trafﬁcofhuman a-mannosidase in plant cells suggests the … · 2013. 4. 25. ·...

Traffic of Human a-Mannosidase in Plant Cells Suggeststhe Presence of a New Endoplasmic Reticulum-to-VacuolePathway 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 specifictransport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a humana-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be usedin enzyme replacement therapy. Although ubiquitously expressed, a-mannosidases are targeted to lysosomes or vacuolesthrough different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1reaches 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 targetingsignal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminaldomain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plant-secreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence inplants 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 exoglyco-sidases responsible for the removal of a-linked Manresidues in the catabolism of glycoproteins (Danielet al., 1994). These enzymes are secretory proteins thatperform their function within the lysosomes in mam-malian cells and into the vacuoles of yeast (Saccha-romyces cerevisiae) and plant cells. Moreover, acidica-mannosidases have also been described in microor-ganisms (Santacruz-Tinoco et al., 2010). The secretoryproteins normally move from the endoplasmic reticu-lum (ER) to the target compartment using either vesi-cles or direct connections between compartments(Vitale and Hinz, 2005). These types of proteins needan N-terminal signal peptide to be inserted into the ER,which is removed in the ER lumen by signal pepti-dases. Once in the ER, secretory proteins, in the ab-sence of other types of sorting signals, are secretedout of the cell (Jurgens, 2004). With regard to acidica-mannosidases, while the primary structure of theseproteins is highly conserved among various kingdoms,

the way in which they are targeted to their finalcompartment inside the cell differs in eukaryotic cells.In animal cells, these hydrolases are transported tolysosomes thanks to trans-Golgi mannose 6-phosphatereceptors (MPRs) that recognize the phosphorylationof a specific residue of Man (Man-6-P) in the glucidicstructure of the protein. Hence, the phosphorylatedoligosaccharide side chains act as targeting signals forthe lysosomal compartment (Thomas, 2001; Hansenet al., 2004). Two types of MPRs have been identifiedwith molecular masses of 46 kD (cation-dependentMPR) and 300 kD (cation-independent MPR). MPRsare also present on the cell surface, and at least thecation-independent MPR is capable of endocytosingextracellular lysosomal hydrolases (Díaz and Pfeffer,1998). In yeast, these enzymes reach the vacuolar lo-calization by both cytoplasm-to-vacuole targeting andautophagy pathways (Hutchins and Klionsky, 2001).In plants, vacuolar a-mannosidase follows the classicsecretory pathway involving the ER-Golgi system toreach 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 enzyme-replacement therapy for a-mannosidosis, which is arare lysosomal storage disease caused by mutations inthe MAN2B1 gene (De Marchis et al., 2011). In thehuman cells, MAN2B1 is synthesized as a high-Mrprecursor that is posttranslationally modified by N-glycosylation, disulfide bridge formation, proteolysis,zinc binding, and homodimer formation (Tollersrudet al., 1997). Similarly, in transgenic plants, recombi-nant MAN2B1, provided with a plant signal peptide,is synthesized as a 110-kD precursor that undergoes

1 This work was supported by the Fondazione Cassa di Risparmiodi Perugia (project no. 2012.0197.021, Ricerca Scientifica e Tecnolog-ica), by European Cooperation in Science and Technology ActionFA0804 (Molecular Farming: Plants as a Production Platform forHigh Value Proteins), and by an Institute of Plant Genetics researchercontract and a University of Perugia doctoral fellowship to F.D.M.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed 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

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specific posttranslational modifications including N-glycosylation and proteolytic maturation in the vacu-ole, producing four processed forms with apparentmolecular masses of 70, 40, 32, and 18 kD. Unexpect-edly, recombinant MAN2B1 in tobacco, instead ofbeing secreted due to the absence in plants of MPRs(Gaudreault and Beevers, 1984), is targeted to thevacuole (De Marchis et al., 2011). Conversely, anotherhuman lysosomal enzyme, glucocerebrosidase, whenproduced in Arabidopsis (Arabidopsis thaliana) seeds,is mainly secreted in the apoplast, and only a minorfraction of the protein is detected in protein storagevacuoles (PSVs; He et al., 2012). Indeed, to facilitateglucocerebrosidase targeting to the vacuoles of carrot(Daucus carota) cells, Shaaltiel and colleagues (2007)added a seven-amino acid vacuole-targeting signal tothe C terminus of the protein. Therefore, in this study,we tried to understand which route is used by thesoluble lysosomal MAN2B1 in tobacco to reach thevacuoles.

Mammalian lysosomes are considered equivalent toplant lytic vacuoles (LVs), but plants also contain PSVsfor reserve accumulation, even if the distinction be-tween different vacuoles is debated (Frigerio et al.,2008). In plants, regardless of the type of vacuole (LVor PSV), soluble vacuolar proteins reach the vacuolethrough the Golgi apparatus (Hwang, 2008). Thetransport of most secretory proteins from the ER to theGolgi complex is coat protein II mediated beforereaching their final destinations. From the Golgi ap-paratus, vacuolar proteins reach the vacuole eitherthrough electron-opaque vesicles or via clathrin-coatedvesicles (Vitale and Hinz, 2005). Plant vacuolar sortingsignals and vacuolar sorting receptors that enable thistraffic have recently been described (Hwang, 2008; DeMarcos Lousa et al., 2012). There are certainly excep-tions to this main vacuolar sorting mechanism, char-acterized by proteins that travel directly from the ER tothe vacuole, bypassing the Golgi system, but thesepolypeptides are either membrane proteins or proteinsthat form insoluble aggregates. For example, the vacuo-lar storage proteins of pumpkin (Cucurbita maxima) reachPSVs via precursor-accumulating vesicles, bypassing theGolgi complex (Hara-Nishimura et al., 1998). In addition,the route that bypasses the Golgi system seems to belinked to the specific transport of proteins that form largeaggregates (Herman and Schmidt, 2004; Herman, 2008).Cereal prolamins, when aggregated in the ER in largepolymers, can also be transported directly from the ER toPSVs, apparently by autophagy (Levanony et al., 1992;Reyes et al., 2011). Moreover, many vacuolar enzymesare stored in ER-derived vesicles, which, under certaincircumstances such as programmed cell death or seedgermination, are directly fused with the vacuolar com-partment (Hayashi et al., 2001; Rojo et al., 2003).

We show that MAN2B1, when expressed in tobacco,reaches the vacuole of leaf cells while bypassing theGolgi and that the N-terminal domain of MAN2B1has a cryptic vacuolar targeting signal. Indeed, theremoval of 200 amino acids from the N terminus

prevents MAN2B1 vacuolar delivery, and, when fusedwith a secreted protein, this N-terminal domain is ableto redirect this protein to the vacuole by a transportmechanism without involving the Golgi apparatus.Therefore, this study describes an alternative routefollowed by plant soluble vacuolar proteins to reachthe vacuole directly from the ER, without passingthrough the Golgi complex.

RESULTS

MAN2B1 Targeting to the Vacuole Is Not Mediated byGolgi Delivery and Is Independent of the Presence ofN-Linked Glycans

When MAN2B1 is expressed in tobacco plants, thepolypeptide is synthesized in the ER as a precursorwith a molecular mass around 110 kD, then it reachesthe vacuole to be fragmented, as in the lysosome, insmaller functional polypeptides. MAN2B1 is a glyco-protein and, in animal cells, carries both complex-typeand high-Man-typeN-linked oligosaccharides (Tollersrudet al., 1997). MAN2B1 is also glycosylated in plants,but, as demonstrated by De Marchis and colleagues(2011), only high-Man-type N-linked glycans arefound, indicating that the protein does not undergoany changes mediated by Golgi enzymes. In order toensure that the precursor MAN2B1 traffic does notinvolve the Golgi apparatus, MAN2B1-transgenic to-bacco leaf protoplasts were incubated in the absence orpresence of the fungal toxin brefeldin A (BFA) andthen pulse-chase analyzed. Before the BFA treatment,these transgenic protoplasts were also transientlytransformed with plasmid pDHA.T343F in order toexpress a bean (Phaseolus vulgaris) vacuolar storageprotein, phaseolin, as a control. Phaseolin is a secretoryvacuolar protein that traffics to the vacuole, followingthe classical route through the Golgi complex. BFAnegatively affects Golgi-mediated protein traffic, andin tobacco cells, phaseolin transport to the vacuole isinhibited in the presence of this toxin (Pedrazzini et al.,1997). After each chase point, protoplasts were ho-mogenized in reducing conditions and immunopreci-pitated with anti-MAN2B1 or anti-phaseolin antiserum(Fig. 1A). In the absence of BFA, phaseolin is targeted tothe vacuole, where it is processed: the signal corre-sponding to the intact protein decreases (Fig. 1A, toppanel, arrowhead), whereas the vacuolar fragmentsderived from its proteolytic maturation increase overthe time (Fig. 1A, top panel, vertical bar). As expected,in the presence of BFA, phaseolin transport to thevacuole is interrupted, with the consequent disap-pearance of the vacuolar fragments and the corre-sponding increase of the intact protein half-life (Fig.1A, top panel, arrowhead). Conversely, the 110-kDMAN2B1 precursor remains substantially unaffectedby the addition of BFA (Fig. 1A, bottom panel, arrow).To confirm the pulse-chase analysis results, aliquots ofBFA-treated and nontreated protoplasts underwent

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microscopy analyses. Phaseolin immunolocalizationindicates that the protein is mainly detectable as alarge, condensed structure (Fig. 1, B–D), which isknown to localize in the vacuole (Frigerio et al., 2001b),whereas in the presence of BFA, phaseolin transport tothe vacuole is interrupted and the protein is retained inthe ER, as shown by the labeling of the typical plantER network (Fig. 1, E–G). On the other hand, MAN2B1localization 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 routeto vacuoles that does not pass through the Golgiapparatus.To further demonstrate that the MAN2B1 precursor

traffic does not involve the Golgi apparatus, proteinsextracted from MAN2B1-transgenic tobacco leaveswere loaded on an isopycnic Suc gradient (Fig. 2A). Asalready reported by De Marchis et al. (2011), MAN2B1is found in the vacuolar fractions, whereas the ER-marker BiP (for binding protein) is mainly detectablein the ER fractions (Fig. 2B), even though a very smallamount of BiP is also present in the vacuole, as dem-onstrated previously (Pimpl et al., 2006). The vacuolarfractions of the gradient were subjected to endogly-cosidase H (Endo-H) treatment. Endo-H cleaves the

high-Man-type N-linked glycans added to the glyco-protein in the ER, but it does not digest complex gly-cans derived from glycan modification in the Golgiapparatus. Endo-H digestion, followed by SDS-PAGEand western blot with the anti-MAN2B1 antibody,clearly shows that both the precursor and almost allthe polypeptides derived from its maturation in thevacuole are still sensitive to the action of this enzyme(Fig. 2C). Since the enzymes responsible for glyco-protein acquisition of complex glycan structures arelocated in the Golgi, this strongly suggests that thetraffic to the vacuole of the MAN2B1 precursor poly-peptide does not depend on Golgi-mediated delivery.

Moreover, to investigate if MAN2B1 transport to thevacuole involves its N-linked glycans, transgenic leafprotoplasts were incubated in the absence or presenceof tunicamycin and then pulse-chase analyzed (Fig. 3).Tunicamycin is an inhibitor of N-linked glycosylationin the ER. In the absence of the inhibitor, as alreadyobserved in Figure 1A (bottom panel), the recovery ofthe immunoselected MAN2B1 precursor decreasesover time, as a result of the precursor vacuolar delivery(Fig. 3, lanes 1–4). As expected, in the presence oftunicamycin, the glycosylation of the MAN2B1 pre-cursor is inhibited (Fig. 3; compare the size difference

Figure 1. Vacuolar delivery of MAN2B1 is not affected by BFA. A, Transgenic tobacco protoplasts expressing MAN2B1 tran-siently transformed with a bean vacuolar storage protein, phaseolin, were treated with the fungal toxin BFA (+BFA) or remainedwithout treatment (2BFA). Then, protoplasts were pulse labeled for 1 h with a mixture of [35S]Met and [35S]Cys and chased forthe indicated periods of time. After each chase point, protoplasts were homogenated in reduced conditions, immunopreci-pitated with anti-MAN2B1 or anti-phaseolin antiserum, and analyzed by SDS-PAGE and fluorography. Co, Protoplasts from awild-type plant. On the left, the arrowhead indicates phaseolin, the arrow indicates the MAN2B1 precursor, the vertical barindicates phaseolin fragments, and the asterisk represents a nonspecific band that cross reacted with the antiserum. Numbers onthe right indicate the positions of molecular mass markers in kD. B to M, Aliquots of BFA-treated or untreated protoplasts fromthe same experiment described in A were fixed, permeabilized, and subjected to immunofluorescence. The images showfluorescence originating from the anti-MAN2B1 or anti-phaseolin antiserum detected using FITC-conjugated anti-rabbit sec-ondary antibody (green signal), 49,6-diamidino-2-phenylindole (DAPI) nuclear stain (blue signal), and the overlay of the twofluorescent images. Bars = 50 mm.

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of the polypeptides in lanes 1 and 5), but the 110-kDprecursor traffic seems to be unchanged (Fig. 3, lanes5–8). This last result strongly suggests that MAN2B1N-linked glycans play no specific role in targeting tothe vacuole.

The N-Terminal Domain of MAN2B1 Has a Role in ItsIntracellular Traffic

Vacuolar sorting of a secretory protein is an activetransport that often involves definite amino acid se-quences recognized by a specific molecular mechanism(Wang et al., 2011). The mechanism that deliversMAN2B1 inside lysosomes in animal cells is based onthe presence of phosphorylated Man residues on theglycosylated polypeptide, which are recognized byMPRs located in the trans-Golgi network. We havealready shown that N-linked glycans do not act astargeting signals for MAN2B1 vacuolar delivery;therefore, other vacuolar sorting signals should bepresent in the enzyme molecule. Since MAN2B1 doesnot contain known plant vacuolar sorting signals, acomparative analysis of the amino acid sequences ofvacuolar or lysosomal a-mannosidases from differentplant and animal species was performed in order tofind conserved domains (Supplemental Fig. S2). Wefocused our attention on the N-terminal part of the

a-mannosidase enzyme, which is one of the mostconserved domains, because it could somehow be in-volved in MAN2B1 targeting to the vacuole. To verifythis hypothesis, the MAN2B1 protein was deprived ofthe first 200 N-terminal amino acids immediately afterthe signal peptide sequence, and the mutant proteingenerated was termed DN-aman. Transiently trans-formed tobacco plants expressing DN-aman were an-alyzed by immunofluorescence, and the anti-MAN2B1antibody revealed a different protein localization in-side the cells in comparison with protoplasts transientlyexpressing the intact MAN2B1 (Fig. 4A). MAN2B1 wasdetected in spherical structures known to be derivedfrom the aggregation in the vacuole of the enzymeprocessed fragments (De Marchis et al., 2011). Con-versely, DN-aman showed a reticular localization typ-ical of ER-resident proteins (Fig. 4A). To demonstrateunequivocally that DN-aman is localized to the vacuole,transgenic tobacco protoplasts expressing DN-amanwere transiently transformed with a construct encodingphaseolin as a vacuolar marker. Phaseolin is a homo-trimeric soluble protein that accumulates in the PSVs ofbean seeds. This protein is also transported to the vac-uoles in vegetative tissues of transgenic plants, where itaccumulates in the form of stable fragments of about 20to 30 kD (Frigerio et al., 1998). Total proteins extractedfrom these protoplasts were loaded on an isopycnic Sucgradient, and each fraction was analyzed by SDS-PAGE

Figure 2. MAN2B1 glycans are not modified by Golgi enzymes. A and B, Total proteins extracted from leaves of MAN2B1-transgenic tobacco plants were fractionated by centrifugation on an isopycnic Suc gradient. Total proteins from each fractionwere 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 otherone remained untreated. Proteins were analyzed by SDS-PAGE and immunoblot using anti-MAN2B1 antiserum. P, MAN2B1precursor. Black arrowheads indicate glycosylated polypeptides, and gray arrowheads indicate the corresponding deglycosy-lated 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 Bindicate the gradient fractions, which are the same for A and B. The vacuolar and ER fractions are underlined. The positions ofmolecular mass markers in kD are indicated by numbers on the side of the panels.


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and western blot with anti-MAN2B1, anti-BiP (as an ERmarker), and anti-phaseolin antisera. This experimentconfirmed that DN-aman was present only in the pro-tein fractions corresponding to ER localization (Fig. 4B).MAN2B1 precursor maturation, and the consequentformation of proteolytically derived fragments, indicateMAN2B1 localization inside the vacuole (De Marchiset al., 2011). Therefore, to further demonstrate that DN-aman was not localized in the vacuole, western-blotanalysis was performed on proteins extracted fromtransiently transformed protoplasts. As expected,MAN2B1-transformed protoplasts, alongside the110-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 kDbecome visible after prolonged exposure of the film).Conversely, DN-aman-transformed protoplasts showedonly the DN-aman 90-kD precursor (Fig. 4C, lane 2,white arrowhead), and no other polypeptide fragmentswere detectable. Hence, it is evident from these resultsthat the DN-aman protein is not able to reach the vac-uole but is localized inside the ER. However, we did notknow if DN-aman is a stable polypeptide or an unstablemutated protein that is rapidly degraded by the ERprotein quality control. To answer this question, stabletobacco plants expressing DN-aman were generated,and a pulse-chase experiment was performed on DN-aman transgenic protoplasts, indicating that the trun-cated protein is not secreted outside the cells and that ithas a lifetime of about 2 h (Supplemental Fig. S3).To understand if the MAN2B1 N-terminal domain

of 200 amino acids contains a vacuolar localizationdeterminant, a chimera protein was created linkingthis domain to the N terminus of a mutated version ofphaseolin named D418, immediately after its signalpeptide, to obtain the ManD418 fusion protein. Pha-seolin reaches the PSVs of bean seeds using as vacu-olar sorting signal four C-terminal amino acid residues

(AFVY). In D418, the removal of the AFVY stretchensures the complete protein secretion outside the cellof transgenic tobacco leaves (Frigerio et al., 1998). If theMAN2B1 N-terminal domain contained a vacuolarsorting signal, its addition to D418 would redirect thefusion protein ManD418 to the vacuole, preventing itssecretion outside the cell. To verify this hypothesis,protoplasts from tobacco leaves were transientlytransformed with plasmids pDHA.D418 and pDHA.ManD418, coding for D418 and ManD418 polypep-tides, respectively. After 24 h of incubation at 25°C,total proteins were extracted both from protoplastsand from their culture medium and analyzed bywestern blot with the anti-phaseolin antiserum (Fig. 5,A and B). Both proteins are correctly synthesizedinside the tobacco cells, and the molecular mass ofManD418 (66 kD) is the result of the D418 molecularmass (46 kD) added of the 200 MAN2B1 amino acids(Fig. 5A). Moreover, as expected, D418 has a greaterpresence in the medium than in the cells (Fig. 5A,compare lanes 3 and 6), whereas ManD418 is not de-tectable in the medium fraction (Fig. 5A, lane 5). Thesame protoplasts, analyzed by immunofluorescencewith the anti-phaseolin antibody, show that the fusionprotein is localized in discrete aggregates (Fig. 5C),resembling those of MAN2B1 localized in the vacuole(Fig. 4A). Conversely, as demonstrated by Park andcolleagues (2004), D418 is only visible in the ER beforebeing secreted outside the cell, and an ER-similarnetwork is shown in Figure 5C. These results suggestthat the MAN2B1 N-terminal domain can modify theintracellular 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 poly-peptide were generated. When total proteins extractedfrom these plants were subjected to protein blot,together with proteins extracted from tobacco plantstransformed with the wild-type phaseolin, a correctlysynthesized 66-kD ManD418 protein was evidenced bythe anti-phaseolin antiserum (Fig. 6). Alongside theManD418 polypeptide, specific fragments of about 20to 25 kD, very similar to those present in phaseolin-transformed cells, were detected (Fig. 6, vertical bar).Phaseolin polypeptides must fold and assemble in atrimeric structure before being delivered to the vacuole(Lawrence et al., 1990). This oligomerization makes theprotein resistant to full degradation operated by vac-uolar enzymes, resulting in the detection of fragmentswith a molecular mass of 20 to 30 kD (Ceriotti et al.,1991). Therefore, the presence in the ManD418 sampleof fragments similar to those of phaseolin indicates thatalso ManD418 is able to reach the vacuolar compart-ment. The ManD418 20-kD fragments are not detectablewhen the protein is transiently expressed (Fig. 5A, lane2), likely because under these conditions there is notenough time for their accumulation in the vacuole.

Figure 3. MAN2B1 targeting to the vacuole does not depend on thepresence of N-linked glycans. Protoplasts expressing MAN2B1 weretreated with tunicamycin (+TM) or not treated (2TM). Then, proto-plasts were pulse labeled for 1 h with a mixture of [35S]Met and [35S]Cys and chased for the indicated periods of time. Homogenated cellswere immunoprecipitated with anti-MAN2B1 antiserum and analyzedby SDS-PAGE and fluorography. The arrowhead marks the position ofglycosylated MAN2B1 precursor, while the arrow indicates the cor-responding deglycosylated protein. Numbers on the right indicate thepositions of molecular mass markers in kD.






To confirm the vacuolar localization of ManD418fragments, leaves from transgenic plants expressingManD418 or phaseolin were homogenated with buffercontaining 12% Suc and then subjected to an isopycnicSuc gradient in order to separate the different organ-elles according to their densities. Proteins from eachfraction of the gradient, separated by SDS-PAGE, weredetected with the anti-phaseolin antiserum (Fig. 7, Aand B). Moreover, the fractions obtained from theManD418 gradient were also immunoblotted with theanti-BiP antiserum (Fig. 7C). Both ManD418 and pha-seolin proteins, as well as the ER marker BiP, are lo-calized around fraction 11 (density of 1.17 g mL21; Fig.2), corresponding to an ER localization, whereas theirproteolytic fragments are detectable in the first frac-tions of the gradient, in which cytoplasmic and solublevacuolar proteins are usually recovered (Fig. 7, A andB). These data suggest an active transport of ManD418to the vacuole. To further demonstrate ManD418

vacuolar localization, leaf tissues of untransformedand transformed plants expressing ManD418 wereanalyzed by immunoelectron microscopy using ananti-phaseolin antiserum (Fig. 8). In ManD418transformed plants, there is no evidence of any largeaggregate resembling those described by Frigerioand colleagues (1998) for phaseolin in transgenictobacco plants, but small clusters labeled by theanti-phaseolin antiserum are detected in the vacuole(Fig. 8, A and E–G), whereas no specific labeling isobserved in the ER or the Golgi apparatus (Fig. 8,A and B). Even if these structures rarely appear invacuoles of ManD418 transformed cells, they are notpresent in the wild-type vacuoles, where only aweak cross contamination is detectable (Fig. 8, Cand D). To be sure that the observed ManD418immunogold labeling was significant, a statisticalanalysis of the labeling patterns was performed (Fig.8H). While there was no difference for immunogold

Figure 4. The N-terminal domain of MAN2B1 is necessary for vacuolar localization. A, Tobacco protoplasts transientlyexpressing the intact MAN2B1 or a mutated form deprived of the first 200 amino acids (DN-aman) were fixed and subjected toimmunofluorescence with anti-MAN2B1 antiserum, followed by incubation with secondary FITC-conjugated goat anti-rabbitantibody. Bars = 50 mm. B, Total proteins derived from transgenic tobacco protoplasts expressing DN-aman and transientlytransformed with a plasmid encoding the bean vacuolar storage protein phaseolin were fractionated by centrifugation on anisopycnic Suc gradient, and each fraction was analyzed by protein blot and visualized using anti-MAN2B1, anti-phaseolin, oranti-BiP antiserum. The top of the gradients is at the left; numbers on the top indicate the gradient fractions; the vertical barindicates 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 anti-MAN2B1 antiserum. The black arrowhead indicates MAN2B1, the white arrowhead indicates DN-aman, the gray arrowheadsindicate 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.


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labeling of various organelles between transformedand untransformed cells, a significantly higherdensity (1.7 6 0.19 particles mm22) of gold particleswas detected in the vacuoles of transformed cellswith respect to the vacuoles of wild-type cells (0.75 60.15 particles mm22). This suggests that ManD418 frag-ments are likely present in the vacuoles both as smallaggregates and in a soluble form. Therefore, it is pos-sible to conclude that in tobacco leaves, the MAN2B1N-terminal domain is able to redirect the secreted formof phaseolin D418 to the vacuole.

ManD418 Is Properly Folded, and Its Vacuolar TransportBypasses the Golgi Complex

In yeast, plants, and animals, the defective secretoryproteins are degraded by the ubiquitin/proteasome

system after retrotranslocation from the ER to the cy-tosol (Anelli and Sitia, 2008). However, in plant cells, amechanism has been recently described that providesthe degradation of unfolded polypeptides throughtheir transport from the ER to the vacuole, and itis inhibited by BFA (Foresti et al., 2008). To excludeManD418 vacuolar transport from being part of a plantvacuolar sorting pathway devoted to the disposalof defective secretory proteins, a characterization ofManD418 conformation was carried out to search foralterations of its quaternary structure. The larger do-main of the fusion protein consists of D418, which isnormally folded into trimers (Frigerio et al., 1998);therefore, it is reasonable to suggest that ManD418 canalso form trimers. In order to understand if ManD418is able to form polymers, leaves from transgenicManD418 plants were homogenated, and protein ex-tracts were subjected to a Suc sedimentation velocity

Figure 5. The secretion of phaseolin D418 isblocked by the addition of the MAN2B1 N-terminal domain. A, Total proteins, extracted fromtobacco protoplasts transiently expressing a se-creted form of phaseolin (D418) or the sameprotein fused to the MAN2B1 N-terminal domain(ManD418), and the corresponding media wereanalyzed by SDS-PAGE and protein blot. Proteinbands on the membrane were detected by re-versible Ponceau S stain. The black arrowheadindicates phaseolin D418, and the white arrow-head indicates the ManD418 fusion protein. B,Anti-phaseolin antiserum was used for the im-munoblotting. Numbers on the left indicate thepositions of molecular mass markers in kD. C, Thesame protoplasts used in A were fixed and sub-jected to immunofluorescence with anti-phaseolinantiserum, followed by incubation with secondaryFITC-conjugated goat anti-rabbit antibody. SR1,Wild-type plant. Bars = 50 mm.





gradient. Then, proteins from each gradient fractionwere separated by SDS-PAGE and visualized withthe anti-phaseolin antiserum. The ManD418 66-kDprotein reached on the gradient a peak of migrationaround 200 to 250 kD, suggesting that it assemblesinto trimers, while vacuolar fragments derived fromManD418 proteolysis formed a peak around 150 to200 kD, indicating their probable hexameric structure(Fig. 9). These results demonstrate that ManD418is correctly folded into trimers; thus, it is not a mis-folded polypeptide, and its transport to the vacuoleis not due to a sorting mechanism for defectiveproteins.

We showed that MAN2B1 reaches the vacuolewithout following the classic route involving theGolgi apparatus and that the N-terminal part of theprotein has an important role in this sorting. There-fore, ManD418 also could be addressed to the vac-uolar compartment with the same mechanism asMAN2B1. To confirm this hypothesis, transgenictobacco protoplasts expressing ManD418 were sub-jected to radioactive labeling in the absence orpresence of BFA, homogenated, and immunopreci-pitated with anti-phaseolin antiserum. BFA has noeffect on ManD418 traffic to the vacuole, because thefusion protein decreases over time, with the sametrend, in the absence or presence of the ER-Golgitraffic inhibitor (Fig. 10A). These data were confirmedby an immunolocalization experiment on the sameprotoplasts with the anti-phaseolin antiserum. Thepattern of ManD418 localization does not change inthe presence of BFA but remains mainly localized insmall aggregates (Fig. 10B). Moreover, the Endo-H

digestion of vacuolar and ER ManD418 fractions de-rived from the isopycnic gradient (Fig. 7, fractions 2and 11, respectively) shows that not only the intactManD418 but also its vacuolar fragments are sensitiveto the action of the enzyme (Fig. 10C, fractions 11and 2, respectively), indicating that ManD418 reachesthe vacuole without being modified by the Golgienzymes.

DISCUSSION

In recent years, the pathway that transports proteinsfrom the ER to the vacuole has been extensivelycharacterized, and in almost all cases, it involves themembranes of the Golgi complex (Vitale and Hinz,2005; Wang et al., 2010). The few examples of proteinsthat reach the vacuoles while bypassing the Golgiapparatus are ascribed to membrane proteins or topolypeptides that form insoluble aggregates (Levanonyet al., 1992; Hara-Nishimura et al., 1998; Herman andSchmidt, 2004). Moreover, the direct transport of pro-teins from the ER to the vacuole can be adopted bycells in particular physiological situations such as seedgermination (Chrispeels and Herman, 2000) or apo-ptosis (Hayashi et al., 2001). A recent study has pro-vided evidence for another route in the aleurone layer

Figure 6. ManD418 expressed in stable transgenic tobacco plantsundergoes proteolysis. Total proteins extracted from leaves of a wild-type plant (SR1) or from tobacco plants expressing wild-type phaseolinor ManD418 were analyzed by SDS-PAGE and protein blot using anti-phaseolin antiserum. The vertical bar indicates vacuolar fragments.The black arrowhead indicates phaseolin, and the white arrowheadindicates the ManD418 fusion protein. Numbers on the right indicatethe positions of molecular mass markers in kD.

Figure 7. ManD418 fragments are recovered in the vacuolar fractions.Total proteins extracted from leaves of transgenic tobacco plantsexpressing ManD418 or phaseolin were extracted with homogenationbuffer containing 12% Suc. Homogenates were fractionated by cen-trifugation on an isopycnic Suc gradient, and each fraction was ana-lyzed by protein blot and visualized using anti-phaseolin (A and B) oranti-BiP (C) antiserum. Vertical bars indicate vacuolar fragments.Numbers on the right indicate the positions of molecular mass markersin kD; numbers on the top indicate the gradient fractions. The vacuolarand ER fractions are underlined.


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of maize (Zea mays) seeds that transports zeins,a-globulin, and legumin-1 directly from the ER to thePSV without involving the Golgi apparatus (Reyeset al., 2011). The authors suggested the existence incereals of an atypical autophagic process that deliversER proteins directly to PSVs, sequestering them intocomplex prevacuolar compartments. However, thiskind of transport also appears to involve protein ag-gregates and represents a specialized case described inone cell type of the cereal endosperm. This studysuggests the existence in plants of an alternative typeof vacuolar traffic without involving the Golgi com-plex, which can be used by cells to transport soluble

and correctly folded vacuolar proteins. This transporthas been revealed by studying the secretory behavior ofa human soluble a-mannosidase, MAN2B1, producedin tobacco plants for biotechnological purposes (DeMarchis et al., 2011). In animal cells, this glycoprotein istargeted to lysosomes with a mechanism involvingtrans-Golgi MPRs, which recognize Man-6-P-containingglycans (Ghosh et al., 2003). Due to the lack of ananalogous mechanism in plant cells, soluble MAN2B1expressed in tobacco transgenic plants, in the absenceof any other sorting signal, should have been secretedin the apoplast. Conversely, this recombinant protein,which is correctly folded and does not form large

Figure 8. ManD418 fragments are localizedwithin the vacuole. Thin sections prepared fromyoung leaves of transgenic tobacco plantsexpressing ManD418 (A, B, and E–G) or with awild-type plant (C and D) were incubated withthe anti-phaseolin antiserum, followed by incu-bation with secondary goat anti-rabbit 15-nmgold complex. Aggregates derived fromManD418fragments (marked with arrows) were visible onlyin the vacuole of transformed plants as shown inA, whereas they were not detectable in other or-ganelles. Some of these aggregates are shown in Eto G. E is an enlargement of A. These smallclusters were not present in the wild-type vacu-oles (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 immu-nogold labeling in the vacuole of transformedcells with respect to the vacuole of wild-type cells(wt). Sixty vacuole sectors were analyzed for eachplant. The bars show mean values of numericaldensities (number of particles mm22) of 15-nmgold particles in the vacuoles. Error bars representSD. Statistical analysis was performed using a two-tailed unpaired Student’s t test, and the differencebetween the two means was considered statisti-cally significant at P , 0.05.





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 arehow can the human enzyme be targeted to the vac-uoles, which kind of route is involved, which is thevacuolar sorting determinant, and where is it locatedin the protein sequence?

Here, we have shown that, in tobacco leaf cells, thesoluble human enzyme is able to reach the vacuolewhile bypassing the Golgi complex, and this vacu-olar targeting is independent of the presence ofMAN2B1 N-linked glycans. This last result was ex-pected, because soluble plant vacuolar proteins donot use modified glycans as sorting signals (Voelkeret al., 1989) but contain short peptide sequences asvacuolar sorting signals (De Marcos Lousa et al.,2012, and refs. therein). Hence, we were looking forsuch sorting signals on the MAN2B1 protein se-quence, but no peptide sequence could be identi-fied using those published in recent reviews (Vitaleand Hinz, 2005; Hwang, 2008). Conversely, we weresurprised to find that MAN2B1 cannot be targetedto the vacuole using the same Golgi-mediated routeutilized by endogenous tobacco a-mannosidases. Infact, the “hidden” vacuolar targeting signal presentin the sequence of MAN2B1 should be similar to thatlocated in the tobacco a-mannosidases. The use of analternative vacuolar sorting mechanism for MAN2B1in plant cells could be explained by the large diver-sity of a-mannosidase transport mechanisms thatoriginated during evolution in different kingdoms(Faye et al.,1998; Hutchins and Klionsky, 2001;Hansen et al., 2004). By multiple sequence align-ment between different a-mannosidase proteins, we

decided to investigate the presence of an unknownvacuolar sorting signal in a large domain of 200amino acids comprising a conserved N-terminal re-gion of a-mannosidases. We show here that a de-leted MAN2B1 mutant (DN-aman), lacking the first200 N-terminal amino acids, is unable to reach thevacuole and is retained in the ER. This could meanthat the deleted N-terminal domain contains thesignal for MAN2B1 vacuolar delivery. Indeed, weexpected that DN-aman would be secreted out of thecell without being retained in the ER. DN-aman ERretention has two possible reasons. The first is thatremoval of the N-terminal domain produces a poly-peptide not properly folded that is retrotranslocatedto the cytosol and degraded by the proteasome(Plemper and Wolf, 1999). The other possibility isthat the removal of the 200 amino acids alters thestructure of the protein, which may become partiallyactive and therefore binds to the glycosylated pro-teins present in the ER, similar to what has beenobserved in the transport and processing of conca-navalin A in jack bean (Canavalia ensiformis). Thetransport of this glycosylated protein from the ER tothe vacuole is blocked by the removal of the glycan(Faye and Chrispeels, 1987; Vitale et al., 1993), andthe unglycosylated precursor acquires the capacityfor binding to ER glycoproteins (Min et al., 1992;Sheldon and Bowles, 1992).

To further demonstrate the presence of a vacuolarsorting signal in the first 200 N-terminal amino acidsof MAN2B1, we linked this domain to the D418protein (ManD418), which in tobacco is secreted outof the cell. D418 is a mutated form of phaseolincharacterized by the absence of the tetrapeptideAFVY, which is responsible for protein delivery tothe vacuole in tobacco cells (Frigerio et al., 1998,2001a). In this case, the ManD418 fusion protein,instead of being secreted, was mainly localized in theER as an intact polypeptide, but its processed frag-ments were detected in the vacuole, indicating thatthe MAN2B1 200-amino acid domain can at leastpartially redirect the secreted protein D418 to thisorganelle. The route followed by ManD418 from theER to the vacuole is not a degradation pathway likethe one described in plant cells by Foresti and col-leagues (2008), because ManD418 is correctly folded.The intact fusion protein is assembled into trimersin the ER, and its vacuolar fragments are oligomers,likely hexamers. Moreover, as with MAN2B1, thetransport mechanism of ManD418 bypasses the Golgicomplex, as suggested by the ManD418 vacuolarfragments that are still sensitive to Endo-H digestionas well as by the experiments in the presence of BFA.Therefore, it is reasonable to suppose that within theN-terminal domain of MAN2B1 resides a sequence ofamino acids that acts as a cryptic vacuolar sortingsignal.

An alternative explanation for our data is thatMAN2B1 and ManD418 proteins enter the cis-Golgiapparatus and bypass only the medial/trans-Golgi

Figure 9. ManD418 can form oligomers. Total proteins from tobaccoleaves expressing ManD418 were fractionated by centrifugation on aSuc sedimentation velocity gradient. Each fraction was analyzed byprotein blot and visualized using anti-phaseolin antiserum. The top ofthe gradient is on the left, and numbers on the top indicate the mo-lecular mass of sedimentation markers in kD. The white arrowheadindicates ManD418 trimers, the asterisk represents a nonspecific bandthat cross reacted with the antiserum, and the vertical bar indicatesManD418 vacuolar fragments. Numbers on the right indicate the po-sitions of molecular mass markers in kD.


De Marchis et al.




cisternae. In this way, their high-Man-type N-glycansare not changed into complex N-glycans by theenzymes located in the medial/trans-Golgi appa-ratus, a maturation process that makes them in-sensitive to Endo-H digestion. A phenomenon likethis has been described for a mutated version ofphaseolin that is retained in the ER through theinsertion of an amino acid signal (KDEL) at the Cterminus (Frigerio et al., 2001b). Phaseolin-KDEL ismostly localized in the ER, but a very small amountreaches the vacuole, where it is fragmented. Also inthis case, as for the MAN2B1 and ManD418 vacuolarfragments, the glycans of the phaseolin-KDEL vac-uolar fragments are not modified by the Golgi en-zymes; instead, from our results, phaseolin-KDELtransport to the vacuole seems to be blocked bythe action of BFA. In addition, the proportions ofMAN2B1 and ManD418 polypeptides that reach thevacuoles appear to be much bigger than thosedescribed by Frigerio and colleagues (2001b), sug-gesting that they are two different sorting mecha-nisms. Yet, we are still not able to immunoprecipitatethe MAN2B1 and ManD418 vacuolar fragments inpulse-chase experiments. This means that it is im-possible to quantify the turnover of these proteins inrelation 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 aconsequence, the secretory pathway to this organ-elle is much more complex in plants with respect toother organisms with vacuoles. In particular, protein

transport from the ER directly to the vacuole, with-out involving the predominant traffic through theGolgi complex, seems to have an important andunique biological significance in plant cells (Hermanand Schmidt, 2004). Recently, the existence of anew type of compartment, defined by plant-specificAtg8-interacting proteins, has been suggested (Honiget al., 2012). Two plant-specific proteins, termedATI1 and ATI2, have been shown to associate withnovel bodies that move on the ER network and reachthe LV, indicating that they may operate in the se-lective turnover of specific proteins. The authorssuggest that this kind of transport is probably con-stitutively active at a basal level and can increaseduring plant starvation. In the same way, we cansuppose that the expression of the human proteinMAN2B1 (and the fusion protein ManD418) in plantcell suggests the existence of a novel route for solu-ble proteins, which transports polypeptides from theER directly to the vacuoles and operates at leastin leaf cells. MAN2B1 likely has a vacuolar sortingdeterminant that is somehow recognized by thistransport machinery. However, in our work, there isno evidence of the actual route taken by MAN2B1.Therefore, it would be interesting to study in thefuture which is the minimal MAN2B1 N-terminalsequence that represents the vacuolar sorting sig-nal, if there are specific vacuolar receptors, whichendogenous plant proteins use this new route, and ifspecific compartments like autophagic bodies, pre-vacuolar compartments, or vesicles are involved inthis route.

Figure 10. ManD418 transport bypasses the Golgi complex. A, Transgenic tobacco protoplasts expressing ManD418 were pulselabeled for 1 h with a mixture of [35S]Met and [35S]Cys and chased for the indicated periods of time in the presence or absenceof BFA. Homogenated cells were immunoprecipitated with anti-phaseolin antiserum and analyzed by SDS-PAGE and fluo-rography. Co, Protoplasts from a wild-type plant. B, Protoplasts from the same experiment described in A were fixed andsubjected to immunofluorescence with rabbit anti-phaseolin antiserum, followed by incubation with the secondary FITC-conjugated goat anti-rabbit antibody. Nuclei were counterstained with 49,6-diamidino-2-phenylindole. Bars = 50 mm. C, Totalproteins from an isopycnic gradient of ManD418 (Fig. 7, fractions 2 and 11) were treated with or without Endo-H and thenanalyzed 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 mo-lecular mass markers in kD.





MATERIALS AND METHODS

Plasmid Construction and Tobacco Transformation

A scheme of pDHA.DN-aman and pDHA.ManD418 vectors is representedin Supplemental Figure S1. Other DNA vectors (pDHA.T343F andpDHA.D418) used in this study are described by Pompa et al. (2010). Both thepDHA.DN-aman and pDHA.ManD418 plasmids were derived from thepDHA.(sp1)Man2B1 vector (De Marchis et al., 2011). For pDHA.DN-amanconstruction, the pDHA.(sp1)Man2B1 vector was digested with XbaI/SacIIrestriction enzymes followed by filling of sticky ends using the Klenowfragment before ligation. This caused the deletion of 200 N-terminal aminoacids from the a-mannosidase complete protein sequence immediately afterthe signal peptide. For transgenic plant production, the fragment excised byEcoRI digestion from pDHA.DN-aman including the 35S promoter, the se-quence coding for the DN-aman protein, and the 35S terminator was intro-duced into the EcoRI site of the pGreenII binary vector (Hellens et al., 2000),thus obtaining pGreen.DN-aman. In pDHA.ManD418, the D418 complemen-tary DNA, except for the part coding for the signal peptide, was PCR amplifiedfrom the pDHA.D418 expression vector (Pompa et al., 2010) using the forwardprimer (59-GACCCGCGGGTACTTCACTCCGGGAGGAGGAAGAGAGC-39)and the reverse primer (59-CGGGCATGCCTAACCCTTTCTTCCCTTTTGCTGT-TCCTG-39) to insert a SacII and a SphI restriction site (underlined) at the 59 and 39end of the D418 gene, respectively. The resulting PCR product was cleaved withSacII and SphI and cloned into pDHA.(sp1)Man2B1 opened with the samerestriction enzymes. pGreen.ManD418 was obtained as described above forpGreen.DN-aman. Strain GV3101 of Agrobacterium tumefaciens was transformedby electroporation with pGreen.DN-aman or pGreen.ManD418 vector and used toproduce transgenic tobacco (Nicotiana tabacum ‘Petit Havana SR1’) plants as de-scribed by De Marchis et al. (2011).

Protoplast Preparation, Pulse-Chase Labeling,and Immunoprecipitation

Protoplasts were prepared from young leaves of transgenic plantsexpressing Man2B1, DN-aman, or ManD418 and subjected to pulse-chaselabeling with Pro-Mix (a mixture of [35S]Met and [35S]Cys; Amersham Bio-sciences, now part of GE Healthcare; http://www.3.gehealthcare.com), asdescribed by Pompa et al. (2010). For transient protein expression, protoplastswere isolated from small leaves of DN-aman or wild-type tobacco plants andsubjected to polyethylene glycol-mediated transfection using 40 mg of theindicated plasmid DNA as described by Pompa and Vitale (2006). Afterovernight recovery, protoplasts were subjected to pulse-chase labeling as de-scribed above. Immunoprecipitation of radioactive proteins from protoplasthomogenates was performed according to Pompa and Vitale (2006) usingrabbit polyclonal antisera raised against Man2B1 or phaseolin. The immuno-precipitates were analyzed by SDS-PAGE. After electrophoresis, gels weretreated with Amplify fluorography reagent (GE Healthcare), dried, and ex-posed for fluorography. When indicated, 10 mg mL21 BFA (from a 2 mg mL21

stock solution in ethanol; Boehringer Ingelheim; http://www.boehringer-ingelheim.com) or equivalent quantities of the respective solvents for thecontrol were added to the incubation medium 45 min before labeling and werekept at the same concentration throughout the pulse chase. Protoplasthomogenation was performed by adding 2 volumes of ice-cold homogenationbuffer (150 mM Tris-HCl, 150 mM NaCl, 1.5 mM EDTA, 1.5% Triton X-100, pH7.5, and 4% b-mercaptoethanol [2-ME]) supplemented with Complete prote-ase inhibitor cocktail (Roche; http://www.roche.com) to frozen samples.

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 mLof cell suspension was spread onto poly-Lys-coated slides (Sigma), and cellswere allowed to adhere for 30 min at room temperature. Cells were fixed for30 min at room temperature in MaCa buffer containing 4% (w/v) parafor-maldehyde. Cells were then permeabilized by being washed three times withTSW 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 rabbitanti-phaseolin or anti-Man2B1 antiserum (both at 1:1,000 dilution) occurred inthe same buffer for 1 h at room temperature. After three washes in TSW, cellswere incubated for 1 h at room temperature with fluorescein isothiocyanate(FITC)-conjugated anti-rabbit secondary antibody (BB International) at a

dilution of 1:200. After three final washes in TSW, cells were mounted inVectashield-49,6-diamidino-2-phenylindole (Vector Laboratories). Cells werevisualized with a Zeiss PALM Microbeam Axio-observer.Z1 fluorescencemicroscope equipped with a 633 oil-immersion objective. Images were col-lected with an AxioCam MRm 60N-C 1”1, ox camera (Zeiss) and visualizedwith Axiovision software.

Subcellular Fractionation

For the isopycnic gradient, young leaves of transgenic tobacco expressingphaseolin, ManD418, or DN-aman were homogenized with homogenationbuffer (12% Suc, 10 mM KCl, 100 mM Tris-Cl, pH 7.8, and 2 mM MgCl2) withoutdetergent. A continuous Suc gradient between 16% and 55% was made usingthe same buffer, and 600 mL of the homogenate was loaded on top of thegradient. After centrifugation at 141,000g for 4 h at 4°C in a Beckman SW28rotor (Beckman Coulter; http://www.beckmancoulter.com), fractions of750 mL were collected. An equal aliquot of each fraction (usually 40 mL) wastreated with loading buffer with 2-ME and then analyzed by SDS-PAGE andprotein blot as described above with anti-phaseolin or anti-Man2B1 (1:10,000)antiserum. For velocity centrifugation on Suc gradients, young leaves oftransgenic tobacco expressing ManD418 were homogenized using homoge-nation buffer (200 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, and 100 mM Tris-Cl, pH 7.8). The homogenate was loaded on a linear 5% to 25% (w/v) Sucgradient 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 BeckmanSW28 rotor, fractions of 750 mL were collected. An equal aliquot of eachfraction (40 mL) was treated with loading buffer with 2-ME and then analyzedby SDS-PAGE and protein blot.

Protein Analysis

Total proteins from leaves were extracted by homogenization with homoge-nation buffer (200 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, 100 mM Tris-Cl, pH7.8, and 4% 2-ME) supplemented with Complete protease inhibitor cocktail(Roche). For protoplast homogenation, the buffer contained 1% Triton X-100 in-stead 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 elec-trotransferred to Hybond-P membrane (Amersham Biosciences, now part of GEHealthcare) and revealed using anti-phaseolin (Pompa et al., 2010; 1:10,000 di-lution) antiserum and the Super- Signal West Pico Chemiluminescent Substrate(Pierce; http://www.thermoscientificbio.com), according to the manufacturer’sprotocol. Protein Mr markers (Fermentas; http://www.thermoscientificbio.com)were used as molecular mass markers. For Endo-H treatment of proteins derivedfrom isopycnic Suc gradient, 750 mL of each fraction, containing the proteins ofthe 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 boiledfor 15 min. Bovine serum albumin (100 mg mL21) was then added to a finalconcentration 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. Sampleswere split into two tubes and incubated with 20 milliunits of Endo-H (BoehringerMannheim), or with water as a control, at 37°C for 4 h. Total proteins were thenprecipitated adding 1 volume of cold 30% TCA, and the protein pellet waswashed twice with ice-cold acetone and then dissolved in SDS-PAGE loadingbuffer. Samples were then analyzed by SDS-PAGE followed by immunoblot asdescribed.

Electron Microscopy

Small pieces of young leaves derived from wild-type or transgenic plantsexpressing ManD418 were fixed in 1.6% (w/v) paraformaldehyde mixed with1.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH 6.9, for 1 h at roomtemperature. After being washed with 0.1 M phosphate buffer, the sampleswere dehydrated in ethanol and embedded overnight in LR Empty resin at 60°C.Ultrathin sections (70–80 nm) were cut using a Leica Microsystems UltracutE, mounted on 300-mesh nickel grids, and immunogold labeled. Grids werefloated on drops of double-distilled water, phosphate-buffered saline (PBS),normal goat serum diluted 1:10 in PBS for 10 min, and 5% bovine serumalbumin in PBS for 10 min. They were then incubated with anti-phaseolinantiserum (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


De Marchis et al.



http://www3.gehealthcare.com

http://www.boehringer-ingelheim.com

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anti-rabbit secondary antibody (1:25 dilution) conjugated with 15-nm goldparticles (BB International). The grids were washed in drops of 0.1% BSAc inPBS and in PBS and double-distilled water, poststained in uranyl acetate, andexamined with an electron microscope (EM 400 T; Philips). For statisticalanalysis of ManD418 immunogold labeling, numerical densities of gold par-ticles were measured as indicated by Philimonenko et al. (2000). For each plant(one transformed and one wild type), two resin-embedded leaf sections andfive grids were obtained from each embedded leaf. From these 10 grids, about50 random digital electron microscope images (five per grid) were taken inorder to count gold particles in the cytoplasm, vacuole, ER, Golgi apparatus,chloroplast, and mitochondria. For each organelle, from 30 to 60 image sectorswere analyzed, and the data are presented as mean values of 15-nm goldparticles mm22 6 SD. A two-tailed unpaired Student’s t test was used forstatistical analysis, and the results were considered statistically significant atP , 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 lysosomala-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 criticalreview and also for kindly providing anti-phaseolin antibody. We also thankDr. Tommaso Beccari for kindly providing anti-MAN2B1 antibody.

Received January 15, 2013; accepted February 28, 2013; published February 28,2013.

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