ABP1 Mediates Auxin Inhibition of Clathrin-Dependent Endocytosis in Arabidopsis

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1 ABP1 Mediates Auxin Inhibition of Clathrin-Dependent Endocytosis in Arabidopsis Stéphanie Robert, 1,2,11 Jürgen Kleine-ehn, 1,2,11 Elke Barbez, 1,2 Michael Sauer, 1,2,12 Tomasz Paciorek, 1,2 Pawel Baster, 1,2 Steffen anneste, 1,2 Jing Zhang, 1,2 Sibu Simon, 3 Milada Covanová, 3 Kenichiro Hayashi, 4 Pankaj Dhonukshe, 5 Zhenbiao Yang, 6 Sebastian Y. Bednarek, 7 Alan M. Jones, 8 Christian Luschnig, 9 Fernando Aniento, 10 Eva Zazímalová, 3 and Jirí Friml 1,2, * 1 Department of Plant Systems Biology, IB, 9052 Gent, Belgium 2 Department of Plant Biotechnology and Genetics, Ghent University, 9052 Gent, Belgium 3 Institute of Experimental Botany, ASCR, Praha 6, Czech Republic 4 Department of Biochemistry, Okayama University of Science, Okayama , Japan 5 Department of Biology, Utrecht University, 3584 CH Utrecht, The Netherlands 6 Department of Botany and Plant Sciences and Center for Plant Cell Biology, Institute for Integrative Genome Biology, University of California, Riverside, Riverside, CA 92521, USA 7 Department of Biochemistry, University of Wisconsin, Madison, Madison, WI , USA 8 Departments of Biology and Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA 9 Institute for Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences, BOKU, 1190 Wien, Austria 10 Departamento de Bioquímica y Biología Molecular, Universidad de alencia, Burjassot, Spain 11 These authors contributed equally to this work 12 Present address: Centro Nacional de Biotecnología Consejo Superior de Investigaciones Científicas Departamento de Genética Molecular de Plantas c/ Darwin n 3, Lab. 316 Campus de Cantoblanco, Madrid, Spain *Correspondence: jiri.friml@psb.vib-ugent.be DOI /j.cell SUMMARY Spatial distribution of the plant hormone auxin regulates multiple aspects of plant development. These self-regulating auxin gradients are established by the action of PIN auxin transporters, whose activity is regulated by their constitutive cycling between the plasma membrane and endosomes. Here, we show that auxin signaling by the auxin receptor AUXIN-BINDING PROTEIN 1 (ABP1) inhibits the clathrin-mediated internalization of PIN proteins. ABP1 acts as a positive factor in clathrin recruitment to the plasma membrane, thereby promoting endocytosis. Auxin binding to ABP1 interferes with this action and leads to the inhibition of clathrin-mediated endocytosis. Our study demonstrates that ABP1 mediates a nontranscriptional auxin signaling that regulates the evolutionarily conserved process of clathrin-mediated endocytosis and suggests that this signaling may be essential for the developmentally important feedback of auxin on its own transport. INTRODUCTION The plant signaling molecule auxin is an important regulator of plant developmental processes, including embryogenesis, organogenesis, tissue patterning, and growth responses to external stimuli (Santner and Estelle, 2009; anneste and Friml, 2009). Current models on auxin signaling and action focus on the paradigm that auxin regulates the expression of subsets of genes, thus eliciting different cellular and, consequently, developmental responses. Nuclear auxin signaling involves the F box protein transport inhibitor response 1 (TIR1), which acts as an auxin coreceptor (Kepinski and Leyser, 2005; Dharmasiri et al., 2005a, 2005b; Tan et al., 2007), and downstream Aux/ IAA and ARF transcriptional regulators (Dharmasiri and Estelle, 2004). This pathway controls a remarkable number of auxinmediated processes, but some rapid cellular responses to auxin are not associated with TIR1-based signaling (Badescu and Napier, 2006; Schenck et al., 2010). Decades ago, the plant-specific protein AUXIN-BINDING PROTEIN 1 (ABP1) was proposed to be an auxin receptor (Hertel et al., 1972; Löbler and Klämbt, 1985). ABP1 in both monocot and dicot plant species shows physiological affinities toward natural and synthetic auxin ligands (Jones, 1994). ABP1, despite carrying a KDEL-endoplasmic reticulum (ER) retention motif, is secreted to some extent to the extracellular space where it is active (Jones and Herman, 1993; Tian et al., 1995; Henderson et al., 1997). ABP1 is essential for embryogenesis (Chen et al., 2001) and postembryonic shoot and root development (Braun et al., 2008; Tromas et al., 2009) and mediates auxin effect on cell elongation, but the underlying mechanism remains unclear (Jones et al., 1998; Leblanc et al., 1997). An important regulatory level in auxin action is its differential distribution within tissues (anneste and Friml, 2009). Such auxin gradients result from local auxin biosynthesis and directional, Cell 143, , October 1, 2010 ª2010 Elsevier Inc. 111

2 Figure 1. Auxin-Mediated Inhibition of Endocytosis by Nontranscriptional, TIR1- Independent Mechanism (A C) Time lapse showing BFA-induced increase of PIN2-GFP endosomal signal and its intracellular accumulation within minutes (A). NAA treatment effectively and rapidly inhibits BFAinduced PIN2-GFP internalization (B) also when protein synthesis is inhibited by cycloheximide (CHX) (C). (D H) BFA treatment for 90 min induces intracellular accumulation of PIN1 (D). Auxins, such as NAA (30 min pretreatment), inhibit BFA-induced PIN1 internalization in the wild-type (E); in the TIR1-mediated auxin signaling-deficient mutants, such as overexpressors of stabilized IAA17 (HS::axr3-1; induced for 2 hr at 37 C) (F); in the tir/afb quadruple mutant (G); and after MG132- mediated inhibition of proteasome function (H). See also Figure S1. (I M) Auxin treatments for 3 hr, such as NAA (J), but not BFA alone (I), induce transcriptional auxin response monitored by DR5::GUS in the wild-type (J), but not in the HS::axr3-1 (K) and tir/afb quadruple (L) mutants or after MG132 treatment (M). See also Figure S1. Arrows mark PIN proteins internalized into BFA compartments. Arrowheads highlight PIN retention at the plasma membrane. Scale bar, 10 mm. intercellular auxin transport (Petrásek and Friml, 2009) that is triggered by a network of carrier proteins (Swarup et al., 2008; Geisler et al., 2005; Petrásek et al., 2006; ieten et al., 2007; Yang and Murphy, 2009). The directionality of auxin flow depends on the polar plasma membrane distribution of PIN- FORMED (PIN) auxin efflux carriers (Wisniewska et al., 2006). In addition to PIN phosphorylation that directs PIN polar targeting (Friml et al., 2004; Michniewicz et al., 2007), PIN activity can be regulated by constitutive endocytic recycling from and to the plasma membrane (Geldner et al., 2001; Friml et al., 2002; Dhonukshe et al., 2007). Auxin itself inhibits the internalization of PIN proteins, increasing their levels and activity at the plasma membrane (Paciorek et al., 2005). The molecular mechanism of this auxin effect remains unknown, but it has been proposed to account for a feedback regulation of cellular auxin homeostasis and for multiple auxin-mediated polarization processes (Leyser, 2006). Here, we show that auxin regulation of PIN internalization involves the ABP1-mediated signaling pathway that targets clathrin-mediated endocytosis at the plasma membrane. RESULTS Auxin Inhibits PIN Internalization by a Rapid, Nontranscriptional Mechanism PIN proteins dynamically cycle between the endosomes and the plasma membrane (Geldner et al., 2001; Dhonukshe et al., 2007). Plasma membrane-localized PIN1 rapidly internalizes in response to the vesicle trafficking inhibitor brefeldin A (BFA) (Geldner et al., 2001), and this intracellular PIN accumulation is inhibited by auxins (Paciorek et al., 2005). In addition, auxin mediates with slower kinetics the degradation of PIN proteins (Sieberer et al., 2000; Abas et al., 2006). The auxin effects on PIN internalization and PIN degradation involve distinct mechanisms (Sieberer et al., 2000; Paciorek et al., 2005; Abas et al., 2006). These processes can be largely distinguished by BFA treatments at 25 and 50 mm that inhibit preferentially recycling or also vacuolar targeting for degradation, respectively (Sieberer et al., 2000; Abas et al., 2006; Kleine-ehn et al., 2008). We addressed the characteristics of the auxin signaling mechanism for inhibiting PIN internalization. It is experimentally established that the auxin regulation based on nuclear signaling requires at least min for execution (Badescu and Napier, 2006), whereas auxin inhibited the PIN2-GFP internalization more rapidly (<5 min) (Figures 1A and 1B). This suggests that this process does not involve auxin-dependent regulation of gene expression. Consistently, chemical inhibition of transcription (cordycepine or actinomycin D treatment) (Figure S1 available online) or de novo protein synthesis (cycloheximide treatment) (Figure 1C) does not prevent the auxin-mediated inhibition of PIN internalization. Auxin Inhibits PIN Internalization by a TIR1-Independent Pathway To elucidate the molecular mechanism by which auxin inhibits PIN internalization, we first tested the involvement of the TIR1- mediated signaling by genetical or chemical interference with different steps of this pathway. We analyzed (1) the quadruple tir1/afb mutant deficient in most of the TIR1/AFB auxin receptors function, (2) dominant lines conditionally expressing the stabilized transcriptional inhibitor IAA17 (HS::axr3-1), (3) stabilized mutations in other Aux/IAA-encoding genes (axr2-1, axr3-1, shy2-2, and slr-1), and (4) silenced lines for multiple ARFs (2X35S::miRNA160), as well as (5) seedlings treated with the proteasome inhibitor MG132 that interferes with auxin-mediated degradation of Aux/IAA repressors (Figures 1D 1H and Figure S1). These manipulations have all been shown to strongly inhibit TIR1-mediated transcriptional auxin responses (Timpte et al., 1994; Fukaki et al., 2002; Tian et al., 2002; Knox et al., 2003; Dharmasiri et al., 2005b). Moreover, interference with the TIR1 pathway can be visualized (Figures 1I 1M and Figure S1) by monitoring the activity of the synthetic auxin-responsive promoter DR5, which is an indicator for TIR1-dependent gene 112 Cell 143, , October 1, 2010 ª2010 Elsevier Inc.

3 A anti-pin1 + anti-pin2 Col-0 G BFA[25] B C D E F Col-0 NAA[10] 5 min BFA[25] Col-0 NAA[10] 15 min BFA[25] NAA[10] 30 min BFA[25] Col-0 H I J K L Col-0 NAA[10] 60 min BFA[25] Col-0 NAA[10] 120 min BFA[25] Figure 2. Auxin Effect on BFA-Induced PIN Internalization Kinetics of auxin effect on 25 mm BFA-induced PIN internalization with different time points of auxin pretreatment (0, 5, 15, 30, 60, and 120 min) in the wild-type (A F) and in the quadruple tir/afb mutant (G L). Note the comparable sensitivity of the quadruple tir/afb mutant and wild-type to auxin effect on PIN internalization. Auxin effect on PIN protein internalization was immediate (within minutes) but transient: prolonged auxin treatments from 1 to 2 hr resulted in reduced inhibition of PIN internalization (arrows). Scale bar, 10 mm. See also Figure S2. anti-pin1 + anti-pin2 tir1/afb1,2,3 tir1/afb1,2,3 tir1/afb1,2,3 expression (Ulmasov et al., 1997). As expected, treatments with different auxins increased the DR5::GUS expression in the wildtype root, but following interference with the TIR1 pathway, auxin was ineffective in inducing DR5 activity (Figures 1I 1M). In contrast, all of these manipulations did not interfere with the auxin inhibition of PIN internalization as monitored by BFAinduced intracellular PIN1 accumulation (Figures 1D 1H and Figure S1). In addition, the kinetics of the auxin effect on endocytosis in the quadruple tir1/afb mutant was indistinguishable from that of the wild-type (Figures 2A 2L and Figure S2). Together, these findings show that the auxin effect on PIN internalization does not require TIR1-mediated auxin signaling. This conclusion is seemingly contradictory to a previous report that proposed TIR1 involvement in auxin effect on BFA-induced PIN internalization (Pan et al., 2009). However, given the experimental conditions used (BFA at 50 mm), the Pan et al. report primarily addressed the auxin effect on PIN vacuolar trafficking that, in terms of kinetics and molecular mechanisms involved, is distinct from the regulation of PIN internalization (Figure S2 and Figure S3). Auxin Effects on Transcription and PIN Internalization Involve Distinct Perception Mechanisms To independently test whether auxin regulation of gene expression and inhibition of PIN internalization require independent signaling pathways, we tested a number of structural analogs of the natural auxin indole-3-acetic acid (IAA) for both effects. As expected, most analogs tested affected both gene expression and PIN internalization, albeit often at different effective concentrations. Importantly, we also identified auxin-like compounds that were specific for one or the other process only. For example, a-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA) (Figure S3) did not induce the expression of DR5rev::GFP reporter (Figure 3C) nor transcription of auxininducible genes related to the TIR1-dependent signaling pathway (Figure S3). However, similar to classical auxins, PEO- tir1/afb1,2,3 tir1/afb1,2,3 IAA inhibited the BFA-induced PIN internalization (Figure 3H). In contrast, another auxin analog, 5-fluoroindole-3-acetic acid (5-F-IAA), activated DR5rev::GFP already at 5 mm(figure 3D and Figure S3) but failed to inhibit PIN internalization, even at 25 mm(figure 3I). These nonoverlapping effects of compounds structurally related to auxin suggest that auxin perception upstream of either regulation of gene expression or PIN internalization involves distinct auxinbinding sites, confirming independently that auxin utilizes different signaling pathways for mediating these effects. tir1/afb1,2,3 abp1 Knockdown Lines Have Decreased PIN Internalization As the effect of auxin on PIN internalization is not mediated by TIR1-dependent signaling, we addressed the possible role of the putative auxin receptor, ABP1 (Jones, 1994; Napier et al., 2002). To test the involvement of ABP1 in PIN1 internalization, we monitored PIN subcellular dynamics in conditional immunomodulation and antisense abp1 knockdown lines (Braun et al., 2008; Tromas et al., 2009). Following downregulation of ABP1, the intracellular accumulation of PIN proteins in response to BFA treatment was diminished (Figures 4A 4D and data not shown). Similarly, pulse-labeling and time-lapse monitoring intracellular fluorescence revealed that uptake of the endocytic tracer FM4-64 was clearly reduced in roots of both immunomodulated and antisense abp1 knockdown lines as compared to the wildtype (Figure S4 and data not shown). In addition, a genetic interaction between abp1 knockdown lines and pin mutants (pin1-1 or eir1-1) was demonstrated by the enhancement of the single mutant phenotypes (Figure S4). Thus, the ABP1 function is required for PIN internalization and overall endocytosis, indicating that ABP1 plays a positive role in regulating endocytosis in plants. ABP1 Gain-of-Function Alleles Have Increased PIN Internalization Next, we tested the effect of ABP1 gain of function on PIN internalization. ABP1 is predominantly located in the lumen of the ER due to a C-terminal ER retention signal (KDEL), but some ABP1 is secreted and has been shown to be closely associated with the plasma membrane (Jones and Herman, 1993; Henderson et al., 1997; Shimomura et al., 1999). Cell 143, , October 1, 2010 ª2010 Elsevier Inc. 113

4 Untr. DR5rev::GFP + anti-pin2 anti-pin1 Untr. NAA[5] PEO[25]5FIAA[5] A B C D E F H BFA[25] PEO[5]/BFA[25] NAA[5]/BFA[25] To investigate the potential role of ABP1 outside of the ER lumen, tobacco (Nicotiana tabacum; Bright Yellow 2 (BY-2)) suspension-cultured cells were transfected with PIN1 (35S::PIN1-RFP) and the Arabidopsis ABP1 variant lacking the KDEL ER retention signal (35S::ABP1 DKDEL -GFP). When the full-length ABP1 protein was expressed (35S::ABP1-GFP), the G I FIAA[25]/BFA[25] Figure 3. Distinct Auxin Perception Mechanisms for the Regulation of Transcription and Endocytosis (A D) Activity of auxin-responsive promoter DR5rev::GFP (A) induced by treatment with auxin analogs, such as NAA (B) and 5-F-IAA (D) at 5 mm for 3 hr, but not by PEO-IAA even at concentrations up to 25 mm (C). (E) Relative DR5rev::GFP signal of meristematic cells versus nonmeristematic cells. n = 3 independent experiments with at least 21 roots analyzed for each assay. See also Figure S3. (F I) BFA-induced internalization of PIN1 and PIN2 (F) inhibited by NAA (G) and PEO-IAA (H) at 5 mm, but not by 5-F-IAA (all 30 min pretreated), even at concentrations up to 25 mm (I). Arrows mark PIN proteins internalized into BFA compartments. Arrowheads mark the PIN retention at the plasma membrane. Scale bar, 10 mm. Error bars represent standard deviation. *p < * N ] P EO[25 ] AA[50 * FIAA[5 5 ] PIN1-RFP localized largely to the plasma membrane, similarly to the control experiments (Figures 4E, 4F, and 4H). In contrast, coexpression of PIN1-RFP with the secreted ABP1 DKDEL -GFP version resulted in a strong internalization of PIN1-RFP (Figures 4G and 4H), indicating that ABP1 exported from ER regulates endocytosis. When introduced into Arabidopsis seedlings, ABP1 DKDEL -GFP expression led to auxin-related phenotypes, such as three cotyledons, shorter roots, and reduced apical dominance, but frequently resulted into seedling lethality or sterile development already in the T1 generation (Figure 4I and data not shown). To further characterize the role of ABP1 gain of function in PIN1 internalization, we monitored the subcellular dynamics of PIN1 proteins in the seedlings moderately expressing ABP1 DKDEL -GFP. In accordance with the transient BY-2 assays, the ABP1 DKDEL -GFP expression increased PIN1 internalization in Arabidopsis root cells treated with 25 mm BFA for 30 min (Figures 4J 4L). In summary, ABP1 gain of function induces PIN internalization, whereas reduced expression of ABP1 leads to reduced PIN internalization. These results strongly suggest that ABP1 acts as a positive effector of endocytosis in plants. Auxin Negatively Regulates ABP1 Action on PIN Internalization To study the potential role of ABP1 in mediating auxin inhibition of PIN internalization, we tested the auxin effect in BY-2 cells coexpressing PIN1-RFP and ABP1 DKDEL -GFP (Figure 5). Of note, NAA treatment counteracted the positive effect of secreted ABP1 on PIN internalization, leading to a preferential retention of PIN proteins at the plasma membrane (Figure 5E). In contrast, the structurally similar auxin analog 5-F-IAA, which promotes auxin-dependent gene transcription but does not inhibit PIN1 endocytosis (Figure 3 and Figure S3), showed also no detectable effect on ABP1-mediated PIN internalization (Figure 5F). This observation is consistent with the reported weak affinity for 5-F-IAA of the plasma membrane-associated auxin-binding site, which is likely related to ABP1 (Zazímalová and Kutácek, 1985). These results, as well as similarities between knockdown lines and auxin treatment, suggested a model in which auxin inhibits ABP1-mediated stimulation of PIN internalization. To test this scenario, we used the abp1-5 mutant allele (Xu et al., 2010) with a point mutation in the conserved auxin-binding pocket (Napier et al., 2002). Conversion of the conserved histidine to tyrosine (H94Y) weakens the Pi interaction between the side-chain ring and the indole ring and is, therefore, predicted to reduce the auxin-binding affinity without major steric hindrance or changes in domain structure (Woo et al., 2002). In contrast to ABP1 knockdown lines that showed an auxin-like inhibitory effect on PIN internalization, the abp1-5 allele was partially resistant to auxin with respect to its effect on PIN internalization. Auxins, such as NAA or IAA, in abp1-5 root cells were much less effective in inhibiting BFA-induced internalization of PIN proteins than the wild-type roots (Figures 5H 5L). Next, we deleted the KDEL ER retention signal in the abp1-5 mutant sequence. Similarly to ABP1 DKDEL -GFP, the overexpression of ABP1-5 DKDEL induced the PIN1-RFP internalization in tobacco BY-2-cultured cells. But, in contrast to ABP1 DKDEL - GFP, the ABP1-5 DKDEL -promoted PIN1 internalization was not 114 Cell 143, , October 1, 2010 ª2010 Elsevier Inc.

5 A B C E F G I J K H L D Figure 4. Positive ABP1 Role in PIN Internalization (A D) Reduced BFA-induced PIN1 internalization in inducible abp1 knockdown lines SS12S (B) and SS12K (C) as compared to the induced wild-type (A). Number of BFA compartments was reduced after ABP1 downregulation in immunomodulation (SS12S and SS12K) (D). alues in (D) represent the relative mean surface area (pixels 2 ) in comparison with the wild-type for each individual experiment. n > 3 independent experiments with a least 60 cells measured for each assay. See also Figure S4. (E H) Cotransfection of tobacco BY-2 cells with PIN1-RFP (0.05 mg) (in red) and ER marker HDEL- GFP (0.05 mg) (E), full-length ABP1-GFP (0.5 mg) (F), and ABP1 with deleted ER retention signal (ABP1 DKDEL -GFP) (0.05 mg) (G) (all in green). In contrast to the full-length ABP1-GFP, the secreted ABP1 DKDEL -GFP induced pronounced PIN internalization. Percentage of cells displaying severe (green), mild (red), or no detectable (blue) PIN1- RFP internalization (H). n > 3 independent experiments and at least 60 cells counted for each assay. (I L) Phenotypes of 4-day-old 35S::ABP1 DKDEL - GFP stable transformed Col-0 seedlings. Primary root growth defects and aberrant cotyledon number observed in the primary transformants. See also Figure S4. (I) BFA-induced internalization of PIN1 within 30 min is promoted in 35S::ABP1 DKDEL -GFP seedlings (K) versus the Col-0 control (J). Relative number of BFA bodies per cell (L). n = 3 independent experiments on two different transformants and at least 150 cells counted for each assay. Arrows mark PIN protein internalization. Scale bar, 10 mm. Error bars represent standard deviation. *p < 0.05; **p < counteracted by exogenous auxin application, indicating an auxin resistance due to a decreased affinity of auxin binding to the auxin-binding pocket in the ABP1-5 DKDEL modified version. This result shows that mutations in the auxin-binding pocket of ABP1 led to a decrease in auxin sensitivity of auxin-mediated inhibition of PIN internalization, supporting our hypothesis that auxin binding to ABP1 inhibits the positive action of ABP1 on endocytosis. Auxin Specifically Targets Clathrin-Based Mechanism of Endocytosis Previous work using single cells suggested that PIN proteins are cargos of endocytic mechanism involving the vesicle coat protein clathrin (Ortiz-Zapater et al., 2006; Dhonukshe et al., 2007). Thus, we examined the role of clathrin in PIN internalization in planta by conditionally overexpressing the C-terminal part of clathrin heavy chain (termed HUB1) that exerts a dominant negative effect on clathrin function by binding and consequently depleting clathrin light chains (Liu et al., 1995). This interference with the clathrin function inhibited the BFA-induced PIN internalization, confirming that PIN proteins are internalized in Arabidopsis root cells by the clathrin-based mechanism of endocytosis (Figure S5). To specifically test whether auxin inhibits clathrin-mediated endocytosis, we monitored the internalization of a well-established and specific cargo of clathrin-dependent endocytosis, the human transferrin receptor (htfr) and its ligand transferrin. In Arabidopsis protoplasts, which heterologously expressed htfr, exogenously applied transferrin was efficiently internalized (Figures 6A), as shown previously (Ortiz-Zapater et al., 2006). As expected, this internalization was completely blocked by tyrphostin A23, a known inhibitor of clathrin-mediated processes (Banbury et al., 2003; Konopka et al., 2008) (Figure 6B and Figure S5). Physiological levels of natural (IAA; data not shown) and synthetic (NAA; Figure 6C) auxins rapidly and efficiently inhibited transferrin internalization in htfr-expressing Arabidopsis protoplasts, demonstrating that auxin-mediated inhibition of endocytosis targets a general clathrin mechanism and is not cargo specific. In contrast, NAA was ineffective in inhibiting the htfr internalization in HeLa cells (data not shown), suggesting that the effect of auxin on the clathrin endocytotic pathway requires plantspecific factors. These auxin effects on internalization of both endogenous and heterologous cargos of the clathrin pathway suggest that auxin targets the clathrin-mediated mechanism of endocytosis. Cell 143, , October 1, 2010 ª2010 Elsevier Inc. 115

6 PIN1-RFP HDEL-GFP PIN1-RFP Δ KDEL-GF P A BP 1 anti-pin1 PIN1-RFP ABP1-5ΔKDEL-GFP A D B H I J K Col-0 M Untr. abp1-5 BFA[25] Untr. E NAA [10] N Col-0 C F 5FIAA [10] abp1-5 NAA[5]/BFA[25] NAA [10] G 100 O Percentages of cells displaying PIN1 internalization Untr. NAA 5FIAA Untr. NAA 5FIAA HDEL-GFP ABP1ΔKDEL-GFP severe mild no PIN1 internalization L Number of B FA bodies per cell Col-0 abp1-5 untr. abp1-5 Col-0 ** abp1-5 BFA NAA/BFA Percentages of cells displaying PIN1 internalization abp1-5 NAA severe mild no PIN1 internalization ** Figure 5. ABP1 Involvement in Auxin-Mediated Inhibition of PIN Protein Internalization (A G) Cotransfection of tobacco BY-2 cells with PIN1-RFP (0.05 mg) (in red) (A-F) and ER marker HDEL-GFP (0.05 mg) (A and B) or ABP1 DKDEL - GFP (0.05 mg) (D F) (all in green). After transfection, BY-2 cells were treated with NAA (B and E) or 5-F-IAA (C and F). NAA, but not 5-F-IAA, suppressed the ABP1 DKDEL -dependent effect on PIN1 internalization. Percentage of cells displaying severe (green), mild (red), or not detectable (blue) PIN1-RFP internalization (G). n > 3 independent experiments with at least 60 cells counted for each assay. (H L) BFA-induced PIN internalization in wild-type (H) and abp1-5 lines with mutation in auxin-binding site of ABP1 (I). Whereas NAA (5 mm, 30 min pretreatment) reduced the BFA-induced PIN protein internalization in the wild-type (J), the abp1-5 mutant seedlings were partially resistant to this auxin effect (K). Average number of BFA bodies per root cell in BFA- or NAA/BFA-treated wild-type and abp1-5 mutant seedlings (L). n = 3 independent experiments with at least 150 cells counted for each assay. (M O) Cotransfection of tobacco BY-2 cells with PIN1-RFP (0.05 mg) (in red) (A F) and mutated ABP1-5 DKDEL (0.05 mg) (in green) (M and N). After transfection, BY-2 cells were treated with NAA (N). NAA did not suppress the positive effect of ABP1-5 DKDEL with mutated auxin-binding site on PIN1 internalization. Percentage of cells displaying severe (green), mild (red), or not detectable (blue) PIN1-RFP internalization (O). n > 3 independent experiments, and at least 60 cells counted for each assay. Arrows mark PIN proteins internalized into BFA compartments. Arrowheads mark the PIN retention at the plasma membrane. Scale bar, 10 mm. Error bars represent standard deviation. *p < 0.05; **p < Auxin Interferes with Clathrin Recruitment to the Plasma Membrane To address a possible mode of auxin action on clathrin-mediated endocytosis, we tested for an auxin effect on clathrin localization. As previously described (Konopka et al., 2008), clathrin light chain fused to GFP (CLC-GFP) is associated with intracellular endomembranes (presumably TGN) and with dynamic foci at the plasma membrane (Figure 6D). The amount of clathrin detected at the plasma membrane was variable and strongly depended on growth conditions. Nonetheless, both anti-chc immunolocalizations (Figure S6) and time-lapse visualizations of CLC-GFP revealed that auxin treatments led to a decrease in the fluorescence associated with the plasma membrane but had no detectable effect on clathrin association with intracellular endomembranes (Figures 6D 6G and 6K and Figure S6). The effect of auxin on clathrin recruitment to the plasma membrane was rapid and transient and displayed kinetics similar to those of the auxin-mediated inhibition of PIN internalization (Figure S6). In contrast, auxin did not visibly affect other regulators of the early and late endosomal trafficking (Figure S5), including RabF2b (Rab5/Ara7) that is required for PIN internalization, presumably at later steps of endocytosis (Ueda et al., 2001; Dhonukshe et al., 2008). In addition, PEO-IAA, the effective inhibitor of PIN protein internalization, also showed an effect on clathrin incidence at the plasma membrane (Figures 6I and 6K), whereas 5-F-IAA, which is ineffective in the inhibition of PIN protein internalization, showed no detectable effect on CLC incidence at the plasma membrane (Figures 6J and 6K). These experiments demonstrated that auxin specifically interferes with the clathrin recruitment to the plasma membrane, providing a plausible mechanism for auxin effect on the endocytosis of PIN1 and other cargos. Auxin Negatively Regulates ABP1 Action on Clathrin-Dependent PIN Internalization Next, we addressed the potential role of ABP1 in mediating auxin effect on the clathrin-dependent endocytosis. First, we tested the effect of the interference with the clathrin function on ABP1-mediated PIN1 internalization. In BY-2 cells, the ABP1- mediated internalization of PIN1 proteins was abrogated by the inhibition of clathrin-mediated endocytosis either by expression of the dominant-negative clathrin HUB1 (35S::HUB1-GFP) orby treatment with tyrphostin A23 (Figures 7A 7D), indicating that 116 Cell 143, , October 1, 2010 ª2010 Elsevier Inc.

7 Transferrin CLC-GFP A B Tyr23[350] C F NAA[10] NAA[30] Untr. 30 min 60 min 120 min D E F G H Untr. CLC-GFP I PEO[30] J 5FIAA[30] 30 min K Percentage of cells showing CLC-GFP 100 at the PM ** ** Untr. NAA PEO 5FIAA Figure 6. Auxin Effect on Clathrin-Dependent Endocytosis and Clathrin Recruitment to the Plasma Membrane (A C) Heterologous expression of human transferrin receptor in protoplast enabled Alexa633-labeled transferrin internalization (A). Transferrin uptake was blocked by both tyrphostin A23 (B) and auxin (NAA) (C). See also Figure S5. Arrowheads mark internalized proteins. (D K) Clathrin light-chain GFP (CLC-GFP) localization at the trans-golgi network (TGN) and the plasma membrane (D). After auxin treatment for 30 (E) to 60 min (F), the CLC-GFP transiently disappeared from the plasma membrane but stayed at the TGN. After longer auxin treatments (2 hr), CLC-GFP reappeared at the plasma membrane (G). Arrowheads mark CLC- GFP intensity at the plasma membrane. See also Figure S5. (H J) PEO-IAA (30 mm for 30 min) inhibited the CLC-GFP localization at the plasma membrane (I), whereas treatment with 5-F-IAA (30 mm for 30 min) had no visible effect (J, arrowheads). (K) Percentage of cells showing CLC-GFP labeling at the plasma membrane in untreated seedlings and treated with NAA, PEO-IAA, and 5-F-IAA for 30 min. The percentage of cells showing a plasma membrane localization of CLC-GFP was calculated for at least 21 roots for each condition. Arrowheads mark CLC-GFP intensity at the plasma membrane. Scale bar, 10 mm. Error bars represent standard deviation. **p < the functional clathrin machinery is required for ABP1 effect on PIN internalization. In addition, the effect of ABP1 downregulation on the clathrin abundance at the plasma membrane was examined. The plasma membrane association of clathrin was strongly reduced in both immunomodulated (Figures 7E 7H and Figure S6) and antisense abp1 knockdown lines (data not shown) when compared to wildtype or noninduced controls. The auxin effect on clathrin abundance at the plasma membrane was significantly lower in abp1-5 mutant seedlings than in wild-type seedlings (Figures 7I 7M). Remarkably, these results correlate well with the auxin resistance observed in the abp1-5 line for the effect on PIN internalization. These multiple lines of observation clearly linked ABP1 action and clathrin mechanism of PIN internalization: (1) the positive effect of ABP1 on PIN protein internalization requires the clathrin-dependent endocytosis; (2) ABP1 action is required for clathrin localization at the plasma membrane; and (3) a mutation in the auxin-binding pocket of ABP1 conveys decreased auxin sensitivity of auxin effect on clathrin abundance at the plasma membrane. All of these results suggest that auxin binding to ABP1 inhibits the positive action of ABP1 on clathrin-mediated endocytosis. DISCUSSION Nonnuclear Auxin Signaling Targets Clathrin- Dependent Mechanism of Endocytosis in Plants In plants, the existence of endocytosis has been a matter of debates for decades, but in recent years, its physiological importance has become increasingly obvious, and a number of endocytic cargos have been identified (Robinson et al., 2008). The pronounced inhibition of the bulk of the endocytic processes after interference with the clathrin pathway (Dhonukshe et al., 2007) and its accessory protein (such as dynamin-related proteins) (Collings et al., 2008; Konopka et al., 2008) suggests that most endocytic processes in plants depend on an evolutionarily conserved mechanism involving clathrin. We demonstrated through multiple approaches that clathrinmediated endocytosis is rapidly inhibited by auxin and that auxin promotes the rapid disappearance of plasma membrane-associated clathrin. Of note, this auxin signaling does not involve the molecular components of the nuclear TIR1/AFB pathway (Kepinski and Leyser, 2002; Dharmasiri and Estelle, 2004) and does not require gene transcription or protein synthesis. This auxin effect on endocytosis is not specific to PIN proteins but regulates a number of endogenous and heterologous cargos. Cell 143, , October 1, 2010 ª2010 Elsevier Inc. 117

8 Figure 7. ABP1 Mediates Auxin Effect on Clathrin-Dependent PIN Internalization (A D) Cotransfection of tobacco BY-2 cells. ABP1 DKDEL -GFP-dependent (green) promotion of PIN1-RFP (red) internalization (A) is reduced after inhibition of clathrin-dependent endocytosis by HUB-GFP (green) (B) or tyrphostin A23 (C). Percentage of cells displaying severe (green), mild (red), or no detectable (blue) PIN1-RFP internalization (D). n > 3 independent experiments and at least 60 cells counted for each assay. Arrows mark PIN proteins internalization. Arrows indicate PIN internalization. (E H) Localization of clathrin as visualized by CLC- GFP at the TGN and the plasma membrane (E, arrowheads). In the abp1 knockdown immunomodulation lines, CLC-GFP labeling remained at the TGN but decreased at the plasma membrane (F and G). Percentage of the cells showing CLC-GFP localization at the plasma membrane (H). n = 3 independent experiments with at least 18 roots analyzed for each assay. See also Figure S6. Arrowheads mark CLC-GFP at the plasma membrane. (I M) Localization of clathrin as visualized by immunodetection with an anti-chc antibody at the TGN and the plasma membrane in Col-0 and the abp- 1-5 lines mutated in the auxin-binding site. In the abp1-5 mutant, depletion of clathrin from the plasma membrane was less sensitive to NAA. (M) Percentage of the cells showing CHC localization at the plasma membrane. n = 3 independent experiments with at least 15 roots analyzed for each assay. Arrowheads mark CHC immunolabeled intensity at the plasma membrane. Scale bar, 10 mm. Error bars represent standard deviation. *p < 0.05; **p < These observations strongly suggest that nontranscriptional auxin signaling interferes specifically with the general process of clathrin-mediated endocytosis in plant cells. ABP1 Acts as an Auxin-Sensitive, Positive Regulator of Clathrin-Mediated Endocytosis To identify the molecular mechanism underlying auxin effect on endocytosis, we tested the involvement of the putative auxin receptor ABP1 that is essential, but the mechanism of its action remained unclear (Badescu and Napier, 2006). Our loss- and gain-of-function analyses show that ABP1 acts as a positive regulator of clathrin-mediated endocytosis. ABP1 seems to be a plant-specific regulatory element of the evolutionarily conserved clathrin-mediated endocytic mechanism. Because ABP1 binds auxin with high affinity (Jones, 1994; Napier et al., 2002), it is suggestive that auxin mediates its effect on clathrinmediated endocytosis via ABP1. In this scenario, given the positive effect of ABP1 but the negative effect of auxin on endocytosis, auxin binding to ABP1 inhibits rather than activates the ABP1 action in endocytosis. This model (see Graphical Abstract) is supported by several independent lines of evidence: (1) the stereo-selectivity of auxins correlates with ABP1 binding (Zazímalová and Kutácek, 1985) and inhibition of endocytosis; (2) both increasing auxin or decreasing the active pool of ABP1 diminishes the clathrin incidence at the plasma membrane and inhibits the clathrin-dependent endocytosis; (3) increasing levels of secreted ABP1 lead to enhanced endocytosis that can be reversed by auxin treatment; and (4) an ABP1 with a mutated auxin-binding site is less effective in mediating auxin effect on clathrin incidence at the plasma membrane and on inhibition of endocytosis. These observations and, in particular, the remarkable difference between the knockdown abp1 and abp1-5 mutants provide strong support for the model that auxin binding to ABP1 interferes with its positive action on clathrin-mediated endocytosis. However, it remains open by which mechanism this regulation occurs. Physiological Role of the ABP1 Pathway for Regulation of Clathrin-Dependent Endocytosis Our studies here have primarily focused on PIN auxin transporters as targets for auxin- and ABP1-mediated regulation of endocytosis. By this mechanism, auxin increases the incidence of PIN proteins at the cell surface, stimulating auxin efflux (Paciorek et al., 2005) and providing developmentally important feedback of auxin on the rate of its intercellular flow. However, a number of additional membrane proteins and other cargos of clathrin-mediated endocytosis might be regulated in a similar manner. A more general auxin effect on clathrin-dependent endocytosis might be related to its phylogenetically ancient role in the control of cell expansion (Lau et al., 2009), whereby ABP1 also plays a crucial role (Jones et al., 1998). During this process, when the cell surface rapidly increases, generally the endocytosis rate is attenuated to retain the essential signaling 118 Cell 143, , October 1, 2010 ª2010 Elsevier Inc.

9 and structural components at the cell surface. Of note, ABP1 has also been connected to the ROP-GTPase pathway involved in the interdigitating growth of epidermal pavement cells (Xu et al., 2010), but the mechanistic link of this ABP1 function with its role in the clathrin-mediated endocytosis is still missing. Future work that builds on the proposed framework of the ABP1 action in clathrin-mediated endocytosis is necessary in order to understand how and with which components of the clathrin machinery ABP1 communicates. The intriguing possibility that the ABP1-mediated regulation of endocytosis is a part of a long-looked mechanism for auxin-mediated cell expansion and tissue polarization also remains open. EXPERIMENTAL PROCEDURES Material and Growth Conditions Arabidopsis thaliana (L.) Heyhn. seedlings, Columbia ecotype (Col-0), were grown on vertical half-strength Murashige and Skoog (0.5 MS) agar plates at 22 C for 4 days. BFA (Molecular Probes and Sigma), tyrphostin A23 (Sigma), tyrphostin A51 (Sigma), cycloheximide (Sigma), cordecypin (Sigma), or actinomycin (Sigma) were used from 50 mm dimethylsulfoxide stock solutions and added to the liquid 0.5 MS growth medium for the indicated times, if not mentioned otherwise: 90 min with 25 mm BFA; 30 min with 5, 10, or 30 mm of NAA; 30 min with 30 mm tyrphostin A23 or tyrphostin A51; and 30 min with 50 mm cordycepin or actinomycin followed eventually by 90 min NAA or NAA/BFA cotreatment. In control treatments, equal amounts of solvent were used. Transferrin Uptake Assays in Arabidopsis Transferrin uptake in Arabidopsis protoplasts expressing htfr was assayed as described (Ortiz-Zapater et al., 2006) with transferrin-alexa Fluor 546 (500 mg/ml, 28 C, 45 min). Tyrphostin A23 (350 mm) or auxin (10 mm NAA or 10 mm IAA) were added 15 min before transferrin and remained present during the internalization period. Immunodetection and Microscopy Immunofluorescence in Arabidopsis roots was analyzed as described (Sauer et al., 2006). The anti-pin1 antibody (1:1000) (Benková et al., 2003), the anti- PIN2 antibody (1:1000) (Abas et al., 2006), and the anti-chc antibody (1:400) (Kim et al., 2001) were used, and the fluorochrome-conjugated secondary antibodies Alexa488 and the anti-rabbit-cy3 (1:600) (Dianova) were used. Live-cell microscopy was done as described (Kleine-ehn et al., 2008). SUPPLEMENTAL INFORMATION Supplemental Information includes Extended Experimental Procedures, six figures, and one table and can be found with this article online at doi: / j.cell ACKNOWLEDGMENTS The authors thank the anonymous reviewer for helpful comments and Martine De Cock for help in preparing the manuscript. We are very grateful to X.Y. Chen, M. Estelle, T. Gaude, N. Geldner, O. Leyser, C. Perrot-Rechenmann, and J.W. Reed for sharing published materials and Drs. Jin-Gui Chen and Ming-Jing Wu for generating abp1 mutants. This work was supported by the Odysseus program of the Research Foundation Flanders, the Ministry of Education of the Czech Republic (project LC06034), and the Ministerio de Educación y Ciencia (grant BFU ). E.B. is indebted to the Agency for Innovation by Science and Technology for a predoctoral fellowship and S.. to EMBO for a long-term fellowship (ATLF ). M.S. was supported by HFSP long-term and Marie Curie IEF fellowships. A.M.J is supported by grants from The National Institute of General Medical Sciences (GM65989), The Department of Energy (DE-FG02-05er15671), and The National Science Foundation (MCB , ). P.D. is supported by NWO- ENI grant. Received: August 25, 2009 Revised: May 10, 2010 Accepted: September 14, 2010 Published: September 30, 2010 REFERENCES Abas, L., Benjamins, R., Malenica, N., Paciorek, T., Wisniewska, J., Moulinier- Anzola, J.C., Sieberer, T., Friml, J., and Luschnig, C. (2006). 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11 Wisniewska, J., Xu, J., Seifertová, D., Brewer, P.B., Ruzicka, K., Blilou, I., Rouquié, D., Benková, E., Scheres, B., and Friml, J. (2006). Polar PIN localization directs auxin flow in plants. Science 312, 883. Woo, E.-J., Marshall, J., Bauly, J., Chen, J.-G., enis, M., Napier, R.M., and Pickersgill, R.W. (2002). Crystal structure of auxin-binding protein 1 in complex with auxin. EMBO J. 21, Xu, T., Wen, M., Nagawa, S., Fu, Y., Chen, J.-G., Wu, M.-J., Perrot-Rechenmann, C., Friml, J., Jones, A.M., and Yang, Z. (2010). Cell surface- and Rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis. Cell 143, this issue, Yang, H., and Murphy, A.S. (2009). Functional expression and characterization of Arabidopsis ABCB, AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe. Plant J. 59, Zazímalová, E., and Kutácek, M. (1985). In vitro binding of auxin to particulate fractions from the shoots of dark-grown wheat seedlings. Plant Growth Regul. 3, Cell 143, , October 1, 2010 ª2010 Elsevier Inc. 121

12 Supplemental Information EXTENDED EXPERIMENTAL PROCEDURES Material The transgenic lines used have been described previously: HS::axr3-1 (Knox et al., 2003); tir1/afb1/afb2/afb3 (Dharmasiri et al., 2005a); RabF2b-YFP, RabC1-YFP, TI12-YFP and RabA1g-YFP (Geldner et al., 2009); CLC-GFP (Konopka et al., 2008); snx1-1 (Jaillais et al., 2006); axr3-1 (Rouse et al., 1998); axr2-1 (Nagpal et al., 2000); 35S(2x)::miRNA160 (Wang et al., 2005); vps29-3 (Kleine-ehn et al., 2008), abp1 knockdown lines (SS12S, SS12K and AS; Braun et al., 2008), DR5::GUS (Ulmasov et al., 1997); pin1-1 (Okada et al., 1991); eir1-1 (Luschnig et al., 1998); and DR5rev::GFP (Friml et al., 2003). The abp-1-5 mutant, provided by the ABRC stock center (# CS91358), led to a change of a highly conserved histidine to a tyrosine (H94Y) in the BOX A of the ABP1 protein. abp1-5 had been backcrossed 5 times with Col-0 and genotyped by restriction digestion of PCR fragments (primers: Atabpe1x2FW 5 0 -TGACCTTCCT- CAGGATAACTATGG-3 0 and Atabp1ex4R 5 0 -CCAACACCTGCAGGTCCTCATGAC-3 0 ). A restriction fragment length polymorphism upon RsaI digestions allowed identification of abp1-5. Three fragments were generated in wild-type (523, 207, and 73 bp) whereas four fragments were observed in abp1-5 (523, 165, 73, and 45 bp). The inducible HUB line was constructed with pintam and dominant negative HUB with the GAL4/UAS-based transactivation system (Dhonukshe et al., 2007). HUB expression was induced on solid media containing 2 mm 2-hydroxytamoxifen for the time indicated. ABP1-GFP was cloned by inserting GFP into ABP1 after the glycine 120 by primer extension PCR with one or two glycines flanking the GFP coding sequence, respectively. 35S::ABP1-GFP functionality was tested by complementation of abp1/abp1. The 35S::ABP1 DKDEL -GFP construct was made based on the aforementioned primers, replacing the C-terminal KDEL motif of ABP1 with a stop codon. Similarly, ABP1-5 DKDEL was cloned by amplifying ABP1 from abp1-5 lacking the KDEL ER retention motif. In Situ isualization of Auxin-Induced Gene Expression Four-day-old DR5rev::GFP or DR5::GUS seedlings were transferred to sterile 0.5 MS liquid medium supplemented with either 5 or 25 mm of the analyzed compounds and incubated for 3 hr. The compounds tested were 5-chloro-indole-3-acetic acid (5-Cl-IAA) 5- bromo-indole-3-acetic acid (5-Br-IAA), indole-3-carboxylic acid (I3CA), 4-chloro-indole-3-acetic acid (4-Cl-IAA), purchased from Ol- ChemIm Ltd, ( indole-3-acetic acid (IAA), 5-fluoroindole-3-acetic acid (5-F-IAA), 6-chloroindole-3-acetic acid (6-Cl-IAA), naphthalene-1-acetic acid (1-NAA), naphthalene-2-acetic acid (2-NAA), naphthalene, indole, phenyl acetic acid (PAA), indole-3-butyric acid (IBA), 2,4-dichloro-phenoxy acetic acid (2,4-D), 2,4,5-trichloro-phenoxy acetic acid (2,4,5-T), 2,4-dichloro-phenoxy propionic acid (2,4-DP), indole-3-acetyl-l-alanine (IAA-L-Ala), indole-3-propionic acid (IPA), benzoic acid (BA), indole-3-lactic acid (ILA) (Sigma, and a-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA) (Hayashi et al., 2008). Synthesis of PEO-IAA PEO-IAA was synthesized by the nucleophilic addition of indole to trans-3-benzoylacrylic acid using essentially the same reaction procedure as in (Abubshait SA, 2007). Briefly, equal moles of indole and trans-3-benzoylacrylic acid were refluxed in benzene for 12 hr. The crude solid was precipitated after cooling on ice, and then recrystallized from benzene to give pure PEO-IAA as colorless crystal (79% Yield): m.p C; IR y max: 3400, 3055, 1711, 1677, 1453 cm -1 ; 1 H NMR (400 MHz, acetone-d 6 ): d 3.41(1H, dd, J = 17.8, 4.1 Hz), 4.13 (1H, dd, J = 17.8, 11.0 Hz), 4.57 (1H, dd, J = 11.0, 4.1 Hz), 7.06 (1H, t, J = 8.2 Hz), 7.13(1H, t, J = 8.2 Hz), 7.37 (1H, s), 7.41 (1H, d, J = 8.2 Hz), 7.51 (2H, dd, J = 8.2, 7.8 Hz), 7.57 (1H, t, J = 7.8 Hz), 7.80 (1H, d, J = 8.3 Hz), 8.05 (2H, d, J = 8.2 Hz), (1H, brs, COOH); 13 C NMR (100 MHz, acetone-d 6 ): d 38.51, 42.35, , , , , , , , , , , , , , ; FAB-MS: m/z 294 [M+H] + ; HRFAB-MS: m/z [M+H] +, calcd. for (C 18 H 16 NO 3 ). Transient Transformation of Tobacco BY-2 Cells Ten ml of 3-day-old cells were harvested on filter paper by vacuum filtration and kept on solid BY-2 medium. The cells were transformed via particle bombardment with a PDS-1000/He biolistic system (Biorad) according to the manufacturer s recommendations. To coat the gold particles with DNA, 2 ml of plasmid DNA (0.05 mg/ml of each construct to transform) was added to 6.25 ml of 1.6-mm diameter gold particles and the suspension was supplemented with 2.5 ml spermidine (0.1 M stock solution) and 6.25 ml CaCl 2 (2.5 M stock solution). The particles were pelleted by centrifugation, washed twice with 70% and 100% ethanol. The pellet was suspended in 10 ml of 100% ethanol. Cells were bombarded under a pressure of 1100 psi. After transformation, 1 ml of auxin-free medium (mock) or enriched with 10 mm NAA or 10 mm 5-F-IAA was added to the cells. The plates were sealed with parafilm and kept in darkness for 18 hr at 25 C. Samples were analyzed via confocal microscopy (Zeiss 710). Quantification of FM4-64 Uptake The mean fluorescence intensity of vesicles inside the cell excluding the plasma membrane was measured with the ImageJ software (National Institutes of Health, Quotients of intracellular and plasma membrane fluorescence intensities were calculated for 10 to 15 cells from at least three different roots. The average of three independent experiments was calculated. The value of each phenotype was standardized to corresponding wild-type controls. SS12S and SS12K abp1 knockdown lines were Cell 143, , October 1, 2010 ª2010 Elsevier Inc. S1

13 induced for 24 hr before measurements with an 8% ethanol solution kept in a vial at the bottom of vertically oriented plates (Braun et al., 2008). Data were analyzed by Student s t test ( Quantitative RT PCR Total RNA was extracted with the RNeasy kit (QIAGEN). Poly(dT) cdna was prepared from total RNA with Superscript III (Invitrogen). Quantitative RT-PCR was done with LightCycler 480 SYBR Green I Master reagents (Roche Diagnostics) and a LightCycler 480 Real- Time PCR System (Roche Diagnostics). Targets were quantified with specific primer pairs designed with Beacon Designer 4.0 (Premier Biosoft International). Data were analyzed with qbase v1.3.4 (Hellemans et al., 2007). Expression levels were normalized to the non-auxin-responsive genes PEROXIN4 (At5g25760), UBIQUITIN-SPECIFIC PROTEASE 8 (At5g22030) and an unknown protein (At4g16100). For primer sequences, see Table S1. SUPPLEMENTAL REFERENCES Abubshait, S.A. (2007). An efficient synthesis and reactions of novel indolylpyridazinone derivatives with expected biological activity. Molecules 12, Friml, J., ieten, A., Sauer, M., Weijers, D., Schwarz, H., Hamann, T., Offringa, R., and Jürgens, G. (2003). Efflux-dependent auxin gradients establish the apicalbasal axis of Arabidopsis. Nature 426, Geldner, N., Anders, N., Wolters, H., Keicher, J., Kornberger, W., Muller, P., Delbarre, A., Ueda, T., Nakano, A., and Jürgens, G. (2003). The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 112, Geldner, N., Dénervaud-Tendon,., Hyman, D.L., Mayer, U., Stierhof, Y.-D., and Chory, J. (2009). Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J. 59, Hayashi, K.-i., Tan, X., Zheng, N., Hatate, T., Kimura, Y., Kepinski, S., and Nozaki, H. (2008). Small-molecule agonists and antagonists of F-box protein-substrate interactions in auxin perception and signaling. Proc. Natl. Acad. Sci. USA 105, Hellemans, J., Mortier, G., De Paepe, A., Speleman, F., and andesompele, J. (2007). qbase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8, R19. Jaillais, Y., Fobis-Loisy, I., Miège, C., Rollin, C., and Gaude, T. (2006). AtSNX1 defines an endosome for auxin-carrier trafficking in Arabidopsis. Nature 443, Luschnig C, Gaxiola RA, Grisafi P, Fink GR.(1998). EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana. Genes Dev. 12, Nagpal, P., Walker, L.M., Young, J.C., Sonawala, A., Timpte, C., Estelle, M., and Reed, J.W. (2000). AXR2 encodes a member of the Aux/IAA protein family. Plant Physiol. 123, Okada, K., Ueda, J., Komaki, M.K., Bell, C.J., and Shimura, Y. (1991). Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation. Plant Cell 3, Peyroche, A., Antonny, B., Robineau, S., Acker, J., Cherfils, J., and Jackson, C.L. (1999). Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex: involvement of specific residues of the Sec7 domain. Mol. Cell 3, Rouse, D., Mackay, P., Stirnberg, P., Estelle, M., and Leyser, O. (1998). Changes in auxin response from mutations in an AUX/IAA gene. Science 279, Wang, J.-W., Wang, L.-J., Mao, Y.-B., Cai, W.-J., Xue, H.-W., and Chen, X.-Y. (2005). Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis. Plant Cell 17, Zeeh, J.-C., Zeghouf, M., Grauffel, C., Guibert, B., Martin, E., Dejaegere, A., and Cherfils, J. (2006). Dual specificity of the interfacial inhibitor brefeldin a for arf proteins and sec7 domains. J. Biol. Chem. 281, S2 Cell 143, , October 1, 2010 ª2010 Elsevier Inc.

14 Figure S1. Aux/IAA-Independent Signaling for Auxin Effect on PIN Internalization, Related to Figure 1 (A G) BFA-induced internalization of PIN1 in wild-type (A) and TIR1-Aux/IAA signaling-deficient transgenic seedlings: axr3-1 (B), axr2-1 (C), 2X35S::miRNA160 (D), slr-1 (E), tir1/afb1,2,3 (F) and HS::axr3-1(G). (H N) BFA-induced PIN accumulations inhibited by NAA (30 min pretreatment) in wild-type (H) and in TIR1-Aux/IAA signaling-deficient transgenic seedlings, such as axr3-1 (I), axr2-1 (J), 2X35S::miRNA160 (K), slr-1 (L), tir1/afb1,2,3(m) and HS::axr3-1 (N). (O T) Interference with MG132-dependent proteasome activity (O and P) and cordecypin- (Q and S) or actinomycin-dependent (R and T) transcription did not affect auxin effect on BFA-induced PIN internalization. After inhibition of the proteasome activity by MG132 for 30 min, cotreatment with BFA-induced PIN protein internalization (O) and the effect of auxin on PIN internalization still occurred (P). Upon incubation with the transcription inhibitors cordycepine and actinomycin D for 30 min, auxin did not induce the activity of auxin-responsive promoter DR5rev::GFP (Q S), but still inhibited PIN protein internalization after 3 hr of treatment (T and U). Arrows mark PIN proteins internalized in BFA compartments and arrowheads highlight PIN protein retention at the plasma membrane. Scale bar: 10 mm. Cell 143, , October 1, 2010 ª2010 Elsevier Inc. S3

15 Figure S2. Auxin Effect on BFA-Induced PIN Internalization and Degradation, Related to Figure 2 BFA is a well-characterized inhibitor of a subclass of ARF-GEF, regulators of vesicle budding. BFA sensitivity of different ARF-GEFs depends on a single amino acid in the ARF-GEF sequence and the actual interacting ARF partner (Peyroche et al., 1999; Geldner et al., 2003; Zeeh et al., 2006). Thus, different ARF-GEFmediated subcellular vesicle trafficking processes can show clear differences in BFA sensitivity. For instance, 25 mm BFA appears to specifically interfere with the recycling processes to the plasma membrane, whereas 50 mm BFA affects also the degradation pathway to the vacuole (Kleine-ehn et al., 2008). Therefore we used this differential effect to distinguish the auxin effect on PIN internalization and PIN vacuolar targeting. (A H) Auxin effect on PIN internalization and degradation visualized by the presence or absence of PIN proteins in BFA bodies: NAA (30 min pretreatment) inhibited BFA-induced PIN1 and PIN2 intracellular accumulation at both 25 mm and 50 mm BFA in wild-type seedlings (A-D). In mutants with an increased PIN turnover in lytic vacuoles, such as snx1-1 (Kleine-ehn et al., 2008) (E-H), NAA prevented the BFA-induced PIN1 and PIN2 intracellular accumulation only at 25 mm(e and F), but not at 50 mm BFA (G and H), indicating that BFA treatment at 50 mm visualizes PIN proteins that are designated for degradation, while BFA incubation at 25 mm predominantly affects PIN recycling. (I T) Analysis of the effect of auxin (10 mm) and BFA (50 mm) cotreatment for PIN degradation over time in wild-type (I-N) and in a tir/afb quadruple mutant (O-T). The rate of PIN protein turnover, as visualized by an increased accumulation of PIN proteins in 50 mm BFA-induced compartments, was higher in the tir/afb quadruple mutant than in the wild-type. (U) Average number of BFA bodies per cell under the conditions described in Figure 2. n = 3 independent experiments with at least 60 cells counted for each assay. Error bars represent standard deviation. * and ** mark statistically significant p values at < 0.05 and < 0.001, respectively. Arrows mark PIN proteins internalized in BFA compartments and arrowheads highlight PIN protein retention at the plasma membrane. Scale bar: 10 mm. S4 Cell 143, , October 1, 2010 ª2010 Elsevier Inc.

16 A (Phenyl Ethyl-2-One)-Indole-3-Acetic Acid (PEO-IAA) 5-Fluoroindole-3-Acetic Acid (5-F-IAA) B GATA23 IAA1 IAA IAA5 PIN Figure S3. a-(phenyl Ethyl-2-one)-Indole-3-Acetic Acid (PEO-IAA) and 5-Fluoro-IAA (5-F-IAA), Related to Figure 3 (A) Chemical structure of the auxin analogs PEO-IAA and 5-F-IAA. (B) Effect of mock, NAA, PEO-IAA and 5-F-IAA on transcript levels of auxin-regulated genes, such as GATA23, IAA1, IAA19, IAA5, and PIN1 at two different time points (2 hr and 4 h). Data were normalized to non-auxin responsive genes PEROXIN4 (At5g25760), UBIQUITIN-SPECIFIC PROTEASE 8 (At5g22030), and an unknown protein (At4g16100). Transcript levels of auxin-inducible genes were still responsive to 5-F-IAA, but not to PEO-IAA in accordance with Figure 3. Error bars represent standard deviation. Cell 143, , October 1, 2010 ª2010 Elsevier Inc. S5

17 Figure S4. ABP1 Downregulation Inhibits Endocytosis, Related to Figure 4 (A C) Inhibited uptake of the endocytotic tracer FM4-64 in the inducible abp1 knock-down lines SS12S (B) and SS12K (C) when compared to the induced wildtype (A). Quantification of FM4-64 uptake in the wild-type and in conditional abp1 knock-down lines (D). n > 3 independent experiments with at least 90 cells analyzed for each assay. (E F) Genetic interaction between an abp1 knock-down line and pin mutants: defects in gravity responses of eir-1-1 (E) and in root elongation of pin1-1 (F) were enhanced by ABP1 downregulation. Measurements have been done on 7 to 40 roots for each assay. Scale bar: 10 mm. Error bars represent standard deviation. * and ** mark statistically significant p values at < 0.05 and < 0.001, respectively. S6 Cell 143, , October 1, 2010 ª2010 Elsevier Inc.

18 Figure S5. PIN Proteins Internalized through a Clathrin-Dependent Pathway, Related to Figure 6 Elevated HUB1 levels tagged with red fluorescent protein (RFP) in inducible pintam > > RFP-HUB1 lines correlate with reduced PIN internalization upon BFA treatment. Cell 143, , October 1, 2010 ª2010 Elsevier Inc. S7

19 (A D) PIN1 localization in non-induced (A and B) and induced for 30 hr (C and D) dominant-negative clathrin HUB1 lines: BFA-induced PIN1 internalization (B) was inhibited by HUB1 induction (D). (E G) BFA-induced PIN1 internalization (E) was inhibited by tyrphostin A23 (30 min pretreatment) (F), but not by the inactive analog tyrphostin A51 (G). (H) isualization of pintam > > RFP-HUB after induction with 2 mm tamoxifen for 12 hr. Arrows mark PIN proteins internalized into BFA compartments and arrowheads PIN protein retention at the plasma membrane. (I) BFA-induced PIN1 internalization (top): tyrphostin A23, when applied prior to BFA, strongly reduced the BFA-induced PIN internalization (middle). This effect was reversible when tyrphostin A23 was removed and replaced by BFA for another 30 min (bottom). (J Q) Auxin treatment (30 min) did not affect localization of RabF2b (J and N), RabC1 (K and O), TI12 (L and P) and RabA1g (M and Q) fused to yellow fluorescent protein (YFP). Arrows mark PIN proteins internalized in BFA compartments and arrowheads highlight PIN protein retention at the plasma membrane. Scale bar: 10 mm. S8 Cell 143, , October 1, 2010 ª2010 Elsevier Inc.

20 Figure S6. Auxin Affects Clathrin Heavy Chain, but Not Other Endosomal Regulators, Related to Figure 7 (A) Co-localization of a CLC-GFP fusion protein with immunodetected CHC. Seedlings expressing CLC::CLC:GFP were used for immunolabeling with an anti- CHC antibody (detected with Cy3 secondary antibody). Signals detected upon excitation with either 488 nm (GFP, in green) or 561 nm (Cy3, in red) indicated pronounced protein co-localization (see overlay). (B) Quantification CHC at the plasma membrane in wild-type and abp1 knock-down lines upon auxin-treatment for 30 min or in untreated controls. Percentage of cells per root displaying CHC localized to the plasma membrane. n = 3 independent experiments with at least 90 cells analyzed for each assay. Arrowheads show clathrin at the plasma membrane. Scale bar: 10 mm. Error bars represent standard deviation. * and ** mark statistically significant p values at < 0.05 and < 0.001, respectively. Cell 143, , October 1, 2010 ª2010 Elsevier Inc. S9