# Plasma membrane localization of Solanum tuberosum remorin from group 1, homolog 3 is mediated by conformational changes in a novel C-terminal anchor and required for the restriction of potato virus X movement

Artemis Perraki, Jean-Luc Cacas, Jean-Marc Crowet, Laurence Lins, Michel Castroviejo, Sylvie German-Retana, Sébastien Mongrand, Sylvain Raffaele

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Artemis Perraki, Jean-Luc Cacas, Jean-Marc Crowet, Laurence Lins, Michel Castroviejo, et al. Plasma membrane localization of Solanum tuberosum remorin from group 1, homolog 3 is mediated by conformational changes in a novel C-terminal anchor and required for the restriction of potato virus X movement. Plant Physiology, 2012, 160 (2), pp.624-637. 10.1104/pp.112.200519. hal-02648092

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1 Running head: Membrane anchor of StREM1.3 Remorin

2

3 **Correspondence:**

4 Dr Sébastien MONGRAND, phD

5 Address: Laboratoire de Biogenèse Membranaire (LBM)

6 CNRS UMR 5200 / Université Bordeaux Segalen

7 146, rue Léo Saignat - Case 92

8 33076 Bordeaux Cédex

9 Email: sebastien.mongrand@u-bordeaux2.fr

10 Telephone: + 33 (0) 5 57 57 14 35

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12 **Journal research area:**

13 Biochemical Processes and Macromolecular Structures

14 Cell Biology
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Plasma membrane localization of StREM1.3 Remorin is mediated by conformational changes in a novel C-terminal anchor and required for the restriction of PVX movement

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Artemis Perraki, Jean-Luc Cacas, Jean-Marc Crowet, Laurence Lins, Michel Castroviejo, Sylvie German-Retana, Sébastien Mongrand¹* and Sylvain Raffaele¹

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Laboratoire de Biogenèse Membranaire, (LBM) Unité Mixte de Recherche 5200 (UMR 5200) Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France (SR, AP, JLC and SM)

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The Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK (SR)

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Centre de Biophysique Moléculaire Numérique, ULg, Gembloux Agro Bio Tech, Passage des Déportés, 2, B-5030 Gembloux, Belgium (JMC and LL)

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Laboratoire de Microbiologie Fondamentale et Pathogénicité Unité Mixte de Recherche 5234 (UMR 5234) Centre National de la Recherche Scientifique (CNRS) 146 rue Léo Saignat 33076 Bordeaux Cedex, France (MC)

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Equipe de Virologie, INRA and Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, BP81, 33883 Villenave d'Ornon Cedex, France. (SGR)
Footnotes :

40 This work was supported by a Marie Curie IEF to S.R. (contract 255104). We
41 acknowledge the French ‘Agence Nationale pour la Recherche’ (ANR) for financial
42 support (contracts NT09_517917 PANACEA to S.M. and JLC). L.L. is senior
43 research assistant at the Belgian National Funds for Research (FNRS). LL and JMC
44 thank IAP project (iPROS) for financial support.

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46 1 - SR and SM must be considered as co-last authors

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48 SR Present address: Laboratoire des Interactions Plantes Micro-organismes, Chemin
de Borde Rouge BP 52627, 31326 Castanet Tolosan, France.

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51 JLC Present address: Université de Bourgogne, UMR Plante-Microbe-Environnement
52 1088, Institut National de la Recherche Agronomique (INRA), 5184 CNRS, 21065
53 Dijon Cedex, France.
54 **ABSTRACT**

The formation of plasma membrane (PM) micro-domains plays a crucial role in
the regulation of membrane signalling and trafficking. Remorins are a plant-specific
family of proteins organized in six phylogenetic groups, and Remorins of the group 1
are among the few plant proteins known to specifically associate with membrane
rafts. As such, they are valuable to understand the molecular bases for PM lateral
organization in plants. However, little is known about the structural determinants
underlying group 1 Remorins specific association with membrane rafts. We used a
structure-function approach to identify a short C-terminal anchor (RemCA)
indispensable and sufficient for tight direct binding of *Solanum tuberosum* REMORIN
1.3 (StREM1.3) to the PM. RemCA switches from unordered to an alpha-helical
structure in a non-polar environment. Protein structure modelling indicates that
RemCA folds into a tight hairpin of amphipathic helices. Consistently, mutations
reducing RemCA amphipathy abolished StREM1.3 PM localization. Furthermore,
RemCA directly binds to biological membranes *in vitro*, shows higher affinity for
Detergent-Insoluble Membranes (DIM) lipids, and targets YFP to DIMs *in vivo*.
Mutations in RemCA resulting in cytoplasmic StREM1.3 localization abolish
StREM1.3 function in restricting potato virus X movement. The mechanisms
described here provide new insights on the control and function of lateral segregation
of plant PM.
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# INTRODUCTION

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Protein-lipid interactions are increasingly recognised as key regulatory processes for signal perception and cellular signalling cascades (Cho and Stahelin, 2005). During signal transduction and trafficking, a number of soluble proteins dynamically associate with plasma membranes (PMs) to deliver their cargo and to recruit pathway components to the sites of action (Seong et al., 2011). For such proteins, membrane association can be critical for function (Porter and Koelle, 2010).

PM targeting of peripheral proteins is achieved through (i) binding to integral membrane proteins, (ii) post-translational modifications, or (iii) directly by intrinsic membrane anchor domains. Post-translational modifications function as auxiliary modifications for transient or weak association of soluble proteins to the intracellular face of the PM. In plants, these include N-myristoylation, S-palmitoylation, prenylation by farnesyl or geranylgeranyl moieties, or attachment of glycosylphosphatidylinositol (GPI) anchors (Thompson and Okuyama, 2000). GPI-anchors, for example, tightly associate proteins to the extracellular face of PMs by interaction of the inositol head group of the membrane lipid phosphatidylinositol with a glucosamine residue linked to the C-terminal amino acid of the protein (Paulick and Bertozzi, 2008). As an alternative mechanism, globular structures either recognize phospholipids in a stereospecific manner or associate with membranes by their biophysical properties (reviewed in Lemmon, 2008). Other proteins expose unstructured clusters of basic and hydrophobic residues to mediate PM binding (McLaughlin et al., 2002; McLaughlin and Murray, 2005).

Selective recognition of membrane compartments or domains by protein anchors can be critical in triggering the appropriate downstream trafficking and signalling events (for review: Gruenberg, 2003; De Matteis and Godi, 2004). Membrane domain selectivity can be specified by the anchoring post-translational modification or by a protein anchor domain. For instance proteins carrying GPI-anchors are over-represented in membrane rafts, indicating that addition of this lipid anchor directs proteins to these micro-domains (Cordy et al., 2003; Kierszniowska et al., 2009). Membrane rafts are enriched in highly-saturated long chain sphingolipids, sterols and saturated phospholipids, creating tightly-packed domains, designated as ‘Liquid-ordered’. These lipids display a stronger affinity to saturated acyl chains as
found in GPI anchored and acylated proteins (Brown, 2006). The composition of membrane rafts also prevents solubilisation by detergent at low temperature with non-ionic detergent, and allows the partial purification of raft in so called Detergent-Insoluble Membrane (DIM) fractions, supposedly biochemical counterparts of membrane rafts. Many signalling proteins are found in membrane rafts supporting the hypothesis that they serve as key platforms for cellular signal transduction and cell-to-cell communication (Lingwood and Simons, 2010; Simon-Plas et al., 2011). For example, in human cells, key soluble signalling components such as the Ser/Thr kinase Akt (protein kinase B) are recruited to membrane rafts where they activate signal transduction cascades (Lasserre et al., 2008). Nevertheless, few protein motifs were described to contribute to raft targeting (Rossin et al., 2010) while the six amino acid long raft target signal from human tyrosine phosphatase Src homology 2-containing phosphatase 1 (SHP-1) is the only known motif sufficient to anchor soluble proteins specifically to domains of the intracellular face of the PM (Sankarshanan et al., 2007). However, the anchoring mechanism itself remains to be unravelled. In plants even though DIMs also exist (Mongrand et al., 2010) and functional PM domains have been reported (Bhat et al., 2005), the molecular basis for specific targeting and binding of proteins to membrane rafts has never been described.

Remorins form a diverse family of plant-specific proteins organized in six distinct phylogenetic groups (Raffaele et al., 2007). Remorins from the group 1 have been reported to localize to the PM despite their overall hydrophilic nature (Reymond et al., 1996; Raffaele et al., 2007). Moreover, group 1 Remorins almost exclusively associate to DIMs and localize to membrane micro-domains in a sterol-dependent manner (Lefebvre et al., 2007; Kierszniowska et al., 2009; Raffaele et al., 2009). The function of Remorins is mostly unknown, but we showed in a previous study that StREM1.3 (*Solanum tuberosum* Remorin from group 1, homolog 3, initially described in (Reymond et al., 1996) regulates cell-to-cell propagation of the potato virus X (PVX) likely by directly interacting with the viral movement protein TGBp1 (Raffaele et al., 2009). StREM1.3 localizes to the inner leaflet of PMs and along plasmodesmata, bridges connecting neighbour cells essential for cell-to-cell communication in plants (Maule, 2008). Other members of the Remorin family group 1 are likely involved in innate immune responses (Liu et al., 2009; Widjaja et al., 2009; Keinath et al., 2010). Remorins from group 2 are involved in the control of
infection by symbiotic bacteria at nodular infection threads and the peribacteroid
membrane (Lefebvre et al., 2010) These data suggest general roles for Remorins in
regulating signalling in plant-microbe interactions (Jarsch and Ott, 2011).

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Elucidating the mechanisms driving StREM1.3 association with PM micro-
domains therefore provides a unique opportunity for understanding the regulation
and function of membrane lateral segregation in plants. StREM1.3 does not contain
predictable transmembrane or membrane associated domains. The bases for its
association to PMs and selective targeting to DIMs are unknown. Here we identified a
novel membrane anchor domain required for StREM1.3 tight and direct association
with detergent insoluble fraction of the PM. We combined biophysics, in silico
analysis and directed mutagenesis to unravel the molecular bases of StREM1.3
membrane binding and its biological significance in the control of PVX propagation.

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## RESULTS

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StREM1.3 is a strongly associated peripheral membrane protein

To identify structural domains potentially involved in PM localization of
StREM1.3, we performed *in silico* domain analysis and secondary structure
prediction. Proteins of the Remorin family typically contain a variable N-terminal
region (Marín and Ott, 2012) and a conserved C-terminal domain, termed Remorin_C
(PF03763), of approximately 120 amino acid (aa) residues that encompasses a
predicted coiled-coil domain. In StREM1.3, the Remorin_C domain extends from aa
85 to 195 with a coiled-coil domain predicted between aa 116 to 152 (Figure 1A).
StREM1.3 C-terminal region is predicted to be mostly alpha-helical composed of a 84
aa long coiled alpha helix ranging from position 85 to 169. This helix is predicted to
be interrupted at Glycine residue 171. A short (~12 aa) alpha helix is predicted
between residues 172 and 187. Finally the most C-terminal residues are predicted to
form a random coil except for residues 192 to 195 where a short alpha helix is
predicted. The N-terminal region of StREM1.3 contains the proline-rich Remorin_N
domain (PF03766) extending between aa 27 to 84 and is mostly unordered (Marín
and Ott, 2012). No recognizable transmembrane domains or any other membrane-
targeting signals could be detected, indicating that StREM1.3 is a peripheral
membrane protein with an atypical membrane-binding domain.

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To test the nature of Remorin association with biological membranes we purified native microsomes and PMs from tobacco leaves and tested its affinity by removal of extrinsic membrane proteins. In accordance with published data PM stripping resulted in ~50% loss of total protein, representing peripheral proteins with weak affinity to the PM (Santoni et al., 2003). Typical of peripheral membrane proteins is Actin, which was almost completely removed from the PM after treatment (Figure 1B). However, endogenous StREM1.3 remained almost entirely associated with the PM, similar to true integral proteins such as the aquaporin PIP2 that was used as an integral membrane protein control. Similar results were obtained when purified PMs were washed under high ionic strength conditions, increasing EDTA concentrations, and increasing pH for 30 minutes (Figure S1A). These results suggest that the association of StREM1.3 with the membrane is not limited to electrostatic interactions.

**Remorin C-terminal Anchor (RemCA) is required and sufficient for plasma membrane targeting in vivo.**

A series of full length and truncated protein variants was generated and fused to G/YFP to analyse the structural requirements for StREM1.3 PM localization. The full-length protein localized to the PM (Figure 1C). This localization is consistent with immunolocalization studies reported earlier (Raffaele et al., 2009; Lefebvre et al., 2010) suggesting that the fluorescent tag did not affect localization of the fusion proteins. Deletion of the C-terminal residues (StREM1.3¹⁻¹⁷⁰) resulted in an entire loss of PM association indicating the presence of a PM anchor motif within these residues (Figure 1C). We will refer to this 28 amino acid region as the “Remorin C-terminal Anchor” (RemCA). This hypothesis was confirmed by reciprocal experiments where expression of soluble YFP tagged with RemCA led to clear YFP association with the PM (Figure 1C). We designed a variety of different constructs to express the fluorescently tagged RemCA but we were not able to prevent a partial cleavage of the tag, resulting in a residual nucleo-cytoplasmic fluorescence in vivo. To confirm that RemCA is sufficient to anchor soluble proteins to the PM, we performed biochemical fractionation experiments on cells expressing StREM1.3 G/YFP fusion constructs (Figure 1D). These experiments showed that the GFP:StREM1.3 is fully microsomal whereas YFP:StREM1.3¹⁻¹⁷⁰ is entirely soluble. The YFP:RemCA fusion was recovered exclusively in the microsomal fraction, with only cleaved YFP in the
210 soluble fraction. These results demonstrate that RemCA is required for StREM1.3
211 PM localization and sufficient to fully associate soluble proteins with the PM.
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213 **Non-polar environments trigger conformational changes in RemCA.**

214 To analyse the structural properties of RemCA we obtained chemically
215 synthesized tag-free ultra pure peptides that were assessed using circular dichroism
216 (CD) spectroscopy. CD spectra were acquired in 100% 2,2,2-Trifluoroethanol (TFE),
217 a solvent mimicking biological membranes hydrophobicity (causing partial
218 desolvation of the peptide, a situation that occurs in membranes and during protein-
219 protein and protein-lipid interactions), or in semi-hydrophobic solvent (50% TFE, 50%
220 Tris-HCl). CD spectra showed distinct minima at 208 and 222 nm, with a marked
221 maximum at 195 nm, consistent with RemCA forming an alpha helical structure
222 (Figure 2A). By contrast the spectrum of RemCA in aqueous solution (100% Tris-
223 HCl) showed a reduced minimum at 208 nm and reduced concavity around 195 nm,
224 indicating that alpha helical contributions to the overall RemCA structure are reduced
225 under these conditions. We estimated RemCA protein secondary structure
226 composition from CD spectra (Figure 2B) and found that it undergoes transition from
227 15% alpha-helical content in aqueous solution to 50% in hydrophobic non-polar
228 solvents. Such estimations remain challenging and should thus be taken with
229 caution. Nevertheless, these data imply that the RemCA peptide is largely unordered
230 in aqueous solution and folds into a mostly alpha helical structure in hydrophobic
231 environments.

232 To gain further insights into the structural mechanisms underlying RemCA
233 binding to PMs, we performed homology 3D modelling of this peptide using the I-
234 TASSER server. The predicted structure for RemCA corresponds to two alpha
235 helices arranged as a tight hairpin (Figure 2C, Supplemental File 1). This structure
236 is reminiscent of a recently published structure for Influenza hemagglutinin fusion
237 peptide (HAfp) (Lorieau et al., 2010) (Figure S2). The predicted structure has a high
238 helical content, therefore probably corresponding to the conformation adopted by
239 RemCA in hydrophobic environments. This hypothesis is consistent with the structure
240 of the HAfp which forms a tight hairpin of alpha helices in the presence of lipids
241 (Lorieau et al., 2010). The distribution of the relative hydrophobicity of amino-acids
242 reveal an accumulation of hydrophobic aa at the centre of the hairpin, with the side
243 chains of hydrophilic charged amino acids pointing outward (Figure 2C). The turn
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connecting the two alpha helices is composed of the palindromic neutral sequence Ala-Thr-Gly-Thr-Ala.

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**Amphipathy of RemCA alpha helices is required for Remorin anchoring to the PM in vivo.**

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The predicted hydrophobic core at the centre of the RemCA hairpin and solvent-exposed charged surfaces (Figure 2C) implies an asymmetry in the distribution of hydrophobic and charged residues around the alpha-helices, a property known as amphipathy. Amphipathic helices are crucial for transient anchoring of some peripheral membrane proteins, notably viral fusion proteins (e.g Lins and Brasseur, 2008; Drin and Antonny, 2009). We used the heliquest server to calculate the difference, named hydrophobic moment (µH), in hydrophobicity between the two faces of each alpha helix forming RemCA. The E<sup>172</sup>-A<sup>186</sup> helix had a µH of 0.153, similar to values obtained for viral fusion peptides (e.g 0.171 for the Simian influenza virus -SIV- fusion peptide), whereas helical fragment P191-F198 had a µH of 0.661, with each helix organized with clearly distinct charged and hydrophobic faces (Figure 3A).

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To test the importance of alpha-helices amphipathy in the function of RemCA, we designed a StREM1.3* variant harbouring a mutated RemCA* with reduced amphipathy (Figure 3B, Supplemental File 2). The transient expression of a YFP:StREM1.3* in tobacco leaves showed that the mutations completely abolished membrane localization, leading to a fully cytoplasmic protein (Figure 3C). Loss of PM association was confirmed biochemically by protein fractionation where the YFP:StREM1.3* protein was exclusively detected in the soluble fraction, while endogenous Remorin remained in the PM fraction (Figure 3D).

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**RemCA directly binds to lipid bilayers in vitro.**

To test whether RemCA anchors StREM1.3 to the PM independently of post-translational modifications or association with other proteins, recombinant 6His:StREM1.3 (+), 6His:StREM1.3<sup>1-170</sup> (-, deletion of RemCA), and 6His:StREM1.3* (*, mutated RemCA, see above) were purified from *E. coli*, and tested for binding to liposomes mimicking plant PM lipid composition *in vitro*. Liposomes were separated at the top of a sucrose gradient by centrifugation. The presence of StREM1.3 in the liposome-bound or unbound fraction was assessed by Western Blot and lipids were
278 analysed by TLC (Figures 4A and S3) (Figure. StREM1.3 bound to liposomes in the
279 absence of any other proteins (Figure 4A). Although similar amounts of liposomes
280 were isolated for each sample (Figure S3B), binding was completely abolished in
281 StREM1.3<sup>1-170</sup> and StREM1.3* variants (Figure 4A). Furthermore, and in agreement
282 with our data obtained using purified PMs, treatment with denaturing agents did not
283 significantly reduce the amount of 6His:StREM1.3 bound to liposomes (Figure S1B).
284 These results demonstrate that RemCA confers direct membrane binding to
285 StREM1.3. Loss of membrane binding in StREM1.3* indicates that amphipathy plays
286 a crucial role in this process.

287 Insertion of a peptide into membranes results in a partial destabilization of
288 artificial bilayers and can thus be taken as a measure to discriminate between
289 physical insertion and outer association (Longo et al., 1997). To test whether RemCA
290 induces membrane destabilization, we performed lipid-mixing assays in which
291 application of purified peptides to a mixture of liposomes, labelled or not by the
292 lipophilic fluorescent dye octadecyl rhodamine chloride (R18), leads to a de-
293 quenching of fluorescence. We confirmed the functionality of this assay by the
294 application of the well-known Simian immunodeficiency virus (SIV) fusion peptide
295 (Lorin et al., 2008) to liposomes (see materials and methods). By contrast liposome
296 treatment with TFE, used as a solvent for the peptides, did not result in lipid mixing.
297 Application of synthetic ultra pure RemCA peptides resulted in a ~40% de-quenching
298 of fluorescence relative to the values obtained with the SIV fusion peptide (Figure
299 4B) indicating destabilization of the membrane and insertion of the peptide in the
300 bilayer.

301 To verify that the increase in fluorescence was due to liposome destabilization rather
302 than liposome aggregation, we performed liposome leakage assays, in which
303 liposomes entrapping the fluorescent dye 1-hydroxypyrene-3,6,8-trisulfonate (HPTS)
304 and the corresponding cationic quencher α,α'-dipyridinium p-xylene dibromide (DPX)
305 were used. Application of fusion peptides leads to a de-quenching of fluorescence
306 due to physical separation of both components (Ellens et al., 1985). We used
307 liposomes composed of either negatively charged lipids or neutral lipids. As
308 expected, the use of the detergent TX100 entirely disrupted the liposomes, leading to
309 maximal fluorescence (Figure 4C). Application of the SIV fusion peptide induced
310 ~90% leakage of negatively charged liposomes, and only ~9% leakage of neutral
311 liposomes, well above leakage induced by solvents used for the peptides (leakage
312 <2% by either DMSO or TFE for negatively charged and neutral liposomes
313 respectively). Application of synthetic RemCA peptide yielded in ~5% of leakage of
314 negatively charged liposomes, but did not cause any significant leakage of neutral
315 liposomes (Figure 4C). These experiments demonstrate that RemCA physically
316 inserts into membranes independently of other proteins, and predominantly binds
317 negatively charged lipids, suggesting a differential affinity for classes of lipids.

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319 **RemCA confers lipid-specific binding to StREM1.3**

320 To test whether RemCA binds selectively to certain lipids, we probed
321 immobilized lipids with purified recombinant 6His:StREM1.3 and 6His:StREM1-170 in
322 lipid overlay assays. StREM1.3 showed differential affinity to 5 of the 7 lipids tested
323 (Figure 5A). Affinity of StREM1.3 was strong to PI(3,4)P₂ phosphoinositol (3,4)-
324 bisphosphate and sphingosine-1-phosphate (S1P), moderate to phosphatidic acid
325 (PA) and PI(3,5)P₂, and weak to phosphatidylserine (PS). Binding to these
326 immobilized lipids was dependent on RemCA since it was almost completely
327 abolished in the StREM1.31-170 protein, with only a weak residual binding to PI(3,4)P₂
328 remaining. Similar results were obtained with the purified recombinant
329 6His:StREM1.3* mutant (Figure S4A). These results indicate that RemCA has
330 affinity for minor anionic phospholipids and negatively charged lipids, and that
331 RemCA confers selectivity to the range of lipids bound in vitro. Interestingly, these
332 lipids are enriched in membrane rafts from plants (Vermeer et al., 2009; Furt et al.,
333 2010) or animal cells (Hope and Pike, 1996; Pike et al., 2002), suggesting that
334 RemCA lipid-specificity contributes to driving the segregation of StREM1.3 into
335 membrane micro-domains.

336 To support this hypothesis, we first tested the binding of 6His:StREM1.3 to
337 liposomes with lipid composition mimicking either the PM or DIMs *in vitro* (see
338 materials and methods). Although the total amount of liposomes pulled down was
339 similar for all samples (Figure S4B), the relative quantification of 6His:StREM1.3
340 bound demonstrated a higher affinity for DIM-like liposomes compared to PM-like
341 liposomes (Figure 5B).

342 Finally, to test the specificity of RemCA-mediated binding *in vivo*, we extracted
343 DIMs from PM purified from tobacco plants expressing RemCA tagged with the
344 soluble YFP. As expected, ~80% of total proteins were excluded from the DIM
345 fraction (Figure 5C, S4C) (see also Raffaele et al., 2009) but the endogenous
tobacco Remorin proteins were almost exclusively found in DIM fractions (Figure 5C). Strikingly, the majority of YFP:RemCA proteins were also detected in DIMs (Figure 5C). These results demonstrate that RemCA is sufficient to target soluble proteins specifically to DIMs of plant PM.

## StREM1.3 forms homo-trimers *in vitro* independently of RemCA

Previous work showed by direct electron microscopy observations and chemical crosslinking with glutaraldehyde that group1 Remorins forms homo-polymeric filaments (Bariola et al., 2004). To test whether RemCA plays a role in the assembly or stability of these higher-order polymers, we used exclusion column chromatography to estimate the native molecular weight (MW) of 6His:StREM1.3 and 6His:StREM1.3* recombinant proteins. Chromatography revealed a peak of elution at ~1.25 mL (Figure S5A). Western blot analysis after denaturing SDS-PAGE of the corresponding fractions confirmed that 6His:StREM1.3 eluted at this volume (Figure S5B). Calibration of the gel filtration column with standards of known MW allowed to estimate 6His:StREM1.3 native MW to ~120 kDa, suggesting that 6His:StREM1.3 forms meta-stable trimers *in vitro* (Figure 6A). Neither monomers nor dimers were detected by gel filtration. Chromatography performed with 6His:StREM1.3* protein showed similar elution pattern, indicating that a non-functional RemCA does not alter the assembly of StREM1.3 into trimers. Interestingly, as discussed previously (Figure S2), RemCA shows structural similarities with influenza virus hemagglutinin (HA) fusion peptide. Influenza HA adopts a central trimeric alpha-helical coiled-coil structure, and the formation of trimers of fusion peptide hairpins is critical for membrane fusion (Cross et al., 2001). To get insights on the possible consequences of StREM1.3 trimer formation on its membrane-binding properties, we used homology modelling and geometry based docking to produce a 3D model of StREM1.3 C-terminal domain (E87-F198) trimer (Figure 6B). This model suggests that RemCAs are organized circularly, forming a hydrophobic core at the centre of RemCA trimer of hairpins and exposing patches of positively charged residues all around the C-terminus of the trimer. This conformation would create a strong hydrophobic moment directed inward RemCA trimer, and likely increases the strength and lipid-specificity of the membrane binding.
Mutations in RemCA* abolish StREM1.3 restriction of PVX cell to cell movement

To test for the biological significance of StREM1.3 PM localization, we exploited the previously reported role of StREM1.3 in restricting the cell-to-cell propagation of the potato virus X (PVX) in tomato leaves (Raffaele et al., 2009). Here, we transiently co-expressed in *N. benthamiana* leaves 35S:StREM1.3 or 35S:StREM1.3* with a highly diluted suspension of *Agrobacterium tumefaciens* harbouring an infectious PVX:GFP clone, so that only isolated individual cells were infected with the PVX:GFP. The accumulation of PVX:GFP in distinct infection foci was visible 5 days post infection (dpi). StREM1.3 or StREM1.3* expression was verified by Western blot (Figure S6). The effect of the expression of the StREM1.3 proteins was assessed by visualizing the virus infection foci and counting the number of fluorescent cells of at least 30 infection foci by comparison with plants infiltrated with *A. tumefaciens* carrying the PVX:GFP clone only. At 5 dpi, plants infiltrated with *A. tumefaciens* carrying PVX:GFP alone showed infection foci covering 17 cells in average (Figure 7A, B). As expected, the co-expression of StREM1.3 lead to a significant decrease in PVX:GFP spreading, with foci covering 12 cells in average (Figure 7A, B). By contrast, co-expression of StREM1.3* did not significantly alter the size of the PVX:GFP infection foci (Figure 7A, B). StREM1.3 was shown to directly interact with the TGBp1 viral movement protein during PVX infection (Raffaele et al., 2009). To determine if the loss of activity in StREM1.3* was due a loss of interaction with TGBp1, we tested the interaction of the StREM1.3* and StREM1.3<sup>1-170</sup> mutants with TGBp1 in a Split Ubiquitin assay. In both case, a clear interaction was observed, similar to the wild type StREM1.3 (Figure 7C). These results clearly demonstrate that RemCA-mediated PM anchoring of StREM1.3 is required for the function of this protein in the control of virus propagation and indicate that this activity takes place at the PM.

DISCUSSION

In this paper we showed that StREM1.3 directly binds to the PM via a short C-terminal anchor domain (RemCA). This domain is unordered in aqueous solution and spontaneously folds, probably into a tight hairpin of amphipathic alpha helices, in non-polar environments. The amphipathy of RemCA helices is required for StREM1.3 PM localization. RemCA selectively binds to lipids enriched in DIMs and targets the
soluble YFP to DIMs *in vivo*. StREM1.3 forms trimers *in vitro*. This does not require a
functional RemCA and may increase the stability of PM anchoring *in vivo*. PM
anchoring is crucial for StREM1.3 function in restricting PVX movement since
mutations in RemCA abolishing PM binding also abolish StREM1.3 function towards
PVX. Molecular mechanisms controlled by raft-mediated signalling and invasion of
host cells via membrane micro-domains may be altered by directly targeting proteins
to these sites. Using RemCA as an anchor may thus provide the opportunity to
modulate raft-mediated signalling processes or alter host resistance towards
pathogenic microbes.

Molecular bases for the assembly of Remorin micro-domains at the PM: a
model.

Our results indicate that the folding of RemCA into amphipathic alpha helices,
probably organized as a tight hairpin, is required for StREM1.3 PM anchoring and
function. Our CD data showed that RemCA switches from unordered to alpha helical
folding in non-polar environments. RemCA folding could be triggered by proximity
with the hydrophobic vicinity of the PM (Figure 8A). Similar observations were made
for Fis1 Tail-anchor protein (Suzuki et al., 2003), the ArfGAP1 lipid sensor motif
(Bigay et al., 2005), and for the amphipathic membrane-binding helix of the epsin N-
terminal homology (ENTH) family of clathrin adaptors (Ford et al., 2002).
Alternatively, the formation of StREM1.3 trimers would result in the formation of a
hydrophobic core, thereby creating an environment favourable to RemCA folding
(Figure 8B, C). Association of group 1 Remorins into higher-order complexes has
been reported in vitro (Bariola et al., 2004) and in vivo (Raffaele et al., 2009;
Lefebvre et al., 2010), but whether this is triggered by external signals remains
unknown. We propose that RemCA anchoring occurs as described for other lipid-
binding proteins (Stahelin et al., 2003; Peter et al., 2004) in a two-step manner. First,
positively charged residues E172-A186 increase the affinity of RemCA for negatively
charged lipids of the inner leaflet of membrane rafts (Figure 8A, D). This destabilizes
the internal monolayer of the PM and allows RemCA to insert deeper in the
membrane (Figure 8B, E). The complete folding of RemCA results in the formation of
a tight hairpin of alpha helices defining a hydrophobic pocket at its core. As described
for Influenza HAfp (Han et al., 2001), formation of this hairpin could result in a deeper
insertion of RemCA explaining the intrinsic-like behaviour of Remorins (see Figure
1B). Considering that Influenza HAfp is non fusogenic if not properly kinked (Lai and Tamm, 2007), the tight closure of RemCA hairpin of helices might be required for stable anchoring in the PM. Finally, the possibility that the formation of StREM1.3 multimers occurs at the PM, eventually under control of external signals (Figure 8B, E), would allow the aggregation of membrane rafts into higher order clusters, as suggested during the response to pathogen (Raffaele et al., 2009).

## 454 Biophysical properties defining RemCA lipid-specificity

In this work, we have shown that RemCA alone is able to anchor soluble hydrophilic proteins to the PM, such as G/YFP (Figure 1). We cannot exclude that additional structural features of StREM1.3 protein modulate this activity, such as post-translational modifications (e.g. phosphorylation), and the formation of StREM1.3 multimers that would exacerbate local gradients of hydrophobicity and charge. It is remarkable that RemCA preferentially binds to negatively-charged lipids abundant in membrane rafts and target the soluble YFP to DIMs. A number of globular domains is known to bind to the surface of the PM by specifically recognizing certain lipids, particularly discriminating between different phospholipid types (Lemmon, 2008; Kutateladze, 2010). A combination of steric recognition of specific lipid polar head groups, electrostatic attraction to negatively charged lipids and membrane destabilization-mediated penetration govern the function of these domains. Some of those such as the Annexin, ENTH, ANTH ('AP180 N-terminal Homology'), BAR and PX ('Phox homology') domains adopt an alpha-helical fold. Overall the hydrophobicity, the hydrophobic moment, the net charge and helix-breaking amino acids contribute to the membrane binding properties of amphipathic helices (Drin and Antonny, 2009). In small GTPases, polybasic clusters provide PM specificity by binding to negatively charged PI(4,5)P₂ and PI(3,4,5)P₃ of the PM (Heo et al., 2006). The charge of the polar face of ArfGAP1 amphipathic helices has been associated with the ability to perceive membrane curvature, with the introduction of lysine residues at the polar-non polar interface reducing curvature sensitivity and allowing binding to large liposomes (Drin et al., 2007). It is therefore tempting to speculate that RemCA membrane binding could sense membrane curvature as a mean to specifically target certain domains in the membrane. In addition, large hydrophobic residues on both sides of the kink are required for setting the angle of the hairpin structure and for function in Influenza HAfp (Lai and Tamm, 2007). The
L174, L180, I194 and F198 could play this role in RemCA. The overall conformation of RemCA as well as individual residue side chains are likely to contribute to its membrane binding ability, and further investigations will be required to dissect how this activity is mediated.

485

## Towards understanding the molecular bases of specific association with membrane rafts

RemCA drives the soluble YFP to DIM PM fractions, and lipids bound by RemCA are enriched in DIMs. Indeed, polyphosphoinositides and sphingolipids are enriched in membrane rafts from plants (Vermeer et al., 2009; Furt et al., 2010) and animal cells (Hope and Pike, 1996), the latter being also enriched in PS (Pike et al., 2002). The ability to specifically target DIMs might therefore be related to the differential affinity of RemCA to certain negative lipids, as known for SNARE proteins (Mima and Wickner, 2009). However, It should be noted that the presence of a protein in DIMs is not sufficient to associate it with membrane rafts (Munro, 2003; Simons and Gerl, 2010; Tanner et al., 2011). In the case of StREM1.3, we previously reported that enrichment in DIMs correlate with clustering into microdomains of 70 nm in the PM detected by immunogold-labeling (Raffaele et al., 2009). Importantly, both DIM enrichment and clustering at the PM were phytosterol-dependent (Kierszniowska et al., 2009; Raffaele et al., 2009; Tanner et al., 2011). Since lipid-lipid interactions are sufficient to drive the segregation of liquid-ordered and liquid-disordered phases in model membranes (Dietrich et al., 2001; Baumgart et al., 2003), it can be hypothesized that RemCA selective lipid binding properties could be sufficient to restrict the localization of StREM1.3 to membrane rafts.

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Nevertheless, it is reasonable to assume that in addition to RemCA other domains or motifs may determine the organization of StREM1.3 into PM microdomains. Indeed, the association of Remorins with DIMs is regulated quantitatively by signals such as cold (Minami et al., 2009) and pathogen-associated molecular patterns (PAMPs) (Keinath et al., 2010). The molecular bases of this regulation are not fully understood but may likely involve protein-protein interactions or post translational modifications. It is also not known whether the high-ordered organization of Remorins into multimers is regulated by external signals. In any case, protein-protein interactions mediated by the coiled-coil or the N-terminal domain of Remorins
514 (Lefebvre et al., 2010; Tóth et al., 2012) likely modulate the localization of Remorins
515 *in vivo*.

516

517

518 Implications for the mechanisms underlying PVX cell-to-cell movement

519 In a previous study we reported that StREM1.3 restricts potato virus X (PVX)
520 cell-to-cell movement independently of viral replication and that StREM1.3 physically
521 associates *in vivo* and *in vitro* with the triple gene bloc (TGB) protein 1 of the virus
522 (Raffaele et al., 2009). Here we show that restriction of PVX movement is abolished
523 in StREM1.3* cytoplasmic mutant. We also show that deletion or mutation of RemCA
524 do not alter StREM1.3 interaction with PVX TGBp1 in a split ubiquitin assay, which is
525 consistent with evidences that RemCA would insert into the PM. This indicates that
526 RemCA is not directly involved in the interaction with TGBp1 but rather that
527 StREM1.3–TGBp1 interaction takes place at the PM. The sub-cellular localization of
528 TGBp1 is complex and depends on the other TGB proteins, viral RNA, and on the
529 host plant species (Verchot-Lubicz et al., 2010). When expressed alone in N.
530 benthamiana, TGBp1 is mostly nuclear and cytoplasmic. However in the presence of
531 viral RNA, TGBp1 could traffic along the ER with TGBp2 and TGBp3 granules, and
532 reach plasmodesmata. Given that StREM1.3 is anchored on the cytosolic face on the
533 PM, it could associate with cytoplasmic TGBp1 at the periphery of the cell, explaining
534 why we could detect association when TGBp1 was expressed alone. Future work will
535 investigate the specificity of StREM1.3 association with TGBp1 and the impact of
536 viral RNA on this interaction. The recent report of the role of *Arabidopsis*
537 synaptotagmin 1 (SYT1) in the control of Begomovirus and Tobamovirus cell-to-cell
538 movement (Lewis and Lazarowitz, 2010) shed new light on the involvement host
539 membrane proteins in virus movement. SYT1 resides to the cortical endoplasmic
540 reticulum, endosomes, in DIMs at the PM and in PDs (Schapire et al., 2008; Minami
541 et al., 2009; Lewis and Lazarowitz, 2010; Yamazaki et al., 2010), and regulates
542 endocytosis and PM repair processes (Schapire et al., 2008; Yamazaki et al., 2008;
543 Lewis and Lazarowitz, 2010). These studies support a link between DIMs and PDs,
544 and provide evidence for a role of DIM proteins in cell-to-cell communication via PDs.

545

546 Considering the diversity of proteins in the Remorin family (Raffaele et al.,
547 2007), there is probably a corresponding diversity of sub-cellular localizations and
functions to be discovered. Analysing the variability the RemCA region in relation with
Remorins sub-cellular location, the variability of the membrane lipid composition
according to the nature of membrane compartments and plant species, and specific
functions devoted to each protein in the Remorin family will provide valuable insights
in the mechanisms regulating lateral heterogeneity of plant PM.

553

## MATERIALS AND METHODS

### Molecular cloning and sequence analyses

StREM1.3 was cloned from Potato *Solanum tuberosum* cv. Desiree cDNAs by
standard Gateway techniques using the pDONR221 entry vector and primers
containing the recombination site sequences and the gene specific sequences 5'-
ATGGCAGAATTGGAAGCT-3' (forward) and 5'-TCAAAATATTCCAAGGAT-3'
(reverse for N-terminal fusion), 5'-AAATATTCCAAGGAT-3' (reverse for C-terminal
fusion), or 5'- TCAACGTTTAGCTTCAATCAT-3' (reverse for YFP :StREM1-170). A
forward primer containing the gene specific sequence 5'-GGAGAAGATCTTCTC-3'
was used to clone the RemCA constructs. StREM1.3* was generated by PCR
amplification using a reverse primer containing the sequence 5'-
TCAAAATATTACAGAGATTTTCTTTGGAGAAGTTACAGTGGAACGGACTTTTGC-
3'. Mutated residues were selected at the C-terminus of RemCA to facilitate PCR-
based mutations. The position of Glycines and the aromatic residue were shown to
be important in HA fusion peptide function (Cross et al., 2001), therefore G188V and
G196V were introduced to preserve small size while introducing hydrophobic amino-
acids and Y184V to remove the aromatic residue. A186S and A190S were
introduced to preserve the tiny size of amino acids while removing hydrophobic
residues and L195S allowed reducing further the local hydrophobicity of RemCA
peptide. These mutations allowed reducing the hydrophobic moment from 0.182 to
0.112 for the whole RemCA peptide. These entry clones were recombined into the
pDEST17 (6His fusions), pH7YWG2, pH7WGY2 or pk7WGF2 (YFP/GFP fusions)
vectors. Secondary structure predictions were ran on SSPro
(http://download.igb.uci.edu/sspro4.html) and JPred 3
(http://www.compbio.dundee.ac.uk/www-jpred/) servers. Coiled-coil domains were
predicted using Marcoil1.0 server (http://www.isrec.isb-
sib.ch/webmarcoil/webmarcoilC1.html).. Amphipathic helix analyses were performed
with heliquest server (http://heliquest.ipmc.cnrs.fr/) and 3D modelling via the I-
582 TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) (Roy et al., 2010).
583 StREM1.3 C-terminal domain models were assembled into a trimer using the
584 SymmDock server (Schneidman-Duhovny et al., 2005). Models were rendered using
585 UCSF Chimera (Pettersen et al., 2004).
586

586

587 Plant transformation and fluorescence microscopy

588 Four week old tobacco plants (Nicotiana tabacum cv. Xanthi) were used for
589 Agrobacterium tumefaciens (strain GV3101)-mediated transient expression (Batoko
590 et al., 2000). Confocal imaging was performed two days after agroinfiltration using
591 Leica TCS SP2/5 confocal microscopes with 20x to 63x oil/water immersion
592 objectives. Laser and image settings were as described by (Raffaele et al., 2009).
593

593

594 Preparation of pure PM and Detergent Insoluble Membrane (DIM)

595 Microsomal fractions and PMs from tobacco leaves were purified described in
596 (Mongrand et al., 2004). For DIM preparation, TX100 (10 % w/v) was added to a ratio
597 detergent-to-PM proteins of 10 (at 1% w/v final concentration), and the PM
598 preparations were solubilized at 4°C for 30min. Treated PMs were brought to a final
599 concentration of 48 % sucrose (w/w), overlaid successively with layers of 40, 35 and
600 30 % sucrose in TBS buffer (w/w), and then centrifuged for 16 h at 200,000 g in
601 TST41 rotor (SORVALL). DIMs could be recovered above the 30-35 % layers as an
602 opaque band and this fraction was washed in 4 ml of TBS buffer to remove residual
603 sucrose. The protein concentration was determined with a BCA protein assay to
604 avoid TX100 interference, using bovine serum albumin as a protein standard.
605

605

606 Protein analysis, Western blots and protein purification

607 6-Histidine tagged constructs were expressed in E. coli BL21 DE3 cells. Cells were
608 lysed by ultasonication, and the crude extract was centrifuged at 10,000g for 15 min.
609 6-Histidine tagged proteins were mostly recovered in the soluble fraction. Soluble
610 proteins were then and purified using fast flow chelating sepharose resin (Amersham)
611 according to manufacturer's instructions. Ultrapure RemCA peptides were obtained
612 by de novo peptide synthesis (GL Biochem Shanghai Ltd; www.glschina.com).
613 Western blot analyses were performed using either anti-REM (Raffaele et al., 2009),
614 anti-PMA (Lefebvre et al., 2004), anti-PIP (Santoni et al., 2003), anti-actin or
615 commercial anti-GFP (Millipore/Roche) antibodies.
616

## 617 Extrinsic PM proteins stripping

Stripping on PM preparations and microsomal fractions was performed as described in (Santoni et al., 2003) with minor modifications. Membranes (200 µg) were incubated in 40 ml/1 ml of 5 mM EDTA, 5 mM EGTA, 4 M urea and 5 mM Tris-HCl, pH 9.5, for 5 min on ice before being centrifuged for 20 min at 100,000 g. The subsequent pellet was suspended in 20 mM NaOH, and centrifuged at 100,000 g for 20 min. The membranes were then washed in 2 mM EDTA, 2 mM EGTA, 100 mM NaCl and 5 mM Tris-HCl, pH 8, centrifuged at 100 000 g for 20 min, and finally resuspended in loading buffer for SDS-PAGE and Western blot where samples were loaded at equal volumes. The mean protein-purification yield was ~50%. A negative control treated with TBS was added (mock), yielding 95% recovery. Total protein amounts were quantified by Bradford or by image quantification of the Coomassie-stained gels.

630

## 631 Liposome and lipid binding assays

Liposomes mimicking the lipid content of tobacco PM were freshly prepared in TBS buffer according to (Doms et al., 1985). For PM-like liposome, the lipid content was phosphatidylethanolamine (PE)/ phosphatidylcholine (PC)/ phosphatidic acid (PA) / glucosylcerebrosides (gluCER) / tobacco mix of free sterols / acyl steryl glucosides (ASG)/ steryl glucosides (SG) at molar ratio 1/0.6/0.2/0.6/0.8/0.5/0.5 and for DIM-like liposomes same lipids at molar ratio 1/0.6/0.2/1.2/2.5/3/1 (Mongrand et al., 2004; Furt et al., 2010). Signals were quantified by the ImageJ software. Standard plant lipids were purchased from SIGMA and MATREYA. 5 µg of purified 6His:StREM1.3, 6His:StREM1.3<sup>1-170</sup> or 6His:StREM1.3* was incubated for 30 min at 30°C with 5 mM liposomes. The samples were further processed in 62% sucrose, and placed at the bottom of a discontinuous sucrose gradient (35/30/15/5% w/w). After centrifugation, liposomes floating at the 15-5% interface (top of the gradient) and fractions at the bottom of the gradient were collected, proteins were precipitated with 15% trichloracetic acid (TCA) and analysed by western blot. To control for liposome recovery, lipids were extracted and analysed by Thin Layer Chromatography as described previously (Mongrand et al., 2004).

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648

## 649 Circular dichroism (CD) measurements
CD spectra were recorded on a Jasco J-815 CD spectrometer with 10-mm path length quartz cuvettes. Ten scans were taken and automatically averaged in the wavelength range from 190 to 250 nm. Peptide secondary structures were determined using CDpro software package, which involved CDSSTR, SELCON3, and CONTINLL methods (Adam et al., 2004; Zakharov et al., 2004). Percentages were calculated by averaging the percentages provided by the three methods. The peptides stock solutions used for the measurements were diluted in 1 mM Tris buffer at pH 7.4, or in TFE.

## Liposome leakage assays

Membrane perturbation and vesicle release can be measured by the assay of (Ellens et al., 1985) based on the quenching of HPTS by DPX. HPTS and DPX were co-encapsulated in the aqueous phase of the same liposomes. When leakage of vesicle content occurs, the quenching by DPX stops and subsequently the fluorescence of HPTS increases. Small unilamellar vesicles (SUVs) were used in our experiments. These vesicles were prepared from a solution of multilamellar vesicles (MLV) obtained after hydration for 1 h at 37°C of dry lipid films. These films were mixtures by weight of 26.6 % PC, 26.6 % SM, 26.6 % PE, and 20.2 % CHOL for neutral liposomes and 30 % PC, 30 % PE, 2.5 % PI, 10 % PS, 5 % SM, and 22.5% CHOL for negatively charged liposomes. Large unilamellar vesicles (LUVs) were prepared by the extrusion technique of Mayer et al. The MLV suspension was submitted to five successive cycles of freezing and thawing and thereafter extruded 10 times through stacked polycarbonate filters (pore size, 0.08 µm), under a nitrogen pressure of 20 bars using an extruder (Lipex Biomembranes, Vancouver Canada). The concentration of the liposome suspensions was determined by phosphorus analysis. The peptides, dissolved in TFE or HFP/TFE, were added to a mixture of liposomes with encapsulated HPTS and DPX. HPTS fluorescence was measured on a Perkin-Elmer LS-50B fluorimeter (excitation, 360 nm; emission, 520 nm). Liposomes were prepared as described above but were rehydrated with 1 ml of 12.5 mM HPTS (45 mM NaCl), 45 mM DPX (20 mM NaCl), and 10 mM Tris-HCl at pH 7.4 and passed through a Sephadex G-75 column to removed un-encapsulated material.

## Lipid mixing assays
The induction of vesicular lipid mixing by the different peptides was tested with liposomes (SUVs) made of PC/PE/SM/chol at 26.6/26.6/26.6/20.2 molar ratio and PC/PE/PI/PS/SM/CHOL at a molar ratio 30/30/2.1/10/5/22.5. Labelled liposomes were obtained by incorporating R18 in the dry lipid film at a concentration of 6.3 % of the total lipid weight. Labelled and unlabelled liposomes were mixed at a weight ratio of 1:4 and a final concentration of 50 mM in 10mM Tris-HCl, 150 mM NaCl, 0.01% EDTA, 1 mM NaN₃ (pH 8). Mixing of liposome membranes was followed by measuring the fluorescence increase of R18, a lipid soluble probe, occurring after the fusion of labelled and unlabelled liposomes, as described in (Lins et al., 2002). Incubation of labelled and unlabelled vesicles in buffer alone did not modify the fluorescence intensity. Fluorescence was recorded at room temperature (excitation: 560 nm, emission: 590 nm) on an LS-50B PerkinElmer fluorimeter.

## Size-exclusion chromatography

Size-exclusion chromatography was performed on an AKTApurifier apparatus (GE Healthcare). 6His:StREM1.3 and 6his:StREM1.3* purified from *E. Coli* cultures (1.2g/l) were centrifuged for 5 min at 20,000 g before being processed. The molecular size of the proteins was analysed by chromatography on a Superdex 75 10/30 column (GE Healthcare) equilibrated with 20 mM of HEPEs, pH 7,4, 300mM NaCl. Proteins (200 µl) were eluted with a flow rate of 0.2 ml/min and recorded by continuously monitoring the absorbance at 280nm. The column was calibrated with the standard proteins described in Fig.6.

## Viral spreading and split ubiquitin assays

To assess spreading of PVX:GFP in *N. benthamiana* leaves *Agrobacterium tumefaciens* strain GV3101 carrying the respective StREM1.3 constructs were infiltrated at a final OD600= 0.2 together with the same strain carrying the plasmid pGr208, which expresses the PVX:GFPcDNA, as well as the helper plasmid pSoup (Peart et al., 2002) at final OD600 = 0.001. Spreading of PVX:GFP was visualized by confocal laser-scanning microscopy at 5 days post infection and the number of fluorescent cells of at least 30 PVX:GFP infection foci were counted. Protein extracts from the infiltrated *N. benthamiana* leaves were subjected to cell fractionation and Western Blot. The experiment was repeated 3 times with same results. Split ubiquitin assays were performed as described in (Raffaele et al., 2009) with yeasts grown on
717 SD medium supplement with uracile (50 mg.L⁻¹), leucine (380 mg.L⁻¹), lysine (76 mg.L⁻¹) and 5'fluoro-orotic acid (1 g.L⁻¹).

719

720 ACKNOWLEDGEMENTS

721 We would like to thank Mohammed-Amine Belka, Christelle Flore and Jeanny
722 Laroche-Traineau their valuable contributions. We are very grateful to Claudia Popp
723 and Thomas Ott (LMU Munich) for inspiring discussions, shared data, contagious
724 enthusiasm, and a lot of help. We thank all other members of the labs of Sébastien
725 Mongrand (LBM Bordeaux) and Thomas Ott (LMU Munich) for fruitful discussions.
726 We acknowledge the platform Métabolome- Fluxome-Lipidome of Bordeaux
727 (http://www.biomemb.cnrs.fr/INDEX.html)

728 https://www.bordeaux.inra.fr/umr619/RMN_index.htm) for contribution to lipid
729 analysis and the unit of plant imaging of Bordeaux Imaging Center (BIC) for confocal
730 imaging. This work has been funded by the French National Agency for Research,
731 ANR program (NT09_517917, ANR PANACEA to SM, JLC).

732 AUTHOR CONTRIBUTION

733 A.P., L.L., S.M. and S.R. designed research. A.P., J.L.C., J.M.C., S.M. and S.R.
734 performed research. J.M.C., L.L. and S.G-R. contributed new reagents and analytic
735 tools. A.P., J.M.C, S.M. and S.R. analysed data. S.M. and S.R. wrote the paper. All
736 authors read, edited and approved the manuscript.

737

738 CONFLICT OF INTEREST

739 The authors declare no conflict of interest.
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941
942 FIGURE LEGENDS

943

944 Figure 1. A C-terminal anchor peptide (RemCA) from StREM1.3 Remorin is required and sufficient for strong plasma membrane (PM) anchoring in vivo. (A)

946 Predicted secondary structure and protein domains in StREM1.3. Numbers indicate amino acid position. (B) Stripping of peripheral PM proteins is not sufficient to wash out StREM1.3. Purified PMs from tobacco leaves were washed according to Santoni et al. 2003 (Stripped) or treated with TBS (Mock). In average, 50% (n=3) of the PM-associated proteins, such as Actin, were stripped whereas the quantity of Remorin protein associated to the PM was unaffected, as for the Plasma membrane Intrinsic Protein 2 (PIP2) aquaporin intrinsic PM protein. (C) Subcellular localization of YFP-tagged StREM1.3, truncated StREM1.3 comprising amino-acids 1 to 170, and truncated StREM1.3 comprising amino acids 171 to 198 (RemCA peptide only) transiently expressed in tobacco epidermal cells. Scale bars indicate 20 µm. (D) Cell fractionation of the constructs shown in C. Crude protein extracts (CE) from tobacco leaf cells expressing each construct were fractionated into soluble (sol.) and microsomal (µ) compartments by centrifugation (15µg of proteins per lane). In spite of various attempts to solve this problem, we were not able to prevent partial cleavage of the YFP:RemCA construct, resulting in a free YFP signal as shown.

961

962 Figure 2. Non-polar environments trigger conformational changes in the RemCA peptide. (A) Circular dichroism spectra of synthetic ultrapure RemCA peptide in various environments. The spectral signatures of alpha helices (at 195, 208 and 222nm) are indicated by arrows. Spectra were recorded in aqueous (100% Tris-HCl), semi-hydrophobic (50% TFE) and hydrophobic (100% TFE) environments.

967 (B) Estimated distribution of secondary folds in RemCA peptide in the different environments assayed. (C) Sequence and predicted structure of the RemCA peptide. 968 Homology modeling predicts a tight hairpin of alpha helices for RemCA, consistent 969 with the circular dischroism spectra collected in hydrophobic environments. The 970 hydrophobicity of residues (Kyte-Doolittle scale) is indicated by a color-code. TFE, 971 2,2,2 trifluoroethanol. 972

973

974 Figure 3. Amphipathy of RemCA alpha-helices plays a critical role in anchoring StREM1.3 Remorin to the plasma membrane. (A) A helical wheel projection of

975
976 RemCA as a hairpin of α-helices showing amphipathy of each helix. Overall helix
977 hydrophobicity (H) and hydrophobic moment (μH) are indicated in the middle of the
978 helices, with an arrow showing the direction and intensity of the hydrophobic
979 moments. (B) Ribbon and surface models of the RemCA and mutated RemCA*
980 peptides with hydrophobicity (Kyte-Doolittle scale) shown by a color-code. The
981 surface model of RemCA shows the predicted hydrophobic core in red, and charged
982 surfaces in blue. Substitutions introduced in the RemCA* mutant aimed at reducing
983 the amphipathy of the α-helices by altering the distribution of hydrophobic residues
984 (Y184V, A190S, and L195S) and positively charged residues (A186S, G188V and
985 G196V). (C) Maximum projection of confocal scans through *N. benthamiana*
986 epidermal cells expressing YFP:StREM1.3* mutant, with plastids shown in red. Scale
987 bar indicates 20 µm. (D) Cell fractionation analysis of cells expressing GFP tagged
988 StREM1.3 with native (+) and mutated (*) RemCA sequence, confirming that
989 mutations introduced in StREM1.3* abolish PM localization.
990

991 **Figure 4.** RemCA confers intrinsic lipid binding to StREM1.3 Remorin. (A) 992 Affinity purified 6His:StREM1.3 (WT) binds to protein-free liposomes *in vitro* in a 993 RemCA-dependent manner. Liposome binding is abolished in 6His:StREM1.3 994 recombinant protein lacking RemCA (ΔCA) or harbouring a mutated RemCA (*). The 995 soluble (Sol.) and liposome-bound (Bound) fractions recovered after centrifugation 996 are shown. Lipid mixing assays (B) and liposome leakage assays (C) show that 997 RemCA ultrapure peptides insert into liposome membranes containing negatively 998 charged lipids, as indicated by an increase in fluorescence de-quenching. By 999 contrast, liposome leakage assays (C) showed that RemCA is not able to insert into 1000 neutral liposome membranes. SIV, Simian influenza virus fusion peptide; TFE, 1001 trifluoroethanol; TX100, Triton X-100. Solvent used in (C) was dimethyl sulfoxide for 1002 negatively charged liposomes and TFE for neutral liposomes. Error bars show 1003 standard deviation of 6-8 independent measurements.
1004

1005 **Figure 5.** RemCA targets soluble proteins to the PM in a lipid-specific manner.
1006 (A) Lipid overlay assays performed with 6His tagged StREM1.3 and StREM1.3<sup>1-170</sup>
1007 show a differential affinity of StREM1.3 for various classes of lipids, dependent on
1008 RemCA. The right panel shows Western Blot of the protein input material illustrating
1009 equal loading for StREM1.3 and StREM1.3<sup>1-170</sup>. PA, phosphatidic acid; PIP<sub>3</sub>,
phosphoinositol 3,4,5, triphosphate; PIP₂, Phosphoinositol bisphosphate, the position
of phosphate groups is indicated below; S1P, sphingosine 1-phosphate. (B)
6His:StREM1.3 shows higher affinity for liposomes with a composition mimicking
detergent insoluble membranes (DIM) compared to plasma membrane (PM -
described in materials and methods). Histograms show quantifications and standard
error based on Western blot signals for three independent experiments. Differential
affinity is significant with p<0.05 in a Student t-test. (C) RemCA-tagged GFP
transiently expressed in tobacco leaves segregates into the DIM fractions of the PM,
similar to the endogenous Remorin. Total proteins were quantified by bicinchoninic
acid assay (BCA) relative to total proteins in purified PM (error bars show standard
deviation from three independent replicates). µ, microsomal fraction; DSM, detergent
soluble membranes.

1022

Figure 6. StREM1.3 Remorin forms homo-trimers independently of RemCA *in vitro*. (A) Exclusion column chromatography of 6His:StREM1.3, 6His:StREM1.3* and elution standards allows to estimate the native MW of 6His:StREM1.3 to ~120 kDA, suggesting that it assembles into trimers, and shows that mutations introduced in 6His:StREM1.3* do not alter the ability to form trimers. (B) A trimer model of the C-terminal domain of StREM1.3. Views from the C-terminal end of the trimer with residues colored according to hydrophobicity (Kyte-Doolittle scale) reveal the formation of a hydrophobic core (in red) at the centre of the trimer, with positive surface patches surrounding the RemCA region, in blue. Standards used in A: 1, blue dextran (2MDa); 2, Thyroglobuline (670kDa); 3, Catalase (232kDa); 4, Ovalbumine (43kDa); 5, Chymotrypsinogen (25kDa); 6, soybean Trypsin inhibitor (21.5kDa); 7, Ribonuclease (13.7kDa); 8, Cytochrome C (12.3kDa).

1035

Figure 7. RemCA-mediated plasma membrane binding is required for StREM1.3 restriction of PVX cell to cell propagation. (A) Expression of PVX:GFP alone and in combination with StREM1.3 or the mutated StREM1.3* variant unable to bind to the plasma membrane. Images of infection foci were taken at 5 days post infection (dpi) of *N. benthamiana* leaves. Scale bars indicate 150µm. (B) Quantification of PVX cell to cell spreading showing the average number of cells in the infection foci at 5 dpi. For each treatment, 90 pictures were analysed, with error bars showing standard deviation. Significance was assessed by Dunn's multiple comparison test (***,
p<0.001; n=3). (C) Split ubiquitin assays showing that StREM1.3<sup>1-170</sup> and StREM1.3* interact with PVX TGBp1 movement protein similar to the wild type StREM1.3.

1046

Figure 8. Model for the molecular mechanisms leading to the assembly of StREM1.3 micro-domains at the plasma membrane (PM). Conformational changes in RemCA, leading to folding into amphipathic alpha helices, are required to anchor StREM1.3 to the PM. These conformational changes are triggered by non-polar environments, such as the vicinity of the PM (A). Helix E172-A186 folds exposing clusters of positively charged residues (+). These residues establish electrostatic interactions with the negatively charged lipids of the inner leaflet of membrane rafts, destabilizing the PM and allowing insertion of RemCA in the bilayer (B). Hydrophobicity triggers RemCA hairpin formation, creating an hydrophobic pocket (small red star) that deepens and strengthen insertion in the membrane. Alternatively, an external signal may trigger the formation of StREM1.3 trimers (C). The hydrophobic core of the trimer (large yellow star) favours folding of helix E172-A186 of RemCA (D), leading to PM anchoring as previously described. The formation of trimers, either prior to or after PM anchoring, is likely to increase the strength of the lipid binding, may promote membrane domain aggregation, lead to membrane deformation or mediate the assembly of protein complexes at the PM via Remorin protein-protein interaction domains (E).

1064

## SUPPLEMENTAL DATA

1066

Figure S1. Membrane washing experiments show that StREM1.3 strongly binds to purified plasma membrane and liposomes. (A) Anti-Remorin Western blot on tobacco leaf purified plasma membranes incubated for 30 min in water, in Tris-HCl buffer saline (TBS) containing NaCl, urea, TBS of increasing pH and TBS containing Ethylene diamine tetra acetic acid (EDTA). (B) Anti-Remorin Western blot on liposomes supplemented with 1 µg of purified 6His:StREM1.3 (input). These samples were incubated in TBS in H2O or in TBS containing 1 M NaCl or 6 M Urea for 30 min at 4°C, and centrifuged.

1075

Figure S2. Sequence and structure comparison between StREM1.3 RemCA and Influenza hemagglutinin fusion peptide (HAfp). StREM1.3 homology model (in
blue) was manually superimposed with HAfp structure (in yellow, Lorieau *et al.*, 2010). Kyte-Doolittle scale is used to illustrate the hydrophobicity of residues along the alignment and in one of the structure views.

**Figure S3. Controls for the production of purified recombinant proteins and liposome binding assays.** (A) 6His-tagged StREM1.3 constructs were expressed in BL21 DE3 E. Coli cells, with induction controlled by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG - top left panel). Red arrowheads indicate the size expected for StREM1.3 constructs. Cells were lysed by ultrasonication (CE) and centrifuged. StREM1.3 variants were mostly recovered in the supernatant (Sup.) but not the pellet (Pel.). Soluble proteins were then processed using fast flow chelating sepharose resin with imidazole concentrations as indicated on the figure (mM). (B) To control the recovery of the liposomes, the lipids of half of the liposomes supplemented with StREM1.3, StREM1.3<sup>1-170</sup> or StREM1.3* were extracted and analysed by Thin Layer Chromatography as described in Mongrand *et al.*, 2004. Lipids were identified by comparison with commercial standards. ASG, acyl steryl glucoside; DIM, detergent insoluble membranes; gluCER, glucosyl ceramide; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SG, Steryl glucoside.

**Figure S4. Controls for lipid-specific binding of StREM1.3.** (A) Lipid overlay assay comparing phospholipid binding properties of recombinant 6His:StREM1.3 and 6His:StREM1.3*. Total protein used in the assay is shown in the top panel. (B) To control for the recovery of the liposomes in the liposome binding assays, the lipids of half the liposomes mimicking PM or DIM lipid composition were extracted and analyzed by Thin Layer Chromatography as described in Mongrand *et al.* 2004. Lipids were identified by comparison with commercial standards. ASG, acyl steryl glucoside; DIM, detergent insoluble membranes; gluCER, glucosyl ceramide; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SG, Steryl glucoside. (C) Segregation of the PMA proton pump and Remorin along a sucrose gradient showing distribution of these proteins between Detergent Insoluble Membranes (DIM, fraction 1) and Detergent Soluble Membranes (DSM, fractions 2 to 4).
1112 **Figure S5. StREM1.3 Remorin forms trimers in vitro.** (A) Exclusion column 1113 chromatogram obtained for 6His:StREM1.3. (B) Fractions A8, A9, B4, B5, C3 and C4 1114 were analysed by SDS-PAGE (denaturing conditions) followed by anti-Remorin 1115 western blot showing that StREM1.3 elutes in fractions B4 and B5. 1116

1117 **Figure S6. Western Blot controls for the expression of StREM1.3 and 1118 StREM1.3* in PVX:GFP propagation experiments.** Protein extracts (10 µg) from 1119 infected tobacco leaves with PVX:GFP alone or co-expressing StREM1.3 or 1120 StREM1.3* were used for Western Blot analysis. 1121

1122 **Supplemental File 1. Protein Databank file containing atom coordinates for the 1123 predicted StREM1.3 structure.** 1124

1125 **Supplemental File 2. Protein Databank file containing atom coordinates for the 1126 predicted StREM1.3* structure.**
A

<figure><img src="image_2.png" /></figure>

B

<figure><img src="image_3.png" /></figure>

C

<figure><img src="image_4.png" /></figure>

D

<figure><img src="image_5.png" /></figure>

**Figure 1. A C-terminal anchor peptide (RemCA) from StREM1.3 Remorin is required and sufficient for strong plasma membrane (PM) anchoring in vivo.** (A) Predicted secondary structure and protein domains in StREM1.3. Numbers indicate amino acid position. (B) Stripping of peripheral PM proteins is not sufficient to wash out StREM1.3. Purified PMs from tobacco leaves were washed according to Santoni *et al*. 2003 (Stripped) or treated with TBS (Mock). In average, 50% (n=3) of the PM-associated proteins, such as Actin, were stripped whereas the quantity of Remorin protein associated to the PM was unaffected, as for the Plasma membrane Intrinsic Protein 2 (PIP2) aquaporin intrinsic PM protein. (C) Subcellular localization of YFP-tagged StREM1.3, truncated StREM1.3 comprising amino-acids 1 to 170, and truncated StREM1.3 comprising amino acids 171 to 198 (RemCA peptide only) transiently expressed in tobacco epidermal cells. Scale bars indicate 20 µm. (D) Cell fractionation of the constructs shown in C. Crude protein extracts (CE) from tobacco leaf cells expressing each construct were fractionated into soluble (sol.) and microsomal (µ) compartments by centrifugation (15µg of proteins per lane). In spite of various attempts to solve this problem, we were not able to prevent partial cleavage of the YFP:RemCA construct, resulting in a free YFP signal as shown.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 2.** Non-polar environments trigger conformational changes in the RemCA peptide. (A) Circular dichroism spectra of synthetic ultrapure RemCA peptide in various environments. The spectral signatures of alpha helices (at 195, 208 and 222nm) are indicated by arrows. Spectra were recorded in aqueous (100% Tris-HCl), semi-hydrophobic (50% TFE) and hydrophobic (100% TFE) environments. (B) Estimated distribution of secondary folds in RemCA peptide in the different environments assayed. (C) Sequence and predicted structure of the RemCA peptide. Homology modeling predicts a tight hairpin of alpha helices for RemCA, consistent with the circular dischroism spectra collected in hydrophobic environments. The hydrophobicity of residues (Kyte-Doolittle scale) is indicated by a color-code. TFE, 2,2,2 trifluoroethanol.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Figure 3. Amphipathy of RemCA alpha-helices plays a critical role in anchoring StREM1.3 Remorin to the plasma membrane.** (A) A helical wheel projection of RemCA as a hairpin of α-helices showing amphipathy of each helix. Overall helix hydrophobicity (H) and hydrophobic moment (μH) are indicated in the middle of the helices, with an arrow showing the direction and intensity of the hydrophobic moments. (B) Ribbon and surface models of the RemCA and mutated RemCA* peptides with hydrophobicity (Kyte-Doolittle scale) shown by a color-code. The surface model of RemCA shows the predicted hydrophobic core in red, and charged surfaces in blue. Substitutions introduced in the RemCA* mutant aimed at reducing the amphipathy of the α-helices by altering the distribution of hydrophobic residues (Y184V, A190S, and L195S) and positively charged residues (A186S, G188V and G196V). (C) Maximum projection of confocal scans through *N. benthamiana* epidermal cells expressing YFP:StREM1.3* mutant, with plastids shown in red. Scale bar indicates 20 µm. (D) Cell fractionation analysis of cells expressing GFP tagged StREM1.3 with native (+) and mutated (*) RemCA sequence, confirming that mutations introduced in StREM1.3* abolish PM localization.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Figure 4.** RemCA confers intrinsic lipid binding to StREM1.3 Remorin. (A) Affinity purified 6His:StREM1.3 (WT) binds to protein-free liposomes *in vitro* in a RemCA-dependent manner. Liposome binding is abolished in 6His:StREM1.3 recombinant protein lacking RemCA (ΔCA) or harbouring a mutated RemCA (*). The soluble (Sol.) and liposome-bound (Bound) fractions recovered after centrifugation are shown. Lipid mixing assays (B) and liposome leakage assays (C) show that RemCA ultrapure peptides insert into liposome membranes containing negatively charged lipids, as indicated by an increase in fluorescence de-quenching. By contrast, liposome leakage assays (C) showed that RemCA is not able to insert into neutral liposome membranes. SIV, Simian influenza virus fusion peptide; TFE, trifluoroethanol; TX100, Triton X-100. Solvent used in (C) was dimethyl sulfoxide for negatively charged liposomes and TFE for neutral liposomes. Error bars show standard deviation of 6-8 independent measurements.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Figure 5. RemCA targets soluble proteins to the PM in a lipid-specific manner. (A)** Lipid overlay assays performed with 6His tagged StREM1.3 and StREM1.3<sup>1-170</sup> show a differential affinity of StREM1.3 for various classes of lipids, dependent on RemCA. The right panel shows Western Blot of the protein input material illustrating equal loading for StREM1.3 and StREM1.3<sup>1-170</sup>. PA, phosphatidic acid; PIP<sub>3</sub>, phosphoinositol 3,4,5, triphosphate; PIP<sub>2</sub>, Phosphoinositol bisphosphate, the position of phosphate groups is indicated below; S1P, sphingosine 1-phosphate. (B) 6His:StREM1.3 shows higher affinity for liposomes with a composition mimicking detergent insoluble membranes (DIM) compared to plasma membrane (PM - described in materials and methods). Histograms show quantifications and standard error based on Western blot signals for three independent experiments. Differential affinity is significant with p<0.05 in a Student t-test. (C) RemCA-tagged GFP transiently expressed in tobacco leaves segregates into the DIM fractions of the PM, similar to the endogenous Remorin. Total proteins were quantified by bicinchoninic acid assay (BCA) relative to total proteins in purified PM (error bars show standard deviation from three independent replicates). µ, microsomal fraction; DSM, detergent soluble membranes.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Figure 6.** StREM1.3 Remorin forms homo-trimers independently of RemCA in vitro. (A) Exclusion column chromatography of 6His:StREM1.3, 6His:StREM1.3* and elution standards allows to estimate the native MW of 6His:StREM1.3 to ~120 kDA, suggesting that it assembles into trimers, and shows that mutations introduced in 6His:StREM1.3* do not alter the ability to form trimers. (B) A trimer model of the C-terminal domain of StREM1.3. Views from the C-terminal end of the trimer with residues colored according to hydrophobicity (Kyte-Doolittle scale) reveal the formation of a hydrophobic core (in red) at the centre of the trimer, with positive surface patches surrounding the RemCA region, in blue. Standards used in A: 1, blue dextran (2MDa); 2, Thyroglobuline (670kDa); 3, Catalase (232kDa); 4, Ovalbumine (43kDa); 5, Chymotrypsinogene (25kDa); 6, soybean Trypsin inhibitor (21.5kDa); 7, Ribonuclease (13.7kDa); 8, Cytochrome C (12.3kDa).</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**Figure 7.** RemCA-mediated plasma membrane binding is required for StREM1.3 restriction of PVX cell to cell propagation. (A) Expression of PVX:GFP alone and in combination with StREM1.3 or the mutated StREM1.3* variant unable to bind to the plasma membrane. Images of infection foci were taken at 5 days post infection (dpi) of *N. benthamiana* leaves. Scale bars indicate 150µm. (B) Quantification of PVX cell to cell spreading showing the average number of cells in the infection foci at 5 dpi. For each treatment, 90 pictures were analysed, with error bars showing standard deviation. Significance was assessed by Dunn's multiple comparison test (***, p<0.001; n=3). (C) Split ubiquitin assays showing that StREM1.3<sup>1-170</sup> and StREM1.3* interact with PVX TGBp1 movement protein similar to the wild type StREM1.3.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Figure 8.** Model for the molecular mechanisms leading to the assembly of StREM1.3 micro-domains at the plasma membrane (PM). Conformational changes in RemCA, leading to folding into amphipathic alpha helices, are required to anchor StREM1.3 Remorin to the PM. These conformational changes are triggered by non-polar environments, such as the vicinity of the PM (A). Helix E172-A186 folds exposing clusters of positively charged residues (+). These residues establish electrostatic interactions with the negatively charged lipids of the inner leaflet of membrane rafts, destabilizing the PM and allowing insertion of RemCA in the bilayer (B). Hydrophobicity triggers RemCA hairpin formation, creating an hydrophobic pocket (small red star) that deepens and strengthen insertion in the membrane. Alternatively, an external signal may trigger the formation of StREM1.3 trimers (C). The hydrophobic core of the trimer (large yellow star) favours folding of helix E172-A186 of RemCA (D), leading to PM anchoring as previously described. The formation of trimers, either prior to or after PM anchoring, is likely to increase the strength of the lipid binding, may promote membrane domain aggregation, lead to membrane deformation or mediate the assembly of protein complexes at the PM via Remorin protein-protein interaction domains (E).</figcaption></figure>