The Arabidopsis ALF 4 protein is a regulator of SCF E3 ligases
2017; Springer Nature; Volume: 37; Issue: 2 Linguagem: Inglês
10.15252/embj.201797159
ISSN1460-2075
AutoresRammyani Bagchi, Charles W. Melnyk, Gideon Christ, Martin Winkler, Kerstin Kirchsteiner, Mohammad Salehin, Julia Mergner, Michael Niemeyer, Claus Schwechheimer, Luz Irina A. Calderón Villalobos, Mark Estelle,
Tópico(s)Plant nutrient uptake and metabolism
ResumoArticle12 December 2017free access Source DataTransparent process The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases Rammyani Bagchi Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Charles W Melnyk orcid.org/0000-0003-3251-800X Sainsbury Laboratory, University of Cambridge, Cambridge, UK Search for more papers by this author Gideon Christ Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle, Germany Search for more papers by this author Martin Winkler Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle, Germany Institute of Biology, Structural Biology/Biochemistry, Humboldt-University Berlin, Berlin, Germany Search for more papers by this author Kerstin Kirchsteiner Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Mohammad Salehin Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Julia Mergner Plant Systems Biology, Technische Universität München, Freising, Germany Search for more papers by this author Michael Niemeyer Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle, Germany Search for more papers by this author Claus Schwechheimer Plant Systems Biology, Technische Universität München, Freising, Germany Search for more papers by this author Luz Irina A Calderón Villalobos Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle, Germany Search for more papers by this author Mark Estelle Corresponding Author [email protected] orcid.org/0000-0002-2613-8652 Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Rammyani Bagchi Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Charles W Melnyk orcid.org/0000-0003-3251-800X Sainsbury Laboratory, University of Cambridge, Cambridge, UK Search for more papers by this author Gideon Christ Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle, Germany Search for more papers by this author Martin Winkler Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle, Germany Institute of Biology, Structural Biology/Biochemistry, Humboldt-University Berlin, Berlin, Germany Search for more papers by this author Kerstin Kirchsteiner Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Mohammad Salehin Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Julia Mergner Plant Systems Biology, Technische Universität München, Freising, Germany Search for more papers by this author Michael Niemeyer Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle, Germany Search for more papers by this author Claus Schwechheimer Plant Systems Biology, Technische Universität München, Freising, Germany Search for more papers by this author Luz Irina A Calderón Villalobos Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle, Germany Search for more papers by this author Mark Estelle Corresponding Author [email protected] orcid.org/0000-0002-2613-8652 Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Author Information Rammyani Bagchi1, Charles W Melnyk2,†, Gideon Christ3, Martin Winkler3,4, Kerstin Kirchsteiner1, Mohammad Salehin1, Julia Mergner5,†, Michael Niemeyer3, Claus Schwechheimer5, Luz Irina A Calderón Villalobos3 and Mark Estelle *,1 1Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA, USA 2Sainsbury Laboratory, University of Cambridge, Cambridge, UK 3Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle, Germany 4Institute of Biology, Structural Biology/Biochemistry, Humboldt-University Berlin, Berlin, Germany 5Plant Systems Biology, Technische Universität München, Freising, Germany †Present address: Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden †Present address: Proteomics and Bioanalytics, Technische Universität München, Freising, Germany *Corresponding author. Tel: +1 858 246 0453; E-mail: [email protected] EMBO J (2018)37:255-268https://doi.org/10.15252/embj.201797159 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 cullin-RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin-conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins. Synopsis ALF4, the Arabidopsis thaliana ortholog of the cullin-ring ubiquitin ligase regulator glomulin, stabilizes CUL1 in vivo, thereby decreasing the levels of Aux/IAA proteins and modulating the auxin hormone response. The Arabidopsis ALF4 protein is an ortholog of human glomulin (GLMN). ALF4 binds to the RBX1 subunit of cullin-ring ligases and inhibits SCFTIR1 in vitro. Loss of ALF4 results in stabilization of the SCF substrates IAA7 and RGA in vivo. Global levels of ubiquitinated proteins are reduced in the alf4 mutant. Introduction Ubiquitin–protein conjugation is a highly regulated process that involves ubiquitin-activating and conjugating enzymes (E1 and E2), as well as a ubiquitin ligase (E3). The E3 ligase coordinates with the E2 enzyme to conjugate ubiquitin to lysine residues in the substrate protein. The cullin-RING ligases (CRLs) are a large class of E3 ligases that consist of a cullin, a RING protein called RING BOX1 (RBX1), and a substrate adapter protein (Hua & Vierstra, 2011). In humans, CRLs have been implicated in a wide variety of cellular processes, including those related to cancer, while in plants they have a central role in diverse developmental and physiological processes (Hua & Vierstra, 2011; Kelley & Estelle, 2012; Zheng et al, 2016). The Skp1-Cullin1-F-box (SCF) E3s are a subclass of CRLs in which the substrate adapter consists of Skp1 (ASK in plants) and an F-box protein. Although there are many F-box proteins in all eukaryotes, the family has dramatically expanded in plants (~700 in Arabidopsis), suggesting that SCFs have been co-opted for many cellular and developmental programs (Gagne et al, 2002). SCF regulation is a highly dynamic process that involves several proteins and protein complexes (Deshaies & Joazeiro, 2009; Hua & Vierstra, 2011; Lydeard et al, 2013). These E3s are activated by conjugation of the ubiquitin-related protein RELATED TO UBIQUITIN (RUB), or NEDD8 in animals, to the C-terminus of the cullin subunit. Neddylation causes dramatic conformational changes in CUL1 and RBX1 that allow the RING domain on RBX1 to interact with the E2 (Duda et al, 2008). On the other hand, SCFs are inhibited by the COP9 SIGNALOSOME (CSN) through its de-neddylating activity as well as by direct binding to the SCF (Enchev et al, 2012). Another protein, CULLIN-ASSOCIATED NEDD8-DISSOCIATED PROTEIN 1 (CAND1), binds to the cullin and is important for substrate adapter exchange (Pierce et al, 2013; Wu et al, 2013; Zemla et al, 2013). The human disease glomuvenous malformation, characterized by cutaneous lesions, is caused by mutations in the Glomulin (GLMN) gene. In the familial form of this disease, affected individuals typically carry one loss-of-function glmn allele and experience a second somatic glmn mutation in the affected tissue (Duda et al, 2012; Tron et al, 2012). The glmn null mice die as embryos, suggesting that the gene is probably essential in humans (Tron et al, 2012). Recent studies indicate that GLMN regulates CRLs by binding to RBX1 and preventing the E2-conjugating enzyme from engaging the CRL (Duda et al, 2012; Tron et al, 2012). In human cells, one known consequence of glmn mutations is a decrease in the amount of the F-box protein Fbw7 and an increase in the level of Fbw7 substrates cyclin E and c-Myc (Tron et al, 2012). There are several well-characterized SCFs in plants, including SCFTIR1 and SCFSLY1 (Schwechheimer & Willige, 2009; Salehin et al, 2015; Lavy & Estelle, 2016). SCFTIR1 promotes the degradation of transcriptional repressors called Aux/IAA proteins in response to the hormone auxin, while SCFSLY1 promotes degradation of another class of transcriptional regulators, the DELLA proteins, in response to the hormone gibberellic acid (GA). Strikingly, several SCF subunits, as well as regulators of SCF activity, were originally identified through screens for auxin-resistant mutants in Arabidopsis (Walker & Estelle, 1998). The Arabidopsis aberrant lateral root formation 4 (alf4) mutant exhibits a number of auxin-related defects but its role in auxin signaling is unknown. The mutant was isolated in a screen for defects in root architecture, particularly a dramatic reduction in lateral root formation (Celenza et al, 1995; DiDonato et al, 2004). In addition, ALF4 is required for protoplast regeneration, callus formation, and efficient graft formation (Chupeau et al, 2013; Melnyk et al, 2015; Shang et al, 2016). Here, we demonstrate that ALF4 inhibits SCF ligases and is related to mammalian GLMN. Further, we show that Aux/IAA and DELLA proteins accumulate in the alf4 mutant. These results suggest that the developmental defects ascribed to the mutant are at least partly due to defects in hormone signaling. Results The alf4 mutants are resistant to auxin and display defects in root and shoot growth Previous studies established that the alf4-1 mutant has a normal primary root, but is deficient in lateral root initiation (Celenza et al, 1995; DiDonato et al, 2004). We confirmed this phenotype with three alf4 alleles containing deletions or T-DNA insertions (Figs 1A and B, and EV1). All three lines had normal or near-normal primary root elongation but formed dramatically fewer lateral roots. In addition, all three alleles were affected in shoot development (Fig 1C and D). The rosettes of mutant plants were much smaller than the wild-type control and had distorted leaves and twisted petioles. Rarely did the alf4-2 or alf4-063 mutants survive long enough to flower on soil, but alf4-1 produced a short and largely infertile inflorescence. Based on these results and previous observations, it is likely that alf4-1, a 12-bp deletion mutant, is not a null allele (DiDonato et al, 2004). To determine whether the effect of the mutation on the shoot was due to a reduced root system, we grafted alf4 scions onto Col-0 wild-type root stocks. As shown in Fig 1C and D, the wild-type root stock enhanced growth of the mutant shoot and increased fertility of the alf4-1 shoot. However, scions from alf4-2 and alf4-063 remained severely affected, indicating that ALF4 function was required in the shoot. Figure 1. The alf4 mutants exhibit a pleiotropic phenotype A, B. Primary root length (A) and lateral root number (B) of wild-type and mutant seedlings (different letters represent significant differences within a time point, mean ± SE, n = 34–49 roots/treatment, ANOVA with Tukey's post hoc test, P < 0.01). C. Wild-type and mutant plants 35 days after grafting. The top row are ungrafted, while the bottom row are mutant scion grafted onto a Col-0 root stock. D. Wild-type and mutant plants 70 days after grafting. E. Effect of auxin on wild-type and alf4-1 root growth. Five-day-old seedlings were transferred to fresh medium ± IAA and allowed to grow for 3 days. Growth is presented as the percentage of the DMSO control treatment for each genotype. Each value represents the mean, and error bars represent standard deviation (n ≥ 10). F. Gravitropic response in wild-type and alf4-1 seedlings. Seedlings grown on agar medium were rotated 90 degrees at t = 0. The angle of curvature from the horizontal was measured at the times indicated. Each point represents the mean of six measurements. Error bars represent the standard deviation (n ≥ 10). G. Expression of the auxin-responsive marker, pDR5:GFP, (green signal) imaged in the presence or absence of synthetic auxin NAA for 24 h. Roots were counterstained with propidium iodide (red signal). H. Quantification of GFP in (G) (different letters represent significant differences between groups, mean ± SE, n = 9–15 root tips/treatment, ANOVA with Tukey's post hoc test, P < 0.01). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Genomic locations of the Arabidopsis ALF4 mutationsA schematic of the ALF4 genomic region showing the alf4-1 deletion (green letters) and the alf4-2 and alf4-063 SALK T-DNA insertions. UTRs are red, exons orange with uppercase letters, introns blue with lowercase letters. T-DNA insertion information based on sequencing from the left border. Download figure Download PowerPoint The phenotype of the alf4 mutant suggests a defect in auxin signaling. To address this possibility, we examined the effects of auxin on primary root growth in alf4-1 and Col-0 plants and found that the mutant was resistant to low concentrations of IAA (Fig 1E). Further, alf4-1 plants exhibited a delayed gravitropic response, consistent with a defect in auxin signaling (Fig 1F). To determine whether ALF4 was required for the transcriptional response to auxin, we introduced the pDR5:GFP reporter into the alf4-1 mutant. We found that GFP signal was reduced in the mutant compared to the wild type in the absence and presence of the auxin 1-naphthyl acetic acid (NAA) (Fig 1G and H). In contrast, the response to the cytokinin N6-benzyladenine (BA) using the pARR5:GFP cytokinin reporter was largely unaffected in the alf4 mutant (Fig EV2). These results suggest that the pleiotropic phenotype exhibited by the alf4 mutants may be partly due to reduced auxin response. Click here to expand this figure. Figure EV2. ALF4 does not affect cytokinin response and is not substantially affected by auxin or cytokinin treatments Expression of the auxin-responsive marker, pDR5:GFP (green signal), imaged in the presence or absence of synthetic auxin NAA for 24 h. These images are the same as those presented in Fig 1G, but without the red channel. Expression of the cytokinin responsive marker pARR5:GFP in Col-0 and alf4-1 roots in the presence or absence of synthetic cytokinin (BA) for 24 h. Roots are counterstained with propidium iodide (red signal). BA treatment caused a strong ARR5 response in both wild-type plants and alf4-1 plants, whereas DMSO did not (mean ± SE, n = 5–10 root tips/treatment, ANOVA with Tukey's post hoc test, P < 0.01). Levels of pALF4:ALF4-GFP in root tips were not substantially affected by treatment with NAA, BA, or the auxin transport inhibitor NPA compared to DMSO controls. Increases in root vascular signal upon NAA treatment coincided with lateral root induction. Download figure Download PowerPoint Expression of ALF4 in the root The ALF4 gene is broadly expressed throughout the plant (DiDonato et al, 2004). To further examine expression of ALF4 in the root, we analyzed the previously published pALF4:ALF4-GFP line (DiDonato et al, 2004). We found that ALF4 protein was present in the nuclei of cells in the primary root tip, particularly the epidermal cells, and in the vascular tissue (Fig 2A and F). Consistent with the lateral root defect, ALF4 protein begins to accumulate in the lateral root primordium and continues to increase in levels throughout formation of the lateral root meristem and emergence of the lateral root (Fig 2B–E). To determine whether the ALF4 gene was regulated by auxin or cytokinin, we also treated the pALF4:ALF4-GFP line with NAA, the auxin transport inhibitor naphthylphthalamic acid (NPA), and BA. We did not observe a clear effect of these treatments on expression of the transgene (Fig EV2). In addition, publically available data show that ALF4 is not regulated by GA (http://bar.utoronto.ca) (Winter et al, 2007). Figure 2. ALF4 protein accumulates during lateral root formationpALF4:ALF4-GFP seedlings display ALF4-GFP (green signal) and are counterstained with propidium iodide (red signal). Scale bar is 50 μm. A. ALF4 is expressed in the vasculature. B–E. ALF4 protein accumulates in the lateral root primordium and the emerging lateral root. Asterisks highlight the location of the emerging lateral root. F. ALF4 protein is also present throughout the primary root tip. Download figure Download PowerPoint The ALF4 protein is related to GLMN and interacts with RBX1 The GLMN protein was recently shown to be an important regulator of cullin-RING E3 ligases in mammals (Duda et al, 2012; Tron et al, 2012). GLMN interacts with RBX1 and prevents binding of the E2 protein. Structural studies showed that GLMN consists of a series of helical repeats similar to HEAT repeats (Duda et al, 2012). The protein has two such domains, bisected by a single helix that is perpendicular to the other helices, while the RBX1-binding domain is in the C-terminal HEAT repeat domain. An amino acid alignment of GLMN and ALF4 revealed that the two proteins are ~25% identical along their entire length. Importantly, several key residues known to contribute to the interaction between GLMN and RBX1 are conserved in ALF4 (Fig EV3). In addition, the Phyre2 protein structure prediction server predicted that ALF4 was a helical repeat protein with an overall organization that is very similar to GLMN (Fig EV4) (Kelley et al, 2015). Click here to expand this figure. Figure EV3. Alignment of the ALF4 and human GLMN sequenceShading indicates conserved amino acids. Residues shown to be important for interaction between GLMN and RBX1 are indicated with black asterisks. ALF4 residues mutated to generate ALF4A484 and ALF4A614 are indicated with red asterisks. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Structure of GLMN and ALF4 X-ray structure of the Glomulin–RBX1–CUL1 complex. Adapted from PDB:4F52 originally published in Duda et al (2012). ALF4 schematic of the homology model generated with Phyre2 (webportal for protein modeling, prediction, and analysis; Kelley et al, 2015). 79% of ALF4 residues were modeled with > 90% confidence (red, see confidence key). 129 residues were modeled ab initio (blue). Download figure Download PowerPoint To determine whether ALF4 interacted with Arabidopsis RBX1, we performed a series of in vitro and in vivo experiments. Yeast two-hybrid assays demonstrated a strong interaction between the two proteins in this assay (Fig 3A). To further assess this interaction, we generated two ALF4 protein variants where the conserved K484 and R614 amino acids were replaced with alanine. Both residues contribute to the interaction between GLMN and RBX1 (Duda et al, 2012). In addition, we generated a mutant lacking the C-terminal 94 amino acids (ALF41-532stop). In the yeast assay, the strength of the interaction between ALF4A484A614 and RBX1 was similar to that of wild-type ALF4. In contrast, ALF41-532stop did not interact with RBX1, indicating that the C-terminal region of ALF4 is, as in the case of GLMN, important for RBX1 binding (Fig 3A). Figure 3. ALF4 interacts with RBX1 The ALF41-532stop mutant displays reduced interaction with RBX1 in comparison with full-length ALF4 protein in a yeast two-hybrid assay. Blue color represents X-GAL staining. EV, empty vector. RBX1 co-immunoprecipitates with ALF4 in extracts prepared from 14-day-old pALF4:ALF4-GFP plants. The pEF1a-GFP line serves as a control. In vitro pulldown of HIS-ALF4 and ALF4 mutants with GST-tagged RBX1 or GST alone. ALF4 variants do not interact with RBX1. BIFC assay testing the interaction of ALF4 or ALF4 mutants in pCYCE(R) vector with RBX1 cloned in pVYNE(R). Scale bars are 50 μm. Microscale thermophoresis (MST) analysis of ALF4 binding to Cul1–RBX. Thermophoresis curves for protein binding over a temperature gradient and over time (upper panel), and fitted curves plotting normalized fluorescence against concentration of ligands (lower panel). HsCul1–RBX1 interacts with ALF4 with a Kd = 346.04 ± 77.05 nM. Measurements were performed with a dilution series of ALF4 concentrations from 7 μM to 0.21 nM, and constant levels of fluorescently labeled Cul1–RBX1 (10 nM). Dissociation constant was calculated from three independent biological replicates. Binding of Cul1–RBX1 to not-charged E2 (UBC8) (without ubiquitin) serves as a negative control (blue). In the upper panel start (0 s) and end (21 s) of the temperature gradient were indicated with pink and green boxes, respectively. Error bars to correspond s.e.m. of three independently collected MST traces. See Figs EV5 and EV6 for MST raw data. Source data are available online for this figure. Source Data for Figure 3 [embj201797159-sup-0003-SDataFig3.pdf] Download figure Download PowerPoint To confirm the interaction between ALF4 and RBX1, we performed a co-immunoprecipitation experiment using the pALF4:ALF4-GFP line and an antibody directed against a peptide from human RBX1 that recognizes Arabidopsis RBX1 (Xu et al, 2002; Gilkerson et al, 2009). The results in Fig 3B show that RBX1 is recovered in an immunoprecipitation of ALF4-GFP, indicating that these two proteins are interacting in the plant extract. We extended this finding using an in vitro pulldown experiment. As expected, wild-type ALF4 clearly interacted with RBX1 in vitro. However, neither ALF4A484A614 nor ALF41-532stop were recovered in this GST-RBX1 pulldown assay, confirming that K484 and R614 are important for RBX1 binding (Fig 3C). To demonstrate an interaction in vivo, we performed a BiFC (bimolecular fluorescence complementation) experiment using RBX1 with wild-type and mutant ALF4. Similar to the yeast two-hybrid and pulldown experiments, only the wild-type ALF4 protein displayed a robust interaction with RBX1 (Fig 3D). The mutant variants interacted weakly (ALF4A484A614) or not at all (ALF41-532stop). Finally, to quantify ALF4–RBX1 interaction when RBX1 is associated with cullin in solution, we carried out microscale thermophoresis (MST) (Fig 3E). For this experiment, recombinant, purified MmRBX1–HsCUL1 (Li et al, 2005) was fluorescently labeled and incubated with ALF4 at a range of concentrations. We used the mouse RBX1 and human CUL1 proteins for this experiment because their expression had been optimized (Li et al, 2005). Within the concentration range 7 μM to 0.21 nM, ALF4 exhibited an affinity for MmRBX1–HsCUL1 with a Kd of 346.04 ± 77.05 nM (Figs EV5 and EV6). ALF4 clearly interacted with Cul1–RBX1, and given their structural similarities, the ALF4–RBX1 interaction likely resembles that of GLMN-RBX1. Click here to expand this figure. Figure EV5. Raw data of MST measurements for the interaction between labeled HsCul1–MmRBX1 vs. AtALF4 isoform F4JWD6 A–C. Three independent biological replicates of MST measurements depict ALF4 (red) binding to Cul1–RBX1. These measurements were combined for calculation of the dissociation constant (see Fig 3C). For each replicate, the capillary scan (up), MST traces (middle), and binding curves (low) are shown. Download figure Download PowerPoint Click here to expand this figure. Figure EV6. Raw data of MST measurements for the interaction between labeled HsCul1–MmRBX1 vs. AtUBC8 A–C. Three independent biological replicates of MST measurements depict uncharged UBC8 (without ubiquitin) (green) does not bind to Cul1–RBX1. These measurements were used as a control for ALF4–Cul1–RBX1 interaction in Fig 3C. For each replicate, the capillary scan (up), MST traces (middle), and binding curves (low) are shown. Download figure Download PowerPoint The alf4 mutant stabilizes the SCFTIR1 and SCFSLY substrates IAA17 and RGA Since ALF4 may regulate CRL assembly or activity, we examined the levels of SCFTIR1 and SCFSLY1 substrates, the Aux/IAA and DELLA repressors of the auxin and gibberellin pathways, respectively. To determine the effects of alf4 on DELLA proteins, we examined the turnover of the DELLA protein REPRESSOR OF GA1-3 (RGA) in the wild type and alf4-063 mutant after inhibition of protein biosynthesis with cycloheximide (CHX). Immunoblots showed that RGA strongly accumulated in the alf4 background (Fig 4A). In addition, RGA levels in alf4 plants were not reduced within 30 min of CHX treatment in alf4, whereas the protein was degraded to 60% of its initial levels in the wild type (Fig 4A and B). Figure 4. SCF substrates accumulate in the alf4 mutant Total protein extracts prepared from 13-day-old wild-type and alf4-063 seedlings, separated by SDS–PAGE, and probed with anti-RGA antibody. Background cross-reacting bands are indicated by asterisks. 8-day-old wild-type and mutant seedlings were treated with 50 μM cycloheximide (CHX) for up to 30 min as indicated in the figure. Total protein extracts were separated by SDS–PAGE and probed with anti-RGA antibody. Asterisks indicate background cross-reacting bands. The anti-CDC2 immunoblot serves as loading control. Relative RGA signal intensity was measured using MultiGAUGE and plotted on the right. Confocal images showing IAA17-GFP levels in wild-type and alf4-1 roots. Seedlings were treated with 5 μM dexamethasone for 4 h followed by treatment with 10 μM IAA for the indicated time. Scale bars are 50 μm. IAA17-GFP levels measured using ImageJ software. Data were collected from 4 roots for each time point. Error bars represent standard deviation. The difference between Col-0 and alf4-1 is significant P < 0.001, Student's t-test (two-tailed) for each of the time points (t = 0, t = 60′ and t = 180′). Values above the bar are the fraction of IAA17-GFP remaining relative to time zero. Relative IAA17-GFP transcript levels in 7-day-old seedlings after treatment with dexamethasone for 4 h. Data shown are from three biological replicates. Error bars represent standard deviation. Differences are not significant, Student's t-test (two-tailed). Source data are available online for this figure. Source Data for Figure 4 [embj201797159-sup-0004-SDataFig4.pdf] Download figure Download PowerPoint To determine the role of ALF4 in Aux/IAA degradation, we used a pDEX:IAA17-GFP construct to examine IAA17 levels after auxin treatment in alf4-1 compared to wild-type controls. After a 4-h dexamethasone treatment, the amount of IAA17-GFP was clearly higher in the root tip of alf4-1 plants compared to the wild type (Fig 4C and D). Examination of IAA17-GFP levels after auxin treatment revealed that the protein was relatively stable in alf4-1 plants compared to the wild type. Importantly, IAA7-GFP transcript abundance was similar in the two lines. Because accumulation of Aux/IAA proteins results in auxin resistance, these results are consistent with reduced auxin response observed in the alf4 mutant (Salehin et al, 2015). If ALF4 functions like GLMN and inhibits CRL activity, it is counterintuitive that SCF substrates should be stabilized in the alf4 mutant. One possibility is that loss of ALF4 leads to changes in the abundance of SCF subunits. To assess this possibility, we first examined CUL1 levels in wild-type and alf4-1 plants, in the absence and presence of the proteasome inhibitor MG132. The immunoblot in Fig 5A shows that the levels of unmodified and neddylated CUL1 are reduced in the mutant compared to the wild type. Treatment with MG132 increased the amount of both forms in the mutant and wild type, indicating that CUL1 is a substrate for the proteasome. However, we note that CUL1 levels in alf4 plants are not restored to wild-type levels by MG132. To determine whether CUL1 stability is affected in the mutant, we treated seedlings with CHX and collected samples at time intervals thereafter. The data in Fig 5B and C show that CUL1 stability is substantially reduced in the mutant. In contrast, we find that TIR1 abundance, as detected by examining a fusion of TIR1 to the fluorescent protein VENUS, are increased in the mutant (Fig 5D and E). Since TIR1-VENUS transcript accumulation is not affected in the mutant (Fig 5F), the increase in protein levels is probably due to increased stability. Figure 5. Stability of SCF subunits of the alf4 mutant Total protein extracts prepared from 7-day-old wild-type and alf4-1 seedlings treated or untreated with 100 μM MG132 were
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