Download Trafficking of the human transferrin receptor in plant cells: effects of

The Plant Journal (2006) 48, 757–770
doi: 10.1111/j.1365-313X.2006.02909.x
Trafficking of the human transferrin receptor in plant cells:
effects of tyrphostin A23 and brefeldin A
Elena Ortiz-Zapater, Esther Soriano-Ortega, Marı́a Jesús Marcote, Dolores Ortiz-Masiá and Fernando Aniento*
Departamento de Bioquı́mica y Biologı́a Molecular, Facultad de Farmacia, Universidad de Valencia, Avda Vicente Andrés
Estellés s/n, 46100-Burjassot (Valencia), Spain
Received 9 June 2006; revised 26 July 2006; accepted 8 August 2006.
*For correspondence (fax þ34 963544917; e-mail [email protected]).
Summary
Plant cells possess much of the molecular machinery necessary for receptor-mediated endocytosis (RME), but
this process still awaits detailed characterization. In order to identify a reliable and well-characterized marker
to investigate RME in plant cells, we have expressed the human transferrin receptor (hTfR) in Arabidopsis
protoplasts. We have found that hTfR is mainly found in endosomal (Ara7- and FM4-64-positive) compartments, but also at the plasma membrane, where it mediates binding and internalization of its natural ligand
transferrin (Tfn). Cell surface expression of hTfR increases upon treatment with tyrphostin A23, which inhibits
the interaction between the YTRF endocytosis signal in the hTfR cytosolic tail and the l2-subunit of the AP2
complex. Indeed, tyrphostin A23 inhibits Tfn internalization and redistributes most of hTfR to the plasma
membrane, suggesting that the endocytosis signal of hTfR is functional in Arabidopsis protoplasts. Coimmunoprecipitation experiments show that hTfR is able to interact with a l-adaptin subunit from Arabidopsis
cytosol, a process that is blocked by tyrphostin A23. In contrast, treatment with brefeldin A, which inhibits
recycling from endosomes back to the plasma membrane in plant cells, leads to the accumulation of Tfn and
hTfR in larger patches inside the cell, reminiscent of BFA compartments. Therefore, hTfR has the same
trafficking properties in Arabidopsis protoplasts as in animal cells, and cycles between the plasma membrane
and endosomal compartments. The specific inhibition of Tfn/hTfR internalization and recycling by tyrphostin
A23 and BFA, respectively, thus provide valuable molecular tools to characterize RME and the recycling
pathway in plant cells.
Keywords: receptor-mediated endocytosis, plant cell, transferrin receptor, endocytosis signal, Arabidopsis
thaliana, tyrphostin.
Introduction
Receptor-mediated endocytosis (RME) is a well-established
process in animal cells and is mediated by clathrin-coated
vesicles (CCVs) and other less well-characterized organelles.
In clathrin-mediated RME, ligands bound to their receptors
become internalized via clustering in clathrin-coated pits. A
large number of membrane proteins that traffic through the
clathrin pathway are sorted into coated pits and vesicles by
adaptor complexes, a group of proteins that recognize signals in the cytosolic portion of membrane proteins and also
interact with clathrin (Aniento et al., 2003; Boehm and
Bonifacino, 2001; Bonifacino and Traub, 2003; Conner and
Schmid, 2003; Kirchausen, 1999, 2000; Schmid, 1997). AP2,
the clathrin adaptor specifically involved in endocytosis, is a
heterotetrameric complex composed of a-, b2-, l2- and r2ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd
subunits. The l2-adaptin recognizes tyrosine-based sorting
signals of the form YXX/ (where / is a bulky hydrophobic
residue); the b2-adaptin contains binding sites for a second
sorting signal, the dileucine-based motif, but is also the
main binding partner for clathrin (Bonifacino and Traub,
2003; Conner and Schmid, 2003; Kirchausen, 1999, 2000;
Schmid, 1997). Typically, endocytosed molecules, including
recycling receptors with their bound ligands and downregulated receptors, are delivered to early/sorting endosomes
(EEs), where sorting occurs efficiently. After receptor–ligand
uncoupling at the mildly acidic luminal pH, recycling receptors are rapidly (half-life of approximately 2.5 min) segregated away from their ligands and transported along the
recycling route, and ligands follow the degradation pathway
757
758 Elena Ortiz-Zapater et al.
together with downregulated receptors (Gruenberg, 2001;
Marcote et al., 2000).
Although it has been questioned in the past, there is
compelling evidence that endocytosis is a basic cellular
process that also occurs in plant cells, where it acts in the
internalization of molecules from the plasma membrane,
signal transduction, downregulation of plasma membrane
receptors and polar tip growth (Bahaji et al., 2001, 2003;
Baluska et al., 2002, 2004; Barth and Holstein, 2004; Battey
et al., 1999; Emans et al., 2002; Geldner et al., 2001,
2003; Grebe et al., 2003; Holstein, 2002; Homann and Thiel,
1999; Low and Chandra, 1994; Marcote et al., 2000; Meckel
et al., 2004; Murphy et al., 2005; Tse et al., 2004; Ueda et al.,
2001, 2004). However, clathrin-mediated RME has not yet
been proven unequivocally, although receptor-like kinases
have been proposed as candidates for internalization via
clathrin-mediated endocytosis (Holstein, 2002). Interestingly, Shah et al. (2002) elegantly demonstrated that the
kinase-associated protein phosphatase KAPP regulates
endocytosis of AtSERK1, a leucine-rich repeat (LRR) Ser/
Thr receptor-like kinase. Heterodimerization and endocytic internalization of brassinosteroid receptors BRI1 and
AtSERK3 (BAK1) have also been reported (Russinova et al.,
2004). Co-expression of BRI1 and SERK3 resulted in accelerated endocytosis, suggesting that SERK3 changes the
equilibrium between the plasma membrane-localized BRI1
homodimers and internalized BRI1–SERK3 heterodimers.
Endocytic trafficking of other receptor-like kinases such as
CLV1 (CLAVATA 1) and SRK may be expected because their
kinase domains interact with an endosomal sorting nexin
(Vanoosthuyse et al., 2003). On the other hand, biotin and
biotinylated markers have been shown to enter rice cells by a
process with the characteristics of RME, although a putative
biotin receptor has not yet been identified (Bahaji et al.,
2001, 2003). Finally, it has been recently reported that the
pattern recognition receptor FLS2 in Arabidopsis, which, in
the absence of ligand, is present at the plasma membrane, is
internalized into intracellular compartments after stimulation with the flagellin epitope flg22 (Robatzek et al., 2006).
Clathrin-coated pits and vesicles are abundant at the
plasma membrane of plant cells (Beevers, 1996; Holstein,
2002; Robinson et al., 1998). In addition, plant cells contain
much of the molecular machinery involved in RME, including clathrin heavy (Blackbourn and Jackson, 1996) and light
chains (Scheele and Holstein, 2002), and several adaptin
isoforms, namely two a-adaptins (Barth and Holstein, 2004;
Holstein, 2002), three c-adaptins (Boehm and Bonifacino,
2001; Schledzewski et al., 1997), five b-adaptins (Boehm and
Bonifacino, 2001), five l-adaptins (Happel et al., 2004) and
five r-adaptins (Boehm and Bonifacino, 2001; MaldonadoMendoza and Nessler, 1996; Roca et al., 1998). However, the
composition of plant adaptor complexes has not been
elucidated, and little is known about adaptin functions in
plants. A plant orthologue of mammalian a-adaptin (AtaC-
Ad), which plays a crucial role in endocytosis, has been
shown to bind several mammalian network proteins, and it
also interacts with At-AP180, a monomeric adaptor homologue from Arabidopsis that functions as a plant clathrin
assembly protein (Barth and Holstein, 2004). On the other
hand, direct involvement of CCVs in RME has not yet been
demonstrated, and very little is known about the interaction
between adaptins and putative sorting receptors. In addition, there are only few examples of sorting motifs shown to
function as endocytosis signals and to recruit adaptins from
the AP2 complex in plant cells. As regards, LeEix2, the
receptor for the fungal elicitor ethylene-inducing xylanase, is
a cell surface glycoprotein possessing a tyrosine-based
endocytosis signal: a point mutation (Tyr993 to Ala) within
its endocytosis signal abolished the ability of LeEix2 to
induce a hypersensitive response (Ron and Avni, 2004). The
products of the race-specific Ve1 and Ve2 disease-resistance
genes from tomato contain a typical acidic or tyrosine-based
sorting motif (Kawchuk et al., 2001), while a member of the
Leucine-rich repeat (LRR) subfamily of Receptor protein
kinase (RPK) contains a YXX/ motif within its cytoplasmic
tail that binds to the receptor-binding domain of the
Arabidopsis lA-adaptin (Holstein, 2002).
Given the similarities in the molecular machinery involved
in RME in plant and animal cells, we decided to express in
Arabidopsis protoplasts one of the best characterized receptors involved in RME in animal cells, the human transferrin
receptor (hTfR). The hTfR is a type II plasma membrane
protein-mediating cellular iron uptake via binding and
internalization of the serum ion transport protein transferrin
(Tfn; Dautry-Varsat and Lodish, 1984). hTfR is a homodimeric
glycoprotein with a molecular mass of 90–95 kDa per
subunit. The receptor monomer consists of a 61-residue
N-terminal cytoplasmic domain, a single 28-residue hydrophobic transmembrane domain and a 671-residue extracellular domain (McClelland et al., 1984; Schneider et al.,
1982). The subunits are covalently linked via two disulphide
bonds (Jing and Trowbridge, 1987). Regardless of ligand
binding, hTfR is constitutively internalized from the plasma
membrane via CCVs. This step involves a well-characterized
tyrosine-based endocytosis signal (YTRF), present in the
cytosolic domain of each monomer, which interacts with the
l2-subunit of the AP2 adaptor complex (Ohno et al., 1995).
Tfn and TfR are first delivered to EEs and are then recycled
back to the cell surface. Recycling is thought to be mediated
by vesicular carriers that transfer receptors to a tubulovesicular compartment referred to as a recycling endosome
(RE; Trowbridge et al., 1993; Yamashiro et al., 1984). In
contrast to peripheral tubulovesicular EEs, REs are composed of tubular membranes only and are concentrated
predominantly in the perinuclear region (Gruenberg, 2001).
Unlike many other ligands, Tfn remains bound to its receptor
throughout the recycling pathway and is released upon
returning to the cell surface (Ciechanover et al., 1983).
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
Receptor-mediated endocytosis in plant cells 759
Recycling Tfn may not simply pass sequentially through EEs
and REs, as recycling is typically biphasic with both initial
rapid and later slow components (Daro et al., 1996). At least
in polarized MDCK cells, the two rates reflect a rapid passage
of the majority (65%) of Tfn directly back from EEs to the
plasma membrane or a slower route that requires additional
passage through REs (Sheff et al., 1999).
In the present study, we have transiently expressed the
hTfR in Arabidopsis protoplasts and tested its functionality.
We have found that hTfR is mainly found in endosomal
(Ara7- and FM4-64-positive) compartments, but also partially at the plasma membrane, where it mediates binding and
internalization of its natural ligand Tfn. Cell surface expression of hTfR increases upon treatment with tyrphostin A23,
which has been shown to inhibit the interaction between the
YTRF endocytosis signal in the hTfR cytosolic tail and the l2subunit of the AP2 complex. Indeed, treatment with this drug
blocks Tfn internalization and redistributes most of hTfR to
the plasma membrane, suggesting that the endocytosis
signal of hTfR is functional in Arabidopsis protoplasts. In this
respect, co-immunoprecipitation experiments showed that
hTfR is able to interact with a l-adaptin subunit from
Arabidopsis cytosol, a process that is blocked by tyrphostin
A23. In contrast, treatment with brefeldin A (BFA), which
has been shown to inhibit recycling from endosomes back to
the plasma membrane in plant cells (Baluska et al., 2002;
Boonsirichai et al., 2003; Geldner et al., 2001, 2003; Grebe
et al., 2003), leads to the accumulation of Tfn and hTfR in
larger patches, reminiscent of BFA compartments. Therefore, hTfR has the same trafficking properties in Arabidopsis
protoplasts as it has in animal cells, cycling between the
plasma membrane and endosomal compartments, and thus
provides a valuable marker to characterize RME in plant
cells. In addition, the use of tyrphostin A23, which specifically inhibits Tfn/hTfR internalization, together with BFA,
which inhibits their recycling to the plasma membrane,
provides valuable molecular tools to dissect the internalization/recycling pathway in plant cells.
Results
Expression of the hTfR in Arabidopsis protoplasts
hTfR was expressed under the control of the 35S promoter in
Arabidopsis protoplasts using polyethylene glycol-mediated
transfection (Axelos et al., 1992). After 22 h, protein extracts
from control and transfected protoplasts were analysed by
SDS–PAGE and Western blot analysis using a monoclonal
antibody against the hTfR. Under denaturing and reducing
electrophoresis conditions (Figure 1a, þb-ME), the hTfR was
detected as a homogeneous band of the expected molecular
weight, which is around 95 kDa for the monomer. To test for
dimer formation, samples were prepared under non-reducing electrophoresis conditions (Figure 1b, )b-ME). Most of
(a)
(b)
Figure 1. Western blot analysis of Arabidopsis protoplasts expressing hTfR.
Arabidopsis protoplasts were transformed with the cDNA of the human
transferrin receptor (hTfR) or with the pDH51 vector (control). After incubation
for 22 h, protoplasts were collected by centrifugation, extracted in lysis buffer
and treated with standard Laemmli sample buffer containing b-mercaptoethanol (þb-ME; a) or with buffer lacking b-ME ()b-ME; b). HTfR was detected by
SDS–PAGE and Western blot analysis with a monoclonal antibody against
hTfR.
the hTfR was then detected as a band of about 190 kDa, the
expected molecular weight for the hTfR dimer. Even under
these conditions, a small fraction of hTfR was still found as a
monomer, probably because of diffusion of b-mercaptoethanol from adjacent lanes during SDS–PAGE. Therefore,
Arabidopsis protoplasts express hTfR and the protein normally forms a dimer, as in human cells.
Subcellular localization of the human transferrin receptor in
Arabidopsis protoplasts
The subcellular distribution of the hTfR in transfected Arabidopsis protoplasts was analysed by immunofluorescence,
using a polyclonal (anti-CD71) antibody against the hTfR and
an Alexa 488-labelled secondary antibody. Samples were
then analysed by confocal microscopy. As shown in
Figure 2, hTfR was found mainly in vesicular structures
distributed throughout the cytoplasm, and only partially at
the plasma membrane. To test for cell surface expression of
the receptor and for its correct orientation at the plasma
membrane, intact (non-permeabilized) protoplasts were
incubated with the anti-CD71 antibody, which recognizes the
extracellular domain of hTfR, and therefore can bind to
receptor molecules at the plasma membrane. The incubation was performed at 4C to avoid internalization of receptor and antibody, and was followed by incubation (also at
4C) with an Alexa 488-conjugated antirabbit IgG. As shown
in Figure 3, the anti-CD71 antibody revealed the presence of
hTfR at the plasma membrane of transfected protoplasts
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
760 Elena Ortiz-Zapater et al.
(a)
(b)
(a)
(b)
mock
(c)
(c)
(d)
(d)
hTfR
(e)
(e)
(f)
Figure 2. Subcellular localization of hTfR in Arabidopsis protoplasts.
Arabidopsis protoplasts were transformed with the cDNA of the human
transferrin receptor (hTfR), fixed and analysed by immunofluorescence using
a polyclonal antibody against hTfR (anti-CD71) and an Alexa 488-labeled
secondary antibody. Samples were analysed using a Leica confocal microscope. Protoplasts transfected with the pDH51 vector showed no fluorescence
under the same experimental conditions used to analyse protoplasts expressing hTfR. (a, c and e) Nomarsky images; (b, d and f) confocal images.
(Figure 3d). This was not due to non-specific binding of the
antibody to the cell surface, as no signal was detected, under
the same experimental conditions, in control protoplasts
(Figure 3b). These data indicate that at least a fraction of
hTfR expressed in Arabidopsis protoplasts is present at the
plasma membrane, and that the extracellular domain that is
involved in ligand binding is correctly exposed at the cell
surface. The distribution of hTfR in Arabidopsis protoplasts
is consistent with that observed in several animal cell lines,
and may reflect its continuous internalization to and recycling from endosomal compartments (Ciechanover et al.,
1983; Damke et al., 1994; Hao and Maxfield, 2000; Mayor
et al., 1993; Mellman, 1996; Sheff et al., 1999; Warren et al.,
1997). Indeed, hTfR co-localized extensively with the GTPase
Ara7 (Figure 4a), a marker of endosomal compartments in
plant cells. In particular, Ara7-positive endosomes have
been proposed to be the site of GNOM-dependent recycling
of plasma membrane proteins (Ueda et al., 2004). In
(f)
hTfR
+ A23
Figure 3. Cell surface expression of human transferrin receptor (hTfR) in
Arabidopsis protoplasts.
The presence of hTfR at the plasma membrane was tested in non-permeabilized Arabidopsis protoplasts expressing (c–f; hTfR) or not (a and b; mock)
the hTfR, by incubation at 4C with a polyclonal antibody against an
extracellular epitope of the hTfR (anti-CD71) and an Alexa Fluor 488-conjugated antirabbit IgG. (a, c and e) Transmission images; (b, d and f) confocal
images; (e and f) protoplasts were incubated for 2 h at 28C in the presence of
350 lM tyrphostin A23 prior to incubation with the antibodies.
contrast, almost no co-localization was observed between
hTfR and the ER marker BiP (Figure 4b). Therefore, the cytosolic vesicular structures where hTfR is mainly found under steady-state conditions correspond to endosomal
compartments and not to ER membranes.
Uptake of transferrin in Arabidopsis protoplasts expressing
the human transferrin receptor
We next investigated whether hTfR was functional in Arabidopsis protoplasts. The expected function of the hTfR is
binding and internalization of the serum ion transport protein Tfn. Therefore, we tested whether protoplasts expressing hTfR had the ability to internalize fluorescently labelled
Tfn. As shown in Figure 5(b), Tfn–Alexa 546 was efficiently
internalized in protoplasts expressing hTfR. Internalized
ligand was observed in cytosolic vesicular structures (Figure 5b) in a pattern similar to that observed for hTfR.
Transferrin uptake was temperature-dependent, as no
internalization occurred when the incubation was performed
at 4C. Under these conditions, Tfn was found to bind to the
plasma membrane (Figure 5a). Neither internalization nor
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
Receptor-mediated endocytosis in plant cells 761
(a)
(a)
Control
BFA
hTfR
Ara7
Overlay
(b)
Tfn-546
hTfR
Overlay
Tfn-488
FM4-64
Overlay
(b)
hTfR
BiP
Overlay
Figure 4. Human transferrin receptor (hTfR) co-localizes with the GTPase
Ara7 but not with the ER marker BiP in Arabidopsis protoplasts.
Arabidopsis protoplasts were transformed with the cDNA of the hTfR, fixed
and analysed by immunofluorescence using a monoclonal antibody against
the hTfR and polyclonal antibodies against Ara7 (a) or BiP (b) and Alexa 488/
546-labeled secondary antibodies. Samples were analysed using a Leica
confocal microscope.
(a) 4°C
(b) 28°C
Figure 5. Uptake of transferrin–Alexa 546 in Arabidopsis protoplasts expressing human transferrin receptor (hTfR).
Protoplasts expressing the hTfR were incubated in the presence of transferrin–Alexa 546 for 1 h at 4C (a) or 28C (b) and analysed using a Leica confocal
microscope. Protoplasts transfected with the pDH51 vector showed no
fluorescence under the same experimental conditions used to analyse
protoplasts expressing hTfR.
binding of Tfn to the plasma membrane were observed in
control protoplasts (either untransfected or transfected with
the empty vector) under the same experimental conditions
(data not shown). As Tfn is expected to remain bound to its
receptor during the processes of internalization and recyc-
Figure 6. Internalized transferrin co-localizes with human transferrin receptor (hTfR) and internalized FM4-64.
(a) Protoplasts expressing the hTfR were incubated in the presence of
transferrin–Alexa 546 (Tfn-546) as shown in Figure 5. Where indicated (BFA),
protoplasts were pre-incubated for 30 min in the presence of 200 lM
brefeldin A prior to the addition of transferrin. After the incubation,
protoplasts were analysed by immunofluorescence using the anti-CD71
antibody and an Alexa Fluor 488-labeled antirabbit IgG.
(b) Protoplasts expressing hTfR were incubated in the presence of transferrin–
Alexa 488 (Tfn-488) and FM4-64 for 15 min at 28C and then analysed using a
confocal microscope.
ling, we tested for co-localization between internalized Tfn
and hTfR, using Tfn–Alexa 546 and detecting hTfR by
immunofluorescence with an Alexa 488-labelled secondary
antibody. As shown in Figure 6(a), an almost complete colocalization was observed between internalized Tfn and
hTfR. In addition, we tested whether Tfn uses the same
endocytic route followed by the FM dye FM4-64. To this end,
protoplasts expressing hTfR were incubated for 15 min at
28C in the presence of FM4-64 and Tfn–Alexa 488 and
analysed by confocal microscopy. As shown in Figure 6(b),
internalized Tfn co-localized extensively with FM4-64.
Therefore, hTfR is functional in Arabidopsis protoplasts, and
can mediate binding of its natural ligand, Tfn, at the plasma
membrane, and its subsequent internalization into endosomal (Ara7- and FM4-64-positive) compartments.
Effect of tyrphostins A51 or A23 and BFA on transferrin–
Alexa 546 uptake
In animal cells, the internalization of Tfn and hTfR depends
on the interaction between the l2-subunit of the AP2 adaptor
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
762 Elena Ortiz-Zapater et al.
complex and a tyrosine-based internalization motif (YTRF) in
the hTfR cytosolic domain (Collawn et al., 1990, 1993). In
order to verify the functionality of the internalization motif of
hTfR in Arabidopsis protoplasts, we tested the effect of tyrphostin A23, a tyrosine analogue that has been shown to
inhibit Tfn internalization by interacting with the l2-subunit
of AP2 and therefore interfering with the interaction between
the YTRF motif and the AP2 complex (Banbury et al., 2003;
Crump et al., 1998). When protoplasts expressing hTfR were
pre-incubated with tyrphostin A23 for 15 min at 28C, before
the addition of Tfn–Alexa 546, there was an almost complete block in Tfn uptake. Under these conditions, Tfn was
found to bind to the plasma membrane (Figure 7c,d), as
observed upon incubation at 4C, but could not be detected
in intracellular compartments. Therefore, tyrphostin A23
does not prevent receptor–ligand binding, but inhibits
internalization of receptor–ligand complexes. As a control,
protoplasts expressing hTfR were pre-treated with tyrphostin A51, which is also a tyrosine analogue but has a third
hydroxyl group that prevents it from interacting with the
l2-subunit of AP2 (Banbury et al., 2003). In contrast to
tyrphostin A23, tyrphostin A51 did not have any discernible
effect on Tfn internalization (Figure 7a,b), which occurred
with similar efficiency as in the absence of tyrphostins
(Figure 5). Both tyrphostins A23 and A51 are efficient
inhibitors of tyrosine kinase activity, and both tyrphostins
were used at a concentration (350 lM) many times higher
than their IC50 for inhibition of epidermal growth factor
receptor tyrosine kinase activity (0.8 and 35 lM, respectively). The fact that only A23 inhibited Tfn internalization
indicates that it exerts its effect by a mechanism other than
inhibition of tyrosine kinase activity.
We next tested the effect of BFA, which has been shown to
inhibit recycling from endosomes back to the plasma
membrane in plant cells, and thus leads to accumulation
of rapidly recycling plasma membrane proteins in BFAinduced compartments (Baluska et al., 2002; Boonsirichai
et al., 2003; Geldner et al., 2001, 2003; Grebe et al., 2003).
In contrast to the punctate distribution observed after Tfn
internalization in untreated protoplasts, internalized Tfn
accumulated in larger patches upon BFA treatment, where
it co-localizes with hTfR (Figure 6a, BFA) and also with
internalized FM4-64 (data not shown). This suggests that
hTfR behaves in Arabidopsis protoplasts as other rapidly
recycling plasma membrane proteins do (Murphy et al.,
2005), and probably also accumulates in BFA compartments
upon inhibition of recycling.
Effect of tyrphostin A23 or BFA on transferrin receptor
distribution
The effects of tyrphostin A23 and BFA on Tfn internalization
suggest that hTfR cycles between the plasma membrane
and endosomal compartments in Arabidopsis protoplasts,
(a)
(b)
A51
(c)
(d)
A23
(e)
(f)
Figure 7. Effect of tyrphostin A23 on Tfn uptake and the subcellular distribution of hTfR in Arabidopsis protoplasts.
Protoplasts expressing hTfR were incubated in the presence of 350 lM
tyrphostin A51 (a and b) or tyrphostin A23 (c and d) for 15 min at 28C. Then,
transferrin–Alexa 546 was added, the protoplasts were incubated for 45 min
at 28C and analysed using a Leica confocal microscope. (e and f) Protoplasts
expressing hTfR were incubated in the presence of tyrphostin A23 for 2 h at
28C. After the incubation, protoplasts were washed, fixed and processed for
immunofluorescence using the anti-CD71 antibody and an Alexa Fluor 488labeled antimouse IgG. The left panels show the confocal images, while the
right panels are the transmission images.
as occurs in animal cells (for a review see Alarcón and
Fresno, 1998). Therefore, we investigated whether interfering with its internalization from the plasma membrane or its
recycling from endosomal compartments had a discernible
effect on the steady-state distribution of hTfR. As shown in
Figure 3(e,f), treatment of Arabidopsis protoplasts for 2 h at
28C in the presence of tyrphostin A23 led to a significant
increase in cell surface expression of hTfR, a possible consequence of the inhibition of hTfR internalization without
interfering with recycling to the plasma membrane. Immunofluorescence in permeabilized cells confirmed that treatment with A23 produces a redistribution of hTfR. In contrast
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
Receptor-mediated endocytosis in plant cells 763
to the situation in untreated protoplasts, where the majority
of hTfR was found in intracellular vesicular structures, a
significant fraction of hTfR was redistributed to the plasma
membrane after treatment with tyrphostin A23 (Figure 7e,f),
suggesting that the intracellular compartments where hTfR
is found in untreated protoplasts correspond to endosomal
compartments (as demonstrated by the co-localization with
Ara7 and FM4-64). In contrast, treatment of Arabidopsis
protoplasts with BFA leads to the accumulation of hTfR in
large patches in the cytoplasm, where it co-localizes with
internalized Tfn (Figure 6a, BFA), probably reflecting its
accumulation in BFA compartments. This is consistent with
the view that both receptor and ligand remain together
during their intracellular trafficking, and that interference
with the trafficking of the receptor has the same effect on the
trafficking of the ligand.
Effect of tyrphostin A23 and/or BFA on the internalization of
FM4-64
We next tested whether tyrphostin A23 caused a general
block in membrane internalization, which could be responsible for the inhibition of the internalization of Tfn and hTfR
by tyrphostin A23. To this end, we used the membrane
marker FM4-64. The marker was inserted into the plasma
membrane by 15 min incubation at 4C, and excess marker
was removed by washing. Protoplasts were then incubated
for 15 min on ice in the absence or the presence of tyrphostin A23, and then incubated for 45 min at 28C to allow
membrane internalization. Both in the control (Figure 8a)
and in the A23-treated protoplasts (Figure 8b), FM4-64 was
efficiently internalized into endosomal compartments. We
could not detect any discernible effect of A23 on FM4-64
internalization, in contrast with the effect it has on Tfn
uptake. We also tested the effect of tyrphostin A23 on the
BFA-induced internalization of FM4-64. In the presence of
brefeldin, FM4-64 was seen in internal structures with the
typical ring-shaped morphology reported for BFA compartments (Figure 8c). Again, the addition of tyrphostin A23 did
not have any discernible effect on the formation of these
structures, which were observed both in the absence and the
presence of tyrphostin A23 when BFA was present
(Figure 8d). Therefore, the effect of tyrphostin A23 on the
internalization of Tfn seems to be specific and does not
correlate with a pleiotropic defect in endocytosis.
hTfR interacts with a l-adaptin subunit from Arabidopsis
The results presented here suggest that hTfR can use the
endocytic machinery of Arabidopsis protoplasts, probably
via recruitment of cytosolic adaptins at the plasma membrane. In order to test this possibility, hTfR was immunoprecipitated from human fibroblasts and then incubated in
the presence of a cytosolic extract from Arabidopsis cells.
A23
Control
(a)
(b)
(c)
(d)
BFA
BFA + A23
Figure 8. Effect of tyrphostin A23 and/or brefeldin A on the internalization of
FM4-64.
Arabidopsis protoplasts were incubated for 15 min at 4C in the presence of
50 lM FM4-64. After removal of excess marker, viable protoplasts were
recovered by flotation and incubated with (c and d) or without (a and b)
200 lM BFA for 15 min at 4C. Where indicated, 350 lM tyrphostin A23 was
added (b and d) and the protoplasts were incubated for a further 15 min at 4C.
Finally, the protoplasts were incubated for 45 min at 28C to allow the
internalization of the dye, and analysed by confocal microscopy.
Figure 9. The human transferrin receptor (hTfR)recruits a l-adaptin subunit
from Arabidopsis cytosol.
The hTfR was immunoprecipitated from human fibroblasts and incubated in
the absence ()At cytosol) or presence of Arabidopsis cytosol, with (þA23) or
without (control) 350 lM tyrphostin A23. Immunoprecipitates were analysed
by Western blotting with an antibody against the hTfR or Arabidopsis
lA-adaptin.
Immunoprecipitates were then tested for the presence of
hTfR and l-adaptin by Western blot analysis with the CD71
antibody against hTfR or with an antibody against Arabidopsis lA-adaptin (Happel et al., 2004). As shown in Figure 9,
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
764 Elena Ortiz-Zapater et al.
the lA-adaptin antibody recognized a band of the expected
molecular weight in the immunoprecipate containing hTfR.
This l-adaptin subunit had not been recruited from human
fibroblasts, as no signal was detected when the immunoprecipitate was incubated in the absence of Arabidopsis
cytosol. In human cells, this interaction involves a tyrosinebased (YTRF) endocytosis signal, and is inhibited in the
presence of tyrphostin A23 (Banbury et al., 2003). Therefore,
we tested the effect of this drug in the interaction between
hTfR and Arabidopsis lA-adaptin. As shown in Figure 9, the
presence of tyrphostin A23 completely blocked the interaction between hTfR and l-adaptin. Altogether, these data
suggest that the tyrosine-based endocytosis signal in the
cytosolic tail of hTfR is functional in Arabidopsis protoplasts,
where it probably interacts with a l-adaptin subunit from
Arabidopsis cytosol in order to be recruited into CCVs at the
plasma membrane.
Discussion
Tyrosine-based sorting signals, adaptins and tyrphostins
Sorting of membrane proteins into CCVs depends on certain
motifs within their cytosolic domains. One such motif contains a critical tyrosine residue within the sequence YXX/,
where / represents a bulky hydrophobic residue (Marks
et al., 1996; Trowbridge et al., 1993). Tyrosine-based motifs
conforming to this consensus sequence can interact directly
with the medium (l)-chain subunits of heterotetrameric
adaptor complexes involved in several intracellular trafficking pathways (Conner and Schmid, 2003; Kirchausen, 1999;
Robinson, 2004). The l-subunit from all four adaptor complexes has been shown to interact with the YXX/ motif, with
the precise sequence and context of this motif determining
the specificity of the interaction (Dell’Angelica et al., 1997;
Stephens and Banting, 1998). These interactions are critically dependent on the tyrosine residue (Boll et al., 1996;
Ohno et al., 1995; Stephens et al., 1997). During RME, the
AP2 adaptor complex facilitates incorporation of transmembrane proteins containing YXX/ motifs into CCVs
formed at the plasma membrane. Examples of these motifs
are the YTRF endocytosis signal of the hTfR or the YQRL
motif of TGN38 (Kirchausen, 1999; Robinson, 2004).
Candidate molecules for RME in plant cells have been
shown to contain YXX/ motifs (Holstein, 2002), and, in one
case, the LeEix2 receptor for the fungal elicitor ethyleneinducing xylanase, a tyrosine-based motif has been demonstrated to function as a endocytosis signal (Ron and Avni,
2004). There are also a few examples in plants showing the
interaction between tyrosine-based motifs and l-adaptins.
As regards, lA-adaptin, one of the five l-adaptins from
Arabidopsis thaliana, has been shown to bind the consensus
tyrosine motif YXX/ from the pea vacuolar sorting receptor
(VSR) PS1, as well as from the mammalian TGN38 protein.
Moreover, the tyrosine residue was revealed to be crucial for
binding of the complete cytoplasmic tail of VSR PS1 to the
plant lA-adaptin (Happel et al., 2004). A YXX/ motif in a
member of the LRR subfamily of RPKs has also been shown
to bind lA-adaptin (Holstein, 2002).
In this study, we have shown that the hTfR, which contains
a tyrosine-based endocytosis signal (YTRF) in its cytosolic
tail, is able to interact with a l-adaptin subunit from
Arabidopsis cytosol. The fact that lA-adaptin is found
mainly at the trans-Golgi network in Arabidopsis cells under
steady-state conditions (Happel et al., 2004) is not in conflict
with our results. YXX/ motifs can interact with the l-subunit
of several adaptor complexes, although with preferences for
residues in the X position (Robinson, 2004). For instance, the
YQRL motif of TGN38, which binds Arabidopsis lA-adaptin
(Happel et al., 2004), has been shown to interact with the
l-subunit of AP1, AP2 and AP3 in animal cells, although it
shows the highest affinity towards l2-adaptin (Ohno et al.,
1995; Stephens and Banting, 1998). On the other hand, all of
the amino acids crucial for binding of the tyrosine motif are
highly conserved in the five plant l-adaptin sequences
(Happel et al., 2004). Although we can neither quantify the
relative affinity of the interaction, nor exclude interaction
with other l-adaptins present in the cytosolic extract, our
results clearly show that the hTfR has the ability to recruit
Arabidopsis lA-adaptin. Elucidation of the correct in vivo
binding partner can only be achieved by comparative
binding studies with the five plant l-adaptins. However,
the present results serve to demonstrate the conserved
features of the interaction between tyrosine-based motifs in
plasma membrane receptors and l-adaptin subunits.
The YXX/ internalization motif is remarkably similar to
sequences in which the tyrosine residue can be phosphorylated, and, once phosphorylated, bind to Src homology 2
(SH2) domains (Zhou et al., 1993). It is now clear that
although both tyrosine kinases and medium subunits of
adaptor complexes recognize essentially the same motif, the
two can be discriminated in that very few tyrosine-based
motifs that have been shown to interact with l-chains can
also act as substrates for tyrosine kinases (Chuang et al.,
1997; Shiratori et al., 1997; Stephens and Banting, 1997).
Furthermore, although both l-chains and tyrosine kinases
accommodate the tyrosine side chains as part of their
interaction with the YXX/ motif, there is no great similarity
between the YXX/-binding sites on l-chains and those on
tyrosine kinases (Owen and Evans, 1998).
Tyrphostins are structural analogues of tyrosine. They
were initially developed as substrate-competitive inhibitors
of the epidermal growth factor tyrosine kinase (Gazit et al.,
1989; Lyall et al., 1989; Yaish et al., 1988). Tyrphostins have
subsequently been used to investigate the physiological role
of many different tyrosine kinases. Some tyrphostins
have also been reported to inhibit endocytosis and autophagy (Holen et al., 1995) and vesicle formation from the
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
Receptor-mediated endocytosis in plant cells 765
trans-Golgi network (Austin and Shields, 1996), thus implying a possible role for tyrosine kinases in these processes.
Molecular modelling of tyrphostins into the l2 tyrosinebinding pocket has revealed that the phenyl ring of
tyrphostin A23 is accommodated within the tyrosine-binding
cleft in l2, whereas that of tyrphostin A51 is not (Banbury
et al., 2003). Tyrphostin A23 is 3,4-dihydroxylated on the
phenyl ring, while tyrphostin A51 is a 3,4,5-trihydroxyphenyl
compound. The reason for the failure of tyrphostins with
three-ring hydroxyl groups to inhibit the interaction between
YXX/ motifs and l2 results from the fact that the third
hydroxyl group would necessarily be forced into the hydrophobic part of the cleft (Banbury et al., 2003). Thus, it appears
that the tyrosine-binding cleft in l2 can accommodate a 3,4dihydroxy derivative, but not a 3,4,5-trihydroxy derivative, of
a phenyl ring. In fact, the addition of an extra hydroxyl group
in the 3-position of the phenyl ring is beneficial for the
interaction with l2. Thus, a 3,4-dihydroxyphenyl compound
(such as tyrphostin A23) is predicted, by molecular modelling studies, to both fit well in the tyrosine-binding cleft of l2
and be stabilized in that binding by hydrogen bonding and
other interactions (Banbury et al., 2003).
By inhibiting the interaction between l2 and YXX/ motifs,
tyrphostins (in particular A23) are potentially very useful and
specific inhibitors of RME. Indeed, tyrphostin A23 (but not
tyrphostin A51) specifically inhibits internalization of the
hTfR both in animal cells (Banbury et al., 2003) and in
Arabidopsis protoplasts (this paper), as well as the internalization of TGN38 (Banbury et al., 2003), which also depends
on a tyrosine-based endoytosis signal (YQRL) to be included
in CCVs at the plasma membrane. In contrast, tyrphostin A23
has no effect in fluid-phase endocytosis (as monitored by the
internalization of fluorescent dextran; Banbury et al., 2003),
or in internalization of the lipid probe FM4-64 (Figure 8).
Interfering with the ability of the l2-subunit of AP2 to
interact with sorting signals in plasma membrane receptors
should not affect clathrin-coated pit formation, as it has been
shown that the formation of clathrin-coated pits and vesicles
is independent of receptor internalization signal levels
(Santini and Keen, 1996; Santini et al., 1998). In addition,
AP2 depletion in mammalian tissue culture cells has been
shown to block uptake of the Tfn receptor but not that of the
EGF or LDL receptors (Hinrichsen et al., 2003; Motley et al.,
2003). Therefore, tyrphostin A23 should specifically interfere
with sorting of hTfR into CCVs, as suggested by the
inhibition by tyrphostin A23 of the interaction between hTfR
and Arabidopsis lA-adaptin. As candidate molecules for
RME in plant cells also contain YXX/ motifs (possibly
working as endocytosis signals; Holstein, 2002), it is conceivable that tyrphostin A23 can also be used as an specific
inhibitor for RME in plant cells, at least for the internalization
of proteins bearing these motifs (Aniento and Robinson,
2005). Tyrphostin A23 (and perhaps other tyrphostins) may
also inhibit other membrane traffic events dependent on
tyrosine-based sorting motifs and l-adaptins. One likely
candidate would be transport of the VSR, which depends on
the interaction between its YXX/ motif and lA-adaptin
(Happel et al., 2004).
Trafficking of the human transferrin receptor in Arabidopsis
protoplasts
Our results show that hTfR expressed in Arabidopsis protoplasts forms a dimer and is transported to the plasma
membrane, where it is correctly oriented, its extracellular
epitope being accessible in non-permeabilized cells to an
antibody raised against this epitope. To perform its expected
function, receptor molecules at the plasma membrane
should bind its ligand, Tfn, and be internalized into endosomal compartments. Experiments using fluorescently labelled Tfn show that Tfn does indeed first bind to the plasma
membrane of transfected protoplasts, when the incubation
is carried out at 4C, a condition that allows receptor–ligand
binding but not plasma membrane internalization. Upon
incubation at 28C, Tfn is internalized into intracellular
compartments, where it co-localizes extensively with the Tfn
receptor.
hTfR has been previously expressed in Saccharomyces
cerevisiae, and has been used as a model for heterologous
expression of a membrane protein in yeast (Terng et al.,
1998). Although the protein is functional and can bind Tfn
in vitro, the major part of the expressed TfR in yeast is
localized in the endoplasmic reticulum, probably because of
its inability to be transported from the ER to the plasma
membrane (Prinz et al., 2003). Immunofluorescence in
Arabidopsis protoplasts shows that, under steady-state
conditions, most of hTfR is found in endosomal compartments and not in the ER. This suggests that, once synthesized in the ER, hTfR is properly targeted to the plasma
membrane of Arabidopsis protoplasts. The presence of
most of the hTfR in endosomal compartments may reflect
the kinetics of internalization and recycling of hTfR, which in
human cells has a constitutive role in Tfn recycling (Ciechanover et al., 1983; Damke et al., 1994; Mellman, 1996;
Sheff et al., 1999, 2002; Warren et al., 1997). Using radiolabelled Tfn to measure the proportion of hTfRs on the surface
of HeLa cells, it has been estimated that 80% of the receptor
resides in intracellular membranes, and only 20% is at the
plasma membrane under steady-state conditions (Damke
et al., 1994; Warren et al., 1997, 1998). Experiments with
non-permeabilized cells show that, indeed, a fraction of the
receptor is present at the plasma membrane under steadystate conditions, where it can bind its natural ligand Tfn or
the anti-CD71 antibody.
The effects of tyrphostin A23 and BFA on the distribution
of Tfn and hTfR in Arabidopsis protoplasts, as well as the colocalization with Ara7 or FM4-64, suggest that hTfR is mainly
found in endosomal compartments under steady-state con-
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
766 Elena Ortiz-Zapater et al.
ditions, and that hTfR cycles between the plasma membrane
and endosomes, as occurs in animal cells (Ciechanover
et al., 1983; Damke et al., 1994; Mellman, 1996; Sheff et al.,
1999, 2002; Warren et al., 1997). Interfering with internalization (upon A23 treatment) causes accumulation of the
receptor at the plasma membrane, suggesting that recycling
remains unaffected. This is in agreement with kinetic
measurements for internalization and recycling of hTfR,
which show that nearly half of the internalized hTfR recycles
with a half-life of about 1.5 min (Mayor et al., 1993), and that
the exchange of membrane between the plasma membrane
and the endosomes is very extensive (Ciechanover et al.,
1983; Damke et al., 1994; Hao and Maxfield, 2000; Murphy
et al., 2005; Sheff et al., 1999). A similar shift in the distribution of hTfR to the plasma membrane has been reported in
dynamin mutants (Damke et al., 1994) or after saturation of
TfR endocytosis by hTfR over-expression (Warren et al.,
1997, 1998). In contrast, interfering with recycling (upon BFA
treatment) causes accumulation of the receptor in intracellular compartments. A similar effect has been observed after
Nef expression: Nef reduces the rate of recycling of hTfR to
the plasma membrane, causing hTfR to accumulate in EEs
and reducing its expression at the cell surface (Madrid et al.,
2005).
(Gruenberg, 2001). While Ara6-rich endosomes are likely to
be located at a later stage of the endocytic pathway (probably equivalent to multi-vesicular bodies and/or late endosomes in animal cells; for a review see Marcote et al., 2000),
Ara7/Rha1-rich endosomes probably represent an earlier
compartment involved in recycling to the plasma membrane
(Ueda et al., 2004). Indeed, our data show extensive colocalization of hTfR with Ara7. However, it is not clear whether plant cells contain a recycling compartment that is different to sorting endosomes. hTfR could therefore be used
to monitor transit through different endosomal compartments in the recycling (not the degradative) part of the
endocytic pathway. In this respect, pulse–chase experiments
with fluorescent Tfn and double-labelling with specific Rab
GTPases could be used to further dissect the pathway.
In conclusion, the use of the hTfR, together with specific
inhibitors of internalization (tyrphostin A23) or recycling
(BFA), provides very valuable tools to explore and characterize RME and the recycling pathway in plant cells. In
addition, specific tyrphostins may also be useful to investigate the internalization of endogenous receptors and to
characterize other membrane trafficking events that are
dependent on tyrosine-based sorting signals.
Experimental procedures
hTfR as a marker to characterize receptor-mediated endocytosis and recycling to the plasma membrane in plant cells
The results presented here suggest that the hTfR expressed
in Arabidopsis protoplasts makes use of the same endocytic
machinery as in animal cells, most probably by using its
well-characterized YTRF internalization signal to recruit
cytosolic adaptins from plant cells. Heterologous interactions between sorting signals and coat proteins have been
already shown, such as the interaction between the cytosolic
tail of mammalian TGN38 and Arabidopsis lA-adaptin
(Happel et al., 2004), or the interaction between yeast and
human cytosolic tails containing di-lysine motifs and plant
coatomer (Contreras et al., 2004). Therefore, hTfR may be a
valuable tool to study RME in plant cells. An advantage of
using hTfR as a reporter molecule is the ability to monitor
trafficking using the natural ligand Tfn. While fluorescent
derivatives of Tfn are useful to follow its intracellular traffic,
radioactively labelled Tfn may be used for biochemical
assays. Tfn is endocytosed via a constitutively internalized
receptor and remains bound to the receptor. The route
followed by hTfR is well established in animal cells, and
involves passage through early and recycling endosomes.
Indeed, hTfR has been extensively used as a marker to
dissect trafficking along the endocytic pathway in animal
cells, in particular through the recycling pathway. Ueda et al.
(2004) have suggested the existence of different subpopulations of endosomes in plant cells, based on their different
composition in Rab GTPases, as is the case in animal cells
Media and solutions
Proto medium (per l). 30 ml stock A (65.5 g l)1 KNO3; 4.4 g l)1
CaCl2Æ2H2O; 3.7 g l)1 MgSO4Æ7H2O; 1.7 g l)1 KH2PO4); 0.3 ml
stock B (6.2 g l)1 H3BO3; 22.3 g MnSO4Æ4H2O; 10.6 g l)1 ZnSO4Æ7H2O; 0.83 g l)1 KI; 0.25 g l)1 Na2MoO4Æ2H2O; 0.025 g l)1 CoCl2;
0.025 g l)1 CuSO4Æ5H2O); 2 ml stock C (2.78 g l)1 FeSO4Æ7H2O;
3.72 g l)1 Na2EDTAÆ2H2O); 1 ml stock VT (0.5 g l)1 nicotinic
acid; 0.5 g l)1 pyridoxine HCl; 0.4 g l)1 thiamine HCl); 0.1 g myoinositol, 154 g sucrose; adjusted to pH 5.7 with KOH and autoclaved.
PEG solution (per 100 ml). 25 g PEG-6000, 8.2 g mannitol,
2.36 g Ca(NO3)2Æ4H2O; adjusted to pH 9 with a fresh 0.1 N NaOH
solution (stored in aliquots at )20C and readjusted to pH 9 just
before use); filter-sterilized through a 0.22 lM nitrocellulose filter.
Ca(NO3)2 solution (per l). 64.9 g Ca(NO3)2Æ4H2O; adjusted to pH
5.7–6.0 with KOH and autoclaved.
W5 medium. 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose; adjusted to pH 5.7 or 7.0 and autoclaved.
FM4-64- and Alexa-labelled transferrin. FM4-64 (Invitrogen
S.A., Barcelona, Spain) was stored as a 2 mM stock solution in Me2SO
at )20C and added to solutions just before use. Lyophilized Alexa
488- or 546-labeled Tfn (Invitrogen S.A.) was reconstituted with bidistilled water to give a final concentration of 5 mg ml)1 in PBS, and
added to protoplasts in Proto medium just before use.
Tyrphostins and BFA. Tyrphostins A51 and A23 (Sigma-Aldrich)
were stored as 350 mM (1000-fold) stock solutions in Me2SO at
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
Receptor-mediated endocytosis in plant cells 767
)20C and added to solutions just before use. BFA was stored as a
5 mg ml)1 stock solution in MeOH at )20C.
Lysis buffer. 25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM
EDTA, 10% glycerol, 1% Triton X-100.
Antibodies
Monoclonal and polyclonal antibodies against hTfR were obtained
from Abcam (Cambridge, UK) and Santa Cruz Biotechnology
(Santa Cruz, CA, USA), respectively. The antibodies against lAadaptin (Happel et al., 2004), Ara7 (Ueda et al., 2001) and BiP
(Pedrazzini et al., 1997) were generous gifts from Susanne Holstein (University of Heidelberg, Germany), Takashi Ueda (University of Tokyo, Japan) and Alessandro Vitale (Instituto di
Biologia e Biotenologia Agraria, Milan, Italy), respectively.
Construction of pDH51-TR
The coding region of the hTfR was cloned into the plant expression
vector pDH51 cut with XbaI and SalI. Previously, the cDNA of TR
(contained in the pGEM-TR-T7 vector) was cloned into the pBK-CMV
vector (Stratagene, La Jolla, CA, USA) to generate XbaI and SalI
restriction sites at the 5¢- and 3¢-ends of the TR cDNA, respectively.
Protoplast isolation and PEG-mediated transfection
Protoplasts were isolated from A. thaliana (LT87) cell suspension
cultures as previously described (Axelos et al., 1992). For transfection of protoplasts, 100 ll of protoplast suspension, containing 106
protoplasts in Proto medium, were mixed with 30–50 lg DNA from
the pDH51-TR (or pDH51) plasmid and 4 lg of pDH51-LUC DNA as
an internal control. PEG solution (200 ll) was added drop by drop to
the side of 15 ml conical centrifuge tubes, with gentle swirling in
order to avoid a rapid increase of PEG concentration in the medium.
The mixture was incubated at room temperature for 20 min, then
diluted with 5 ml of Ca(NO3)2 solution in order to stop the PEG
treatment. After a thorough mixing, the suspension was incubated
for an additional 10 min and centrifuged at 80 g for 5 min. The
supernatant was discarded and the protoplast pellet was gently
resuspended in 1 ml of Proto medium supplemented with 5 lM
NAA and 1 lM kinetin. The centrifuge tube was maintained in a
horizontal position and the suspension was incubated at 20C in the
dark. At the end of the expression period, usually 20–22 h, each
sample was thoroughly mixed with 1.5 ml of W5 medium and
centrifuged at 100 g for 10 min. The protoplast pellets were washed
again twice with W5 medium, and finally resuspended in the desired volume of W5 or Proto medium. An aliquot of the transfected
protoplasts was extracted in lysis buffer and used to measure luciferase activity. All transient expression experiments were repeated at least three times with similar results.
Internalization of Alexa-labeled transferrin or FM4-64
Protoplasts (5 · 105 to 5 · 106) were incubated in 200 ll of Proto
medium in the absence or presence of 350 lM tyrphostins A23 or
A51 or 200 lM BFA for 15 min at 28C. Then, Tfn–Alexa Fluor 546
(or 488; final concentration: 0.5 mg ml)1) or FM4-64 (final concentration: 50 lM) were added and the protoplasts were incubated
for 1 h at 4C or 28C. After the incubation, protoplasts were
washed three times with W5 medium and analysed by confocal
microscopy.
Cell surface expression of the human transferrin receptor
To test for cell surface expression of the hTfR, control or transfected
protoplasts were treated with 350 mM tyrphostin A23 for 1 h at 28C,
or left untreated. They were then incubated overnight at 4C with a
polyclonal antibody against an extracellular epitope of the hTfR
(CD71; 10 lg ml)1 in Proto medium). After three washes in W5
medium, protoplasts were incubated for 2 h at 4C with an Alexa
Fluor 488-conjugated antirabbit IgG (10 lg ml)1 in Proto medium),
washed again in W5 medium and analysed by confocal microscopy.
Immunofluorescence
Arabidopsis protoplasts transfected with the hTfR were fixed with
1% paraformaldehyde and 0.2% glutaraldehyde in W5 medium for
1 h at room temperature under gentle agitation. Fixed protoplasts
were allowed to settle onto poly-L-lysine-coated multi-well slides for
30 min at room temperature, washed three times in PBS, and then
incubated overnight in the presence of freshly prepared 0.1% NaBH4
in PBS to permeabilize cells and reduce autofluorescence. The protoplasts were then incubated in blocking solution (1% BSA/PBS) for
1 h at 37C, and then for 2 h at 37C in the presence of a 1/50 dilution
of the primary antibodies in 0.1% BSA/PBS. After three washes in
PBS, the protoplasts were incubated in the dark for 2 h at 37C with
Alexa-Fluor 488 or 546 goat antirabbit or antimouse immunoglobulin G (Invitrogen S.A.) diluted 1/100 in 0.1% BSA/PBS, washed four
times in PBS and mounted in Slow Fade mounting medium without
glycerol (Molecular Probes Europe) before observation.
Confocal laser scanning microscopy
Protoplasts were observed with a Leica TCS-SP confocal microscope equipped with argon ion, krypton and helium–neon lasers
(Leica, Heidelberg, Germany). Images were acquired with a 1.4
numerical aperture · 100 oil-immersion HCX PL APO CS 100
objective. Alexa Fluor 488 was excited with the 488 nm argon laser
line, and confocal sections were collected using a 510–580 nm
emission setting. Alexa Fluor 546 was excited with the 543 nm laser
line, and confocal sections were collected using a 560–620 nm
emission setting. FM4-64 was excited using the 543 nm laser line,
and confocal sections were collected using a 650–850 nm emission
setting. For co-localization studies, confocal images were acquired
serially and overlaid using the Leica confocal software. In all cases,
protoplasts transfected with the empty vector (and therefore not
expressing the hTfR) were analysed in parallel, to verify the specificity of labelling, including the experiments on Tfn internalization,
cell surface expression of hTfR and immunofluorescence. In the
latter, control protoplasts were incubated with both the primary and
the secondary antibodies, and the samples analysed in the confocal
microscope to verify the absence of labelling under the same conditions used to analyse the protoplasts expressing hTfR. Control
wells in which the primary antibody was omitted were also included
for every experiment.
Immunoprecipitation of the human transferrin receptor and
interaction with an Arabidopsis l-adaptin subunit
The polyclonal antibody against the hTfR (CD71) was covalently
linked to AminoLink Plus gel (ProFoundTM co-immunoprecipitation kit; Pierce, Rockford, IL, USA) following the instructions from
the manufacturer, and incubated with an extract from human
fibroblasts (0.5 mg protein and 20 ll of antibody-coupled gel in
0.5 ml lysis buffer per point). Immunoprecipitates were washed four
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
768 Elena Ortiz-Zapater et al.
times with lysis buffer and incubated in the absence or presence of
Arabidopsis cytosol (2 mg ml)1), with or without 350 lM tyrphostin
A23. Beads were washed four times with lysis buffer and analysed
by Western blot analysis with an antibody against hTfR or lAadaptin (Happel et al., 2004).
Acknowledgements
We thank Erwin Knecht (Centro de Investigacion principe Felipe,
Valencia, Spain) and David G. Robinson (University of Heidelberg,
Germany) for critically reading the manuscript. The pGEM-TR-T7
vector that contains the cDNA of the human transferrin receptor was
a generous gift from Dr Marino Zerial (Max Planck Institute of
Molecular Cell Biology and Genetics, Dresden, Germany). We thank
Takashi Ueda (Tokyo, Japan), Susanne Holstein (Heidelberg,
Germany) and Alessandro Vitale (Instituto di Biologia e Biotecnologia Agraria, Milan, Italy) for their generous gifts of antibodies. We
acknowledge Dr Dolors Ludevid (Institut de Biologia Molecular,
Barcelona, Spain) for the generous gift of Arabidopsis cell suspension cultures (LT87) and the pDH51 vector. We thank Enrique
Navarro (Servei Central de suport a la Investigació Experimental
(SCSIE), Universidad de Valencia) for his expert advice with the
confocal microscope. This work was supported by grants from the
Ministerio de Ciencia y Tecnologı́a (grant numbers BMC2002-03993
and BFU2005-00071) to F.A. E.O.-Z. is a research fellow of the
Ministerio de Ciencia y Tecnologı́a (Beca F.P.U.). M.J.M. has a
contract from the Ministerio de Ciencia y Tecnologı́a (Contrato
Ramón y Cajal). D.O.-M. is a research fellow of the Universidad de
Valencia (Beca V Segles).
References
Alarcón, B. and Fresno, M. (1998) Transferrin receptor (CD71). In
Encyclopedia of Immunology (Delves, P.J. and Roitt, I.M., eds).
London: Academic Press, pp. ??–??.
Aniento, F. and Robinson, D.G. (2005) Testing for endocytosis in
plants. Protoplasma (in press).
Aniento, F., Helms, B. and Memon, A. (2003) How to make a vesicle:
coat protein–membrane interactions. In The Golgi Apparatus and
the Plant Secretory Pathway (Robinson, D.G., ed.). Oxford:
Blackwell Publishing, pp. 39–62.
Austin, C.D. and Shields, D. (1996) Formation of nascent secretory
vesicles from the trans-Golgi network of endocrine cells is
inhibited by tyrosine kinase and phosphatase inhibitors. J. Cell
Biol. 135, 1471–1483.
Axelos, M., Curie, C., Mazzolini, L., Bardet, C. and Lescure, B. (1992)
A protocol for transient gene expresión in Arabidopsis thaliana
protoplasts isolated from cell suspension cultures. Plant Physiol.
Biochem. 30, 123–128.
Bahaji, A., Cornejo, M.J., Ortiz-Zapater, E., Contreras, I. and
Aniento, F. (2001) Uptake of endocytic markers by rice cells: variations related to the growth phase. Eur. J. Cell Biol. 80, 178–186.
Bahaji, A., Aniento, F. and Cornejo, M.J. (2003) Uptake of an endocytic marker by rice cells: variations related to osmotic and saline
stress. Plant Cell Physiol. 44, 1100–1111.
Baluska, F., Hlavacka, A., Samaj, J., Palme, K., Robinson, D.G.,
Matoh, T., McCurdy, D.W., Menzel, D. and Volkmann, D. (2002) Factin-dependent endocytosis of cell wall pectins in meristematic
root cells. Insights from brefeldin A-induced compartments. Plant
Physiol. 130, 422–431.
Baluska, F., Samaj, J., Hlavacka, A., Kendrick-Jones, J. and
Volkmann, D. (2004) Actin-dependent fluid-phase endocytosis in
inner cortex cells of maize root apices. J. Exp. Bot. 55, 463–473.
Banbury, D.N., Oakley, J.D., Sessions, R.B. and Banting, G. (2003)
Tyrphostin A23 inhibits internalization of the transferrin receptor
by perturbing the interaction between tyrosine motifs and the
medium chain subunit of the AP-2 adaptor complex. J. Biol.
Chem. 278, 12022–12028.
Barth, M. and Holstein, S.E. (2004) Identification and functional
characterization of Arabidopsis AP180, a binding partner of plant
alphaC-adaptin. J Cell Sci. 117, 2051–2062.
Battey, N.H., James, N.C., Greenland, A.J. and Brownlee, C. (1999)
Exocytosis and endocytosis. Plant Cell, 11, 643–659.
Beevers, L. (1996) Clathrin-coated vesicles in plants. Int. Rev. Cytol.
167, 1–35.
Blackbourn, H.D. and Jackson, A.P. (1996) Plant clathrin heavy
chain. Sequence analysis and restricted localisation in growing
pollen tubes. J. Cell Sci. 109, 777–787.
Boehm, M. and Bonifacino, J.S. (2001) Adaptins: the final recount.
Mol. Biol. Cell, 12, 2907–2920.
Boll, W., Ohno, H., Songyang, Z., Rapoport, I., Cantley, L.C.,
Bonifacino, J.S. and Kirchhausen, T. (1996) Sequence requirements for the recognition of tyrosine-based endocytic signals by
clathrin AP-2 complexes. EMBO J. 15, 5789–5795.
Bonifacino, J.S. and Traub, L.M. (2003) Signals for sorting of
transmembrane proteins to endosomes and lysosomes. Annu.
Rev. Biochem. 72, 395–447.
Boonsirichai, K., Sedbrook, J.C., Chen, R., Gilroy, S. and Masson,
P.H. (2003) Altered response to gravity is a peripheral membrane
protein that modulates gravity-induced cytoplasmic alkalinization
and lateral auxin transport in plant statocytes. Plant Cell, 15,
2612–2625.
Chuang, E., Alegre, M.L., Duckett, C.S., Noel, P.J. and VanderHeiden,
M.G. (1997) Interaction of CTLA-4 with the clathrin-associated
protein AP50 results in ligand-independent endocytosis that limits
cell surface expression. J. Immunol. 159, 144–151.
Ciechanover, A., Schwartz, A.L., Dautry-Varsat, A. and Lodish, H.F.
(1983) Kinetics of internalization and recycling of transferrin and
the transferrin receptor in a human hepatoma cell line. J. Biol.
Chem. 258, 9681–9689.
Collawn, J.F., Stangel, M., Kuhn, L.A., Esekogwu, V., Jing, S.Q.,
Trowbridge, I.S. and Tainer, J.A. (1990) Transferrin receptor
internalization sequence YXRF implicates a tight turn as the
structural recognition motif for endocytosis. Cell, 63, 1061–1072.
Collawn, J.F., Lai, A., Domingo, D., Fitch, M., Hatton, S. and
Trowbridge, I.S. (1993) YTRF is the conserved internalization
signal of the transferrin receptor, and a second YTRF signal at
position 31–34 enhances endocytosis. J. Biol. Chem. 268, 21686–
21692.
Conner, S.D. and Schmid, S.L. (2003) Regulated portals of entry into
the cell. Nature, 422, 37–44.
Contreras, I., Ortiz-Zapater, E. and Aniento, F. (2004) Sorting signals
in the cytosolic tail of membrane proteins involved in the interaction with plant ARF1 and coatomer. Plant J. 38, 685–698.
Crump, C., Williams, J.L., Stephens, D.J. and Banting, G. (1998)
Inhibition of the interaction between tyrosine-based motifs and
the medium chain subunit of the AP-2 adaptor complex by specific tyrphostins. J. Biol. Chem. 273, 28073–28077.
Damke, H., Baba, T., Warnock, D.E. and Schmid, S.L. (1994) Induction of mutant dynamin specifically blocks endocytic coated
vesicle formation. J. Cell Biol. 127, 915–934.
Daro, E., Van der Sluijs, P., Galli, T. and Mellman, I. (1996) Rab4 and
cellubrevin define different early endosome populations on the
pathway of transferrin receptor recycling. Proc. Natl Acad. Sci.
USA, 93, 9559–9564.
Dautry-Varsat, A. and Lodish, H.F. (1984) How receptors bring proteins and particles into cells. Sci. Am. 250, 48–54.
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
Receptor-mediated endocytosis in plant cells 769
Dell’Angelica, E.C., Ohno, H., Ooi, C.E., Rabinovich, E., Roche, K.W.
and Bonifacino, J.S. (1997) AP-3: an adaptor-like protein complex
with ubiquitous expression. EMBO J. 16, 917–928.
Emans, N., Zimmermann, S. and Fischer, R. (2002) Uptake of a
fluorescent marker in plant cells is sensitive to brefeldin A and
wortmannin. Plant Cell, 14, 71–86.
Gazit, A., Yaish, P., Gilon, C. and Levitzki, A. (1989) Tyrphostins: I.
Synthesis and biological activity of protein tyrosine kinase
inhibitors. J. Med. Chem. 32, 2344–2352.
Geldner, N., Friml, J., Stierhof, Y.D., Jurgens, G. and Palme, K.
(2001) Auxin transport inhibitors block PIN1 cycling and vesicle
trafficking. Nature, 413, 425–428.
Geldner, N., Anders, N., Wolters, H., Keicher, J., Kornberger, W.,
Muller, P., Delbarre, A., Ueda, T., Nakano, A. and Jurgens, G.
(2003) The Arabidopsis GNOM ARF-GEF mediates endosomal
recycling, auxin transport, and auxin-dependent plant growth.
Cell, 112, 219–230.
Grebe, M., Xu, J., Mobius, W., Ueda, T., Nakano, A., Geuze, H.J.,
Rook, M.B. and Scheres, B. (2003) Arabidopsis sterol endocytosis
involves actin-mediated trafficking via ARA6-positive early
endosomes. Curr. Biol. 13, 378–387.
Gruenberg, J. (2001) The endocytic pathway: a mosaic of domains.
Nat. Rev. Mol. Cell Biol. 2, 721–730.
Hao, M. and Maxfield, F.R. (2000) Characterization of rapid membrane internalization and recycling. J. Biol. Chem. 275,
15279–15286.
Happel, N., Honing, S., Neuhaus, J.M., Paris, N., Robinson, D.G. and
Holstein, S.E.H. (2004) Arabidopsis muA-adaptin interacts with
the tyrosine motif of the vacuolar sorting receptor VSR-PS1. Plant
J. 37, 678–693.
Hinrichsen, L., Harborth, J., Andrees, L., Weber, K. and Ungewickell,
E.J. (2003) Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and
receptor trafficking in HeLa cells. J. Biol. Chem. 278, 45160–45170.
Holen, I., Stromhaug, P.E., Gordon, P.B., Fengsrud, M., Berg, T.O.
and Seglen, P.O. (1995) Inhibition of autophagy and multiple
steps in asialoglycoprotein endocytosis by inhibitors of tyrosine
protein kinases (tyrphostins). J. Biol. Chem. 270, 12823–12831.
Holstein, S.E.H. (2002) Clathrin and plant endocytosis. Traffic, 3,
614–620.
Homann, U. and Thiel, G. (1999) Unitary exocytotic and endocytotic
events in guard-cell protoplasts during osmotically driven volume changes. FEBS Lett. 460, 495–499.
Jing, S.Q. and Trowbridge, I.S. (1987) Identification of the intermolecular disulfide bonds of the human transferrin receptor and
its lipid-attachment site. EMBO J. 6, 327–331.
Kawchuk, L.M., Hachey, J., Lynch, D.R. et al. (2001) Tomato Ve
disease resistance genes encode cell surface-like receptors. Proc.
Natl Acad. Sci. USA 98, 6511–6515.
Kirchausen, T. (1999) Adaptors for clathrin-mediated traffic. Annu.
Rev. Cell Biol. 15, 705–732.
Kirchausen, T. (2000) Clathrin. Annu. Rev. Biochem. 69, 699–727.
Low, P.S. and Chandra, S. (1994) Endocytosis in plants. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 45, 609–631.
Lyall, R.M., Zilberstein, A., Gazit, A., Gilon, C., Levitzki, A. and
Schlessinger, J. (1989) Tyrphostins inhibit epidermal growth
factor (EGF)-receptor tyrosine kinase activity in living cells and
EGF-stimulated cell proliferation. J. Biol. Chem. 264, 14503–
14509.
Madrid, R., Janvier, K., Hitchin, D., Day, J., Coleman, S., Noviello, C.,
Bouchet, J., Benmerah, A., Guatelli, J. and Benichou, S. (2005)
Nef-induced alteration of the early/recycling endosomal compartment correlates with enhancement of HIV-1 infectivity. J. Biol.
Chem. 280, 5032–5044.
Maldonado-Mendoza, I.E. and Nessler, C.L. (1996) Cloning and
expression of a plant homologue of the Golgi-associated clathrin
assembly protein AP19 from Camtotheca acuminata. Plant Mol.
Biol. 32, 1149–1153.
Marcote, M.J., Gu, F., Gruenberg, J. and Aniento, F. (2000) Membrane transport in the endocytic pathway: animal versus plant
cells. Protoplasma, 210, 123–132.
Marks, M.S., Woodruff, L., Ohno, H. and Bonifacino, J.S. (1996)
Protein targeting by tyrosine- and di-leucine-based signals: evidence for distinct saturable components. J. Cell Biol. 135,
341–354.
Mayor, S., Presley, J.F. and Maxfield, F.R. (1993) Sorting of membrane components from endosomes and subsequent recycling to
the cell surface occurs by a bulk flow process. J. Cell Biol. 121,
1257–1269.
McClelland, A., Kühn, L.C. and Ruddle, F.H. (1984) The human
transferrin receptor gene: genomic organization, and the complete primary structure of the receptor deduced from a cDNA
sequence. Cell, 39, 267–274.
Meckel, T., Hurst, A.C., Thiel, G. and Homann, U. (2004) Endocytosis
against high turgor: intact guard cells of Vicia faba constitutively
endocytose fluorescently labelled plasma membrane and GFPtagged Kþ-channel KAT1. Plant J. 39, 182–193.
Mellman, I. (1996) Endocytosis and molecular sorting. Annu. Rev.
Cell Dev. Biol. 12, 575–625.
Motley, A., Bright, N.A., Seaman, M.N. and Robinson, M.S. (2003)
Clathrin-mediated endocytosis in AP-2-depleted cells. J. Cell Biol.
162, 909–918.
Murphy, A.S., Bandyopadhyay, A., Holstein, S.E.H. and Peer, W.A.
(2005) Endocytotic cycling of PM proteins. Annu. Rev. Plant Biol.
56, 221–251.
Ohno, H., Stewart, J., Fournier, M.-C., Bosshart, H., Rhee, I.,
Miyatake, S., Saito, T., Gallusser, A., Kirchhausen, T. and
Bonifacino, J.S. (1995) Interaction of tyrosine-based sorting
signals with clathrin-associated proteins. Science, 269, 1872–
1875.
Owen, D.J. and Evans, P.R. (1998) A structural explanation for the
recognition of tyrosine-based endocytotic signals. Science, 282,
1327–1332.
Pedrazzini, E., Giovinazzo, G., Bielli, A., de Virgilio, M., Frigerio, L.,
Pesca, M., Faoro, F., Bollini, R., Ceriotti, A. and Vitale, A. (1997)
Protein quality control along the route to the plant vacuole. Plant
Cell, 9, 1869–1880.
Prinz, B., Stahl, U. and Lang, C. (2003) Intracellular transport of a
heterologous membrane protein, the human transferrin receptor,
in Saccharomyces cerevisiae. Int. Microbiol. 6, 49–55.
Robatzek, S., Chinchilla, D. and Boller, T. (2006) Ligand-induced
endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. Genes Dev. 20, 537–542.
Robinson, M.S. (2004) Adaptable adaptors for coated vesicles.
Trends Cell Biol. 14, 167–174.
Robinson, D.G., Hinz, G. and Holstein, S.E.H. (1998) The molecular
characterization of transport vesicles. Plant Mol. Biol. 38, 47–76.
Roca, R., Stiefel, V. and Puigdomenech, P. (1998) Characterization of
the sequence coding for the clathrin coat assembly protein AP17
(sigma 2) associated with the plasma membrane from Zea mays
and constitutive expression of its gene. Gene, 208, 67–72.
Ron, M. and Avni, A. (2004) The receptor for fungal elicitor ethyleneinducing xylanase is a member of a resistance-like family in tomato. Plant Cell, 16, 1604–1615.
Russinova, E., Borst, J.W., Kwaaitaal, M., Cano-Delgado, A., Yin, Y.,
Chory, J. and De Vries, S.C. (2004) Heterodimerization and
endocytosis of Arabidospis brassinosteroid receptors BRI1 and
AtSERK3 (BAK1). Plant Cell, 16, 3216–3229.
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770
770 Elena Ortiz-Zapater et al.
Santini, F. and Keen, J.H. (1996) Endocytosis of activated receptors
and clathrin-coated pit formation: deciphering the chicken or egg
relationship. J. Cell Biol. 132, 1025–1036.
Santini, F., Marks, M.S. and Keen, J.H. (1998) Endocytic clathrincoated pit formation is independent of receptor internalization
signal levels. Mol. Biol. Cell, 9, 1177–1194.
Scheele, U. and Holstein, S.E.H. (2002) Functional evidence for the
identification of an Arabidopsis clathrin light chain polypeptide.
FEBS Lett. 514, 355–360.
Schledzewski, K., Brinkmann, H., LaBrie, S.T., Crawford, N.M. and
Mendel, R.R. (1997) Gamma-adaptin from Arabidopsis thaliana:
molecular cloning and characterization of the gene. In Current
Topics in Plant Biochemistry, Physiology and Molecular Biology
(16th Annual Missouri Symposium) (Baskin, T. and Rogers, J.C.,
eds). Columbia: University of Missouri, pp. 59–60.
Schmid, S.L. (1997) Clathrin-coated vesicle formation and protein
sorting: an integrated process. Annu. Rev. Biochem. 66, 511–548.
Schneider, C., Sutherland, R., Newman, R. and Greaves, M. (1982)
Structural features of the cell surface receptor for transferrin that
is recognized by the monoclonal antibody OKT9. J. Biol. Chem.
257, 8516–8522.
Shah, K., Russinova, E., Gadella, T.W. Jr, Willemse, J. and De Vries,
S.C. (2002) The Arabidopsis kinase-associated protein phosphatase controls internalisation of the somatic embryogenesis
receptor kinase. Genes Dev. 16, 1707–1720.
Sheff, D., Daro, E.A., Hull, M. and Mellman, I. (1999) The receptor
recycling pathway contains two distinct populations of early
endosomes with different sorting functions. J. Cell Biol. 145, 123–
139.
Sheff, D., Pelletier, L., O’Connell, C.B., Warren, G. and Mellman, I.
(2002) Transferrin receptor recycling in the absence of perinuclear
recycling endosomes. J. Cell Biol. 156, 797–804.
Shiratori, T., Miyatake, S., Ohno, H., Nakaseko, C., Isono, K.,
Bonifacino, J.S. and Saito, T. (1997) Tyrosine phosphorylation
controls internalization of CTLA-4 by regulating its interaction
with clathrin-associated adaptor complex AP-2. Immunity, 6, 583–
589.
Stephens, D.J. and Banting, G. (1997) Insulin dependent tyrosine
phosphorylation of the tyrosine internalisation motif of TGN38
creates a specific SH2 domain binding site. FEBS Lett. 416,
27–29.
Stephens, D.J. and Banting, G. (1998) Specificity of interaction
between adaptor-complex medium chains and the tyrosine-
based sorting motifs of TGN38 and lgp120. Biochem. J. 335,
567–572.
Stephens, D.J., Crump, C.M., Clarke, A.R. and Banting, G. (1997)
Serine 331 and tyrosine 333 are both involved in the interaction
between the cytosolic domain of TGN38 and the mu2 subunit of
the AP2 clathrin adaptor complex. J. Biol. Chem. 272, 14104–
14109.
Terng, H.J., Gessner, R., Fuchs, H., Stahl, U. and Lang, C. (1998)
Human transferrin receptor is active and plasma membrane-targeted in yeast. FEMS Microbiol. Lett. 160, 61–67.
Trowbridge, I.S., Collawn, J.F. and Hopkins, C.R. (1993) Signaldependent membrane protein trafficking in the endocytic pathway. Annu. Rev. Cell Biol. 9, 129–161.
Tse, Y.C., Mo, B., Hillmer, S., Zhao, M., Lo, S.W., Robinson, D.G. and
Jiang, L. (2004) Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells. Plant Cell,
16, 672–693.
Ueda, T., Yamaguchi, M., Uchimiya, H. and Nakano, A. (2001) Ara6,
a plant-unique novel type Rab GTPase, functions in the endocytic
pathway of Arabidopsis thaliana. EMBO J. 17, 4730–4741.
Ueda, T., Uemura, T., Sato, M.H. and Nakano, A. (2004) Functional
differentiation of endosomes in Arabidopsis cells. Plant J. 40,
783–789.
Vanoosthuyse, V., Tichtinsky, G., Dumas, C., Gaude, T. and Cock,
J.M. (2003) Interaction of calmodulin, a sorting nexin and kinaseassociated protein phosphatase with the Brassica oleracea S locus receptor kinase. Plant Physiol. 133, 919–929.
Warren, R.A., Green, F.A. and Enns, C.A. (1997) Saturation of the
endocytic pathway for the transferrin receptor does not affect the
endocytosis of the epidermal growth factor receptor. J. Biol.
Chem. 272, 2116–2121.
Warren, R.A., Green, F.A., Stenberg, P.E. and Enns, C.A. (1998)
Distinct saturable pathways for the endocytosis of different
tyrosine motifs. J. Biol. Chem. 273, 17056–17063.
Yaish, P., Gazit, A., Gilon, C., Levitzki, A., Lyall, R.M. and Zilberstein,
A. (1988) Blocking of EGF-dependent cell proliferation by EGF
receptor kinase inhibitors. Science, 242, 933–935.
Yamashiro, D.J., Tycko, B., Fluss, S.R. and Maxfield, F.R. (1984)
Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi
compartment in the recycling pathway. Cell, 37, 789–800.
Zhou, S.Y., Shoelson, S.E., Chaudhuri, M., Gish, G., Pawson, T. and
Haser, W.G. (1993) SH2 domains recognize specific phosphopeptide sequences. Cell, 72, 767–778.
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 757–770