Journal of Experimental Botanydoi:10.1093/jxb/erv373

RESEARCH PAPER

The Qb-SNARE Memb11 interacts specifically with Arf1 in the Golgi apparatus of Arabidopsis thaliana

Claireline Marais1, Valérie Wattelet-Boyer1, Guillaume Bouyssou1, Agnès Hocquellet2, Jean-William Dupuy3, Brigitte Batailler4, Lysiane Brocard4, Yohann Boutté1, Lilly Maneta-Peyret1 and Patrick Moreau1,4,*1 CNRS-University of Bordeaux, UMR 5200 Membrane Biogenesis Laboratory, INRA Bordeaux Aquitaine, 33140 Villenave d’Ornon, France2 University of Bordeaux- INP Bordeaux, BPRVS, EA4135, F-33000 Bordeaux, France3 Proteome platform, Functional Genomic Center of Bordeaux, University of Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France4 Bordeaux Imaging Center, UMS 3420 CNRS, US4 INSERM, University of Bordeaux, 33000 Bordeaux, France

* To whom correspondence should be addressed. E-mail: patrick.moreau@u-bordeaux.fr

Received 19 March 2015; Revised 6 July 2015; Accepted 7 July 2015

Editor: Chris Hawes

Abstract

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are critical for the func-tion of the secretory pathway. The SNARE Memb11 is involved in membrane trafficking at the ER-Golgi interface. The aim of the work was to decipher molecular mechanisms acting in Memb11-mediated ER-Golgi traffic. In mam-malian cells, the orthologue of Memb11 (membrin) is potentially involved in the recruitment of the GTPase Arf1 at the Golgi membrane. However molecular mechanisms associated to Memb11 remain unknown in plants. Memb11 was detected mainly at the cis-Golgi and co-immunoprecipitated with Arf1, suggesting that Arf1 may interact with Memb11. This interaction of Memb11 with Arf1 at the Golgi was confirmed by in vivo BiFC (Bimolecular Fluorescence Complementation) experiments. This interaction was found to be specific to Memb11 as compared to either Memb12 or Sec22. Using a structural bioinformatic approach, several sequences in the N-ter part of Memb11 were hypoth-esized to be critical for this interaction and were tested by BiFC on corresponding mutants. Finally, by using both in vitro and in vivo approaches, we determined that only the GDP-bound form of Arf1 interacts with Memb11. Together, our results indicate that Memb11 interacts with the GDP-bound form of Arf1 in the Golgi apparatus.

Key words: Arf1, Golgi, GTPase, Memb11, protein interaction, secretory pathway, SNARE.

Introduction

SNARE (soluble N-ethylmaleimide-sensitive factor attach-ment protein receptor) proteins are components of the molecu-lar machinery involved in the vesicular secretory pathway of eukaryotic cells. They contribute to the fusion of membranes and are essential for numerous plant physiological functions

(Lipka et al., 2007; Moreau et al., 2007; Bassham et al., 2008; Kim and Brandizzi, 2012). Most of them have a C-terminal (C-ter) transmembrane domain and at least one coil-coiled domain (~70 amino acids), also called the SNARE domain, in the cytosolic part of the protein. Within the SNARE domain,

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a variable hydrophilic residue characterizes four different types of SNAREs: Qa-, Qb-, Qc- and R-SNAREs. The fusion event requires usually one SNARE of each group (three Q-SNAREs and one R-SNARE). In Arabidopsis thaliana, several SNAREs have been identified at the ER-Golgi interface (Lipka et  al., 2007) and by homology with yeast, three SNAREs could potentially be involved in the fusion of COPII vesicles to the cis-Golgi membrane: Memb11 (At2g36900, Qb-SNARE), Sec22 (At1g11890, R-SNARE) and SYP31 (At5g05760, Qa-SNARE). Furthermore, these three SNAREs have been shown to be involved in ER-Golgi trafficking (Uemura et al., 2004; Chatre et al., 2005; Sanderfoot, 2007; Bubeck et al., 2008).

Honda et al. (2005) proposed that in mammals, membrin (the orthologue of Memb11) could also act as a cis-Golgi recruiter of Arf1. Indeed, Arf1 (a small GTP binding pro-tein) is mostly cytosolic in its inactivated form Arf1-GDP and needs to be recruited to the membrane surface to be activated as Arf1-GTP. The authors showed that: (i) the interaction of this protein with membrin leads to its recruitment and (ii) it was dependent on the presence of the 110MXXE113 motif in the sequence of Arf1. They proposed a model according to which Arf1-GDP is recruited to the cis-Golgi membrane by membrin and then would be activated as Arf1-GTP (Honda et al., 2005). In plants, Arf1 localizes over Golgi cisternae by IEM (Stierhof and El Kasmi, 2010) and in A.  thaliana the presence and the importance of the motif MXXE in Arf1 for its localization to the Golgi apparatus has been shown (Matheson et al., 2008). However, we know nothing about the molecular mechanisms involved in the recruitment of Arf1 at the Golgi membrane. Therefore, it is questioned whether Arf1 could interact with Memb11 and if it leads to its recruitment to the Golgi in A.  thaliana. Two isoforms of membrin are present in A. thaliana, Memb11 and Memb12 (At5g50440), with a high identity [~82%, estimated with the Needleman and Wunsch algorithm, (http://mobyle.pasteur.fr/cgi-bin/portal.py#forms::needle)] which may interact with Arf1. To investigate the putative interaction of either Memb11 or Memb12 with Arf1 in A.  thaliana, an immunoprecipita-tion (IP) approach was first developed with anti-Memb11 antibodies. The co-IP of both Memb11 and Memb12 with Arf1 suggested a possible interaction between these proteins. These interactions have then been addressed in vivo by a bi-molecular fluorescence complementation (BiFC) approach.

Since only Memb11 was found to interact in vivo with Arf1, an in silico bioinformatic approach was performed on Memb11 and Memb12 to determine critical amino acid sequences. Then, BiFC experiments with chimeric proteins allowed us to determine which sequences of Memb11 were required for the interaction with Arf1. In the last part of the study we have tested both GDP- and GTP-bound forms of Arf1 to determine which one was interacting with Memb11.

Since Arf1 was also found to be able to interact with other proteins of the secretory pathway (Contreras et al., 2004; Min et al., 2007, 2013; Xu and Liu, 2012; Montesinos et al., 2014), the overall results have been discussed considering both Arf1’s ability to participate with multiple protein complexes and the potential dual role of Memb11 as a SNARE and/or as Arf1 recruiter.

Materials and methods

Plant materials and growth conditionsAll A. thaliana lines are Columbia-0 (Col-0) background except for the suspension cell culture for which the ecotype is Landsberg erecta. For the Golgi apparatus localization, the transgenic fusion-protein marker line p35S::N-ST-mRFP (rat N-α-2,6-sialyltransferase) (Viotti et al., 2010) was used.

Suspension cell cultures of A. thaliana were grown as described in Bayer et al. (2004) except that cells were sub-cultured once instead of twice a week (20 ml into 200 ml of fresh media).

For immunolocalisation and BiFC experiments, plants were grown in vitro on 4.4 g/l Murashige and Skoog nutrient mix, 0.7% w/v agar plant, 1% w/v sucrose, 0.05% w/v MES [2-(N-morpholino)ethanesulfonic acid] and buffered to pH 5.7 with KOH. For IP, plants were grown in liquid medium with 2.15 g/l Murashige and Skoog nutrient mix, 2% w/v glucose, 0.39% w/v MES and buffered to pH 5.7 with KOH.

Seeds were surface-sterilized with 3.2% v/v chloride hydroxide for 10 min and then rinsed six times with distillated water. Seeds were left for the stratification at 4°C for 2 d in water. Plants were grown in culture room under conditions of 40% relative humidity, 16/8 h light/dark cycle and 22/20°C. The hydroponic culture was in the same conditions with a rotation of 200 rpm.

Plasmid construction for the Rapid Translation SystemFor Rapid Translation System (RTS) production, open reading frames (ORFs) of Memb11 and ARF1A1C were amplified from A. thaliana cDNA using the primers listed in Supplementary Table S1. Memb11 was cloned without the transmembrane domain for only the first 200 amino acids because this hydrophobic domain was difficult to express in vitro. Memb11 and ARF1A1C were inserted into the pIVEX2.3 vector (Roche, www.roche.com) using NcoI and SmaI sites. This plasmid contains the T7 promoter/terminator and the 6-His tag.

Plasmid construction for yeast expression and BiFC assayORFs were amplified from A.  thaliana cDNA using the primers listed in Supplementary Table S1. For the different hybrid mutants (Memb11/Memb12, Memb12/Memb11, Memb121-11/Memb11, Memb121-25/Memb11 and Memb121-38/Memb11) and the point-mutated proteins (Memb11-ΔKARD, Memb11-ΔESSSMDSP, ARF1A1C -Q71L and ARF1A1C -T31N), OE-PCR (overlap exten-sion polymerase chain reaction) was used to create the new genes; the primers needed are listed in Supplementary Table S1. The cor-responding PCR fragments were cloned into the pDONR™221 ENTRY vector (Invitrogen, http://www.lifetechnologies.com) by GATEWAY® recombinational cloning technology, and subsequently transferred into the appropriate destination vector by LR clon-ing. For yeast expression, the vectors pYES2:GW and pYES3:GW (Invitrogen, www.lifetechnologies.com) were used and transformed into Saccharomyces cerevisiae strain INVSc1 (Invitrogen, http://www.lifetechnologies.com). For the BiFC experiments in planta the vectors pBiFP1, pBiFP2, pBiFP3 and pBiFP4 (Azimzadeh et  al., 2008) were used and transformed into Agrobacterium tumefaciens cells of strain C58C1 GV3101 (Koncz and Schell, 1986) with pMP90 helper plasmid.

Anti-Memb11 antibodies productionMemb11∆TMD-6His (Memb11 without its transmembrane domain and coupled to a 6 His tag) was expressed with the pET-15b vec-tor in Escherichia coli C41 cells and produced in a 2 l bioreactor Biostat B.  One gram of fresh biomass was suspended in 5 ml of 10 mM Tris buffer (pH 8), 1 mM EDTA and 1 mM PMSF. After sonication, the material was centrifuged at 3000 ×g for 5 min at 4°C

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and then the supernatant was centrifuged at 10 000 ×g for 15 min at 4°C. Inclusion bodies were suspended in 20 mM Tris buffer (pH 8), 0.5 M NaCl, 6 M guanidine-HCl, 5 mM imidazole and 1 mM PMSF. Solubilization of proteins was performed for at least 1 h at 4°C. The subsequent homogenate was then centrifuged at 10 000 ×g for 15 min at 4°C and the supernatant (solubilized inclusion bodies) was loaded onto a Ni2+ chelating Sepharose fast flow column (Akta Explorer, GE Healthcare) for protein purification by immobilized metal affinity chromatography (IMAC). After on-column buffer exchange in the presence of urea (8 M), an on-column refolding was included in the purification process. A step using 25 mM imi-dazole was performed to eliminate unspecific proteins. A one-step elution with 0.5 M imidazole was then applied to obtain the puri-fied Memb11∆TMD-6His protein. The purity and the efficiency of the protein production were controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Then the protein extract was used by Covalab (Lyon, France) to produce the anti-Memb11 antibodies.

IP on plant extractPlants were ground in IP buffer [100 mM NaCl, 50 mM HEPES, 10 mM Potassium acetate, 5 mM EDTA, 400 mM sucrose (pH 6.5), 1% v/v Triton X-100, 1 mM PMSF, and 1× protease inhibitor cock-tail] on ice and the lysate was solubilized at 4°C for 2 h on a rotating platform with 1% v/v Triton X-100. Cellular debris were removed by centrifugation at 3000 rpm for 4 min at 4°C. The supernatant was then used to perform the IP.

One hundred microlitres of µMACSTM protein A  micro beads (Miltenyi Biotec) and 10 µl of purified polyclonal antibodies (anti-Memb11 or pre-immune) were added to 1 ml of supernatant. Then the protocol according to the manufacturer was followed (http://www.miltenyibiotec.com) with little modification for the washing buffer (the IP buffer was used).

IP with peptides from RTSProteins were expressed with the RTS. The pIVEX2.3 plasmid (1.5  µg) with the gene of interest were added to 100  µl of reac-tion mix composed of the initial mix [0.05% w/v NaNo3, 2% v/v PEG 8000, 150.8 mM potassium acetate, 7.1 mM Mg(OAC)2, 0.1 M HEPES, 1× complete EDTA-free anti-protease (Roche, www.roche.com), 0.01% v/v folic acid, 2 mM DTT, 1× NTP mix, 20 mM phos-phoenolpyruvic acid (PEP), 20 mM acetyl phosphate, 1× complete amino acid mix, 1 M amino acid mix (RCWMDE)] and 0.4 mg/ml pyruvate kinase, 1.2 mg/ml tRNA, 1.4 U/µl T7 RNA polymerase and 35% v/v S30 bacteria extract. This reaction mix was separated from 1.7 ml of nutritive mix (initial mix with 35% v/v S30 buffer) with a dialysis membrane (exclusion limit of 10 KDa). After 22 h of incu-bation at 28°C with a rotation of 50 rpm, peptides were collected in the reaction mix.

To purify the peptides, 100  µl of IMAC Sepharose 6 gel (GE Healthcare Life Sciences, www.gelifesciences.com) were used. Before starting the purification, the gel was washed twice with 500 µl H2O and equilibrated three times with 500  µl of PNI buffer (20 mM Phosphate, 0.5 M NaCl, 5mM imidazole and buffered to pH 7.4). The reaction mix (100 µl) was added to 800 µl of PNI buffer and to the gel. After 5 min at room temperature, the gel was washed three times with 1 ml of PNI buffer before elution of the peptides with 100 µl of PNI buffer with 500 mM imidazole.

Each experiment was performed in two successive steps. First, GDP or GTP loading of ARF1A1C was carried out by adding a solution of GDP or GTP-γ-S according to Prouzet-Mauléon et al. (2008). GDP or GTP-γ-S [25 mM in 20 mM Tris-HCl, 25 mM NaCl, 0.1 mM DTT, 10 mM EDTA (pH 7.6)] were incubated with 10 µM of ARF1A1C peptides for 10 min at room temperature. The reac-tion was stopped by adding 36 mM ice-cold MgCl2, and the solu-tion was kept on ice. Then, for the IP, 100 µl of µMACSTM protein A microbeads (Miltenyi Biotec) and 10 µl of purified anti-Memb11

polyclonal antibodies were added to 900 µl TBS buffer (30% Trizma base, 80% NaCl, 2% KCl, 1× protease inhibitor cocktail and buff-ered to pH7.4 with HCl), 36 mM MgCl2, 20 ng ARF1A1C peptides with GDP or GTP-γ-S and 15 ng of Memb11 peptides. The solution was incubated for 2 h at 4°C on a rotating platform and then the pro-tocol according to the manufacturer was followed (Miltenyi Biotec, http://www.miltenyibiotec.com), except that the washing buffer was exchanged by TBS buffer with 1% v/v Triton X-100.

IP on yeast extractOptical density (OD) at 600 nm of cells grown in pre-culture medium with raffinose was measured; cells were seeded in 50 ml cul-ture medium with galactose in order to obtain an OD 600 nm of 4 and grown for 6.5 h at 30°C to induce the expression of the different genes introduced with the plasmids pYES2:GW and pYES3:GW. Cells were harvested and washed with distilled water, centrifuged at 5000 ×g for 5 min at 4°C and the pellets were frozen at −80 °C until use. Cells were disrupted in 50 µl lysis buffer [50 mM TrisHCl (pH 7.2), 1% v/v Triton X-100, 500 mM NaCl, 10 mM MgCl2, 1 mM PMSF and protease inhibitor mixture] with glass beads at 4°C using a Mini-Beadbeater (Biospec Products, http://www.biospec.com). The supernatants were collected and mixed with the lysis buffer used to rinse the glass beads (three times, 100 µl). Cell lysates were cen-trifuged at 500 ×g for 5 min at 4°C. The supernatants were collected and kept frozen at −80 °C. The protein’s concentrations were deter-mined by a BCA dosage.

For the IP, 100 µl of µMACSTM protein A microbeads (Miltenyi Biotec) and 10  µl of purified anti-Memb11 polyclonal antibodies were added on 900 µl TBS buffer (Trizma base 30%, NaCl 80%, KCl 2%, 1× protease inhibitor cocktail and buffered to pH 7.4 with HCl) and 500 ng of yeast proteins. The solution was incubated for 2 h at 4°C on a rotating platform after which the protocol according to the manufacturer was followed (http://www.miltenyibiotec.com), except that the washing buffer was exchanged by TBS buffer with 1% v/v Triton X-100.

Western blotProteins from IP were solubilized with 1× Laemmli buffer (Laemmli, 1970) for 5 min at 99°C. Samples were subjected to Western blotting by standard procedures (12% poly-acrylamides gel and PVDF mem-brane) and visualized with the Western Lightning Plus–ECL (enhanced chemiluminescence) kit (PerkinElmer, http://www.perkinelmer.com). The anti-Memb11 antibody and the serum anti-Arf1 (AS08 325, Agrisera, www.agrisera.com) were used at a dilution of 1/4000 and 1/1000, respectively. The anti-rabbit antibody coupled with the HRP (Biorad, www.bio-rad.com) was used at a dilution of 1/50000.

Mass spectrometry analysesSample preparation Each SDS-PAGE band was cut into 1 × 1 mm gel pieces. Gel pieces were destained in 25 mM ammonium bicarbo-nate (NH4HCO3), 50% acetonitrile (ACN) and shrunk in ACN for 10 min. After ACN removal, gel pieces were dried at room tempera-ture. Proteins were digested by incubating each gel slice with 10 ng/µl of trypsin (T6567, Sigma-Aldrich) in 40 mM NH4HCO3, 10% ACN, rehydrated at 4°C for 10 min, and finally incubated overnight at 37°C. The resulting peptides were extracted from the gel by three steps: a first incubation in 40 mM NH4HCO3, 10% ACN for 15 min at room temperature and two incubations in 47.5% ACN, 5% for-mic acid for 15 min at room temperature. The three collected extrac-tions were pooled with the initial digestion supernatant, dried in a SpeedVac, and resuspended with 25 μl of 0.1% formic acid before nanoLC-MS/MS analysis.

nanoLC-MS/MS analysis Online nanoLC-MS/MS analyses were performed using an Ultimate 3000 system (Dionex, www.dionex.com) coupled to a nanospray LTQ Orbitrap XL mass spectrometer

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(Thermo Fisher Scientific, www.thermofisher.com). Ten microlitres of each peptide extract were loaded on a 300 µm ID × 5 mm PepMap C18 precolumn (LC Packings, Dionex, www.dionex.com) at a flow rate of 20 µl/min. After 5 min desalting, peptides were online sepa-rated on a 75 µm ID × 15 cm C18PepMapTM column (LC packings, Dionex, www.dionex.com) with a 5–40% linear gradient of solvent B (0.1% formic acid in 80% ACN) in 45 min. The separation flow rate was set at 200 nl/min. The mass spectrometer operated in posi-tive ion mode at a 1.8 kV needle voltage and a 32 V capillary voltage. Data were acquired in a data-dependent mode alternating an FTMS scan survey over the range m/z 300–1700 with the resolution set to a value of 60 000 at m/z 400 and six ion trap MS/MS scans with collision-induced dissociation (CID) as activation mode. MS/MS spectra were acquired using a 3 m/z unit ion isolation window and a normalized collision energy of 35. Mono-charged ions and unas-signed charge-state ions were rejected from fragmentation. Dynamic exclusion duration was set to 30 s.

Database search and results processing Data were searched by SEQUEST algorithms through Bioworks 3.3.1 interface (Thermo Finnigan) against an Arabidopsis thaliana TAIR9 database (32 782 entries, August 2009). The DTA generation allowed the averaging of several MS/MS spectra corresponding to the same precursor ion with a tolerance of 1.4 Da. Spectra from the precursor ion higher than 3500 Da or lower than 500 Da were rejected. The mass accuracy of the peptide precursor and peptide fragments was set to 10 ppm and 0.5 Da, respectively. Only b- and y-ions were considered for mass cal-culations. Oxidation of methionine oxidation (+16) was considered as differential modifications. Two missed trypsin cleavages were allowed. Tryptic peptides were validated using the following criteria: DeltaCN ≥0.1, Xcorr ≥1.50 (single charge), 2.00 (double charge), 2.50 (triple charge), 3.00 (≥ quadruple charge), and peptide probability ≤0.001. Proteins were validated as soon as two different peptides are validated.

BiFC experimentsFor BiFC experiments, cotyledons from 4-day-old A.  thaliana seedlings were transformed with Agrobacterium tumefaciens. The protocol from Marion et al. (2008) was followed with few modifica-tions. Seeds were sown on sterile filters with a pore size of 500 µm (Fisher Scientific, ref.: 11755498, https://fishersci.com). A.  tumefa-ciens cells containing the different constructs were grown for 30 h at 30°C in 20 ml Luria-Bertani (LB) culture medium. Cells were col-lected after centrifugation (3700 ×g for 15 min) and resuspended in 2 ml of agro-infiltration solution (20 g/l glucose, 2.15 g/l MS, 3.91 g/l MES, 200 µM acetosyringone and buffered to pH 5.7). For BiFC co-infiltration, each culture was equally diluted in 4 ml to achieve the final infiltration concentration at the appropriate OD600 (0.3 for each strain). Agro-infiltration was performed by covering the 4-day-old seedlings with the A. tumefaciens solution and applying a vacuum (10 mm Hg) twice for 1 min. Excess infiltration medium was subse-quently removed and the plates were transferred to the culture room for 3 d. During the observation, only the abaxial part of the cotyle-don was transformed.

Fluorescence detection by confocal laser-scanning microscopy (CLSM) was performed by using a Leica TCS SP2 (http://www.leica-microsystems.com). The fluorescence signal was excited at 514 nm with the argon ion laser for eYFP and at 543 nm with the He/Ne laser for mRFP. Observation windows were of 580–610 nm and 630–680 nm, respectively. For the ‘multilabelling’ studies, detec-tion was in sequential mode. All the confocal images were captured in line-scanning mode with a line average of 4.

Immunoelectron microscopyFor immunogold labelling, 10-day-old meristematic zone of Arabidopsis roots and Arabidopsis suspension cells were high-pressure frozen with a Leica EMPACT system (http://www.leica-microsystems.com). They were submerged in BSA solution (20%

w/v BSA, 2.15 g/l MS, 3.91 g/l MES and buffered to pH 5.7) in a flat copper carrier with an aclar disc and frozen in a high-pressure freezer (EMPACT-1 Leica). The subsequent cryo-substitution was performed in a Leica AFS2 freeze substitution unit in acetone sup-plemented with 2% w/v osmium tetroxide (OsO4) and 0.1% w/v ura-nyl acetate at −90°C for 60 h. Temperature was then raised up to −50°C at a rate of 3°C/h and samples kept at −50°C for 38 h. They were subsequently washed three times for 20 min in 100% acetone, and three times for 20 min in 100% ethanol. Embedding in Lowicryl HM20 resin was performed for 1 h and then for 2 h in 100% HM20 at −50°C with intermediate steps of 2 h in 25%, 2 h in 50% and over-night in 75% HM20 in ethanol. Polymerization was done under UV for 48 h at −50°C. Immunogold labelling was performed as previ-ously described (Kang et  al., 2011) with few modifications. 70 nm sections of the samples were mounted on nickel slot grids coated with parlodion and probed with the anti-Memb11 antibody (1 h) diluted at 1/20, and with the secondary antibody GAR10 (goat anti-rabbit antibody coupled with 10 µm diameter gold beads, Tebu-Bio, www.tebu-bio.com) diluted at 1/30 (1 h). The solution in which the samples were blocked, rinsed and the antibodies diluted was PBSTB [phosphate buffered saline (PBS) (0.15 M NaCl, 7.5 mM Na2HPO4, 0.25 mM NaH2PO4), 0.2% v/v Tween 20, 1% w/v BSA].

Samples were viewed with a MET Philips CM10 80kV with AMT ×60 camera (Elexience).

Bioinformatic analysesThe protein sequence alignment between Memb11 and Memb12 was performed with the Needleman-Wunsch global alignment algo-rithm with Needle software (http://mobyle.pasteur.fr/cgi-bin/portal.py#forms::needle).

The secondary structures of Memb11 and Memb12 were predicted by PSI-Pred (http://bioinf.cs.ucl.ac.uk/psipred/). The SNARE motif was identified in the Pfam 27.0 database (http://pfam.sanger.ac.uk/) and the transmembrane domain by the following software: THMM 2 (http://www.cbs.dtu.dk/services/TMHMM/), TMPred (http://www.ch.embnet.org/software/TMPRED_form.html), MEMSAT 3 and MEMSAT-SVM (http://bioinf.cs.ucl.ac.uk/psipred/).

The 3D structure of The N-ter part of Memb11 (amino acids 1–139) and Memb12 (amino acids 1–133) were modelled by I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) and those pre-dictions were verified by two software programs: PROCHECK (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html) and QMEAN (http://swissmodel.expasy.org/qmean/cgi/index.cgi). Molecular graphics and analyses were performed with the UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311) (http://www.cgl.ucsf.edu/chimera/).

Results

Memb11 locates on cis-Golgi cisternae in Arabidopsis roots and suspension cells

Polyclonal anti-Memb11 antibodies raised against the cyto-solic part of Memb11 were produced as described in the experimental section and were first tested by Western blots on homogenates of 14-day-old seedlings of Arabidopsis thali-ana. As shown in Supplementary Fig. S1A, only one band was revealed at a slightly higher molecular weight (~30 kDa) than expected (~25 kDa) but the mass spectrometry analysis confirmed the presence of Memb11. As Memb12 (82% iden-tical to Memb11) was also detected in the mass spectrometry analysis, we have performed a competitive enzyme-linked immunosorbent assay (ELISA) with purified Memb11 and

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Memb12 peptides to estimate the specificity of the antibod-ies to these peptides. This assay showed that the Memb11 curve decreased more rapidly than that of Memb12 and we estimated that the antibody recognized more efficiently (10–20 times) Memb11 than Memb12 (Supplementary Fig. S1B). Moreover, publicly available microarrays and RNAseq databases clearly indicate that Memb11 transcripts are largely more abundant than Memb12 transcripts (data from Genevestigator, https://www.genevestigator.com/gv/). The higher efficiency of the antibodies with Memb11 than with Memb12 and the fact that Memb11 is more expressed than Memb12 strongly suggested that anti-Memb11 antibodies resulted in the labelling of Memb11 rather than Memb12. The expression of fluorescent constructs has shown that these SNAREs reside in the Golgi (Uemura et  al., 2004; Chatre et al., 2005; Geldner et al., 2009). To determine the precise localization of Memb11 in the Golgi we performed immu-nolocalizations with anti-Memb11 antibodies on high-pres-sure frozen, freeze-substituted A.  thaliana suspension cells. Electron microscopy revealed that Memb11 was associated with the Golgi apparatus and precisely localized to cis-Golgi cisternae (Fig. 1). Similar observations were obtained on root apex from 10-day-old A. thaliana seedlings (data not shown). Memb11 is not detected on other membranes suggesting a rather specific localization of Memb11 at cis-Golgi.

ARF1 co-immunoprecipitates together with Memb11 and Memb12

To investigate whether Arf1 and Memb11 could be part of a common protein complex, we first tested if we could detect Arf1 in the close membrane environment of Memb11. To address this we performed co-IP using anti-Memb11 anti-bodies without adding cross-linking reagents.

Eighteen IP with anti-Memb11 antibodies and 10 IP with control rabbit serum were performed from 14-day-old A. thaliana seedling homogenates. For each assay, we first ran Western blots after SDS-PAGE. The anti-Memb11 antibod-ies revealed a band at ~30 kDa in the seedling homogenates and in anti-Memb11 IP fractions but not in the control IP fractions (Fig. 2A). Moreover, immunoblots performed with anti-Arf1 antibodies revealed the presence of Arf1 in anti-Memb11 IP but not in control IP (Fig.  2B). These results suggest that Memb11 and/or Memb12 are closely associated to Arf1.

Using mass spectrometry on anti-Memb11 IP fractions, we could detect Memb11 in 100% of the IP performed. Memb12 was revealed as well in 94% of the anti-Memb11 IP fractions but both proteins were never detected in the samples issued from the control IP. These results are in agreement with the results obtained by Western blot (Fig. 2A, B). In 83% of the IP realized with the anti-Memb11 antibodies, mass spec-trometry analyses showed the presence of peptides from Arf1 which could originate from the six isoforms A–F (respectively At1g23490, At5g14670, At2g47170, At1g70490, At3g62290 and At1g10630; Vernoud et  al., 2003). As a point of com-parison we also looked at the frequency of IP of two other proteins: the SNARE Sec22, which is another SNARE of the ER-Golgi interface (Chatre et al., 2005), and the GTPase Sar1 (At1g56330), which is an element of the COPII machin-ery. The SNARE Sec22 was not detected and compared to the 83% frequency of Arf1 co-IP with Memb11, a relatively low frequency of co-IP was obtained for the GTPase Sar1 (only 15%).

Despite the cyclic location of Arf1, its interaction with Memb11 and/or Memb12 seems to be strong enough to co-immunoprecipitate the complex and this prompted us to ver-ify in vivo by BiFC whether or not this interaction could be confirmed.

Memb11 but not Memb12 interacts with ARF1A1C in vivo

The IP experiments revealed the presence of the six GTPases isoforms of the Arf1 subclass. However it was impossible to determine if one was preferentially co-immunoprecipitated because these proteins share ~99% identity. Amongst the six isoforms of Arf1 potentially interacting with Memb11, we found ARF1A1C, which has been localized at the Golgi com-plex and TGN (Robinson et al., 2011 and references therein), and is involved in the intracellular trafficking through the Golgi (Lee et al., 2002; Takeuchi et al., 2002; Xu and Scheres, 2005; Tanaka et al., 2014). Hence, ARF1A1C was the best candidate for testing the interaction with Memb11 and we designed several constructs to investigate potential inter-action of the SNAREs Memb11, Memb12 and Sec22 with the Arf1 isoform ARF1A1C. Given that the three SNAREs carry a transmembrane domain at their C-ter extremity we fused one half of the YFP (YN155: amino acids 1–154, or YC155: amino acids 155–238) to the N-ter side of these pro-teins: YN155-SNARE or YC155-SNARE (Supplementary Fig. S2). Oppositely, ARF1A1C is linked to the membrane

Fig. 1. Immunolocalization of Memb11 in A. thaliana cultured cells by transmission electron microscopy. The black arrow indicates the cis to trans orientation of the Golgi stacks. Scale bar, 100 nm.

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through its N-ter extremity. Thus, we fused the other half of YFP to the C-ter extremity of ARF1A1C: ARF1A1C-YC155 or ARF1A1C-YN155 (Supplementary Fig. S2). In order to design negative controls, we fused the YFP halves to the opposite extremity of the different proteins: SNARE-YN155 or SNARE-YC155 and YN155-ARF1A1C or YC155-ARF1A1C (Supplementary Fig. S2).

Co-infiltrations of A. thaliana cotyledons with the differ-ent constructs were realized and fluorescence was monitored

after 48 h. Punctuate signals with a size similar to that of the Golgi bodies were observed for the following expressed couples: YN155-Memb11/ARF1A1C-YC155 and YC155-Memb11/ARF1A1C-YN155 (Fig. 2C). On the contrary, all the couples tested with the negative controls described above did not show any fluorescence. In order to confirm that the punctuated fluorescence observed for the positive couples was effectively associated with Golgi bodies, we performed BiFC experiments with them in an A. thaliana line expressing

Fig 2. IP and BiFC studies reveal the Memb11-Arf1 interaction. Western blot with anti-Memb11 antibodies (A) and with anti-Arf1 antibodies (B) after the IP performed with anti-Memb11 antibodies. H, homogenate; IP-NI, non-immune IP; IP-Memb11, IP performed with the anti-Memb11 antibodies. (C) Pictures showing the BiFC fluorescence obtained with the couple Memb11-ARF1A1C (YN155-Memb11/ARF1A1C-YC155 and YC155-Memb11/ARF1A1C-YN155). Scale bars, 10 µm. (D) BiFC performed on A. thaliana seedlings expressing the Golgi marker N-ST-mRFP. Scale bar, 10 µm. (E) Frequency of BiFC observed for the 3 SNAREs Memb11, Memb12 and Sec22 with ArfA1c. The number of cotyledons observed for each couple was as follows: Memb11/ARF1A1C (n=32), Memb12/ARF1A1C (n=28) and Sec22/ARF1A1C (n=28). The values of error bars are 0.7, 0.4 and 0.6, respectively, and correspond to the standard deviation of the means.

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the Golgi marker N-ST-mRFP. We observed co-labelling between the N-ST-mRFP and the YFP fluorescence due to BiFC (Fig.  2D), indicating that Memb11 and ARF1A1C interacted at the Golgi bodies.

Then we addressed whether ARF1A1C could also interact with the SNAREs Memb12 or Sec22. To perform a statis-tically efficient comparison, we observed a high number of cotyledons per condition (Fig. 2E). An average of 4.8 fluores-cent cells per cotyledon were found for the positive Memb11/ ARF1A1C couples whereas the other couples Memb12/ ARF1A1C and Sec22/ ARF1A1C gave respectively 0.5 and 0.29 fluorescent cells per cotyledon (Fig. 2E). These results indicated that there was some specificity for the interaction of Memb11 with ARF1A1C. We therefore concluded that Memb11 and Arf1 can interact at the level of the Golgi bod-ies in A. thaliana cotyledons. The observed specificity of the interaction of Arf1 with Memb11 was somehow surprising when considering the high identity between Memb11 and Memb12 (~82%). In order to better understand this differ-ential interaction and determine eventual specific sequences required for the specificity of the interaction, we developed an in silico structural study of both proteins.

In silico structural modelling of Memb11 and Memb12 revealed distinct accessible residues in the N-ter part

We predicted the secondary structures of Memb11 and Memb12 using the PSIPRED v3.3 software which allows pre-dictions with a Q3 (percentage of correctly classified residues in control sequence) of 81.4% ±0.6% (Buchan et al., 2010). This first analysis revealed that Memb11 and Memb12 con-tain only coils and α-helices (Supplementary Fig. S3). The SNARE domain at the C-ter side of the protein made of a unique α-helix of 66 amino acids (potentially involved in SNARE interactions) matches very well with the V-SNARE_C family from the Pfam 27.0 databank (Supplementary Fig. S3) (Marchler-Bauer et al., 2013). The SNARE domains of Memb11 and Memb12 are between amino acids 134 and 199, and amino acids 128 and 193, respectively. By combin-ing TMHMM2, TMPred, MEMSAT3 and MEMSAT-SVM prediction softwares, we determined that Memb11 could have its transmembrane domain (TMD) between the amino acids 202/204 and 221/223 (with a TMD length of 18–22 amino acids), and Memb12 could have its TMD between the amino acids 196 and 198 and 215 and 217 (also with a TMD length of 18–22 amino acids) (Supplementary Fig. S3). Although a strong structural similarity was confirmed between the two proteins, important differences appeared in the N-ter regions (first 140 amino acids) and particularly in the two first coils of the proteins (Supplementary Fig. S3). Hence, we restricted the 3D modelling to the N-ter part of Memb11 and Memb12. For this, we applied the I-TASSER (iterative threading assem-bly refinement) software which combines «threading» and ab initio approaches (Roy et al., 2010). We obtained several models for each protein fragment and selected only those giv-ing C-scores higher than −3. As a consequence, we retained three putative models for the N-ter of Memb11 with C-scores between −2.82 and −1.93 and two putative models for the

N-ter of Memb12 with C-scores of −2.93 and −2.68. To fur-ther sort these different models, we used the PROCHECK and QMEAN software to test our models. The PROCHECK software evaluates the stereochemical qualities of the models by analysing the geometry of each amino acid, and the results are given as Ramachandran plots (Laskowski et  al., 1993, 1996). The QMEAN software takes into account different criteria such as the torsion angles (phi, psi and omega), the energy required for atom interactions and the solvent acces-sibility (Benkert et al., 2008, 2009, 2011).

By combining these three softwares (I-TASSER, PROCHECK and QMEAN) we propose the 3D structures for the N-ter parts of Memb11 (first 139 amino acids) and Memb12 (first 133 amino acids) (Fig. 3). The comparison of the two N-ter structures of Memb11 and Memb12 (Fig. 3A) reveals that 14 amino acids (coloured in purple for Memb11 and fuchsia for Memb12) are distinct and some are present in accessible regions (Fig. 3B). Indeed, the amino acids K25-D28-R32 and S43-P44 of Memb11, respectively in the first and second α-helices are different in Memb12 (R22-N25-K29 and P37-T38) and are all exposed outside of the structures. Furthermore, in the two first coils Memb11 possesses two additional sequences of amino acids (E7-G8-G9 and S39-S40-M41), which are exposed outside too. As a result, dif-ferences are mostly located around the second coil and are accessible. Finally, the orientations of the last α-helix (begin-ning of the SNARE domain) are opposite and could induce some steric hindrance for accessibility of amino acids in the first α-helices and the second coil in the case of Memb12 (Fig.  3C). All these features indicated that the regions and amino acids of Memb11 described above could be critical for the specific interaction with Arf1.

Finally, since Honda et  al. (2005) have proposed that in mammals, membrin can act as a cis-Golgi recruiter of Arf1, and that rat Arf1 and ARF1A1C share 87.8% identity, it was interesting to compare rat membrin structure with that of Memb11. We also performed an in silico structural model-ling of rat membrin. As shown in Supplementary Fig. S4, the structure of the N-ter of rat membrin is very close to that of Memb11. In addition, although the amino acids in and around the second coil are different, the overall charges are significantly similar. As a consequence, both the similarities in the structure and the number of charged amino acids in this area of the N-ter of rat membrin and Memb11 are com-patible with the hypothesis of a role of the N-ter of Memb11 for the interaction with ARF1A1C.

Molecular characterization of Memb11/Arf1 interaction

Based on the structural in silico approaches, we designed several Memb11 and Memb12 chimeras by interchanging sequences corresponding to the amino acids expected to be critical for the interaction of Memb11 with Arf1. Seven chimeric constructs of Memb11-Memb12 were established (Fig. 4A) and tested in vivo by BiFC for their interaction with Arf1. We designed two chimeras called Memb11-Memb12 and Memb12-Memb11 where we respectively fused amino acids 1–139 of Memb11 to amino acids 134–219 of Memb12,

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and amino acids 1–133 of Memb12 to amino acids 140–225 of Memb11 in order to swap the N-ter domain to the begin-ning of the SNARE domain between the two proteins. We first confirmed their sub-cellular localization at the Golgi complex (Supplementary Fig. S5A, B). Interestingly, the chimera Memb12-Memb11 behaved as Memb12 and did not show a high interaction with Arf1 (Fig.  4B). On the contrary, Memb11-Memb12 with the N-ter domain of Memb11 interacted with Arf1 with an efficacy close to that of Memb11 (Fig. 4B). These results clearly indicated that the N-ter domain of Memb11 was critical for the interaction of Memb11 with Arf1 and confirmed in silico predictions. We next swapped small portions of Memb11 N-ter part with cor-responding portions from Memb12 N-ter part. We therefore created the following mutants called Memb121-11-Memb11 (amino acids 1–11 of Memb12 fused with amino acids 15–225 of Memb11), Memb121-25-Memb11 (amino acids 1–25 of Memb12 fused with amino acids 29–225 of Memb11) and Memb121-38-Memb11 (amino acids 1–38 of Memb12 fused with amino acids 45–225 of Memb11). We first confirmed

that these constructs were correctly targeted to the Golgi except for the Memb121-25/Memb11 mutant, which did not locate to this organelle (Supplementary Fig. S5C–E). Hence, this chimera was not suitable for testing in vivo interaction with ARF1A1C. Memb121-11-Memb11 and Memb121-38-Memb11, which were correctly located to the Golgi, did not interact with Arf1 indicating that the amino acid sequences 1–14 and 1–44 from Memb11 were required for the interac-tion between Memb11 and Arf1 (Fig. 4B).

Finally, we designed a Memb11 mutant, called Memb11-ΔKARD, where the 25KARD28 sequence from Memb11 was substituted by the RARN sequence from Memb12. Additionally, we also designed another Memb11 mutant in which the sequence 37ESSSMDSP44 was replaced by the sequence DSDPT from Memb12 and called Memb11-ΔESSSMDSP. The two mutants were correctly targeted to the Golgi (Supplementary Fig. S5F, G). A decreased interaction with Arf1 was observed for the Memb11-ΔESSSMDSP chi-mera while no interaction could be detected for the Memb11-ΔKARD chimera (Fig. 4B).

Fig. 3. 3D structure of Memb11 and Memb12 determined in silico. Presentation of the 3D structures (A) and the protein molecular surfaces (B) of the N-ter part of Memb11 (amino acids: 1–139) and Memb12 (amino acids: 1–133). (C) Alignment of the two 3D structures of Memb11 and Memb12 shows the main differences in terms of amino acids and orientation of the SNARE domains. The amino acids that are different between Memb11 and Memb12 are coloured purple for Memb11 and fuchsia for Memb12. The visualizations were realized with the software UCSF Chimera.

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Altogether our results on the different versions of the N-ter part of Memb11 clearly identified the first 44 amino acids of the N-ter part, and especially the 25KARD28 and 37ESSSMDSP44 sequences, required for interaction of Memb11 with Arf1 (Figs 3, 4).

GDP-bound but not GTP-bound form of Arf1 interacts with Memb11

In order to determine which form (GDP-bound or GTP-bound) of ARF1A1C interacts in vitro with Memb11, we first synthesized the cytosolic part of Memb11 and the entire ARF1A1C protein through the RTS, which allows the in vitro production of proteins via E. coli extracts (Fig. 5A). We per-formed IP experiments with the anti-Memb11 antibodies in the presence of either GTP-γ-S (no hydrolysable analogue of GTP) or GDP (Fig. 5A). We found that when Memb11 was immunoprecipitated in the presence of GTP-γ-S, ARF1A1C did not co-immunoprecipitate with Memb11 (Fig.  5B).

However when Memb11 was immunoprecipitated in the pres-ence of GDP, we clearly detected ARF1A1C (Fig. 5B). These results indicate that Memb11 and ARF1A1C only interact when ARF1A1C was bound to GDP.

However, since this system was performed in vitro with pro-teins synthesized in a prokaryotic system, we tried to confirm these results by expressing the whole proteins in yeast (eukar-yotic system) (Fig. 6A). We performed IP of Memb11 using anti-Memb11 antibodies in yeast. We first tested whether anti-Memb11 antibodies would react with any yeast proteins and we could not detect any signal in yeast extract reveal-ing that the antibodies did not cross-react with yeast pro-teins (Fig. 6B). Specific plasmids were designed for Memb11, Memb12, the native form of ARF1A1C and its GTP-(Q71L) or GDP- (T31N) bound blocked forms (Fig.  6A). Our results showed that ARF1A1C was immunoprecipitated together with Memb11 when ARF1A1C was expressed as the GDP-bound blocked form but not when it was expressed as the GTP-bound blocked form (Fig. 6B). In addition, the

Fig. 4. BiFC studies with Memb11 mutant forms. (A) Presentation of the different mutant forms derived from Memb11 and Memb12 that were used with ARF1A1C. (B) Results of BiFC obtained with the different mutants of Memb11 and Memb12 compared to those obtained with Memb11, taking the latter as the reference equal to 1. The number of cotyledons observed for each couple and the values of error bars (corresponding to the standard deviation of the means) were as follows: Memb11/ARF1A1C (n=169), Memb12/ARF1A1C (n=118; 0.06), Memb12-Memb11/ARF1A1C (n=72; 0.15), Memb11-Memb12/ARF1A1C (n=72; 0.24), Memb121-11-Memb11/ARF1A1C (n=72; 0.04), Memb121-38-Memb11/ARF1A1C (n=72; 0.02), Memb11-∆ESSSMDSP/ARF1A1C (n=78; 0.07) and Memb11-ΔKARD/ARF1A1C (n=72; 0.04).

Fig. 5. In vitro analysis of the interaction of the GDP- and GTP-bound forms of ARF1A1C with Memb11. (A) Presentation of the experimental approach. (B) Western blots with the anti-Memb11 and the anti-Arf1 antibodies after the IP performed with the anti-Memb11 antibodies.

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ARF1A1C GDP-bound blocked form was only detected in IP when Memb11 was expressed but not when Memb12 was expressed (Fig. 6).

Therefore, our results clearly confirm that Memb11 but not Memb12 interacted with the GDP-bound form of ARF1A1C.

Discussion

Honda et  al. (2005) proposed a model in which membrin (the orthologue of Memb11) can act as a cis-Golgi recruiter of Arf1. Since Arf1 is located over Golgi cisternae in plants (Stierhof and El Kasmi, 2010) and Memb11 was found to be required at the ER-Golgi interface (Chatre et al., 2005), it was reasonable to question whether Arf1 could also interact with Memb11 to be recruited to the Golgi in A. thaliana. Thanks to IP and BiFC studies performed in vivo, we determined that Memb11 interacted with ARF1A1C at the Golgi appa-ratus. To better understand this interaction, we determined the regions of interest in Memb11 that are indispensable for Arf1 interaction. In silico structural studies were conducted to characterize the residues present in Memb11 but not in Memb12 that could be critical for the interaction with Arf1. It was found that, although the structure of Memb11 is close to that of Memb12 by exclusively containing α-helices and coils, Memb11 has several amino acids that are potentially highly accessible for interaction.

Different parts of Memb11 were then swept by parts of Memb12 and these chimeras were tested by BiFC with Arf1. It appeared that several amino acids in the first 44 were required for the interaction of Memb11 with Arf1. These amino acids, only present in Memb11 and determined by in

silico structural studies to be exposed outwardly of the 3D structures of the protein, are K25, D28 and R32 in the sur-face of the first α-helix and the amino acids S39, S40 and M41 in the second coil. In addition, although Memb11 and rat membrin (which interacts with Arf1; Honda et al., 2005) share only 23.9% identity, there is a significant conservation of the structure with charged amino acids around the sec-ond coil (Supplementary Fig. S4), which argues in favour of a role of this domain in Memb11-ARF1A1C and rat mem-brin-Arf1 interactions. The rat and human membrins show a 92.6% similarity with a total conservation of the charged amino acids around the second coil. Therefore, it is tempt-ing to conclude that this protein area is universally critical for interactions between membrin and Arf1 proteins. In addi-tion, the orientation of the SNARE domain in Memb11 seemed to be also important for the interaction. Recognition of Memb11 by Arf1 could therefore be both sequence- and structure-specific.

The observation that Memb12 did not interact with ARF1A1C needs additional comments. Memb12 is also local-ized to the Golgi apparatus when transiently over-expressed in A.  thaliana protoplasts (Uemura et  al., 2004) or stably expressed in A. thaliana (Geldner et al., 2009). Regarding its role, it was observed that the RNA of Memb12 was the target of miRNA* decreasing its translation after a pathogen attack (Pseudomonas syringae pv. tomato; Zhang et al., 2011). The consequence of this reduction was an increase of the exocy-tosis of the PR1 protein, which is engaged in a plant defence reaction (Zhang et al., 2011). This observation could indicate that there is a specific secretion pathway for PR1 during the activation of plant defence in which Memb12 is involved, that Memb12 may function as a negative regulator of exocytosis

Fig. 6. In vivo analysis of the interaction of the GDP- and GTP- bound blocked forms of ARF1A1C with Memb11 expressed in yeast. (A) Presentation of the experimental approach. (B) Western blots with the anti-Memb11 and the anti-Arf1 antibodies after the IP performed with the anti-Memb11 antibodies. T31N and Q71L correspond respectively to the GDP- and GTP-bound blocked forms of ARF1A1C.

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by an i-SNARE-like mechanism, for example (Varlamov et al., 2004; Di Sansebastiano, 2013), and/or that Memb12 is engaged in any other undefined function related to the secre-tory pathway. Indeed, it seems that the distinct cellular func-tions of Memb11 and Memb12 depend on the specificity of their interaction with different partners.

Our BiFC data did not exclude the possibility that Memb11 and Arf1 are associated in multi-protein complexes. As suggested by Rein et  al. (2002), there is probably more than one way to recruit Arf1 to membranes and therefore to the Golgi membranes. It was shown in humans that the cyto-plasmic domain of the p23 protein (p24 family) can interact with a sequence of 22 amino acids at the C-ter of Arf1-GDP (Gommel et  al., 2001). Furthermore, this interaction was recently observed by crystallographic studies (Zheng et  al., 2013). It was also found that p24 proteins can interact with Arf1 in plant cells (Contreras et al., 2004) and for example that p24δ5 interacts with Arf1 at acidic pH, which is compat-ible with an interaction in the Golgi (Montesinos et al., 2014). In addition, it was also observed in animals that the sequence 110MXXE113 of Arf1-GDP is critical for its localization at the Golgi apparatus and its interaction with membrin (Honda et  al., 2005). These two sequences, separated by 46 amino acids, define two zones in Arf1-GDP for interactions with several effectors. In A. thaliana, the MXXE sequence was also shown to be necessary for the recruitment of Arf1-GDP to the cis-Golgi membrane (Matheson et  al., 2008). This sug-gested that the sequence of Arf1 interacting with Memb11 was also MXXE in A.  thaliana. The Arf-GAPs Agd8 and Agd9 (soluble proteins) can also have a role in targeting Arf1-GDP to the membrane of the Golgi apparatus (Min et al., 2013). Furthermore, a sequence in the N-ter part of Agd8 and Agd9 is close to the BoCCS sequence of their homo-logues ArfGap2 and ArfGap3 from animal (Min et al., 2013) and BoCCS was shown to allow the interaction with membrin (Schindler et al., 2009). So, it is possible that Agd8 and Agd9 interact with Memb11 prior or after binding to Arf1-GDP in order to reach the Golgi membrane. Nevertheless, Memb11 could interact directly with Arf1-GDP as we showed in the in vitro IP experiments. Therefore, these data indicate that several Arf-GAPs, Memb11 and orthologues of the p24 pro-tein family could participate to the recruitment of Arf1-GDP to the Golgi membrane to activate the COPI machinery in A. thaliana.

In addition, Arf1 interaction with specific cargoes has also to be taken into account (Xu and Liu, 2012; Montesinos et al., 2014). Independently of its role in the recruitment of the COPI machinery, Arf1 can bind the TGN membrane by its myristoylation and induce the formation of constitutive secretory vesicles (Barr and Huttner, 1996) and by this way can interact with another pool of proteins. Moreover, in yeast and mammalian cells, it was shown that Arf1 recruits PI4 kinase and elicit phosphatidylinositol-4-phosphate formation (de Matteis and Godi, 2004). Arf1 locates to distinct mem-brane compartments (Golgi apparatus, TGN, endosomes and plasma membrane) and could interact with a large number of proteins. It would be interesting to determine the possi-ble interactions between all the putative partners potentially

forming different multi-protein complexes engaged in various functions (Anders and Jürgens, 2008; Matheson et al., 2008; Gendre et al., 2015; Tanaka et al., 2014; Yorimitsu et al., 2014 and references therein).

Another question arises from the results showing that the over-expression of Memb11 induces a secretion block and the relocation of Golgi markers into the ER (Chatre et al., 2005). It was concluded that Memb11, as a SNARE, was critical for anterograde transport between the ER and the Golgi (Chatre et al., 2005). However, it was also shown that anterograde and retrograde pathways between the ER and the Golgi apparatus were coupled (Stefano et al., 2006). So if the retrograde pathway (COPI) is blocked, the anterograde pathway (COPII) should also be altered. The disturbance of the anterograde pathway induced by over-expressing Memb11 could be directly related to the role of Memb11 as a SNARE or indirectly a result of the titration of Arf1 by Memb11. Memb11 (mostly localized at the cis-Golgi) could therefore function both as a SNARE in membrane fusion at the ER-Golgi interface, and as a ‘receptor/regulator’ of Arf1 for modulating the COPI machinery in such compart-ments. However, as no SNARE complex has been character-ized at the ER-Golgi interface in plants yet, we do not know whether Memb11 is a member of a SNARE complex at the cis-Golgi apparatus. Since Arf1 has a wide membrane locali-zation (Golgi apparatus, TGN, endosomes) and appears to be involved in multiple trafficking processes, future studies will be required to further investigate the two possible functions of Memb11 and the role of its specific interaction with Arf1 in early Golgi compartments.

The divergence observed between Memb11 and Memb12 function, despite their sequence identity, again feeds the discussion on SNARE redundancy. Until recently, the large amount of SNARE proteins in plants as opposed to yeast or mammals suggests that members of the same family can have redundant functions. This seems to be true for some of them like VAMP721 with VAMP722 in the immune response (Collins et al., 2003; Assaad et al., 2004; Kwon et al., 2008) or the SYP2 family (Shirakawa et al., 2010; Uemura et al., 2010).

The SYP4 family is the source of divergent results. On one hand, immunogold labelling assays observed by electron microscopy indicate the presence of SYP41 and SYP42 in dif-ferent domains of the TGN (Bassham et al., 2000), and the lethality of the single knock-out (KO) mutants also argues in favour of essential specificity (Sanderfoot et al., 2001). On the other hand, functional redundancy requires at least a double mutation in order to observe a phenotype. Triple mutation is indispensable to induce a gametophytic lethality (Uemura et al., 2012). In vitro lipid mixing assays highlight the capacity of SYP42 and SYP43 to substitute for SYP41 in the SNARE complex with SYP61 and VTI12 (Kim and Bassham, 2013). However, these results highlight a substantial overlap in func-tion of the SYP4 isoforms.

In the VTI1 family, VTI11 and VTI12 share 60% of sequence identity but are involved in different pathways. VTI11 is involved in trafficking towards the lytic vacuole and is localized to the TGN, PVC and the central vacuole. VTI12 seems to be

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involved in the trafficking of storage proteins and is localized at the TGN (Sanmartín et al., 2007). Although in vitro assays showed that they can substitute for each other at a molecu-lar level in SNARE complexes (Surpin et al., 2003; Kim and Bassham, 2013), the different in vivo localization of the two pro-teins prevent a total overlap of function (Niihama et al., 2005).

At the plasma membrane, SYP121 and SYP122 (sharing 64% of sequence identity) are involved in the transport of salicylic acid and jasmonic acid but do not have the same role in non-host resistance (Assaad et al., 2004; Zhang et al., 2007; Pajonk et al., 2008). However they may have an overlapping function in plant growth and development (Assaad et  al., 2004; Kim and Brandizzi, 2012). On the other hand SYP121 but not SYP122 is important for plasma membrane localiza-tion of the KAT1 K+ channel (Sutter et al., 2006). SYP121 interacts with the regulatory K+ channel subunit KC1, which affects the activity of KAT1 (Honsbein et  al., 2009, 2011; Grefen et  al., 2010). Moreover, VAMP721 and VAMP722, which are known to form a SNARE complex with SYP121 (Kwon et  al., 2008; Karnik et  al., 2013), also interact with the K+ channel (Zhang et al., 2015). SYP121, VAMP721 and VAMP722 have therefore a dual role as SNAREs in mem-brane fusion and as part of SNARE-K+ channel protein complex for the regulation of K+ exchange.

The t-SNAREs SYP51 and SYP52 are closely related, shar-ing 82% of sequence identity, and exhibit different functional specificities. It was shown that these t-SNAREs are necessary for the traffic to the central vacuole from small vacuoles for SYP51 or from PVCs for SYP52 (De Benedictis et al., 2013). They also may act as an i-SNARE at the tonoplast. The authors speculated that SYP51 and SYP52 reach the tono-plast by default when their respective SNARE complexes are saturated at the pre-vacuolar compartments. Then, as a nega-tive feedback effect, SYP51 and SYP52 respectively block the fusion with small vacuoles and PVCs.

Like Memb11 and Memb12, SYP51/SYP52 and SYP121/SYP122 have different roles despite their high identity. In addition, a dual role, as observed for SYP121, could be applied to Memb11. Indeed, we assume it acts as a SNARE for ER-Golgi transport (Chatre et  al., 2005; Bubeck et  al., 2008) and participates to the Golgi recruitment of Arf1.

Supplementary data

Supplementary data are available at JXB online.Supplementary Table S1. All primers used in the study.Supplementary Fig. S1. Properties of the anti-Memb11

antibody.Supplementary Fig. S2. Schematic representations of the

different couples tested by BiFC.Supplementary Fig. S3. 2D representations of Memb11

and Memb12 N-ter parts.Supplementary Fig. S4. Comparison of the structures of

the N-ter of rat membrin with Memb11.Supplementary Fig. S5. Controls of the sub-cellular

localizations of the different mutants between Memb11 and Memb12 used for BiFC assays.

AcknowledgmentsThe authors thank William Nicolas for careful reading of the manuscript. We thank Marie-France Giraud (IBGC, UMR 5095 CNRS-University of Bordeaux) for her help with the in vitro production of Memb11 and Memb12 peptides. Thanks to Patricia Thebault and the CBiB of CGFB (http://www.cgfb.u-bordeaux2.fr/fr/synthese-bioinformatique) for their help with the bio-informatic approach, Majid Noubany and Xavier Santarelli (University of Bordeaux, Bordeaux INP) for their help with the production and purifica-tion of Memb11∆TMD-6His, Didier Thoraval and François Doignon for their help to set up the in vivo yeast expression system, and to the proteomic platform of CGFB (http://www.cgfb.u-bordeaux2.fr/en/synthese-proteome) for the mass spectrometry analyses of immunoprecipitated proteins. Imaging was done at the Bordeaux Imaging Center (http://www.bic.u-bordeaux2.fr) of the University of Bordeaux, a member of the France-BioImaging National infrastructure (http://france-bioimaging.org/). This work was sup-ported by CNRS (Centre National de la Recherche Scientifique), University of Bordeaux and ANR-10-INBS-04 France-BioImaging. CM was supported by a PhD fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche (France).

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