Flavonol‐mediated stabilization of PIN efflux complexes regulates polar auxin transport
2020; Springer Nature; Volume: 40; Issue: 1 Linguagem: Inglês
10.15252/embj.2020104416
ISSN1460-2075
AutoresWilliam Teale, Taras Pasternak, Cristina Dal Bosco, Alexander Dovzhenko, Krystyna Kratzat, Wolfgang Bildl, Manuel Schwörer, Thorsten Falk, Benedetto Ruperti, Jonas V. Schaefer, Mojgan Shahriari, Lena Pilgermayer, Xugang Li, Florian Lübben, Andreas Plückthun, Uwe Schulte, Klaus Palme,
Tópico(s)Plant nutrient uptake and metabolism
ResumoArticle13 November 2020Open Access Source DataTransparent process Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport William D Teale Corresponding Author William D Teale [email protected] orcid.org/0000-0003-0956-7372 Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Taras Pasternak Taras Pasternak Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Cristina Dal Bosco Cristina Dal Bosco Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Alexander Dovzhenko Alexander Dovzhenko orcid.org/0000-0002-9940-3074 Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Krystyna Kratzat Krystyna Kratzat Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Wolfgang Bildl Wolfgang Bildl Institute of Physiology II, Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Manuel Schwörer Manuel Schwörer Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Thorsten Falk Thorsten Falk Institute for Computer Science, University of Freiburg, Freiburg, Germany Search for more papers by this author Benadetto Ruperti Benadetto Ruperti Department of Agronomy, Food, Natural resources, Animals and Environment—DAFNAE, University of Padova, Padova, Italy Search for more papers by this author Jonas V Schaefer Jonas V Schaefer High-Throughput Binder Selection Facility, Department of Biochemistry, University of Zurich, Zurich, Switzerland Search for more papers by this author Mojgan Shahriari Mojgan Shahriari Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Lena Pilgermayer Lena Pilgermayer Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Xugang Li Xugang Li Sino German Joint Research Center for Agricultural Biology, and State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai'an, China Search for more papers by this author Florian Lübben Florian Lübben orcid.org/0000-0003-2398-4519 Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Andreas Plückthun Andreas Plückthun orcid.org/0000-0003-4191-5306 High-Throughput Binder Selection Facility, Department of Biochemistry, University of Zurich, Zurich, Switzerland Search for more papers by this author Uwe Schulte Uwe Schulte Institute of Physiology II, Faculty of Medicine, University of Freiburg, Freiburg, Germany Logopharm GmbH, Freiburg, Germany Signalling Research Centres BIOSS and CIBSS, Freiburg, Germany Search for more papers by this author Klaus Palme Corresponding Author Klaus Palme [email protected] orcid.org/0000-0002-2728-3835 Institute of Biology II, University of Freiburg, Freiburg, Germany Signalling Research Centres BIOSS and CIBSS, Freiburg, Germany Search for more papers by this author William D Teale Corresponding Author William D Teale [email protected] orcid.org/0000-0003-0956-7372 Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Taras Pasternak Taras Pasternak Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Cristina Dal Bosco Cristina Dal Bosco Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Alexander Dovzhenko Alexander Dovzhenko orcid.org/0000-0002-9940-3074 Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Krystyna Kratzat Krystyna Kratzat Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Wolfgang Bildl Wolfgang Bildl Institute of Physiology II, Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Manuel Schwörer Manuel Schwörer Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Thorsten Falk Thorsten Falk Institute for Computer Science, University of Freiburg, Freiburg, Germany Search for more papers by this author Benadetto Ruperti Benadetto Ruperti Department of Agronomy, Food, Natural resources, Animals and Environment—DAFNAE, University of Padova, Padova, Italy Search for more papers by this author Jonas V Schaefer Jonas V Schaefer High-Throughput Binder Selection Facility, Department of Biochemistry, University of Zurich, Zurich, Switzerland Search for more papers by this author Mojgan Shahriari Mojgan Shahriari Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Lena Pilgermayer Lena Pilgermayer Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Xugang Li Xugang Li Sino German Joint Research Center for Agricultural Biology, and State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai'an, China Search for more papers by this author Florian Lübben Florian Lübben orcid.org/0000-0003-2398-4519 Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Andreas Plückthun Andreas Plückthun orcid.org/0000-0003-4191-5306 High-Throughput Binder Selection Facility, Department of Biochemistry, University of Zurich, Zurich, Switzerland Search for more papers by this author Uwe Schulte Uwe Schulte Institute of Physiology II, Faculty of Medicine, University of Freiburg, Freiburg, Germany Logopharm GmbH, Freiburg, Germany Signalling Research Centres BIOSS and CIBSS, Freiburg, Germany Search for more papers by this author Klaus Palme Corresponding Author Klaus Palme [email protected] orcid.org/0000-0002-2728-3835 Institute of Biology II, University of Freiburg, Freiburg, Germany Signalling Research Centres BIOSS and CIBSS, Freiburg, Germany Search for more papers by this author Author Information William D Teale *,1, Taras Pasternak1, Cristina Dal Bosco1, Alexander Dovzhenko1, Krystyna Kratzat1, Wolfgang Bildl2, Manuel Schwörer1, Thorsten Falk3, Benadetto Ruperti4, Jonas V Schaefer5, Mojgan Shahriari1, Lena Pilgermayer1, Xugang Li6, Florian Lübben1, Andreas Plückthun5, Uwe Schulte2,7,8 and Klaus Palme *,1,8 1Institute of Biology II, University of Freiburg, Freiburg, Germany 2Institute of Physiology II, Faculty of Medicine, University of Freiburg, Freiburg, Germany 3Institute for Computer Science, University of Freiburg, Freiburg, Germany 4Department of Agronomy, Food, Natural resources, Animals and Environment—DAFNAE, University of Padova, Padova, Italy 5High-Throughput Binder Selection Facility, Department of Biochemistry, University of Zurich, Zurich, Switzerland 6Sino German Joint Research Center for Agricultural Biology, and State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai'an, China 7Logopharm GmbH, Freiburg, Germany 8Signalling Research Centres BIOSS and CIBSS, Freiburg, Germany *Corresponding author. Tel: +49 761 20367861; E-mail: [email protected] *Corresponding author. Tel: +49 761 2032954; E-mail: [email protected] The EMBO Journal (2021)40:e104416https://doi.org/10.15252/embj.2020104416 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 Abstract The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue-native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo- and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole-3-acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex. Synopsis PIN-FORMED (PIN) transporters regulate distribution of the phytohormone auxin in plant tissues. The current study shows that PIN core complexes are formed by PIN homo- and heterodimers, which are stabilized by the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) and natural flavonols. Quantitative mass spectrometry identifies PIN protein homo- and heterodimers at the plasma membrane, with PIN1 homodimers most prevalent NPA and naturally-occurring flavonols stabilize PIN dimers Auxin treatment or PIN1 phosphomimetic amino acid substitutions reduce formation of PIN complexes Antibody-mediated blocking of PIN1-PIN1 interactions renders plants less sensitive to NPA Introduction In plants, concentration gradients of auxin direct cell identity and growth (Benkova et al, 2003). Although PIN auxin efflux carriers, polarly localized major facilitator superfamily (MFS) proteins, establish these gradients in response to developmental and environmental cues, they are not solely responsible for the regulated transport of auxin out of the cell (Paponov et al, 2005; Geisler et al, 2016). For many years, specific inhibitors have played a key role in the identification and characterization of the wider polar auxin transport (PAT) machinery (Morgan & Soding, 1958; Hertel et al, 1983). One such compound, 1-naphthylphthalamic acid (NPA), inhibits a second group of dedicated auxin efflux carriers belonging to the ABCB family of multidrug efflux proteins. NPA disrupts the association between ABCB19 and the immunophilin-like FKBP42 TWISTED DWARF1 (TWD1) (Bailly et al, 2008); however, several lines of evidence suggest that this interaction is not the only way in which NPA inhibits PAT. The complicated nature of cellular auxin efflux regulation is illustrated by the observation that NPA-treated plants resemble the pin1 but not the abcb19 phenotype in diverse species (Katekar & Geissler, 1980; Okada et al, 1991; Reinhardt et al, 2000; Xu et al, 2005). Furthermore, NPA binds to ABCB proteins at a concentration of 10 nM, (Zhu et al, 2016) but only inhibits auxin transport when present in excess of 100 nM (Michalke et al, 1992). Thirdly, in Arabidopsis, abcb19 and pin1 both display reductions in basipetal auxin transport capacity of over 50% when compared to wild type (Okada et al, 1991; Noh et al, 2001), implying that their mechanisms at least partially overlap. Indeed, the proteins have been shown to interact in plants, with ABCB19 stabilizing PIN1 in specific membrane microdomains (Titapiwatanakun et al, 2009) lending weight to the hypothesis that they both represent components of a common auxin efflux protein complex (Blakeslee et al, 2007). The ability of PIN1 and ABCB19 together to stimulate cellular auxin efflux in an NPA-sensitive manner has been demonstrated after their co-expression in human cells (Rojas-Pierce et al, 2007). However, to date, no careful characterization of the stoichiometry of native PIN1/ABCB19-containing protein complexes has been carried out. ABCB19 interacts with TWD1 in the absence of NPA with the function of this interaction appearing to be related to the proper trafficking of ABCB19 to the plasma membrane (Bailly et al, 2008; Wu et al, 2010). However, despite such detailed insights, an overall understanding of the relationship between NPA and the auxin efflux complex remains elusive (Geisler et al, 2016). Two NPA-binding sites of low and high affinity at the plasma membrane have been proposed, but it is the estimated dissociation constant of the low-affinity site (approximately 1 µM) which correlates closely with the concentration of NPA which inhibits auxin efflux (Michalke et al, 1992). However, it is ABCB19 (with a relatively high affinity for NPA) and not PIN1 (not a high-affinity NPA-binding protein) which has been shown most convincingly to be the target for NPA-mediated auxin transport inhibition. NPA is a particularly important inhibitor as it gives easy experimental access to an endogenous mechanism which regulates PAT. It does this by competing for membrane-binding sites with flavonols such as kaempferol and quercetin (Rubery, 1990). In general, those flavonols which most efficiently inhibit auxin transport also show the greatest ability to displace NPA from cell membranes (Jacobs & Rubery, 1988), and tt4, an Arabidopsis genotype unable to synthesize flavonols, displays higher rates of PAT (Buer et al, 2013). Although the currently available data suggest that flavonols inhibit ABCB-mediated auxin efflux (Geisler et al, 2005), they also indicate that this interaction is embedded into a more intricate regulatory mechanism, possibly also involving the direct inhibition of MFS proteins. In this report, we investigate the relationship between NPA and the PIN proteins, showing that (i) NPA directly inhibits PIN1-mediated cellular auxin efflux, (ii) the functional core of the PIN protein complex contains only trace amounts of ABCB proteins, but (iii) comprises a PIN dimer which is stabilized by both NPA and flavonols, and (iv) this stabilization is necessary for the inhibition of PIN-mediated auxin efflux by NPA and flavonols, thus revealing a crucial regulatory mechanism. Results In order to establish whether NPA is able to inhibit PIN1-dependent cellular auxin efflux in plants, an efflux assay was designed in which a nuclear auxin sensor was co-expressed with PIN1 in Arabidopsis leaf protoplasts (Wend et al, 2013). Here, auxin-mediated degradation of an AUX/IAA domain II-linked firefly luciferase sensor, normalized with a translationally fused Renilla luciferase, enabled the relative quantification of internal auxin concentration between populations of cells incubated in solutions containing different concentrations of the natural auxin, indole-3-acetic acid (IAA). These measurements enabled the relative efflux capacity of the cells to be inferred. All assay conditions have been previously optimized elsewhere (Wend et al, 2013). In untransformed cells, NPA did not significantly alter the accumulation of intracellular IAA (as measured by the stabilization of firefly luciferase activity) when protoplasts were incubated in external IAA concentrations ranging between 1 nM and 10 µM, indicating that no NPA-sensitive background auxin transport activity could be measured. However, PIN-dependent IAA efflux, which counteracted the intracellular accumulation of IAA, occurred in protoplasts transiently expressing PIN1 after incubation in IAA solutions at concentrations higher than 1 nM (Fig 1A). This activity of PIN1 was largely inhibited by the application of NPA, over nearly the whole range of IAA concentrations investigated (Fig 1A). Figure 1. NPA inhibits PIN1-dependent cellular auxin efflux A. Normalized firefly luciferase luminescence (compared to Renilla luciferase) as a function of external IAA concentration. Arabidopsis protoplast cells were transiently transformed with either AtPIN1 or GFP (in the case of the control) both under the control of a constitutive CaMV 35S promoter. Where indicated, 10 µM NPA was added. B. Normalized firefly luciferase luminescence (compared to Renilla luciferase) as a function of external NPA concentration in the presence of 100nM IAA in control GFP (blue or red)- or PIN1 (green or purple)-transformed protoplasts. Data information: Each point is the mean of six measurements normalized to the firefly luciferase signal from cells with no external auxin added. n = 6, error bars indicate standard error. Download figure Download PowerPoint An NPA concentration of around 5 µM was sufficient to fully inhibit PIN1-dependent auxin transport in this system (Fig 1B). These values correspond well with both the Kd of the interaction between NPA and the low-affinity microsomal NPA-binding site (Michalke et al, 1992) and the concentrations at which NPA inhibits PAT in plants (Hertel & Leopold, 1962). We therefore conclude that in its native environment of the plant plasma membrane, PIN1 is able to effect the cellular efflux of IAA, and this efflux is inhibited by NPA. PINs are not the only plant proteins which transport auxin in an NPA-sensitive manner. ABCB19, ABCB1 and ABCB21-dependent auxin efflux are also inhibited by NPA, and knockout plants have a reduced capacity for PAT (Noh et al, 2001; Bailly et al, 2008; Jenness et al, 2019). As ABCB19 has previously been shown to interact with PIN1 (Blakeslee et al, 2007), we set out to test specifically whether ABCB19 and PIN1 were core components of the same complex. As a first approach, Arabidopsis plasma membrane proteins were solubilized under low (ComplexioLyte 47 [CL47]) or high (ComplexioLyte 27 [CL27]) stringency conditions (optimized mixtures of non-ionic and partially ionic detergents, respectively; Logopharm GmbH) and resolved by blue-native polyacrylamide gel electrophoresis (BN-PAGE). An optimum solubilization protein–detergent ratio of 1:7 (w/w) was determined in titration experiments to exclude size shift artefacts that could result from incomplete solubilization (Fig EV1A). Subsequent SDS–PAGE separation showed PIN1 to be a constituent of a homogenous protein complex focusing at an apparent molecular size of around 350 kDa at low stringency (Fig 2A). Upon more stringent solubilization with CL27, dissociation into smaller subcomplexes occurred, with more dispersed populations of PIN1 detected at between 100 kDa and 300 kDa (Fig 2B). Click here to expand this figure. Figure EV1. Examination of the PIN protein complex A. Native PIN1 protein complex analysis by blue-native PAGE. Arabidopsis microsomes containing 30µg protein were solubilized by ComplexioLyte 27 at the concentrations indicated. Western blots used an anti-PIN1 antibody. B. Mass spectrometry-based QconCAT quantification of PIN subtypes and PGP19 in individual PIN-GFP immunoprecipitates. C, D. After transient expression in tobacco leaves, PIN1-GFP interacts with (C) 3xHA-PIN3 and (D) 3xHA-PIN1. In each case, Western blots used an anti-HA antibody. Cont, control; I, input; FT, flow-through; W, last column wash; E, elution. Control pull-downs on the right in both images labelled No bait lack PIN1-GFP. Download figure Download PowerPoint Figure 2. Definition and composition of the PIN1 core complex A, B. Native PAGE separation reveals distinct detergent-sensitive PIN1 complexes. Plasma membrane preparations from dark-grown Arabidopsis cell suspension cultures were solubilized with (A) ComplexioLyte 27 (a mixture of ionic and non-ionic detergents) and (B) ComplexioLyte 47 (lower stringency, non-ionic detergent) (both Logopharm); first dimension BN-PAGE, second dimension SDS–PAGE, blots stained with anti-PIN1 antibody revealing distinct PIN1 complex populations at the indicated positions. Values are given in KDa. C. Molecular abundance (abundancenormspec values calculated as summarized peptide PVs divided by the number of MS-accessible protein-specific amino acids) of PIN subunits and ABCB19 in anti-GFP affinity purification of CL47-solubilized PIN1-GFP-expressing roots with a GFP-specific DARPin. Note the significant heteromerization of several endogenous PINs with PIN1-GFP and the absence of other abundant interaction partners (ABC19B shown as an example). Download figure Download PowerPoint To identify the protein composition of the larger, low stringency-solubilized PIN assembly, we affinity-purified GFP-fused PIN proteins from Arabidopsis roots after expression from their native promoters and after the induction of discrete lateral roots and analysed the captured proteins by label-free quantitative mass spectrometry (LC-MS/MS). This approach included GFP-PINs 1, 3, 4 and 7 proteins, which are all exclusively localized to the plasma membrane, share similar expression domains and display considerable functional redundancy in the Arabidopsis root apical meristem, as well as GFP-PIN2 (Blilou et al, 2005). Proteins were solubilized and subjected to affinity purification (AP) with an immobilized anti-GFP monoclonal antibody or GFP-specific designed ankyrin repeat protein (DARPin) (Dreier et al, 2011; Brauchle et al, 2014); the DARPin, a small, stable GFP-binding protein, significantly out-performed more traditional antibody-based purifications in our analysis. Proteins that bound a negative control bait protein (the plasma membrane-bound LTi6b-GFP) in a parallel experiment were not considered as PIN interactors. Under these conditions, all GFP-tagged PIN proteins could be purified together with lesser amounts of endogenous untagged PIN subtypes and a limited set of other proteins including PGP19 (Fig EV1B). Determination of molecular abundance using concatenated protein standards for peptide intensity calibration (label-free QconCAT (Schwenk et al, 2014)) confirmed that PIN1-GFP co-purified only substoichiometric amounts of endogenous PINs and PGP19 (Fig 2C). These results suggest that native PIN 1, 2, 3, 4 and 7 core complexes may exist as detergent-labile dimers or tetramers in mostly homomeric configuration which do not contain PGP19. Formation of PIN1 heteromers was recapitulated by pull-down of either 3xHis-tagged PIN1 or PIN3 together with GFP-PIN1, after all constructs were transiently expressed in tobacco leaves (Fig EV1C and D). Endogenous PIN1 was detected in Western blots of PIN1-GFP (where the band intensities between PIN1 and PIN1-GFP proteins were similar), PIN3-GFP, PIN4-GFP and PIN7-GFP (Fig EV1B, lower panel). We next tested whether PIN1 complex formation was independent from other plant factors by heterologous reconstitution of the PIN1 complex in a human cell line. BN-PAGE analysis of PIN1-transfected HEK cells solubilized with CL27 showed two PIN1 signals, one at 100 kDa representing PIN1 monomers and one around 230 kDa likely resulting from PIN1 dimers (Fig 3A). However, PIN1 dimer formation/stability was significantly less in HEK cells than in Arabidopsis microsomes, prepared from MM2d cells, a dark-grown Arabidopsis cell culture consistently able to yield large amounts of homogenous material (Menges et al, 2002). We therefore hypothesized the existence of a plant-specific factor which stabilized PIN1 dimers. Figure 3. NPA and quercetin both stabilize PIN1 complex formation A. BN-PAGE was performed with PIN1-containing microsomes prepared from either PIN1-expressing HEK293T cells or a dark-grown Arabidopsis cell suspension culture. Prior to solubilization with 1% dodecyl maltoside, microsomes were incubated with either 10 µM NPA or 10 µM quercetin. B. PIN1 dimer stability induced by NPA after expression in HEK cells. Relative distribution between monomer and dimer after NPA treatment is given relative to distribution of untreated samples after solubilization with 50% (v/v) CL27. Each measurement given (three for each concentration) represents the mean of three gel lanes for wild type (circles) or triple S2523, S253E and S261E phosphomimetic sequences (crosses) (example images are given in the figure inset). Download figure Download PowerPoint We next tested the influence of various auxin transport inhibitors and modulators of IAA transport on the stability of solubilized PIN1 dimers. Incubation of PIN1-expressing HEK membranes with 10 µM NPA or the flavonol quercetin (also at 10 µM), an endogenous functional analogue of NPA (Brown et al, 2001), prior to solubilization stabilized the PIN1 complex at 230 kDa, an identical size to the PIN1 complex found in plants and solubilized under identical conditions (Fig 3A). We therefore conclude that, in plants, the core PIN1 complex comprises flavonol-stabilized PIN dimers. Further experiments showed that naturally occurring flavonols varied in their ability to stabilize PIN interactions, but incubation with quercetin glycoside, a related compound which does not inhibit PAT, did not stabilize PIN1 dimers (Appendix Fig S1) (Jacobs & Rubery, 1988). The specificity of dimer stabilization was next tested as plant-derived microsome preparations were incubated with a range of compounds prior to their solubilization. As shown in Appendix Fig S2, at 50% CL27, PIN1 was distributed between 230 kDa complex and a monomer migrating at 100 kDa. However, in the presence of 10 µM NPA, PIN1 was localized exclusively to the 230 kDa complex. At concentrations of 10 µM, incubation in the presence of tryptophan, the PAT inhibitor 2,3,5 triiodobenzoic acid (TIBA) or the exocytosis inhibitor brefeldin A (BFA) did not alter the distribution of PIN1 between the 230 kDa and 100 kDa populations. We therefore conclude that the stabilization of the endogenous PIN1 auxin efflux core complex is specifically dependent on NPA and the PAT-inhibiting flavonols to which it is functionally related. To test whether this stabilization was specific to PIN1, we transiently expressed PIN4 in HEK293 cells before incubation with NPA, which resulted in the stabilization of PIN4 protein complexes (Appendix Fig S1). The stabilization of protein interactions by NPA is therefore likely to be a shared feature within the PIN1, PIN2, PIN3, PIN4 and PIN7 subfamily (Paponov et al, 2005). The regulation of function by substrate concentration is an established model of transporter feedback control (Zahniser & Doolen, 2001). We therefore wanted to know whether IAA effected its own efflux by counteracting the ability of NPA to stabilize PIN-PIN interactions (Paciorek et al, 2005). To this end, microsomes from HEK cells expressing PIN1 were solubilized and BN-PAGE was separated in the presence of IAA and NPA (Fig EV2A). PIN1 dimers were stabilized from 1 µM NPA. However, in the presence of IAA, the lowest concentration at which PIN1 dimers were observed was 6 µM NPA. At 10 µM NPA, the proportion of preserved PIN1 dimers was decreased by the addition of IAA (Fig EV2A). The presence of 10 µM IAA inhibited NPA-induced dimer stabilization, a process which was sensitive at a lower NPA concentration (Fig EV2B). We therefore conclude that IAA and NPA are able to act in an antagonistic manner on the stability of PIN1 dimers. Click here to expand this figure. Figure EV2. IAA counteracts the NPA-induced stabilization of PIN1 interactions A. Microsomes from PIN1-expressing HEK cells were incubated with either NPA (concentrations as indicated) or NPA in the presence of 100 µM IAA. Protein complexes were solubilized and separated under native conditions as described before. Western blotting was performed with an anti-PIN1 monoclonal antibody. B. The titration was also performed in the presence of 0.1 µM NPA and IAA (concentrations as indicated). Protein complexes were solubilized and separated under native conditions as described before. Western blotting was performed with an anti-PIN1 monoclonal antibody. Download figure Download PowerPoint The cytosolic domain of PIN1 is reversibly phosphorylated by AGC kinases at several positions (Michniewicz et al, 2007). The consequence of this phosphorylation is twofold: it changes the apical–basal polarity of the plasma membrane localization (Friml et al, 2004) and increases the rate of auxin efflux (Zourelidou et al, 2014). We therefore next introduced residues to mimic phosphorylated serine residues to test whether phosphorylation of the PIN1 cytosolic domain could lead to dimer instability. Three phosphoserines were identified in the PIN1 cytosolic domain in our MS/MS analysis of the affinity-purified solubilized PIN1 complex: Ser252, Ser253 and Ser261. After replacing each with glutamic acid, the full-length PIN1 sequences were expressed in HEK cells, and protein extracts were treated with NPA and solubilized as described above. 50 µM NPA failed to fully stabilize dimers of PIN1 proteins carrying phosphomimetic point mutations at all concentrations tested (Fig 3B). These data support the hypothesis that PIN1 phosphorylation functions, at least in part, by reducing the affinity with which core complexes enter into a stable inactive conformation. In order to ascertain whether PIN dimer stability can be regulated in living plants, PIN interactions in Arabidopsis root meristems were measured with a quantitative 3-D proximity ligation assay (PLA; Pasternak et al, 2018) after the exogenous application of NPA and IAA (both at 10 µM). Proximity ligation assays use complementary oligonucleotides fused to antibodies to determine the frequency with which proteins of interest find themselves in close proximity. A matrix of PIN interactions was tested in order to assess the reliability of the assay in Arabidopsis roots, with the prediction that fewer interactions should occur when target protein pairs have increasingly discrete expression domains (Fig EV3). Click here to expand this figure. Figure EV3. Proximity ligation assay marked heterodimers increase in frequency as co-expression domain size increasesA–R Co-immunolocalization of PINs (A–E); family members as indicated
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