JXB Advance Access originally published online on March 28, 2008
Journal of Experimental Botany 2008 59(6):1327-1340; doi:10.1093/jxb/ern039
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Peripheral membrane proteins mediate binding of vacuolar storage proteins to membranes of the secretory pathway of developing pea cotyledons
1Strukturelle Zellphysiologie, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Georg-August Universität Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany
2Biochemie der Pflanze, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Georg-August-Universität Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen, Germany
3Zellbiologie, Heidelberger Institut für Pflanzenwissenschaften, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany
* To whom correspondence should be addressed. E-mail: giselbert.hinz{at}uni-heidelberg.de
Received 22 January 2008; Revised 22 January 2008 Accepted 23 January 2008
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Abstract |
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In developing pea cotyledons, storage proteins are sorted via dense vesicles into the protein storage vacuole. Formation of these unique transport vesicles is characterized by aggregation of their cargo proteins. Protein sorting into dense vesicles is pH dependent. In order to gain insight into the molecular basis of storage protein sorting, a membrane binding assay was developed which allows for a detailed biochemical analysis of binding events. Employing this assay it was possible to show that storage proteins bind in a pH-dependent manner to the membranes of the secretory pathway with a pH optimum in the range of the lumenal pH of the Golgi cisternae. Through reconstitution experiments, it was possible to demonstrate further that this recruitment occurs via the interaction of peripheral rather than intrinsic membrane proteins. Results of co-immunoprecipitation experiments point to interactions between different storage proteins in the secretory system. These results are discussed in terms of the aggregation-mediated sorting of storage proteins into maturing dense vesicles.
Key words: Dense vesicles, Golgi apparatus, legumin, pea, receptor, sorting
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Introduction |
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The nutritional value of plant seeds strongly depends on their content of specialized proteins, termed seed storage proteins. These proteins are deposited during the maturation phase of the seed in specialized organelles, the protein storage vacuoles (PSV). Like the lytic vacuoles, PSV are part of the secretory system of the cell and receive their proteins through the secretory pathway. Despite the importance of these proteins for human nutrition, transport of seed storage proteins into the PSV is only partially understood.
Segregation of vacuolar hydrolases destined for the plant lytic vacuole is mediated by the high affinity interaction of conserved sequence-specific vacuolar sorting determinants (ssVSD) (Matsuoka and Neuhaus, 1999) within the protein with a vacuolar sorting receptor protein (termed either BP-80, AtELP, or AtVSR) in the membrane of the trans-Golgi network (Jiang and Rogers, 2003). These ligand/receptor complexes are then recruited into a highly conserved class of vacuolar transport vesicles, the clathrin-coated vesicles (CCV) (Robinson et al., 1998a; Rouillé et al., 2000). Some proteins of the PSV, such as ricin, also possess such an ssVSD (Joliffe et al., 2004) and may, therefore, also be sorted via the AtVSR/CCV pathway into the PSV (Shimada et al., 2003; Joliffe et al., 2004; Vitale and Hinz, 2005).
However, evidence is accumulating that other sorting mechanisms into the PSV may also exist in addition to this AtVSR1/CCV-mediated pathway. The most abundant seed storage proteins, like, for instance, legumin in pea and field bean, glycinin in soybean, or phaseolin in garden bean, do not possess such conserved ssVSD. They possess either C-terminal hydrophobic VSD (ctVSD), or protein structure-dependent VSD (psVSD), sharing little or no conserved sequence homologies (Matsuoka and Neuhaus, 1999; Vitale and Hinz, 2005). Although results showing that sorting of storage proteins bearing ctVSD is saturable (Neuhaus et al., 1994; Frigerio et al., 1998) have led to the assumption that sorting of these proteins might also be mediated by a receptor, these proteins do not bind, or bind with low affinity, to AtVSR1 (Kirsch et al., 1994, 1996; Cao et al., 2000). Furthermore, split GFP fluorescence life-imaging experiments have shown that a reporter bearing the ctVSD of chitinase does not bind to AtVSR1 in vivo (Park et al., 2007). Recent reports indicate that an alternative plant vacuolar sorting receptor, which seems to be responsible for the sorting of proteins bearing a ctVSD, AtRMR1, does indeed exist (Jiang et al., 2000; Jiang and Rogers, 2003; Park et al., 2005, 2007).
Morphological observations of the sorting of storage proteins in developing pea and Arabidopsis cotyledons have led to the postulation of an additional vesicular sorting pathway into the PSV. The precursors of pea seed storage proteins, prolegumin, vicilin, and sucrose-binding protein (SBP) homologue and the Arabidopsis storage protein cruciferin are not transported via CCV but rather via a separate class of vacuolar transport vesicles, the dense vesicles (DV), instead (Hohl et al., 1996; Wenzel et al., 2005; Hinz et al., 2007). Moreover, sorting of storage proteins into DV is spatially segregated from the AtVSR-mediated sorting of proteins into CCV. Whereas AtVSR and CCV are both detectable at the trans-cisternae of the Golgi-stack, sorting of storage proteins into DV, by contrast, occurs in the cis half of the stack (Robinson et al., 1997; Hillmer et al., 2001; Hinz et al., 2007), and co-localizes with AtRMR1 but not with AtVSR1 (Hinz et al., 2007).
Recent results published by Otegui et al. (2006) also indicate the presence of two different vesicular pathways in Arabidopsis seeds. Genetic evidence further supports this notion. Rojo and co-workers (Sanmartin et al., 2007) could demonstrate that the two vacuolar sorting pathways in Arabidopsis are dependent on two distinct SNARE proteins, VTI11 and VTI12, where VTI11 is involved in targeting into the lytic vacuole and VTI12 in sorting into the storage vacuole.
The formation of DV is only poorly understood. Independent observations have led to the conclusion that the formation of DV seems to be more complicated and that sorting of proteins into DV might not occur exclusively through ‘classical’ ligand/receptor interactions such as those described for the vacuolar sorting receptor (VSR)-mediated sorting of vacuolar hydrolases into CCV (Rouillé et al., 2000). Results of Holkeri and Vitale (2001), demonstrating that the three sorting determinants present in the phaseolin trimer—the 7S storage globulin of bean seeds—act cumulatively, gave a first hint that binding of the storage proteins to putative receptors might not occur with the same stoichiometry as described for the ssVSD/CCV pathway.
Based on the observations that prolegumin forms membrane-associated, high salt-resistant aggregates and that SBP forms detergent-insoluble complexes in the membrane of the Golgi apparatus (Hinz et al., 1997; Wenzel et al., 2005), it seems that protein aggregation also plays a role in DV formation. Sorting of prolegumin, SBP, and vicilin into budding DV at the periphery of the cis-Golgi is accompanied by the condensation of these proteins, leading to the formation of large osmiophilic, electron-opaque aggregates, about 100 nm in diameter, within the lumen of the DV (Hohl et al., 1996; Robinson et al., 1997; Hinz et al., 1999; Wenzel et al., 2005). In addition, the cargo shows a stratified distribution within the lumen of the DV (Wenzel et al., 2005): Whereas legumin is concentrated in the lumen, SBP is concentrated around the legumin at the membrane of the DV. Because it is very unlikely that a soluble ligand can bind to a membrane receptor when located inside a large stratified protein conglomerate, it seems reasonable to assume that, at least within the DV, not every single cargo protein is associated with a single receptor molecule in the membrane. Instead, aggregation might be part of the sorting process. It might be triggered by the interaction of the storage proteins with other protein(s), via homotypic interactions of the precursor polypeptides, by the pH and ionic milieu of the lumen of the DV, or through a combination of these factors.
Recent studies about the interaction of phaseolin, the major storage protein from garden bean, with membranes further support the assumption (Castelli and Vitale, 2005) that aggregation might play an important role in the sorting process. The ctVSD-mediated interaction of phaseolin with the membranes of the secretory system leads to the formation of carbonate-resistant and sodium dodecyl sulphate (SDS)-insoluble complexes. Formation of these complexes was disturbed after deletion of the ctVSD.
To gain insight into the molecular basis of storage protein sorting into nascent DV, a membrane binding assay was developed which allows for a biochemical analysis of binding events. Employing this assay, the binding behaviour of three different storage proteins, prolegumin, vicilin, and SBP, to the membranes of the pea cotyledon secretory pathway was investigated.
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Materials and methods |
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Plant material
Pea (Pisum sativum L., var. Kleine Rheinländerin) plants were grown hydroponically in a greenhouse. Cotyledons with 8- and 9-mm-long axis diameters were collected (20–22 d after flowering) (Hoh et al., 1995).
Isolation of Golgi-enriched membrane fractions
All the following experiments were performed at 4 °C with addition of protease inhibitors.
Golgi-enriched fractions were isolated from pea cotyledons according to an established, standard protocol via isopycnic gradient centrifugation as described previously (Hinz et al., 1997, 1999; Robinson and Hinz, 2001). Sixty grams of testa-free cotyledons were homogenized in a mixer with 2 ml g–1 fresh weight of slushy frozen medium A of 0.3 M sorbitol, 50 mM MOPS–KOH, pH 6.5, 3 mM EDTA, 0.5 mM MgCl2, and the protease inhibitors 2 µg ml–1 leupeptin, 2 µg ml–1 aprotinin, 1 µg ml–1 trans-epoxysuccinyl-L-leucylamido-(4-guanido)-butan (E-64), 0.7 µg ml–1 pepstatin, and 1 mM o-phenanthroline. The homogenate was filtered through two layers of paperfleece (Schleicher und Schüll, Dassel, Germany) and then centrifuged for 10 min at 200 g. The supernatant was centrifuged for 20 min at 16 000 g. The supernatant was then layered onto a sucrose step gradient [7 ml 20%, 7 ml 35%, 7 ml 41% and 5 ml 55% sucrose (w/w) in medium A (50 mM MOPS pH 6.5, 3 mM EDTA, 0.5 mM MgCl2)] and centrifuged for 180 min at 110 000 g in a swing-out rotor (AH 629; Kendro, Newtown, USA). The 20–35% sucrose interphase was harvested, pooled, diluted at least 2-fold in medium A, and sedimented for 60 min at 120 000 g in a swing-out rotor. The sediment was immediately frozen in liquid nitrogen and stored at –80 °C.
Membrane binding assay
Forty milligrams of isolated Golgi membranes were stripped with 10 ml of 50 mM TRIS-HCl pH 7.5 and 800 mM NaCl including one freeze/thawing step, divided in four equal portions, and then pelleted at 100 000 g for 45 min in a Beckman TL-100.3 rotor. Each of the sediments was resuspended in 0.25 ml of the appropriate binding buffers (acidic buffers, 50 mM MES–TRIS and 150 mM NaCl, or neutral buffers, 50 mM TRIS-HCl and 150 mM NaCl). The pH values and the ionic conditions of the supernatants were adjusted by dialysing against the appropriate buffers. After dialysis, precipitated protein was sedimented by centrifugation for 45 min at 100 000 g. Equal amounts of volume from the supernatants and the resuspended aggregates were precipitated with methanol/CHCl3 (Wessel and Flügge, 1984) and analysed on SDS–PAGE.
In the binding assay, 1 ml of dialysed and cleared supernatant was incubated with 0.1 ml of high salt-washed membranes at the appropriate pH values for 3 h on a rotator. Controls were performed by incubating the supernatant alone without added membranes (1, control) and by incubating the membranes with buffer instead of cleared high-salt supernatant (2, control). The suspensions were then centrifuged at 100 000 g for 45 min. Equal amounts of volume from the supernatant and the resuspended membrane sediment were precipitated and analysed as described for the first binding assay.
For the reconstitution of membrane binding, first the Golgi membranes were stripped with 1 M Na2CO3, pH 11.5 (Hatefi and Hanstein, 1976; Castelli and Vitale, 2005) including three freeze/thawing steps, and the supernatant dialysed against pH 6.0 or pH 7.5. The resulting aggregates were pelleted by centrifugation for 45 min at 100 000 g. Equal amounts of the supernatants and of the pH-adjusted carbonate-stripped membranes were incubated overnight at pH 6.0 or pH 7.5, followed by centrifugation at 100 000 g for 45 min. To the resuspended membrane sediments high-salt supernatant was added. Incubation, centrifugation, and analysis were performed as described for the first binding assay.
For the isopycnic gradient centrifugation, 180 µl of membranes achieved after the membrane binding assay at pH 6.0 were layered on top of 4.2 ml linear sucrose gradients (10–50%, w/w) and centrifuged for 15 h at 120 000 g in a swing-out rotor (Sorvall-AH-650). One hundred microlitres of each of the 10 fractions, collected from the top of the gradients, were precipitated with methanol/chloroform and analysed on SDS–PAGE.
Antibodies and immunoprecipitation
Polyclonal antibodies raised in rabbits against the pea legumin (Hinz et al., 1999) were used in a dilution of 1:15 000 for western blots. Polyclonal antibodies raised in rabbits against the Vicia faba SBP were used in a dilution of 1:5000 on western blots (Heim et al., 2001; Wenzel et al., 2005). Polyclonal antibodies against vicilin were raised in rabbits and used in a dilution of 1:5000 on western blots (Hohl et al., 1996). Polyclonal antibodies against the RMR protein were raised in rabbits against the lumenal domain of the protein, affinity purified against the peptide (Jiang et al., 2000), and used in a concentration of 1 µg ml–1 on western blots. Polyclonal antibodies against the Arabidopsis AtVSR1 were raised in rabbits and used in a dilution of 1 µg ml–1 (Tse et al., 2004).
Legumin antiserum or pre-immune serum was covalently coupled to protein A–Sepharose Cl4B fast flow (Sigma) via dimethyl-pimelidate as cross-linker according to the protocol published in Harlow and Lane (1999). Coupled antibodies equivalent to 10 µl of serum were added to a mixture of cleared high-salt supernatant and cleared carbonate supernatant (1+1) at pH 6.0. The solution was incubated at 4 °C overnight and the beads were washed five times in buffers containing 50 mM MES–TRIS, pH 6.0, 150 mM NaCl, 1% NP-40, and 0.1% SDS. The final pellet was resuspended in Laemmli sample buffer (2.5% SDS), boiled for 5 min, and the Sepharose beads removed by pelleting for 5 min at 2000 g.
Gel electrophoresis, protein gel blotting, and protein determination
Prior to SDS–PAGE, proteins were precipitated with methanol/CHCl3 according to Wessel and Flügge (1984). SDS–PAGE was performed according to Laemmli (1970). The gels were stained with Coomassie according to the method of Neuhoff (1985).
Proteins were electroblotted onto nitrocellulose using a semi-dry blotting device from BioRad in a transfer-buffer (0.025 M TRIS, 0.192 M glycine) containing 10% (w/v) methanol for up to 90 min at 2 mA cm–2. After blocking (1% BSA, 5% fat-free dry milk), the blots were probed with appropriate primary and secondary antibodies. Secondary antibodies were coupled to horseradish peroxidase (Sigma). Visualization of the bound antibodies was with an enhanced chemoluminescence kit (Pierce).
Protein was measured according to the methods of Bradford (1976) and Peterson (1977).
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Results |
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Storage proteins bind in a pH-dependent manner to isolated membranes
In this report, the binding to isolated membrane fractions of three pea seed storage proteins, legumin, vicilin, and SBP, was investigated. The antibodies against legumin and vicilin were raised against purified fractions of mature proteins. Both proteins are coded for by small multigene families. The antisera, therefore, recognize multiple legumin or vicilin polypetides (Hohl et al., 1996; Hinz et al., 1999). The antiserum against SBP was raised against the N-terminal 194 amino acid peptide of the V. faba homologue (Heim et al., 2001). In pea the 60 kDa proSBP is processed into two polypeptides, a 48 kDa N-terminal peptide and a 16 kDa C-terminal peptide (Castillo et al., 2000). The antiserum, therefore, recognizes the 60 kDa proSBP and the 48 kDa N-terminal peptide (Wenzel et al., 2005).
The experiments were carried out with an enriched Golgi fraction, and not with isolated DV. Because of the very efficient lateral sorting of prolegumin and vicilin into the growing DV, it is very likely that, if a sorting receptor exists, it must already be present in the cisternae of the Golgi stack (Robinson et al., 1997; Hillmer et al., 2001). The membrane fraction was isolated following an established, routinely used standard protocol (Hinz et al., 1997, 1999; Robinson and Hinz, 2001). In these fractions, Golgi membranes are highly enriched as compared with the total membrane sediments, the amount of vacuolar membranes is strongly reduced, and ER membranes are the major contaminants (Hinz et al., 1997, 1999).
Binding experiments were carried out according to the following protocol. In the first step, isolated membrane vesicles were washed with high salt in order to strip off endogenous soluble and peripheral membrane proteins. In the second step, the strip supernatant was dialysed to adjust the pH and ionic composition of the solution. In the third step, the stripped membranes were re-incubated with the dialysed strip supernatant. Finally the membranes were sedimented by high-speed centrifugation and the distribution of the storage proteins between the resulting supernatant and pellet fractions was analysed by SDS–PAGE and western blotting. Recruitment to the membrane was evaluated by monitoring the disappearance of the storage proteins from the supernatant after adding the membranes, and their relative increase in the membrane sediment. To prevent proteolytic degradation of the proteins during the incubation period, the experiments were carried out on ice and a protease inhibitor mix was added (Robinson and Hinz, 2001).
Because the pH in the lumen of the Golgi apparatus as compared with the ER is assumed to be slightly acidic, in the range of pH 6.2–6.7 (Zhang et al., 1993; Llopis et al., 1998; Miesenböck et al., 1998; Porchia et al., 2002), the first experiment was performed at a pH of 6.0. However, during dialysis of the high-salt strip supernatant against the acidic binding buffer, progressive clouding of the solution was observed. Isoelectric precipitation might be a reason for this insolubility, as described for mature legumin (Gueguen et al., 1988). To test for this, dialysis at different pH values was performed. After dialysis the samples were centrifuged and the soluble and insoluble proteins were analysed by SDS–PAGE and western blotting. Coomassie staining of the gel, as shown in Fig. 1B, revealed a differential precipitation behaviour of the proteins; some proteins precipitated at all pH values tested (e.g. the protein marked with an asterisk), some remained in the supernatant (e.g. the protein marked with a dot), and some showed a pH-dependent precipitation (e.g. the protein marked with a diamond). Similar to mature legumin (Gueguen et al., 1988) precipitation of prolegumin was much more pronounced at an acidic pH value as compared with pH 6.9 and pH 7.3, as shown in Fig. 1A. SBP showed a similar precipitation behaviour, but only one of the vicilin polypeptides (60 kDa) showed a pH-dependent precipitation as prolegumin and SBP did.
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Because a certain amount of prolegumin, SBP, and 60 kDa vicilin, respectively, remained in the supernatant after sedimenting the insoluble proteins (Fig. 1A), the following binding experiments were performed with this cleared high-salt strip supernatant.
In a first experiment, a test was carried out to see if the high salt-washed membranes were able to recruite storage proteins out of the cleared high-salt strip supernatant at a pH of 6.0. Two controls were used: (i) cleared supernatant was incubated without added membranes to monitor the disappearance of the storage proteins from the supernatant; and (ii) the membranes were incubated in buffer instead of cleared high-salt supernatant to follow the increase of the storage protein concentration in the membrane fraction.
As shown in Fig. 2A, after incubation with the membranes, the amount of legumin, vicilin, and SBP in the supernatant was strongly reduced as compared with the control without added membranes (Fig. 2A, lane 2 versus the control, lane 1). The disappearance of the storage proteins from the supernatant was not due to unspecific precipitation, because the proteins were nearly absent in the sediment of the first control without added membranes (Fig. 2A, lane 5). The loss of SBP and vicilin from the supernatant was paralleled by an increase of both proteins in the membrane fraction after incubation (Fig. 2A, lane 3 versus the control, lane 4). The binding efficacy of prolegumin varied between different experiments and it was less pronounced as compared with the binding of SBP and vicilin. However, several independent experiments (data not shown) confirmed the binding of prolegumin to the membranes.
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Coomassie staining of the gel revealed the specificity of the membrane recruitment: out of the bulk of proteins present in the supernatant only some minor proteins were recruited to the membranes together with the storage proteins (Fig. 2B, proteins marked with asterisks).
To verify whether the proteins really bound to the membranes or formed unspecific aggregates only, the membranes were resuspended after the binding experiment, layered on top of a linear sucrose gradient, and re-centrifuged under isopycnic conditions. As shown in Fig. 3A, all three proteins peaked in the gradient. No significant unspecific pellet was detectable.
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Because the membrane fraction used contained Golgi as well as ER membranes, the question arose whether the observed binding was due to the ER or the Golgi fraction. Because attempts to isolate ER fractions depleted of Golgi vesicles failed, the pH dependence of membrane binding was tested in more detail. As shown in Fig. 4A, binding was indeed pH dependent; binding of vicilin and SBP was pronounced at a pH of 6.1 and 6.5, respectively. By contrast, at pH 6.9, 7.3 (Fig. 4A), and 7.5 (Fig. 4B) binding of vicilin was totally, and that of SBP mostly, abolished. This observed pH dependence is in agreement with the slightly acidic environment in the lumen of the Golgi apparatus as compared with the neutral ER lumen (Zhang et al., 1993; Llopis et al., 1998; Miesenböck et al., 1998; Porchia et al., 2002).
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Extrinsic membrane proteins participate in prolegumin binding
To answer the question whether the interaction with peripheral membrane proteins was responsible for the observed binding, membranes were treated with 1 M sodium carbonate, pH 11.5 (Hatefi and Hanstein, 1976). These carbonate-stripped membranes were then incubated at pH 6.0 with the cleared high-salt supernatant which had successfully been used in the experiments described above. As shown in Fig. 5, binding was inhibited and nearly all proteins remained in the supernatant. Only a small proportion of SBP associated with the membranes.
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This inhibition of binding could be due to the fact that the carbonate-treated but not the high salt-washed membranes were depleted of some peripheral membrane protein(s) which were essential for the binding reaction. These proteins, therefore, would not be present in the high-salt supernatant and thus be missing in the experiment performed with high-salt supernatant and carbonate-stripped membranes. To test this, a reconstitution assay was performed prior to the membrane binding experiment: the carbonate-washed membranes were re-incubated with the dialysed and cleared carbonate strip supernatant, thus leading to a possible re-association of proteins out of the carbonate supernatant with the membranes. Because binding had been proved to be pH dependent, the re-association of proteins out of this cleared carbonate supernatant was carried out at pH 6.0 or at pH 7.5. As shown in Fig. 6A, out of the carbonate supernatant neither prolegumin, vicilin, nor SBP became recruited to the carbonate-stripped membranes (western blots, lane 2 versus the control, lane 1). Possibly, the storage proteins became denatured after carbonate treatment as described for mature legumin (Gueguen et al., 1988). Coomassie staining of the gel revealed that proteins with apparent molecular masses of 12 kDa and 31 kDa disappeared partly from the supernatant independent of the pH of the medium (panels A and B, proteins marked with asterisks). This was not due to unspecific precipitation, because these proteins did not pellet in the control without added membranes (Fig. 6A, B, lane 5). This observation strengthened the assumption that a successful in vitro re-association of extrinsic membrane proteins with carbonate-stripped membranes was indeed possible.
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To test whether storage proteins were able to bind to these reconstituted membranes, these membranes were in turn re-incubated with high-salt strip supernatant at pH 6.0. As shown in Fig. 7A, after incubation with reconstituted membranes, all three storage proteins were as efficiently removed from the high-salt supernatant (lane 3 versus the control, lane 1) as was the case after incubation with high-salt-stripped membranes (lane 2 versus lane 1). Binding was independent whether the reconstitution had been performed at pH 6.0 or at pH 7.5 Fig. 7A, B).
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Two type I membrane proteins of the secretory pathway are discussed as putative storage protein receptors: AtVSR1 (Shimada et al., 2003; Fuji et al., 2007) and AtRMR1 (Jiang et al., 2000; Jiang and Rogers, 2003; Park et al., 2005, 2007). Because the membranes were stripped with very high stringency these two proteins may have been removed from the membrane and the reconstitution may be due to re-association of either of the two proteins, or both. To test for this, the carbonate-stripped membranes were probed with antibodies against both proteins. The antibody against AtRMR1 was raised against the lumenal domain of the 50 kDa RMR-JR702 isoform (Jiang et al., 2000). As shown in Fig. 8, it specifically recognizes a homologue with an apparent molecular mass in SDS–PAGE of about 55 kDa in pea. After stringent carbonate stripping, both proteins, cross-reacting with antibodies directed against AtVSR1 and AtRMR1, were still detectable in the membrane pellet.
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Storage proteins form heterologous aggregates in the secretory pathway
Because prolegumin was only weakly recruited to the membranes, as compared with SBP or vicilin, it is possible that prolegumin was indirectly recruited to the membranes via interactions with other proteins like SBP. An indication that this might indeed be the case stems from the observation that prolegumin forms higher molecular weight aggregates within the DV as compared with the endoplasmic reticulum (Hinz et al., 1999). To test for the possibility that storage proteins might form heterologous oligomers, prolegumin was immunoprecipitated using a mixture of high-salt and carbonate supernatant at pH 6.0 as antigen solution. After immunoprecipitation, western blots of the precipitated proteins were probed with legumin, vicilin, and SBP antisera to see whether other storage proteins were co-immunoprecipitated. The western blots are shown in Fig. 9. The smeary high molecular weight bands in the western blots probably correspond to the unspecific labelling of IgG aggregates because they are present in all lanes including the non-immune serum control (Fig. 9). All three proteins are synthesized as high-molecular-weight precursor polypeptides which are cleaved to the mature proteins at different stages of development (Croy et al., 1980a, b; Castillo et al., 2000; Heim et al., 2001). As compared with the non-immune serum control, the legumin antibody recognized a single immunoprecipitated polypeptide of 60 kDa which corresponds to the molecular mass of prolegumin (Croy et al., 1980a; Hinz et al., 1999). The vicilin antibody recognized several co-immunoprecipitated polypetides with apparent molecular masses in SDS of about 70, 60, 50, 30, and 20 kDa, respectively. These polypeptides correspond to uncleaved (the 70, 60, and 50 kDa) and cleaved (30 and 20 kDa) vicilin polypeptides (Croy et al., 1980b; Hohl et al., 1996). The SBP antibody recognized two polypeptides with apparent molecular masses in SDS of about 60 kDa and 48 kDa which correspond to the molecular masses of the uncleaved (60 kDa) and the cleaved (48 kDa) SBP polypetides (Castillo et al., 2000; Heim et al., 2001; Wenzel et al., 2005). The SBP antibody used was raised against SBP from V. faba (Heim et al., 2001). Because of the very similar molecular masses of SBP as compared with the vicilin polypeptides, Heim et al. (2001) confirmed the specificity of the serum and could exclude a cross-reactivity of the SBP antiserum with a purified vicilin fraction. The specificity of the co-immunoprecipitation was further confirmed by the observation that the legumin antibody which was employed for the immunoprecipitation recognized the 60 kDa prolegumin only but none of the other proteins which were recognized by the vicilin or SBP antibodies in the western blots. Furthermore, the 70 kDa as well as the low-molecular-weight vicilin polypeptides were not recognized by the SBP antibody.
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Discussion |
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In this report, an in vitro binding assay was presented which allows for biochemical investigations of DV formation in their native tissue, the seed. It circumvents, therefore, problems arising from the expression of seed proteins in heterologous non-seed tissues, probably not bearing a storage vacuole at all (as discussed by Robinson et al., 2005). Pure, in vitro transcribed/translated proteins were not employed for these experiments, because it was not known in advance whether other soluble proteins of the Golgi apparatus might also participate in membrane binding, as is the case for sorting of secretory proteins into secretory granules in mammalian glands (Dartsch et al., 1998). In addition, this technique allows the binding of different storage proteins to be followed in parallel.
We believe that the results achieved by this assay are reliable for the following reasons. (i) A proteolytic degradation of the proteins in the supernatant during the incubation period could be excluded because experiments were carried out on ice, a protease inhibitor mix was added (Robinson and Hinz, 2001), and proteolytic breakdown products and unspecific precipitates were not observed. (ii) Sedimentation of the storage proteins depended on the presence of membranes. (iii) Only a small subset of the proteins present in the supernatant were recruited to the membranes. (iv) The proteins were found again in the membrane fraction after isopycnic re-centrifugation of the membranes after the binding assay. (v) SBP and vicilin showed an increase in the pellet fraction after the incubation with the membranes.
One of the results achieved by this experiment was that storage proteins tend to precipitate at a slightly acidic pH (Fig. 1), as assumed to be present in the Golgi apparatus (Zhang et al., 1993; Llopis et al., 1998; Miesenböck et al., 1998; Porchia et al., 2002). For prolegumin it might be due to isoelectric precipitation, as described for mature legumin (Gueguen et al., 1988) and for the soy bean storage protein conglycicin (Mori et al., 2004). Accordingly, the isoelectric point of pea SBP is also about pH 6.0. Precipitation seems to be concentration dependent; below a threshold concentration the proteins remained soluble (Fig. 1). Whether this precipitation reflects a physiologically relevant aggregation process remains to be elucidated.
Aggregation seems not to be a prerequisite for binding of the storage proteins to the Golgi membranes, because binding occurred after the insoluble precipitates had been removed out of the supernatant. Several reports support the assumption that aggregation might indeed be involved in DV formation. Morphological observations on developing pea seeds have demonstrated that a perturbation in the electrochemical gradient of the Golgi apparatus leads to secretion of the proteins into the apoplast (Craig and Goodchild, 1984). This mis-targeting, in turn, is correlated with a swelling of the DV attached to the Golgi cisternae and a dispersal of the electron-opaque protein aggregates in their lumen (Robinson et al., 1998b). Furthermore, when present in DV, prolegumin forms higher molecular weight aggregates as compared with the 9S trimer present in the ER and Golgi cisternae (Hinz et al., 1999). In addition, the major seed storage protein of garden bean, phaseolin, transiently forms SDS-insoluble aggregates when attached to the membranes of the secretory pathway (Castelli and Vitale, 2005). Finally, storage proteins seem to form heterologous oligomers, as demonstrated by immunoprecipitation experiments in this report (Fig. 9). This result is supported indirectly by reports demonstrating that the solubility of the storage proteins strongly depends on the relative composition of these proteins in the secretory system, thus pointing to the importance of protein–protein interactions for the sorting process (Kinney et al., 2001; Mori et al., 2004).
Storage proteins seemed to have distinct binding affinities for the Golgi membranes; binding of prolegumin was relatively weak as compared with vicilin and SBP (Figs 2, 4). This might indicate that prolegumin does not bind to the Golgi membranes in the same way as SBP or vicilin do. Putatively, sorting of prolegumin might depend more on aggregation as compared with the sorting of SBP or vicilin. This would also explain the stratification of storage proteins in the lumen of DV where prolegumin in the electron-opaque lumen is surrounded by a protein sphere consisting mainly of SBP (Wenzel et al., 2005).
Binding of the storage proteins to the membranes was optimal at slightly acidic pH (Fig. 4) which is in agreement with the observation that storage proteins are mis-targeted into the apoplast after disruption of the electrochemical gradient by ionophores (Craig and Goodchild, 1984; Robinson et al., 1998b). Binding of lytic proteases bearing an ssVSD to VSR also shows a relatively sharp pH optimum of around pH 6 (Kirsch et al., 1996; Jiang and Rogers, 2003), which is in the range of the observed lumenal pH of the Golgi apparatus. The same holds true for the binding of phaseolin to RMR (Park et al., 2005).
The binding assay was employed to investigate whether the recruitment of storage proteins to the membranes might be mediated by membrane proteins of the pea Golgi apparatus. However, the results achieved by this assay indicate that the presence neither of BP-80/VSR nor of RMR was sufficient for the binding reaction: although both proteins were still detectable, binding of storage proteins to carbonate-stripped membranes was abolished (Figs 5, 8).
The presence of peripheral membrane proteins seemed to be crucial for the binding competence of the Golgi membranes (Fig. 7). Quite interestingly, whereas binding of the storage proteins is pH dependent, the reconstitution of the binding capacity itself, i.e. the association of the putative assembly factors with the pea Golgi membrane, seems to be pH independent (Fig. 7). Indeed, proteins with a similar molecular mass of 12 kDa and 31 kDa seem to be recruited to the carbonate-stripped membranes out of the carbonate supernatant at pH 6.0 or pH 7.5 (Fig. 6). The association of these proteins with the membranes might already occur in the ER.
There are examples in the literature that binding of proteins to the membranes of the secretory pathway indeed depends on the presence of peripheral membrane proteins. It has been discussed that the formation of DV in plants somehow resembles the formation of immature secretory granules in mammalian endocrine and exocrine glands (Saalbach et al., 1991; as reviewed in Robinson et al., 2005). This process may depend on condensation and the presence of so-called assembly factors in the trans-Golgi which promote membrane association and the aggregation process. These assembly factors seem to be exclusive for a given cell type and they may also fulfil different functions beside granule formation. They might themselves be regulated secretory proteins, like the chromogranins or carboxypeptidase E, or even chaperones (as reviewed in Arvan, 2004; Arvan et al., 2002; Day and Gorr, 2003; Borgonovo et al., 2006). A very elaborate mechanism has been described in rat pancreatic acinar cells, where the lumenal leaflet of the secretory granules is surrounded by a proteoglycan matrix which is linked to certain lectins. These lectins, in turn, recruit the secretory proteins to the membrane of the growing granule (Dartsch et al., 1998; Kleene et al., 1999; Schmidt et al., 2000). With respect to plant seeds this model is very appealing. One might speculate that the peripheral membrane proteins involved in the reconstitution of the binding activity of pea Golgi membranes might also be some kind of assembly factor.
Based on this, the following hypothesis for the sorting of storage proteins in the Golgi apparatus in developing pea cotyledons may now be put forward. Probably, several more or less independent sorting events may take place which may include CCV- and DV-mediated pathways in parallel (Vitale and Hinz, 2005). After their arrival in the cis-Golgi apparatus, storage proteins, like SBP and vicilin, bind to membrane proteins like RMR and/or other putative peripheral membrane proteins. These protein complexes may then serve as aggregation nuclei for other storage proteins, like prolegumin which does not show the same strong membrane-binding behaviour as SBP or vicilin. These complexes are then sorted laterally into forming DV buds at the rim of the Golgi cisternae. Inside the growing DV the concentration reaches a threshold which, together with the slightly acidic pH, induces aggregation, finally forming the electron-opaque DV core. These aggregates may then serve as a trap for free storage proteins leading to the formation of a concentration gradient which further drives the influx of protein into the forming DV. Sorting would therefore depend on several events: binding followed by lateral sorting, aggregate formation, as well as a passive concentration-driven import into the growing DV. After reaching the trans-Golgi apparatus, DV are often observed bearing a clathrin cap (Hohl et al., 1996; Robinson and Hinz, 1997; Hinz et al., 2007), as is the case in the maturation of secretory granules where these clathrin caps serve as a rescue mechanism for vacuolar hydrolases (Kuliawat et al., 1997; Kakhlon et al., 2006). Therefore, also, DV formation may not be exclusive. Proteins bearing an ssVSD may accidentally be trapped in the DV and retrieved by budding CCV at the trans-Golgi apparatus.
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Acknowledgements |
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The authors are indebted to Dr DG Robinson (University Heidelberg, Germany) and to Dr John C Rogers (NSF, Washington DC, USA) for critically reading the manuscript and, in addition, Dr John C Rogers for the gift of the AtRMR1 antibody. The authors are also indebted to Dr U Wobus (IPK Gatersleben, Germany) for the gift of the VfSBPL and to Dr L Jiang (Hong Kong) for the gift of the AtVSR1 antibody. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG-Ro-440-13-1) to DG Robinson.
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