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Journal of Experimental Botany, Vol. 67, No. 15 pp. 4435–4449, 2016 doi:10.1093/jxb/erw222 Advance Access publication 4 June 2016 DARWIN REVIEW Receptor-mediated sorting of soluble vacuolar proteins: myths, facts, and a new model David G. Robinson1,* and Jean-Marc Neuhaus2 1 2 Centre for Organismal Studies (COS), University of Heidelberg, Germany Institute of Biology, Laboratory of Cell and Molecular Biology, University of Neuchatel, Switzerland * Correspondence: [email protected] Received 11 April 2016; Accepted 17 May 2016 Editor: Donald Ort, University of Illinois Abstract To prevent their being released to the cell exterior, acid hydrolases are recognized by receptors at some point in the secretory pathway and diverted towards the lytic compartment of the cell (lysosome or vacuole). In animal cells, the receptor is called the mannosyl 6-phosphate receptor (MPR) and it binds hydrolase ligands in the trans-Golgi network (TGN). These ligands are then sequestered into clathrin-coated vesicles (CCVs) because of motifs in the cytosolic tail of the MPR which interact first with monomeric adaptors (Golgi-localized, Gammaear-containing, ARF-binding proteins, GGAs) and then with tetrameric (adaptin) adaptor complexes. The CCVs then fuse with an early endosome, whose more acidic lumen causes the ligands to dissociate. The MPRs are then recycled back to the TGN via retromer-coated carriers. Plants have vacuolar sorting receptors (VSRs) which were originally identified in CCVs isolated from pea (Pisum sativum L.) cotyledons. It was therefore assumed that VSRs would have an analogous function in plants to MPRs in animals. Although this dogma has enjoyed wide support over the last 20 years there are many inconsistencies. Recently, results have been published which are quite contrary to it. It now emerges that VSRs and their ligands can interact very early in the secretory pathway, and dissociate in the TGN, which, in contrast to its mammalian counterpart, has a pH of 5.5. Multivesicular endosomes in plants lack proton pump complexes and consequently have an almost neutral internal pH, which discounts them as organelles of pH-dependent receptor–ligand dissociation. These data force a critical re-evaluation of the role of CCVs at the TGN, especially considering that vacuolar cargo ligands have never been identified in them. We propose that one population of TGN-derived CCVs participate in retrograde transport of VSRs from the TGN. We also present a new model to explain how secretory and vacuolar cargo proteins are effectively separated after entering the late Golgi/TGN compartments. Key words: Clathrin-coated vesicles (CCV), multivesicular bodies (MVBs), nanobody technology, organellar pH, retromer, transGolgi network (TGN), vacuolar sorting receptors (VSRs). Abbreviations: AP (1,2…), Adaptor protein complex (1,2…); BFA, Brefeldin A; CCVs, Clathrin-coated vesicles; CD-MPR, Cation-dependent mannosyl 6-phosphate receptor; CI-MPR, Cation-independent mannosyl 6-phosphate receptor; CNX, Calnexin; COP (I, II), Coat protein-coated vesicle (I, II); CTx, Cholera toxin; DV, Dense vesicles; EE, Early endosome; EGF, Epidermal growth factor; ER, Endoplasmic reticulum; ERD2, Endoplasmic reticulum retention defective 2; FLIM, Fluorescence Lifetime Imaging Microscopy; FRET, Förster Resonance Energy Transfer; GGAs, Golgi-localized, Gamma-ear-containing, ARF-binding proteins; LE, Late endosome; LPVC, Late prevacuolar compartment; MPR, Mannosyl 6-phosphate receptor; MVBs, Multivesicular bodies; PA, Protease associated (domain); PEx, Pseudomonas toxin; PM, Plasma membrane; PSV, Protein storage vacuole; PVCs, Prevacuolar compartments; SNX, Sorting nexin; STx, Shiga toxin; TGN, trans-Golgi network; VSD, Vacuolar sorting determinant; VSRs, Vacuolar sorting receptors; WLS, Wntless © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected] 4436 | Robinson and Neuhaus Introduction Acid hydrolases, which are destined for the lytic compartment of the cell, and extracellular (i.e. secretory) proteins are both synthesized in the endoplasmic reticulum (ER) and are exported to the Golgi apparatus via COPII vesicles (Barlowe and Miller, 2013). However, they exit the Golgi via different mechanisms, which ensures that hydrolytic enzymes are not released to the cell exterior. This is true for all eukaryotic cells, and involves the participation of transmembrane receptors which recognize and bind the acid hydrolases, thereby diverting them from the bulk flow of secretory proteins to the plasma membrane. However, it should be emphasized that animals and fungi belong to the same major group of eukaryotes, the Opisthokonta, and mechanisms common to them cannot automatically be generalized to the other four major groups, one of which (the Archaeplastida) includes plants. Plants possess a family of hydrolase receptors known as vacuolar sorting receptors (VSRs). It has been held by many (e.g. De Marcos Lousa et al., 2012; Xiang et al., 2013) that they, and the compartments in which they have been detected, operate in the same fashion as the lysosomal hydrolase receptors of mammalian cells to divert soluble vacuole-destined proteins away from the flow of secretory proteins. However, we have expressed doubts on several occasions that this scenario might not apply to plants (e.g. Robinson et al., 2012; Robinson, 2014), but in the absence of in situ receptor–ligand binding data, this controversy could not be resolved. In this review, we carefully and critically examine the evidence usually put forward in support of the notion that soluble vacuolar cargo ligands are sorted in the trans-Golgi network (TGN), where they interact with VSRs and become sequestered into clathrin-coated vesicles (CCV), which then fuse with multivesicular bodies/prevacuolar compartments (MVBs/PVCs). The latter would therefore represent organelles in which VSR–ligand dissociation occurs and from which VSRs are recycled to the TGN. In contrast, it has been claimed that VSR–ligand interactions already occur in the lumen of the ER. This alternative hypothesis for protein sorting in the plant endomembrane system has recently received considerable support from Künzl et al. (2016), who have been able to monitor compartment-specific VSR–ligand interactions in vivo using genetically encoded nanobody-assembled VSR sensors. Receptor-mediated sorting of hydrolases to the lytic compartments of non-plant cells Mammalian cells The mannosyl 6-phosphate receptor (MPR) is responsible for the segregation of lysosomal acid hydrolases in animal cells, and has acted as a paradigm for receptor-mediated protein trafficking in numerous textbooks (for a comprehensive review see Braulke and Bonifacino, 2009; for an overview see Robinson et al., 2012). There are two types of MPR: the 46 kDa cation-dependent form (CD-MPR), present as a homodimer and with one M6P binding site; and the monomeric 300 kDa cation-independent form (CI-MPR), with M6P binding sites in two of its fifteen repeated domains. Whereas both MPR types participate in protein sorting at the TGN, only the CI-MPR is responsible for retrieving missorted hydrolases at the plasma membrane (PM). MPRs are prevented from interacting in the early secretory pathway (ER and cis/medial-Golgi cisternae) with their cargo ligands, which are ‘capped’ by the addition of GlcNac1-phosphate to α-1,6-linked terminal mannose residues. The ligands only become ‘demasked’ at the TGN through the action of a so-called uncovering enzyme, which reveals the cryptic M6P signal by hydrolysing the N-acetylglucosamine1-phosphodiester bond (Rohrer and Kornfeld, 2001). Thus, through this seemingly complicated mechanism, cargo recognition by modifying enzymes (in the cis-Golgi) and sorting by receptors (in the TGN) are spatially separated events. Receptor–ligand complexes are then collected into CCVs, a process involving recognition of sorting signals in the cytosolic domain of the MPR firstly by monomeric adaptors (Golgi-localized, Gamma-ear-containing, ARF-binding proteins [GGAs]) then by tetrameric AP-1 adaptors (Bonifacino, 2004). After their release from the TGN, the CCVs then fuse with an early endosome (EE) (Carreno et al., 2004). As the EE gradually matures into a late endosome (LE), the ligands dissociate from the MPRs (van Weering et al., 2010). Cargo hydrolases are finally released into the interior of the lysosome through fusion of the multivesicular LE with the lysosomal boundary membrane. In order for the process to run economically, MPRs are recycled numerous times from the maturing EE/LE to the TGN. This involves the aggregation of MPRs into retromercoated domains of tubular EE domains, followed by retrograde trafficking of retromer carriers to the TGN from the maturing endosome (Seaman, 2012). Compartmental pH plays a key role in this process: the measured pH of the TGN in the mammalian cell (pH 6.3, Machen et al., 2003) corresponds to the optimal pH for MPR–ligand binding (pH 6.4–6.0, Tong and Kornfeld, 1989), whereas the decreased pH in maturing endosomes (pH 6.0–4.8, Huotari and Helenius, 2011) causes dissociation. Drosophila has a single MPR homologue, LERP, that has five luminal domains, interacts with GGAs, and partially restores lysosomal sorting in MPR-deficient mammalian cells, but does not bind mannosyl 6-phosphate, which Drosophila does not appear to use (Dennes et al., 2005). LERP-deficient Drosophila has only a partial lysosomal sorting defect, suggesting other receptors exist as well (Hasanagic et al., 2015). Mammalian cells also have other receptors which sort a smaller number of proteins to the lysosome. The most studied are sortilin and related proteins (SorLA, SorCS1, etc.). They are type I proteins similar to yeast Vps10p (see below), but with a single VPS10 domain (whereas Vps10p has two). They interact with GGAs and mediate transport from the Golgi to endosomes and also function at the PM as neuropeptide receptors, interacting with AP-2 for their internalization (Nielsen et al., 2001). Sortilin is synthesized as an inactive precursor, unable to bind ligands in the ER. Activation occurs TGN and vacuolar sorting receptors | 4437 post-translationally owing to the cleavage of the propeptide by furin in the TGN (Petersen et al., 1999). Yeast The best-studied VSR in yeast is Vps10p, which is required for the proper sorting of carboxypeptidase Y. It has no sequence homology to MPRs, but is a type I membrane protein with a cytosolic tail containing similar sorting determinants (such as the tyrosine-based motif YXXΦ). Indeed, a chimeric CI-MPR with the cytosolic tail of Vps10p correctly traffics and sorts lysosomal enzymes in animal cells and also interacts with GGAs (Dennes et al., 2002). The luminal domain of Vps10p contains two VPS10 domains and is needed for ligand binding. Because the sorting signal is not a modified glycan, but a polypeptide motif, nothing prevents binding already taking place in the ER. Vps10p also shuttles repeatedly between the late Golgi and a prevacuolar compartment (Cooper and Stevens, 1996). There is also a distant homologue of CD-MPRs, Mrl1p, in fungi (budding yeast, fission yeast, Botrytis cinerea). In budding yeast, Mrl1p appears to contribute to vacuolar sorting of proteinases A and B (Whyte and Munro, 2001). However, nothing is known about its localization or trafficking. Plant VSRs Based on the hypothesis of a conserved sorting mechanism for acid hydrolases in all eukaryotic cells, plant vacuolar receptors were expected to traffic along with their ligands, via CCVs, from the (trans-) Golgi to an intermediate compartment between the Golgi and the vacuoles. Therefore VSRs were looked for and found in a purified CCV fraction from developing pea (Pisum sativum L.) cotyledons (Kirsch et al., 1994). An 80 kDa protein (BP-80) was retained on an affinity column with a coupled proaleurain peptide at pH 7.1 and released at low pH (pH 4). Interestingly, given that the pH optimum for VSR–ligand binding lies around pH 6, these are suboptimal isolation conditions. Pea BP-80 was subsequently cloned and homologues were identified in Arabidopsis thaliana (Paris et al., 1997). Soon, other groups also cloned VSRs from Arabidopsis (AtELP; Ahmed et al., 1997; Laval et al., 1999) and pumpkin (Cucurbita; PV72 and PV82; Shimada et al., 1997). VSRs are type I membrane proteins with a large luminal domain, a single transmembrane helix, and a short cytosolic tail (Fig. 1A). The luminal domain comprises a proteaseassociated (PA) domain also found in some animal proteases and receptors as well as in RMR proteins and related animal proteins (Wang et al., 2011), a large VSR-specific domain, and three epidermal growth factor (EGF) repeats. According to Cao et al. (2000), only the last of these (with a B.2 consensus) has a Ca2+-binding motif. The PA domain binds a part of the vacuolar sorting determinant (VSD) of the cargo proteins, causing a conformational change which allows the VSR-specific domain to bind (or contribute to the binding of) the conserved Leu/Ile motif of these sequence-specific VSDs Fig 1. (A) The domain structure of a VSR showing the high and low affinity ligand binding sites. PA, protease-associated domain, EGF, epidermal growth factor domain. (B–D) Fluorescent VSR reporter constructs: GFP-BP80 lacking the ligand binding domain (see Tse et al., 2004) (B); a ligand-binding VSR construct (see Saint-Jean et al., 2010) (C); and organelle-specific VSRs for the determination of ligand binding through FRET-FLIM (D). A GFP-specific nanobody serves as a linker between the ligand binding domains and GFP tagged onto either a type I or type II membrane marker. The ligand is tagged with RFP (see Künzl et al., 2016). (E) Amino acid sequence of the cytosolic tail of a VSR, with the sorting motifs indicated in red. (Luo et al., 2014). There are also three N-glycosylation sites: one in the PA domain, one in the VSR-specific domain, and one in the first EGF repeat. Mutation of the last one and of at least one of the other two causes a significant loss of binding affinity for the ligands, without affecting the trafficking of the VSR (Shen et al., 2014). The cytosolic tail also harbours Tyr and acidic dileucine-like motifs (see below). The Tyr motif interacts with the µ subunit of an AP adaptin complex (Happel et al., 2004). The cytosolic tail also associates with EPSIN1, clathrin, AP-1, and VTI11 (Song et al., 2006). As is the case for MPRs (Punnonen et al., 1996), VSRs (i.e. AtVSR1) also form homomeric complexes, either trimers or dimers, with an unrelated third protein. This requires membrane anchoring and the cytosolic tail, that is, some amino acids within a nine amino acid–long stretch including the Tyr of the Tyr motif (Kim et al., 2010). VSRs are found in all land plants, as well as in several algae (chlorophytes, e.g. Chlamydomonas, Ostreococcus, and Micromonas) but not in rhodophytes or glaucophytes. A VSR is also present in the genome of the most simple eukaryotic organism, Ostreococcus tauri, a chlorophyte and the smallest known eukaryote (Becker and Hoef-Emden, 2009). Whatever function a VSR has in this organism, it does not require post-Golgi compartments because its extremely reduced secretory system lacks both multivesicular endosomes as well a lysosomal/vacuolar compartment (Henderson et al., 2007). Apart from these viridiplantae, VSRs have even been found in several stramenopiles (e.g. Phaeodactylum, Phytophthora; De Marcos Lousa et al., 2012). These belong to another major group of eukaryotes and have probably acquired their VSR genes through horizontal gene transfer or secondary endosymbiosis (Yoon et al., 2002). Angiosperms and gymnosperms have three subfamilies of VSRs (Neuhaus and Paris, 2005; De Marcos Lousa et al., 4438 | Robinson and Neuhaus 2012). Their most obvious differences are in the terminal part of the cytosolic tails, beyond the Tyr motifs, which might modulate their intracellular localization. Indeed, a map of the organellar proteome of Arabidopsis has identified four different VSRs at three separate locations by principal components analysis. One of them is very close to a V0a subunit of a V-ATPase subunit, pointing to a TGN location (Dunkley et al., 2006). This suggests a functional specialization of different isoforms. Indeed, PV72, a VSR of the subfamily VSR1, has been identified (and cloned) in precursor-accumulating vesicles in pumpkin seeds (Shimada et al., 1997). Its ligand binding is Ca2+ sensitive but pH insensitive. This isoform is specifically expressed in maturing seeds (Shimada et al., 2002), and was suggested to recycle between the precursor-accumulating vesicles and the Golgi complex (Watanabe et al., 2002). A VSR of the same subfamily was found to be important in Arabidopsis for storage protein accumulation in protein storage vacuoles (PSVs), and ligand binding was also Ca2+ dependent (Shimada et al., 2003). AtVSR3 is very similar to BP-80 (subfamily VSR2) and is expressed specifically in Arabidopsis guard cells. Its RNAi-mediated suppression prevents guard cells from responding to abscisic acid (Avila et al., 2008). In a screen for mutations affecting vacuolar sorting of the artificial storage cargo VAC2 [a fusion of CLAVATA3 with the C-terminal VSD of barley (Hordeum vulgare) lectin that causes premature meristem termination], a loss-offunction mutant in the AtVSR4 gene was identified (Zouhar et al., 2010). When T-DNA mutants in each of the other VSRs were tested, it was found that VSRs of both subfamilies VSR1 (AtVSR1 and 2, the latter only being expressed in flowers) and VSR2 (AtVSR4 and 3, which are encoded by a gene tandem and are almost identical) are involved in VAC2 sorting in meristems in proportion to their expression levels. AtVSR1 is the most important receptor for proper sorting of 12S and 2S albumins to PSVs in developing seeds, with minor contributions from AtVSR3 and 4. However, seedling germination is strongly affected in double mutants. In fact, silencing the subfamilies VSR1 and VSR2 completely inhibits germination (Okmeni Nguemelieu, 2006). In leaves, the predominantly expressed AtVSR1 and 4 both contribute to vacuolar sorting of AtAleurain, a lytic vacuole protease, but not of other vacuolar cargos (AtCPY, VPEɣ, or TGG2). The VSR subfamily 3, with AtVSR5, 6, and 7, has more divergent sequences and is only expressed in roots. However, none of these VSRs complement mutations in the three other genes even when expressed with an AtVSR1 or 4 promoter. Thus, the VSR subfamily 3 is suggested to have a different function and presumably interacts with different cargos than the other two subfamilies (Zouhar et al., 2010). De Marcos Lousa et al., 2012). However, a closer look at the original paper does not allow such a clear statement. For the isolation of their CCVs from developing pea cotyledons, Kirsch and co-workers used a Ficoll/D2O gradient procedure previously introduced by Depta and Robinson (1986). At best, these fractions have only an 85–90% purity (see Fig. 2, and Depta et al., 1991; Hoh et al., 1991), and the PM part of the contaminants can lead to erroneous conclusions about the ability of CCVs to produce callose (Depta et al., 1987). Interestingly, in the Kirsch et al. (1994) paper, they use a lighter Ficoll/D2O fraction that is more enriched in BP80 than the ‘purified CCV fraction’. The authors state that the low density fraction was enriched in Golgi vesicles. Exactly what kind of vesicles is not explained, but it is possible that contamination of the CCV fraction through such vesicles was the real source of VSRs. Using the same isolation procedure, Harley and Beevers (1989) reported that the pea cotyledon CCV fraction was virtually uncontaminated by Golgi membranes, as monitored for using the marker inosine diphosphatase. However, the storage protein pea lectin was detected in the CCV fraction. The storage globulins vicilin and legumin were also detected in pea cotyledon CCV fractions with an estimated purity of 90% (Robinson et al., 1989; Hoh et al., 1991). Again, the question is, are these proteins really sequestered by the CCV or are they present as contaminants? At the time these papers were published, the existence of a special type of transport vesicle for storage proteins, the socalled dense vesicle (DV), had not been discovered. In contrast to CCVs, DVs form at the periphery of cis-Golgi cisternae where the storage proteins accumulate and gradually form electron-dense aggregates (Hohl et al., 1996; Hillmer et al., 2001). As a consequence of cisternal maturation, the DVs eventually reach the TGN where they are released (Robinson and Hinz, 1999). It is therefore quite possible that contaminating DVs were the source of the storage polypeptides detected in CCV fractions. This is supported by the significant observation that DVs at the TGN are invariably seen to bud off CCVs, which, as seen by immunogold labelling, do not contain storage proteins (see Fig. 3 and figure 3b–d in Hohl et al., 1996, and figure 7b–e in Robinson and Hinz, 1997). VSRs and CCVs: old data revisited Ever since their discovery in 1994 (Kirsch et al., 1994), it has become customary in publications dealing with vacuolar protein sorting to state that VSRs are enriched in CCVs and have been isolated from them (e.g. Masclaux et al., 2005; Fig. 2. A typical example of a negatively stained sample of CCV isolated from developing pea cotyledons via the Ficoll/D2O centrifugation method of Depta and Robinson (1986). Such fractions are not homogeneous for CCV and contain 10–20% smooth-surfaced vesicular contaminants. Bar = 100 nm. TGN and vacuolar sorting receptors | 4439 Fig. 3. DVs and CCVs in cells of developing pea cotyledons. (A) A Golgi stack with DVs at cis-, median, and TGN regions. The DVs (arrows) are recognizable through their dense osmiophilic contents. CCVs (arrowheads) are seen in the immediate vicinity of DVs at the TGN. PSV, protein storage vacuole. (B, C) CCVs budding from DVs. C is an anti-vicilin immunogoldlabelled example showing that the storage globulins are restricted to the DV. Bars = 100 nm. So do DVs carry VSRs? Contradictory reports exist as to the presence of VSRs in DVs: whereas Otegui et al. (2006) published convincing images of VSR labelling of DVs, Hinz et al. (2007) were unable to obtain a similar result. However, genetic analyses strongly point to the participation of VSRs in storage protein sorting (see above and Zouhar et al., 2010). Moreover, mutations in genes for three of the four subunits of the AP-4 complex have been found to affect VSR1mediated sorting of 12 S seed storage proteins (Fuji et al., 2016). Although these proteins are segregated from secretory proteins in the cis-Golgi through the formation of insoluble aggregates, it is quite possible that VSRs are required for the initial sequestration event triggering condensation. The budding of CCVs from DVs is reminiscent of the situation in mammalian cells which are engaged in regulated secretion and produce large secretory granules. Immature secretory granules, which form at the TGN, have surface domains coated with clathrin and the AP1-adaptor complex, and bud off CCV (Klumperman et al., 1998). As suggested by Molinete et al. (2000) and Burgess et al. (2011) for secretory granules, this suggests that CCVs may function in recovering missorted proteins (e.g. hydrolases plus their receptors) from the DV at the TGN in developing legume cotyledons. It would therefore be an indication that CCVs at the TGN of plant cells perform a retrograde rather than an anterograde trafficking function (see also below). Finally, there is another set of observations regarding VSRs which is difficult to reconcile with the notion that VSRs are exclusively incorporated into anterograde trafficking CCVs at the TGN. VSRs have been detected at the PM of growing pollen tubes by immunogold electron microscopy (Wang et al., 2011) and also in tobacco leaf (Nicotiana tabacum) epidermal cells after transiently expressing a fluorescently tagged full-length functional VSR (Saint-Jean et al., 2010). The latter paper is particularly important because it provides evidence for the endocytosis of the PM-located VSRs. Transgenic Arabidopsis plants stably expressing the same fluorescent VSR reporter also showed a partial distribution of the receptor at the PM, particularly in root tissues: in upper root regions, the reporter almost exclusively labelled the PM (Saint-Jean et al., 2010). When treated with the fungal toxin brefeldin A (BFA), the recycling pathway for endocytosed proteins is inhibited and a so-called BFAbody is formed, consisting of TGN elements and endosomes (Langhans et al., 2011). Under these conditions, the VSR reporter accumulated in root cells in BFA bodies, indicating that it is constitutively endocytosed. As described above, VSRs have a tyrosine motif (YXXΦ) in their cytosolic tail which has been shown to interact with μ-adaptins. In mammalian cells, this is a classical endocytosis motif (Traub, 2005) and is used to internalize MPRs at the PM, which serves to rid mannose 6-phosphate–modified hydrolases and other cargos from the extracellular space (Braulke and Bonifacino, 2009). It could therefore be argued (see Saint-Jean et al., 2010) that CCVs containing VSRs are endocytic in nature rather than being vectors for biosynthetic traffic to the vacuole. In summary, one must say that while some of the literature dealing with CCV and VSRs is ambiguous, the balance is tipped in favour of VSRs being incorporated into CCVs. However, one must hasten to add that there is actually no evidence for the presence of cargo ligands in CCVs isolated from plant cells. Studies on VSR reporter mutants: a re-evaluation MPRs and sortilins contain several sorting motifs in their cytosolic tails: for the CI-MPR, a C-terminal acidic cluster/ dileucine motif (DDSDEDLLHI) and casein kinase 2 sites are required for GGA binding and recruitment to clathrincoated pits at the TGN (Tortorella et al., 2007). The MPR subsequently interacts with an AP-1 adaptor via the same dileucine motif, the phosphorylated casein kinase 2 sites, and a tyrosine motif (YSKV; Ghosh and Kornfeld, 2004; Stöckli et al., 2004). Sorting motifs have also been identified in the much shorter cytosolic tails of VSRs. Several groups have generated fluorescent VSR reporter proteins by replacing the luminal binding domain (LBD) of the VSR with GFP (or another fluorescent protein), which is then directly fused to the transmembrane and cytosolic domains of VSRs (Fig. 1B). These reporter proteins accumulated in the PVC (Tse et al., 2004; daSilva et al., 2005; Miao and Jiang, 2007) but some also reached the vacuole, where a ‘core’ GFP fragment was detected. The GFP-BP80a, b, d, and f (four of the Arabidopsis VSRs) were found to compete in tobacco protoplasts with endogenous VSRs, supposedly by preventing their recycling after having released their cargo towards the vacuole. This indicates that the recycling pathway is selective and saturable (daSilva et al., 2005). Amino acids conserved in the cytosolic tails of VSRs have been mutated to Ala and the mutants were tested for 4440 | Robinson and Neuhaus localization and competition with endogenous VSRs. Several mutants (within the three highlighted motifs in Fig. 1E) had reduced competitivity. In GFP-VSR reporters (Fig. 1B), mutation of the Tyr (Y612A) and the Leu (L615A) of the YXXΦ motif both reduced competition, but Y612A reduced leakage to the vacuole while L615A increased it. The Y612A mutant localized mainly to the TGN, as shown by fluorescent co-localization with the TGN marker YFP-SYP61 and by immunogold electron microscopy, but it was also found in PVCs (Foresti et al., 2010). In addition, it also localized partially to the PM. In contrast, the L615A mutant accumulated in a compartment distinct from Golgi, TGN, or PVC and leaked to the vacuole. This new compartment was also labelled with Venus-Rha1 (a Rab5 GTPase) and was interpreted as a late PVC (LPVC), free from wild-type VSRs and ready to fuse with the vacuole. The Tyr was thus interpreted to be an anterograde sorting signal from TGN to PVC, whereas the Leu constituted a retrograde sorting signal from the PVC to the TGN (Foresti et al., 2010). A similar study was made with different mutations in the cytosolic tail of a pea BP80 GFP-reporter (also lacking the luminal domains, Fig 1B) that included the equivalent Y612A mutation and three different acidic residues. The latter mutations had only weak effects, increasing the leakage to the vacuole 3–4 fold, except for E604A (EXXXIM; first highlighted motif in Fig. 1E), which accumulated in the Golgi (co-localization with ERD2-CFP; Saint-Jean et al., 2010). The Y612A mutant caused partial redistribution to the PM. Combining E604A and Y612A resulted in Golgi labelling. The dileucinelike motif (IleMet) was also mutated to alanines (IMAA), which led to strong labelling of the vacuole. Transient expression of this mutant gave rise to punctate structures that colocalized with vacuolar cargo Aleu-CFP and were thus of a prevacuolar nature. Combining the IMAA and Y612A mutations caused increased labelling of the PM but no vacuolar labelling. The few remaining dots were mostly Golgi. So, once again, the Tyr motif was interpreted as an anterograde TGN to PVC signal, but it is possibly also involved in endocytosis. In contrast, the IleMet motif was suggested to act as a retrograde sorting motif from PVC to TGN, but also as an endocytosis signal along with E604 (acidic dileucine-like motif, EXXXIM; Saint-Jean et al., 2010). The IleMet motif was also confirmed as an endocytosis signal in an assay for secretion of vacuolar cargo when the Tyr motif was mutated (Gershlick et al., 2014). This explanation for the behaviour of the VSR reporter mutants assumes the correctness of the MPR model, that is, anterograde transport via CCVs out of the TGN which depends upon a Tyr motif/clathrin adaptor interaction, and retrograde transport which requires another type of motif and a non-clathrin carrier. It predicts that VSRreporters lacking the Tyr motif are not sequestered into CCVs and therefore accumulate at the TGN. In contrast, recycling from the MVB/PVC is not inhibited so that, gradually, VSRs no longer become detectable in this compartment. However, there is an alternative interpretation of these data. If, as we have already suggested, TGN-derived CCVs serve to cycle VSRs upstream to the ER/cis-Golgi, the same phenotype will be observed: VSR-reporters with a mutated Tyr motif would also accumulate in the TGN. Given that MVBs/PVCs are continually being formed (at the TGN) and consumed (through fusion with the vacuole), MVBs arising during the course of the expression period will also gradually lack the VSR-reporters. This scenario also allows one to understand how the VSR Tyr motif mutants can be diverted to the PM or the vacuole: VSR mutants which cannot be incorporated into CCVs at the TGN will eventually enter secretory vesicles by default. The reduction in VSR reporter labelling of the MVB/PVC is more difficult to explain, but can be understood if the Tyr motif is considered to be required primarily for the segregation of VSR–ligand complexes into sorting domains in the ‘early TGN’ before the ‘late TGN’ receptor–ligand dissociation and CCV formation occurs. That is, Tyr motifs are also necessary for anterograde trafficking of VSRs to the MVB/PVC but without using CCVs as a transport vector (see section ‘A new model for the sorting of secretory and vacuolar cargo proteins at the trans-Golgi/early TGN’ for a more detailed discussion of this possible mechanism). VSRs interact with their cargo ligands early in the secretory pathway We have previously pointed out that there is no obvious similarity between VSRs and MPRs in terms of their structure nor in their ligand-binding properties (Robinson et al., 2012; Robinson, 2014). In contrast to MPRs, binding motifs for lytic vacuole cargo molecules are present in the primary sequence of the ligand (usually NPIR, often at the N-terminus; Robinson et al., 2005). They also do not appear to be shielded from receptor interaction during their passage through the lumen of the ER or early Golgi compartments. On the contrary, soluble vacuolar proteins accumulate in the lumen of the ER when the LBD of a VSR is expressed as a fusion construct with an ER (HDEL)–retention signal (Watanabe et al., 2004; daSilva et al., 2005), or when a construct is expressed where the LBD is fused to the transmembrane domain of an ER-resident protein (calnexin, Niemes et al., 2010a). More recently Gershlick et al. (2014) have reported on experiments performed with ligand reporter constructs having an NPIR (a vacuolar sorting signal) at the N-terminus and HDEL (a classical ER retrieval signal) at the C-terminus. The transport of the dual signal reporters was compared with reporters containing a single N- or C-terminus signal. Their data suggest that VSRs and ERD2, the receptor for retrieval of HDELcontaining proteins, compete for the dual signal reporter. Because HDEL-cargos interact with ERD2 in the cis-Golgi cisternae (Phillipson et al., 2001), it was concluded that this was also the location for the initial VSR–ligand interactions. Although nothing prevents newly synthesized VSRs from binding their ligands in the ER (contrary to MPRs or sortilins), the cis-Golgi, rather than the ER, may indeed be the point of entry for VSRs recycled from the TGN (see below), making this location the major site for cargo ligand binding in the secretory pathway. It is, however, unclear why this compartment should be the cargo-charging site for VSRs, unless their glycans must have first matured in the Golgi. In favour TGN and vacuolar sorting receptors | 4441 of the ER instead of the cis-Golgi is the potential for selection and enrichment of vacuolar cargos in COPII vesicles. This is supported by an early observation (Di Sansebastiano et al., 2001) in a transient expression assay in tobacco protoplasts of a much faster ER export of Aleu-GFP (a VSR cargo) compared with GFP-Chi (another vacuolar reporter though not thought to be a VSR cargo) or secGFP (secreted by default, not a VSR cargo). Contrary to these other reporters, Aleu-GFP was not detected in the ER but instead in rapidly produced punctate fluorescent signals, possibly reflecting the labelling of ER exit sites (ERES) and/or overlying cis-Golgi. VSR–ligand interactions are also modulated by calcium, probably due to the conformational changes induced by Ca2+binding to EGF repeats in the LBD (Cao et al., 2000; Watanabe et al., 2002). On the one hand, high Ca2+ concentrations can prevent ligand dissociation at pH 4; on the other hand, ligand release can be triggered owing to the lack of Ca2+ (Watanabe et al., 2002). It is therefore likely that the removal of Ca2+ through the inclusion of EGTA in the elution medium, rather than the low pH, was the factor triggering ligand release in the original experiments of Kirsch et al. (1994). Nevertheless, the ER is well known to have the highest luminal calcium concentrations (0.2–5 mM) among the endomembranes of animal cells (Montero et al., 1997; Rizzuto and Pozzan, 2006). Although actual values are not available, the ER in plant cells is also assumed to be rich in Ca2+ owing to the presence of Ca2+ pumps (Bonza and De Michelis, 2011) and calcium-binding ER-resident proteins (Christensen et al., 2010). In contrast, the Golgi apparatus has lower levels of luminal Ca2+ (0.7 μM; Ordenes et al., 2012). Presumably, Ca2+ concentrations in the plant MVB/PVC are even lower. In comparison to the ER, the low Ca2+ concentrations in these two compartments are not conducive to VSR–ligand binding. The TGN is not where VSRs bind their cargo ligands Central to our understanding of how VSRs function in plant cells is the question whether cargo ligand-VSR interactions are maintained in the acidic TGN. An answer to this question has recently been provided in an elegant study using nanobody fusion proteins (Künzl et al., 2016). These authors generated compartment-specific VSR sensors by fusing a nanobody sequence to the soluble LBD of a VSR. Because the nanobody (a 13 kDa VHH domain of an alpaca heavy chain antibody) was raised against GFP it can bind compartment-specific N-terminal respectively C-terminal GFPtagged type I or type II membrane markers. If this sensor is co-expressed with an RFP-tagged cargo ligand, and the LBD binds the ligand, the two fluorophores will localize as punctae. Because the distance separating the two fluorophores is less than 10 nm, the interaction can be validated by Förster Resonance Energy Transfer (FRET), and can be recorded with Fluorescence Lifetime Imaging Microscopy (FLIM), because the energy transfer that occurs shortens the fluorescence lifetime of the GFP. FLIM also provides an indication of the luminal pH of the compartment in question. The fluorescent cargo ligand used was aleurain (aleu)RFP, and GFP-calnexin (CNX) and SYP61-GFP were used as membrane markers for the ER and the TGN respectively. Co-localization of fluorescence signals and FRET was recorded when GFP-CNX+LBD-Nb was co-expressed with aleu-RFP, but not when sialyl transferase (ST)GFP+LBD-Nb was co-expressed with aleu-RFP, indicating that VSRs bind cargo ligands in the lumen of the ER but not in the TGN. Co-expression of the Golgi-based VSR marker mannosidase1 (Man1)-GFP+LBD-Nb with aleu-RFP also gave positive results for VSR–ligand binding. Negative binding results were also obtained with the MVB/LE marker GFP-BP80. Indeed, after treatment with wortmannin, which causes the MVB to enlarge, the latter compartment showed a clear spatial separation of the fluorescent membrane-bound and soluble cargo markers. The fidelity of the negative binding data for the TGN was tested by treating tobacco leaf protoplasts with BFA, which disassembles the Golgi apparatus and causes Golgi and TGN proteins to accumulate in the ER. Under these conditions, aleu-RFP binds to the SYP61-GFP VSR sensor. The interpretation that VSR-ligand binding starts in the ER, is maintained during trafficking through the Golgi stack, but ceases in the TGN/EE, also finds support in FLIM measurements. Fluorescence lifetime is an intrinsic property of a fluorophore and is pH-dependent, being longest at neutral pH and shortest at acidic pHs. Accordingly, Künzl et al. (2016) found that the fluorescence lifetimes of the VSR sensors were longest for the ER- and MVB-based sensors, and shortest for the TGN-based sensor. They showed that the fluorescence lifetime decreased along an ER-GATGN/EE gradient. VSR-ligand dissociation does not occur in MVBs/PVCs According to the MPR model, MPR–ligand complexes are transported to the EE. After pH-dependent release of ligands, the MPRs are then recycled from the EE via sorting nexin 1/retromer-containing carriers (Mari et al., 2008). The sorting motif is a phenylalanine–tryptophan (Phe–Trp) aromatic motif (Diaz and Pfeffer, 1998). Similar motifs are also present in sortilins and in yeast Vps10p. The MPR model further predicts that VSRs will accumulate in either a ligandbinding or a ligand-releasing compartment, depending on the relative speeds of anterograde versus retrograde transport. Indeed, VSRs have been localized several times and in different plants by immunofluorescence or immunoelectron microscopy in the (trans-)Golgi, but mostly in a post-Golgi, PEP12-containing compartment, and identified functionally as a PVC (Jiang and Rogers, 1998; Li et al., 2002; Miao et al., 2006; Otegui et al., 2006; Wang et al., 2007) and structurally as an MVB (Tse et al., 2004). Wortmannin treatment of plant cells leads to a fusion of MVBs, and VSRs have also been detected in these enlarged MVBs. Fusion proteins with luminal GFP linked to the transmembrane domain and cytosolic tail of various VSRs have been co-localized with endogenous VSRs in MVBs/PVCs as detected by antibodies (Tse et al., 4442 | Robinson and Neuhaus 2004). GFP-BP-80/GFP-VSR has thus become an accepted visual marker for the MVB/PVC. However, co-localization of VSRs and GFP-VSR is not complete because some VSR compartments contain no fluorescent reporter. This was rationalized as a possible differentiation of PVCs with VSRs from different subfamilies (Miao et al., 2006). As with MPRs, the pH optimum for VSR–ligand binding in vitro lies around 6.0 (Kirsch et al., 1994). It was therefore assumed that the pH in the plant TGN would be similar to this and that the pH in the LE/PVC would be considerably more acidic (Hwang, 2008). Surprisingly, recent measurements of pH in these two compartments are not compatible with this scenario. It turns out that the pH in the plant TGN lies around 5.5 (Luo et al., 2015) and the transition from TGN to LE/PVC involves an alkalization rather than acidification, with measured values of 6.8 for the PVC, and even 7.1 for the LPVC (Martinière et al., 2013; Schumacher, 2014). These values are, however, in keeping with the fact that the LE/PVC in plants lack proton-pumping complexes. As Viotti et al. (2013) have shown, both V-H+-ATPase and V-H+-PPase are transferred directly to the tonoplast from the ER during vacuole biogenesis. Moreover, the TGN-specific VHA-a1 ATPase does not appear to leave the TGN during MVB biogenesis (Scheuring et al., 2011). As is now well known, and unlike the situation in animal cells, the plant TGN also functions as an EE. The pH values in the apoplast and the TGN/ EE (Luo et al., 2015) are similar, suggesting that extracellular ligands which bind to cell surface receptors and are endocytosed do not dissociate in the EE as a result of decreased pH (Luo et al., 2015). Rather, as indicated above, the binding and release of ligands to/from VSRs may depend more on the Ca2+ concentration than on pH. VSR recycling: the unknowns Historically speaking, the PVC in plant cells was discovered through analyses of an Arabidopsis homologue to yeast Pep12p/mammalian syntaxin 7, a t-SNARE that localizes to late endosomes and vacuolar/lysosomal membranes (Fischer von Mollard et al., 1997; Mullock et al., 2000). AtPEP12 functionally complements a yeast pep12 mutant (Bassham et al., 1995) and also localizes to a post-Golgi compartment as well as the tonoplast of plants (Da Silva Conceiçao et al., 1997; Sanderfoot et al., 1998; Uemura et al., 2010). Subcellular fractionation of Arabidopsis root tissue reveals that AtPEP12 is present in three distinct fractions. VSR1 (AtELP) is also detected in these fractions, as well as in a light density fraction, which is presumably the TGN (Da Silva Conceiçao et al., 1997). This suggests that, in addition to the tonoplast, AtPEP12 resides on two different classes of endosomal compartments. One of these has been identified as an MVB (Tse et al., 2004), but the nature of the other remains unclear. It is possible, if not likely, that these two AtPEP12positive fractions represent different stages in endosomal maturation. Given that both are VSR positive, are they and the TGN equally capable of recycling VSRs? Could instead the VSRs in one or more of these compartments merely represent non-functional receptors awaiting eventual degradation through fusion with the vacuole? At the moment we cannot estimate how much of a delay there is between ligand release at the TGN and receptor recycling. Presumably some VSRs are carried forward as the TGN matures, but what proportion and for how long? It has always been assumed that VSRs in MVBs will be recycled, but there is no direct experimental evidence for this and whether retromer-coated carriers are responsible is also unclear. Retromer-mediated retrieval of ligand-free MPRs in animal cells is coupled to a switch from Rab5- to Rab7-type GTPases that occurs as the EE mature into the LE (Seaman, 2012; van Weering et al., 2012b). It has been reported that a related Rab7 GTPase is also required for the membrane recruitment of the retromer core in plants (Zelazny et al., 2013). However, the transition from Rab5 to Rab7 GTPases in plants is related to MVB/PVC fusion with the vacuole rather than endosomal maturation (Cui et al., 2014; Singh et al., 2014). According to Foresti et al. (2010), the last LE compartment prior to fusion with the tonoplast (termed the late prevacuolar compartment, LPVC) no longer possesses VSRs, which the authors assume have been retrieved earlier in endosome maturation. So, apparently VSRs are no longer present when the Rab conversion occurs. This has recently been confirmed in a detailed proteomic analysis of TGN/EE and other endosomal compartments (Heard et al., 2015). Evidence for the participation of retromer in protein transport to the vacuole is based on essentially two types of observations: (1) VSR1 can be immunoprecipitated with VPS35 (a retromer core subunit) antibodies (Oliviusson et al., 2006); and (2) traffic to the protein storage vacuole is perturbed in vps35, vps27, and snx1/2a/2b single/double and triple mutant plants (Shimada et al., 2006; Yamazaki et al., 2008; Pourcher et al., 2010). The exact location of the retromer core subunit in plant cells has not been unequivocally demonstrated, with conflicting reports on TGN and MVB locations (Robinson et al., 2012; Robinson et al., 2015). However, in a recent detailed proteomic study, VPS35 was mainly present on LPVC membranes (Heard et al., 2015). If this is true, one must conclude that whatever the function of the retromer core really is, it is not involved in VSR recycling, given that VSRs are absent from this endosomal compartment. It is known that the formation of tubular recycling carriers from maturing endosomes in animal cells cannot occur through recruitment of the retromer core subunit alone; for this, the sorting nexins (SNXs) are also required (van Weering et al., 2012a). In plants, the SNXs are not present on the LVPC and instead are found mainly on the TGN or developing MVB (Stierhof et al., 2013; Heard et al., 2015). Interestingly, SNX mutants do not appear to prevent the endocytic traffic of internalized PM transporters (IRT1 and PIN2) to the vacuole (Ivanov et al., 2014; Kleine-Vehn et al., 2008). Moreover, transient expression of SNX1 and SNX2a mutants in tobacco leaf protoplasts also has no effect on the transport of soluble vacuolar cargo to the vacuole (Niemes et al., 2010b). These observations suggest that, whatever function the SNXs have at the TGN, they are not involved in post-Golgi trafficking to the vacuole, and they perform this function without the retromer core. TGN and vacuolar sorting receptors | 4443 Retrograde transport from the TGN to the ER has frequently been studied in mammalian cells, often involving the internalization and trafficking of exotoxins from the PM. The most frequently investigated toxins are of bacterial (cholera toxin, CTx; Pseudomonas exotoxin, PEx; and Shiga toxin, STx), or plant (ricin) origin. All have a catalytic (toxic) A chain associated with either one (PEx, ricin) or five (CTx, STx) B chains. They all eventually reach the ER and are translocated into the cytosol where cell/organismal death is triggered by interaction with the protein translational machinery (PEx, STx) or by activation of adenylate cyclase, which induces intestinal chloride secretion leading to massive diarrhoea (Spooner et al., 2006; Sandvig et al., 2010). There would appear to be several possibilities for retrograde transport of exotoxins from the TGN to the ER. CTx and PEx have C-terminal KDEL-like motifs and use a COPI-vesicle– mediated ERD2 receptor retrieval mechanism (Jackson et al., 1999). Interfering with COPI coat assembly prevents retrograde traffic (Majoul et al., 1998). The second pathway used by STx is COPI independent, requires Rab6, and uses detergent-resistant membrane microdomains (Girod et al., 1999; White et al., 1999). In the third pathway, CTx is supposed to bypass the Golgi complex entirely, moving directly from the TGN to the ER. This conclusion was based on the observation that exo2treatment, which disassembles the Golgi but does not affect the TGN, does not prevent retrograde transport to the ER (Feng et al., 2004). Finally, although ricin lacks a KDEL retrieval motif it appears to interact in the Golgi with calreticulin, which does have a KDEL-motif (Day et al., 2001). The exotoxins are all soluble proteins and therefore trafficking studies involving them may not be that useful when trying to explore possible mechanisms for retrograde transport of VSRs. However, an example for the retrograde transport of a membrane protein from the TGN to the ER in mammalian cells has recently been published (Yu et al., 2014). This involves a multi-spanning membrane protein called WLS or Wntless, which normally acts as a carrier protein for the anterograde transport of lipidated Wnts (Wingless-related integration site in Wnt signalling) molecules from the ER to the PM through the secretory pathway. WLS is recycled from the PM, first to endosomes then to the TGN. Yu et al. (2014) have identified an ER-targeting signal at the C-terminus distinct from but related to the classical dilysine retrieval motif for membrane proteins (Nilsson et al., 1989). Experiments with ARF1 mutants led Yu et al. (2014) to conclude that WLS also uses COPI vesicles to progress upstream from the TGN to the ER. VSRs do not have ER retrieval motifs at their cytosolic C-terminus, which projects into the cytosol, so we can rule out hijacking of the ERD2 retrieval system for retrograde TGN to ER transport of VSRs. Nevertheless, that COPI vesicles are observed at the TGN suggests they could be agents of retrograde trafficking through the Golgi stack (Staehelin and Kang, 2008; Kang et al., 2011). On the other hand, we have pointed to circumstantial evidence in support of CCVretrieving VSRs from DVs at the TGN in storage protein–producing cells of developing seeds. This might also happen to VSRs in vegetative cells transporting hydrolases to lytic-type vacuoles. In this respect, one notes with interest that recent studies on yeast cells suggest that recycling of Kex2 from the TGN upstream to late Golgi elements seems to involve a population of CCVs containing the adaptor AP-1 (see Papanikou et al., 2015). Notwithstanding the controversy surrounding the exact localization of the core retromer subunit (see above), the demonstration that VPS35 (a retromer core subunit) and VSR can co-immunoprecipitate (Oliviusson et al., 2006) is an indication that retromer-coated carriers may still be candidates for retrograde recycling of VSRs from the TGN. Post-Golgi transport of soluble proteins to the vacuole: a receptor-independent process? Once lysosomal acid hydrolases have separated from their receptors – during the transition from EE to LE – they remain in the lumen of the LE as it completes its maturation. They finally reach their target destination through fusion of the LE with the lysosome. Thus, the later stage of acid hydrolase transport to the lysosome is a receptor-independent process. Endosomal maturation is well accepted in the animal literature, and involves gradual modifications in phosphoinositides, Rab GTPases, and sorting nexins (van Weering et al., 2010; Huotari and Helenius, 2011; Scott et al. 2014). In the case of the plant cell, it entails drastic changes in the morphology and function of the TGN. In essence, it describes the development and detachment of an MVB from the TGN, and it is in opposition to earlier concepts whereby the TGN and MVB are discrete, long-living compartments that exchange receptors and ligands via vesicles. The formation of MVBs at the plant TGN is a consequence of cisternal maturation in the Golgi stack and therefore represents just one segment of membrane flow from the ER to the vacuole. This can be nicely illustrated by perturbing TGN function by applying the V-ATPase inhibitor concanamycin A. This leads on the one hand to a pile-up of trans-cisternae which do not leave the stack, and on the other hand to a depletion in the number of free MVBs (Scheuring et al., 2011). Curiously, for an ongoing process and despite the use of rapid freezing technology for EM sample preparation, it has proven very difficult to obtain images showing the process of MVB formation at the TGN. Fragmentation of the TGN into vesicle clusters has been documented by Staehelin and coworkers in electron tomographic studies of Golgi structure in Arabidopsis root cells (Staehelin and Kang, 2008; Kang et al., 2011), but MVB formation is speculated rather than being depicted: ‘TGN fragmentation into SVs [secretory vesicles] and CCVs was typically accompanied by the appearance of membrane fragments … these fragments may become precursors of MVBs’ (Kang et al., 2011). To increase the chances of capturing events in MVB formation, Scheuring et al. (2011) looked at Arabidopsis root cells recovering from concanamycin A treatment. Unusual, pleomorphic MVBs, often with bottleneck protuberances, were observed in the immediate vicinity of Golgi stacks, but really convincing direct connections to TGN elements were not shown. MVB formation in plants remains an unsolved problem. 4444 | Robinson and Neuhaus If the TGN is indeed a compartment where newly synthesized vacuolar cargo ligands are released from their receptors, it follows that post-TGN transport of soluble proteins to the vacuole is a passive, bulk-flow process coupled to endosomal maturation. One would therefore predict that soluble endocytic cargo molecules internalized at the PM would, on reaching the TGN, also move passively downstream to the vacuole. Künzl et al. (2016) have provided evidence in support of this contention. These authors have isolated triple (3x) RFP secreted by protoplasts as a fluorescent reporter protein for endocytic uptake. Having been secreted, this reporter has no vacuolar sorting signals and cannot therefore interact with a VSR. This reporter, as well as an anti-GFP nanobody fusion construct, 3xRFP-Nb, was successfully endocytosed and reached the vacuole. Traffic through the TGN and MVB was confirmed by co-expressing the Nb reporter with the corresponding compartmental markers SYP61-GFP (TGN) or GFP-BP80 (MVB). This is yet another piece of evidence in favour of the notion that post-Golgi transport of soluble cargo to the vacuole does not require VSRs. A new model for the sorting of secretory and vacuolar cargo proteins at the trans-Golgi/early TGN In order to reconcile the results discussed above, we propose a new model based on the ultrastructural data of Scheuring et al. (2011) and Kang et al. (2011), who performed electron tomography on Arabidopsis root meristem cells. These cells produce a lot of cell wall material, which explains the abundance of secretory vesicles. Our model (see Fig. 4) may be more applicable to cells producing more proteins and fewer polysaccharides. The essence of our model is that there is a maturation sequence from Golgi via the TGN to the PVC and that CCVs are not involved in anterograde transport. We propose that the segregation of secretory and vacuolar cargos relies on lateral segregation into domains within the maturing TGN compartment. This is the step where we propose that the Tyr motifs in VSRs are required to segregate VSR–cargo complexes away from forming secretory vesicles. These motifs are typically recognized by μ-subunits of the AP adaptor complexes, which then recruit clathrin coats. However, not all adaptins interact with clathrin. This is the case with AP-4 and AP-5 in animal cells. In plants, AP-4, like AP-1, binds to the Tyr motif of VSR1 (Song et al., 2006; Gershlick et al., 2014) and is required for the correct targeting of storage globulins to the PSV in Arabidopsis (Fuji et al., 2016). Assuming that plant AP-4 also does not interact with clathrin, we propose that the Tyr motif in VSRs is nevertheless involved in anterograde cargo transport by retaining VSR–cargo complexes within a domain of TGN so that they do not get included in secretory vesicles. Once the latter have been formed, the VSRs can release their cargo, which might be induced by a lowered Ca2+ concentration. At this point, the VSRs can be recycled to an upstream compartment while the cargo can remain in the TGN as it matures into a MVB, and then into a late PVC, and finally into vacuoles. Recycling of VSRs to Fig. 4. A new model for the VSR-dependent sorting of vacuolar proteins. (A) Vegetative tissues: anterograde transport is based on the continuous maturation from Golgi cisternae via TGN to late PVC, rather than vesicle-mediated transport between the TGN and MVB/PVC. Cargo proteins (red dots) are transported from the ER to the cis-Golgi by COPII vesicles either by default or already bound to VSRs. When the trans-Golgi cisterna turns into Golgiassociated TGN (Ga-TGN, Kang et al., 2011), the cargo-VSR complexes are prevented from entering the secretory vesicles (SV) targeted to the PM by interaction with an AP-4 coat (violet line). In the remaining free TGN, a lower Ca2+ concentration causes release of the cargo proteins and the VSRs can then be retrieved by either an AP-1-CCV or an SNX-dependent, but retromer-independent mechanism (red coat, recycling carrier [RC]) and recycled back to either the cis-Golgi or ER. The TGN matures into a PVC, still containing some of the VSRs, and finally into an LPVC, ready to fuse with the lytic vacuole. Some VSRs may be transported by SVs to the PM, either by leakage or in a regulated process, for example in upper root tissues (Saint-Jean et al., 2010). VSRs are then endocytosed and transported by AP-2(?)-CCVs to the TGN. (B) In seeds: VSRs may contribute to nucleate storage protein aggregation in the Golgi and may be included into the nascent DVs. VSRs are retrieved by AP-1-CCVs and recycled back to either the cis-Golgi or ER. In pumpkin, the DVs form at the ER as PAC vesicles. DVs fuse with PVC before fusing with the PSV. TGN and vacuolar sorting receptors | 4445 an earlier compartment, for example, the ER or cis-Golgi, could be achieved by either an SNX-dependent but retromerindependent mechanism or by AP-1–dependent CCVs. The transition of AP-4 to AP-1 binding could be regulated by phosphorylation or dephosphorylation or by dimerization of the VSRs. Conclusions and perspectives The general assumption that physiological processes that are outwardly similar in all eukaryotes should operate in the same way is not surprising but is not necessarily true. In the case of the secretory and endocytic pathways, it is even more understandable considering the inter-kingdom homologies in vesicle coat proteins (Paul and Frigerio, 2007). Therefore, and taking into account the huge number of publications on secretion and endocytosis in mammals and yeast, it has been difficult to resist applying a non-plant template for the interpretation of data obtained on these processes in plant cells. Nevertheless, as emphasized on numerous occasions (e.g. Robinson et al., 2007; Richter et al., 2009; Contento and Bassham, 2012; Jürgens et al., 2015), the membrane compartments of the higher plant secretory and endocytic pathways in plants are organized differently to their non-plant counterparts. Physiologically, the cargos passing through the plant secretory system are also very different to those in animal and yeast cells, a fact perhaps best exemplified by the transport and deposition of seed storage proteins (Robinson and Hinz, 1999). And, as most recently speculated by Luo et al. (2015), the similar pH values in the apoplast and the TGN/EE of plant cells is such that a dissociation of endocytosed PM-located receptor–ligand complexes in the plant EE and the consequential recycling of the receptor to the PM, as known for mammalian cells, is unlikely to occur in plants. Taken together, these differences should have generated more caution among plant scientists when reading publications dealing with the MPR sorting system for lysosomal acid hydrolases. That VSRs and MPRs are structurally unrelated, and the sorting motifs in their respective ligands are not identical, should have lead to even more scepticism about the apparent similarities in the two protein sorting systems. As described above, recently published data on VSR– ligand interactions have convincingly demonstrated that receptor–ligand binding is initiated in the ER/cis-Golgi and terminates in the TGN. The absence of proton-pumping complexes together with in situ measurements of organellar pH discounts the MVB/LE/PVC as being a compartment where pH-dependent VSR–ligand dissociation occurs. Much weight has been given in the past to interactions between the cytoplasmic domain of VSRs and clathrin adaptors as evidence for the anterograde transport of soluble vacuolar cargo ligands out of the TGN. However, not only has the actual presence of such ligands in the lumen of a CCV never been demonstrated, but clathrin–VSR interactions could equally well point to a role for CCVs in retrograde trafficking of VSRs from the TGN. Thus, there is in our opinion no hard evidence to support the notion that VSRs first interact with their ligands in the TGN and deliver them via CCVs to the MVB/PVC, where the ligands would dissociate due to an acidic pH. It is time to discard this dogma! Based on our new model for VSR-function, we propose that research on plant VSRs in the immediate future should be focused on three major problems: 1.The identification and characterization of the domains of the TGN responsible for the temporal separation of secretory vesicle and retrograde VSR carrier formation. The question as to the timing of retrograde VSR traffic in relation to TGN status (Golgi-attached or Golgi-free) also needs to be addressed. 2.The identity of the retrograde carrier (clathrin- versus retromer-coated) needs to be established beyond doubt, as well as the nature of the cytoskeletal element(s) responsible for upstream trafficking. 3. Determining the exact location in the early secretory pathway (ER or cis-Golgi) for the delivery of recycled VSRs. Should the ER be the target for retrograde VSR traffic, the next question would be: are the import sites randomly distributed throughout the ER or associated together with ERESs and COPII-import sites as part of the secretory unit (see Lerich et al., 2012; Robinson et al., 2015 for a description of the secretory unit concept)? If, instead, the cis-Golgi turns out to be the port of re-entry into the secretory pathway, our previous concept of cis-cisternal formation, based solely on COPII vesicle fusion (Donohoe et al., 2013), would need to be modified to include retrograde VSR-containing vesicles. Investigations on retrograde trafficking of membrane proteins present technical difficulties that will demand new experimental strategies. A way must be found to post-translationally label TGN-located VSRs to distinguish them from anterograde trafficking (newly synthesized/re-usable) VSRs. 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