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Keywords:

  • albumin;
  • ricin;
  • sorting receptor;
  • storage proteins;
  • vacuole

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

We have studied the transport of proricin and pro2S albumin to the protein storage vacuoles of developing castor bean (Ricinus communis L.) endosperm. Immunoelectron microscopy and cell fractionation reveal that both proteins travel through the Golgi apparatus and co-localize throughout their route to the storage vacuole. En route to the PSV, the proteins co-localize in large (>200 nm) vesicles, which are likely to represent developing storage vacuoles. We further show that the sequence-specific vacuolar sorting signals of both proricin and pro2SA bind in vitro to proteins that have high sequence similarity to members of the VSR/AtELP/BP-80 vacuolar sorting receptor family, generally associated with clathrin-mediated traffic to the lytic vacuole. The implications of these findings in relation to the current model for protein sorting to storage vacuoles are discussed.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

Developing castor bean (Ricinus communis L.) seeds accumulate lipids and storage proteins in their endosperm. The castor bean endosperm is a living tissue which, upon germination, undergoes programmed cell death to ensure full mobilization of the storage material to the cotyledons (Gietl and Schmid, 2001; Schmid et al., 2001). In developing endosperm, the major storage proteins: 7S lectins [namely ricin (RCAII) and R. communis agglutinin (RCAI)], 2S albumins and 11S globulins, accumulate in protein storage vacuoles (PSV, previously named protein bodies or aleurone grains; Tully and Beevers, 1976; Youle and Huang, 1976). The R. communis PSV contains phytin globoids and a single large protein crystalloid within a soluble protein matrix. The insoluble crystalloid is composed of 11S globulin while the protein matrix is a mixture of 7S lectins and 2S albumins (Fukusawa et al., 1988; Tully and Beevers, 1976). Each of these storage proteins is co-translationally translocated into the lumen of the endoplasmic reticulum (ER) and subsequently transported to the PSV. N-glycosylated lectins travel through the Golgi apparatus, where their glycans are modified and, in the case of ricin, specific fucose residues are added (Lord and Harley, 1985).

We have recently studied the intracellular sorting of both castor bean ricin and 2S albumin in heterologous tobacco mesophyll protoplasts (Brown et al., 2003; Frigerio et al., 1998). Ricin is a heterodimeric glycoprotein composed of a catalytic, ribotoxic A chain (RTA) disulphide-linked to a galactose-binding B chain (RTB). It is made as a single polypeptide precursor in which RTA and RTB are joined by a 12 amino acid linker that is removed upon vacuolar delivery. 2S albumin is also synthesized as a single precursor protein, subsequently processed in PSV into two, different heterodimeric storage proteins, each composed of a large and a small subunit (Brown et al., 2003; Irwin et al., 1990). In transfected protoplasts, as in the native tissue, both proteins reach the vacuole, where they are processed to their mature forms (Brown et al., 2003; Frigerio et al., 1998).

We have characterized the vacuolar sorting signals of both the ricin and albumin precursors. For proricin, the signal resides within a 12-residue linker propeptide that lies between the A and the B chain (Frigerio et al., 2001). For proalbumin, the signal is again contained within an internal propeptide (PPII), in this case separating small subunit I from large subunit I (Brown et al., 2003). NMR structural analysis shows that this propeptide resides in a relatively unstructured loop which is exposed on the surface of the protein (Pantoja-Uceda et al., 2003). Both the proricin and proalbumin sorting signals appear to be of the sequence-specific type (ssVSS; Matsuoka and Neuhaus, 1999), which have previously only been associated with Golgi-mediated traffic to the lytic vacuole (LV). ssVSS contain a hydrophobic residue in the context of an NPIR-like sequence which has been shown to interact with receptor proteins of the vacuolar sorting receptor (VSR) family (Ahmed et al., 1997; Kirsch et al., 1994; Paris and Neuhaus, 2002; Shimada et al., 1997). Accordingly, mutation of a single isoleucine (for proricin) or leucine (for proalbumin) residue within these motifs causes the precursors to be secreted by tobacco protoplasts. Both sorting signals are also sufficient to target reporter proteins to the vacuole (Brown et al., 2003; Frigerio et al., 2001; Jolliffe et al., 2003).

A novel transport route for non-glycosylated proteins including 2S albumin has been described in developing castor bean endosperm and pumpkin cotyledons (Hara-Nishimura et al., 1998). In this route, large precursor-accumulating vesicles (PAC) containing proalbumin bud directly from the ER, seemingly bypassing the Golgi complex to fuse with PSV. A working model for targeting in castor bean endosperm has correspondingly been proposed whereby 2S albumin exits the ER in PAC, which subsequently receive Golgi-modified glycoproteins, such as proricin, into their periphery before they reach PSV (Hara-Nishimura et al., 1998). Indeed, two putative receptors of the VSR class, PV72/82, have been isolated from the membranes of PAC (Shimada et al., 1997, 2002). PV72 has been shown to be present in the Golgi (Shimada et al., 2002), and to interact with various sequences derived from pumpkin 2S albumin (Shimada et al., 1997, 2002; Watanabe et al., 2002). These findings were rationalized by ascribing a role to PV72 in salvaging any 2S albumin that escapes aggregation in the ER (Shimada et al., 2002). However, the presence of an ssVSS in castor bean 2S albumin suggests that transport of this protein to PSV may be solely receptor-, and thus Golgi-, mediated. In addition, we have recently provided indirect evidence for transport of proalbumin through the Golgi in tobacco protoplasts (Brown et al., 2003). The disparity of these data prompted us to investigate in more detail the transport of proricin and proalbumin in their native tissue.

In this work we show that both proricin and proalbumin indeed co-localize and travel through the Golgi apparatus. Both proteins subsequently appear in large vesicles which are likely to represent developing storage vacuoles. These are distinct from PAC as their lumina contain proteins carrying Golgi-modified glycans. We further show that the sequence-specific sorting signals of both proricin and proalbumin bind in vitro to a protein that shares homology with the VSR/AtELP/BP-80 vacuolar sorting receptor family, previously associated with the clathrin coated vesicle (CCV)-mediated pathway to LV (Bassham and Raikhel, 2000; Vitale and Raikhel, 1999).

Ricin and 2S albumin co-localize in the Golgi and PSV

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

We isolated developing castor bean endosperm at the stage of testa maturation corresponding to the peak of synthesis of all major classes of storage proteins (Lord, 1985b). The endosperm is characterized by regularly shaped cells containing toluidine blue-reactive structures (Figure 1a). Ultrastructural analysis reveals abundant, electron translucent lipid bodies (Figure 1b,c) and a number of more electron-opaque, spherical structures of variable size corresponding to the toluidine blue-reactive structures. These were previously identified as PSV (Figure 1) (Tully and Beevers, 1976; Youle and Huang, 1976). Although both globoids and crystalloids have previously been detected at this stage of development after chemical fixation (Gifford et al., 1982), only the globoids were visible in high pressure frozen sections of the same tissue (Figure 1c, arrowheads).

image

Figure 1. The castor bean endosperm is rich in LB and PSV. (a) Semi-thin section of high-pressure frozen developing castor bean endosperm stained with toluidine blue and visualized by light microscopy. Bar: 20 μm. (b, c) Ultrathin, high-pressure frozen sections of castor bean endosperm visualized by low magnification transmission electron microscopy. LB, lipid bodies; PSV, protein storage vacuoles; N, nucleus; n, nucleolus; arrowheads, globoid cavities. Bars: (b), 5 μm; (c), 1 μm.

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Based on substantial biochemical evidence, ricin and 2S albumin are classified as storage proteins of the PSV matrix (Irwin et al., 1990; Lord, 1985b; Tully and Beevers, 1976; Youle and Huang, 1976). This was confirmed by immunolabelling endosperm sections with anti-ricin A chain (anti-RTA) and anti-2S albumin (anti-2SA) antisera. Figure 2 (lanes 1 and 2) shows that both rabbit and sheep anti-RTA antisera specifically recognized the proricin precursor (grey arrow) and two glycoforms of ricin A chain (empty arrowheads) (Fulton et al., 1986) from a preparation of total endosperm proteins. However, it has previously been demonstrated that the rabbit anti-2SA antiserum, in addition to recognizing 2S albumin (black arrowhead), also recognizes the ricin A chain glycoforms (empty arrowheads) (Figure 2, lane 3; also Brown et al., 2003). We therefore removed the contaminating antibodies by passage through a column bearing immobilized ricin, generating an antiserum which recognized only 2S albumin (Figure 2, lane 4).

image

Figure 2. Evaluation of antisera against ricin and 2S albumin. Total proteins from castor bean endosperm were resolved by SDS-PAGE and immunoblotted with rabbit (lane 1) or sheep (lane 2) anti-RTA antiserum, or crude (lane 3) or purified (lane 4) anti-2SA antiserum. The numbers on the left and right indicate molecular mass markers in kilodaltons. The grey arrow indicates the ricin precursor, open arrowheads the two immunoreactive ricin A chain (RTA) glycoforms, and the closed arrowhead the albumin precursor.

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To immunologically assess the identity of PSV in developing castor bean endosperm, we initially labelled sections with antibodies against α-TIP, a well-established marker for the tonoplast of the PSV (Jauh et al., 1999; Johnson et al., 1990). Anti-α-TIP coupled with 10 nm gold particles clearly not only decorated the outer rim of the electron opaque bodies (Figure 3a), but also the Golgi apparatus (Figure 3b). This pattern has been previously observed in developing pea cotyledons (Hillmer et al., 2001). We therefore conclude that the anti-α-TIP-lined organelles are PSV. Both anti-RTA (Figure 3c, 10 nm gold) and anti-2SA antisera (Figure 3d) label the lumen of the electron opaque bodies. Double immunogold labelling using anti-RTA (coupled with 10 nm gold) and anti-α-TIP (coupled with 20 nm gold) confirmed that the matrix of the PSV was the site of ricin and 2S albumin deposition (Figure 3c, arrowheads indicate α-TIP labelling). Most importantly, both ricin and 2S albumin are also found in the Golgi apparatus (Figure 3, panels e and f). Whilst previously reported for ricin, where the acquisition of Golgi-modified glycans is well documented (Lord, 1985a; Lord and Harley, 1985), these findings now directly demonstrate that proalbumin is also transported through the Golgi complex en route to PSV in castor bean endosperm. Indeed, 94% of the Golgi stacks observed in this study (73 of 78) were labelled with anti-2SA. Double immunogold labelling with anti-RTA (10 nm gold) and anti-2SA (20 nm gold) confirmed the co-localization of these two proteins in both the Golgi and PSV (Figure 4a,b, respectively). Background labelling was consistently very low (see Table S1). In addition to reproducibly detecting 2S albumin in the Golgi, we also observed immunogold labelling in the ER (Figure 4c). Strikingly, the ER in developing endosperm displayed a homogeneous, tubular morphology, in which dense aggregates of the type postulated to form PAC vesicles were never observed. Furthermore, 2S albumin labelling within the ER was always uniformly distributed, in contrast to the clusters observed in the Golgi cisternae (Figures 3f and 4a). This is consistent with the sorting event occurring in the Golgi, rather than the ER. Thus, we conclude that in developing castor bean endosperm both proricin and proalbumin are transported from the ER to the Golgi en route to PSV.

image

Figure 3. Ricin and 2S albumin travel through the Golgi complex and accumulate in PSV. Ultrathin sections of high-pressure frozen castor bean endosperm were immunolabelled with the following antisera: (a, b) Polyclonal rabbit anti-α-TIP antibody (Figure 3a,b) showing labelling of the PSV membrane and Golgi. (c) Polyclonal sheep anti-RTA (10 nm gold) and polyclonal rabbit anti-α-TIP antibody (20 nm gold), showing labelling of the PSV matrix and bounding membrane. (d) Polyclonal rabbit anti-2SA labelling the PSV matrix. (e, f) Labelling of a Golgi stack with polyclonal anti-RTA and anti-2SA, respectively. G, Golgi complex; LB, lipid bodies; PSV, protein storage vacuoles. Bars: (a), 200 nm; (b–f), 100 nm.

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image

Figure 4. Ricin and 2S albumin co-localize in the Golgi and PSV. (a, b) Ultrathin sections of high-pressure frozen castor bean endosperm were double-immunolabelled with sheep anti-RTA (10 nm gold) and rabbit anti-2SA (20 nm gold). Both the Golgi (a) and the PSV (b) are labelled. G, Golgi complex; PSV, protein storage vacuoles. Bars: 100 nm. Inset in (b): a detail of the PSV lumen showing decoration by both 10 and 20 nm gold. Bar: 50 nm. (c) Sections were immunolabelled with rabbit anti-2SA (20 nm gold). Note the tubular appearance of the ER. This image is representative of 39 observations of ER morphology. ER, endoplasmic reticulum; CW, cell wall; LB, lipid bodies. Bar: 100 nm.

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Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

As well as the Golgi and large PSV, the antibodies raised against RTA (coupled with 10 nm gold) and 2S albumin (coupled with 20 nm gold) also label the lumen of large vesicles of 150–400 nm in diameter (Figure 5a). These vesicles possessed a peripheral electron-translucent layer, and were often seen close to PSV [Figure 5b(inset),c]. Ribosomes were never observed around the periphery of these structures, suggesting that they did not originate directly from rough ER. In addition, anti-complex glycan antiserum revealed a distribution indistinguishable from anti-RTA (Figure 5b, 10 nm gold), labelling throughout the lumen of these vesicles (Figure 5b, main panel and inset, 20 nm gold), further indicating that the lumenal cargo has travelled via the Golgi. As these structures appear to have the same distribution of lectins and albumins as PSV, they may therefore represent precursors to storage vacuoles. In further support of this, anti-α-TIP labelled the membrane of these vesicles (Figure 5c). The vesicles were also often found in close proximity to one another and/or to a large storage vacuole, suggesting imminent homotypic fusion. Figure 5(c) shows such an example, where five α-TIP-lined vesicles (black stars) are clustered together near a PSV.

image

Figure 5. Ricin and 2S albumin are found in large, post-Golgi vesicular structures. Ultrathin sections of high-pressure frozen castor bean endosperm were immunolabelled with the following antisera: (a) Sheep anti-RTA (10 nm gold) and rabbit anti-2SA (20 nm gold). Bar: 50 nm. (b) Sheep anti-RTA (10 nm gold) and rabbit anti-complex glycans (20 nm gold). Inset shows a large vesicle in close proximity to a protein storage vacuole (PSV). Bars: 50 nm (main panel) and 100 nm (inset). (c) Rabbit anti-α-TIP. Stars indicate individual, large vesicles. Bar: 200 nm.

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Ricin and 2S albumin co-fractionate with VSR-like proteins

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

The sequence-specific nature of the vacuolar sorting signals of ricin and 2S albumin suggests that both proteins may be ligands for a sorting receptor(s) of the VSR family (previously named BP-80 or ELP; Ahmed et al., 1997; Kirsch et al., 1994; Shimada et al., 1997). As the interaction between such receptors and clathrin adaptor proteins has been demonstrated in vitro (Sanderfoot et al., 1998), this in turn makes them candidates for recruitment into CCV. As neither the clathrin heavy-chain antiserum nor the anti-BP-80 antisera (MAbs 17F9 or 14G7) (Cao et al., 2000) were compatible with the EM methodology described here (data not shown), we instead investigated the potential co-localization of these proteins by subcellular fractionation and Western blotting.

Developing castor bean endosperm was homogenized and subjected to fractionation on a continuous 15–55% isopycnic sucrose gradient. Fractions were resolved by either reducing or non-reducing SDS-PAGE before immunoblotting with a panel of antisera (Figure 6). The majority of the precursor (Figure 6) and mature (data not shown) forms of ricin and 2S albumin are detected at the top of the gradient. This is expected, as fragile PSV break during homogenization in aqueous buffer. There is also a degree of breakage of other endomembranes, as testified by the fact that anti-BiP labels fractions at the top of the gradient, in addition to the microsome peak. Both the ricin and 2S albumin precursors, however, also peak at a fraction of density 1.21 g ml−1. These fractions are clearly distinct from the ER microsomes, as indicated by the position of the molecular chaperone BiP (Figure 6). Significantly, proteins reacting with anti-BP-80 monoclonal antibody 17F9 also coincide with ricin and 2S albumin. Furthermore, this 1.21 g ml−1 fraction is also labelled by an anti-clathrin heavy chain antibody. We conclude that a proportion of proricin and proalbumin is found in cellular fractions that are enriched in both BP-80-like species and clathrin.

image

Figure 6. A proportion of proricin and proalbumin is found in cellular fractions enriched in BP-80-like proteins and clathrin. Total cell homogenates from developing castor bean endosperms were subjected to isopycnic centrifugation on a linear 15–55% (w/w) sucrose gradient. Fractions were resolved on reducing SDS-PAGE (with the exception of the non-reducing SDS-PAGE for the gel to be immunoblotted with anti-BP-80) and immunoblotted with the indicated antisera. Numbers on the left indicate molecular mass markers in kilodaltons. Numbers below selected gradient fractions indicate fraction density in g ml−1.

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The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

The cell fractionation data raise the possibility that proricin and proalbumin may be ligands of VSR (BP-80)-like receptors. To test this hypothesis, we produced affinity columns carrying immobilized peptides corresponding to the proricin linker and proalbumin PPII, both in their wild-type form or with the respective, crucial isoleucine/leucine changed to glycine (Figure 7a). A potential receptor would be expected to bind to the wild-type, but not the mutated propeptides. As a positive control, a column bearing the proaleurain VSS was also generated, as previously used to select BP-80 from pea (Kirsch et al., 1994). Clarified CHAPS extracts of membranes prepared from developing castor bean endosperm were applied to the columns, washed, and eluted at low pH. Eluates were separated by non-reducing SDS-PAGE and visualized with Coomassie (Figure 7b). A protein migrating below the 64 kDa marker bound to the wild-type, but not the I271G ricin linker peptide column (Figure 7b, empty arrowhead, compare lanes 1 and 2). Likewise, a species of comparable mobility bound to the 2S albumin PPII, but not the L58G peptide column (Figure 7b, compare lanes 4 and 5). A protein with an apparent molecular mass of around 50 kDa was also eluted in every case (Figure 7b, black arrowhead) as well as from a column prepared without coupled peptide (data not shown). This protein, identified by mass spectrometry as calreticulin (data not shown), is a contaminant which binds to the column matrix itself.

image

Figure 7. The sorting signals of proricin and proalbumin bind in vitro to BP-80-like proteins. (a) Sequences of the peptides used for in vitro binding experiments. (b) Clarified membrane extracts from developing castor bean endosperm were applied to the indicated peptide columns, washed, and eluted at low pH. Eluates were separated by non-reducing SDS-PAGE and visualized with Coomassie. (c) Gels as for (b) were immunoblotted with monoclonal anti-BP80 17F9 antibody. Immunoreactive bands were visualized by ECL. Numbers on the right indicate molecular mass markers in kilodaltons. (d) Total extracts from castor bean endosperm and pea cotyledons were resolved by non-reducing SDS-PAGE and immunoblotted with monoclonal anti-BP-80 17F9 antibody. Immunoreactive bands were visualized by ECL. Numbers on the left indicate molecular mass markers in kilodaltons. (e) Gels as for (b) were immunoblotted with monoclonal anti-BP-80 14G7 antibody. Immunoreactive bands were visualized by ECL. (f) Total extract from castor bean endosperm was immunoblotted with monoclonal anti-BP-80 14G7 antibody. Immunoreactive bands were visualized by ECL.

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Interestingly, the proteins released from the control proaleurain column were indistinguishable from those released by the wild-type ricin linker or 2S albumin PPII columns at the resolution provided by this SDS-PAGE (Figure 7b, compare lanes 1, 3 and 4). These data suggest that a protein from castor bean, shown to bind the targeting signals of these endogenous PSV residents in an isoleucine/leucine-dependent manner, also bound the ssVSS responsible for targeting proaleurain to LV, and thus may be BP-80-like. This possibility was investigated by Western blotting. Figure 7(c) shows immunoblots of gels loaded with column eluates from castor bean endosperm as above, using a primary antibody raised against a non-reduced epitope that spans the ‘unique’ N-terminal domain of pea BP-80 (MAb17F9) (Cao et al., 2000). The lanes loaded with protein eluted from the linker-, PPII- and proaleurain-affinity columns yielded immunoreactive bands (Figure 7c, empty arrowheads), whereas there were no detectable immunoreactive bands in the eluates from either column bearing the mutated propeptides (Figure 7c, lanes 2 and 5). This concurs both with an earlier biochemical demonstration of the inability of these signals to confer vacuolar targeting in tobacco protoplasts (Brown et al., 2003; Frigerio et al., 2001), and with the Coomassie-stained gels presented in Figure 7(b). In order to provide a size marker for BP-80, total castor bean and pea (Pisum sativum) extracts were immunoblotted with the same antibody. Figure 7(d) shows that a single immunoreactive band from pea and three to four bands from castor bean extracts were revealed that migrated within the range of the castor bean proteins eluted from the ricin and albumin VSS-bearing columns. To corroborate our findings, we also performed immunoblots using a different monoclonal antibody (Mab 14G7) that detects the third EGF repeat in pea BP-80 (Cao et al., 2000). The 14G7 antibody recognizes one of the castor bean polypeptides around 64 kDa both in the column eluate (Figure 7e, lane 1) and in total castor bean extracts (Figure 7f). As two different monoclonal antibodies detect bands in the same size range, this confirms that the castor bean species are indeed BP-80-like. In most gel systems, BP-80 proteins resolve with an apparent Mr of approximately 80 kDa. The apparent size discrepancy detected in our experiment is due to our SDS-PAGE system, which is optimized for the separation of storage globulins and uses a 200:1 acrylamide/bisacrylamide ratio (see Experimental procedures for details). Indeed, when total castor bean homogenates were resolved on conventional SDS-PAGE, (acrylamide/bisacrylamide ratio of 37:1), immunoblotting with 17F9 revealed a compact band migrating at around 80 kDa (see Figure S2).

From the data shown in Figure 7 we conclude that the ssVSS of both ricin and 2S albumin can bind BP-80-like receptor(s) in vitro. More than one immunoreactive species is bound to each wild-type peptide column (Figure 7c, empty arrowheads), with the number and immunoreactivity of these species differing between columns. This suggests the presence of multiple VSR isoforms in castor bean endosperm. We focused on the polypeptide that bound to both the ricin linker and 2S albumin PPII peptides and that produced the strongest Coomassie-stained band (Figure 7b, lane 1, empty arrowhead). After subjecting the excised polypeptide to tryptic digestion, the peptide mixture was analysed by nano-electrospray tandem MS. Several multiple charged peptides were fragmented in order to resolve their amino acid sequence. Three peptides presented high sequence similarity to several known members of the VSR family (Table 1). Specifically, one peptide was identical to the Arabidopsis vacuolar sorting receptor AtELP1 (VSRAt-1; Ahmed et al., 1997) and the remaining two peptides showed more than 80% sequence identity with the same protein. The three peptides span the lumenal domain of the receptor (Table 1). Intriguingly a fourth peptide, WGGYDC, with no apparent homology to other known VSR, was also identified.

Table 1.  Sequence similarity of three peptides from the major proricin linker-binding protein to members of the vacuolar sorting receptor family
SourcePeptide 1Peptide 2Peptide 3Reference
Ricinus communisLNLPSALLTKFVGDGYTHCCLGDTEADVDNThis work
Arabidopsis thaliana (AtVSR1/AtELP)IPSALITKFVGDGYTHCCIGDPEADVENAhmed et al. (1997)
Cucurbita sp. (PV72)IPSALISKFVGDGYTHCCIGDPEADVENShimada et al. (1997)
Pisum sativum (BP-80)IPSALIGKFKGDGYTTCCMGDPNADTENKirsch et al. (1994)
Triticum aestivumIPSVLITKFVGDGYTNCCVGDPEADEENShy et al. (2001)

The deposition of both proricin and proalbumin in the PSV would therefore appear to be a receptor-mediated event, in a manner at least analogous to CCV-mediated traffic to LV.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

We have systematically analysed the transport of two major storage proteins to the PSV of developing castor bean endosperm. The data presented here not only confirm the predictions we made whilst studying the fate of ricin and albumin in tobacco protoplasts (Brown et al., 2003; Frigerio et al., 1998), but also provide further insights into the sorting of seed storage proteins in the native tissue. Namely, we demonstrate a direct link between the targeting of two storage protein precursors to PSV and the interaction of their sorting signals with receptors of the VSR family.

In this study, developing castor bean endosperm was subjected to high-pressure freezing and freeze-substitution, to prevent any artefactual alterations to structure and protein location. The immunogold labelling presented here clearly shows that castor bean proalbumin is present in the Golgi complex.

Further, EM data reveal that the distribution of 2S albumin – a non-glycosylated protein – and the previously characterized glycoprotein, ricin, is identical using these methods. In addition to observing these storage proteins together in the PSV, co-localization was also seen in smaller structures (150–400 nm, Figure 5). These structures could be labelled with antibodies raised against α-TIP, a PSV marker, and were frequently found close to PSV. We believe that the presence of complex glycan-bearing proteins throughout the lumen of these vesicles is sufficient to distinguish them from PAC. Indeed, the data presented here suggest that the entire content of these large vesicles is derived from the Golgi. Furthermore, throughout our study, ribosomes were never seen on their delimiting membranes, again indicating that these structures did not originate directly from the ER. Moreover, we never observed aggregates within the ER, or indeed any evidence of budding from this compartment. Accordingly, these large vesicles should instead be regarded as early PSV, or possibly pre-vacuolar compartments, although further work is required to clarify their precise role in storage protein deposition.

Our data firmly support the idea that in developing castor bean endosperm, all 2S albumin is trafficked to PSV via the Golgi apparatus. The co-localization of 2S albumin and ricin throughout the secretory pathway indicates that both proteins follow the same route to PSV. Indeed, the evidence presented here formally rationalizes, in the native tissue, our previous demonstration that both proteins possess ssVSS (Brown et al., 2003; Frigerio et al., 2001). A family of VSR has now been characterized, of which pea BP-80 (Kirsch et al., 1994) is the prototype. Members of this family are integral membrane proteins which bind with high specificity to NPIR-like vacuolar sorting signals at the trans-Golgi level and recruit clathrin coats through an adaptin-binding tyrosine motif present in their cytosolic tail (Paris and Neuhaus, 2002). VSR have been shown to exist in pea, maize, rice, pumpkin and Arabidopsis (Paris and Neuhaus, 2002). In accordance with this model, castor bean proteins recognized by antibodies raised against BP-80 and clathrin heavy chain co-localize with the precursors of both ricin and 2S albumin when resolved on a sucrose gradient (Figure 6). Furthermore, the vacuolar sorting signals of both ricin and 2S albumin bind in a sequence-specific manner to a BP-80-like protein. The demonstration of a specific interaction with a sorting receptor, and co-localization of ricin and 2S albumin with this receptor and with a clathrin coat protein, may explain the apparent paradox of two bona fide PSV residents possessing ssVSS.

Although VSR have been previously implicated in the transport of other members of the albumin family (Kirsch et al., 1996; Shimada et al., 1997, 2002; Watanabe et al., 2002), the roles of the peptides used to bind the receptors in vitro were not always investigated fully in vivo. Here we have clearly shown that the well-characterized VSS of two castor bean storage proteins (Brown et al., 2003; Frigerio et al., 2001; Jolliffe et al., 2003) interact with a novel member of the recognized VSR family, thus adding R. communis to the growing list of plants in which these receptors have been identified, and fuelling the debate as to the breadth of their function. Indeed a recent report by Shimada et al. (2003) has shown that genetic knockout of one of the seven Arabidopsis isoforms of VSR, namely VSRat-1, led to partial secretion of the precursors of all major classes of storage proteins into the apoplast of mature Arabidopsis seeds. This new finding further strengthens the link between VSR-mediated transport pathways and the sorting of proteins to the PSV in seeds. Together, these findings potentially call for a re-evaluation of the working model for seed storage protein deposition and for the role of VSR in this process. In this respect it will be important to characterize the transport of the remaining class of storage proteins – the 11S globulins – to the PSV of Ricinus endosperm.

2S albumin antibody purification

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

Crude rabbit polyclonal antisera raised against 2S albumin (Brown et al., 2003) was purified to remove contaminating antibodies recognizing ricin agglutinins by passage through a column bearing 1 ml of agarose-bound R. communis agglutinin II (Vector Laboratories, Peterborough, UK). The column was washed with PBS containing 0.1 m lactose, and the antisera applied in 0.1 m lactose to prevent interaction of antibody glycans with the lectin subunit of the immobilized ricin. Flow-through fractions, which contained the anti-2SA antibodies, were collected.

Sucrose gradient fractionation

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

Endosperm isolated from maturing castor bean seeds was homogenized in an ice-cold mortar with ice-cold 100 mm Tris-Cl, pH 7.8, 10 mm KCl, containing 12% (w/w) sucrose and 2 mm MgCl2, using 6 ml of buffer per gram of fresh leaf tissue. The homogenate was centrifuged for 10 min at 1000 g at 4°C; 600 μl of the supernatant was loaded on a 11-ml linear 15–55% (w/w) sucrose gradient. After centrifugation at 35 000 rpm, 4°C for 2 h in a Beckman SW40 rotor (Beckman Coulter, High Wycombe, UK) (154 400 g average), fractions of 600 μl were collected. Immunoblot analysis of 60 μl aliquots of these fractions was performed as described (Pedrazzini et al., 1997), using the indicated antisera.

Affinity chromatography

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

A 2.5 ml volume [2 mg ml−1 in coupling buffer (50 mm Tris; 5 mm EDTA, pH 8.5)] of sulphydryl-containing peptides (synthesized by Alta Biosciences, University of Birmingham, UK) were immobilized on ‘SulfoLink’ coupling gel columns (Pierce, Perbio Science UK, Tattenhall, UK), and the non-specific binding sites blocked, according to the manufacturer's instructions. Endosperm was homogenized in a Petri dish on ice by continuous chopping for 15 min using a hand-held razor blade, after addition of cold grinding buffer (150 mm Tricine, pH 7.5; 1 mm EDTA-Na, pH 7.5; 10 mm KCl; 1 mm MgCl2; 100 mm lactose), supplemented immediately before use with ‘complete’ protease inhibitor cocktail (Roche Diagnostics, Lewes, UK). Pea homogenates were prepared using an ice-cold pestle and mortar by grinding in the same buffer. The resulting suspensions were filtered through four layers of mira cloth, and centrifuged at 10 000 g for 15 min at 4°C. The floating lipid layers were discarded and the underlying supernatants carefully removed to a clean tube. Membranes were pelleted from post-mitochondrial supernatants by centrifugation in 3 ml thick-walled polycarbonate tubes at 100 000 g (Beckman TL-100 ultracentrifuge, TLA-100.3 rotor) at 4°C for 30 min, and the supernatant discarded. Pellets were resuspended in CHAPS buffer (Kirsch et al., 1994: 20 mm Hepes NaOH, pH 7.1; 150 mm NaCl; 1 mm MgCl2; 1 mm CaCl2; 1% (w/v) 3-[(3-cholramidopropyl) dimethylammonio]-1-propanesulphonate (CHAPS)], and incubated at room temperature for 30 min to solubilize integral membrane proteins. Residual membranes were removed by re-centrifugation of the same tubes, as in the first step, and the supernatant was carefully removed to a clean tube. Two-millilitre aliquots of membrane protein extracts were applied to the affinity columns, previously brought to room temperature and equilibrated according to the manufacturer's instructions. Column gel beds were resuspended, and incubated with gentle agitation for 30 min, before being allowed to settle for 30 min. Columns were washed according to the manufacturer's instructions. Bound proteins were released by addition of 10 ml elution buffer [25 mm NaOAc, pH 4.0; 150 mm NaCl; 1 mm EGTA; 1% (w/v) CHAPS], and the eluate collected in 1 ml aliquots. Protein was precipitated from fractions by the addition of an equal volume of cold 30% (w/v) TCA. After mixing, the sample was incubated on ice for a minimum of 30 min. The precipitant was harvested by centrifugation at 17 000 g for 15 min at 4°C, the supernatant discarded, and the pellet washed twice with 1 ml of cold acetone, centrifuging between each wash for 5 min as before. The acetone-washed pellet was then dried in a vacuum desiccator, resuspended in non-reducing loading buffer, and separated by SDS-PAGE. Subsequent protein detection was either by Coomassie-blue staining, or immunoblot as described (Pedrazzini et al., 1997), using monoclonal BP-80 antibody.

Peptide sequencing

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

The band of interest was excised from a Coomassie-stained gel and digested overnight with trypsin as described (Anelli et al., 2002). The unseparated peptide mixture was concentrated and desalted over a capillary column packed with POROS R2 material and eluted directly into the electrospray needle. Nanoelectrospray tandem MS experiments were performed on a Q-Star Pulsar (PE SCIEX Instruments, Toronto, Canada). Several multiple charged peptides were fragmented in order to read the amino acid sequence. One peptide (FVGDGYTHCK) was identical to AtELP (Accession number sptrembl: P93026). Two other sequenced peptides, namely CIGDTEADVDNPVLK and LNIPSALITK, showed high homology to CIGDPEADVENPVLK and IPSALITK, also present in P93026.

High-pressure freezing.

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

Samples for transmission electron microscopy observation were chosen from castor bean endosperm at the testa maturation stage. Thin slices of endosperm (<0.5 mm) were dissected, then 2 mm diameter discs were cut out of the slices using a disposable biopsy punch (Stiefel Laboratories Ltd, Wooburn Green, UK), and placed in an aluminium sample holder. Prior to capping the sample holder, the sample was covered with hexadecene in order to remove any remaining air between the sample and the holder. Pairs of holders were clamped together, and samples were immediately frozen using a BAL-TEC HPM 010 high-pressure freezer (BAL-TEC AG, Balzers, Liechtenstein).

Freeze-substitution

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

Freeze-substitution was carried out in a Reichert AFS (Leica, Vienna, Austria) freeze-substitution system. Sample holders were split open under liquid nitrogen and placed into plastic porous specimen pots containing the substitution medium previously frozen in liquid nitrogen. For standard ultrastructural observations, 2% osmium tetroxide in acetone dried over molecular sieve was used. Plastic specimen pots were put into an universal aluminium container onto the surface of the frozen substitution medium and transferred into the Reichert AFS pre-cooled to −160°C. Sample temperature was increased to −85°C over 5 h. Freeze-substitution was carried out as follows: −85°C for 26 h, 2°C h−1 temperature increase over 12.5 h, −60°C for 8 h, 2°C h−1 temperature increase over 15 h, −30°C for 9 h (Steinbrecht and Müller, 1987; Studer et al., 2001). At the end of the freeze-substitution run, samples were warmed up to 20°C with a temperature increase of 4°C h−1. All subsequent steps were carried out at room temperature.

For immunogold labelling, samples were subjected to the same treatment except that the freeze-substitution medium was 0.5% uranyl acetate in ethanol dried over molecular sieve and after the freeze substitution run, samples were warmed up to −20°C with a temperature increase of 1°C h−1. All subsequent steps were carried out at this temperature.

Embedding and sectioning

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

Samples for standard ultrastructural observations were rinsed in anhydrous acetone, carefully removed from the sample holders, embedded stepwise in Spurr's medium resin (TAAB Laboratories Equipment Limited, Aldermaston, Berkshire, UK) (Spurr, 1969), and polymerized at 70°C for 9 h.

Samples for immunogold labelling were rinsed in cold ethanol and, after careful removal from the sample holders, embedded stepwise in LR White medium resin (Agar Scientific, Stansted, UK) over 1 week. Polymerization was under UV light for 24 h at −20°C and for another 24 h at 0°C.

Ultrathin sections were cut with an RMC MTXL ultramicrotome (Boeckeler Instruments, Tucson, AZ, USA) and collected on formvar-coated 300 mesh hexagon copper grids (Agar Scientific).

Immunogold labelling, staining and observation

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

For immunogold labelling, sections were first blocked for 30 min with a 1:30 dilution of pre-immune serum in TRIS (20 mm TRIS, 15 mm NaN3, 225 mm NaCl), pH6.9, supplemented with 1% BSA (TRIS-BSA). They were then treated with 0.1% Tween20 in TRIS-BSA and 0.02 m glycine in TRIS-BSA, each for 15 min, washed three times for 10 min in TRIS-BSA, and incubated in a dilution of primary antibody at 4°C overnight. The following primary antibodies were used in this study: anti-α-TIP (dilution 1:1000, 1:2000), rabbit anti-RTA (dilution 1:200), sheep anti-RTA (dilution 1:2000, 1:4000), rabbit anti-2SA (dilution 1:2000, 1:8000), rabbit anti-complex glycans (dilution 1:2000, 1:4000).

After washing in TRIS-BSA (three times 15 min), sections were treated with a 1:20 dilution (in TRIS-BSA) of the corresponding secondary antibodies (goat anti-rabbit or donkey anti-sheep) conjugated to 10 nm gold (BBInternational, Cardiff, UK) for 1.5 h at room temperature. Finally, after thorough washing in TRIS-BSA and ultrapure water, sections were dried on filter paper. Occasionally, 1% fish gelatin (Sigma, Dorset, UK) was added to the primary and secondary antibody solutions as well as to the TRIS-BSA washings as an additional blocking agent.

For simultaneous double immunogold labelling, the same protocol was applied except that sections were exposed to mixtures of pre-immune serum (both at a 1:30 dilution in TRIS-BSA) and to mixtures of the primary antibodies (for dilutions see above). Secondary antibodies were also used as a mixture (both at a 1:20 dilution in TRIS-BSA) with one conjugated to 10 nm gold particles and the other one to 20 nm gold particles (see Figures 3–5).

For controls of both single and double immunogold labelling, samples were subjected to the same treatment except that the corresponding pre-immune serum substituted the primary antibody.

All sections were stained with 2% uranyl acetate in 70% ethanol for 15 min, followed by lead citrate for 15 min, and examined with a JEOL JEM-1200EXII (JEOL UK, Hertfordshire, UK) at 80 or 120 kV.

Supplementary material

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

Table S1 Analysis of background immunogold labelling Sets of electron micrograph negatives were digitalized and the areas of Golgi stacks, protein storage vacuoles (PSV), and surrounding cytoplasm were calculated using the ImageJ analysis package (publicly available at http://www.rsb.info.nih.gov/ij/index.html). Labelling of the Golgi and/or PSV was compared with background labelling and is shown as the average number of gold particles per square micrometre ± standard deviation. (*) number of gold particles for α-TIP was calculated for the areas of both Golgi and PSV

Figure S1 Gel mobility of castor bean VSR-like proteins. Total extract from castor bean endosperm was resolved by SDS-PAGE either using either standard (37:1) or 200:1 acrylamide/bisacrylamide ratio and immunoblotted with monoclonal anti-BP-80 17F9 antibody. Immunoreactive bands were visualized by ECL. Numbers on the left indicate molecular mass markers in kilodaltons. Note the gel-type-dependent difference in mobility of the immunoreactive polypeptides.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information
  • Ahmed, S.U., Bar-Peled, M. and Raikhel, N.V. (1997) Cloning and subcellular location of an Arabidopsis receptor-like protein that shares common features with protein-sorting receptors of eukaryotic cells. Plant Physiol. 114, 325336.
  • Anelli, T., Alessio, M., Mezghrani, A., Simmen, T., Talamo, F., Bachi, A. and Sitia, R. (2002) ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family. EMBO J. 21, 835844.
  • Bassham, D. C. and Raikhel, N. V. (2000) Unique features of the plant vacuolar sorting machinery. Curr. Opin. Cell Biol. 12, 491495.
  • Bollini, R. and Chrispeels, M.J. (1979) The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons. Planta, 146, 487501.
  • Brown, J.C., Jolliffe, N.A., Frigerio, L. and Roberts, L.M. (2003) Sequence-specific, Golgi-dependent targeting of the castor bean 2S albumin to the vacuole in tobacco protoplasts. Plant J. 36, 711719.
  • Cao, X., Rogers, S.W., Butler, J., Beevers, L. and Rogers, J.C. (2000) Structural requirements for ligand binding by a probable plant vacuolar sorting receptor. Plant Cell, 12, 493506.
  • Frigerio, L., Vitale, A., Lord, J.M., Ceriotti, A. and Roberts, L.M. (1998) Free ricin A chain, proricin and native toxin have different cellular fates when expressed in tobacco protoplasts. J. Biol. Chem. 273, 1419414199.
  • Frigerio, L., Jolliffe, N.A., Di Cola, A., Hernández Felipe, D., Paris, N., Neuhaus, J.-M., Lord, J.M., Ceriotti, A. and Roberts, L.M. (2001) The internal propeptide of the ricin precursor carries a sequence-specific determinant for vacuolar sorting. Plant Physiol. 126, 167175.
  • Fukusawa, T., Hara-Nishimura, I. and Nishimura, M. (1988) Biosynthesis, intracellular transport and in vivo processing of 11S globulin precursor proteins of developing castor bean endosperm. Plant Cell Physiol. 29, 339345.
  • Fulton, R.J., Blakey, D.C., Knowles, P.P., Uhr, J.W., Thorpe, P.E. and Vitetta, E.S. (1986) Purification of ricin A1, A2, and B chains and characterization of their toxicity. J. Biol. Chem. 261, 53145319.
  • Gietl, C. and Schmid, M. (2001) Ricinosomes: an organelle for developmentally regulated programmed cell death in senescing plant tissues. Naturwissenschaften, 88, 4958.
  • Gifford, D., Greenwood, J. and Bewley, J. (1982) Deposition of matrix and crystalloid storage proteins during protein body development in the endosperm of Ricinus communis L. - cv Hale seeds. Plant Physiol. 69, 14711478.
  • Hara-Nishimura, I., Shimada, T., Hatano, K., Takeuchi, Y. and Nishimura, M. (1998) Transport of storage proteins to protein storage vacuoles is mediated by large precursor-accumulating vesicles. Plant Cell, 10, 825836.
  • Hillmer, S., Movafeghi, A., Robinson, D.G. and Hinz, G. (2001) Vacuolar storage proteins are sorted in the cis-cisternae of the pea cotyledon Golgi apparatus. J. Cell Biol. 152, 4150.
  • Irwin, S.D., Keen, J.N., Findlay, J.B.C. and Lord, J.M. (1990) The Ricinus communis 2S albumin precursor: a single preproprotein may be processed into two different heterodimeric storage proteins. Mol. Gen. Genet. 222, 400408.
  • Jauh, G.-Y., Phillips, T.E. and Rogers, J.C. (1999) Tonoplast intrinsic protein isoforms as markers for vacuolar functions. Plant Cell, 11, 18671882.
  • Johnson, K.D., Höfte, H. and Chrispeels, M.J. (1990) An intrinsic tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GlpF). Plant Cell, 2, 525532.
  • Jolliffe, N.A., Ceriotti, A., Frigerio, L. and Roberts, L.M. (2003) The position of the proricin vacuolar targeting signal is functionally important. Plant Mol. Biol. 51, 631641.
  • Kirsch, T., Paris, N., Butler, J.M., Beevers, L. and Rogers, J.C. (1994) Purification and initial characterization of a potential plant vacuolar targeting receptor. Proc. Natl Acad. Sci. USA, 91, 34033407.
  • Kirsch, T., Saalbach, G., Raikhel, N. V. and Beevers, L. (1996) Interaction of a potential vacuolar targeting receptor with amino- and carboxyl-terminal targeting determinants. Plant Physiol. 111, 469474.
  • Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685.
  • Lainé, A.-C., Gomord, V. and Faye, L. (1991) Xylose-specific antibodies as markers of subcompartmentation of terminal glycosylation in the Golgi apparatus of sycamore cells. FEBS Lett. 295, 179184.
  • Lord, J.M. (1985a) Precursors of ricin and Ricinus communis agglutinin. Glycosylation and processing during synthesis and intracellular transport. Eur. J. Biochem. 146, 411416.
  • Lord, J.M. (1985b) Synthesis and intracellular transport of lectin and storage protein precursors in endosperm from castor bean. Eur. J. Biochem. 146, 403409.
  • Lord, J. and Harley, S. (1985) Ricinus communis agglutinin B-chain contains a fucosylated oligosaccharide side-chain not present on ricin B-chain. FEBS Lett. 189, 7276.
  • Matsuoka, K. and Neuhaus, J.-M. (1999) Cis-elements of protein transport to the plant vacuoles. J. Exp. Bot. 50, 165174.
  • Pantoja-Uceda, D., Bruix, M., Gimenez-Gallego, G., Rico, M. and Santoro, J. (2003) Solution structure of RicC3, a 2S albumin storage protein from Ricinus communis. Biochemistry, 42, 1383913847.
  • Paris, N. and Neuhaus, J.M. (2002) BP-80 as a vacuolar sorting receptor. Plant Mol. Biol. 50, 903914.
  • Pedrazzini, E., Giovinazzo, G., Bielli, A., de Virgilio, M., Frigerio, L., Pesca, M., Faoro, F., Bollini, R., Ceriotti, A. and Vitale, A. (1997) Protein quality control along the route to the plant vacuole. Plant Cell 9, 18691880.
  • Sanderfoot, A.A., Ahmed, S.U., Marty-Mazars, D., Rapoport, I., Kirchhausen, T., Marty, F. and Raikhel, N.V. (1998) A putative vacuolar cargo receptor partially colocalizes with AtPEP12p on a prevacuolar compartment in Arabidopsis roots. Proc. Natl Acad. Sci. USA, 95, 99209925.
  • Schmid, M., Simpson, D.J., Sarioglu, H., Lottspeich, F. and Gietl, C. (2001) The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum. Proc. Natl Acad. Sci. USA, 98, 53535358.
  • Shimada, T., Kuroyanagi, M., Nishimura, M. and Hara-Nishimura, I. (1997) A pumpkin 72-kDa membrane protein of precursor accumulating vesicles has characteristics of a vacuolar sorting receptor. Plant Cell Physiol. 38, 14141420.
  • Shimada, T., Watanabe, E., Tamura, K., Hayashi, Y., Nishimura, M. and Hara-Nishimura, I. (2002) A vacuolar sorting receptor PV72 on the membrane of vesicles that accumulate precursors of seed storage proteins (PAC vesicles). Plant Cell Physiol 43, 10861095.
  • Shimada, T., Fuji, K., Tamura, K., Kondo, M., Nishimura, M. and Hara-Nishimura, I. (2003) Vacuolar sorting receptor for seed storage proteins in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA, 100, 1609516100.
  • Shy, G., Ehler, L., Herman, E. and Galili, G. (2001) Expression patterns of genes encoding endomembrane proteins support a reduced function of the Golgi in wheat endosperm during the onset of storage protein deposition. J Exp Bot 52, 23872388.
  • Spurr, A.R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 3143.
  • Steinbrecht, R. and Müller, M. (1987) Freeze-substitution and freeze-drying. in: Freeze-substitution and freeze-drying, Springer Verlag, Berlin, 149172.
  • Studer, D., Graber, W., Al-Amoudi, A. and Eggli, P. (2001) A new approach for cryofixation by high-pressure freezing. J. Microsc. 203, 285294.
  • Tully, R.E. and Beevers, H. (1976) Protein bodies of castor bean endosperms. Isolation, fractionation and characterisation of protein components. Plant Physiol. 58, 710716.
  • Vitale, A. and Raikhel, N. V. (1999) What do proteins need to reach different vacuoles? Trends Plant Sci. 4, 149155.
  • Watanabe, E., Shimada, T., Kuroyanagi, M., Nishimura, M. and Hara-Nishimura, I. (2002) Calcium-mediated association of a putative vacuolar sorting receptor PV72 with a propeptide of 2S albumin. J Biol Chem 277, 87088715.
  • Youle, R.J. and Huang, A.H.C. (1976) Protein bodies from the endosperm of castor bean. Subfractionation, protein components, lectins and changes during germination. Plant Physiol. 58, 703709.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Ricin and 2S albumin co-localize in the Golgi and PSV
  6. Ricin and 2S albumin accumulate in large, post-Golgi vesicular structures
  7. Ricin and 2S albumin co-fractionate with VSR-like proteins
  8. The sorting signals of ricin and 2S albumin bind to VSR-like proteins in vitro
  9. Discussion
  10. Experimental procedures
  11. Plant material
  12. Antisera
  13. 2S albumin antibody purification
  14. Sucrose gradient fractionation
  15. Electrophoretic separation of proteins
  16. Affinity chromatography
  17. Peptide sequencing
  18. Transmission electron microscopy
  19. High-pressure freezing.
  20. Freeze-substitution
  21. Embedding and sectioning
  22. Immunogold labelling, staining and observation
  23. Acknowledgements
  24. Supplementary material
  25. References
  26. Supporting Information

Figure S1  Estimation of response linearity and detection limits of pure methyl ester (ME) standards analyzed by isobutane CI-GC/MS SIM at the levels of 10 pg, 100 pg and 1 ng (n=3). Note: log/log scale. A) benzoic acid (BA-ME; r2=0.998), B) salicylic acid (SA-ME; r2=0.992), C) cinnamic acid (CA-ME; r2=0.996), D) jasmonic acid (JA-ME; r2=0.990), E) indole-3-acetic acid (IAA-ME; r2=0.991), F) abscisic acid (ABA-ME; r2=0.996), G) linolenic acid (18:3-ME; r2=0.994), H) 12-oxo-phytodienoic acid (OPDA-ME; r2=0.992), and I) coronatine (COR-ME; r2=0.985). Selected ions monitored are given in the lower right hand corner of each figure (A-I). Given the peak area and slope of the regression line, 1 pg of material will be below the detection limit for all compounds with the possible exception of A) BA-ME and D) JA-ME.

Figure S2  Arabidopsis tissue (g FW) requirement (mixture of mock inoculated control and Pst DC3000 infected plants) and detection limit for VPE isobutane CI-GC/MS SIM peak areas of selected analytes using 1 mg, 10 mg, and 100 mg leaf tissue. Note: log/log scale. A) benzoic acid (BA-ME; r2=0.866), B) salicylic acid (SA-ME; r2=0.939), C) cinnamic acid (CA-ME; r2=0.975), D) jasmonic acid (JA-ME; r2=0.973), E) indole-3-acetic acid (IAA-ME; 100 mg requirement), F) abscisic acid (ABA-ME; r2=0.970), G) linolenic acid (18:3-ME; r2=0.935), H) 12-oxo-phytodienoic acid (OPDA-ME; r2=0.964), and I) coronatine (COR-ME; 10 mg requirement). Selected ions monitored are given in the lower right hand corner of each figure (A-I). Circled regions represent 3 important deviations which include the following: 1) shallow BA-ME slope indicates background contamination with BA-ME, 2) no detectable IAA-ME ions in the 1 to 10 mg tissue samples, 3) no detectable COR-ME ions in the 1 mg tissue samples. Based on these results BA-ME levels cannot be accurately reported and the minimum tissue levels required for IAA and COR are 100 mg and 10 mg, respectively. Highly linear recovery from a plant matrix of all spiked free acid analytes as MEs (in the range of 60-1000 pg/injection) has previously been reported (Schmelz et al., 2003). Selected ions monitored are given in the lower right hand corner of each figure (A-I). Note: For the purposes of establishing variability and reproducibility, total selected ion peak area is plotted for the detection limits; thus, the variability between data points represents variation associated with the plant tissue aliquots, weighing and all steps of the VPE CI-GC/MS analysis. VPE conditions were 500 ml min-1, at 200 C for 2 min.

Figure S3  Representative isobutane CI-GC/MS spectra of the predominant [M+H]+ parent ions of A) SA-ME (rt=8.44 min, m/z=153), B) CA-ME (rt=10.10 min,m/z=163), C) JA-ME (rt=trans 12.38 min / cis 12.61 min, m/z=225), D) IAA-ME (rt=13.47 min, m/z=190), E) ABA-ME (rt=15.36 min, m/z=279), F) 18:3-ME (rt=15.53 min, m/z=293), G) OPDA-ME (rt=trans 17.08 min / cis 17.52 min, m/z=307) and H) COR-ME (rt=19.55 min, m/z=334). ABA-ME was the only analyte producing significant [M-H20+H]+ fragmentation; thus, 261 was used for ABA-ME quantification purposes. Identical to the situation with A) SA-ME and F) 18:3-ME, BA-ME, linoleic acid-ME, oleic acid-ME, and steric acid-ME also produced predominantly [M+H]+ parent ions with no significant fragmentation.

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