Journal of Experimental Botany, Vol. 66, No. 21 pp. 6665–6678, 2015
doi:10.1093/jxb/erv373 Advance Access publication 24 July 2015
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,
France
2 University of Bordeaux- INP Bordeaux, BPRVS, EA4135, F-33000 Bordeaux, France
3 Proteome platform, Functional Genomic Center of Bordeaux, University of Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux Cedex,
France
4 Bordeaux Imaging Center, UMS 3420 CNRS, US4 INSERM, University of Bordeaux, 33000 Bordeaux, France
* To whom correspondence should be addressed. E-mail: [email protected]
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 function 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 mammalian 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 hypothesized 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 attachment protein receptor) proteins are components of the molecular 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,
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
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6666 | Marais et al.
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 protein) 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 immunoprecipitation (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 bimolecular 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 conditions
All 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 System
For 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 assay
ORFs 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 pointmutated proteins (Memb11-ΔKARD, Memb11-ΔESSSMDSP,
ARF1A1C -Q71L and ARF1A1C -T31N), OE-PCR (overlap extension polymerase chain reaction) was used to create the new genes;
the primers needed are listed in Supplementary Table S1. The corresponding 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 cloning. 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 production
Memb11∆TMD-6His (Memb11 without its transmembrane domain
and coupled to a 6 His tag) was expressed with the pET-15b vector 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
Memb11-Arf1 interaction in the Golgi | 6667
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 imidazole was performed to eliminate unspecific proteins. A one-step
elution with 0.5 M imidazole was then applied to obtain the purified Memb11∆TMD-6His protein. The purity and the efficiency of
the protein production were controlled by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).
Then the protein extract was used by Covalab (Lyon, France) to
produce the anti-Memb11 antibodies.
IP on plant extract
Plants 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 cocktail] 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 (antiMemb11 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 RTS
Proteins were expressed with the RTS. The pIVEX2.3 plasmid
(1.5 µg) with the gene of interest were added to 100 µl of reaction 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 phosphoenolpyruvic 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 incubation 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 reaction was stopped by adding 36 mM ice-cold MgCl2, and the solution 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 buffered 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 protocol 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 extract
Optical density (OD) at 600 nm of cells grown in pre-culture
medium with raffinose was measured; cells were seeded in 50 ml culture 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 centrifuged at 500 ×g for 5 min at 4°C. The supernatants were collected
and kept frozen at −80 °C. The protein’s concentrations were determined 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 blot
Proteins 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 membrane) 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 analyses
Sample preparation Each SDS-PAGE band was cut into 1 × 1 mm
gel pieces. Gel pieces were destained in 25 mM ammonium bicarbonate (NH4HCO3), 50% acetonitrile (ACN) and shrunk in ACN for
10 min. After ACN removal, gel pieces were dried at room temperature. 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% formic acid for 15 min at room temperature. The three collected extractions 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
6668 | Marais et al.
(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 separated 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 positive 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 unassigned 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 calculations. 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 experiments
For 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 modifications. Seeds were sown on sterile filters with a pore size of 500 µm
(Fisher Scientific, ref.: 11755498, https://fishersci.com). A. tumefaciens cells containing the different constructs were grown for 30 h
at 30°C in 20 ml Luria-Bertani (LB) culture medium. Cells were collected 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 subsequently removed and the plates were transferred to the culture room
for 3 d. During the observation, only the abaxial part of the cotyledon 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, detection was in sequential mode. All the confocal images were captured
in line-scanning mode with a line average of 4.
Immunoelectron microscopy
For immunogold labelling, 10-day-old meristematic zone of
Arabidopsis roots and Arabidopsis suspension cells were highpressure frozen with a Leica EMPACT system (http://www.leicamicrosystems.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 supplemented with 2% w/v osmium tetroxide (OsO4) and 0.1% w/v uranyl 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 overnight in 75% HM20 in ethanol. Polymerization was done under UV
for 48 h at −50°C. Immunogold labelling was performed as previously 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 antirabbit 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 analyses
The protein sequence alignment between Memb11 and Memb12
was performed with the Needleman-Wunsch global alignment algorithm 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 predictions 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 cytosolic 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 thaliana. 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% identical to Memb11) was also detected in the mass spectrometry
analysis, we have performed a competitive enzyme-linked
immunosorbent assay (ELISA) with purified Memb11 and
Memb11-Arf1 interaction in the Golgi | 6669
Memb12 peptides to estimate the specificity of the antibodies 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 immunolocalizations with anti-Memb11 antibodies on high-pressure 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 antibodies 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 antibodies 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 antiMemb11 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 spectrometry 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 comparison 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 machinery. 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 coimmunoprecipitate the complex and this prompted us to verify in vivo by BiFC whether or not this interaction could be
confirmed.
Memb11 but not Memb12 interacts with ARF1A1C
in vivo
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.
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 complex 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 interaction 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 proteins: YN155-SNARE or YC155-SNARE (Supplementary
Fig. S2). Oppositely, ARF1A1C is linked to the membrane
6670 | Marais et al.
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.
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 YC155ARF1A1C (Supplementary Fig. S2).
Co-infiltrations of A. thaliana cotyledons with the different 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 YC155Memb11/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
Memb11-Arf1 interaction in the Golgi | 6671
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 statistically efficient comparison, we observed a high number of
cotyledons per condition (Fig. 2E). An average of 4.8 fluorescent 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 bodies 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 differential 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 predictions 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 contain 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 combining 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 assembly 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 giving 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 further 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 accessibility (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 K25D28-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 S39S40-M41), which are exposed outside too. As a result, differences are mostly located around the second coil and are
accessible. Finally, the orientations of the last α-helix (beginning 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 modelling 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 compatible 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,
6672 | Marais et al.
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.
and amino acids 1–133 of Memb12 to amino acids 140–225
of Memb11 in order to swap the N-ter domain to the beginning 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 corresponding 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 Memb121-38Memb11, 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 interaction between Memb11 and Arf1 (Fig. 4B).
Finally, we designed a Memb11 mutant, called Memb11ΔKARD, 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 Memb11ΔESSSMDSP. The two mutants were correctly targeted to the
Golgi (Supplementary Fig. S5F, G). A decreased interaction
with Arf1 was observed for the Memb11-ΔESSSMDSP chimera while no interaction could be detected for the Memb11ΔKARD chimera (Fig. 4B).
Memb11-Arf1 interaction in the Golgi | 6673
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
37
ESSSMDSP44 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 GTPbound) 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 performed 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 presence 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 proteins synthesized in a prokaryotic system, we tried to confirm
these results by expressing the whole proteins in yeast (eukaryotic 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 revealing that the antibodies did not cross-react with yeast proteins (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), Memb11Memb12/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.
6674 | Marais et al.
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.
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 apparatus. 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 surface 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 second coil (Supplementary Fig. S4), which argues in favour of
a role of this domain in Memb11-ARF1A1C and rat membrin-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 tempting to conclude that this protein area is universally critical
for interactions between membrin and Arf1 proteins. In addition, 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 localized 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 exocytosis 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
Memb11-Arf1 interaction in the Golgi | 6675
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 secretory pathway. Indeed, it seems that the distinct cellular functions 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 cytoplasmic 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 compatible with an interaction in the Golgi (Montesinos et al., 2014).
In addition, it was also observed in animals that the sequence
110
MXXE113 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 suggested 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 Arf1GDP 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 homologues 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 protein 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 membrane compartments (Golgi apparatus, TGN, endosomes and
plasma membrane) and could interact with a large number
of proteins. It would be interesting to determine the possible 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 compartments. However, as no SNARE complex has been characterized 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 localization (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 different 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 function 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
6676 | Marais et al.
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 molecular level in SNARE complexes (Surpin et al., 2003; Kim and
Bassham, 2013), the different in vivo localization of the two proteins 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 localization 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 membrane 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, sharing 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 tonoplast by default when their respective SNARE complexes are
saturated at the pre-vacuolar compartments. Then, as a negative 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.
Acknowledgments
The 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 bioinformatic approach, Majid Noubany and Xavier Santarelli (University of
Bordeaux, Bordeaux INP) for their help with the production and purification 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 supported 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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