Plant Physiology and Biochemistry 73 (2013) 337e343
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Two glycosylated vacuolar GFPs are new markers for ER-to-vacuole sorting Egidio Stigliano b, c, Marianna Faraco a, Jean-Marc Neuhaus b, Anna Montefusco a, Giuseppe Dalessandro a, Gabriella Piro a, Gian-Pietro Di Sansebastiano a, * a b c
DiSTeBA, Università del Salento, via prov. Lecce-Monteroni, 73100 Lecce, Italy Laboratory of Cell and Molecular Biology, University of Neuchâtel, Rue Emile-Argand 11, CH-2000 Neuchâtel, Switzerland CNR-IGV, Institute of Plant Genetics, Thematic Center for the Preservation of Mediterranean Plant Biodiversity, via Nazionale 44, 75025 Policoro, MT, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 June 2013 Accepted 10 October 2013 Available online 23 October 2013
Vacuolar Sorting Determinants (VSDs) have been extensively studied in plants but the mechanisms for the accumulation of storage proteins in somatic tissues are not yet fully understood. In this work we used two mutated versions of well-documented vacuolar fluorescent reporters, a GFP fusion in frame with the C-terminal VSD of tobacco chitinase (GFPChi) and an N-terminal fusion in frame with the sequence-specific VSD of the barley cysteine protease aleurain (AleuGFP). The GFP sequence was mutated to present an N-glycosylation site at the amino-acid position 133. The reporters were transiently expressed in Nicotiana tabacum protoplasts and agroinfiltrated in Nicotiana benthamiana leaves and their distribution was identical to that of the non-glycosylated versions. With the glycosylated GFPs we could highlight a differential ENDO-H sensitivity and therefore differential glycan modifications. This finding suggests two different and independent routes to the vacuole for the two reporters. BFA also had a differential effect on the two markers and further, inhibition of COPII trafficking by a specific dominant-negative mutant (NtSar1h74l) confirmed that GFPChi transport from the ER to the vacuole is not fully dependent on the Golgi apparatus. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: GFP Glycosylation Protoplast Secretion Vacuole ctVSD Golgi
1. Introduction Vacuolar trafficking of soluble proteins is always an interesting field of investigation due to the high potential for biotechnology applications. Although many targeting signals have been identified for the plant secretory pathway [1], the mechanisms of storage protein deposition and of tissue- and species-specific accumulation strategies are not yet fully understood [2,3], so that the targeting of exogenous proteins to unexpected compartments still occurs [4]. Two vacuolar fluorescent reporters, a GFP fusion in frame with the C-terminal Vacuolar Sorting Determinant (ctVSD) of tobacco chitinase (EC 3.2.1.14, GFPChi) and an N-terminal fusion in frame with the sequence-specific Vacuolar Sorting Determinant (ssVSD) of the cysteine protease barley aleurain (EC 3.4.22.16, AleuGFP) have been widely used to characterize alternative vacuolar sorting pathways [5,6]. These two reporters allowed the identification of two different vacuoles, a non-acidic vacuole labelled by GFPChi and an acidic vacuole labelled by AleuGFP [5e7]. Each reporter has * Corresponding author. Tel.: þ39 0832 298713, þ39 0832 298714; fax: þ39 0832 298858. E-mail addresses: [email protected], [email protected] (G.-P. Di Sansebastiano). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.10.010
independent routes to arrive to the vacuole. In Arabidopsis thaliana AleuGFP interacts with AtVSR1 as does aleurain itself [8] and rapidly labels the central vacuole in most tissues while GFPChi and chitinase, whose receptor is probably an AtRMR [9], transit for a longer time in the ER and then in small vacuoles before reaching the central vacuole [5]. Arabidopsis aleurain vacuolar trafficking depends essentially from binding to VSR-1 [8] but it was recently shown that VSR-4 also might participate, demonstrating a certain functional redundancy in the VSR family [10]. Several studies have addressed the pathway of the GFPChi to vacuoles. It has been argued very convincingly that the GFPChi follows the classical pathway passing through Golgi-TGN-PVC, involving the SNARE components AtVPS45 [11] and VTI12 [6] and finally reaching the central vacuole. On the other hand, it was also found that GFPChi could bypass the Golgi-TGN-PVC route. Incubation of Arabidopsis roots expressing GFPChi with BFA did not dramatically alter the typical punctate GFPChi pattern, while AleuGFP targeting was inhibited [12]. As Zouhar and co-workers [11] asserted in their discussion it was still possible to detect a faint fluorescence in GFPChi plants crossed with VPS45-silenced plants, meaning “that less fusion protein was reaching the vacuole while the majority was effectively
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Fig. 1. Confocal images of tobacco protoplasts transiently expressing vacuolar GFP reporters. A) GFPgl133Chi fluorescence labelled the large central vacuole and the ER; B) AleuGFPgl133 labelled the central vacuole and small dot structures corresponding to PVC. “N” indicate the space occupied by the nucleus. Scale bar: 20 mm.
secreted into the apoplast”. Similar observations were also made by Sanmartín [6] in GFPChi plants crossed with vti12 null allele plants. Other typical storage proteins, the 7S and 2S globulins, are delivered to the Protein Storage Vacuole bypassing the classical route in maturing pumpkin seeds, as revealed by electron microscopy. This trafficking is achieved by PAC vesicles (Precursor Accumulating Vesicles), in which storage proteins aggregate [13]. Although functionally similar compartments were described in leaf tissues of three plant species [14], no evidence for the existence of a direct ER-Vacuole (ERV) pathway was provided but a new study of the sorting of human a-mannosidase in plant cells suggests the existence of such a pathway [15]. Bypassing the Golgi apparatus is expected to have a dramatic effect on the glycosylation patterns of vacuolar proteins. In all eukaryotes, proteins N-glycosylation occurs in the ER by the transfer of an oligosaccharide precursor to asparagine side chains in Asn-X-Ser/Thr sequences of nascent polypeptide chains [16,17]. Subsequently, glucose and mannose residues are removed from the N-glycan by ER-resident glucosidases and mannosidases. This process is highly regulated and links glycan modification to protein folding and quality control in the ER. Further processing of N-linked glycans to Man5GlcNAc2 is performed by Golgi-a-mannosidase I [16,18,19] and by glycosyltransferases within the Golgi. In plants, complex glycans formed in the Golgi are normally decorated with b-1,2-xylose and core a-1,3-fucose residues, which are absent or differently linked in mammalian systems. In some cases, plant cells further elongate the fucosylated glycans by adding b-1,3galactose and a-1,4-fucose residues forming Lewis-a epitopes (Lea). On the other hand, plants, like insects, also can modify Nglycans by removing the GlcNAc residues to produce paucimannosidic N-glycans [20]. Little is known about the glycosylation pattern of plant vacuolar proteins, even if the paucimannosidic protein N-glycosylation is considered typical [21]. The development and characterization of a new set of glycosylated vacuolar markers can contribute to a better definition of vacuolar sorting machineries and of glycan modification processes. In the present work we validate the use of two glycosylated vacuolar GFPs and provide evidences for a transport route to the vacuole that bypass the Golgi.
This glycosylation site 133 (gl133) was introduced into two vacuolar reporters, GFPChi and AleuGFP, previously expressed by our group in different plant species and tissues [7,23,24]. Several studies proved that the two reporters are sorted to vacuoles by different sorting machineries [5,6,25]. GFPChi contains the ctVSD of tobacco chitinase A [26] while AleuGFP harbours the N-terminal ssVSD from barley aleurain, containing the specific conserved sequence NPIR [5]. The resulting glycosylated reporters GFPgl133Chi and AleuGFPgl133 were transiently expressed in tobacco protoplasts and their fluorescence pattern was identical to the pattern previously described for their non-glycosylated equivalents. GFPgl133Chi labelled the ER and the central vacuole (Fig. 1A), but 1e5 mm compartments (not shown) can also be recognized in specific cell types [5]. AleuGFPgl133 labelled the central vacuole and PVCs (Fig. 1B). 2.2. Both GFPgl133Chi and AleuGFPgl133 are partially glycosylated In order to control the sorting efficiency we developed a rapid fractionation method for soluble proteins that takes advantage of protoplast fragility. After harvesting the protoplasts they were resuspended in buffer and broken by a single cycle of freezee thawing. Centrifugation separated the vacuolar sap and cellular fraction. The latter was then lysed by further freezeethawing in presence of SDS. We analyse then the three fractions corresponding to the medium (Med), the vacuolar sap (Vac) and the rest of soluble fraction including microsomal and cytosolic proteins (Mic). To validate this fractionation protocol, we used two control markers:
2. Results 2.1. Glycosylated GFPs targeted to neutral vacuoles and to lytic vacuoles We previously showed that an N-glycosylation site at position 133 of a secreted GFP (secGFP) was efficiently glycosylated [22].
Fig. 2. Fractionation of transiently expressed GFP markers. (A) Validation of the fractionation procedure with two well-known markers: GFP-KDEL and secGFP. Protoplasts were separated from the medium (Med) and fractionated in microsomes (Mic) and cell sap (Vac). The method appears adequate to distinguish the distribution of soluble GFPs between the three fractions. (B) Vacuolar targeting of glycosylated GFPs. Protoplasts were fractionated as above. The Vac fraction contains mostly soluble proteins from the vacuolar compartments.
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secGFP and GFP-KDEL [26,27]. Lacking any sorting signal, secGFP is secreted into the incubation medium, while GFP-KDEL, having at its C-terminus the KDEL signal sequence, is retained in the ER. We indeed observed the expected enrichment of secGFP in the Med fraction and of GFP-KDEL in the Mic fraction (Fig. 2A). The latter reporter was also recovered in the vacuolar sap as expected for a protein with the KDEL signal overexpressed to saturation levels [28]. Fractionation and western blot analysis of the glycosylated reporters showed that they were both correctly and efficiently targeted to the vacuolar sap (Vac, Fig. 2B). As shown previously for secGFPgl133 [22], both GFPgl133Chi and AleuGFPgl133 were only partially glycosylated (as evidenced by the presence of two very closed bands) but both glycosylated and non-glycosylated forms were found almost exclusively in the Vac fraction. Only a small fraction of both forms of GFPgl133Chi was also detected in the Mic fraction (The quantity of protein in the Mic fraction may be influenced by overexpression and receptors saturation).
2.3. Glycosylation as an indicator for transit through the Golgi apparatus for vacuolar soluble proteins In the Golgi apparatus, high-mannose-type N-glycans can undergo several modifications that will lead to complex-type N-glycans, which are resistant to endoglycosidase H (EndoH). EndoH resistance thus demonstrates the transit of a glycoprotein through the Golgi. EndoH sensitivity however is less informative, as not all N-glycans are modified in the Golgi [21]. We have already shown that the secreted marker secGFPgl133 recovered from the incubation medium was resistant to EndoH digestion while fraction recovered in the intracellular soluble fraction was sensitive to EndoH [22]. This indicated that the gl133 glycosylation site is compatible with Golgi modifications and that the glycosylated secGFPgl133 passed through the Golgi apparatus to reach its final destination. The EndoH sensitive intracellular secGFPgl133 most probably corresponds to an ER pool of not yet secreted glycoproteins. For the two vacuolar reporters, GFPgl133Chi collected from whole cells was also sensitive to EndoH digestion (Fig. 3A) while AleuGFPgl133 was resistant (Fig. 3B).
Fig. 3. Differential EndoH sensitivity of glycans on GFPgl133Chi (A) and AleuGFPgl133 (B) transiently expressed in protoplasts. The indents indicate the position of the 29 and 37 kDa markers.
2.5. GFPChi and AleuRFP localize on different Golgi domains Before reaching the vacuole, coexpressed AleuRFP and GFPChi transited through distinct small compartments (Fig. 5A). BFA
2.4. Is GFPgl133Chi trafficking to vacuole Golgi-dependent? The GFPChi pathway to the central vacuole is not identical to the pathway of other vacuolar markers such as RFP-AFVY. The C-terminal tetrapeptide AFVY is the phaseolin’s ctVSD and efficiently targets the fusion protein to the central vacuole [29,30]. Observations at confocal microscope after 36 h of expression have brought to light a different transport rate (Suppl. Fig. 1). While RFP-AFVY clearly accumulated in the central vacuole, GFPChi still labelled ER and compartments, which also contained the tetrapeptide reporter. Since the GFPgl133Chi glycans were not modified by Golgi glycosyltransferases to become EndoH resistant [22] we tested the hypothesis that this reporter may bypass the Golgi apparatus. We treated protoplasts with 100 mM Brefeldin A (BFA) [31,32], a fungal macrocyclic lactone that inhibits the activation of ARF1, the GTPase necessary for the COP I-mediated retrograde transport from the Golgi to the ER. BFA also affects vacuolar sorting [23]. After 24 h treatment we imaged cells expressing GFPgl133Chi (Fig. 4A) and AleuGFPgl133 (Fig. 4B) revealing that both markers were visibly affected by BFA as they accumulated in BFA bodies. We also treated protein extracts with EndoH and found both markers to be resistant (Fig. 4C and D).
Fig. 4. Effect of BFA on protoplasts expressing GFPgl133Chi (A) and AleuGFPgl133 (B). Scale bar: 20 mm. EndoH sensitivity assay of glycans on GFPgl133Chi (C) and AleuGFPgl133 (D) transiently expressed in protoplasts in presence of BFA. The indents indicate the position of the 29 and 37 kDa markers.
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affected the organization of these compartments identifying them as Golgi or TGNs. Again, even under the effect of BFA, the labelling by the two markers did not fully overlap (Fig. 5B). The dotted structures labelled by GFPChi did not overlap with the ER marker RFP-KDEL (Fig. 5C) nor did it co-localize with the Golgi marker STRFP (Fig. 5D). This non-co-localization with well-known markers and the consideration that TGN-localized SYP5 is necessary to GFPChi sorting [25] induced us to concentrate our attention on ERto-Golgi transport mechanisms that may differentiate GFPChi from AleuRFP. Sar1 is the small GTPase required for the formation of the COPII coat involved in the anterograde ER-to-Golgi transport. This transport can be specifically inhibited by the expression of the dominant-negative mutant Sar1H74L [33]. Co-infection experiments with GFPChi and NtSar1H74L were performed on Nicotiana benthamiana leaves to reduce the fluorescence detection problems typical of GFPChi expression in Nicotiana tabacum leaves [23]. Even in this specie where GFPChi is more stable, the number of cells with a fluorescent central vacuole was low [23]. A statistically significant increase of vacuoles labelled with GFPChi was observed when
NtSar1h74l was co-expressed (Fig. 6A and B). Counting was actually done on cells additionally expressing the Golgi marker Gonst1-RFP [34] and only cells where NtSar1H74L expression was confirmed by the altered Gonst1-RFP distribution (not shown). 3. Discussion GFPgl133 [22] was validated as a glycosylated reporter comparing the glycosylation patterns of two vacuolar forms: GFPgl133Chi and AleuGFPgl133. Gl133 glycosylation site did not affect GFP folding and sorting. Indeed, both GFPgl133Chi and AleuGFPgl133 displayed fluorescent patterns identical to those previously described for GFPChi and AleuGFP (Fig. 1) [6,25] and were normally sorted to vacuoles. To further investigate the sorting of these vacuolar markers we developed a simple fractionation procedure (Fig. 2A) to analyse by western blot their vacuolar accumulation. Western-blot also confirmed the partial glycosylation of the markers overexpressed in protoplasts similarly to what had previously been shown for secGFPgl133 [22].
Fig. 5. A) Confocal image of a protoplast coexpressing AleuRFP and GFPChi; this section does not cross the central vacuole; B) altered compartments after 12 h treatment with 100 mM BFA on cells co-expressing AleuRFP and GFPChi; C) co-expression of RFP-KDEL and GFPChi; D) co-expression of ST-RFP and GFPChi. Scale bar 20 mm.
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Fig. 6. A) Agroinfiltrated N. benthamiana leaf epidermis expressing the vacuolar reporter GFPChi and the dominant negative mutant NtSar1H74L. B) Percentage of cells showing GFPChi labelling of the central vacuole in presence or absence of Sar1H74L. Bars show the standard deviation. Chi-square significance value: p < 0.001.
The partial glycosylation is probably due to the high transfection rate of exogenous DNA resulting in a massive protein synthesis likely able to saturate the involved glycosyltransferases. The outcome of this process is the synthesis of two forms of the overexpressed proteins, glycosylated and not-glycosylated. This gives us the opportunity to compare the trafficking of the two forms [22]. Once again, protoplasts are a very versatile experimental system [35]. The sensitivity of the two reporters to endoglycosidase H (EndoH) was tested. It is known that, before the action of mannosidase II in the Golgi, N-glycans are sensitive to hydrolysis by EndoH [36]. It is not known where mannosidase II acts in the Golgi stacks, but the medial-Golgi stacks are a likely location [37,38]. Interestingly the effect of EndoH on the two vacuolar reporters was different: GFPgl133Chi was sensitive to EndoH digestion (Fig. 3A) whereas AleuGFPgl133 was not (Fig. 3B). Since Golgi enzymes have no structural obstacles to modify a glycan at the gl133 site, EndoH sensitivity can be taken as a strong indication that the fusion protein did not pass the Golgi. Therefore we hypothesize that vacuolar AleuGFPgl133 passes through the Golgi, whereas GFPgl133Chi does not. Several controls were needed to verify the hypothesis. First GFPgl133Chi chimeras could fail to acquire EndoH resistance because either they did not traffic through the Golgi complex or because the glycan itself was not available for processing. To verify if the glycan in GFPgl133Chi is accessible to glycosyltransferases we performed EndoH digestion on extracts of BFA-treated protoplasts. This fungal toxin inhibits the activation of the Arf protein required for COP I vesicle formation, disturbs the ER-Golgi trafficking and is thus widely used as a vesicle trafficking inhibitor [31,39,40], including vacuolar targeting of proteins with ctVSD [21]. For the glycan modifying enzymes the result is the relocation of Golgi enzymes into the ER [41]. If the glycans of ER-retained proteins are potentially accessible for processing they will be processed by the Golgi enzymes now re-localized in the ER. The treatment of protoplasts with BFA resulted in the inhibition of the export of both GFPgl133Chi and AleuGFPgl133 and their consequent accumulation in the so-called “Golgi bodies” [42] and hybrid ER-Golgi compartment. After the visual confirmation of the BFA effect on protoplasts, we tested the EndoH sensitivity of the expressed GFPgl133Chi and AleuGFPgl133. Treatment of protein extracts from tobacco protoplasts transiently expressing AleuGFPgl133 confirmed that the construct was insensitive to the enzyme (Fig. 4D) as already seen in the previous assay (Fig. 3B). After BFA treatment, GFPgl133Chi also became insensitive to EndoH (Fig. 4C). It is then evident that the GFPgl133Chi glycan did not escape modification to an EndoH-resistant form because did not
transit via the compartment containing the required glycosyltransferases. In fact when GFPgl133Chi and Golgi glycosyltransferases were mixed as a consequence of the BFA treatment, the glycan was modified to an EndoH-resistant form. GFPgl133Chi and AleuGFPgl133 were sorted in the same way of their non-glycosylated variants. These were used for co-localization with different fluorophore. GFPChi was co-expressed with AleuRFP and confirmed that the two markers did not co-localize (Fig. 5A). Even BFA treatment failed to completely merge the two marker distributions (Fig. 5B). While the small punctate compartments labelled by AleuGFP or AleuGFPgl133 are known to be Golgi, TGN and PVCs, the GFPChi- and GFPgl133Chi-labelled compartments had to be characterized. We performed co-localization experiments and observed that the small (
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Two glycosylated vacuolar GFPs are new markers for ER-to-vacuole sorting Egidio Stigliano b, c, Marianna Faraco a, Jean-Marc Neuhaus b, Anna Montefusco a, Giuseppe Dalessandro a, Gabriella Piro a, Gian-Pietro Di Sansebastiano a, * a b c
DiSTeBA, Università del Salento, via prov. Lecce-Monteroni, 73100 Lecce, Italy Laboratory of Cell and Molecular Biology, University of Neuchâtel, Rue Emile-Argand 11, CH-2000 Neuchâtel, Switzerland CNR-IGV, Institute of Plant Genetics, Thematic Center for the Preservation of Mediterranean Plant Biodiversity, via Nazionale 44, 75025 Policoro, MT, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 June 2013 Accepted 10 October 2013 Available online 23 October 2013
Vacuolar Sorting Determinants (VSDs) have been extensively studied in plants but the mechanisms for the accumulation of storage proteins in somatic tissues are not yet fully understood. In this work we used two mutated versions of well-documented vacuolar fluorescent reporters, a GFP fusion in frame with the C-terminal VSD of tobacco chitinase (GFPChi) and an N-terminal fusion in frame with the sequence-specific VSD of the barley cysteine protease aleurain (AleuGFP). The GFP sequence was mutated to present an N-glycosylation site at the amino-acid position 133. The reporters were transiently expressed in Nicotiana tabacum protoplasts and agroinfiltrated in Nicotiana benthamiana leaves and their distribution was identical to that of the non-glycosylated versions. With the glycosylated GFPs we could highlight a differential ENDO-H sensitivity and therefore differential glycan modifications. This finding suggests two different and independent routes to the vacuole for the two reporters. BFA also had a differential effect on the two markers and further, inhibition of COPII trafficking by a specific dominant-negative mutant (NtSar1h74l) confirmed that GFPChi transport from the ER to the vacuole is not fully dependent on the Golgi apparatus. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: GFP Glycosylation Protoplast Secretion Vacuole ctVSD Golgi
1. Introduction Vacuolar trafficking of soluble proteins is always an interesting field of investigation due to the high potential for biotechnology applications. Although many targeting signals have been identified for the plant secretory pathway [1], the mechanisms of storage protein deposition and of tissue- and species-specific accumulation strategies are not yet fully understood [2,3], so that the targeting of exogenous proteins to unexpected compartments still occurs [4]. Two vacuolar fluorescent reporters, a GFP fusion in frame with the C-terminal Vacuolar Sorting Determinant (ctVSD) of tobacco chitinase (EC 3.2.1.14, GFPChi) and an N-terminal fusion in frame with the sequence-specific Vacuolar Sorting Determinant (ssVSD) of the cysteine protease barley aleurain (EC 3.4.22.16, AleuGFP) have been widely used to characterize alternative vacuolar sorting pathways [5,6]. These two reporters allowed the identification of two different vacuoles, a non-acidic vacuole labelled by GFPChi and an acidic vacuole labelled by AleuGFP [5e7]. Each reporter has * Corresponding author. Tel.: þ39 0832 298713, þ39 0832 298714; fax: þ39 0832 298858. E-mail addresses: [email protected], [email protected] (G.-P. Di Sansebastiano). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.10.010
independent routes to arrive to the vacuole. In Arabidopsis thaliana AleuGFP interacts with AtVSR1 as does aleurain itself [8] and rapidly labels the central vacuole in most tissues while GFPChi and chitinase, whose receptor is probably an AtRMR [9], transit for a longer time in the ER and then in small vacuoles before reaching the central vacuole [5]. Arabidopsis aleurain vacuolar trafficking depends essentially from binding to VSR-1 [8] but it was recently shown that VSR-4 also might participate, demonstrating a certain functional redundancy in the VSR family [10]. Several studies have addressed the pathway of the GFPChi to vacuoles. It has been argued very convincingly that the GFPChi follows the classical pathway passing through Golgi-TGN-PVC, involving the SNARE components AtVPS45 [11] and VTI12 [6] and finally reaching the central vacuole. On the other hand, it was also found that GFPChi could bypass the Golgi-TGN-PVC route. Incubation of Arabidopsis roots expressing GFPChi with BFA did not dramatically alter the typical punctate GFPChi pattern, while AleuGFP targeting was inhibited [12]. As Zouhar and co-workers [11] asserted in their discussion it was still possible to detect a faint fluorescence in GFPChi plants crossed with VPS45-silenced plants, meaning “that less fusion protein was reaching the vacuole while the majority was effectively
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Fig. 1. Confocal images of tobacco protoplasts transiently expressing vacuolar GFP reporters. A) GFPgl133Chi fluorescence labelled the large central vacuole and the ER; B) AleuGFPgl133 labelled the central vacuole and small dot structures corresponding to PVC. “N” indicate the space occupied by the nucleus. Scale bar: 20 mm.
secreted into the apoplast”. Similar observations were also made by Sanmartín [6] in GFPChi plants crossed with vti12 null allele plants. Other typical storage proteins, the 7S and 2S globulins, are delivered to the Protein Storage Vacuole bypassing the classical route in maturing pumpkin seeds, as revealed by electron microscopy. This trafficking is achieved by PAC vesicles (Precursor Accumulating Vesicles), in which storage proteins aggregate [13]. Although functionally similar compartments were described in leaf tissues of three plant species [14], no evidence for the existence of a direct ER-Vacuole (ERV) pathway was provided but a new study of the sorting of human a-mannosidase in plant cells suggests the existence of such a pathway [15]. Bypassing the Golgi apparatus is expected to have a dramatic effect on the glycosylation patterns of vacuolar proteins. In all eukaryotes, proteins N-glycosylation occurs in the ER by the transfer of an oligosaccharide precursor to asparagine side chains in Asn-X-Ser/Thr sequences of nascent polypeptide chains [16,17]. Subsequently, glucose and mannose residues are removed from the N-glycan by ER-resident glucosidases and mannosidases. This process is highly regulated and links glycan modification to protein folding and quality control in the ER. Further processing of N-linked glycans to Man5GlcNAc2 is performed by Golgi-a-mannosidase I [16,18,19] and by glycosyltransferases within the Golgi. In plants, complex glycans formed in the Golgi are normally decorated with b-1,2-xylose and core a-1,3-fucose residues, which are absent or differently linked in mammalian systems. In some cases, plant cells further elongate the fucosylated glycans by adding b-1,3galactose and a-1,4-fucose residues forming Lewis-a epitopes (Lea). On the other hand, plants, like insects, also can modify Nglycans by removing the GlcNAc residues to produce paucimannosidic N-glycans [20]. Little is known about the glycosylation pattern of plant vacuolar proteins, even if the paucimannosidic protein N-glycosylation is considered typical [21]. The development and characterization of a new set of glycosylated vacuolar markers can contribute to a better definition of vacuolar sorting machineries and of glycan modification processes. In the present work we validate the use of two glycosylated vacuolar GFPs and provide evidences for a transport route to the vacuole that bypass the Golgi.
This glycosylation site 133 (gl133) was introduced into two vacuolar reporters, GFPChi and AleuGFP, previously expressed by our group in different plant species and tissues [7,23,24]. Several studies proved that the two reporters are sorted to vacuoles by different sorting machineries [5,6,25]. GFPChi contains the ctVSD of tobacco chitinase A [26] while AleuGFP harbours the N-terminal ssVSD from barley aleurain, containing the specific conserved sequence NPIR [5]. The resulting glycosylated reporters GFPgl133Chi and AleuGFPgl133 were transiently expressed in tobacco protoplasts and their fluorescence pattern was identical to the pattern previously described for their non-glycosylated equivalents. GFPgl133Chi labelled the ER and the central vacuole (Fig. 1A), but 1e5 mm compartments (not shown) can also be recognized in specific cell types [5]. AleuGFPgl133 labelled the central vacuole and PVCs (Fig. 1B). 2.2. Both GFPgl133Chi and AleuGFPgl133 are partially glycosylated In order to control the sorting efficiency we developed a rapid fractionation method for soluble proteins that takes advantage of protoplast fragility. After harvesting the protoplasts they were resuspended in buffer and broken by a single cycle of freezee thawing. Centrifugation separated the vacuolar sap and cellular fraction. The latter was then lysed by further freezeethawing in presence of SDS. We analyse then the three fractions corresponding to the medium (Med), the vacuolar sap (Vac) and the rest of soluble fraction including microsomal and cytosolic proteins (Mic). To validate this fractionation protocol, we used two control markers:
2. Results 2.1. Glycosylated GFPs targeted to neutral vacuoles and to lytic vacuoles We previously showed that an N-glycosylation site at position 133 of a secreted GFP (secGFP) was efficiently glycosylated [22].
Fig. 2. Fractionation of transiently expressed GFP markers. (A) Validation of the fractionation procedure with two well-known markers: GFP-KDEL and secGFP. Protoplasts were separated from the medium (Med) and fractionated in microsomes (Mic) and cell sap (Vac). The method appears adequate to distinguish the distribution of soluble GFPs between the three fractions. (B) Vacuolar targeting of glycosylated GFPs. Protoplasts were fractionated as above. The Vac fraction contains mostly soluble proteins from the vacuolar compartments.
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secGFP and GFP-KDEL [26,27]. Lacking any sorting signal, secGFP is secreted into the incubation medium, while GFP-KDEL, having at its C-terminus the KDEL signal sequence, is retained in the ER. We indeed observed the expected enrichment of secGFP in the Med fraction and of GFP-KDEL in the Mic fraction (Fig. 2A). The latter reporter was also recovered in the vacuolar sap as expected for a protein with the KDEL signal overexpressed to saturation levels [28]. Fractionation and western blot analysis of the glycosylated reporters showed that they were both correctly and efficiently targeted to the vacuolar sap (Vac, Fig. 2B). As shown previously for secGFPgl133 [22], both GFPgl133Chi and AleuGFPgl133 were only partially glycosylated (as evidenced by the presence of two very closed bands) but both glycosylated and non-glycosylated forms were found almost exclusively in the Vac fraction. Only a small fraction of both forms of GFPgl133Chi was also detected in the Mic fraction (The quantity of protein in the Mic fraction may be influenced by overexpression and receptors saturation).
2.3. Glycosylation as an indicator for transit through the Golgi apparatus for vacuolar soluble proteins In the Golgi apparatus, high-mannose-type N-glycans can undergo several modifications that will lead to complex-type N-glycans, which are resistant to endoglycosidase H (EndoH). EndoH resistance thus demonstrates the transit of a glycoprotein through the Golgi. EndoH sensitivity however is less informative, as not all N-glycans are modified in the Golgi [21]. We have already shown that the secreted marker secGFPgl133 recovered from the incubation medium was resistant to EndoH digestion while fraction recovered in the intracellular soluble fraction was sensitive to EndoH [22]. This indicated that the gl133 glycosylation site is compatible with Golgi modifications and that the glycosylated secGFPgl133 passed through the Golgi apparatus to reach its final destination. The EndoH sensitive intracellular secGFPgl133 most probably corresponds to an ER pool of not yet secreted glycoproteins. For the two vacuolar reporters, GFPgl133Chi collected from whole cells was also sensitive to EndoH digestion (Fig. 3A) while AleuGFPgl133 was resistant (Fig. 3B).
Fig. 3. Differential EndoH sensitivity of glycans on GFPgl133Chi (A) and AleuGFPgl133 (B) transiently expressed in protoplasts. The indents indicate the position of the 29 and 37 kDa markers.
2.5. GFPChi and AleuRFP localize on different Golgi domains Before reaching the vacuole, coexpressed AleuRFP and GFPChi transited through distinct small compartments (Fig. 5A). BFA
2.4. Is GFPgl133Chi trafficking to vacuole Golgi-dependent? The GFPChi pathway to the central vacuole is not identical to the pathway of other vacuolar markers such as RFP-AFVY. The C-terminal tetrapeptide AFVY is the phaseolin’s ctVSD and efficiently targets the fusion protein to the central vacuole [29,30]. Observations at confocal microscope after 36 h of expression have brought to light a different transport rate (Suppl. Fig. 1). While RFP-AFVY clearly accumulated in the central vacuole, GFPChi still labelled ER and compartments, which also contained the tetrapeptide reporter. Since the GFPgl133Chi glycans were not modified by Golgi glycosyltransferases to become EndoH resistant [22] we tested the hypothesis that this reporter may bypass the Golgi apparatus. We treated protoplasts with 100 mM Brefeldin A (BFA) [31,32], a fungal macrocyclic lactone that inhibits the activation of ARF1, the GTPase necessary for the COP I-mediated retrograde transport from the Golgi to the ER. BFA also affects vacuolar sorting [23]. After 24 h treatment we imaged cells expressing GFPgl133Chi (Fig. 4A) and AleuGFPgl133 (Fig. 4B) revealing that both markers were visibly affected by BFA as they accumulated in BFA bodies. We also treated protein extracts with EndoH and found both markers to be resistant (Fig. 4C and D).
Fig. 4. Effect of BFA on protoplasts expressing GFPgl133Chi (A) and AleuGFPgl133 (B). Scale bar: 20 mm. EndoH sensitivity assay of glycans on GFPgl133Chi (C) and AleuGFPgl133 (D) transiently expressed in protoplasts in presence of BFA. The indents indicate the position of the 29 and 37 kDa markers.
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affected the organization of these compartments identifying them as Golgi or TGNs. Again, even under the effect of BFA, the labelling by the two markers did not fully overlap (Fig. 5B). The dotted structures labelled by GFPChi did not overlap with the ER marker RFP-KDEL (Fig. 5C) nor did it co-localize with the Golgi marker STRFP (Fig. 5D). This non-co-localization with well-known markers and the consideration that TGN-localized SYP5 is necessary to GFPChi sorting [25] induced us to concentrate our attention on ERto-Golgi transport mechanisms that may differentiate GFPChi from AleuRFP. Sar1 is the small GTPase required for the formation of the COPII coat involved in the anterograde ER-to-Golgi transport. This transport can be specifically inhibited by the expression of the dominant-negative mutant Sar1H74L [33]. Co-infection experiments with GFPChi and NtSar1H74L were performed on Nicotiana benthamiana leaves to reduce the fluorescence detection problems typical of GFPChi expression in Nicotiana tabacum leaves [23]. Even in this specie where GFPChi is more stable, the number of cells with a fluorescent central vacuole was low [23]. A statistically significant increase of vacuoles labelled with GFPChi was observed when
NtSar1h74l was co-expressed (Fig. 6A and B). Counting was actually done on cells additionally expressing the Golgi marker Gonst1-RFP [34] and only cells where NtSar1H74L expression was confirmed by the altered Gonst1-RFP distribution (not shown). 3. Discussion GFPgl133 [22] was validated as a glycosylated reporter comparing the glycosylation patterns of two vacuolar forms: GFPgl133Chi and AleuGFPgl133. Gl133 glycosylation site did not affect GFP folding and sorting. Indeed, both GFPgl133Chi and AleuGFPgl133 displayed fluorescent patterns identical to those previously described for GFPChi and AleuGFP (Fig. 1) [6,25] and were normally sorted to vacuoles. To further investigate the sorting of these vacuolar markers we developed a simple fractionation procedure (Fig. 2A) to analyse by western blot their vacuolar accumulation. Western-blot also confirmed the partial glycosylation of the markers overexpressed in protoplasts similarly to what had previously been shown for secGFPgl133 [22].
Fig. 5. A) Confocal image of a protoplast coexpressing AleuRFP and GFPChi; this section does not cross the central vacuole; B) altered compartments after 12 h treatment with 100 mM BFA on cells co-expressing AleuRFP and GFPChi; C) co-expression of RFP-KDEL and GFPChi; D) co-expression of ST-RFP and GFPChi. Scale bar 20 mm.
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Fig. 6. A) Agroinfiltrated N. benthamiana leaf epidermis expressing the vacuolar reporter GFPChi and the dominant negative mutant NtSar1H74L. B) Percentage of cells showing GFPChi labelling of the central vacuole in presence or absence of Sar1H74L. Bars show the standard deviation. Chi-square significance value: p < 0.001.
The partial glycosylation is probably due to the high transfection rate of exogenous DNA resulting in a massive protein synthesis likely able to saturate the involved glycosyltransferases. The outcome of this process is the synthesis of two forms of the overexpressed proteins, glycosylated and not-glycosylated. This gives us the opportunity to compare the trafficking of the two forms [22]. Once again, protoplasts are a very versatile experimental system [35]. The sensitivity of the two reporters to endoglycosidase H (EndoH) was tested. It is known that, before the action of mannosidase II in the Golgi, N-glycans are sensitive to hydrolysis by EndoH [36]. It is not known where mannosidase II acts in the Golgi stacks, but the medial-Golgi stacks are a likely location [37,38]. Interestingly the effect of EndoH on the two vacuolar reporters was different: GFPgl133Chi was sensitive to EndoH digestion (Fig. 3A) whereas AleuGFPgl133 was not (Fig. 3B). Since Golgi enzymes have no structural obstacles to modify a glycan at the gl133 site, EndoH sensitivity can be taken as a strong indication that the fusion protein did not pass the Golgi. Therefore we hypothesize that vacuolar AleuGFPgl133 passes through the Golgi, whereas GFPgl133Chi does not. Several controls were needed to verify the hypothesis. First GFPgl133Chi chimeras could fail to acquire EndoH resistance because either they did not traffic through the Golgi complex or because the glycan itself was not available for processing. To verify if the glycan in GFPgl133Chi is accessible to glycosyltransferases we performed EndoH digestion on extracts of BFA-treated protoplasts. This fungal toxin inhibits the activation of the Arf protein required for COP I vesicle formation, disturbs the ER-Golgi trafficking and is thus widely used as a vesicle trafficking inhibitor [31,39,40], including vacuolar targeting of proteins with ctVSD [21]. For the glycan modifying enzymes the result is the relocation of Golgi enzymes into the ER [41]. If the glycans of ER-retained proteins are potentially accessible for processing they will be processed by the Golgi enzymes now re-localized in the ER. The treatment of protoplasts with BFA resulted in the inhibition of the export of both GFPgl133Chi and AleuGFPgl133 and their consequent accumulation in the so-called “Golgi bodies” [42] and hybrid ER-Golgi compartment. After the visual confirmation of the BFA effect on protoplasts, we tested the EndoH sensitivity of the expressed GFPgl133Chi and AleuGFPgl133. Treatment of protein extracts from tobacco protoplasts transiently expressing AleuGFPgl133 confirmed that the construct was insensitive to the enzyme (Fig. 4D) as already seen in the previous assay (Fig. 3B). After BFA treatment, GFPgl133Chi also became insensitive to EndoH (Fig. 4C). It is then evident that the GFPgl133Chi glycan did not escape modification to an EndoH-resistant form because did not
transit via the compartment containing the required glycosyltransferases. In fact when GFPgl133Chi and Golgi glycosyltransferases were mixed as a consequence of the BFA treatment, the glycan was modified to an EndoH-resistant form. GFPgl133Chi and AleuGFPgl133 were sorted in the same way of their non-glycosylated variants. These were used for co-localization with different fluorophore. GFPChi was co-expressed with AleuRFP and confirmed that the two markers did not co-localize (Fig. 5A). Even BFA treatment failed to completely merge the two marker distributions (Fig. 5B). While the small punctate compartments labelled by AleuGFP or AleuGFPgl133 are known to be Golgi, TGN and PVCs, the GFPChi- and GFPgl133Chi-labelled compartments had to be characterized. We performed co-localization experiments and observed that the small (
