Traffic of Human a-Mannosidase in Plant Cells Suggests the Presence of a New Endoplasmic Reticulum-to-Vacuole Pathway without Involving the Golgi Complex1[W] Francesca De Marchis, Michele Bellucci, and Andrea Pompa* Istituto di Genetica Vegetale, Consiglio Nazionale delle Ricerche, 06128 Perugia, Italy
The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human a-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, a-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plantsecreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins.
Acidic a-mannosidases (EC 3.2.1.24) are exoglycosidases responsible for the removal of a-linked Man residues in the catabolism of glycoproteins (Daniel et al., 1994). These enzymes are secretory proteins that perform their function within the lysosomes in mammalian cells and into the vacuoles of yeast (Saccharomyces cerevisiae) and plant cells. Moreover, acidic a-mannosidases have also been described in microorganisms (Santacruz-Tinoco et al., 2010). The secretory proteins normally move from the endoplasmic reticulum (ER) to the target compartment using either vesicles or direct connections between compartments (Vitale and Hinz, 2005). These types of proteins need an N-terminal signal peptide to be inserted into the ER, which is removed in the ER lumen by signal peptidases. Once in the ER, secretory proteins, in the absence of other types of sorting signals, are secreted out of the cell (Jurgens, 2004). With regard to acidic a-mannosidases, while the primary structure of these proteins is highly conserved among various kingdoms, 1 This work was supported by the Fondazione Cassa di Risparmio di Perugia (project no. 2012.0197.021, Ricerca Scientifica e Tecnologica), by European Cooperation in Science and Technology Action FA0804 (Molecular Farming: Plants as a Production Platform for High Value Proteins), and by an Institute of Plant Genetics researcher contract and a University of Perugia doctoral fellowship to F.D.M. * Corresponding author; e-mail
[email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Andrea Pompa (
[email protected]). [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.113.214536
the way in which they are targeted to their final compartment inside the cell differs in eukaryotic cells. In animal cells, these hydrolases are transported to lysosomes thanks to trans-Golgi mannose 6-phosphate receptors (MPRs) that recognize the phosphorylation of a specific residue of Man (Man-6-P) in the glucidic structure of the protein. Hence, the phosphorylated oligosaccharide side chains act as targeting signals for the lysosomal compartment (Thomas, 2001; Hansen et al., 2004). Two types of MPRs have been identified with molecular masses of 46 kD (cation-dependent MPR) and 300 kD (cation-independent MPR). MPRs are also present on the cell surface, and at least the cation-independent MPR is capable of endocytosing extracellular lysosomal hydrolases (Díaz and Pfeffer, 1998). In yeast, these enzymes reach the vacuolar localization by both cytoplasm-to-vacuole targeting and autophagy pathways (Hutchins and Klionsky, 2001). In plants, vacuolar a-mannosidase follows the classic secretory pathway involving the ER-Golgi system to reach their final destination (Faye et al., 1998). Recently, a functional human a-mannosidase (MAN2B1) has been expressed in stably transformed tobacco (Nicotiana tabacum) plants to develop an enzymereplacement therapy for a-mannosidosis, which is a rare lysosomal storage disease caused by mutations in the MAN2B1 gene (De Marchis et al., 2011). In the human cells, MAN2B1 is synthesized as a high-Mr precursor that is posttranslationally modified by Nglycosylation, disulfide bridge formation, proteolysis, zinc binding, and homodimer formation (Tollersrud et al., 1997). Similarly, in transgenic plants, recombinant MAN2B1, provided with a plant signal peptide, is synthesized as a 110-kD precursor that undergoes
Plant PhysiologyÒ, April 2013, Vol. 161, pp. 1769–1782, www.plantphysiol.org Ó 2013 American Society of Plant Biologists. All Rights Reserved.
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specific posttranslational modifications including Nglycosylation and proteolytic maturation in the vacuole, producing four processed forms with apparent molecular masses of 70, 40, 32, and 18 kD. Unexpectedly, recombinant MAN2B1 in tobacco, instead of being secreted due to the absence in plants of MPRs (Gaudreault and Beevers, 1984), is targeted to the vacuole (De Marchis et al., 2011). Conversely, another human lysosomal enzyme, glucocerebrosidase, when produced in Arabidopsis (Arabidopsis thaliana) seeds, is mainly secreted in the apoplast, and only a minor fraction of the protein is detected in protein storage vacuoles (PSVs; He et al., 2012). Indeed, to facilitate glucocerebrosidase targeting to the vacuoles of carrot (Daucus carota) cells, Shaaltiel and colleagues (2007) added a seven-amino acid vacuole-targeting signal to the C terminus of the protein. Therefore, in this study, we tried to understand which route is used by the soluble lysosomal MAN2B1 in tobacco to reach the vacuoles. Mammalian lysosomes are considered equivalent to plant lytic vacuoles (LVs), but plants also contain PSVs for reserve accumulation, even if the distinction between different vacuoles is debated (Frigerio et al., 2008). In plants, regardless of the type of vacuole (LV or PSV), soluble vacuolar proteins reach the vacuole through the Golgi apparatus (Hwang, 2008). The transport of most secretory proteins from the ER to the Golgi complex is coat protein II mediated before reaching their final destinations. From the Golgi apparatus, vacuolar proteins reach the vacuole either through electron-opaque vesicles or via clathrin-coated vesicles (Vitale and Hinz, 2005). Plant vacuolar sorting signals and vacuolar sorting receptors that enable this traffic have recently been described (Hwang, 2008; De Marcos Lousa et al., 2012). There are certainly exceptions to this main vacuolar sorting mechanism, characterized by proteins that travel directly from the ER to the vacuole, bypassing the Golgi system, but these polypeptides are either membrane proteins or proteins that form insoluble aggregates. For example, the vacuolar storage proteins of pumpkin (Cucurbita maxima) reach PSVs via precursor-accumulating vesicles, bypassing the Golgi complex (Hara-Nishimura et al., 1998). In addition, the route that bypasses the Golgi system seems to be linked to the specific transport of proteins that form large aggregates (Herman and Schmidt, 2004; Herman, 2008). Cereal prolamins, when aggregated in the ER in large polymers, can also be transported directly from the ER to PSVs, apparently by autophagy (Levanony et al., 1992; Reyes et al., 2011). Moreover, many vacuolar enzymes are stored in ER-derived vesicles, which, under certain circumstances such as programmed cell death or seed germination, are directly fused with the vacuolar compartment (Hayashi et al., 2001; Rojo et al., 2003). We show that MAN2B1, when expressed in tobacco, reaches the vacuole of leaf cells while bypassing the Golgi and that the N-terminal domain of MAN2B1 has a cryptic vacuolar targeting signal. Indeed, the removal of 200 amino acids from the N terminus 1770
prevents MAN2B1 vacuolar delivery, and, when fused with a secreted protein, this N-terminal domain is able to redirect this protein to the vacuole by a transport mechanism without involving the Golgi apparatus. Therefore, this study describes an alternative route followed by plant soluble vacuolar proteins to reach the vacuole directly from the ER, without passing through the Golgi complex.
RESULTS MAN2B1 Targeting to the Vacuole Is Not Mediated by Golgi Delivery and Is Independent of the Presence of N-Linked Glycans
When MAN2B1 is expressed in tobacco plants, the polypeptide is synthesized in the ER as a precursor with a molecular mass around 110 kD, then it reaches the vacuole to be fragmented, as in the lysosome, in smaller functional polypeptides. MAN2B1 is a glycoprotein and, in animal cells, carries both complex-type and high-Man-type N-linked oligosaccharides (Tollersrud et al., 1997). MAN2B1 is also glycosylated in plants, but, as demonstrated by De Marchis and colleagues (2011), only high-Man-type N-linked glycans are found, indicating that the protein does not undergo any changes mediated by Golgi enzymes. In order to ensure that the precursor MAN2B1 traffic does not involve the Golgi apparatus, MAN2B1-transgenic tobacco leaf protoplasts were incubated in the absence or presence of the fungal toxin brefeldin A (BFA) and then pulse-chase analyzed. Before the BFA treatment, these transgenic protoplasts were also transiently transformed with plasmid pDHA.T343F in order to express a bean (Phaseolus vulgaris) vacuolar storage protein, phaseolin, as a control. Phaseolin is a secretory vacuolar protein that traffics to the vacuole, following the classical route through the Golgi complex. BFA negatively affects Golgi-mediated protein traffic, and in tobacco cells, phaseolin transport to the vacuole is inhibited in the presence of this toxin (Pedrazzini et al., 1997). After each chase point, protoplasts were homogenized in reducing conditions and immunoprecipitated with anti-MAN2B1 or anti-phaseolin antiserum (Fig. 1A). In the absence of BFA, phaseolin is targeted to the vacuole, where it is processed: the signal corresponding to the intact protein decreases (Fig. 1A, top panel, arrowhead), whereas the vacuolar fragments derived from its proteolytic maturation increase over the time (Fig. 1A, top panel, vertical bar). As expected, in the presence of BFA, phaseolin transport to the vacuole is interrupted, with the consequent disappearance of the vacuolar fragments and the corresponding increase of the intact protein half-life (Fig. 1A, top panel, arrowhead). Conversely, the 110-kD MAN2B1 precursor remains substantially unaffected by the addition of BFA (Fig. 1A, bottom panel, arrow). To confirm the pulse-chase analysis results, aliquots of BFA-treated and nontreated protoplasts underwent Plant Physiol. Vol. 161, 2013
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Figure 1. Vacuolar delivery of MAN2B1 is not affected by BFA. A, Transgenic tobacco protoplasts expressing MAN2B1 transiently transformed with a bean vacuolar storage protein, phaseolin, were treated with the fungal toxin BFA (+BFA) or remained without treatment (2BFA). Then, protoplasts were pulse labeled for 1 h with a mixture of [35S]Met and [35S]Cys and chased for the indicated periods of time. After each chase point, protoplasts were homogenated in reduced conditions, immunoprecipitated with anti-MAN2B1 or anti-phaseolin antiserum, and analyzed by SDS-PAGE and fluorography. Co, Protoplasts from a wild-type plant. On the left, the arrowhead indicates phaseolin, the arrow indicates the MAN2B1 precursor, the vertical bar indicates phaseolin fragments, and the asterisk represents a nonspecific band that cross reacted with the antiserum. Numbers on the right indicate the positions of molecular mass markers in kD. B to M, Aliquots of BFA-treated or untreated protoplasts from the same experiment described in A were fixed, permeabilized, and subjected to immunofluorescence. The images show fluorescence originating from the anti-MAN2B1 or anti-phaseolin antiserum detected using FITC-conjugated anti-rabbit secondary antibody (green signal), 49,6-diamidino-2-phenylindole (DAPI) nuclear stain (blue signal), and the overlay of the two fluorescent images. Bars = 50 mm.
microscopy analyses. Phaseolin immunolocalization indicates that the protein is mainly detectable as a large, condensed structure (Fig. 1, B–D), which is known to localize in the vacuole (Frigerio et al., 2001b), whereas in the presence of BFA, phaseolin transport to the vacuole is interrupted and the protein is retained in the ER, as shown by the labeling of the typical plant ER network (Fig. 1, E–G). On the other hand, MAN2B1 localization in small and delimited structures (Fig. 1, H–J) remains the same after BFA addition (Fig. 1, K–M), suggesting the existence of a MAN2B1 route to vacuoles that does not pass through the Golgi apparatus. To further demonstrate that the MAN2B1 precursor traffic does not involve the Golgi apparatus, proteins extracted from MAN2B1-transgenic tobacco leaves were loaded on an isopycnic Suc gradient (Fig. 2A). As already reported by De Marchis et al. (2011), MAN2B1 is found in the vacuolar fractions, whereas the ERmarker BiP (for binding protein) is mainly detectable in the ER fractions (Fig. 2B), even though a very small amount of BiP is also present in the vacuole, as demonstrated previously (Pimpl et al., 2006). The vacuolar fractions of the gradient were subjected to endoglycosidase H (Endo-H) treatment. Endo-H cleaves the Plant Physiol. Vol. 161, 2013
high-Man-type N-linked glycans added to the glycoprotein in the ER, but it does not digest complex glycans derived from glycan modification in the Golgi apparatus. Endo-H digestion, followed by SDS-PAGE and western blot with the anti-MAN2B1 antibody, clearly shows that both the precursor and almost all the polypeptides derived from its maturation in the vacuole are still sensitive to the action of this enzyme (Fig. 2C). Since the enzymes responsible for glycoprotein acquisition of complex glycan structures are located in the Golgi, this strongly suggests that the traffic to the vacuole of the MAN2B1 precursor polypeptide does not depend on Golgi-mediated delivery. Moreover, to investigate if MAN2B1 transport to the vacuole involves its N-linked glycans, transgenic leaf protoplasts were incubated in the absence or presence of tunicamycin and then pulse-chase analyzed (Fig. 3). Tunicamycin is an inhibitor of N-linked glycosylation in the ER. In the absence of the inhibitor, as already observed in Figure 1A (bottom panel), the recovery of the immunoselected MAN2B1 precursor decreases over time, as a result of the precursor vacuolar delivery (Fig. 3, lanes 1–4). As expected, in the presence of tunicamycin, the glycosylation of the MAN2B1 precursor is inhibited (Fig. 3; compare the size difference 1771
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Figure 2. MAN2B1 glycans are not modified by Golgi enzymes. A and B, Total proteins extracted from leaves of MAN2B1transgenic tobacco plants were fractionated by centrifugation on an isopycnic Suc gradient. Total proteins from each fraction were analyzed by SDS-PAGE and immunoblot using anti-MAN2B1 (A) or anti-BiP (B) antiserum. tot, Total protein extract. C, Vacuolar fraction 2 from the isopycnic gradient (A) was divided into two aliquots, one was treated with Endo-H and the other one remained untreated. Proteins were analyzed by SDS-PAGE and immunoblot using anti-MAN2B1 antiserum. P, MAN2B1 precursor. Black arrowheads indicate glycosylated polypeptides, and gray arrowheads indicate the corresponding deglycosylated form. The top of the gradients is at the left; numbers on the top in A indicate density (g mL21); numbers on the bottom in B indicate the gradient fractions, which are the same for A and B. The vacuolar and ER fractions are underlined. The positions of molecular mass markers in kD are indicated by numbers on the side of the panels.
of the polypeptides in lanes 1 and 5), but the 110-kD precursor traffic seems to be unchanged (Fig. 3, lanes 5–8). This last result strongly suggests that MAN2B1 N-linked glycans play no specific role in targeting to the vacuole.
The N-Terminal Domain of MAN2B1 Has a Role in Its Intracellular Traffic
Vacuolar sorting of a secretory protein is an active transport that often involves definite amino acid sequences recognized by a specific molecular mechanism (Wang et al., 2011). The mechanism that delivers MAN2B1 inside lysosomes in animal cells is based on the presence of phosphorylated Man residues on the glycosylated polypeptide, which are recognized by MPRs located in the trans-Golgi network. We have already shown that N-linked glycans do not act as targeting signals for MAN2B1 vacuolar delivery; therefore, other vacuolar sorting signals should be present in the enzyme molecule. Since MAN2B1 does not contain known plant vacuolar sorting signals, a comparative analysis of the amino acid sequences of vacuolar or lysosomal a-mannosidases from different plant and animal species was performed in order to find conserved domains (Supplemental Fig. S2). We focused our attention on the N-terminal part of the 1772
a-mannosidase enzyme, which is one of the most conserved domains, because it could somehow be involved in MAN2B1 targeting to the vacuole. To verify this hypothesis, the MAN2B1 protein was deprived of the first 200 N-terminal amino acids immediately after the signal peptide sequence, and the mutant protein generated was termed DN-aman. Transiently transformed tobacco plants expressing DN-aman were analyzed by immunofluorescence, and the anti-MAN2B1 antibody revealed a different protein localization inside the cells in comparison with protoplasts transiently expressing the intact MAN2B1 (Fig. 4A). MAN2B1 was detected in spherical structures known to be derived from the aggregation in the vacuole of the enzyme processed fragments (De Marchis et al., 2011). Conversely, DN-aman showed a reticular localization typical of ER-resident proteins (Fig. 4A). To demonstrate unequivocally that DN-aman is localized to the vacuole, transgenic tobacco protoplasts expressing DN-aman were transiently transformed with a construct encoding phaseolin as a vacuolar marker. Phaseolin is a homotrimeric soluble protein that accumulates in the PSVs of bean seeds. This protein is also transported to the vacuoles in vegetative tissues of transgenic plants, where it accumulates in the form of stable fragments of about 20 to 30 kD (Frigerio et al., 1998). Total proteins extracted from these protoplasts were loaded on an isopycnic Suc gradient, and each fraction was analyzed by SDS-PAGE Plant Physiol. Vol. 161, 2013
Protein Trafficking from Endoplasmic Reticulum to Vacuole
Figure 3. MAN2B1 targeting to the vacuole does not depend on the presence of N-linked glycans. Protoplasts expressing MAN2B1 were treated with tunicamycin (+TM) or not treated (2TM). Then, protoplasts were pulse labeled for 1 h with a mixture of [35S]Met and [35S] Cys and chased for the indicated periods of time. Homogenated cells were immunoprecipitated with anti-MAN2B1 antiserum and analyzed by SDS-PAGE and fluorography. The arrowhead marks the position of glycosylated MAN2B1 precursor, while the arrow indicates the corresponding deglycosylated protein. Numbers on the right indicate the positions of molecular mass markers in kD.
and western blot with anti-MAN2B1, anti-BiP (as an ER marker), and anti-phaseolin antisera. This experiment confirmed that DN-aman was present only in the protein fractions corresponding to ER localization (Fig. 4B). MAN2B1 precursor maturation, and the consequent formation of proteolytically derived fragments, indicate MAN2B1 localization inside the vacuole (De Marchis et al., 2011). Therefore, to further demonstrate that DNaman was not localized in the vacuole, western-blot analysis was performed on proteins extracted from transiently transformed protoplasts. As expected, MAN2B1-transformed protoplasts, alongside the 110-kD precursor (Fig. 4C, lane 3, black arrowhead), exhibited vacuolar fragments of 70 and 32 kD (Fig. 4C, lane 3, gray arrowheads; fragments of 40 and 18 kD become visible after prolonged exposure of the film). Conversely, DN-aman-transformed protoplasts showed only the DN-aman 90-kD precursor (Fig. 4C, lane 2, white arrowhead), and no other polypeptide fragments were detectable. Hence, it is evident from these results that the DN-aman protein is not able to reach the vacuole but is localized inside the ER. However, we did not know if DN-aman is a stable polypeptide or an unstable mutated protein that is rapidly degraded by the ER protein quality control. To answer this question, stable tobacco plants expressing DN-aman were generated, and a pulse-chase experiment was performed on DNaman transgenic protoplasts, indicating that the truncated protein is not secreted outside the cells and that it has a lifetime of about 2 h (Supplemental Fig. S3). To understand if the MAN2B1 N-terminal domain of 200 amino acids contains a vacuolar localization determinant, a chimera protein was created linking this domain to the N terminus of a mutated version of phaseolin named D418, immediately after its signal peptide, to obtain the ManD418 fusion protein. Phaseolin reaches the PSVs of bean seeds using as vacuolar sorting signal four C-terminal amino acid residues Plant Physiol. Vol. 161, 2013
(AFVY). In D418, the removal of the AFVY stretch ensures the complete protein secretion outside the cell of transgenic tobacco leaves (Frigerio et al., 1998). If the MAN2B1 N-terminal domain contained a vacuolar sorting signal, its addition to D418 would redirect the fusion protein ManD418 to the vacuole, preventing its secretion outside the cell. To verify this hypothesis, protoplasts from tobacco leaves were transiently transformed with plasmids pDHA.D418 and pDHA. ManD418, coding for D418 and ManD418 polypeptides, respectively. After 24 h of incubation at 25°C, total proteins were extracted both from protoplasts and from their culture medium and analyzed by western blot with the anti-phaseolin antiserum (Fig. 5, A and B). Both proteins are correctly synthesized inside the tobacco cells, and the molecular mass of ManD418 (66 kD) is the result of the D418 molecular mass (46 kD) added of the 200 MAN2B1 amino acids (Fig. 5A). Moreover, as expected, D418 has a greater presence in the medium than in the cells (Fig. 5A, compare lanes 3 and 6), whereas ManD418 is not detectable in the medium fraction (Fig. 5A, lane 5). The same protoplasts, analyzed by immunofluorescence with the anti-phaseolin antibody, show that the fusion protein is localized in discrete aggregates (Fig. 5C), resembling those of MAN2B1 localized in the vacuole (Fig. 4A). Conversely, as demonstrated by Park and colleagues (2004), D418 is only visible in the ER before being secreted outside the cell, and an ER-similar network is shown in Figure 5C. These results suggest that the MAN2B1 N-terminal domain can modify the intracellular traffic of extracellular secretory proteins, likely redirecting them to the vacuole. ManD418 Is Targeted to the Vacuole
To clearly characterize the fusion protein ManD418, stable transgenic tobacco plants expressing this polypeptide were generated. When total proteins extracted from these plants were subjected to protein blot, together with proteins extracted from tobacco plants transformed with the wild-type phaseolin, a correctly synthesized 66-kD ManD418 protein was evidenced by the anti-phaseolin antiserum (Fig. 6). Alongside the ManD418 polypeptide, specific fragments of about 20 to 25 kD, very similar to those present in phaseolintransformed cells, were detected (Fig. 6, vertical bar). Phaseolin polypeptides must fold and assemble in a trimeric structure before being delivered to the vacuole (Lawrence et al., 1990). This oligomerization makes the protein resistant to full degradation operated by vacuolar enzymes, resulting in the detection of fragments with a molecular mass of 20 to 30 kD (Ceriotti et al., 1991). Therefore, the presence in the ManD418 sample of fragments similar to those of phaseolin indicates that also ManD418 is able to reach the vacuolar compartment. The ManD418 20-kD fragments are not detectable when the protein is transiently expressed (Fig. 5A, lane 2), likely because under these conditions there is not enough time for their accumulation in the vacuole. 1773
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Figure 4. The N-terminal domain of MAN2B1 is necessary for vacuolar localization. A, Tobacco protoplasts transiently expressing the intact MAN2B1 or a mutated form deprived of the first 200 amino acids (DN-aman) were fixed and subjected to immunofluorescence with anti-MAN2B1 antiserum, followed by incubation with secondary FITC-conjugated goat anti-rabbit antibody. Bars = 50 mm. B, Total proteins derived from transgenic tobacco protoplasts expressing DN-aman and transiently transformed with a plasmid encoding the bean vacuolar storage protein phaseolin were fractionated by centrifugation on an isopycnic Suc gradient, and each fraction was analyzed by protein blot and visualized using anti-MAN2B1, anti-phaseolin, or anti-BiP antiserum. The top of the gradients is at the left; numbers on the top indicate the gradient fractions; the vertical bar indicates phaseolin vacuolar fragments of 20 to 30 kD. tot, Total protein extract. The vacuolar and ER fractions are underlined. C, Total proteins derived from the same protoplasts used in A were analyzed by SDS-PAGE and protein immunoblot using antiMAN2B1 antiserum. The black arrowhead indicates MAN2B1, the white arrowhead indicates DN-aman, the gray arrowheads indicate MAN2B1 vacuolar fragments, and the asterisk represents a nonspecific band that cross-reacted with the antiserum. Numbers on the right indicate the positions of molecular mass markers in kD. SR1, Wild-type plant.
To confirm the vacuolar localization of ManD418 fragments, leaves from transgenic plants expressing ManD418 or phaseolin were homogenated with buffer containing 12% Suc and then subjected to an isopycnic Suc gradient in order to separate the different organelles according to their densities. Proteins from each fraction of the gradient, separated by SDS-PAGE, were detected with the anti-phaseolin antiserum (Fig. 7, A and B). Moreover, the fractions obtained from the ManD418 gradient were also immunoblotted with the anti-BiP antiserum (Fig. 7C). Both ManD418 and phaseolin proteins, as well as the ER marker BiP, are localized around fraction 11 (density of 1.17 g mL21; Fig. 2), corresponding to an ER localization, whereas their proteolytic fragments are detectable in the first fractions of the gradient, in which cytoplasmic and soluble vacuolar proteins are usually recovered (Fig. 7, A and B). These data suggest an active transport of ManD418 to the vacuole. To further demonstrate ManD418 1774
vacuolar localization, leaf tissues of untransformed and transformed plants expressing ManD418 were analyzed by immunoelectron microscopy using an anti-phaseolin antiserum (Fig. 8). In ManD418 transformed plants, there is no evidence of any large aggregate resembling those described by Frigerio and colleagues (1998) for phaseolin in transgenic tobacco plants, but small clusters labeled by the anti-phaseolin antiserum are detected in the vacuole (Fig. 8, A and E–G), whereas no specific labeling is observed in the ER or the Golgi apparatus (Fig. 8, A and B). Even if these structures rarely appear in vacuoles of ManD418 transformed cells, they are not present in the wild-type vacuoles, where only a weak cross contamination is detectable (Fig. 8, C and D). To be sure that the observed ManD418 immunogold labeling was significant, a statistical analysis of the labeling patterns was performed (Fig. 8H). While there was no difference for immunogold Plant Physiol. Vol. 161, 2013
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Figure 5. The secretion of phaseolin D418 is blocked by the addition of the MAN2B1 Nterminal domain. A, Total proteins, extracted from tobacco protoplasts transiently expressing a secreted form of phaseolin (D418) or the same protein fused to the MAN2B1 N-terminal domain (ManD418), and the corresponding media were analyzed by SDS-PAGE and protein blot. Protein bands on the membrane were detected by reversible Ponceau S stain. The black arrowhead indicates phaseolin D418, and the white arrowhead indicates the ManD418 fusion protein. B, Anti-phaseolin antiserum was used for the immunoblotting. Numbers on the left indicate the positions of molecular mass markers in kD. C, The same protoplasts used in A were fixed and subjected to immunofluorescence with anti-phaseolin antiserum, followed by incubation with secondary FITC-conjugated goat anti-rabbit antibody. SR1, Wild-type plant. Bars = 50 mm.
labeling of various organelles between transformed and untransformed cells, a significantly higher density (1.7 6 0.19 particles mm22) of gold particles was detected in the vacuoles of transformed cells with respect to the vacuoles of wild-type cells (0.75 6 0.15 particles mm22). This suggests that ManD418 fragments are likely present in the vacuoles both as small aggregates and in a soluble form. Therefore, it is possible to conclude that in tobacco leaves, the MAN2B1 N-terminal domain is able to redirect the secreted form of phaseolin D418 to the vacuole.
ManD418 Is Properly Folded, and Its Vacuolar Transport Bypasses the Golgi Complex
In yeast, plants, and animals, the defective secretory proteins are degraded by the ubiquitin/proteasome Plant Physiol. Vol. 161, 2013
system after retrotranslocation from the ER to the cytosol (Anelli and Sitia, 2008). However, in plant cells, a mechanism has been recently described that provides the degradation of unfolded polypeptides through their transport from the ER to the vacuole, and it is inhibited by BFA (Foresti et al., 2008). To exclude ManD418 vacuolar transport from being part of a plant vacuolar sorting pathway devoted to the disposal of defective secretory proteins, a characterization of ManD418 conformation was carried out to search for alterations of its quaternary structure. The larger domain of the fusion protein consists of D418, which is normally folded into trimers (Frigerio et al., 1998); therefore, it is reasonable to suggest that ManD418 can also form trimers. In order to understand if ManD418 is able to form polymers, leaves from transgenic ManD418 plants were homogenated, and protein extracts were subjected to a Suc sedimentation velocity 1775
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digestion of vacuolar and ER ManD418 fractions derived from the isopycnic gradient (Fig. 7, fractions 2 and 11, respectively) shows that not only the intact ManD418 but also its vacuolar fragments are sensitive to the action of the enzyme (Fig. 10C, fractions 11 and 2, respectively), indicating that ManD418 reaches the vacuole without being modified by the Golgi enzymes. DISCUSSION
Figure 6. ManD418 expressed in stable transgenic tobacco plants undergoes proteolysis. Total proteins extracted from leaves of a wildtype plant (SR1) or from tobacco plants expressing wild-type phaseolin or ManD418 were analyzed by SDS-PAGE and protein blot using antiphaseolin antiserum. The vertical bar indicates vacuolar fragments. The black arrowhead indicates phaseolin, and the white arrowhead indicates the ManD418 fusion protein. Numbers on the right indicate the positions of molecular mass markers in kD.
gradient. Then, proteins from each gradient fraction were separated by SDS-PAGE and visualized with the anti-phaseolin antiserum. The ManD418 66-kD protein reached on the gradient a peak of migration around 200 to 250 kD, suggesting that it assembles into trimers, while vacuolar fragments derived from ManD418 proteolysis formed a peak around 150 to 200 kD, indicating their probable hexameric structure (Fig. 9). These results demonstrate that ManD418 is correctly folded into trimers; thus, it is not a misfolded polypeptide, and its transport to the vacuole is not due to a sorting mechanism for defective proteins. We showed that MAN2B1 reaches the vacuole without following the classic route involving the Golgi apparatus and that the N-terminal part of the protein has an important role in this sorting. Therefore, ManD418 also could be addressed to the vacuolar compartment with the same mechanism as MAN2B1. To confirm this hypothesis, transgenic tobacco protoplasts expressing ManD418 were subjected to radioactive labeling in the absence or presence of BFA, homogenated, and immunoprecipitated with anti-phaseolin antiserum. BFA has no effect on ManD418 traffic to the vacuole, because the fusion protein decreases over time, with the same trend, in the absence or presence of the ER-Golgi traffic inhibitor (Fig. 10A). These data were confirmed by an immunolocalization experiment on the same protoplasts with the anti-phaseolin antiserum. The pattern of ManD418 localization does not change in the presence of BFA but remains mainly localized in small aggregates (Fig. 10B). Moreover, the Endo-H 1776
In recent years, the pathway that transports proteins from the ER to the vacuole has been extensively characterized, and in almost all cases, it involves the membranes of the Golgi complex (Vitale and Hinz, 2005; Wang et al., 2010). The few examples of proteins that reach the vacuoles while bypassing the Golgi apparatus are ascribed to membrane proteins or to polypeptides that form insoluble aggregates (Levanony et al., 1992; Hara-Nishimura et al., 1998; Herman and Schmidt, 2004). Moreover, the direct transport of proteins from the ER to the vacuole can be adopted by cells in particular physiological situations such as seed germination (Chrispeels and Herman, 2000) or apoptosis (Hayashi et al., 2001). A recent study has provided evidence for another route in the aleurone layer
Figure 7. ManD418 fragments are recovered in the vacuolar fractions. Total proteins extracted from leaves of transgenic tobacco plants expressing ManD418 or phaseolin were extracted with homogenation buffer containing 12% Suc. Homogenates were fractionated by centrifugation on an isopycnic Suc gradient, and each fraction was analyzed by protein blot and visualized using anti-phaseolin (A and B) or anti-BiP (C) antiserum. Vertical bars indicate vacuolar fragments. Numbers on the right indicate the positions of molecular mass markers in kD; numbers on the top indicate the gradient fractions. The vacuolar and ER fractions are underlined. Plant Physiol. Vol. 161, 2013
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Figure 8. ManD418 fragments are localized within the vacuole. Thin sections prepared from young leaves of transgenic tobacco plants expressing ManD418 (A, B, and E–G) or with a wild-type plant (C and D) were incubated with the anti-phaseolin antiserum, followed by incubation with secondary goat anti-rabbit 15-nm gold complex. Aggregates derived from ManD418 fragments (marked with arrows) were visible only in the vacuole of transformed plants as shown in A, whereas they were not detectable in other organelles. Some of these aggregates are shown in E to G. E is an enlargement of A. These small clusters were not present in the wild-type vacuoles (C) or other subcellular organelles (D). Ch, Chloroplast; Cw, cell wall; Cy, cytoplasm; Go, Golgi apparatus; M, mitochondrion, Nu, nucleus; V, vacuole. Bars = 500 nm (A–D) and 100 nm (E–G). H, Quantitative analysis of ManD418 immunogold labeling in the vacuole of transformed cells with respect to the vacuole of wild-type cells (wt). Sixty vacuole sectors were analyzed for each plant. The bars show mean values of numerical densities (number of particles mm22) of 15-nm gold particles in the vacuoles. Error bars represent SD. Statistical analysis was performed using a twotailed unpaired Student’s t test, and the difference between the two means was considered statistically significant at P , 0.05.
of maize (Zea mays) seeds that transports zeins, a-globulin, and legumin-1 directly from the ER to the PSV without involving the Golgi apparatus (Reyes et al., 2011). The authors suggested the existence in cereals of an atypical autophagic process that delivers ER proteins directly to PSVs, sequestering them into complex prevacuolar compartments. However, this kind of transport also appears to involve protein aggregates and represents a specialized case described in one cell type of the cereal endosperm. This study suggests the existence in plants of an alternative type of vacuolar traffic without involving the Golgi complex, which can be used by cells to transport soluble Plant Physiol. Vol. 161, 2013
and correctly folded vacuolar proteins. This transport has been revealed by studying the secretory behavior of a human soluble a-mannosidase, MAN2B1, produced in tobacco plants for biotechnological purposes (De Marchis et al., 2011). In animal cells, this glycoprotein is targeted to lysosomes with a mechanism involving trans-Golgi MPRs, which recognize Man-6-P-containing glycans (Ghosh et al., 2003). Due to the lack of an analogous mechanism in plant cells, soluble MAN2B1 expressed in tobacco transgenic plants, in the absence of any other sorting signal, should have been secreted in the apoplast. Conversely, this recombinant protein, which is correctly folded and does not form large 1777
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Figure 9. ManD418 can form oligomers. Total proteins from tobacco leaves expressing ManD418 were fractionated by centrifugation on a Suc sedimentation velocity gradient. Each fraction was analyzed by protein blot and visualized using anti-phaseolin antiserum. The top of the gradient is on the left, and numbers on the top indicate the molecular mass of sedimentation markers in kD. The white arrowhead indicates ManD418 trimers, the asterisk represents a nonspecific band that cross reacted with the antiserum, and the vertical bar indicates ManD418 vacuolar fragments. Numbers on the right indicate the positions of molecular mass markers in kD.
aggregates, is localized in vacuoles, both in leaf LVs (De Marchis et al., 2011) and in seed PSVs (Supplemental Fig. S4). Thus, the open questions are how can the human enzyme be targeted to the vacuoles, which kind of route is involved, which is the vacuolar sorting determinant, and where is it located in the protein sequence? Here, we have shown that, in tobacco leaf cells, the soluble human enzyme is able to reach the vacuole while bypassing the Golgi complex, and this vacuolar targeting is independent of the presence of MAN2B1 N-linked glycans. This last result was expected, because soluble plant vacuolar proteins do not use modified glycans as sorting signals (Voelker et al., 1989) but contain short peptide sequences as vacuolar sorting signals (De Marcos Lousa et al., 2012, and refs. therein). Hence, we were looking for such sorting signals on the MAN2B1 protein sequence, but no peptide sequence could be identified using those published in recent reviews (Vitale and Hinz, 2005; Hwang, 2008). Conversely, we were surprised to find that MAN2B1 cannot be targeted to the vacuole using the same Golgi-mediated route utilized by endogenous tobacco a-mannosidases. In fact, the “hidden” vacuolar targeting signal present in the sequence of MAN2B1 should be similar to that located in the tobacco a-mannosidases. The use of an alternative vacuolar sorting mechanism for MAN2B1 in plant cells could be explained by the large diversity of a-mannosidase transport mechanisms that originated during evolution in different kingdoms (Faye et al.,1998; Hutchins and Klionsky, 2001; Hansen et al., 2004). By multiple sequence alignment between different a-mannosidase proteins, we 1778
decided to investigate the presence of an unknown vacuolar sorting signal in a large domain of 200 amino acids comprising a conserved N-terminal region of a-mannosidases. We show here that a deleted MAN2B1 mutant (DN-aman), lacking the first 200 N-terminal amino acids, is unable to reach the vacuole and is retained in the ER. This could mean that the deleted N-terminal domain contains the signal for MAN2B1 vacuolar delivery. Indeed, we expected that DN-aman would be secreted out of the cell without being retained in the ER. DN-aman ER retention has two possible reasons. The first is that removal of the N-terminal domain produces a polypeptide not properly folded that is retrotranslocated to the cytosol and degraded by the proteasome (Plemper and Wolf, 1999). The other possibility is that the removal of the 200 amino acids alters the structure of the protein, which may become partially active and therefore binds to the glycosylated proteins present in the ER, similar to what has been observed in the transport and processing of concanavalin A in jack bean (Canavalia ensiformis). The transport of this glycosylated protein from the ER to the vacuole is blocked by the removal of the glycan (Faye and Chrispeels, 1987; Vitale et al., 1993), and the unglycosylated precursor acquires the capacity for binding to ER glycoproteins (Min et al., 1992; Sheldon and Bowles, 1992). To further demonstrate the presence of a vacuolar sorting signal in the first 200 N-terminal amino acids of MAN2B1, we linked this domain to the D418 protein (ManD418), which in tobacco is secreted out of the cell. D418 is a mutated form of phaseolin characterized by the absence of the tetrapeptide AFVY, which is responsible for protein delivery to the vacuole in tobacco cells (Frigerio et al., 1998, 2001a). In this case, the ManD418 fusion protein, instead of being secreted, was mainly localized in the ER as an intact polypeptide, but its processed fragments were detected in the vacuole, indicating that the MAN2B1 200-amino acid domain can at least partially redirect the secreted protein D418 to this organelle. The route followed by ManD418 from the ER to the vacuole is not a degradation pathway like the one described in plant cells by Foresti and colleagues (2008), because ManD418 is correctly folded. The intact fusion protein is assembled into trimers in the ER, and its vacuolar fragments are oligomers, likely hexamers. Moreover, as with MAN2B1, the transport mechanism of ManD418 bypasses the Golgi complex, as suggested by the ManD418 vacuolar fragments that are still sensitive to Endo-H digestion as well as by the experiments in the presence of BFA. Therefore, it is reasonable to suppose that within the N-terminal domain of MAN2B1 resides a sequence of amino acids that acts as a cryptic vacuolar sorting signal. An alternative explanation for our data is that MAN2B1 and ManD418 proteins enter the cis-Golgi apparatus and bypass only the medial/trans-Golgi Plant Physiol. Vol. 161, 2013
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Figure 10. ManD418 transport bypasses the Golgi complex. A, Transgenic tobacco protoplasts expressing ManD418 were pulse labeled for 1 h with a mixture of [35S]Met and [35S]Cys and chased for the indicated periods of time in the presence or absence of BFA. Homogenated cells were immunoprecipitated with anti-phaseolin antiserum and analyzed by SDS-PAGE and fluorography. Co, Protoplasts from a wild-type plant. B, Protoplasts from the same experiment described in A were fixed and subjected to immunofluorescence with rabbit anti-phaseolin antiserum, followed by incubation with the secondary FITCconjugated goat anti-rabbit antibody. Nuclei were counterstained with 49,6-diamidino-2-phenylindole. Bars = 50 mm. C, Total proteins from an isopycnic gradient of ManD418 (Fig. 7, fractions 2 and 11) were treated with or without Endo-H and then analyzed by SDS-PAGE and protein blot using anti-phaseolin antiserum. Black arrowheads indicate glycosylated polypeptides, and white arrowheads indicate the corresponding deglycosylated forms. Numbers on the right indicate the positions of molecular mass markers in kD.
cisternae. In this way, their high-Man-type N-glycans are not changed into complex N-glycans by the enzymes located in the medial/trans-Golgi apparatus, a maturation process that makes them insensitive to Endo-H digestion. A phenomenon like this has been described for a mutated version of phaseolin that is retained in the ER through the insertion of an amino acid signal (KDEL) at the C terminus (Frigerio et al., 2001b). Phaseolin-KDEL is mostly localized in the ER, but a very small amount reaches the vacuole, where it is fragmented. Also in this case, as for the MAN2B1 and ManD418 vacuolar fragments, the glycans of the phaseolin-KDEL vacuolar fragments are not modified by the Golgi enzymes; instead, from our results, phaseolin-KDEL transport to the vacuole seems to be blocked by the action of BFA. In addition, the proportions of MAN2B1 and ManD418 polypeptides that reach the vacuoles appear to be much bigger than those described by Frigerio and colleagues (2001b), suggesting that they are two different sorting mechanisms. Yet, we are still not able to immunoprecipitate the MAN2B1 and ManD418 vacuolar fragments in pulse-chase experiments. This means that it is impossible to quantify the turnover of these proteins in relation to their vacuolar transport. The plant cell is the only one with two types of vacuoles (LVs and PSVs) having distinctive features (Frigerio et al., 2008; Rojo and Denecke, 2008). As a consequence, the secretory pathway to this organelle is much more complex in plants with respect to other organisms with vacuoles. In particular, protein Plant Physiol. Vol. 161, 2013
transport from the ER directly to the vacuole, without involving the predominant traffic through the Golgi complex, seems to have an important and unique biological significance in plant cells (Herman and Schmidt, 2004). Recently, the existence of a new type of compartment, defined by plant-specific Atg8-interacting proteins, has been suggested (Honig et al., 2012). Two plant-specific proteins, termed ATI1 and ATI2, have been shown to associate with novel bodies that move on the ER network and reach the LV, indicating that they may operate in the selective turnover of specific proteins. The authors suggest that this kind of transport is probably constitutively active at a basal level and can increase during plant starvation. In the same way, we can suppose that the expression of the human protein MAN2B1 (and the fusion protein ManD418) in plant cell suggests the existence of a novel route for soluble proteins, which transports polypeptides from the ER directly to the vacuoles and operates at least in leaf cells. MAN2B1 likely has a vacuolar sorting determinant that is somehow recognized by this transport machinery. However, in our work, there is no evidence of the actual route taken by MAN2B1. Therefore, it would be interesting to study in the future which is the minimal MAN2B1 N-terminal sequence that represents the vacuolar sorting signal, if there are specific vacuolar receptors, which endogenous plant proteins use this new route, and if specific compartments like autophagic bodies, prevacuolar compartments, or vesicles are involved in this route. 1779
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MATERIALS AND METHODS Plasmid Construction and Tobacco Transformation A scheme of pDHA.DN-aman and pDHA.ManD418 vectors is represented in Supplemental Figure S1. Other DNA vectors (pDHA.T343F and pDHA.D418) used in this study are described by Pompa et al. (2010). Both the pDHA.DN-aman and pDHA.ManD418 plasmids were derived from the pDHA.(sp1)Man2B1 vector (De Marchis et al., 2011). For pDHA.DN-aman construction, the pDHA.(sp1)Man2B1 vector was digested with XbaI/SacII restriction enzymes followed by filling of sticky ends using the Klenow fragment before ligation. This caused the deletion of 200 N-terminal amino acids from the a-mannosidase complete protein sequence immediately after the signal peptide. For transgenic plant production, the fragment excised by EcoRI digestion from pDHA.DN-aman including the 35S promoter, the sequence coding for the DN-aman protein, and the 35S terminator was introduced into the EcoRI site of the pGreenII binary vector (Hellens et al., 2000), thus obtaining pGreen.DN-aman. In pDHA.ManD418, the D418 complementary DNA, except for the part coding for the signal peptide, was PCR amplified from the pDHA.D418 expression vector (Pompa et al., 2010) using the forward primer (59-GACCCGCGGGTACTTCACTCCGGGAGGAGGAAGAGAGC-39) and the reverse primer (59-CGGGCATGCCTAACCCTTTCTTCCCTTTTGCTGTTCCTG-39) to insert a SacII and a SphI restriction site (underlined) at the 59 and 39 end of the D418 gene, respectively. The resulting PCR product was cleaved with SacII and SphI and cloned into pDHA.(sp1)Man2B1 opened with the same restriction enzymes. pGreen.ManD418 was obtained as described above for pGreen.DN-aman. Strain GV3101 of Agrobacterium tumefaciens was transformed by electroporation with pGreen.DN-aman or pGreen.ManD418 vector and used to produce transgenic tobacco (Nicotiana tabacum ‘Petit Havana SR1’) plants as described by De Marchis et al. (2011).
Protoplast Preparation, Pulse-Chase Labeling, and Immunoprecipitation Protoplasts were prepared from young leaves of transgenic plants expressing Man2B1, DN-aman, or ManD418 and subjected to pulse-chase labeling with Pro-Mix (a mixture of [35S]Met and [35S]Cys; Amersham Biosciences, now part of GE Healthcare; http://www.3.gehealthcare.com), as described by Pompa et al. (2010). For transient protein expression, protoplasts were isolated from small leaves of DN-aman or wild-type tobacco plants and subjected to polyethylene glycol-mediated transfection using 40 mg of the indicated plasmid DNA as described by Pompa and Vitale (2006). After overnight recovery, protoplasts were subjected to pulse-chase labeling as described above. Immunoprecipitation of radioactive proteins from protoplast homogenates was performed according to Pompa and Vitale (2006) using rabbit polyclonal antisera raised against Man2B1 or phaseolin. The immunoprecipitates were analyzed by SDS-PAGE. After electrophoresis, gels were treated with Amplify fluorography reagent (GE Healthcare), dried, and exposed for fluorography. When indicated, 10 mg mL21 BFA (from a 2 mg mL21 stock solution in ethanol; Boehringer Ingelheim; http://www.boehringeringelheim.com) or equivalent quantities of the respective solvents for the control were added to the incubation medium 45 min before labeling and were kept at the same concentration throughout the pulse chase. Protoplast homogenation was performed by adding 2 volumes of ice-cold homogenation buffer (150 mM Tris-HCl, 150 mM NaCl, 1.5 mM EDTA, 1.5% Triton X-100, pH 7.5, and 4% b-mercaptoethanol [2-ME]) supplemented with Complete protease inhibitor cocktail (Roche; http://www.roche.com) to frozen samples.
dilution of 1:200. After three final washes in TSW, cells were mounted in Vectashield-49,6-diamidino-2-phenylindole (Vector Laboratories). Cells were visualized with a Zeiss PALM Microbeam Axio-observer.Z1 fluorescence microscope equipped with a 633 oil-immersion objective. Images were collected with an AxioCam MRm 60N-C 1”1, ox camera (Zeiss) and visualized with Axiovision software.
Subcellular Fractionation For the isopycnic gradient, young leaves of transgenic tobacco expressing phaseolin, ManD418, or DN-aman were homogenized with homogenation buffer (12% Suc, 10 mM KCl, 100 mM Tris-Cl, pH 7.8, and 2 mM MgCl2) without detergent. A continuous Suc gradient between 16% and 55% was made using the same buffer, and 600 mL of the homogenate was loaded on top of the gradient. After centrifugation at 141,000g for 4 h at 4°C in a Beckman SW28 rotor (Beckman Coulter; http://www.beckmancoulter.com), fractions of 750 mL were collected. An equal aliquot of each fraction (usually 40 mL) was treated with loading buffer with 2-ME and then analyzed by SDS-PAGE and protein blot as described above with anti-phaseolin or anti-Man2B1 (1:10,000) antiserum. For velocity centrifugation on Suc gradients, young leaves of transgenic tobacco expressing ManD418 were homogenized using homogenation buffer (200 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, and 100 mM TrisCl, pH 7.8). The homogenate was loaded on a linear 5% to 25% (w/v) Suc gradient made in 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, and 50 mM Tris-Cl, pH 7.5. After centrifugation at 141,000g for 24 h at 4°C in a Beckman SW28 rotor, fractions of 750 mL were collected. An equal aliquot of each fraction (40 mL) was treated with loading buffer with 2-ME and then analyzed by SDS-PAGE and protein blot.
Protein Analysis Total proteins from leaves were extracted by homogenization with homogenation buffer (200 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, 100 mM Tris-Cl, pH 7.8, and 4% 2-ME) supplemented with Complete protease inhibitor cocktail (Roche). For protoplast homogenation, the buffer contained 1% Triton X-100 instead of 0.2%. The homogenate was centrifuged at 12,000g for 10 min at 4°C. Supernatant was analyzed by protein blot after SDS-PAGE. Proteins were electrotransferred to Hybond-P membrane (Amersham Biosciences, now part of GE Healthcare) and revealed using anti-phaseolin (Pompa et al., 2010; 1:10,000 dilution) antiserum and the Super- Signal West Pico Chemiluminescent Substrate (Pierce; http://www.thermoscientificbio.com), according to the manufacturer’s protocol. Protein Mr markers (Fermentas; http://www.thermoscientificbio.com) were used as molecular mass markers. For Endo-H treatment of proteins derived from isopycnic Suc gradient, 750 mL of each fraction, containing the proteins of the vacuole or ER, were mixed with 0.5 volume of denaturing buffer (0.5% SDS, 1% b-mercaptoethanol, and 100 mM Tris-Cl, pH 8.0), and the mixture was boiled for 15 min. Bovine serum albumin (100 mg mL21) was then added to a final concentration of 0.8 mg mL21, and samples were incubated at 37°C for 15 min. Sodium citrate, pH 5.5, was added to a final concentration of 0.25 M. Samples were split into two tubes and incubated with 20 milliunits of Endo-H (Boehringer Mannheim), or with water as a control, at 37°C for 4 h. Total proteins were then precipitated adding 1 volume of cold 30% TCA, and the protein pellet was washed twice with ice-cold acetone and then dissolved in SDS-PAGE loading buffer. Samples were then analyzed by SDS-PAGE followed by immunoblot as described.
Electron Microscopy Immunocytochemistry Protoplasts were resuspended in MaCa buffer (0.5 M mannitol, 20 mM CaCl2, and 0.1% MES, pH 5.7) at a concentration of 5 3 105 cells mL21; 300 mL of cell suspension was spread onto poly-Lys-coated slides (Sigma), and cells were allowed to adhere for 30 min at room temperature. Cells were fixed for 30 min at room temperature in MaCa buffer containing 4% (w/v) paraformaldehyde. Cells were then permeabilized by being washed three times with TSW buffer (10 mM Tris-HCl, pH 7.4, 0.9% NaCl, 0.25% gelatin, 0.02% SDS, and 0.1% Triton X-100) for 10 min at room temperature. Incubation with rabbit anti-phaseolin or anti-Man2B1 antiserum (both at 1:1,000 dilution) occurred in the same buffer for 1 h at room temperature. After three washes in TSW, cells were incubated for 1 h at room temperature with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody (BB International) at a 1780
Small pieces of young leaves derived from wild-type or transgenic plants expressing ManD418 were fixed in 1.6% (w/v) paraformaldehyde mixed with 1.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH 6.9, for 1 h at room temperature. After being washed with 0.1 M phosphate buffer, the samples were dehydrated in ethanol and embedded overnight in LR Empty resin at 60°C. Ultrathin sections (70–80 nm) were cut using a Leica Microsystems Ultracut E, mounted on 300-mesh nickel grids, and immunogold labeled. Grids were floated on drops of double-distilled water, phosphate-buffered saline (PBS), normal goat serum diluted 1:10 in PBS for 10 min, and 5% bovine serum albumin in PBS for 10 min. They were then incubated with anti-phaseolin antiserum (1:1,000 dilution) in 0.1% bovine serum albumin acetylated (BSAc; Aurion) in PBS for 1 h at room temperature. After being washed with 0.1% BSAc in PBS, the sections were incubated in the same buffer with goat Plant Physiol. Vol. 161, 2013
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anti-rabbit secondary antibody (1:25 dilution) conjugated with 15-nm gold particles (BB International). The grids were washed in drops of 0.1% BSAc in PBS and in PBS and double-distilled water, poststained in uranyl acetate, and examined with an electron microscope (EM 400 T; Philips). For statistical analysis of ManD418 immunogold labeling, numerical densities of gold particles were measured as indicated by Philimonenko et al. (2000). For each plant (one transformed and one wild type), two resin-embedded leaf sections and five grids were obtained from each embedded leaf. From these 10 grids, about 50 random digital electron microscope images (five per grid) were taken in order to count gold particles in the cytoplasm, vacuole, ER, Golgi apparatus, chloroplast, and mitochondria. For each organelle, from 30 to 60 image sectors were analyzed, and the data are presented as mean values of 15-nm gold particles mm22 6 SD. A two-tailed unpaired Student’s t test was used for statistical analysis, and the results were considered statistically significant at P , 0.05.
Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Vectors used in this study. Supplemental Figure S2. Sequence alignment of vacuolar and lysosomal a-mannosidases. Supplemental Figure S3. DN-aman half-life. Supplemental Figure S4. Seed MAN2B1 immunolocalization.
ACKNOWLEDGMENTS We thank Dr. Alessandro Vitale for his helpful comments and critical review and also for kindly providing anti-phaseolin antibody. We also thank Dr. Tommaso Beccari for kindly providing anti-MAN2B1 antibody. Received January 15, 2013; accepted February 28, 2013; published February 28, 2013.
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Plant Physiol. Vol. 161, 2013
