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• W2111280003 abstract "Article1 May 2012free access Source Data Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation Sina Henrichs Sina Henrichs Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, SwitzerlandSharing co-authorship Search for more papers by this author Bangjun Wang Bangjun Wang Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, Switzerland Department of Biology—Plant Biology, University of Fribourg, Fribourg, Switzerland Department of Plant Biology and Biotechnology, University of Copenhagen, Frederiksberg, DenmarkSharing co-authorship Search for more papers by this author Yoichiro Fukao Yoichiro Fukao Plant Global Educational Project, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan Search for more papers by this author Jinsheng Zhu Jinsheng Zhu Department of Biology—Plant Biology, University of Fribourg, Fribourg, Switzerland Search for more papers by this author Laurence Charrier Laurence Charrier Department of Biology—Plant Biology, University of Fribourg, Fribourg, Switzerland Search for more papers by this author Aurélien Bailly Aurélien Bailly Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, Switzerland Department of Biology—Plant Biology, University of Fribourg, Fribourg, Switzerland Search for more papers by this author Sophie C Oehring Sophie C Oehring Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, SwitzerlandPresent address: Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland Search for more papers by this author Miriam Linnert Miriam Linnert Germany, Signaltransduktion, Max-Planck-Forschungsstelle für Enzymologie der Proteinfaltung, Halle (Saale) Search for more papers by this author Matthias Weiwad Matthias Weiwad Germany, Signaltransduktion, Max-Planck-Forschungsstelle für Enzymologie der Proteinfaltung, Halle (Saale) Search for more papers by this author Anne Endler Anne Endler Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, SwitzerlandPresent address: Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany Search for more papers by this author Paolo Nanni Paolo Nanni Functional Genomics Center Zurich, UZH/ETH Zürich, Zürich, Switzerland Search for more papers by this author Stephan Pollmann Stephan Pollmann Ruhr-Universität Bochum, Lehrstuhl für Pflanzenphysiologie, Bochum, GermanyPresent address: Centro de Biotecnología y Genómica de Plantas, Madrid, Spain Search for more papers by this author Stefano Mancuso Stefano Mancuso Department of Plant, Soil and Environmental Science, University of Florence, Sesto Fiorentino, Italy Search for more papers by this author Alexander Schulz Alexander Schulz Department of Plant Biology and Biotechnology, University of Copenhagen, Frederiksberg, Denmark Search for more papers by this author Markus Geisler Corresponding Author Markus Geisler Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, Switzerland Department of Biology—Plant Biology, University of Fribourg, Fribourg, Switzerland Search for more papers by this author Sina Henrichs Sina Henrichs Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, SwitzerlandSharing co-authorship Search for more papers by this author Bangjun Wang Bangjun Wang Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, Switzerland Department of Biology—Plant Biology, University of Fribourg, Fribourg, Switzerland Department of Plant Biology and Biotechnology, University of Copenhagen, Frederiksberg, DenmarkSharing co-authorship Search for more papers by this author Yoichiro Fukao Yoichiro Fukao Plant Global Educational Project, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan Search for more papers by this author Jinsheng Zhu Jinsheng Zhu Department of Biology—Plant Biology, University of Fribourg, Fribourg, Switzerland Search for more papers by this author Laurence Charrier Laurence Charrier Department of Biology—Plant Biology, University of Fribourg, Fribourg, Switzerland Search for more papers by this author Aurélien Bailly Aurélien Bailly Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, Switzerland Department of Biology—Plant Biology, University of Fribourg, Fribourg, Switzerland Search for more papers by this author Sophie C Oehring Sophie C Oehring Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, SwitzerlandPresent address: Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland Search for more papers by this author Miriam Linnert Miriam Linnert Germany, Signaltransduktion, Max-Planck-Forschungsstelle für Enzymologie der Proteinfaltung, Halle (Saale) Search for more papers by this author Matthias Weiwad Matthias Weiwad Germany, Signaltransduktion, Max-Planck-Forschungsstelle für Enzymologie der Proteinfaltung, Halle (Saale) Search for more papers by this author Anne Endler Anne Endler Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, SwitzerlandPresent address: Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany Search for more papers by this author Paolo Nanni Paolo Nanni Functional Genomics Center Zurich, UZH/ETH Zürich, Zürich, Switzerland Search for more papers by this author Stephan Pollmann Stephan Pollmann Ruhr-Universität Bochum, Lehrstuhl für Pflanzenphysiologie, Bochum, GermanyPresent address: Centro de Biotecnología y Genómica de Plantas, Madrid, Spain Search for more papers by this author Stefano Mancuso Stefano Mancuso Department of Plant, Soil and Environmental Science, University of Florence, Sesto Fiorentino, Italy Search for more papers by this author Alexander Schulz Alexander Schulz Department of Plant Biology and Biotechnology, University of Copenhagen, Frederiksberg, Denmark Search for more papers by this author Markus Geisler Corresponding Author Markus Geisler Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, Switzerland Department of Biology—Plant Biology, University of Fribourg, Fribourg, Switzerland Search for more papers by this author Author Information Sina Henrichs1, Bangjun Wang1,2,3, Yoichiro Fukao4, Jinsheng Zhu2, Laurence Charrier2, Aurélien Bailly1,2, Sophie C Oehring1, Miriam Linnert5, Matthias Weiwad5, Anne Endler1, Paolo Nanni6, Stephan Pollmann7, Stefano Mancuso8, Alexander Schulz3 and Markus Geisler 1,2 1Molecular Plant Physiology, Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zurich, Switzerland 2Department of Biology—Plant Biology, University of Fribourg, Fribourg, Switzerland 3Department of Plant Biology and Biotechnology, University of Copenhagen, Frederiksberg, Denmark 4Plant Global Educational Project, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan 5Germany, Signaltransduktion, Max-Planck-Forschungsstelle für Enzymologie der Proteinfaltung, Halle (Saale) 6Functional Genomics Center Zurich, UZH/ETH Zürich, Zürich, Switzerland 7Ruhr-Universität Bochum, Lehrstuhl für Pflanzenphysiologie, Bochum, Germany 8Department of Plant, Soil and Environmental Science, University of Florence, Sesto Fiorentino, Italy *Corresponding author. Department of Biology—Plant Biology, University of Fribourg, 3 Rte. Albert Gockel, Fribourg 1700, Switzerland. Tel.: +41 26 300 8827; Fax: +41 26 300 9740; E-mail: [email protected] The EMBO Journal (2012)31:2965-2980https://doi.org/10.1038/emboj.2012.120 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Polar transport of the plant hormone auxin is controlled by PIN- and ABCB/PGP-efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co-immunoprecipitation (co-IP) and shotgun LC–MS/MS analysis, we identified PID as a valid partner in the interaction with TWD1. In-vitro and yeast expression analyses indicated that PID specifically modulates ABCB1-mediated auxin efflux in an action that is dependent on its kinase activity and that is reverted by quercetin binding and thus inhibition of PID autophosphorylation. Triple ABCB1/PID/TWD1 co-transfection in tobacco revealed that PID enhances ABCB1-mediated auxin efflux but blocks ABCB1 in the presence of TWD1. Phospho-proteomic analyses identified S634 as a key residue of the regulatory ABCB1 linker and a very likely target of PID phosphorylation that determines both transporter drug binding and activity. In summary, we provide evidence that PID phosphorylation has a dual, counter-active impact on ABCB1 activity that is coordinated by TWD1–PID interaction. Introduction Plant development and physiology depends on a unique, plant-specific process, the cell-to-cell or polar transport of auxin (PAT). PAT is controlled by efflux provided by members of the PIN-(PIN-FORMED) and B subfamily of ABC transporters (ABCBs), formerly called PGPs/MDRs (P-GLYCOPROTEIN, MULTIDRUG-RESISTANCE). PIN-efflux carriers show mainly polar locations in PAT tissues and are thought to be the determinants of a ‘reflux loop’ in the root apex; their loss-of-function mutants are therefore characterized by strong developmental phenotypes (Blilou et al, 2005; Vieten et al, 2007). ABCB isoforms have been identified as primary, active (ATP-dependent) auxin pumps showing late developmental loss-of-function phenotypes (Geisler et al, 2005; Blakeslee et al, 2007; Mravec et al, 2008). Despite their predominnatly apolar locations, they have been demonstrated to contribute to PAT and long-range auxin transport (Geisler et al, 2003; Bouchard et al, 2006; Bailly et al, 2008). ABCB- and PIN-mediated auxin efflux can function independently and play identical cellular but separate developmental roles (Mravec et al, 2008). The current picture that emerges is that multilaterally expressed ABCBs minimize apoplastic reflux (Bailly et al, 2012a), while polar PINs provide a specific, vectorial auxin stream (Mravec et al, 2008). However, ABCBs and PINs are also capable of interactive and coordinated transport of auxin (Blakeslee et al, 2007). On the posttranscriptional level, PAT has been shown to be controlled by protein–protein interaction, modulatory drugs and protein phosphorylation. The immunophilin-like FKBP42, TWISTED DWARF1 (TWD1), has been characterized as a central regulator of ABCB-mediated auxin transport by means of protein–protein interaction (Bailly et al, 2006). Positive regulation of ABCB1/PGP1- and ABCB19/PGP19/MDR1-mediated auxin transport (referred to as ABCBs hereafter) accounts for overlapping phenotypes between twd1 and abcb1 abcb19 (Bouchard et al, 2006; Bailly et al, 2008). ABCB1 and ABCB19 have been identified recently as binding proteins of the synthetic auxin-efflux inhibitor, 1-N-Naphtylphthalamic acid (NPA) (Murphy et al, 2002; Geisler et al, 2005; Rojas-Pierce et al, 2007; Nagashima et al, 2008; Kim et al, 2010). In addition, TWD1 binds to NPA and NPA binding disrupts TWD1–ABCB1 interaction (Murphy et al, 2002; Bailly et al, 2008). This leads to disruption of ABCB1 activity, suggesting that TWD1 and ABCB1 represent essential components of the NPA-sensitive-efflux complex (Bailly et al, 2008). On the contrary, several lines of evidence suggest that PIN proteins do not themselves act as direct targets of NPA (Lomax et al, 1995; Luschnig, 2001; Kim et al, 2010). The serine–threonine kinase PINOID (PID) and the trimeric serine–threonine protein phosphatase 2A (PP2A) direct the polar targeting of PIN proteins (Friml et al, 2004; Michniewicz et al, 2007). The current model suggests that PID and PP2A antagonistically determine the fate of PIN cargoes for trafficking to the appropriate membrane by (de)phosphorylating conserved motifs of the hydrophilic loop of PIN proteins (Kleine-Vehn et al, 2009; Dhonukshe et al, 2010; Huang et al, 2010; Ding et al, 2011). The regulatory A subunit, PP2AA1, called ROOTS CURL IN NPA1 (RCN1), is a negative regulator of basipetal transport in the root and as a consequence rcn1 roots exhibit a significant delay in gravitropism, consistent with an increased basipetal auxin transport (Sukumar et al, 2009; Rashotte et al, 2001). Importantly, the rcn1 gravitropic phenotype can be rescued by low concentrations of NPA, a concentration that is sufficient to block gravitropism in wild-type seedlings (Muday and DeLong). On the other hand, acropetal auxin transport is unaffected in rcn1, but shows a dramatic loss of NPA inhibition. Interestingly, rcn1 pin2 double-mutant analyses indicate that elevated basipetal transport in rcn1 does not require PIN2, leading to the suggestion that an NPA-binding protein is involved in this process (Rashotte et al, 2001). PID belongs to the AGC family of serine/threonine kinases, and forms—together with AGC3-4/PID2, WAG1 and WAG2—the clade AGC3 (Galvan-Ampudia and Offringa, 2007). PID loss- or gain-of-function changes the apical (shoot-wards) or basal (root-wards) cellular localization of PIN proteins influencing the direction of the auxin movement (Friml et al, 2004). As a consequence, in the pid mutant, PIN1 localizes to the basal membrane of epidermal cells, which in turn redirects auxin away from the meristem and prevents the initiation of new lateral organs. This results in a pin-shaped inflorescence (Christensen et al, 2000). On the other hand, PID overexpression leads to a basal-to-apical switch of PIN1, PIN2 and PIN4 in root cortex and lateral root cap cells, and finally to a collapse of the root meristem probably due to auxin depletion (Michniewicz et al, 2007). WAG1WAG2 loss-of-function mutations show an auxin-dependent root waving phenotype, and root curling is more resistant to NPA (Santner and Watson, 2006). Recently, PID, WAG1 and WAG2 were shown to phosphorylate PIN carriers at a conserved TPRXS(N/S) motif in the central hydrophilic loop leading to PIN recruitment into the apical recycling pathway (Dhonukshe et al, 2010; Huang et al, 2010). Moreover, disruption of PID and its three closest homologues completely abolishes the formation of cotyledons (Cheng et al, 2008). These findings, together with the fact that WAG1 and WAG2 are apolar and plasma-membrane-associated, suggested that AGC3 kinases act in the same or in a parallel regulatory pathway of PAT (Santner and Watson, 2006). Very recently, photoreceptor AGC4 kinase PHOTOTROPIN1 (phot1) was shown to phosphorylate nucleotide-binding domains (NBDs) of ABCB19 inhibiting its efflux activity (Christie et al, 2011). Genetic and pharmacological analyses revealed that PID and PP2A antagonistically regulate basipetal auxin transport and gravitropic response in the root tip (Sukumar et al, 2009). Loss of PID activity alters the PIN2-mediated basipetal auxin transport and impedes the gravitropic response, without causing an obvious change in PIN2 cellular polarity. This finding indicates that PID promotes and enhances root gravitropism, but is not absolutely required. Furthermore, PID appears to have a specific regulatory effect on the basipetal transport machinery in the root, since the acropetal transport is unaffected in pid. All these data are widely consistent with the concept of PID as a positive regulator of IAA efflux (Lee and Cho, 2006). The direct effect of PID on individual transporter activities, however, has not yet been addressed. Here, we identify and characterize PID as a relevant partner of the TWD1/ABCB subcomplex. Our data suggest that PID, besides its function as a molecular switch of PIN polarity, has a direct, dual impact on ABCB-mediated auxin-efflux activity. Transporter regulation is reversed by binding of the protein-kinase inhibitor, quercetin, a modulator of auxin transport. Results Identification of PINOID as a partner in the ABCB–TWD1 auxin-efflux complex With the aim of identifying novel components of the auxin-efflux complex, characterized by ABCB1 and TWD1, we employed an IP approach followed by shotgun mass spectrometry analysis of TAPa-tagged TWD1 (TAPa-TWD1). Originally, we chose second-generation TAPa tagging, thus offering the possibility of IgG-BD-, 6xHIS- and 9xMYC epitope purification, that had been optimized for the identification of Arabidopsis protein complexes by two-step affinity purification. However, as IP using the HIS-tag only gave poor protein retention and the IgG-BD interfered with the MS analysis, we employed an one-step MYC IP. As starting material, we used total microsomes of 9-dag (days after germination) Arabidopsis roots, thus reducing typical nonspecific contaminants (like ribosomal proteins, chaperons and Rubisco; Supplementary Table S1). To gain further specificity of TWD1-interacting proteins, we subtracted identified vector control (35S:TAPa) proteins from pulled-down TAPa-TWD1 proteins, allowing elimination of proteins binding to the TAPa-tag alone (Supplementary Table S1). Finally, proteins with a score above 30 were considered as significant partners (Figure 1A). This procedure was repeated with each of the two independent transformants resulting in essentially the same TWD1 partners. Figure 1.PINOID physically interacts with the TWD1 immuno-complex. (A) TWD1 partners identified by co-IP using TAPa-TWD1 followed by shotgun MS/MS analysis. MASCOT-identified vector control (35S-TAPa) proteins were subtracted manually from TAPa-TWD1 proteins, and proteins with a score above 30 were considered as significant partners (see Supplementary Table S1 for complete listing and Supplementary Figure S1 for PID peptide sequences). (B, C) Expression of TWD1, PID and ABCB1 proteins overlap in primary root tips based on Arabidopsis gene Expression Database (AREXDB; B) and on confocal microscopy analysis of 35S:TWD1-YFP, 35S:PID-GFP and 35S:ABCB1-YFP (C). Upper row, confocal mid-plane of root tips (scale bar: 50 μm); lower row, close-up of cortical cells (bar, 20 μm). (D) BRET analysis of microsomes prepared from N. benthamiana leaves co-transfected with TWD1-Rluc (35S:TWD1-Rluc) and PID-GFP (35S:PID-GFP). Both TWD1 (TWD1-YFP) and PID (PID-GFP) colocalize on the plasma membrane of transfected protoplasts (inset). Positive BRET ratios that are comparable with established TWD1-Rluc/ABCB1-YFP interaction (Bailly et al, 2012a) are not found with negative plasma-membrane controls, such as PIRK-YFP, suggesting physical interaction in planta. (E) Immobilized PID-GST but not GST or glutathion-sepharose beads are able to pull-down significant amounts of purified TWD11-337in vitro indicated by an asterisk (T, total; N, non-bound; B, bound fractions). (F) ABCB1-GFP (ABCB1:ABCB1-GFP) colocalizes with PID-VENUS (PID:PID-VENUS) in epidermal layers of the root tip (upper row, confocal mid-plane of root tips (scale bar, 50 μm); lower row, epidermal planes (bar, 20 μm). Figure source data can be found with the Supplementary data. Source Data for Figure 1E [embj2012120-sup-0002.pdf] Download figure Download PowerPoint Besides TWD1 as an obvious dominant pulled-down protein (protein score of 129, 13.4% coverage; Figure 1A), we found a so-far uncharacterized polynucleotide adenylyltransferase-like protein (At3G48830) and an unknown protein (At4G25920). In addition, two protein kinases, the histidine-like kinase AHK5/CYTOKININ INDEPENDENT2 (At5G10720) that regulates root elongation (Iwama et al, 2007) and the ACG kinase PINOID (At2G34650), and the catalytic domain of the PP2C-type protein phosphatase, AP2C1 (At2G30020), were also identified. AP2C1 is known to be involved in innate immunity responses by the negative regulation of the map kinases MPK4 and MPK6 (Schweighofer et al, 2007). PINOID (PID) is a well-known key player in polar auxin transport regulation (Galvan-Ampudia and Offringa, 2007) and was therefore chosen for further analysis. TWD1/ABCB1 and PID show overlapping locations, mostly in epidermal and cortical cell layers, which is illustrated by confocal microscope analysis of PID-GFP, TWD1-YFP and ABCB1-YFP lines (Figure 1C). Although expressed under the control of the strong constitive 35SCaMV promoter, locations widely match expression profile predictions, such as those from the Arabidopsis gene Expression Database (AREXDB; Figure 1B), suggesting posttranscriptional modifications. TWD1-PID and ABCB1-PID colocations on the plasma membrane were further substantiated by co-expression in tobacco protoplasts (Figures 1D and 3B, Supplementary Figure S3). TWD1–PID interaction was substantiated by in-planta bioluminescence resonance energy transfer (BRET) measurements (Bailly et al, 2008, 2012a) after co-expressing Renilla luciferase- and GFP-tagged versions of TWD1 (TWD1-Rluc) and PID (PID-GFP), respectively, in Nicotiana. benthamiana leaves. TWD1-Rluc, like Rluc-TWD1 (Bailly et al, 2012a), was shown to be functional by complementation of twd1-3 (Supplementary Figure S1E). Co-transfection of TWD1-Rluc and PID-GFP, widely colocalizing on the plasma membrane (Figure 1D inset), resulted in significant BRET ratios that are comparable to those found for the established TWD1–ABCB interaction (Bailly et al, 2012a), an indication for a physical proximity of <100 Å, and thus interaction. This interaction is specific since single expression of TWD1-Rluc, Rluc or YFP alone or TWD1-Rluc in combination with the non-related, plasma-membrane-bound protein kinase, PIRK (PIRK-YFP), only resulted in negligible BRET ratios. Identification of PID as a TAPa-TWD1 partner does not necessarily imply direct physical interaction as the PID–TWD1 interaction might also be mediated via a third TWD1-interacting protein. Moreover, only one PID peptide with a MASCOT score of 32 was found in the MS analysis (Supplementary Figure S1). Therefore, in order to verify the IP data and to test a direct mode of interaction, we performed in vitro pull-down experiments using recombinant PID-GST (Christensen et al, 2000) and TWD11-337 protein. TWD11-337 purified as described (Kamphausen et al, 2002) contains all functional domains, such as the FKBD, the CaM-binding and TPR domain, except the C-terminal hydrophobic in-plane membrane anchor. Indeed, PID-GST, but not GST alone nor the empty-beads control, was able to pull-down small but significant amounts of TWD11-337 (Figure 1E). In summary, our results demonstrate the utility of the IP approach employed for discovering valid protein–protein interactions of auxin transport complexes in Arabidopsis and suggest a relevant TWD1–PID interaction in planta. PID has a dual impact on ABCB1-mediated auxin efflux PID defines polar PIN locations and thus the directionality of auxin streams by direct PIN phosphorylation (Dhonukshe et al, 2010; Huang et al, 2010). A comparable mechanism has not so far been demonstrated for mainly nonpolar ABCBs or for TWD1, which functions as a regulator of ABCB1-efflux activity (Bouchard et al, 2006; Bailly et al, 2008). Therefore, we quantified ABCB1-mediated auxin efflux in the presence and absence of MYC-tagged PID in yeast. Surprisingly, PID significantly reduced ABCB1, but not vector control (background), IAA efflux by roughly 30% (Figure 2A). This inhibition corresponds to a 70% inhibition of ABCB1-specific efflux and is comparable to that found for ABCB1/TWD1 co-expression in yeast (Bailly et al, 2008). This inhibitory effect, caused by PID, was specific, as it was not found with the mutated, kinase-negative MPID (Christensen et al, 2000) or the non-related GSK-3-like kinase BRASSINOSTEROID-INSENSITIVE2 (BIN2), a negative regulator of brassinosteroid (BR) signalling and cell elongation (Vert, 2008). The specificity of PID-induced inhibition of IAA transport was further underlined by the fact that no effect was found for the unspecific, diffusion control, benzoic acid (BA) (Figure 2A lower panel). Figure 2.PID modulates ABCB1-mediated auxin efflux in yeast. (A) PID specifically inhibits ABCB1-mediated IAA export, while a mutated, inactive PID, MPID or unrelated protein kinase, BIN2, has no significant effect. Reduction of auxin retention (export) was calculated as relative export of initial export where ABCB1 was set to 100% (mean±s.e.; n=4–10). Significant differences (unpaired t-test with Welch's correction, P<0.05) between –PID controls are indicated by asterisks. (B, C) Co-expression with PID, MPID or BIN2 does not significantly alter location (B) and expression (C) of ABCB1-YFP as revealed by confocal microscopy (B) and western analysis (C). Each 20 μg of protein was subjected to PAGE and western analysis using anti-GFP and plasma-membrane marker, anti-PMA1 (H+-ATPase). ABCB1-YFP localizes primarily to raft-like structures and the plasma membrane (see Supplementary Figure S2; Bailly et al, 2008). Bar, 2 μm. Download figure Download PowerPoint Moreover, PID or MPID co-expression did not substantially alter ABCB1-plasma- membrane expression or localization in yeast (Figure 2B and C, Supplementary Figure S2A). ABCB1-YFP localizes primarily to raft-like structures at the boundaries of the plasma membrane as previously shown (Figure 2B; Bailly et al, 2008) and to the plasma membrane as demonstrated by comparison of anti-GFP immune-positive fractions in comparison with the plasma-membrane H+-ATPase, PMA1 (Supplementary Figure S2). To further substantiate our yeast-generated results, we established a novel heterologous plant transport system that allowed quantification of auxin efflux of ABCB1, PID and TWD1 combinations from mesophyll protoplasts that were isolated from co-transfected N. benthamiana leaves. In agreement with described roles for PID on auxin-efflux activity (Lee and Cho, 2006), PID strongly activated ABCB1-catalysed IAA and NAA efflux. This action is specific as PID expression alone enhanced vector control IAA and NAA efflux only slightly, probably by activation of endogenous tobacco ABCB-type auxin exporters. Moreover, closely related AGC3 kinase, WAG1 sharing overlapping functionality and 39% protein sequence identity with PID (Dhonukshe et al, 2010; Huang et al, 2010), had no significant effect on ABCB1. In order to address the role of TWD1 in PID-mediated activation of ABCB1, we quantified auxin efflux from triple-transfected ABCB1/PID/TWD1 protoplasts. Surprisingly, co-expression of ABCB1/PID/TWD1 entirely abolished ABCB1 auxin efflux. Co-expression of ABCB1/TWD1 revealed that at least for IAA a portion of this inhibitory effect is caused by TWD1 itself. However, NAA efflux analysed in parallel clearly demonstrated that PID, in the presence of TWD1, has a significant inhibitory effect on ABCB1 auxin efflux (Figure 3A). The finding that TWD1 only affected IAA but not NAA export, as previously reported for Arabidopsis (Bouchard et al, 2006) was also found for yeast, where TWD1 as reported here has an inhibitory role on ABCB1 (Bouchard et al, 2006; Bailly et al, 2008, 2012a), suggests that TWD1 besides its role as a regulator of activity also has an impact on ABCB1 specificity. Figure 3.PID negatively regulates ABCB1-mediated auxin efflux in planta. (A) Co-transfection of N. benthamiana protoplasts with PID specifically enhances ABCB1-mediated auxin (IAA and NAA) efflux in the absence of TWD1 but triple ABCB1/PID/TWD1 transfection strongly blocks ABCB1 activity (mean±s.e.; n=4). Significant differences (unpaired t-test with Welch's correction, P<0.05) to vector control, ABCB1 and ABCB1/PID are indicated by one, two or three asterisks, respectively. (B) Co-transfection of PID (PID-GFP) and TWD1 (TWD1-GFP or TWD1-YFP) in N. benthamiana protoplasts does not significantly alter ABCB1 (ABCB1-YFP or ABCB1-CFP) location and expression in comparison to vector control co-expression (upper row). Note the colocalization of ABCB1, TWD1 and PID on the plasma membrane of tobacco protoplasts (lower row; insets in upper row show indicated details at higher magnification). For single and double co-expression controls, see Supplementary Figure S3H. Bar, 20 μm. (C) Efflux of native (IAA) and synthetic (NAA) auxin from Arabidopsis PID gain- (35S:PID) and loss-of-function (pid) protoplasts (means±s.e.; n=4; see Supplementary Figure S3C and D for time kinetics). (D) Free IAA levels determined by GC–MS are significantly elevated in the root of PID gain-of-function (35S:PID) and shoot of PID loss-of-function lines (pid+/−), respectively. Data are mean±s.e. (n=4 with each 40–50 seedlings). Note that material for C–D was prepared from heterozygous pid (pid+/−) plants since, due to technical limitations, a determination of homozygosity by shoot phenotyping or genotyping was not possible. Significant differences (unpaired t-test" @default.
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• W2111280003 title "Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation" @default.
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