Retromer recycles vacuolar sorting receptors from the trans-golgi network

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1 The Plant Journal (2010) 61, doi: /j X x Retromer recycles vacuolar sorting receptors from the trans-golgi network Silke Niemes 1, Markus Langhans 1, Corrado Viotti 1, David Scheuring 1, Melody San Wan Yan 2, Liwen Jiang 2, Stefan Hillmer 1, David G. Robinson 1 and Peter Pimpl 1,* 1 Department of Cell Biology, Heidelberg Institute for Plant Sciences, University of Heidelberg, Germany, and 2 Department of Biology and Molecular Biotechnology Program, Centre for Cell and Developmental Biology, The Chinese University of Hong Kong, Shatin NT, Hong Kong, China Received 14 July 2009; revised 14 September 2009; accepted 25 September 2009; published online 4 November *For correspondence (fax ; Peter.Pimpl@zmbp.uni-tuebingen.de). SUMMARY Receptor-mediated sorting processes in the secretory pathway of eukaryotic cells rely on mechanisms to recycle the receptors after completion of transport. Based on this principle, plant vacuolar sorting receptors (VSRs) are thought to recycle after dissociating of receptor ligand complexes in a pre-vacuolar compartment. This recycling is mediated by retromer, a cytosolic coat complex that comprises sorting nexins and a large heterotrimeric subunit. To analyse retromer-mediated VSR recycling, we have used a combination of immunoelectron and fluorescence microscopy to localize the retromer components sorting nexin 1 (SNX1) and sorting nexin 2a (SNX2a) and the vacuolar sorting protein VPS29p. All retromer components localize to the trans-golgi network (TGN), which is considered to represent the early endosome of plants. In addition, we show that inhibition of retromer function in vivo by expression of SNX1 or SNX2a mutants as well as transient RNAi knockdown of all sorting nexins led to accumulation of the VSR BP80 at the TGN. Quantitative protein transport studies and live-cell imaging using fluorescent vacuolar cargo molecules revealed that arrival of these VSR ligands at the vacuole is not affected under these conditions. Based on these findings, we propose that the TGN is the location of retromer-mediated recycling of VSRs, and that transport towards the lytic vacuole downstream of the TGN is receptor-independent and occurs via maturation, similar to transition of the early endosome into the late endosome in mammalian cells. Keywords: sorting nexins, retromer, vacuolar sorting receptor, pre-vacuolar compartment, trans-golgi network, early endosomes. INTRODUCTION Transport to the mammalian lysosome or the vacuole in yeast is selective and involves receptor ligand recognition events within the trans-golgi network (TGN). In mammals the best-studied TGN receptor for this purpose is the mannosyl 6-phosphate receptor (Braulke and Bonifacino, 2008), and in yeast it is Vps10p (vacuolar sorting protein 10p; Bowers and Stevens, 2005). Efficiency of transport to these lytic compartments is achieved by recycling the receptors from an acidic intermediate compartment in which the cargo molecules dissociate from their receptors (Seaman, 2005). In contrast to anterograde receptor ligand trafficking, which involves an interaction between clathrin adaptors and a dileucine motif in the cytoplasmic tail of the receptors (Doray et al., 2002, 2007), receptor recycling back to the TGN in both yeast and mammalian cells employs a different type of cytosolic coat (retromer) and a different recognition domain in the receptor (Burda et al., 2002; Seaman, 2005, 2007; Bonifacino and Rojas, 2006). Retromer is a cytosolic complex with two subunits. The large subunit comprises three proteins: Vps35p, Vps29p, Vps26p (Verges et al., 2007; Bonifacino and Hurley, 2008; Collins et al., 2008). Receptor interaction is mediated by Vps35p (Seaman et al., 1998; Hierro et al., 2007). Vps29p possesses a phosphodiesterase fold and interacts with Vps35p, and thus apppears to be the controlling element in ssembly of the large retromer subunit (Collins et al., 2005; Wang et al., 2005). Vps26p has an arrestin fold, and the C-terminal tail of Vps26 interacts with Vps35p via its C-terminal domain (Shi et al., 2006; Collins et al., 2008). In yeast, the small subunit of retromer comprises the proteins Vps5p and Vps17p (Horazdovsky et al., 1997). These proteins have in common a phox homology (PX) domain that 107 Journal compilation ª 2009 Blackwell Publishing Ltd

2 108 Silke Niemes et al. facilitates binding to phosphoinositides (Carlton et al., 2005), and also a BAR (Bin, amphiphysin, Rvs) domain that, through coiled-coil (CC) interactions, forms a curved dimer with a net positive charge on its concave side. This allows membrane interactions and also induction of membrane curvature (Seaman and Williams, 2002). Surprisingly, there is no clear orthologue of Vps17p in mammals or in plants. Instead, the role of the Vps5p/Vps17p dimer appears to be assumed by members of the sorting nexin (SNX) family (Worby and Dixon, 2002; Verges et al., 2007). In mammals, SNX1 and SNX2 are interchangeable homologues of Vps5p, and SNX5 and SNX6 appear to be the equivalents of Vps17p (Rojas et al., 2007; Wassmer et al., 2007). Arabidopsis thaliana possesses three sorting nexins that have been suggested to form the small subunit of plant retromer (Vanoosthuyse et al., 2003; Jaillais et al., 2006; Phan et al., 2008). A complex resembling the large subunit of retromer has also been described (Oliviusson et al., 2006), and has been reported to localize to a multi-vesicular prevacuolar compartment (PVC) in tobacco (Nicotiana tabacum) BY-2 cells (Oliviusson et al., 2006; Yamazaki et al., 2008), which represents the late endosome in plants. Evidence for the participation of these proteins in the sorting of vacuolar storage proteins has been presented previously (Shimada et al., 2006; Yamazaki et al., 2008), and a role for VPS29 in the endosome-based cycling of PIN auxin efflux transporters has also been proposed (Jaillais et al., 2007). Thus, on the basis of these observations, it appears that retromer in plants is involved in post-golgi trafficking, particularly recycling from the PVC to the TGN. Interestingly, this contrasts with the situation in mammals, where retromer binds to the surface of early endosomes and recycles mannosyl 6-phosphate receptor to the TGN (Arighi et al., 2004; Seaman, 2004; Rojas et al., 2007). Recent research has revealed that the TGN in plant cells assumes the role of an early endosome (Dettmer et al., 2006; Lam et al., 2007a,b; Otegui and Spitzer, 2008; Robinson et al., 2008a). As a consequence of this, and because of the discrepancy in published data concerning the localization of sorting nexins in Arabidopsis roots (compare the results of Jaillais et al., 2006, 2008 with those of Phan et al., 2008), we consider it necessary to re-investigate the situation with respect to sorting nexin/retromer localization. To this end, we have combined transient expression studies using fluorescently tagged sorting nexin constructs with immunofluorescence and immunogold electron microscopy on various cell and tissue types. Our results have identified the TGN rather than the PVC as the location of the sorting nexins and the large subunit of retromer. This defines the TGN as the point of recycling for vacuolar sorting receptors (VSRs). Perturbation of retromer-mediated recycling using various SNX mutants in tobacco leaf protoplasts caused a shift in the steady-state distribution of the VSR from the PVC to the TGN, but did not influence the transport of soluble cargo to the vacuole. These findings indicate that anterograde transport from the TGN to the vacuole does not require VSRs, but instead may occur via maturation. RESULTS Sorting nexins and VPS29 localize to the TGN in plants The Arabidopsis sorting nexins SNX1, SNX2a and SNX2b possess a PX domain and two coiled-coil regions constituting BAR domains at the C-terminus (Figure S1c). SNX1 has a shorter N-terminal domain, reducing the overall sequence similarity to only approximately 33% (see Figure S1b). Nevertheless, there is a high degree of homology in their PX and BAR domains. To characterize the Arabidopsis sorting nexins, we have cloned SNX1, SNX2a and SNX2b and generated fluorescently tagged fusions for in vivo imaging studies. When co-expressed with the cis-golgi marker Man1 RFP (Nebenfuhr et al., 1999) in protoplasts from cultured Arabidopsis cells, SNX2a GFP gave rise to punctate signals that did not overlap with the Golgi signals (Figure 1a c), and only a minority of these signals were adjacent to each other (approximately 7%, see Figure S2b). When SNX2a GFP was co-expressed with the TGN marker YFP SYP61 (Foresti and Denecke, 2008; Zouhar et al., 2009), approximately 90% of the signals overlapped (see Figure S2b), indicating a TGN localization for SNX2a (Figure 1d f). SNX1 has the same subcellular distribution as the signals co-localize with SNX2a (Figure 1g). However, some SNX2a structures appear to lack SNX1. Co-expression of SNX1 mkate and the PVC marker ARA6 RFP resulted in virtually no overlap of signals, indicating that SNX1 is not localized to the PVC (Figure 1h). In contrast, there was a small degree of overlap with the Man1 GFP signal (Figure 1i). In tobacco protoplasts, SNX2a GFP gave a more cytosolic and less punctate pattern, and these punctate signals overlapped with YFP SYP61 rather than Man1 RFP (Figure S3a,b). As in Arabidopsis protoplasts, signals for the two sorting nexins co-localized, but SNX2a GFP and ARA6 RFP did not show any overlap (Figure S3c,d). To ascertain the location of endogenous sorting nexins, we generated a polyclonal antibody using two peptides that are present in the N-terminal domain of SNX2a but not in SNX2b or SNX1 (Figure S4a). Accordingly, this antibody was specific for SNX2a and did not cross-react with either SNX2b or SNX1 (Figure S4b). As protoplasts cannot be permeabilized without bursting, we first performed immunofluorescence on roots from transgenic Arabidopsis plants expressing fluorescent markers for Golgi (ST YFP, Grebe et al., 2003), TGN (VHA-a1 RFP, Dettmer et al., 2006) and PVC (ARA7 GFP, Ueda et al., 2004). No overlap of signals was observed with ARA7 GFP (Figure 2a), but a close association between the SNX2a immunosignal and the ST YFP signal was seen (Figure 2b). We also observed an almost perfect co-localization of VHA-a1 RFP and SNX2a (Figure 2c). In

3 Retromer recycles VSRs from the TGN 109 (a) (b) (c) (d) (e) (f) (g) (h) (i) Figure 1. Sorting nexins localize to the trans-golgi network in Arabidopsis protoplasts. (a f) Transient co-expression of SNX2a GFP with the Golgi marker Man1 RFP (a c) and the TGN marker YFP SYP61 (d f). (g i) Transient co-expression of SNX1 mkate with SNX2a GFP (g), with the PVC marker ARA6 GFP (h) and the Golgi marker Man1 GFP (i). Insets show higher magnification of regions of interest. Scale bars = 5 lm. addition, SNX2a immunofluorescence on transgenic tobacco BY-2 cell lines expressing markers either for the PVC (GFP BP80), the Golgi (GONST1 YFP) or the TGN (SCAMP1 YFP) was performed. There was no overlap between SNX2a and GFP BP80, and only a partial overlap (approximately 12%, see Figure S2b) with the Golgi signals (Figure S3e,f). In contrast, there was an almost perfect overlap (approximately 92%, see Figure S2b) with the TGN signals (Figure S3g). For immunogold electron microscopy (IEM), we used root tips of Arabidopsis and tobacco because structural preservation by high-pressure freezing is better with this tissue than with the more highly vacuolated BY-2 cells. IEM using SNX2a antibodies was specific, with virtually no background labelling (Figure 2d f). Gold particles were present at the trans face of Golgi stacks in both Arabidopsis and tobacco roots (Figure 2d,f). In addition, gold particles decorated numerous vesicles in the core of the brefeldin A (BFA) compartment (Figure 2e), which is an aggregate of TGN- and endosomalderived membranes formed in response to BFA treatment (Robinson et al., 2008b). In contrast, multi-vesicular bodies (PVCs) were not labelled with SNX2a immunogold. We also re-investigated the distribution of the large subunit of retromer by monitoring for VPS29. We therefore performed immunofluorescence analyses with VPS29p antibodies (Oliviusson et al., 2006) in tobacco BY-2 cells as well as IEM on Arabidopsis and tobacco roots. In contrast to previous observations on BY-2 cells (Oliviusson et al., 2006), we saw no overlap with the PVC marker GFP BP80, and only a partial overlap with the Golgi marker GONST1 YFP (Figure 3a,b). However, we did observe a perfect overlap of

4 110 Silke Niemes et al. (a) (b) (d) (e) Figure 2. SNX2a localizes to the TGN in situ. (a c) SNX2a immunofluorescence (red) in Arabidopsis stably expressing markers (green) for the PVC (ARA7 GFP) (a), for the trans-golgi (ST YFP) (b) and for the TGN (VHA-a1 GFP) (c). Insets show higher magnification of regions of interest. Scale bars = 5 lm. (d, e) IEM localization of SNX2a in Arabidopsis roots. SNX2a is detected at the TGN (d), and also in the core of a BFA compartment (e) (90 lm BFA, 1 h). Scale bars = 100 nm. (f) IEM localization of SNX2a to the TGN in tobacco roots. Scale bars = 100 nm. Abbreviations: c, cis-golgi; t, trans-golgi; cw, cell wall; ne, nuclear envelope. The arrowheads indicate labelling by gold particles at the trans- Golgi/TGN and in the BFA compartment. (c) (f) VPS29p immunofluorescence and SCAMP1 YFP fluorescence (Figure 3c). As was the case with SNX2a, IEM of VPS29p in both Arabidopsis and tobacco roots showed TGN localization, with no label on multi-vesicular PVCs (Figure 3d,f). Similarly, this antigen also localized to the core of the BFA compartment (Figure 3e). Together, the confocal laser scanning microscopy and IEM data obtained for both Arabidopsis and tobacco provide unequivocal evidence that the sorting nexins and the large subunit of retromer locate to the TGN rather than the PVC. Steady-state distributions of VSRs between the TGN and the PVC vary The most certain way of determining the location of endogenous BP80-type VSRs in the cell is by immunological methods, e.g. IEM. However, a BP80 construct consisting of GFP fused to the transmembrane domain and cytosolic tail of BP80 (GFP BP80) has been used on a number of occasions to visualize the steady-state distribution of endogenous VSRs (Li et al., 2002; Tse et al., 2004; Jaillais et al., 2008). This truncated reporter contains the trafficking motifs of the wild-type VSR and can compete for transport with endogenous receptors (dasilva et al., 2005), making it a useful tool to monitor VSR transport. In addition, the distribution of this reporter construct faithfully reflects the location of endogenous VSRs as shown by IEM with BP80 antibodies in tobacco BY-2 cells (Tse et al., 2004). In tobacco BY-2 cells, BP80 locates almost exclusively to the multivesicular PVC, and has proved to be a reliable marker for this organelle (Tse et al., 2004; dasilva et al., 2005; Miao et al.,

5 Retromer recycles VSRs from the TGN 111 Figure 3. The retromer subunit VPS29 localizes to the TGN in situ. (a c) VPS29 immunofluorescence in BY-2 cells stably expressing markers for the PVC (GFP BP80) (a), for the Golgi (GONST1 YFP) (b) and for the TGN (SCAMP1 YFP) (c). Scale bars = 5 lm. (d, e) IEM localization of VPS29 in Arabidopsis roots. VPS29 is detected at the TGN (d), and also in the core of a BFA compartment (e) (90 lm BFA, 1 h). Scale bars = 100 nm. (f) IEM localization of VPS29 to the TGN in tobacco roots. Scale bars = 100 nm. The arrowheads indicate labelling by gold particles at the trans-golgi/tgn and in the BFA compartment. (a) (b) (d) (e) (c) (f) 2006). However, in stably transformed Arabidopsis lines, YFP BP80 localized to both wortmannin-responsive structures and the BFA compartment (Jaillais et al., 2008). As the GFP BP80 signals in BY-2 cells were sensitive only to wortmannin, we surmised that GFP BP80 is not restricted to the PVC in Arabidopsis root cells. To test this, we performed IEM on roots of Arabidopsis and tobacco. In both cases, gold label was detected at the trans-golgi/tgn, but also at multivesiculate PVCs (Figure 4). BP80 label was always situated at the periphery of the PVC, and, under the same labelling conditions, the PVC in root cells of Arabidopsis was generally more conspicuously labelled than the PVC in tobacco root cells. Sorting nexin mutants redistribute the vacuolar sorting receptor BP80 To analyse the function of the two sorting nexins in vivo,we prepared sorting nexin mutants that lacked either the coiledcoil domains (SNX1-DCC and SNX2a-DCC), in order to prevent membrane deformation and thus the formation of retromer-coated vesicles, or the N-terminal domain (SNX1- DN and SNX2-DN), in order to inhibit interaction with the large retromer subunit and thus packaging of cargo into these vesicles (Figure S1c). To avoid possible steric hindrance caused by a fluorescence tag, and to ensure that the sorting nexin mutants were still able to interact with the large retromer unit (SNX DCC) or with another sorting nexin (SNX DN) and with the endosomal membrane via their PX domain, we used untagged mutants in these experiments. This is justified, because the rate of co-transfection of various plasmids via electroporation was extremely high. When two plasmids encoding fluorescent proteins were co-transfected, 94% of the transformed cells expressed both proteins, and triple co-transfection yielded only a slightly reduced rate, with 76% of the transformed cells expressing all three proteins (Figure S2a).

6 112 Silke Niemes et al. (a) (b) Figure 4. IEM with BP80 antibodies on root cells of Arabidopsis and tobacco. (a d) Immunogold labelling is seen at both the trans-golgi/tgn and multi-vesicular PVCs. In Arabidopsis (a, c) the label density over the PVC is generally higher than in tobacco (b, d). Scale bars = 100 nm. (c) (d) When tobacco leaf protoplasts were transfected with standard PVC (GFP BP80) and Golgi (Man1 RFP) markers, we obtained separate signals in approximately 85% of the protoplasts and adjacent/co-localizing signals in only 15% (see Figure S2c for statistical analysis) (Figure 5a,d). This situation was unchanged when wild-type sorting nexins were co-expressed (data not shown). In contrast, when the DN mutant of SNX1 or SNX2a was expressed, the Golgi and PVC signals became closely associated (Figure 5b,e). The same effect was observed when the DCC mutant of SNX1 or SNX2a was expressed (Figure 5c,f). In contrast to the control, all sorting nexin mutants changed the distribution of the Golgi and PVC markers to approximately 60% immediately adjacent/co-localized signals, with only approximately 40% of the signals remaining separate from each other (Figure S2c). Thus, a significant difference in the percentage of overlapping signals was observed between control and mutant protoplasts. This raised the question of whether the mutants caused leakage of the Golgi marker to a post-golgi (presumably PVC) location or trapping of the PVC marker at the Golgi. To elucidate the identity of the compartments showing partially overlapping Man1 RFP and GFP BP80 signals as a result of expression of mutant sorting nexins, we treated protoplasts at the end of the expression period with either wortmannin or BFA. The former inhibitor causes the PVC to swell, yielding a ring-like structure (Tse et al., 2004; Robinson et al., 2008a), while BFA in tobacco but not Arabidopsis, results in a redistribution of Golgi membranes into the ER (Ritzenthaler et al., 2002; Robinson et al., 2008b). These effects were confirmed in controls expressing only the marker proteins (Figure 5g,j), and were also observed when wild-type SNX was expressed (data not shown). However, whereas wortmannin had no effect on the punctate signals of GFP BP80 or Man1 RFP in either of the two mutant expression samples (Figure 5h,i), BFA caused the Man1 RFP signals to assume an ER-like distribution in each case (Figure 5k,l). In contrast, only a portion of the GFP BP80 signal shifted to the ER, with the rest remaining punctuate, indicating that it is localized to a part of the Golgi apparatus that is BFA-insensitive, which, in tobacco, can only be the TGN (Robinson et al., 2008b). These results show that expression of sorting nexin mutants causes the vacuolar receptor BP80 to accumulate in the Golgi apparatus/tgn rather than Golgi enzymes being transported forward into the PVC. VSR recycling is not a prerequisite for the formation of PVCs As expression of the sorting nexin mutants caused GFP BP80 to redistribute into the TGN, we wondered whether,

7 Retromer recycles VSRs from the TGN 113 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) Figure 5. Transient expression of sorting nexin mutants in tobacco protoplasts traps BP80 at the TGN. (a f) Effect of sorting nexin mutants on the distribution of Golgi marker Man1 RFP (red) and PVC marker GFP BP80 (green). Co-expression of markers alone (a, d), and together with SNX1 mutants (b, c) and SNX2a mutants (e, f). All SNX mutants shift the distribution of the markers to close proximity to each other. (g l) Protoplasts from the experiments shown in (d f) were incubated with 10 lm wortmannin for 1 h (g i) or with 36 lm BFA for 1.5 h (j l). Both mutants prevent formation of wortmannin-induced ring-like structures, compared to the control (g). Scale bars = 5 lm. Insets show higher magnification of regions of interest. under these circumstances, PVCs were still present. To answer this question, we first expressed the PVC marker ARA6 RFP (Ueda et al., 2004) together with GFP BP80 and either the SNX2a-DCC or SNX2a-DN mutants. In both cases, there was a clear separation of fluorescent signals caused by the mutants (Figure 6e g,i k) compared with the

8 114 Silke Niemes et al. controls (Figure 6a c). Based on the assumption that ARA6 RFP is a bona fide marker for the PVC, which is recruited from the cytosol onto the membrane, this observation provides evidence for the continued presence of PVCs. In control protoplasts, approximately 88% co-localization between the two PVC markers was observed, with 15 21% still colocalized after SNX2a mutant expression (Figure S2d). In a second approach, we expressed an RNAi construct to induce post-translational gene silencing of the SNXs. This construct was directed against the first CC domain of SNX2a (see Figure S1c), but was also expected to affect the other two sorting nexins, due to the high degree of similarity within this domain (Figure S1a,b). To avoid non-specific silencing of other CC domain-containing proteins, a BLAST search against the Arabidopsis genome database was performed, but, apart from a chloroplast-localized protein of unknown function (At5g37050), no further homologous sequences were found. Expression of this RNAi construct led to a dramatic loss of detectable SNX2a in both Arabidopsis and tobacco protoplasts (Figure S5). However, the same phenotype was obtained as when SNX mutants were expressed: a separation of the GFP BP80 and ARA6 RFP signals (Figure 6m o; see Figure S2d for statistical analysis). Thus perturbation of sorting nexin function does not prevent the formation of PVCs. As PVCs in all plant cells are wortmannin-sensitive (Tse et al., 2004; Robinson et al., 2008a), we tested the effects of wortmannin on the ARA6 RFP-positive structures. Unlike the control protoplasts (Figure 6d), neither the GFP BP80 nor the ARA6 RFP signals formed ring structures in protoplasts expressing the SNX mutants after application of wortmannin (Figure 6h,l). In contrast, the ARA6 RFPpositive structures in protoplasts expressing the SNXCCR- NAi construct were sensitive to wortmannin (Figure 6p). Thus, the PVCs that are formed after expression of the SNX mutants are qualitatively different to those of wild-type cells, and to those of cells in which SNX expression is repressed. Inhibition of VSR recycling does not block delivery of soluble cargo to the vacuole As expression of the sorting nexin mutants caused an arrest of BP80 in the TGN, we determined whether the mutants result in a general inhibition of protein export from the TGN, or whether the observed effect is specific for BP80 ligands. Therefore, we performed quantitative protein transport assays using barley a-amylase as a reporter for transport to the cell surface, and a derivative thereof (a-amylase sporamin) carrying the sequence-specific vacuolar sorting signal (NPIR) of sweet potato (Ipomoea batatas) sporamin, to monitor transport to the lytic vacuole (Pimpl et al., 2003). As shown in Figure 7(a), transport of the secretory reporter a-amylase to the cell surface was not significantly reduced when either SNX2a-DN or SN2a-DCC were co-expressed at the same concentration that trapped BP80 at the TGN (compare Figure 5e,f). This was also true for each SNX1 mutant (data not shown). This demonstrates that the arrest of BP80 at the TGN is not due to a general inhibition of protein export. Surprisingly, when the vacuolar reporter a-amylase sporamin was co-expressed under the same conditions as for each of the SNX2a mutants (Figure 7b) or the SNX1 mutants (data not shown), its transport is not affected either. This suggests that transport of soluble cargo molecules to the lytic vacuole still occurs despite the vacuolar sorting receptor BP80 being trapped at the TGN. To confirm this, we co-expressed SNX2a-DCC with GFP sporamin as a vacuolar reporter. This molecule carries the same sequence-specific vacuolar sorting signal as a-amylase sporamin, but also allows precise biochemical analysis of vacuolar transport, as the 40 kda transit form is proteolytically degraded in the lytic vacuole upon arrival, yielding a 28 kda form, termed the GFP core (dasilva et al., 2005). As monitored using GFP antibodies, Figure 7(c) (left panel) shows the distribution of GFP sporamin in the culture medium and the different forms of the reporter in cellular fractions. When GFP sporamin is expressed alone, the majority of the reporter is detected in the vacuolar form, and only a small amount is detected in its transit form. Co-expression of the SNX2a-DCC mutant increased the amount of transit form in medium and cellular fractions, presumably due to slightly reduced transport efficiency. However, the mutant did not prevent the processing of GFP sporamin into the vacuolar GFP core and thus arrival of the reporter at the vacuole, at concentrations (30 lg) that were shown to block the transport of BP80 (Figure 5). To rule out the possibility that this detected GFP core was the result of degradation caused by the release of vacuolar enzymes during sample preparation, protoplasts expressing only a- amylase sporamin were incubated with wortmannin in an additional control experiment. This drug is known to induce the secretion of BP80 ligands, and therefore inhibits delivery to the lytic vacuole (Pimpl et al., 2003, 2006; dasilva et al., 2005). Figure 7(c) (right panel) shows the increased amount of the secreted transit form in the culture medium caused by treatment with 1 30 lm wortmannin. Moreover, the cellular fraction contained only the unprocessed transit form but not the GFP core. This demonstrates that the GFP core, which was detected in the presence of the sorting nexin mutant, is indeed the result of vacuolar delivery. These findings were corroborated by directly visualizing the fluorescent vacuolar reporters aleurain GFP and GFP sporamin in vivo. The typical vacuolar pattern of both reporter molecules (Figure 7d,g) was not abolished by the expression of either SNX2a-DN (Figure 7e,h) or SNX2a-DCC (Figure 7f,i), and the fluorescence in the vacuole of the protoplasts remained visible in all cases. However, this vacuolar signal was somewhat weaker (compared to

9 Retromer recycles VSRs from the TGN 115 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (m) (n) (o) (p) Figure 6. Transport from the TGN towards the PVC is VSR-independent and may occur via maturation during transient expression in tobacco protoplasts. (a c) GFP BP80 (green) is delivered to the PVC as indicated by co-localization with the PVC marker ARA RFP (red) under control conditions. (d) The ARA6 RFP compartment is positively identified as the PVC by the addition of wortmannin (10 lm, 1 h), yielding ring-like structures (see inset). (e l) Co-expression of either SNX2a-DCC (e h) or SNX2a-DN (i l) with GFP BP80 and ARA6 RFP led to clear separation of the fluorescently tagged molecules (g, k), indicating the presence of the PVC even though GFP BP80 is trapped at the TGN and soluble cargo is delivered to the vacuole (compare with Figures 5 and 7). Interestingly, in neither case are the ARA6 RFP signals responsive to wortmannin (h, l). (m p) Co-expression of the SNX RNAi construct with GFP BP80 and ARA6 RFP led to clear separation of the fluorescently tagged molecules (m o). In contrast to the signature of the SNX mutants, these ARA6 RFP signals are still responsive to wortmannin (p), yielding typical ring-like structures. Insets show higher magnification of regions of interest. Scale bars = 5lm. control), and increased labelling at the periphery was seen (Figure 7e,f,h,i), suggesting that SNX2a mutant expression does partially retard transport into the vacuole. The same results were obtained by the co-expression of the SNX1-DN or SNX1-DCC mutants with each of the two fluorescent reporters (data not shown). Taken together, these results show that, while expression of sorting nexin mutants trapped GFP BP80 at the TGN, the overall consequences for vacuolar protein transport are marginal. DISCUSSION Sorting nexins locate to the TGN rather than the pre-vacuolar compartment The PX domain of mammalian sorting nexins binds to phosphoinositol 3-monophosphate (PI-3P), which is enriched in the cytosol-facing leaflet of the early endosome (EE), as well as in the internal vesicles, but with much lower amounts being present in late endosomes (Gillooly et al.,

10 116 Silke Niemes et al. (a) (c) (b) (d) (e) (f) (g) (h) (i) Figure 7. SNX2a mutants do not block vacuolar transport. (a, b) Secretion analysis in tobacco protoplasts using the secretory reporter a-amylase (a) and the vacuolar reporter a-amylase sporamin (b) alone (first column) and co-expressed with mutants of SNX2a as indicated below the axis. Error bars indicate the standard deviation from five independent experiments. (c) Immunodetection using GFP antibodies of the vacuolar reporter GFP sporamin in culture medium and total cell extracts obtained from tobacco protoplasts that were mock-transfected (-), expressed GFP sporamin alone (0) or were co-transfected with increasing concentrations of plasmid encoding SNX2a-DCC, or incubated in various concentrations of wortmannin. Black arrowheads indicate the full-length transport form; the white arrowhead indicates the processed GFP core. In contrast to wortmannin, SNX2a-DCC does not reduce the GFP core. (d i) Expression of the fluorescent vacuolar cargo aleurain GFP (d f) or GFP sporamin (g i) in tobacco protoplasts. Expression of cargo alone (d, g) or together with SNX2a-DN (e, h) or SNX2a-DCC (f). Scale bars = 5 lm. 2000, 2003). In plants, PI-3P is synthesized at the TGN and transported via the PVC to the vacuole (Kim et al., 2001). Correspondingly, two populations of differently sized PI-3Penriched structures have been reported in tobacco BY-2 cells (Vermeer et al., 2006). One of these represents the PVC, while the other was described as being in close proximity to Golgi stacks and presumably represented the TGN. Thus, from the point of view of the subcellular distribution of its lipid-binding partner, sorting nexins in plants probably locate to both the TGN and the PVC. On the other hand, due to the curvature-inducing properties of their BAR domains (Bonifacino and Hurley, 2008), it would be expected that the sorting nexins in plants localize to the TGN, rather than the PVC, which is spherical, multi-vesiculate, and completely lacks tubular protrusions (Tse et al., 2004; Haas et al., 2007). Indeed, in mammals the sorting nexin dimer tends to be

11 Retromer recycles VSRs from the TGN 117 restricted to the tubular extensions of early and recycling endosomes (Carlton et al., 2004, 2005; Rojas et al., 2007; Bonifacino and Hurley, 2008). Previously published data on the subcellular localization of fluorescent protein sorting nexin fusions in Arabidopsis are somewhat contradictory. Whereas SNX2b was found to be equally distributed between the TGN, PVC and an unidentified third endosomal compartment (Phan et al., 2008), SNX1 was localized to both a wortmannin-sensitive endosome and the BFA compartment (Jaillais et al., 2006, 2008). This suggests that SNX1 is present at the PVC, which in plants is a target for wortmannin action (Tse et al., 2004; Robinson et al., 2008a). However, as the core of the BFA compartment contains TGN-derived elements but not multi-vesicular bodies (Robinson et al., 2008a), it could mean that SNX1 is also present at the TGN. In the current study, IEM with SNX2a-specific antibodies has demonstrated unequivocally the presence of this sorting nexin at the TGN in Arabidopsis and tobacco roots, but no label was detected at the multi-vesicular PVC. These in situ observations are in agreement with the data obtained by SNX2a immunofluorescence in tobacco BY-2 cells and Arabidopsis roots stably expressing fluorescent markers for Golgi, PVC and the TGN. In all cases, we observed a clear co-localization of endogenous SNX2a with TGN markers, but not with Golgi or PVC markers. Taken together, the analysis of endogenous and transiently expressed sorting nexins in Arabidopsis and tobacco indicates that the TGN is the principal location for the sorting nexins SNX1 and SNX2a in plant cells. The large retromer subunit also locates predominantly to the TGN In Arabidopsis roots, Vps29p has been reported to co-localize with SNX1 in a wortmannin- and BFA-sensitive compartment that lies downstream of GNOM (Jaillais et al., 2007), a marker for a putative recycling endosome (Geldner et al., 2003). This result is in accordance with previously published data, which indicated that components of the large retromer subunit proteins localized to the PVC in tobacco BY-2 cells (Oliviusson et al., 2006; Yamazaki et al., 2008). However, as the Vps35/29/26 heterotrimer together with the sorting nexins constitute the retromer complex, localization of large retromer subunit proteins to the PVC contradicts the TGN localization of the sorting nexins described above. However, in both previous studies on BY-2 cells, the co-localization of retromer proteins with markers for the PVC (either BP80 or SYP21) was performed using the same protocol for double immunostaining with primary antibodies from the same animal species (Paris et al., 1996). We suspect that insufficient blockage of bound primary antibodies could be a possible reason for these spurious results. In the present investigation, where only single immunostaining in a BY-2 cell line stably expressing the PVC marker GFP BP80 was employed, co-localization of Vps29p and the PVC marker was definitely not observed. In contrast, co-localization of Vps29p and the TGN marker SCAMP was seen in BY-2 cells. In agreement with these observations are the results of IEM in Arabidopsis roots, showing positive labelling of the trans-golgi/tgn as well as the core of the BFA compartment with Vps29p antibodies, but not the multivesicular PVC. Retromer-mediated recycling of VSRs occurs at the TGN In mammalian cells, in which mannosyl 6-phosphate receptors cycle between the TGN and the early endosome, the receptors are more or less equally distributed between these two organelles (Klumperman et al., 1993), but retromer is restricted to the early endosomes (Carlton et al., 2004). By comparison, our localization data show that the distribution of VSR molecules between the early endosome (TGN) and the late endosome (PVC) can vary significantly amongst plant species. Moreover, the almost exclusive presence of VSRs in late endosomes of tobacco BY-2 cells (Tse et al., 2004) contrasts with the strict TGN localization of the retromer components. This situation is difficult to reconcile with the canonical view that the VSR BP80 accompanies vacuolar cargo from the TGN to the PVC, followed by release of ligands and retromer-mediated recycling of the receptor. When receptor recycling is perturbed, the receptors are expected to accumulate at least temporarily at the location where ligands dissociate and from which recycling occurs. In the classic scenario, this is the PVC. However, when retromer function is perturbed by expressing an SNX RNAi construct or mutants of either SNX1 or SNX2, VSRs accumulated at the TGN instead. Thus, the TGN is the organelle from which retromer recycles. Two questions arise from this conclusion. Firstly, where do the receptors recycle to? It has previously been shown that expression of a construct made up of the soluble luminal binding domain of BP80 together with the ER retrieval signal HDEL reduces the wortmannin-induced secretion of soluble vacuolar molecules (dasilva et al., 2005). As this VSR derivative probably cycles between the ER and the cis-golgi, it is possible that VSR ligand interaction occurs at the cis-golgi, or even in the ER. The latter view is supported by work of Watanabe et al. (2002), who, using the same construct, were able to detect the accumulation of vacuolar enzymes in ER fractions. Thus, retromer retrieves VSRs at least to an early Golgi compartment, if not directly to the ER. The second question is why do BP80-type VSRs appear to accumulate in the PVC, as in BY-2 cells? It should be understood that the simple localization of a receptor does not allow predictions to be made as to where ligand binding or release occurs, especially when a GFP BP80 reporter construct is used, which completely lacks ligand-binding capabilities. Thus, if VSRs cease to function in sorting at the

12 118 Silke Niemes et al. TGN, this probably means that the predominance of VSR/ GFP BP80 molecules in the PVC is a reflection of the accumulation of non-recycling receptors, probably destined for degradation in the vacuole. Sorting nexin mutants prevent wortmannin-induced PVC enlargement Wortmannin is a PI-3 kinase inhibitor (Stack and Emr, 1994) that perturbs protein trafficking to the vacuole and leads to secretion of soluble cargo molecules (dasilva et al., 2005). It also causes the release of the sorting nexins, but not the large retromer subunit (Zhong et al., 2002; Arighi et al., 2004; Oliviusson et al., 2006). Wortmannin also leads to enlargement of endosomal compartments, possibly as a result of homeotypic fusion events (Bright et al., 2001; Lam et al., 2007a). We observed that this enlargement is prevented by expression of SNX mutants, but not when their synthesis is prevented during RNAi knockdown. Sorting nexins bind via their PX domains to PI-3P at the TGN/EE, and recruit the large subunit of retromer (Bonifacino and Hurley, 2008). After interacting with the cytoplasmic tail of the receptor and removal of the receptor from the TGN/EE, retromer subunits are released from the surface of the putative retrograde transport vesicle and can then engage in further cycles of recruitment and release. However, when DCC or DN SNX mutants are expressed, they bind to PI-3P via their PX domains, but are not released from the surface of the TGN/EE as they cannot interact with one another or with the large retromer subunit. On the other hand, when an SNX RNAi construct is expressed, there are no sorting nexins to bind (or too few), and there is a surplus of free PI-3P. This may be why wortmannin affects the PVC, rather than the TGN, although the target for wortmannin, PI-3 kinase, is potentially present in both compartments. TGN to vacuole transport does not require VSR-mediated sorting and occurs via maturation of the TGN into the PVC If VSR recycling took place at the TGN, inhibition of receptor transport at the point of recycling should not affect the downstream transport of their ligands, and this is exactly what we have observed (see Figure 7). Neither the expression of SNX1 nor SNX2a mutants prevented arrival of soluble cargo molecules at the vacuole or induced their secretion. Interestingly, over-production of SNX2b was also reported to result in accumulation of the endocytic marker FM4-64 in enlarged endosomal compartments containing the TGN marker SYP41 as well as the PVC marker SYP21 (Phan et al., 2008). Nevertheless, vacuolar delivery of soluble biosynthetic cargo molecules was not inhibited under these conditions. This strongly suggests that VSRs are not required for post-tgn transport of soluble cargo. Our findings are in full agreement with retromer location and function in mammalian cells, and with the receptorindependent transport of acid hydrolases from the early endosome to the late endosome (Braulke and Bonifacino, 2009). Therefore, it is tempting to assume that the equivalent of this transport step in plants is TGN to vacuole transport, which also might occur through organelle maturation. This would mean that the PVC is continually being formed and consumed. As the TGN acts as an EE in plant cells (Dettmer et al., 2006; Lam et al., 2007a), and is also not permanently associated with the Golgi stack (Foresti and Denecke, 2008), this independent organelle might simply be termed an EE rather than TGN/EE. However, its functions are not restricted to endocytosis and receptor recycling, but include secretion to the plasma membrane (Toyooka 2009) as well as biosynthetic transport to the vacuole. A simple term for this versatile compartment is not yet available. If the plant TGN is the equivalent of the early endosome in mammalian cells, it might be questioned whether mammalian TGN functions are performed by trans-most Golgi cisternae in plants in much the same way as the cis cisternae presumably assume some of the functions of the ER-Golgi intermediate compartment (ERGIC), which is absent in plants. EXPERIMENTAL PROCEDURES Plant material Arabidopsis thaliana wild-type Col-0 and transgenic lines expressing VHA-a1 GFP (Dettmer et al., 2006), ST YFP/VHA-a1 GFP (Dettmer et al., 2006) and ARA7 GFP (Ueda et al., 2004) were grown on MS medium containing 0.5% agar. Roots were harvested after 5 days for immunofluorescence or IEM. Nicotiana tabacum var. SR1 was grown as described previously (Pimpl et al., 2006). Suspension cultures of BY-2 tobacco (N. tabacum) and A. thaliana var. Landsberg erecta PSB-D were cultivated as described previously (Miao and Jiang, 2007) and analysed 3 days after sub-culturing. Recombinant plasmid production Arabidopsis coding sequences were amplified by PCR from firststrand cdna prepared from 3-day-old seedlings (Pimpl et al., 2003), using the oligonucleotides shown in Figure S1(d). Recipient plasmids were cut based on the restriction sites of the fragments, and dephosphorylated prior to ligation according to established procedures. The plasmids encoding the markers/reporters used have been described previously: ARA6 RFP (Ueda et al., 2004), ARA6 GFP (Ueda et al., 2001), ST YFP (Brandizzi et al., 2002), Man1 RFP and Man1 GFP (Nebenfuhr et al., 1999), GFP BP80 and GFP sporamin (dasilva et al., 2005), aleurain GFP (Humair et al., 2001) and a- amylase sporamin (Pimpl et al., 2003). The plasmid encoding a- amylase was pamy (Phillipson et al., 2001). Cloning vectors were generated by insertion of an NheI site after the 35S promoter of pamy (pgd5) or an additional SalI site after the amylase insert (psn5). SYP61 (NM_102617) was cloned into the YFP-containing plasmid pds02 (Bubeck et al., 2008), and the resulting plasmid was termed pds13. SNX1 (NM_120696) was cloned into pamy, and the resulting plasmid was termed psn1. For SNX1 XFP fusions, SNX1 was amplified from psn1, and mkate was amplified from ptagfp635-n and ligated into pamy. SNX1-DN/-DCC were amplified from psn1 and ligated into pamy. SNX2a (NM_125230) was cloned into psn5, and the resulting plasmid was termed psn13. SNX2a- DN/-DCC were amplified from psn13 and ligated into psn5. For SNX2a GFP, SNX2a was amplified from psn13 and GFP was

13 Retromer recycles VSRs from the TGN 119 amplified from LeSed5 GFP (Bubeck et al., 2008), followed by ligation into pgd5. SNX2b (NM_120794) was cloned into pamy. SNXCCRNAi was generated by cloning the first coiled-coil domain of SNX2a from psn13 in the sense and antisense orientations, linked by the PDK intron of phannibal, into pgd5. Isolation of protoplasts and transient gene expression Protoplasts were isolated from Arabidopsis suspension-cultured cells 3 days after sub-culturing. The suspension-cultured cells were sedimented at 80 g for 10 min, and resuspended in TEX buffer (Foresti et al., 2006) supplemented with 0.2% macerozyme R10 and 0.4% cellulose R10 (Yakult Honsha Co. Ltd, english/index.html) and incubated at 25 C for 16 h. Protoplasts were washed four times with electroporation buffer (see Bubeck et al., 2008) by flotation at 80 g for 10 min, followed by removal of the liquid medium using a peristaltic pump. Tobacco mesophyll protoplasts were isolated from leaves of 6 8-week-old plants as previously described (Bubeck et al., 2008). Unless otherwise stated, 1 30 lg of plasmid DNA was transfected, and protoplasts were incubated for 16 h. Generation of antibodies, enzyme assays, and gel blot analysis SNX2a antibody was generated by Eurogentec ( eurogentec.com) using peptides CLGVDGGDHPLKISDV and CEDFRSSFSSKPISSD as antigens. a-amylase activity was assayed in culture medium and cells using the Megazyme a-amylase reagent kit ( The secretion index was calculated as the ratio of secreted activity compared to cellular activity. For protein gel blots, cells were extracted; proteins from culture medium were precipitated and processed for SDS PAGE and immunodetection as described previously (Pimpl et al., 2006). Antibodies were used at the following dilutions: SNX2a, 1:2500; VPS29, 1:1000 (Oliviusson et al., 2006); GFP, 1:2500 (Tse et al., 2004). Confocal microscopy and immunofluorescence labelling For immunofluorescence, Arabidopsis plants or BY-2 cells were fixed and processed as previously described (Friml et al., 2003). Samples were incubated overnight at 4 C with SNX2a (1:200) or VPS29 (1:100) antibodies. Alexa-Fluor Ò conjugates 405 and 546 (Invitrogen, were used as secondary antibodies. Imaging was performed using a Zeiss Axiovert LSM 510 meta confocal laser scanning microscope ( and C-Apochromat 63 /1.2 W corr water immersion objective. At the Metadetector, main beam splitters (HFT) 405/514, 458/514 and 488/543 were used. The following fluorophores (excited and emitted by frame switching in the multitracking mode) were used: GFP, 488 nm/ nm; YFP, 514 nm/ nm; RFP, 543 nm/ nm; mkatefp, 543 nm/ nm. Pinholes were adjusted to 1 Airy unit for each wavelength. Post-acquisition image processing was performed using the Zeiss LSM 510 image browser ( ) and Corel- DrawX4 ( ) (Corel, Immunogold electron microscopy Root tips from Arabidopsis and tobacco were high pressure-frozen as described by Bubeck et al. (2008). Freeze substitution was performed in 9.9 ml dry acetone supplemented with 100 ll 20% uranyl acetate in methanol at )85 C for 16 h, before warming up to )50 C over a 5 h period. Roots were infiltrated and embedded in Lowicryl HM20 (Polysciences Inc.; at )50 C, and UV-polymerized for 3 days at )50 C. Ultra-thin sections were incubated with antibodies against SNX2a (1:150), VPS29 (1:50) and BP80 (1:50). Gold-conjugated secondary antibodies (BioCell GAR10; were used at a dilution of 1:50 in PBS supplemented with 1% w/v BSA. Post-staining was performed using aqueous uranyl acetate/lead citrate. Micrographs were obtained with a JEM1400 transmission electron microscope (Jeol, operating at 80 kv using a TVIPS F214 digital camera ( Negatives were either scanned using an Epson Perfection 4990 photo scanner (Epson, epson.com), or micrographs were taken with the TVIPS FastScan F214 digital camera. Images were adjusted for size, contrast and brightness using Photoshop (Adobe Systems, com) to improve the visibility of gold particles. ACKNOWLEDGEMENTS We wish to thank Karin Schumacher (Department of Developmental Biology, University of Heidelberg, Germany) and Thierry Gaude (Reproduction et Développement des Plantes, ENS Lyon, France) for supplying us with seeds of transgenic Arabidopsis lines. We would like to thank Steffi Gold and Barbara Jesenofsky for technical help. The financial support of the German Research Council (PI 769/1-1, RO 440/11-3/14-1), the Research Grants Council of Hong Kong and the Chinese University of Hong Kong Schemes B/C are gratefully acknowledged. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Arabidopsis sorting nexins and oligonucleotides. Figure S2. Statistical analysis. Figure S3. Sorting nexins localize to the trans-golgi network in tobacco. Figure S4. Sequence of Arabidopsis SNX2a and specificity of the SNX2a antibody. Figure S5. Effect and specificity of sorting nexin RNAi knockdown. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. REFERENCES Arighi, C.N., Hartnell, L.M., Aguilar, R.C., Haft, C.R. and Bonifacino, J.S. (2004) Role of the mammalian retromer in sorting of the cation-independent mannose 6 phosphate receptor. J. Cell Biol. 165, Bonifacino, J.S. and Hurley, J.H. (2008) Retromer. Curr. Opin. Cell Biol. 20, Bonifacino, J.S. and Rojas, R. 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