Protein Domains Involved in Assembly in the Endoplasmic Reticulum Promote Vacuolar Delivery when Fused to Secretory GFP, Indicating a Protein Quality Control Pathway for Degradation in the Plant Vacuole
a Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, via Bassini 15, 20133 Milano, Italy, EU
b Istituto di Genetica Vegetale, Consiglio Nazionale delle Ricerche, via della Madonna Alta 130, 06128 Perugia, Italy, EU
1 To whom correspondence should be addressed. E-mail vitale{at}ibba.cnr.it, fax +39 02 23699 411, tel. +39 02 23699 431.
 |
Abstract |
|---|
 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
|---|
The correct folding and assembly of newly synthesized secretory proteins are monitored by the protein quality control system of the endoplasmic reticulum (ER). Through interactions with chaperones such as the binding protein (BiP) and other folding helpers, quality control favors productive folding and sorts for degradation defective proteins. A major route for quality control degradation identified in yeast, plants, and animals is constituted by retrotranslocation from the ER to the cytosol and subsequent disposal by the ubiquitin/proteasome system, but alternative routes involving the vacuole have been identified in yeast. In this study, we have studied the destiny of sGFP418, a fusion between a secretory form of GFP and a domain of the vacuolar protein phaseolin that is involved in the correct assembly of phaseolin and in BiP recognition of unassembled subunits. We show that sGFP418, despite lacking the phaseolin vacuolar sorting signal, is delivered to the vacuole and fragmented, in a process that is inhibited by the secretory traffic inhibitor brefeldin A. Moreover, a fusion between GFP and a domain of the maize storage protein
-zein involved in zein polymerization also undergoes post-translational fragmentation similar to that of sGFP418. These results show that defective secretory proteins with permanently exposed sequences normally involved in oligomerization can be delivered to the vacuole by secretory traffic. This strongly suggests the existence of a plant vacuolar sorting mechanism devoted to the disposal of defective secretory proteins.
Key Words: Endoplasmic reticulum • protein degradation • protein traffic • vacuole
Received for publication July 4, 2008. Accepted for publication September 15, 2008.
|
INTRODUCTION |
|---|
|
TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
|---|
The protein secretory pathway starts from the endoplasmic reticulum (ER), where newly synthesized secretory proteins are inserted, in most cases in a co-translational process. The lumen of the ER is provided with a set of folding helpers that perform two strictly related roles: they favor productive folding and assembly of newly synthesized secretory polypeptides and promote degradation of defective polypeptides that cannot fold correctly. These ER quality control functions require recognition mechanisms capable of distinguishing unfolded polypeptides that are in the process of folding from those that will be unable to fold properly, and mechanisms devoted to the disposal of the latter but not the former (recently reviewed in Anelli and Sitia, 2008, and, for plants, Vitale and Boston, 2008). The best characterized, and possibly most common, pathway of degradation by ER quality control is termed ER-associated degradation (ERAD) and involves retrotranslocation from the ER into the cytosol, ubiquitination, and final degradation by the proteasome (Anelli and Sitia, 2008). This degradation pathway, however, has alternatives. Studies in mammalian and yeast cells showed that certain structurally defective secretory proteins can fail totally or in part to undergo retrotranslocation. In certain cases, these polypeptides form aggregates in the ER that can be disposed by autophagy of portions of the ER (Teckman and Perlmutter, 2000; Kruse et al., 2006). Post-ER quality control can also occur on polypeptides that remain soluble. Perhaps the first evidence was the observation that mutated, thermodynamically unstable forms of the N-terminal domain of the phage
repressor fused to invertase were delivered to the yeast vacuole by a pathway that requires the vacuolar sorting receptor Vps10p (Hong et al., 1996). The choice between autophagy and Vps10-dependent delivery seems to be dictated by the solubility of the defective protein: the soluble form of the Z-variant of human 
-1 proteinase inhibitor uses the Golgi-mediated vacuolar sorting pathway that requires Vps10p, whereas a proportion of molecules that aggregate reach the vacuole by autophagy (Kruse et al., 2006). A number of defective plant secretory proteins are degraded by ERAD (Vitale and Boston, 2008), but studies on the trafficking of the ER chaperone BiP indirectly suggested an alternative pathway involving Golgi-mediated sorting to the vacuole (Pimpl et al., 2006). Furthermore, certain maize storage prolamins (zeins), which in maize endosperm accumulate in ER-located protein bodies, are unstable when expressed alone in transgenic plants and are found in electron-dense aggregates within vacuoles, suggesting disposal by autophagy when the partner prolamin subunits are not co-expressed (Coleman et al., 1996; Bagga et al., 1997).
The trimeric bean vacuolar storage protein phaseolin contains a C-terminal transient tetrapeptide, AFVY, which is necessary for its vacuolar sorting (Bollini et al., 1982; Frigerio et al., 1998): a mutated form devoid of the tetrapeptide correctly assembles into trimers but is secreted instead of being sorted to vacuoles (Frigerio et al., 1998). Assembly into trimers is also a prerequisite for the intracellular traffic of phaseolin. Before assembly, phaseolin monomers interact with BiP (Vitale et al., 1995). Defective mutants of phaseolin that remain monomeric and do not aggregate interact extensively with BiP before being degraded in a process that is not affected by the inhibitor of intracellular traffic brefeldin A, and probably corresponds to ERAD (Pedrazzini et al., 1997). Phaseolin contains two -helical regions, located respectively in the middle of the polypeptide and close to the C-terminus. In phaseolin trimers, the C-terminal domain directly interacts with the middle domain of an adjacent monomer. A fusion (termed sGFP418) was produced between a secretory form of GFP and a fragment of phaseolin that includes its C-terminal -helical domain but does not include the AFVY vacuolar sorting signal (Foresti et al., 2003). We previously showed that sGFP418 undergoes extensive interactions with BiP when expressed in transgenic tobacco, indicating that the domain plays a role in the interactions of phaseolin with BiP before assembly (Foresti et al., 2003). In the present work, we have investigated the final destiny of sGFP418. We show that the protein is efficiently delivered to the vacuole and the GFP portion is released, in a process that is inhibited by brefeldin A. We also show that very similar processing occurs when a fusion between GFP and a domain of the maize storage protein -zein involved in protein body formation is expressed in tobacco. Protein bodies are large polymers formed by cereal storage protein of the prolamin class in the ER and in many cases accumulated there (Shewry and Halford, 2002). These results indicate that protein domains that are involved in oligomerization within the secretory pathway can lead to vacuolar sorting when they remain permanently exposed, supporting the existence of an involvement of the plant vacuole in protein quality control.
|
RESULTS |
|---|
|
TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
|---|
sGFP418 Undergoes Proteolytic Processing that Requires Traffic Along the Secretory Pathway
In protoplasts isolated from leaves of transgenic tobacco, sGFP418 is the major newly synthesized ligand of BiP. This was demonstrated by pulse-labeling with radioactive amino acids followed by immunoprecipitation with anti-GFP or anti-BiP antiserum (Foresti et al., 2003). To investigate the final destiny of this strong BiP ligand, we performed pulse-chase labeling. Protoplasts isolated from leaves of transgenic tobacco expressing sGFP418 were labeled for 30 min with a mixture of 35S Met and 35S Cys followed by 0, 1, 8, or 24 h chase. Immunoprecipitation with anti-GFP antiserum showed a decrease in the abundance of intact sGFP418 during the chase (Figure 1, arrowhead; apparent molecular mass 37 kDa) and the appearance, starting at 8 h chase, of a polypeptide of lower molecular mass that becomes more abundant at 24 h chase (Figure 1, arrow; molecular mass around 28 kDa). The minor polypeptide already detectable at 0 h chase, with molecular mass intermediate between intact sGFP418 and the 28-kDa processing product, is probably the result of minor alternative translation (Foresti et al., 2003) and is therefore not relevant for our study. Immunoprecipitation of proteins present in the protoplast incubation medium indicated that there was no secretion of sGFP418 (Figure 1; the detection of a minor amount of sGFP418 in the medium is due to contamination from protoplasts, because, at each chase point, the relative abundance of the apparently secreted polypeptides parallels the corresponding intracellular patterns). When pulse-chase was performed in the presence of brefeldin A (BFA), this processing event was almost completely inhibited (Figure 1). BFA causes intermixing of the ER and the Golgi complex and inhibits intracellular traffic to vacuoles or secretion. Overall, the results shown in Figure 1 indicate that sGFP418 traffics along the secretory pathway to an intracellular compartment and that this traffic causes proteolytic processing.
|
We next determined if the processing product of sGFP418 remains available for in-vivo interactions with BiP. This was tested by pulse-chase labeling of protoplasts and immunoprecipitation with anti-BiP antiserum. Processed sGFP418 formed during the chase was not co-selected (Figure 2A). We conclude that processing of sGFP418 is accompanied by a release from BiP interactions. The amount of intact sGFP418 co-selected with the chaperone decreases during the chase (Figure 2A), in parallel with the processing detected by immunoprecipitation with anti-GFP antiserum (Figures 1 and 2B): this supports the hypothesis that sGFP418 polypeptides interacting with BiP are not a separate subset of molecules with respect to those that eventually undergo processing. The results strongly suggest that sGFP418 polypeptides undergo cycles of binding and release from BiP that are abolished upon proteolytic processing. Approximate quantification, performed by comparing sGFP418 immunoselected with anti-GFP or anti-BiP antiserum after 1 h pulse-labeling (Figure 2B), indicated that about 25% of total of newly synthesized sGFP418 molecules were associated to BiP at a given time-point. However, this is probably an underestimation: a second round of immunoselection of the same protoplast homogenates (not shown) indicated that a higher proportion of antigen failed to be immunoselected in the first round by the anti-BiP compared to the anti-GFP antiserum. This is most probably because BiP is a very stable and abundant protein, as also indicated by the fact that only a very small proportion of labeled BiP is co-selected with sGFP418 when immunoprecipitation is performed with anti-GFP antiserum (Figure 2B).
|
sGFP418 Is Sorted to the Vacuole
The post-translational processing product of sGFP418 has molecular mass very similar to the vacuolar processing product of sGFPAFVY (Frigerio et al., 2001a). The latter is a fusion between sGFP and the C-terminal AFVY tetrapeptide that constitutes the vacuolar sorting signal of phaseolin (Frigerio et al., 2001a). The release of a GFP ‘core’ has also been observed when secretory GFP was fused to other plant vacuolar sorting signals, and has been considered as an indicator of vacuolar targeting of GFP fusions (daSilva et al., 2005, and references therein). We therefore investigated whether sGFP418 is sorted to the vacuole.
We first determined by protein-blot analysis the steady-state SDS–PAGE pattern of sGFP418 in transgenic tobacco leaves, using as references sGFPAFVY and the ER-located sGFPHDEL (Foresti et al., 2003). Intact sGFP418 was detectable by anti-GFP antiserum, but most of the protein accumulated as the lower molecular mass processed form, which had an electrophoretic mobility indistinguishable from that of the vacuolar, processed form of sGFPAFVY (Figure 3A, arrow). sGFPHDEL was detected as a very abundant intact polypeptide and a much less abundant component that also co-migrated with processed sGFP418, in agreement with previous results obtained by transient expression of sGFPHDEL in tobacco protoplasts (Brandizzi et al., 2003). The processed forms of sGFP418 and sGFPAFVY accumulated to similar amounts, whereas shorter exposure of the blot clearly showed that the accumulation of sGFPHDEL was much higher (Figure 3B). The difference in accumulation is probably due to the ER localization of sGFPHDEL, but we cannot exclude that also different mRNA levels contribute, since we have not performed chase experiments with sGFPHDEL or RNA blot comparisons.
|
To investigate the sub-cellular localization of processed and unprocessed sGFP418, sub-cellular compartments were separated by isopycnic sucrose gradient centrifugation of leaf homogenates, prepared in the absence of detergent. Soluble vacuolar proteins migrate on top of these gradients, because vacuoles break during homogenization (Pedrazzini et al., 1997). Consistently, processed sGFPAFVY is exclusively found in the top fractions, whereas intact sGFPAFVY is also present in sub-cellular fractions that contain BiP, as expected for the newly synthesized form still present in the ER (Frigerio et al., 2001a). Similarly, processed sGFP418 migrated on top of the gradient and unprocessed sGFP418 co-migrated with BiP (Figure 4A and 4B). Intact sGFPHDEL co-migrated with BiP, as expected for an ER-located protein (Figure 4C). These results indicate that unprocessed and processed sGFP418 are in different compartments, consistently with the results of pulse-chase and BFA treatment, and suggest that the processed form is located in the vacuole. The minor, processed form of sGFPHDEL appears similarly located in vacuoles, since it also migrates in the top fractions of the gradient (Figure 4C). It has been reported that recombinant proteins modified by the addition of ER-localization tetrapeptides are in minor amounts delivered to plant vacuoles (Gomord et al., 1997; Frigerio et al., 2001b).
|
Vacuoles were purified from protoplasts isolated from transgenic plants expressing sGFP418. When a total protoplast extract and a vacuolar extract containing the same activity of the vacuolar marker
-mannosidase were compared using anti-BiP antiserum, the results showed that the vacuolar preparation had only very minor contamination by ER-derived microsomes (Figure 4D). Anti-GFP antiserum did not detect sGFP418 in these two samples, indicating that the protein was not abundant enough in the two preparations to be detectable. However, when a four-fold excess of purified vacuoles was analyzed, processed sGFP418, but not the unprocessed precursor, was clearly detected (Figure 4E). Altogether, the results presented in Figures 3 and 4 indicated that the processed form of sGFP418 is located in vacuoles.
A Fusion Between -Zein N-terminal Domains and GFP Undergoes Post-Translational Release of the GFP ‘Core’
In maize seed endosperm, the storage protein -zein forms disulfide-bond linked polymers that are integrated into ER-located protein bodies (PB) together with the other zeins. Gamma-zein forms PB also in leaves of transgenic tobacco (Bellucci et al., 2000). A fragment of -zein roughly corresponding to the N-terminal half of this protein promotes extensive interactions with BiP and polymerization into insoluble ER-located PB when fused to phaseolin in the chimeric protein zeolin, thanks to the formation of inter-chain disulfide bonds (Mainieri et al., 2004; Pompa and Vitale, 2006). Because the -zein fragment and the phaseolin domain used to produce sGFP418 share the ability to promote association to BiP, we produced a fusion between the -zein fragment and GFP and started to investigate its destiny. The chimeric protein (termed zein–GFP) is composed of the first 112 amino acids of -zein, including the N-terminal signal peptide, followed by a flexible linker, a thrombin cleavage site and finally GFP. Zein–GFP was expressed in transgenic tobacco under the control of the 35S CaMV promoter.
Protein blot was first performed, to compare the accumulation, solubility, and banding pattern of zein–GFP with those of other zein-fusions and wild-type tobacco as a control. Two chimeric proteins containing the -zein domain were used for comparison: zein–Nef and zeolin–Nef. Zein–Nef is identical to zein–GFP apart from the replacement of the GFP coding sequence with that of the Human Immunodeficiency Virus protein Nef, which is a cytosolic protein that is degraded by quality control when introduced into the plant ER. Zein–Nef is also degraded in a rapid quality control process that is not inhibited by BFA and therefore most probably corresponds to ERAD, indicating that the zein domain is unable to rescue Nef (de Virgilio et al., 2008). Zeolin–Nef is a fusion between zeolin and Nef. It forms PB, albeit with lower efficiency than zeolin, and is not degraded by quality control, thus accumulating to levels much higher than Nef or zein–Nef (de Virgilio et al., 2008).
Proteins were extracted from leaves of transgenic plants or wild-type tobacco with buffer that was supplemented (Figure 5, lanes R) or not (lanes N) with the reducing agent 2-mercaproethanol. Immunodetection was with anti--zein antiserum. Zein–GFP was detected as a polypeptide with apparent molecular mass around 43 kDa, which was fully solubilized in reducing conditions but insoluble in the absence of reducing agent (Figure 5, empty arrowhead). A faster migrating polypeptide (molecular mass around 37 kDa) was detected also in extracts from plants expressing the other zein fusions, and, more importantly, a component which could represent the same protein was present in most of the different plants, including wild-type, in the fraction that was only solubilized in the presence of SDS (Figure 5, lanes I). It seems therefore that this polypeptide is not related to the recombinant constructs. Both zein–Nef and the very abundant zeolin–Nef had solubility similar to that of zein–GFP (empty and filled circle, respectively, in Figure 5; the presence of a minor polypeptide co-migrating with zein–Nef in the zeolin–Nef extract is due to partial processing that releases the phaseolin domain, thus producing zein–Nef: de Virgilio et al., 2008). Therefore, intact zein–GFP accumulated in transgenic leaves is insoluble unless reduced, like other zein fusions. Zein–GFP accumulates to levels that are higher than those of the very unstable zein–Nef, but much lower than those of the PB-forming zeolin–Nef. RNA blots (not shown) indicated that recombinant mRNA levels were actually higher in zein–GFP than in zeolin–Nef plants, suggesting that zein–GFP was not very stable. This was further investigated by pulse-chase.
|
After pulse-chase labeling, protoplasts were homogenated and immunoprecipitated in the presence or absence of 2-mercaptoethanol. The results show that at 0 h chase, a proportion of intact zein–GFP is not soluble unless reduced, but, during the chase, this insolubility is lost (Figure 6A, compare the different chase points in immunoprecipitations performed in the presence or absence of 2-mercaptoethanol). Similarly to sGFP418, zein–GFP underwent post-translational proteolysis that leads to the formation of the GFP ‘core’ (Figure 6A). No secretion into the incubation medium was detectable (not shown). As expected, the GFP ‘core’ was soluble also in the absence of reducing agent (Figure 6A). Treatment with BFA increased the half-life of zein–GFP about two-fold and almost completely inhibited the formation of the GFP ‘core’ (Figure 6B, and quantitative measurements in 6C).
|
We conclude that zein–GFP, unlike zeolin and zeolin–Nef, is not assembled into stable PB and in relevant part undergoes post-translational, traffic-dependent removal of the GFP portion, strongly suggesting vacuolar delivery. It is also possible that the protein is in part disposed by ERAD, because its degradation is not completely inhibited by BFA.
|
DISCUSSION |
|---|
|
TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
|---|
When a signal peptide for translocation into the ER is added to GFP, the protein is efficiently secreted by plant cells in intact tissue (Boevink et al., 1999; Zheng et al., 2004) or by protoplasts (Frigerio et al., 2001a). Further addition of ER-localization HDEL or KDEL signals to secretory GFP leads to GFP accumulation in the ER (Haseloff et al., 1997), whereas addition of propeptides known to be necessary for the correct sorting of plant vacuolar proteins promotes efficient vacuolar delivery (Frigerio et al., 2001a; daSilva et al., 2005). GFP is therefore a reliable, and indeed commonly used, cargo protein to study sorting in the plant secretory pathway.
A fusion between secretory GFP and the P-domain of the ER folding helper calreticulin (sGFP–P) is not secreted and undergoes extensive interactions with BiP, indicating that it is recognized as a structurally defective protein by the ER quality control system (Brandizzi et al., 2003). This model defective protein is retrotranslocated into the cytosol and the nucleoplasm, and is eventually degraded without the formation of detectable proteolytic fragments, indicating that it is an ERAD substrate (Brandizzi et al., 2003). We have shown here that the secretory GFP fusions sGFP418 and zein–GFP traffic, completely or in substantial part, along the secretory pathway in a BFA-sensitive process and are eventually proteolytically processed into the relatively stable GFP ‘core’ that is typically generated when secretory forms of GFP reach the vacuole. We have also directly shown that the GFP ‘core’ of sGFP418 is indeed located in the vacuole. Neither sGFP418 nor zein–GFP contains known vacuolar sorting signals.
What do the two constructs have in common, and what makes them different from sGFP-P and other defective plant proteins degraded by the BFA-insensitive pathway? One-hour pulse-labeled sGFP418 is extensively associated with BiP, similarly to sGFP-P and unlike other secretory forms of GFP (Foresti et al., 2003). More importantly, assembly-defective forms of phaseolin also associate with BiP before BFA-insensitive degradation (Pedrazzini et al., 1997; Frigerio et al., 2001b). Similarly to sGFP418, these forms have permanently exposed domains that are normally involved in trimer assembly and are devoid of the AFVY vacuolar sorting signal. It is thus clear that BiP interactions are not sufficient per se to determine the destiny of a defective protein in plant cells.
Our data do not allow us to determine what makes sGFP418, unlike sGFP-P and assembly-defective phaseolin constructs, unavailable for ERAD and available for entering intracellular traffic. Once sGFP418 has been transported out of the ER along the secretory pathway, its vacuolar delivery could be mediated either through direct recognition by a vacuolar sorting system or through association with a protein that is recognized by this system. The alternative explanation of autophagic delivery is very unlikely, since autophagy is not inhibited but rather stimulated by BFA (Purhonen et al., 1997; Ding et al., 2007). Furthermore, ER resident proteins are degraded in vacuoles when Arabidopsis cell cultures reach stationary phase, but this process, which has been hypothesized to be due to autophagy, does not involve traffic through the Golgi complex (Tamura et al., 2004). The processing of a very minor proportion of sGFPHDEL that we detected in this study could also be due to autophagy or other, uncharacterized alternative pathways of vacuolar delivery (Frigerio et al. 2001b).
The recent discovery that BiP can be delivered to the vacuole by vesicular traffic led to the hypothesis that it could mediate quality control vacuolar disposal of defective proteins that escape ERAD (Pimpl et al., 2006). This hypothesis implies that when the BiP-ligand complex reaches the Golgi apparatus, it can be either recycled back to the ER because of the BiP HDEL signal or sorted to the vacuole, possibly because BiP also has a (not yet identified) vacuolar sorting signal. This would ensure disposal of BiP ligands that fail to be degraded by ERAD (Pimpl et al., 2006). We have shown here that the GFP ‘core’ of sGFP418 is not associated to BiP. Release from BiP could occur either before or after vacuolar delivery and processing of the GFP construct, the latter alternative being compatible with BiP-mediated vacuolar sorting.
The -zein domains of zein–GFP are not structurally related to the phaseolin domains of sGFP418; however, it should be noticed that they also promote extensive BiP interactions and are involved in the polymerization of a seed storage protein (Mainieri et al., 2004). Indeed, a polypeptide with the same apparent molecular mass of BiP is co-selected with zein–GFP in pulse-chase experiments (Figure 6A, minor polypeptide between the 66 and 97-kDa markers, and compare with the position of BiP in Figure 2); although the identity of this component was not investigated further in this work, it could represent BiP associated to zein–GFP. We have shown that in zein–GFP, the zein domains fail to promote permanent insolubility; this could lead to traffic in association with BiP and final vacuolar sorting. It is therefore tempting to speculate that, if BiP association is responsible for the sorting of sGFP418, this could also be the case for zein–GFP. The observation that BFA does not completely inhibit the degradation of zein–GFP suggests that this protein is subjected to more than one degradation route. It is possible that a proportion of zein–GFP molecules are completely degraded by ERAD or other mechanisms. As we mentioned in the introduction, 2multiple quality control degradation routes for a given protein construct have been already reported, and they have been related to the in-vivo repartition into subsets of misfolded molecules with different solubility properties (Kruse et al., 2006). Zein–GFP molecules detected by protein blot are not soluble unless reduced. These could represent the transiently insoluble, newly synthesized molecules detected by pulse-chase, or a minor proportion of polypeptides stably assembled into PB, or intermediates for degradation by an alternative route with respect to vacuolar delivery and GFP ‘core’ formation.
From a functional point of view, the major difference between the zein and the phaseolin domains that we have fused to sGFP is that the polymerization promoted by the former operates through disulfide bonds instead of hydrophobic interactions and leads to insolubilization and permanent ER retention (Mainieri et al., 2004), whereas phaseolin assembly leads to intracellular traffic (Pedrazzini et al., 1997; Mainieri et al., 2004; Pompa and Vitale, 2006). The common features instead reside in the fact that, in both cases, vacuolar delivery is promoted by domains that have evolved to determine assembly but, in the two constructs, fail to perform this role.
The prolonged exposure of assembly determinants can thus promote vacuolar disposal when the defective molecule is not available for retrotranslocation into the cytosol. As we discussed above, this vacuolar delivery could be mediated through BiP interactions. Direct recognition by a vacuolar sorting mechanism has instead been proposed to explain quality control delivery of a subset of defective proteins in yeast (Hong et al., 1996; Coughlan et al., 2004). The yeast vacuolar sorting receptor Vps10p recognizes various, apparently unrelated, sequences present in the pro-peptides of different vacuolar enzymes. It has been suggested that Vps10 can have affinity for generally flexible sequences that, besides being present in propeptides of vacuolar proteins, could be exposed in defective polypeptides (Hong et al., 1996; Kruse et al., 2006). The vacuolar sorting pathway mediated by Vps10p has many features in common with the BFA-sensitive pathway mediated by plant vacuolar protein receptors of the BP-80/AtVSR class (Bassham and Raikhel, 2000; Jürgens, 2004). The recognition properties of these plant receptors were initially hypothesized to be rather sequence-specific, but more recent genetic evidence for a role of this class of vacuolar receptors in the sorting vacuolar storage proteins could indicate wider recognition properties (Shimada et al., 2003; Craddock et al., 2008). It is thus possible that BP-80/AtVSR receptors are able to recognize defective polypeptides that escape ERAD and expose flexible or unstructured domains. Of course, a third alternative cannot be excluded, involving specific, not yet identified receptors for vacuolar sorting of defective polypeptides. These receptors could recognize exposed hydrophobic sequences already in the ER, after the defective proteins have undergone unproductive cycles of binding and release by BiP.
Whatever the mechanism, our results strongly support the hypothesis that the plant vacuole is involved in the disposal of defective secretory proteins through BFA-sensitive traffic mechanisms.
In light of the results presented here, we can speculate that also the vacuolar delivery of a number of maize zein polypeptides, expressed in transgenic plants in the absence of their partner zeins, could occur through quality control-related traffic rather than by autophagy (Hoffman et al., 1987; Coleman et al., 1996; Bagga et al., 1997). This could also apply to a phaseolin mutated form in which a peptide was inserted to enhance the nutritional value of this seed protein. The mutated phaseolin was delivered to the vacuole via the Golgi complex and degraded there (Hoffman et al., 1988; Pueyo et al., 1995). Upon addition of the HDEL signal, the protein was in part retained in the ER and stabilized, indicating that it is not an ERAD substrate (Pueyo et al., 1995). This phaseolin mutated form still contains the natural vacuolar sorting signal, but its high instability suggests conformational defects that could lead to quality control recognition. A number of wild-type, mutated, or chimeric molecules are therefore available to define in more detail the recognition mechanisms and genes involved in quality control vacuolar delivery in plants.
|
METHODS |
|---|
|
TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
|---|
The material used and described in this work is freely available for non-commercial purposes.
Recombinant DNA and Production of Transgenic Tobacco
The production of transgenic tobacco plants expressing sGFP418, sGFPAFVY, sGFPHDEL, zein–Nef, and zeolin–Nef has been described (Frigerio et al., 2001a; Foresti et al., 2003; de Virgilio et al., 2008). The zein–GFP construct was produced as follows. GFP cDNA was amplified using as template the plasmid PCK.GFP.S65C (Reichel et al., 1996) and primers 5'-GAGCTTGTCGACCTAGTACCAAGAGGTGGTAAAGGAGAAGAACTTTTCACTGG-3', containing the sequence coding for the thrombin cleavage site (LVPRG), and 5'-CGATTCGCATGCTCATCATTATCTAGATCCGGACTTGTATAGTTCA-3', containing a SphI restriction site downstream from three stop codons. The amplified DNA was inserted into the SphI/SalI-restricted plasmid A described in de Virgilio et al. (2008), to generate pDHAzein–GFP. For the production of transgenic plants, the fragment excised by EcoRI digestion of pDHAzein–GFP, including the 35S promoter, the sequence coding for the chimeric zein–GFP protein and 35S terminator, was introduced into the EcoRI site of the binary vector PBI121.1. Strain LBA4404 of Agrobacterium tumefaciens was transformed by electroporation and used to produce transgenic tobacco (Nicotiana tabacum) cv. Petit Havana SR1 as described (Pedrazzini et al., 1997). Briefly, leaf discs from Nicotiana tabacum cv. Petit Havana SR1 were transformed by co-cultivation with A. tumefaciens harboring PBI121.1–zein–GFP. After co-cultivation, the leaf discs were grown at 25°C under full light on the regeneration medium containing 250 mg l–1 cefotaxime, 100 mg l–1 kanamycin. Regenerated shoots of each explant were kept separated in order to guarantee regeneration of independent transformants. After 5 weeks, shoots were plated on half-strength Murashige and Skoog salts, 100 mg l–1 kanamycin, and 250 mg l–1 cefotaxime until the new plants developed. Transformed plants were grown at 25°C in 16 h of light in axenic conditions without antibiotics and propagated every 5–6 weeks.
Leaf Protein Extraction, Sub-Cellular Fractionation, and Protein Gel Blot Analysis
For the extraction of total proteins in non-reducing conditions, young (4–7 cm long) leaves of transgenic tobacco grown in axenic conditions were homogenized in an ice-cold mortar with homogenization buffer (200 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, 100 mM Tris-Cl, pH 7.8, supplemented with Complete protease inhibitor cocktail (Roche, Basel)). For homogenization in reducing conditions, the buffer was supplemented with 4% 2-mercaptoethanol. The homogenate was centrifuged at 1500 g for 10 min at 4°C to discard unbroken tissue and the supernatant supplemented with SDS–PAGE denaturing buffer and analyzed by SDS–PAGE followed by protein blot, using anti-BiP (Pedrazzini et al., 1997; 1:10 000 dilution) or anti--zein (Bellucci et al., 2000; 1:1000 dilution) antiserum or anti-GFP antibodies (Living Colors, Clontech, 1:100 dilution). In some experiments, the material that remained insoluble after extraction in reducing conditions was also analyzed after resuspension in SDS–PAGE denaturing buffer (lanes I in Figure 5).
For isopycnic sucrose gradient fractionation, small leaves were homogenated with ice-cold buffer A (2 mM MgCl2, 10 mM KCl, 100 mM Tris-Cl, pH 7.8) supplemented with 12% (w/w) sucrose, using 1 ml of buffer for 200 mg of leaf. The homogenate was centrifuged at 1000 g, 4°C for 10 min. The supernatant (1 ml) was loaded on 4 ml of a 16–55% (w/w) sucrose gradient made in buffer A. The gradient was centrifuged for 90 min at 154 000 g, 4°C in a SW55 rotor (Beckman). 35 µl of each fraction were analyzed by SDS–PAGE and protein blot, using anti-GFP or anti-BiP antisera as described above. Vacuoles were isolated from leaf protoplasts as described (Dombrowski et al., 1994). The activity of the vacuolar marker -mannosidase was determined by adding 10 µl of a 30-mM solution of p-nitrophenyl alpha-D-mannopyranoside to 50 µl of protoplast homogenate or vacuolar preparation, 100 µl 0.5 M sodium acetate pH 5.0, 340 µl water. After incubation for 10, 30, or 60 min at 30°C, the reaction was stopped by adding 800 µl Na2CO3 and the p-nitrophenol released was quantified by measuring the absorbance at 410 nm.
Pulse-Chase of Leaf Protoplasts and Protein Immunoprecipitation
Protoplasts were prepared from young leaves of transgenic or wild-type tobacco SR1 plants grown in axenic conditions, and were subjected to pulse-chase labeling with Pro-Mix (a mixture of 35SMet and 35SCys; Amersham Biosciences), in the absence or presence of brefeldin A (Roche), as described by Pedrazzini et al. (1997). At the end of each chase point, protoplast and incubation media were collected as described (Pompa and Vitale, 2006), frozen and stored at –80°C. Homogenization of protoplasts and incubation media was performed by adding to frozen samples 2 volumes of ice-cold protoplast homogenization buffer (150 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1.5 mM EDTA, 1.5% Triton X-100, and Complete protease inhibitor cocktail (Roche)). For homogenization in reducing conditions, the homogenization buffer was supplemented with 4% (v/v) 2-mercaptoethanol, which was also included in the immunoprecipitation buffer. Immunoprecipitation was performed as described (Pedrazzini et al., 1997), using rabbit polyclonal antiserum against: GFP (Molecular Probes), tobacco BIP (Pedrazzini et al., 1997), or -zein (Bellucci et al., 2000). The immunoprecipitates were analyzed by SDS–PAGE. 14C-methylated proteins (Sigma-Aldrich) were used as molecular mass markers. After electrophoresis, gels were treated with 2,5diphenyloxazole dissolved in dimethyl sulfoxide, dried and exposed for fluorography. Intensities of fluorography bands were measured using TotalLab software (Nonlinear Dynamics, Newcastle upon Tyne, UK).
|
FUNDING |
|---|
|
TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
|---|
Work supported in part by the EU Research Training Network ‘Biochemical and genetic approaches to study bio-molecular interactions in plants’ (HPRN-CT-2002–00262), the EU Integrated Project ‘Pharma-Planta’ (LSHB-CT-2003–503565) and the EU Marie Curie Research Training Network ‘Vacuolar Transport Equipment for Growth Regulation in Plants’ (MRTN-CT-2006–035833).
|
Acknowledgements |
|---|
We thank Andrea Pompa for the technical assistance. We also thank Aldo Ceriotti and Emanuela Pedrazzini for the stimulating discussions. No conflict of interest declared.
-
Anelli T, Sitia R. Protein quality control in the early secretory pathway. EMBO J. (2008) 27:315–327.[CrossRef][Web of Science][Medline]
Bagga S, Adams H, Rodriquez FD, Kemp JD, Sengupta-Gopalan C. Co-expression of the maize 
- and β-zein genes results in stable accumulation of -zein in ER-derived protein bodies formed by β-zein. Plant Cell (1997) 9:1683–1696.[Abstract]
Bassham DC, Raikhel NV. Unique features of the plant vacuolar sorting machinery. Curr. Opin. Cell Biol. (2000) 12:491–495.
Bellucci M, Alpini A, Paolocci F, Cong L, Arcioni S. Accumulation of maize -zein and -zein:KDEL to high levels in tobacco leaves and differential increase of BiP synthesis in transformants. Theor. Appl. Genet. (2000) 101:796–804.[CrossRef][Web of Science]
Boevink P, Martin B, Oparka K, Cruz SS, Hawes C. Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A. Planta (1999) 208:392–440.
Bollini R, Van der Wilden W, Chrispeels MJ. A precursor of the reserve protein, phaseolin, is transiently associated with the endoplasmic reticulum of developing Phaseolus vulgaris cotyledons. Physiol. Plant (1982) 55:82–92.[CrossRef]
Brandizzi F, Hanton S, daSilva LL, Boevink P, Evans D, Oparka K, Denecke J, Hawes C. ER quality control can lead to retrograde transport from the ER lumen to the cytosol and the nucleoplasm in plants. Plant J. (2003) 34:269–281.[CrossRef][Web of Science][Medline]
Coleman CE, Herman EM, Takasaki K, Larkins BA. The maize -zein sequesters -zein and stabilizes its accumulation in protein bodies of transgenic tobacco endosperm. Plant Cell (1996) 8:2335–2345.[Abstract]
Coughlan CM, Walker JL, Cochran JC, Wittrup KD, Brodsky JL. Degradation of mutated bovine pancreatic trypsin inhibitor in the yeast vacuole suggests post-endoplasmic reticulum protein quality control. J. Biol. Chem. (2004) 279:15289–15297.
Craddock CP, Hunter PR, Szakacs E, Hinz G, Robinson DG, Frigerio L. Lack of a vacuolar sorting receptor leads to non-specific missorting of soluble vacuolar proteins in Arabidopsis seeds. Traffic (2008) 9:408–416.[CrossRef][Web of Science][Medline]
daSilva LL, Taylor JP, Hadlington JL, Hanton SL, Snowden CJ, Fox SJ, Foresti O, Brandizzi F, Denecke J. Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting. Plant Cell. (2005) 17:132–148.
de Virgilio M, De Marchis F, Bellucci M, Mainieri D, Rossi M, Benvenuto E, Arcioni S, Vitale A. The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin. J. Exp Bot. (2008) [Epub ahead of print].
Ding WX, Ni HM, Gao W, Hou YF, Melan MA, Chen X, Stolz DB, Shao ZM, Yin XM. Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival. J. Biol. Chem. (2007) 282:4702–4710.
Dombrowski JE, Gomez L, Chrispeels MJ, Raikhel NV. Targeting of proteins to the vacuole. In: Plant Molecular Biology Manual—Gelvin SB, Schilperoort RA, eds. (1994) 2nd edn. Dordrecht, The Netherlands: Kluwer Academic Publishers. 1–29.
Foresti O, Frigerio L, Holkeri H, de Virgilio M, Vavassori S, Vitale A. A phaseolin domain involved directly in trimer assembly is a determinant for binding by the chaperone BiP. Plant Cell (2003) 15:2464–2475.
Frigerio L, de Virgilio M, Prada A, Faoro F, Vitale A. Sorting of phaseolin to the vacuole is saturable and requires a short C-terminal peptide. Plant Cell (1998) 10:1031–1042.
Frigerio L, Foresti O, Hernández Felipe D, Neuhaus J-M, Vitale A. The C-terminal tetrapeptide of phaseolin is sufficient to target green fluorescent protein to the vacuole. J. Plant Physiol. (2001a) 158:499–503.[CrossRef][Web of Science]
Frigerio L, Pastres A, Prada A, Vitale A. Influence of KDEL on the fate of trimeric or assembly-defective phaseolin: selective use of an alternative route to vacuoles. Plant Cell (2001b) 13:1109–1126.
Gomord V, Denmat L-A, Fitchette-Lainè A-C, Satiat-Jeunemaitre B, Hawes C, Faye L. The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER. Plant J. (1997) 11:313–325.[CrossRef][Web of Science][Medline]
Haseloff J, Siemering KR, Prasher DC, Hodge S. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl Acad. Sci. U S A (1997) 94:2122–2127.
Hoffman LM, Donaldson DD, Herman EM. A modified storage protein is synthesized, processed, and degraded in the seeds of transgenic plants. Plant Mol. Biol. (1988) 11:717–729.[CrossRef][Web of Science]
Hoffman LM, Donaldson DD, Bookland R, Rashka K, Herman EM. Synthesis and protein body deposition of maize 15-KD zein in transgenic tobacco seeds. EMBO J. (1987) 6:3213–3221.[Web of Science][Medline]
Hong E, Davidson AR, Kaiser CA. A pathway for targeting soluble misfolded proteins to the yeast vacuole. J. Cell Biol. (1996) 135:623–633.
Jürgens G. Membrane trafficking in plants. Annu. Rev. Cell Dev. Biol. (2004) 20:481–504.[CrossRef][Web of Science][Medline]
Kruse KB, Brodsky JL, McCracken AA. Characterization of an ERAD gene as VPS30/ATG6 reveals two alternative and functionally distinct protein quality control pathways: one for soluble Z variant of human -1 proteinase inhibitor (A1PiZ) and another for aggregates of A1PiZ. Mol. Biol. Cell. (2006) 17:203–212.
Mainieri D, Rossi M, Archinti M, Bellucci M, De Marchis F, Vavassori S, Pompa A, Arcioni S, Vitale A. Zeolin: a new recombinant storage protein constructed using maize -zein and bean phaseolin. Plant Physiol. (2004) 136:3447–3456.
Pedrazzini E, Giovinazzo G, Bielli A, de Virgilio M, Frigerio L, Pesca M, Faoro F, Bollini R, Ceriotti A, Vitale A. Protein quality control along the route to the plant vacuole. Plant Cell (1997) 9:1869–1880.[Abstract]
Pimpl P, Taylor JP, Snowden C, Hillmer S, Robinson DG, Denecke J. Golgi-mediated vacuolar sorting of the endoplasmic reticulum chaperone BiP may play an active role in quality control within the secretory pathway. Plant Cell (2006) 18:198–211.
Pompa A, Vitale A. Retention of a bean phaseolin/maize gamma–zein fusion in the endoplasmic reticulum depends on disulfide bond formation. Plant Cell (2006) 18:2608–2621.
Pueyo JJ, Chrispeels MJ, Herman EM. Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole. Planta (1995) 196:586–596.[Web of Science][Medline]
Purhonen P, Pursiainen K, Reunanen H. Effects of brefeldin A on autophagy in cultured rat fibroblasts. Eur. J. Cell Biol. (1997) 74:63–67.[Web of Science][Medline]
Reichel C, Mathur J, Eckes P, Langenkemper K, Koncz C, Schell J, Reiss B, Maas C. Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells. Proc. Natl Acad. Sci. U S A (1996) 93:5888–5893.
Shewry PR, Halford NG. Cereal seed storage proteins: structures, properties and role in grain utilization. J. Exp. Bot. (2002) 53:947–958.
Shimada T, Fuji K, Tamura K, Kondo M, Nishimura M, Hara-Nishimura I. Vacuolar sorting receptor for seed storage proteins in Arabidopsis thaliana. Proc. Natl Acad. Sci. U S A (2003) 100:16095–16100.
Tamura K, Yamada K, Shimada T, Hara-Nishimura I. Endoplasmic reticulum-resident proteins are constitutively transported to vacuoles for degradation. Plant J. (2004) 39:393–402.[CrossRef][Web of Science][Medline]
Teckman JH, Perlmutter DH. Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response. Am. J Physiol. Gastrointest. Liver Physiol. (2000) 279:G961–G974.
Vitale A, Boston RS. Endoplasmic reticulum quality control and the unfolded protein response: insights from plants. Traffic (2008) [Epub ahead of print].
Vitale A, Bielli A, Ceriotti A. The binding protein associates with monomeric phaseolin. Plant Physiol. (1995) 107:1411–1418.[Abstract]
Zheng H, Kunst L, Hawes C, Moore I. A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus. Plant J. (2004) 37:398–414.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Pinheiro, M. Samalova, N. Geldner, J. Chory, A. Martinez, and I. Moore Genetic evidence that the higher plant Rab-D1 and Rab-D2 GTPases exhibit distinct but overlapping interactions in the early secretory pathway J. Cell Sci., October 15, 2009; 122(20): 3749 - 3758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Moore and A. Murphy Validating the Location of Fluorescent Protein Fusions in the Endomembrane System PLANT CELL, June 1, 2009; 21(6): 1632 - 1636. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||