Download Receptor-mediated sorting of soluble vacuolar proteins: myths, facts

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.
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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.
As in retrograde transport studies in animal cells, one might
exploit the endocytic pathway to deliver a fluorescent marker
to the TGN/EE, but how a trackable retrograde VSR construct can be assembled remains a challenge for the future.
Acknowledgements
The authors thank Peter Pimpl for his useful comments. JMN was supported
by Grant 31003A_141257 from the Swiss National Science Foundation.
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