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ARF1 Dimerization Is Essential for Vesicle Trafficking and Dependent bioRxiv preprint doi: https://doi.org/10.1101/2020.01.21.913905; this version posted January 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 ARF1 dimerization is essential for vesicle trafficking and dependent on activation by ARF-GEF 2 dimers in Arabidopsis 3 4 Sabine Brumm1, Mads Eggert Nielsen1,2, Sandra Richter1, Hauke Beckmann1, York-Dieter Stierhof3, Manoj 5 K. Singh1, Angela-Melanie Fischer1, Venkatesan Sundaresan4, Gerd Jürgens1,* 6 7 1 Center for Plant Molecular Biology (ZMBP), Developmental Genetics, University of Tübingen, Auf der 8 Morgenstelle 32, 72076 Tübingen, Germany 9 2 University of Copenhagen, Faculty of Science, Section for Plant and Soil Science, Thorvaldsensvej 40, 10 1871 Frederiksberg C, Denmark 11 3 Center for Plant Molecular Biology (ZMBP), Microscopy, University of Tübingen, Auf der Morgenstelle 12 32, 72076 Tübingen, Germany 13 4 Department of Plant Biology and Department of Plant Sciences, University of California, Davis, One 14 Shields Avenue, Davis, CA 95616, USA 15 16 * Corresponding author: 17 Gerd Jürgens 18 Center for Plant Molecular Biology (ZMBP), Developmental Genetics, University of Tübingen, Auf der 19 Morgenstelle 32, 72076 Tübingen, Germany 20 Phone: +49-7071-2978886 21 Email: [email protected] 22 ORCID: 0000-0003-4666-8308 23 24 Short title 25 Activation-dependent ARF1 dimerization 26 27 Material distribution footnote 28 The author responsible for distribution of materials integral to the findings presented in this article is: 29 Gerd Jürgens ([email protected]). 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.21.913905; this version posted January 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 30 Abstract 31 Membrane traffic maintains the organization of the eukaryotic cell and delivers cargo proteins to their 32 subcellular destinations such as sites of action or degradation. Membrane vesicle formation requires ARF 33 GTPase activation by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), 34 resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with 35 monomeric proteins, although ARF-GEFs have been shown to form dimers in vivo. This feature is conserved 36 across the eukaryotes, however its biological significance is unknown. Here we demonstrate ARF1 37 dimerization in vivo and we show that ARF-GEF dimers mediate ARF1 dimer formation. Mutational 38 disruption of ARF1 dimers interfered with ARF1-dependent trafficking but not coat protein recruitment in 39 Arabidopsis. Mutations disrupting simultaneous binding of two ARF1•GDPs by the two SEC7 domains of 40 GNOM ARF-GEF dimer prevented stable interaction of ARF1 with ARF-GEF and thus, efficient ARF1 41 activation. Our results suggest a model of activation-dependent dimerization of membrane-inserted 42 ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary 43 conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur 44 in eukaryotes in general. 45 46 Keywords 47 Membrane trafficking, vesicle formation, ARF1 GTPase, ARF-GEF, dimerization, FRET-FLIM, protein 48 interaction, GNOM, Arabidopsis. 49 50 51 52 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.21.913905; this version posted January 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 53 Introduction 54 Activation of small GTPase ARF1 by guanine-nucleotide exchange factors (ARF-GEFs) plays a pivotal role 55 in membrane traffic across the eukaryotes (Donaldson and Jackson, 2011). GDP-bound ARF1 interacts with 56 the catalytic SEC7 domain of ARF-GEFs on donor membranes, resulting in GDP-GTP exchange on ARF1 57 and membrane insertion of its myristoylated N-terminal hasp (Casanova, 2007; Anders et al., 2008a; Bui et 58 al., 2009). GTP-bound ARF1 interacts with coat proteins involved in vesicle formation and cargo 59 recruitment (D'Souza-Schorey and Chavrier, 2006; Gillingham and Munro, 2007; Singh and Jürgens, 2018). 60 ARF1•GTP forms dimers in vitro, which are required for scission of membrane vesicles from donor 61 membrane (Beck et al., 2008 and 2011). ARF1 dimer formation is disrupted by a Y35A mutation, which 62 reduces the yield of vesicles dramatically in vitro and fails to complement the lethality of arf1 arf2 mutant 63 yeast (Beck et al., 2008). How ARF1 dimers form in vivo has not been addressed but might be related to 64 ARF-GEF action. Large ARF-GEFs such as human GBF1 or Arabidopsis GNOM have a stereotypic domain 65 organization including an N-terminal dimerization (DCB) domain (Casanova, 2007; Anders et al., 2008; 66 Bui et al., 2009). The DCB domain can interact with another DCB domain and with at least one other ARF- 67 GEF domain (Grebe et al., 2000; Ramaen et al., 2007; Anders et al., 2008b). Although conserved across the 68 eukaryotes, the biological significance of ARF-GEF dimerization is not known. Our results presented here 69 suggest that ARF-GEF dimers generate ARF1•GTP dimers during the activation process, allowing 70 productive vesicle formation. 71 72 Results and Discussion 73 In-vivo occurrence and biological significance of ARF1 GTPase dimers 74 To address the issue of ARF1 dimerization, we tested ARF1 wild-type and two variants – activation- 75 deficient ARF1-T31N and hydrolysis-deficient ARF1-Q71L (Dascher and Balch, 1994; Singh and Richter et 76 al., 2018) – for interaction by co-immunoprecipitation (for overview of mutant proteins see Supplemental 77 Figure 1). Both ARF1-T31N and ARF1-Q71L co-precipitated endogenous ARF1, although ARF1 wild-type 78 failed to do so (Figure 1A). However, ARF1-T31N strongly interacted with ARF-GEF GNOM whereas 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.21.913905; this version posted January 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 79 ARF1-Q71L did not, suggesting that ARF1•GDPs might be bridged by ARF-GEF dimer whereas 80 ARF1•GTPs might display interaction independently of ARF-GEF. To test this idea, we made use of the 81 putatively dimerization-deficient ARF1-Y35A mutant (Beck et al., 2008). We generated transgenic 82 Arabidopsis lines that inducibly co-expressed either ARF1-Y35A,Q71L-GFP and ARF1-Y35A,Q71L-RFP or 83 ARF1-Q71L-GFP and ARF1-Q71L-RFP. FRET-FLIM measurements revealed interaction between ARF1- 84 Q71L proteins in seedling root cells, which was prevented by the additional Y35A mutation (Figure 1B). 85 These data suggest that membrane-bound ARF1•GTP molecules form dimers by direct physical interaction. A Figure 1. In-vivo interaction between +GFP beads ARF1•GTP molecules revealed by co- ARF1-YFP ARF1-T N-YFP ARF1-Q L-YFP 31 71 immunoprecipitation and FRET-FLIM IP T M T IP T IP antibodies 180 analysis. SEC7 (A) Estradiol-inducible (20µM 7h) expression of 130 ARF1-YFP, activation-deficient ARF1-T31N- 55 YFP and hydrolysis-deficient ARF1-Q71L-YFP, 40 immunoprecipitation with anti-GFP beads from 35 transgenic Arabidopsis seedling extracts, and ARF1 25 immunoblotting of PAGE-separated precipitates with anti-SEC7(GNOM) and anti-ARF1 antisera. Antisera indicated on the right. T, total extract; IP, 15 immunoprecipitate; M, molecular markers (sizes in kDa indicated on the left). Asterisk, ARF1-YFP fusion proteins; arrow, endogenous ARF1 (both B p=1.06 x 10-6 p=1.08 x 10-14 p=0.63 detected with anti-ARF1 antiserum); SEC7, 3.00 n=19 n=36 n=63 n=77 n=40 n=67 antiserum detecting SEC7 domain of GNOM. 2.95 (B) FRET-FLIM analysis of ARF1-ARF1 interaction in Arabidopsis seedling root cells after 2.90 estradiol induction (20µM 4h). The life time of hydrolysis-deficient ARF1-Q71L-YFP (QL-YFP) 2.85 was reduced whereas ARF1-Q71L-YFP bearing donor lifetime [ns] 2.80 dimerization-disrupting Y35A mutation (QL-YA- GFP) showed normal FRET-FLIM ratios. For 2.75 comparison, the life time of ARF1-YFP was ARF1-YFP ARF1-YFP QL-YFP QL-YFP QL-YA-YFP QL-YA-YFP x x x slightly reduced. Box plots of donor life time in ARF1-RFP QL-RFP QL-YA-RFP ns of at least 19 independent measurements for each sample (exact numbers are indicated by n). Medians are represented by the center lines and notches indicate 95% confidence interval. Tukey whiskers extend to the 1.5xIQR and data points are plotted as bee swarm. Exemplary p values (two tailed t-test assuming equal variances, alpha=0.05) are indicated in the graph. 86 To examine the biological significance of ARF1 dimers, we analyzed the ability of ARF1-Y35A to rescue 87 the secretion of alpha-amylase from tobacco protoplasts inhibited by ARF1-T31N expression (Figure 2A and 88 2B). 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.21.913905; this version posted January 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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