Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin A

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, María Jesús Marcote, Dolores Ortiz-Masiá and Fernando Aniento* Departamento de Bioquímica y Biología 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 fernando.aniento@uv.es). 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 r2- 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 Journal compilation ª 2006 Blackwell Publishing Ltd

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; Maldonado- Mendoza 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).

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 l2- subunit 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) 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 4 C to avoid internalization of receptor and antibody, and was followed by incubation (also at 4 C) 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

760 Elena Ortiz-Zapater et al. (a) (a) mock (c) (d) (c) (d) htfr (e) (f) (e) (f) htfr + A23 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 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 4 C 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 28 C 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, 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 4 C. Under these conditions, Tfn was found to bind to the plasma membrane (Figure 5a). Neither internalization nor

Receptor-mediated endocytosis in plant cells 761 (a) (a) Control BFA htfr Ara7 Overlay Tfn-546 htfr Overlay 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 and Alexa 488/ 546-labeled secondary antibodies. Samples were analysed using a Leica confocal microscope. (a) 4 C Tfn-488 FM4-64 Overlay 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. Protoplasts expressing htfr were incubated in the presence of transferrin Alexa 488 (Tfn-488) and FM4-64 for 15 min at 28 C and then analysed using a confocal microscope. 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 4 C (a) or 28 C 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 recycling, 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 28 C in the presence of FM4-64 and Tfn Alexa 488 and analysed by confocal microscopy. As shown in Figure 6, 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

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 28 C, 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 4 C, 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 IC 50 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) (c) (e) (f) (d) A51 A23 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 28 C. Then, transferrin Alexa 546 was added, the protoplasts were incubated for 45 min at 28 C 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 28 C. After the incubation, protoplasts were washed, fixed and processed for immunofluorescence using the anti-cd71 antibody and an Alexa Fluor 488- labeled 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 28 C 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

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. (a) (c) Control (d) A23 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 4 C, 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 28 C 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. 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 4 C 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 4 C. Where indicated, 350 lm tyrphostin A23 was added (b and d) and the protoplasts were incubated for a further 15 min at 4 C. Finally, the protoplasts were incubated for 45 min at 28 C 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,

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

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 3,4- dihydroxy 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 4 C, a condition that allows receptor ligand binding but not plasma membrane internalization. Upon incubation at 28 C, 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-

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). 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 (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 Media and solutions Proto medium (per l). 30 ml stock A (65.5 g l )1 KNO 3 ; 4.4 g l )1 CaCl 2 Æ2H 2 O; 3.7 g l )1 MgSO 4 Æ7H 2 O; 1.7 g l )1 KH 2 PO 4 ); 0.3 ml stock B (6.2 g l )1 H 3 BO 3 ; 22.3 g MnSO 4 Æ4H 2 O; 10.6 g l )1 ZnSO 4 Æ7- H 2 O; 0.83 g l )1 KI; 0.25 g l )1 Na 2 MoO 4 Æ2H 2 O; 0.025 g l )1 CoCl 2 ; 0.025 g l )1 CuSO 4 Æ5H 2 O); 2 ml stock C (2.78 g l )1 FeSO 4 Æ7H 2 O; 3.72 g l )1 Na 2 EDTAÆ2H 2 O); 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(NO 3 ) 2 Æ4H 2 O; adjusted to ph 9 with a fresh 0.1 N NaOH solution (stored in aliquots at )20 C and readjusted to ph 9 just before use); filter-sterilized through a 0.22 lm nitrocellulose filter. Ca(NO 3 ) 2 solution (per l). 64.9 g Ca(NO 3 ) 2 Æ4H 2 O; adjusted to ph 5.7 6.0 with KOH and autoclaved. W5 medium. 154 mm NaCl, 125 mm CaCl 2,5mM 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 Me 2 SO at )20 C 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 Me 2 SO at

Receptor-mediated endocytosis in plant cells 767 )20 C and added to solutions just before use. BFA was stored as a 5mgml )1 stock solution in MeOH at )20 C. 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 10 6 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(NO 3 ) 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 20 C 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 10 5 to 5 10 6 ) 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 28 C. 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 4 C or 28 C. 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 28 C, or left untreated. They were then incubated overnight at 4 C 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 4 C 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% NaBH 4 in PBS to permeabilize cells and reduce autofluorescence. The protoplasts were then incubated in blocking solution (1% BSA/PBS) for 1 h at 37 C, and then for 2 h at 37 C 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 37 C 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 AminoLinkÒ Plus gel (ProFound TM 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

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 Tecnología (grant numbers BMC2002-03993 and BFU2005-00071) to F.A. E.O.-Z. is a research fellow of the Ministerio de Ciencia y Tecnología (Beca F.P.U.). M.J.M. has a contract from the Ministerio de Ciencia y Tecnología (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) F- actin-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.

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.

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 tyrosinebased 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.