vacuole needs to be investigated. In this report we describe the sorting of the pea sucrose binding protein homolog, which makes up only 2–4% of the total seed protein (11). This protein, of unknown function, is a close homolog of the family of sucrose-binding proteins in soybean that are structurally related to the cupin family of vacuolar storage proteins (12–14). In soybean, three members of this family, GmSBP1-3, are located in the protein storage vacuole (13). The Vicia faba SBP-homolog (VfSBPL) has been identified and cloned from a cotyledonary cDNA library (15). The protein showed an overall homology of 58% at the amino acid level and 68% at the nucleotide level with the soybean protein. It was expressed in cotyledonary tissue and localized in the protein storage vacuole. The pea sucrose binding protein homolog from germinating embryonic axes shows an overall identity of 60% with the SBP from soybean (11,16). This protein also localized to the PSV.

Here we have investigated the relative distribution of legumin and SBP within the secretory system, especially the Golgi apparatus, of developing pea cotyledon cells. In order to do so, we have used the technique of immuno-gold labeling of cryosections (17), which we have previously employed with success for the in situ localization of other DV-related proteins (7,8).

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

VfSBPL antibodies recognize pea SBP

The antibodies used in this investigation were either raised against the N-terminal 199 amino acids of the Vicia faba sucrose binding protein homolog, VfSBPL (15), or raised against the C-terminal 16 kDa peptide p16 from pea (11). As shown in Figure 1, pea and bean sucrose binding protein homologs share an overall identity of 85% at the amino acid level. The N-terminal region, against which the VfSBPL antibody was raised, was 88% identical and the C-terminal region, against which the P-16 antibody was raised, 81% identical. The soybean, VfSBPL, and SBP are soluble proteins with no membrane spanning sequences (11,13,15). Four of the five proteins have three hydrophobic amino acids at the C-terminus (peaSBP (AVV), VfSBPL (AFV), SBPGm2, and SBPGm64 (AVA)) which are similar to the C-terminal vacuolar sorting sequence of the bean storage protein phaseolin (AFVY; (18); and the soybean seed storage protein β-conglycinin α′ subunit (PLSSILRAVY; 19). In addition, all five proteins have a conserved putative N-glycosylation site at position 330.

image

Figure 1. Alignment of pea SBP. The sequence of the pea SBP was aligned to all known members of this family using the ClustalW program. Accession numbers for the sequences were as follows: Vicia faba (SBPVf): CAC27161; Pisum sativum (SBPPs): T06459; Glycine max SBPGm:Q04672; Glycine max SBPGm2: AY234869; Glycine max SBP64: AAF05723. The N-terminal part of the VfSBPL and the C-terminal part of the pea SBP against which the antiserum was raised are shaded in gray. The hydrophobic C-terminal amino acids are printed in bold letters. The conserved putative glycosylation site at position 330 is underlined. The asterisks represent identical amino acids, the double points conserved exchanges, and the single points homolog amino acids.

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To test whether the VfSBPL antibody also recognizes the pea homolog, Western blots were performed with fractions of total seed extract, Golgi, and PSV isolated from maturing pea cotyledons and the labeling pattern of the two antibodies compared. As shown in Figure 2A, the VfSBPL antibody recognized two proteins with apparent molecular masses in SDS-PAGE of about 62 kDa and 48 kDa, respectively, in the pea Golgi and PSV fractions. The 48 kDa band represents the N-terminal peptide (Figure 1). In the total seed extract only the 62 kDa protein was detected. The 62 kDa protein was also detected by the p16 antibody, which additionally labeled a 16 kDa protein, but not the 48 kDa protein (Figure 2A ). The p16 protein was also present in the PSV fraction. This 16 kDa protein represents the C-terminal peptide (Figure 1).

image

Figure 2. Comparison of the VfSBPL and P-16 antibodies. A) 20 μg of each PSV (lanes 1 and 4), Golgi (lanes 2 and 5), and total seed extract (lanes 3 and 6) were separated by SDS-PAGE, electroblotted on nitrocellulose and the blot probed with either the VfSBPL (lanes 1–3) or the P-16 (lanes 4–6) antibodies. B) Time course of SBP appearance during seed development: 20 μg of total seed extract isolated from seeds of different developmental stage was separated by SDS-PAGE, the gels electroblotted on nitrocellulose and the blot probed with the VfSBPL antibody or, as a reference, with the vicilin antibody. The developmental stages were defined by the length of the cotyledon (21). The first traces of vicilin appeared in seeds smaller than 3 mm. The first SBP with a MM of 48 kDa was detectable at the same size. This band disappeared at sizes of 3–4 mm; at 5–6 mm both SBP bands are clearly detectable. C) The PSV fraction is not contaminated with nuclei: 30 μg of total seed extract (lane 1), isolated PSV (lane 2) and isolated nuclei (lane 3) were separated by SDS-PAGE, electroblotted on nitrocellulose and the blot probed with a mouse monoclonal antibody against the Histone A1, clone AE4. The nuclei serving as a reference were isolated from tobacco leaves. The antibody characteristically recognizes a doublet of proteins in the range of 30 kDa (44). This was recognized in the nucleus fraction and in the total seed extract, but not in the fraction of isolated PSV.

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Because it is known that the expression of the seed storage proteins does not occur simultaneously during seed development, the time course of expression of SBP was examined. The expression of the vicilin polypeptides was used as a marker, because their expression precedes that of the legumins during seed development (20). Developmental stages were estimated by the length of the cotyledon (21). As shown in Figure 2B, the SBP protein appeared parallel with the vicilin. However, earlier in seed development there seemed to be transient expression of SBP, as has also been shown in Vicia faba (15).

Because p16 was reported to be localized to the nucleus isolated from germinating pea cotyledons (11,16), we tested whether the protein body fraction used was contaminated with nuclei. This was not the case (Figure 2C). In comparison with the total extract, the histone label in the PSV fraction was below the limit of detection.

To further confirm the specificity of the VfSPBL antibody against the pea homolog, Western blots of 2D-gel electrophoretically separated ER/Golgi fractions were done. Because of the poor solubility of storage pro-proteins in solubilization buffers for iso-electric focusing, 16-BAC-PAGE was performed for the first dimension (22). This method very efficiently resolved proteins showing the same apparent MM in SDS-PAGE in the first dimension (Figure 3A). The VfSBPL antibody only labeled two protein spots with molecular masses of about 62 kDa and 48 kDa (Figure 3B). The spots above 66 kDa and at the top of the gel represent SBP oligomers that were not solubilized in the 16-BAC sample buffer. On Western blots performed with 1D-SDS-PAGE no such bands were detected (Figure 2A). These data indicate that the SBP undergoes aggregation, leading to the formation of tightly associated oligomers that could not be resolved by incubation in cationic detergent.

image

Figure 3. 2D gel electrophoresis of isolated Golgi fractions. 80 μg of Golgi proteins were separated on an acidic, cationic 16-BAC gel in the first dimension. The stained lanes were then cut out of the gel and the proteins were separated on an SDS-PAGE gel in the second dimension and stained in Coomassie (A). SDS-PAGE was electroblotted on nitrocellulose and the blot probed with the VfSBPL antibody (B).

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Pea SBP is restricted to ER/Golgi and the PSV

Immuno-gold localization with VfSBPL antibodies on cryosections was performed in order to localize the SBP in the cotyledonary cells of maturing pea seeds. SBP proteins were detected in the Golgi apparatus and protein storage vacuoles, respectively (Figure 4). No labeling was detected at the plasma membrane (Figure 4e). Because soybean SBP homologs also seem to be localized at the plasma membrane (23) the labeling density at the pea plasma membrane was analyzed in more detail. On different sections, about 8 μm of plasma membrane was analyzed, but no gold-label was detected.

image

Figure 4. Distribution of SBP in developing pea cotyledons. Cryosections of developing pea cotyledons were labeled with either the VfSBPL antibody (a–e) or the pea SBP p16 antibody (f). The VfSBPL antibody labeled the Golgi apparatus (GAPP; a, b, c, e) and the protein storage vacuole (PSV; b,c,d). The plasma membrane (PM; e) was not labeled. The pea SBP p16 antibody labeled the protein storage vacuole (PSV; f) but not the nucleus (N; f). Bars are 200 nm.

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Castillo et al. (11,16), detected pea SBP in nuclei isolated from cells of germinating pea seeds. The in situ distribution of p16 in the cells of pea cotyledons was therefore examined to test whether SBP is localized to the nucleus of developing cotyledonary cells. p16 antiserum specifically labeled the PSV, but a significant labeling of the nucleus in situ with this antibody was not obtained (Figure 4f). Thus, in developing cotyledonary cells SBP seems to be a true vacuolar protein.

SBP is sorted along with legumin into DV and not into CCV

Two types of vacuolar transport vesicles, CCV and DV, were seen at the Golgi apparatus (Figure 5). In order to examine whether SBP is sorted into DV the labeling density of DV and CCV with the VfSBPL antibody was compared. As shown in Figure 5 and Table 1, CCV were not labeled. After statistical analysis of the labeling of 52 Golgi stacks with 212 attached DV and 30 attached CCV, respectively, 159 DV were labeled with 346 gold particles in all, but only 4 CCV with a total of 4 gold particles. The relative labeling density was about 1.6 GP/DV to about 0.13 GP/CCV, respectively. Presuming an average diameter of a DV of 150 nm (6) and an average diameter of 90 nm for the CCV (24), the area of a DV in cross-section would be about 2.8 times larger than that of a CCV. However, the labeling density was about 13 times higher on the DV than on CCV. It therefore seems reasonable to assume that the label on the CCV represents background label.

image

Figure 5. Labeling of dense vesicles or clathrin-coated vesicles with VfSBPL. Cryosections of developing pea cotyledons were double-labeled with the VfSBPL antibody and with the antibody against the major storage protein legumin. Clathrin-coated vesicles were not labeled with VfSBPL or with legumin (a– d). Dense vesicles were labeled with both legumin and VfSBPL. Legumin was labeled with 5 nm gold, VfSBPL with 10 nm gold. Bars are 100 nm.

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Table 1. : Distribution of the SBP-label between DV and CCV
DVCCV
DV totalDV labeledGPGP/DVCCV totalCCV labeledGPGP/CCV
  • 52 individual Golgi stacks have been evaluated.

  • *

    Number of gold points.

212159346*1.63044*0.13

Using double immunogold labeling we analyzed whether SBP is sorted into the same DV as legumin is. Although a different fixation protocol was used, the results achieved for the legumin distribution across the Golgi stack confirmed previously published findings (8): 64% of the legumin label of the stack was in the cis half of the stack, 25% in the median cisternae, and only about 10% in the trans half of the stack (31 stacks with 111/43/18 gold particles). The majority of both labels was in the DV as compared to the labeling of the stack: about 74% of the SBP and about 84% of the legumin label were in the DV and only 24% or 16% of the label, respectively, in the stack (346–111 gold particles of the SBP labeling and 937–172 gold particles of the legumin labeling, respectively). As shown in Figure 5a, SBP and legumin label the same DV. Quantitative analysis of the label is shown in Table 2. About 80% of the labeled DV contained both antigens. All of the SBP label was on DV, which were also labeled with legumin. About 20% of the DV was labeled with the legumin antibody only.

Table 2. : Double labeling of DV and Golgi stacks with SBP and legumin antisera
LabeledDV labeled withDV labeledDV labeled with
DVlegumin and SBPwith SBP onlylegumin only
11087023

Legumin and SBP show a stratified distribution across the lumen of DV

The label with VfSBPL antiserum was not evenly distributed across the lumen of the DV, but seemed to be concentrated at the membrane instead (Figure 4a–c). The same stratification was to be seen after labeling DV with the p16 antiserum (data not shown), strengthening the specificity of the signal. To test whether this could be a fixation or a labeling artifact, double labeling with the legumin antibody was performed. Legumin was equally distributed across the DV (Figures 5 and 6). Statistical analysis of the VfSBPL label of 52 Golgi stacks with 212 attached DV revealed that about 80% of the gold label was near the membrane and only about 20% across the lumen of the DV (Table 3). Legumin, by contrast, showed an equal distribution with 53% of the gold label near the membrane and 47% across the lumen of the DV (Table 3). In order to see whether this stratified distribution might be an artifact caused by steric hindrance of the VfSBPL antibody through the condensed major storage proteins, the distribution of the SBP-labeling across small compact PSV was determined as a control. In these PSV, however, the SBP label was not concentrated at the membrane: statistical analysis revealed that the label was evenly distributed across the lumen of the PSV (Table 4).

image

Figure 6. Distribution of SBP and legumin within dense vesicles. Cryosections of developing pea cotyledons were double-labeled with the VfSBPL antibody and with the antibody against the major storage protein legumin. VfSBPL equally labeled all cisternae of a Golgi stack (b). VfSBPL label was concentrated at the membrane of the dense vesicles (a–c) but not at the membrane of protein storage vacuoles (c). Legumin was equally distributed across the lumen of the dense vesicles (a–c). Legumin was labeled with 5 nm gold, VfSBPL with 10 nm gold. Bars are 200 nm.

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Table 3. : Distribution of SBP and legumin across the lumen of DV
SBP*Legumin**
MembraneLumenMembraneLumen
  • *

    212 individual DV have evaluated for the SBP label.

  • **

    118 individual DV have been evaluated for the legumin label.

  • Number of gold points

27466500437
80.6%19.4%53.4%46.6%
Table 4. : Distribution of SBP across PSV
MembraneLumen
  • 21 compact PSVs with diameters between 300 nm and 1000 nm have been evaluated.

  • *

    Number of gold points.

174*277*
38%62%

Triton X-114 solubilization experiments were performed to test the possibility that SBP, or part of the SBP, behave like membrane-associated proteins in the pea cotyledon Golgi apparatus. Fractions enriched in either Golgi (7) or PSV (6,21) membranes were solubilized in Triton X-114 at a detergent to protein ratio of 10 : 1. The Triton-insoluble proteins were then pelleted by low-spin centrifugation (5 min at 10 000 × g) and the Triton soluble proteins subjected to three times Triton X-114 hydrophobic phase partitioning (25). To mimic the slightly acidic milieu in the Golgi and the protein storage vacuole (26,27), the medium was adjusted to pH 6.0. The gel was loaded with equal volumes except for the detergent phase. Because of the small proportion of membrane protein relative to the aggregated storage proteins in the PSV, 10 times the amount of detergent phases as compared to the other fractions was loaded onto the gel. A significant amount of both the 48 and the 60 kDa were Triton-insoluble in the Golgi or in the PSV fraction (Figure 7). This Triton insolubility is in agreement with the formation of high molecular weight aggregates that were only partially soluble in the cationic detergent 16-BAC, as shown above (Figure 3). In the Golgi fraction the majority of both polypeptides partitioned into the aqueous phase, but some SBP also partitioned into the detergent phase. However, in the PSV fraction the relative amount of SBP present in the detergent phase was below the limit of detection. The partitioning of proteins between the hydrophilic and hydrophobic phases in general, as revealed by protein staining of the gel (Figure 7), confirmed that the observed partitioning of SBP between the hydrophobic and the hydrophilic phase was not due to insufficient phase-partitioning. These results may indicate that, at least in the Golgi apparatus, some of the SBP proteins are indeed recruited to the membranes.

image

Figure 7. Triton X-114 phase partitioning of SBP in either Golgi of PSV fractions. Golgi (lanes 1–5) or PSV (lanes 6–10) fractions were subjected to hydrophobic phase partitioning with Triton X-114. Equal volumes of the fractions were loaded on the gel, except for the detergent phases (lanes 5 and 10), which were 10 times the amount of the other fractions used. The gel was electroblotted on nitrocellulose and the blot probed with the VfSBPL antibody (A). To detect all proteins present, the gels were first stained in silver and then in Coomassie (B). Lane 1: Golgi fraction; lane 6: PSV fraction; lanes 2 and 7: Triton-insoluble sediment, lanes 3 and 8: Triton-soluble supernatant, lanes 4 and 9: hydrophilic upper phase; lanes 5 and 10: hydrophobic detergent phase.

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Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

Localization of SBP in parenchyma cells of developing pea cotyledons

The 60 kDa pea SBP protein is processed into two peptides, a C-terminal peptide with an apparent MM of 16 kDa (p16) and a N-terminal part with an apparent MM of about 48 kDa in SDS-PAGE. Antisera against the VfSBPL labeled the PSV and the Golgi apparatus. There was no significant label at the plasma membrane or in the nucleus. An antiserum against the C-terminal pea p16 peptide also did not label the nucleus. Therefore, SBP appears to be a true vacuolar protein in developing pea cotyledons. This localization is in accordance with data derived from other in situ immuno-localization studies of the homolog SBP proteins, VfSBPL in the faba bean (15) and GmSBP in soybean (13).

Sorting of SBP in the Golgi apparatus

The results reported here indicate that SBP is sorted in DV, that is, along the same route into the PSV as the major storage proteins, legumin and vicilin. Furthermore, SBP-labeled DV were always labeled with legumin, suggesting that there is only one population of DV, transporting all cargo proteins. Until now, no conserved vacuolar sorting signal has been identified in the primary sequence of storage proteins. Common to the pea as well as the faba bean and two of the three soybean sucrose binding protein homologs is the presence of three hydrophobic amino acid residues (AVV for the pea, AFV for the faba bean, AVA for the GmSBP2 and GmSBP3) at the C-terminus that are similar to the C-terminal vacuolar sorting sequences of the garden bean seed storage protein phaseolin (AFVY; 18) and of the soybean seed storage protein β-conglycinin α′ subunit (PLSSILRAVY; 19). One might speculate that a hydrophobic motif at the C-terminus could serve as vacuolar sorting signal. However, as neither legumin or vicilin, or the third soybean homolog, GmSBP1, possess this hydrophobic C-tail, the function of hydrophobic amino acids as a putative vacuolar sorting sequence remains to be elucidated.

Stratification of cargo within the lumen of the DV

A stratified distribution of cargo proteins within the lumen of DV cannot, in our opinion, be explained by a receptor-mediated sorting mechanism as described for CCV. The sorting of storage proteins into the vacuole has been shown to be saturable (18,28), leading to the assumption that this sorting might also depend on the interaction of storage proteins with certain receptor proteins. In accordance with this hypothesis, two membrane proteins have been identified in Arabidopsis thaliana which might play a role in this process: the so-called RMR protein (3,29), and a member of the BP-80/AtELP family, AtVSR1 (30). In the case of the CCV-mediated sorting of mammalian lysosomal hydrolases, receptors are type I membrane proteins that recruit cargo proteins with a stoichiometry of about 1 to the lumenal leaflet of the membrane of the budding CCV (2,31). Similarly, the available data on the sorting of plant vacuolar hydrolases suggest that the plant vacuolar sorting receptors may function in a similar way. Cargo proteins, like the aleurain, have two binding domains which interact simultaneously with two domains of the receptor protein to give a high affinity interaction (3,4,32).

However, the lumen of the DV is completely filled with large electron opaque protein aggregates (6) and sorting of storage proteins into these vesicles is accompanied by an increase in their aggregation state (7), which increases as the DV pass through the Golgi stack (7,9). Crystal structure analysis of the precursor of the soybean 11S globulin, glycinin, a closely related homolog of pea legumin, has revealed that the precursor trimer is arranged around a 3-fold symmetry axis with dimensions of 9.5 × 9.5 × 4.5 nm (33). Under the steric conditions present in the DV it seems very unlikely that a ligand might bind via the same mechanism to a receptor located in the membrane of the DV as to a receptor located in the membrane of CCV. It seems unlikely that every single cargo molecule is bound to a single receptor molecule, and that aggregation might instead be part of the sorting process (5,34).

The observation, made in this report, that the cargo of the DV is stratified within the lumen of the vesicle adds support to this assumption. A stratification of storage protein aggregates has been described earlier. Early in developing pea cotyledons the major storage globulins form stratified protein lumps attached to the tonoplast, correlated with the time-course of their expression (35). These results might support the hypothesis that the stratification of cargo in the lumen of the pea cotyledon DV might reflect the sequence of sorting events of the different storage proteins into the DV while they are transported from the cis- to the trans side of the Golgi stack. Legumin and nonglycosylated vicilin enter the DV at the cis-Golgi cisternae where they aggregate. After the DV reach the medium and trans cisternae of the stack, due to cisternal progression, SBP is sorted into the attached DV. Because the lumen of the DV is already filled SBP stays attached to the DV membrane. This mechanism would certainly exclude the possibility that every single cargo molecule remains attached to a single receptor in the membrane of the DV.

However, stratification might also be the result of interactions between the different storage proteins present, and these interactions in turn might be important for correct sorting. Two reports have revealed that interactions between storage proteins in the secretory pathway do indeed influence the sorting process itself. In Hordeum vulgare and in soybean deletion of one of the storage proteins abolished vacuolar sorting and led to a retention of other storage proteins in the endoplasmic reticulum (36,37). In both cases it is discussed that the deleted storage protein influences the solubility of the other storage protein partners with the deletion leading to a pristine aggregation thus inhibiting the exit out of the ER. Because it is noticeable that SBP is concentrated at the membranes of DV but not of PSV it is tempting to speculate that membrane-attached SBP might influence the aggregation behavior of other storage proteins.

It has also been discussed that sorting of storage proteins in pea morphologically resembles the sorting of regulated secretory proteins into secretory granules in mammalian glands (7,8,38). The condensation of regulated secretory proteins and the formation of immature secretory granules occur within the lumen of the trans-Golgi network. According to the so-called sorting by entry model a subpopulation of the regulated secretory proteins themselves bind to the membrane, constituting a kind of ‘nucleus’ for further aggregation of other secretory proteins (39,40). Several candidate proteins for such unconventional receptors have been described, although their function is still a matter of dispute (41,42). The results obtained by the Triton X-114 phase partitioning together with the observed distribution of SBP in the DV presented in this report might indicate that some of the SBP is recruited to the membrane of the pea cotyledon Golgi. The mechanism of this recruitment remains to be elucidated. Because its biosynthesis starts relatively early during seed development, SBP is already present at the onset of globulin expression. One might speculate that SBP might thus act as a scavenger for the storage proteins, recruiting them to the membrane of nascent DV.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

Plant material

Pea (Pisum sativum L., var. Kleine Rheinländerin) plants were grown hydroponically in a green house. Seeds with a 8–9-mm-long axis diameter (roughly 20–22 days postanthesis) were collected and the testa removed (43).

Antibodies used

Polyclonal antibodies were raised in rabbits against the VfSBPL from Vicia faba (15) and used in a dilution of 1 : 10.000 on Western Blots and 1 : 200 in immunocytochemistry. Polyclonal antibodies were raised in rabbits against the p16 from pea (11,16) and used in a dilution of 1 : 5000 on Western blots and 1 : 200 in immunochemistry. Polyclonal antibodies were raised in rabbits against the α- and β-chains of the mature pea legumin (7) and used in a dilution of 1 : 12.000 on Western blots and 1 : 200 in immunocytochemistry. Polyclonal antibodies were raised in rabbits against the pea vicilin (6) and used in a dilution of 1 : 5000 on Western blots. Anti-Histone A1, clone AE4, mouse monoclonal antibodies (44) were purchased from Upstate Cell Signaling Solutions (Lake Placid, NY) and used in a dilution of 2 μg/mL on Western blots.

Organelle isolation, preparation of total seed extracts, and hydrophobic phase partitioning with Triton X-114

Golgi-enriched fractions were isolated from pea cotyledons by isopycnic gradient centrifugation as described (7). Protein storage vacuoles were isolated from pea cotyledons as described previously (6). Total seed extracts from various stages of pea seed development were isolated as described (21). Nuclei were isolated from tobacco leaves as previously described (45).

For hydrophobic phase partitioning in Triton X-114 (25) isolated Golgi or PSV fractions, respectively, were diluted 10 times in a buffer containing 50 mm Mes/Tris buffer pH 6.0 and 150 mm NaCl, pelleted at 100 000 × g for 60 min in swing out rotor (TST 28/38, Kendro), and the sediment resolved in the same buffer at a concentration of 2 mg/mL. The membranes were solubilized in Triton X-114 for 60 min on a rotator at 4 °C at a detergent to protein ratio of 10 and in the presence of protease inhibitors (2 μg/mL leupeptin, 2 μg/mL aprotinin, 0.7 μg/mL pepstatin, and 1 mm o-phenanthroline). Triton-insoluble material was sedimented at 10 000 × g for 5 min. The Triton-soluble supernatant was subjected to hydrophobic phase partitioning by incubation at a temperature of 38 °C until the solution became turbid (for 5 min). The two phases were partitioned by subsequent centrifugation at 10 000 × g for 5 min at room temperature. To the initial concentration, detergent was added to the hydrophilic upper phase and buffer to the hydrophobic lower phase. The partitioning was repeated twice. Proteins from the thrice partitioned upper and lower phases were precipitated with CHCl3/methanol (46).

Gel electrophoresis, protein gel blotting, protein determination

Prior to SDS-PAGE under reducing conditions (47), proteins were precipitated with CHCl3/methanol (46). Because proteins of pea seeds stain differentially with either silver or Coomassie stains, respectively, gels were first stained with silver (48) and then afterwards with Coomassie (49) in order to monitor all the proteins present. Proteins were electroblotted onto nitrocellulose via semidry blotting in a transfer-buffer containing 10% (w/v) methanol for up to 90 min at 2 mA/cm2. After blocking, the blots were probed with appropriate primary and secondary antibodies. Secondary antibodies were coupled to horseradish peroxidase (Sigma, Taufkirchen, Germany). Visualization of the bound antibodies was with an enhanced chemoluminescence kit (Pierce, Rockford, IL). Protein was measured according to the method of Peterson (50).

2D gel electrophoresis

2D-gel electrophoresis with acid gels containing the cationic detergent 16-BAC in the first dimension was carried out according to Hartinger et al. (22). Separating gels of 0.1 × 5 × □8 cm were prepared from stock solutions leading to final concentrations of 7,5% acrylamide, 0.06% bis-acrylamide, 3 m urea, 75 mm KH2PO4 pH 2.1, 4 mm Na-ascorbate, 8 μm Fe-II-sulfate, 2.5 mm 16-BAC. Polymerization was started by the addition of H2O2 to a final concentration of 0.02%. The focusing gel was prepared from stock solutions leading to a final concentration of: 4% acrylamide, 0.23% bis-acrylamide, 1.6 m urea, 125 mm KH2PO4 pH 4.1, 1.4 mm Na-ascorbate, 8 μm Fe-II-sulfate, and 1.25 mm 16-BAC. Polymerization was started by the addition of H2O2 to a final concentration of 0.04%. Stock solutions of 80 mm Na-ascorbate, 5 mm Fe-II-sulfate, 250 mm 16-BAC, and diluted H2O2 were prepared freshly. 16-BAC was solubilized by heating the solution to 60 °C. The sample buffer was prepared freshly and consisted of 80 mm 16-BAC, 3.75 m urea, 2% glycerin, 37 mm dithiothreitol (DTT), and 0.025% pyronin G. 16-BAC, urea and glycerin were mixed and solubilized by heating to 60 °C; DDT and pyronin G were added afterwards. The mixture was kept at 60 °C until use. Precipitated proteins were solubilized in the sample buffer at 60 °C for 10 min with rigorous vortexing, centrifuged for 1 min at 10 000 × g and loaded on the gel. The gels were run with opposed polarization as compared to SDS-PAGE for 1 h at 10 mA in the focusing gel and 2 h at 20 mm in the running gel. Acidic running gel buffer consists of: 25 mm 16-BAC, 1.5 m glycine, and 5 mm phosphoric acid.

Gels were fixed in 3.5 parts isopropanol, 1 part acidic acid and 5.5 parts H2O for 30 min at room temperature and stained for 12 h in the same solution containing 0.15% Coomassie Brilliant Blue R250. The gel was destained in water, neutralized in 100 mm Tris/HCl pH 6.8 for 3 × 10 min. The lanes containing the separated probes were cut out of the gel and layered on top of a 12% SDS-PAGE gel, overlaid with SDS-PAGE sample buffer and incubated on the gel for up to 15 min. SDS-PAGE was performed as previously described.

Sequence analysis

The sequence alignment was performed using the ClustalW program (EMBL-EBI). The motif search was performed using PROSITE database and search engine (51). Accession numbers for the sequences were as follows: Vicia faba: CAC27161; Pisum sativum: T06459; Glycine max SBPGm:Q04672; Glycine max SBPGm2: AY234869; Glycine max SBP64: AAF05723.

Immunoelectron microscopy of ultrathin cryosections

Immunoelectron microscopy was carried out as described previously (7,52). Cryosections were prepared as described (17). Ultrathin sections were cut in an ultracryomicrotome (Leica, Biel, Switzerland) using a diamond knife (Diatome, Vienna, Austria). For immunolabeling, sections were incubated with antisera against VfSBPL (1 : 200), p16 (1 : 200), and legumin (1 : 200) for 1 h, followed by incubation with protein A-gold (10 nm) for 30 min. For double labeling, after the incubation with the first antibody and incubation with protein A-gold (10 nm), sections were fixed for 5 min with 1% glutaraldehyde. Sections were then quenched with glycine and blocked with BSA, before they were incubated with the second antibody and protein A-gold (5 nm). Sections were contrasted with uranyl acetate/methyl cellulose for 10 min on ice, embedded in the same solution and examined with a Philips CM120 electron microscope.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

The authors are indebted to Dr. D. G. Robinson (Heidelberg, Germany) for critically reading this manuscript. The authors are also indebted to Dr. U. Wobus (IPK, Gatersleben, Germany) for the gift of the VfSBPL antibody, to Dr. L. Franco (Valencia, Spain) for providing us with the pea p16 antibody, and to Dr. N. Happel (Göttingen, Germany) for providing us with the Anti Histone antibody. The authors also wish to thank Dr. W. Dröge-Laser (Göttingen, Germany) who supported us with the isolated nucleus fraction.

References

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References