P Laquel
email: francois.doignon@u-bordeaux.fr

Phosphoinositides containing stearic acid are required for interaction between Rho GTPases and the exocyst to control the late steps of polarised exocytosis

Cell polarity is achieved by regulators such as small G proteins, exocyst members and phosphoinositides, with the latter playing a key role when bound to the exocyst proteins Sec3p and Exo70p, and Rho GTPases. This ensures asymmetric growth via the routing of proteins and lipids to the cell surface using actin cables. Previously, using a yeast mutant for a lysophosphatidylinositol acyl transferase encoded by the PSI1 gene, we demonstrated the role of stearic acid in the acyl chain of phosphoinositides in cytoskeletal organisation and secretion.

Here, we use a genetic approach to characterise the effect on late steps of the secretory pathway.

The constitutive overexpression of PSI1 in mutants affecting kinases involved in the phosphoinositide pathway demonstrated the role of molecular species containing stearic acid in bypassing a lack of phosphatidylinositol-4-phosp hate PI(4)P at the plasma membrane, which is essential for the function of the Cdc42p module. Decreasing the levels of stearic acid-containing phosphoinositides modifies the environment of the actors involved in the control of late steps in the secretory pathway. This leads to decreased interactions between Exo70p and Sec3p, with

Introduction

Maintenance of cell polarity is an essential biological process shared by all eukaryotic cells, ensuring asymmetric growth by routing proteins and lipids to specific sites on the cell surface.

The transport of secretory vesicles and many intracellular organelles is dependent on the presence of polarised actin cables that radiate from the bud cortex and the neck, extending into the mother cell [START_REF] Pruyne | Mechanisms of polarized growth and organelle segregation in yeast[END_REF] . Polarised secretion is spatially and temporally regulated by many different actors, such as small G proteins 2 , exocyst members [START_REF] Wu | The Exocyst at a Glance[END_REF] and phosphoinositides [START_REF] Yakir-Tamang | Phosphoinositides, exocytosis and polarity in yeast: all about actin?[END_REF][START_REF] Johansen | Vesicle trafficking from a lipid perspective: Lipid regulation of exocytosis in Saccharomyces cerevisiae[END_REF] . The quantitative modification of pools of different phosphoinositides affects trafficking processes [START_REF] Mizuno-Yamasaki | Phosphatidylinositol 4phosphate controls both membrane recruitment and a regulatory switch of the Rab GEF Sec2p[END_REF][START_REF] Yakir-Tamang | A phosphatidylinositol-transfer protein and phosphatidylinositol-4-phosphate 5-kinase control Cdc42 to regulate the actin cytoskeleton and secretory pathway in yeast[END_REF][START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF] , due to the control of late steps of the secretory pathway by some Rho GTPases that modulate the docking of vesicles in concert with the exocyst complex [START_REF] Mizuno-Yamasaki | GTPase networks in membrane traffic[END_REF][START_REF] Boyd | Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p[END_REF][START_REF] He | The exocyst complex in polarized exocytosis[END_REF] . Phosphoinositides play a key role in these steps by being simultaneously bound to exocyst subunits Sec3p and Exo70p and to some members of Rho GTPase modules [START_REF] Yakir-Tamang | Phosphoinositides, exocytosis and polarity in yeast: all about actin?[END_REF][START_REF] Martin | PI(4,5)P(2)-binding effector proteins for vesicle exocytosis[END_REF][START_REF] Pleskot | Membrane targeting of the yeast exocyst complex[END_REF] . In a previous study, we characterised a yeast strain carrying a mutation in lysophosphatidylinositol acyltransferase, leading to qualitative modification of the phosphoinositide molecular species. We demonstrated the role of the acyl chain of phosphoinositides, specifically stearic acid, in the organisation of the actin cytoskeleton and the secretory pathway [START_REF] Doignon | Requirement of phosphoinositides containing stearic acid to control cell polarity[END_REF] .

In the present paper, we first characterised the step(s) of the secretory pathway affected by a lack of phosphoinositides containing stearic acid using genetic approaches. The constitutive overexpression of the PSI1 gene in strains carrying mutations in genes encoding kinases involved in the phosphoinositide pathway demonstrated the role of molecular species containing stearic acid in bypassing a lack of PI(4)P, an essential phosphoinositide for the polarisation of the Cdc42p module [START_REF] Meca | Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions[END_REF] . We further show that changes in the composition of the acyl chains of phosphoinositides modify the environment of actors involved in the control of late steps of the secretory pathway. In addition, using a Bimolecular Fluorescence Complementation (BiFC) assay approach, we demonstrate that these changes lead to a decrease in interactions between exocyst components Exo70p and Sec3p, and Rho GTPases (Cdc42p, Rho1p and Rho3p). This marked decrease in the interactions observed between Rho proteins (Rho1p, Rho3p and Cdc42p) and the plasma membrane-bound exocyst subunits (Exo70p and Sec3p) is induced by a disruption in the GTP/GDP ratio of Rho proteins, preventing activation of the exocyst.

Results

Phosphoinositides containing stearic acid are required for the control of late steps of the secretory pathway

Secretion assays have demonstrated that protein trafficking is disturbed in mutants for the lysophosphatidyl acyltransferase psi1∆ [START_REF] Doignon | Requirement of phosphoinositides containing stearic acid to control cell polarity[END_REF] , but do not demonstrate which stage of the trafficking is disturbed. To address this point, we first investigated genetic interactions between the PSI1 and SEC genes involved in the secretory pathway. We created double-mutant strains deficient in PSI1 and harbouring a panel of heat-sensitive sec mutations affecting the activity of the translocon (sec62-1) [START_REF] Reithinger | Sec62 protein mediates membrane insertion and orientation of moderately hydrophobic signal anchor proteins in the endoplasmic reticulum (ER)[END_REF][START_REF] Rothblatt | Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast[END_REF] , signal peptide processing (sec11-7) [START_REF] Bohni | SEC11 is required for signal peptide processing and yeast cell growth[END_REF] , the guanine exchange factor (GEF) of the small GTPase Sar1p involved in anterograde trafficking (sec12-1) [START_REF] Mcmahon | The structure of Sec12 implicates potassium ion coordination in Sar1 activation[END_REF][START_REF] Kaiser | Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway[END_REF] , the GEF of Arf members, the GTPases regulating anterograde and retrograde traffic from the TGN (sec7-1) [START_REF] Richardson | The Sec7 Arf-GEF is recruited to the trans-Golgi network by positive feedback[END_REF][START_REF] Novick | Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway[END_REF] . Furthermore, genetic links with an R-SNARE localised in the ER (endoplasmic reticulum) and the Golgi apparatus and involved in anterograde and retrograde transport by controlling membrane fusion (sec22∆) [START_REF] Adnan | Diverse Role of SNARE Protein Sec22 in Vesicle Trafficking, Membrane Fusion, and Autophagy[END_REF][START_REF] Flanagan | Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion[END_REF] were carried out. We also analysed a putative functional link between PSI1 and the function of the Golgi apparatus via the phosphatidylinositol/phosphatidylcholine transfer protein (sec14-1) [START_REF] Curwin | Localization of lipid raft proteins to the plasma membrane is a major function of the phospholipid transfer protein Sec14[END_REF][START_REF] Bankaitis | The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex[END_REF] and the late steps of trafficking controlled by the Rho protein (cdc42-6) [START_REF] Adamo | Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud[END_REF] or a member of the exocyst (sec6-4) [START_REF] Novick | Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway[END_REF][START_REF] Songer | Sec6p anchors the assembled exocyst complex at sites of secretion[END_REF] .

Finally, a cryosensitive mutant (rho3-V51) [START_REF] Adamo | The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity[END_REF] affecting another member of the Rho GTPase family, also regulating the late stages of exocytosis, was studied. Likewise, mutation of other genes was used to generate double-mutants with the PSI1 deletion. These genes include those involved in ER-to-Golgi apparatus [START_REF] Parlati | Topological restriction of SNARE-dependent membrane fusion[END_REF] and Golgi apparatus-to-ER protein recycling (erd1Δ) [START_REF] Eisfeld | Endocytotic uptake and retrograde transport of a virally encoded killer toxin in yeast[END_REF] , and an endocytosis internalisation step linked to actin patch function (end3Δ) [START_REF] Benedetti | The END3 gene encodes a protein that is required for the internalization step of endocytosis and for actin cytoskeleton organization in yeast[END_REF][START_REF] Kaksonen | A modular design for the clathrin-and actinmediated endocytosis machinery[END_REF] .

Comparisons of the growth of double-mutants (sec, psi1Δ) and single-mutants (sec) were performed using serial dilutions and spotting on complete media at permissive, intermediate and non-permissive temperatures (Figures 1A,B,C). Remarkably, at 30°C, the psi1Δ mutation behaved as a suppressor of sec7-1. Sec7p is a GEF for Arf1p and Arf2p, which are known to facilitate COPI (Coat Protein I) and clathrin-coating dynamics at the Golgi apparatus and function in many different steps of intracellular trafficking, such as retrograde transport from the Golgi apparatus to the ER and anterograde transport to the lysosome and vacuole (Figure 1B) [START_REF] Daboussi | Phosphoinositide-mediated clathrin adaptor progression at the trans-Golgi network[END_REF] .

Growth of the sec22∆ psi1∆ double-mutant was impaired at 37°C relative to the single mutant sec22∆ (Figure 1C) but did not show a decrease in cell viability observed using the methylene blue staining method (data not shown) [START_REF] De Bettignies | RGD1 genetically interacts with MID2 and SLG1, encoding two putative sensors for cell integrity signalling in Saccharomyces cerevisiae[END_REF] . The strains sec62-1 psi1Δ (at permissive and intermediate temperatures, Figure 1A, 1B) and rho3-V51 psi1Δ (at a permissive temperature, Figure 1A) displayed a slight growth defect compared to the single sec mutants. A synergetic effect was observed between the rho3-V51 allele and psi1Δ only at 25°C, based on a cell lethality of 20 ± 2.4% for the double-mutant and 12 ± 1.9% for the rho3 single-mutant. These data suggested that Psi1p may interact functionally in vivo with some proteins involved in exocytosis, mainly during the docking of secretory vesicles to membranes and particularly during the final steps of trafficking. Moreover, the growth of cdc42-6 was strongly affected at 30°C when PSI1 deletion was present alongside this conditional mutation (Figure 1B). Cell lethality at 30°C was estimated at 32 ± 5% for the double-mutant, compared to 8 ± 1% for the single mutant cdc42-6. This synthetic lethality observed between cdc42-6 and psi1Δ revealed that PSI1 and CDC42 are genetically linked and involved in the same pathway(s) regulating secretion.

Growth assays performed on a strain with a mutation in another allele of CDC42 (cdc42-1) only showed a weak effect on secretion but demonstrated impairment of the dynamics of the actin cytoskeleton [START_REF] Adamo | Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud[END_REF][START_REF] Adams | CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Dong | Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast[END_REF] . We also analysed cdc42-1 to determine the specificity of the cdc42-6 allele in showing synthetic lethality with psi1∆. The comparison of the single mutant and double-mutant cdc42-1 psi1Δ did not demonstrate any synergistic effect on growth in the double-mutant (Figure S1). These results show that PSI1 only genetically interacted with the CDC42 allele that is involved in secretion, but not with the allele involved in actin dynamics.

The growth defect observed in cdc42-6 can be rescued by constitutive overexpression of

PSI1

To further investigate the links between PSI1 and genes involved in intracellular trafficking, the PSI1 ORF (Open Reading Frame) was constitutively overexpressed using the pCM189 plasmid [START_REF] Gari | A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae[END_REF] in the different secretory mutants described in section 2.1. No significant growth difference was observed for these secretory mutants, either with the pCM189 plasmid containing the PSI1 gene or with the empty vector, except for the cdc42-6 strain, where the weak growth at 30°C was rescued by constitutive overexpression of the PSI1 gene at the intermediate temperature (30°C) (Figure 2). This result combined with the synthetic lethality between cdc42-6 and psi1∆ suggests a balancing effect between the action of Cdc42p and phosphoinositides with stearic acyl chains at the plasma membrane. Interestingly, previous work showed that the cdc42-6 mutant could also be rescued by overexpression of the kinase encoded by MSS4, which is involved in PI(4,5)P2 biosynthesis [START_REF] Yakir-Tamang | A phosphatidylinositol-transfer protein and phosphatidylinositol-4-phosphate 5-kinase control Cdc42 to regulate the actin cytoskeleton and secretory pathway in yeast[END_REF] .

Constitutive overexpression of PSI1 can suppress the reduced growth of the stt4-4ts

kinase mutant by increasing the level of PIP 36:0

Various studies demonstrate the role of phosphoinositides in intracellular trafficking [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF][START_REF] Martin | PI(4,5)P(2)-binding effector proteins for vesicle exocytosis[END_REF][START_REF] Routt | Nonclassical PITPs activate PLD via the Stt4p PtdIns-4-kinase and modulate function of late stages of exocytosis in vegetative yeast[END_REF][START_REF] He | Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane[END_REF][START_REF] Liu | Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells[END_REF][START_REF] Bloch | Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth[END_REF][START_REF] Angelo | The multiple roles of PtdIns(4)P --not just the precursor of PtdIns(4,5)P2[END_REF] ; therefore, we aimed to analyse the links between the genes involved in phosphoinositide synthesis and the PSI1 gene. The plasmid pCM189 (either the empty vector or carrying the PSI1 gene for constitutive overexpression) was used to transform the conditional mutant strains that affect phosphoinositide synthesis: AAY104 (pik1-83ts) [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF] , AAY102 (stt4-4ts) [START_REF] Routt | Nonclassical PITPs activate PLD via the Stt4p PtdIns-4-kinase and modulate function of late stages of exocytosis in vegetative yeast[END_REF] and AAY202 (mss4-102ts) [START_REF] Stefan | The yeast synaptojanin-like proteins control the cellular distribution of phosphatidylinositol (4,5)-bisphosphate[END_REF] . PSI1 overexpression seems to suppress the growth defect at 30°C of strains when compared with the empty vector, the effect is remarkably strong with stt4-4ts mutant (deficient in plasma membrane PI4-kinase) (Figure 3). The mss4-102ts and the pik1-83ts mutants like the wild-type strain show a much smaller effect, suggesting that the PSI1 overexpression potentially improves the growth of these strains. This could suggest a more general effect of PSI1 overexpression on cell fitness.

To understand the suppressor effect of PSI1 in the stt4-4ts mutant, new strains (either overexpressing PSI1 or not) expressing a PI(4,5)P2 biosensor, GFP-2xPH PLCδ144 , were constructed in stt4-4ts and wild-type backgrounds. In the stt4-4ts mutant at a permissive temperature (25°C), the distribution of the GFP-2xPH PLCδ1 biosensor was shown using confocal microscopy to be located predominantly at the growth site in a similar range (47 ± 4%) to that observed in the wild-type strain (62 ± 9%) (Figures 4A andB). By contrast, after shifting to a non-permissive temperature (30 °C) for one hour, the GFP-2xPH PLCδ1 biosensor revealed drastic modifications in topology for PI(4,5)P2. Notably, the cells had a PI(4,5)P2 topology defect at the growth site, with these molecular species located at the growing site for only 13 ± 5% of the polarised cells. PSI1 overexpression induced PI(4,5)P2 re-localisation to the growth area for 46 ± 5% of the stt4-4ts cells at 30°C, a value similar to that observed under permissive conditions (Figures 4A andB). The ratios of the amount of PI(4,5)P2-specific reporter detected on the plasma membrane at the growing site (bud tip and bud neck) compared to the rest of the mother cell's body was 1.03 ± 0.10 at 30°C for the stt4-4 ts mutant with the empty plasmid pCM189. This reflects a uniform localisation of the probe at the plasma membrane between the bud and the mother cell. This ratio was 1.42 ± 0.18 with the pCM189-PSI1 plasmid, indicating the enrichment of the PI(4,5)P2 probe at growth areas (Figure 4C).

To understand the role of overexpression of the PSI1 gene in rescuing the stt4-4ts mutation, the proportions of different phosphoinositide species were analysed using LC-MS/MS (liquid chromatography-tandem mass spectrometry) (Figures 5A, B andC). A dramatic 35% decrease in PIP 36:0 levels was observed specifically for the stt4-4ts strain transformed with the empty plasmid and grown at a non-permissive temperature (30°C). The levels were restored when the PSI1 gene was constitutively overexpressed at this temperature (Figure 5B). No decrease in the levels of PIP 36:0 was observed at 30°C for the reference strain SEY6210 when transformed with the empty vector (Figure 5E). When the PSI1 gene was overexpressed in the SEY6210 strain, an increase of 81% at 25°C and 93% at 30°C was observed for PIP2 36:1 (Figure 5F). This increase was correlated with the increase in PIP 36:1 (90% at 25°C and 63% at 30°C). More generally, in the SEY6210 parental strain, levels of molecular species of PI, PIP and PIP2 containing stearic acid 34:1; 36:0 and 36:1 were increased following overexpression of PSI1, whereas in the mutant strain stt4-4ts this increase was restricted to PIP 36:0 and PIP2 36:1.

To determine if the suppressive effect of the PSI1 gene overexpression is due only to a qualitative effect of the PIP pool on the cell, we performed steady-state lipid labelling using [1-14 C]acetate. This was carried out in stt4-4ts strains with the PSI1 gene either overexpressed or expressed at normal levels, at permissive and non-permissive temperatures. After the purification of lipids by HPTLC (High-Performance Thin Layer Chromatography) followed by quantification of their radioactivity, we did not detect any increase in PIP levels when PSI1 was overexpressed (Figure S2). This result shows that a qualitative effect of the PIP pool is responsible for the suppressive effect of the PSI1 gene.

Phosphoinositides containing stearic acid are required to maintain certain polarity landmarks associated with a specific membrane environment, but do not cause changes in membrane fluidity

To analyse the association of tagged cellular landmarks involved in cell polarity with features of the membrane environment, crude cell extracts were subjected to a 0-40% discontinuous density gradient of OptiPrep™ by ultracentrifugation, in both a psi1∆/psi1∆ genetic background and in the reference strain. The tagged landmarks were revealed by Western blot using an anti-HA antibody (Figure 6). For both exocyst complex subunits that interact with PI(4,5)P2 through a polybasic region (Exo70p and Sec3p) [START_REF] He | Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane[END_REF][START_REF] Zhang | Membrane association and functional regulation of Sec3 by phospholipids and Cdc42[END_REF][START_REF] Liu | Two subunits of the exocyst, Sec3p and Exo70p, can function exclusively on the plasma membrane[END_REF] and for the landmarks Cdc42p and Rho1p (belonging to the Rho family), a shift in the sedimentation velocity was observed between the mutant strain and the control. This suggests that the membrane environment of these secretory landmarks was modified when stearic acid was absent from the phosphoinositides (Figure 6). In contrast, no disturbance in the sedimentation profile was observed for Rho3p.

PI is a major phospholipid in S. cerevisiae [START_REF] Tuller | Lipid composition of subcellular membranes of an FY1679-derived haploid yeast wild-type strain grown on different carbon sources[END_REF] and it is possible that modification of the acyl chain could alter membrane fluidity. In order to investigate any link between the membrane environment and fluidity, generalised polarisation (GP) indices were calculated using the fluorescent dye laurdan, a sensitive membrane dynamics probe used to measure the degree of membrane order [START_REF] Ishmayana | Further investigation of relationships between membrane fluidity and ethanol tolerance in Saccharomyces cerevisiae[END_REF][START_REF] Owen | Quantitative imaging of membrane lipid order in cells and organisms[END_REF][START_REF] Amaro | Laurdan and Di-4-ANEPPDHQ probe different properties of the membrane[END_REF][START_REF] Klose | Yeast lipids can phase-separate into micrometer-scale membrane domains[END_REF] . The GP indices of the wild-type strain and the psi1∆ mutant deficient for the synthesis of phosphoiniositides with stearic acid were similar, indicating a comparable degree of membrane order (Figure S3). Hence, the secretory pathway in the psi1∆ mutant was not impacted via modification of membrane fluidity.

Interactions between Rho GTPases and members of the exocyst are dependent on the presence of phosphoinositides containing stearic acid

The exocyst complex is composed of eight subunits (Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p and Exo84p) and is involved in the fusion of secretory vesicles with the plasma membrane. Furthermore, the exocyst determines the region of the plasma membrane where exocytosis takes place [START_REF] Wu | The Exocyst at a Glance[END_REF] . Cdc42p interacts with Exo70p 52 and Sec3p [START_REF] Zhang | Membrane association and functional regulation of Sec3 by phospholipids and Cdc42[END_REF] , Rho1p with Sec3p 53 and Rho3p with Exo70p [START_REF] Wu | The Exo70 subunit of the exocyst is an effector for both Cdc42 and Rho3 function in polarized exocytosis[END_REF] . The exocyst subunits (except Sec3p) are associated with the vesicles before they migrate to the bud tip [START_REF] Boyd | Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p[END_REF] . The localisation of Sec3p to the tip of small buds at the plasma membrane is independent of the actin cytoskeleton involved in the secretory pathway [START_REF] Finger | Sec3p is a spatial landmark for polarized secretion in budding yeast[END_REF] .

Two populations of Exo70p exist, the first in transit with vesicles and the second distributed in a manner similar to Sec3p 9 .

To investigate any disruption of the interactions between the Rho GTPases and exocyst subunits Exo70p and/or Sec3p, we established a BiFC assay. Cdc42p, Rho1p and Rho3p were tagged at their N-terminal ends with a C-terminal fragment of the fluorochrome Venus (VC) using a single-step PCR strategy with the pFA6a-His3MX6-PCET1-VC plasmid, the transcription being controlled by the CET1 promoter [START_REF] Sung | Bimolecular fluorescence complementation analysis system for in vivo detection of protein-protein interaction in Saccharomyces cerevisiae[END_REF] . Conversely, Exo70p and Sec3p were tagged at their Cterminal with the N-terminal part of Venus (VN) using the pFA6a-VN-His3MX6 plasmid, allowing regulation of their expression by their native promoters [START_REF] Sung | Bimolecular fluorescence complementation analysis system for in vivo detection of protein-protein interaction in Saccharomyces cerevisiae[END_REF] . We quantitatively analysed the BiFC signal for all constructions co-expressing VC-Rho and exocyst-VN using a flow cytometry-based approach. As shown in Figure 7A, a marked decrease in relative fluorescence intensity compared to the reference strain was observed in each of the four combinations for the psi1∆ mutant (from 78% of decrease for VC-Cdc42/Exo70-VN up to 89% for VC-Rho1/Sec3-VN). This indicates a substantial disruption of the interactions between the Rho GTPases and the exocyst proteins.

In order to determine whether the decrease in fluorescence was due to a limited number of protein-protein interaction events per cell or a limited number of cells in the population exhibiting these interaction events, we conducted confocal microscopy analysis of the cells. For each of the four recombinant strains co-expressing VC-Rho and exocyst-VN, a dramatic decrease (47-93%) in the proportion of cells showing a specific BiFC signal at the growing area was observed in the psi1∆ mutant (Figure 7B). The strongest decrease (93%) was observed for the Rho1p-Sec3p interaction. Thus, in the absence of phosphoinositides with C18:0, fewer cells in the population exhibit an interaction between the Rho proteins and the exocyst subunits known to bind PI(4,5)P2 (Sec3p or Exo70p) [START_REF] Zhang | Membrane association and functional regulation of Sec3 by phospholipids and Cdc42[END_REF][START_REF] Robinson | Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70[END_REF][START_REF] Guo | Spatial regulation of the exocyst complex by Rho1 GTPase[END_REF][START_REF] Wu | The Exo70 subunit of the exocyst is an effector for both Cdc42 and Rho3 function in polarized exocytosis[END_REF] . In order to prove that Rho GTPase/exocyst interactions were dependent on the PSI1 gene, rescue via the overexpression of PSI1 was implemented in strains deleted for PSI1. psi1∆/psi1∆ strains expressing the PSI1 gene carried by pCM189 plasmid, showed a strong increase in the proportion of cells with a specific fluorescent signal at the bud-tip or the bud-neck for all four combinations compared to cells carrying the empty plasmid (Figure S4). This clearly shows that Rho GTPase/Exocyst interactions are dependent on the presence of stearic acid containing phosphoinositides.

As expected, the BiFC fluorescent signal was only observed at the growth area and scored both in the reference strain and the psi1∆ genetic background for each of the four tested combinations (Figure S5A). For the combination VC-Rho3/Exo70-VN, a low fluorescent signal was observed due to the weak pool of Rho3p detected in the bud tip [START_REF] Robinson | Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70[END_REF][START_REF] Wu | The function of two Rho family GTPases is determined by distinct patterns of cell surface localization[END_REF] . No specific fluorescent signal was observed by confocal microscopy at the bud tip or at the bud neck in the strains expressing either VC-CDC42, VC-RHO1, VC-RHO3 paired with VN alone or EXO70-VN, SEC3-VN with VC alone demonstrating that the fluorochrome fragments VC and VN cannot reconstitute a functional fluorochrome by themselves, but require the specific interaction between Rho proteins and exocyst partners (Figure S5B).

In order to prove that differences observed in fluorescence intensity were due to a decrease in the interaction between Rho and the exocyst and not to an effect of the psi1 mutation on the expression of chimeric genes with reporters, RT-qPCR experiments were performed. The results demonstrate that the depletion of phosphoinositides containing stearic acid did not impact the activity of the CET1, EXO70, and SEC3 promoters (Figure S6). Therefore, the decrease of cells in the population with a BiFC signal was induced by a decrease of the Rho/Exocyst interactions.

The localisation of Exo70p and Sec3p is not disturbed in the psi1∆ mutant

Based on the observation that the interaction between the Rho proteins and the exocyst was reduced in the psi1∆ genetic background, we hypothesised that this phenotype could be explained by a mislocalisation of the Rho and exocyst proteins. Therefore, we investigated whether the lack of phosphoinositides containing stearic acid could affect the localisation of either Exo70p or Sec3p by confocal microscopy of strains expressing GFP-tagged proteins (Figure S7A). In three independent experiments, we did not observe any abnormal localisation of either exocyst subunit: Exo70p and Sec3p were distributed in similar proportions at the bud tip, at 77% and 75%, respectively, for the wild-type cells and at 75% and 72%, respectively, for the psi1∆ mutant cells (Figure S7B). Hence, despite the BiFC assay demonstrating that the interactions between Cdc42p and Exo70p and between Cdc42p and Sec3p decreased in psi1∆ background, no alteration in exocyst subunit localisation was highlighted.

Cdc42p cellular distribution was also analysed using a GFP-Cdc42 plasmid. We quantified the localisation of GFP-Cdc42p in wild-type and psi1 mutant cells and measured the GFP fluorescence localised in the early emerging bud cells. A slight difference was highlighted in this case, with 71 ± 3% of psi1Δ cells showing localisation of GFP-Cdc42p to the bud tip compared to 84 ± 4% in the WT strain (Figure S7B). Thus, similarly to the cdc42-6 mutant [START_REF] Adamo | Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud[END_REF] , a slight decrease in localization at the bud tip for Cdc42p was revealed, in contrast to the exocyst proteins Exo70 and Sec3, for which no modification was revealed. This suggest that the lack of interaction of the exocyst proteins with Cdc42p was not induced by a wrong localization of the exocyst proteins, but can be due in part to a slight alteration of the localization of a key factor of polarization, namely Cdc42p.

2.7

The activation states of Rho1p, Rho3p are affected by the lack of stearic acid in phosphoinositides, and perhaps to a lesser extent for Cdc42p.

Previous work has shown that interactions between the Exo70p or Sec3p subunits with Rho proteins are restricted to their GTP-bound active state [START_REF] Guo | Spatial regulation of the exocyst complex by Rho1 GTPase[END_REF][START_REF] Wu | The Exo70 subunit of the exocyst is an effector for both Cdc42 and Rho3 function in polarized exocytosis[END_REF][START_REF] Zhang | Cdc42 interacts with the exocyst and regulates polarized secretion[END_REF] . Our work showed that in a genetic context characterised by the lack of stearic acid in the phosphoinositides, a marked decrease in the interaction between exocyst subunits and key factors in intracellular trafficking occurred. We hypothesised that PI(4,5)P2 containing stearic acid (C18:0) is essential for the control of late steps in trafficking by interacting with key polarisation factors such as Cdc24p, (a RhoGEF of Cdc42p), Rga1/2p and Bem2/3p (several RhoGAPs for Cdc42p [START_REF] Mukherjee | Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes[END_REF][START_REF] Saito | Transbilayer phospholipid flipping regulates Cdc42p signaling during polarized cell growth via Rga GTPase-activating proteins[END_REF] ); Rom2p (the RhoGEF of Rho1p, another member of the Rho family interacting with Sec3p [START_REF] Guo | Spatial regulation of the exocyst complex by Rho1 GTPase[END_REF][START_REF] Manning | The Rho-GEF Rom2p localizes to sites of polarized cell growth and participates in cytoskeletal functions in Saccharomyces cerevisiae[END_REF] ) and/or Rgd1p (the RhoGAP for Rho3p interacting with the exocyst [START_REF] Adamo | The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity[END_REF][START_REF] Prouzet-Mauleon | Phosphoinositides affect both the cellular distribution and activity of the F-BAR-containing RhoGAP Rgd1p in yeast[END_REF][START_REF] Lefebvre | Secretory pathway-dependent localization of the Saccharomyces cerevisiae Rho GTPase-activating protein Rgd1p at growth sites[END_REF] ). Therefore, aiming to detect alterations of the GTP/GDP ratio of Rho GTPases, we performed an in vivo test based on pull-down analysis assays to determine the proportion of the GTP-bound, activated form of the Rho GTPases in the different strains. We used either the RBD (Rho Binding Domain) domain from rhotekin fused to a GST-tag for affinity precipitation of Rho1p [START_REF] Onishi | Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis[END_REF] and Rho3p [START_REF] Claret | Evidence for functional links between the Rgd1-Rho3 RhoGAP-GTPase module and Tos2, a protein involved in polarized growth in Saccharomyces cerevisiae[END_REF] , or the CRIB (Cdc42/Rac Interacting Domain) domain from Ste20p fused to a GSTtag in the case of Cdc42p 66 . 3xHA-Rho-GTP-bound proteins were pulled down from cell extracts prepared from cells grown in SC (Synthetic Complete) lacking uracil and methionine (to induce the production of either 3xHA-Rho1p, 3xHA-Rho3p or 3xHA-Cdc42p). Specific antibodies raised against Pgk1p were used to estimate the total amount of protein loaded onto the gels. RBD-or CRIB-tagged GSTs were used to determine the amount of beads loaded [START_REF] Onishi | Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis[END_REF] (Figure 8A).

The amount of each protein was determined using band intensity quantification. For each experiment, the total amount of Rho proteins was determined and normalised to the total amount of protein loaded. Similarly, the amount of GTP-Rho was determined and normalised to the total amount of GST-RBD or GST-CRIB. The ratios of these normalised values were calculated. For each Rho protein, results from the mutant strain were normalised to the wild-type strain.

The ratio of Rho pull-down to total Rho in extracts from the strains expressing the blocked GTPbound GTPases was 6.70 ± 2.54, 3.24± 2.28 and 1.08 ± 0.33 for Rho1p, Rho3p, and Cdc42p, respectively (Figure 8B), demonstrating the efficiency of the pull-down assay for Rho1p and Rho3p, but less evidently for Cdc42p for which we did not observe a significant accumulation of the blocked GTP-bound GTPase, despite the many analyses carried out and probably related to the use in our hands of the Ste20-CRIB domain. The ratios of the activated Rho proteins to the total Rho proteins in the psi1∆ mutant were 0.72 ± 0.05, 0.62 ± 0.10 and 0.50 ± 0.52 for Rho1p, Rho3p and Cdc42p, respectively, when normalised to the WT strain. This result unambiguously demonstrates that the PSI1 gene deletion, causing a lack of phosphoinositides with stearic acid, induced a decrease in the GTP-forms of at least two Rho proteins namely Rho1 and Rho3. But it also appears that, for Cdc42p, differences between WT and psi1∆ strains were not statistically significant. In other words in our hands the repeatability of the pull down assay for Cdc42p was unsatisfactory. Nevertheless results shown in Figure 8B could suggest that a decrease also in the amount of Cdc42-GTP could occur in psi1∆ strain.

Discussion

Our results show that the presence of PI C18:0 catalysed by the PSI1 gene product is necessary for the correct functioning of the secretory pathway at the vesicle docking step. The strongest genetic interaction was between PSI1 and a specific allele of CDC42, mainly affecting trafficking (cdc42-6), but not with another allele causing disturbance in actin polarisation (cdc42-1) [START_REF] Adamo | Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud[END_REF][START_REF] Dong | Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast[END_REF] . This shows that the PSI1 deletion preferentially affects Cdc42p functions involving secretory pathway partners, rather than protein partners involved in actin cytoskeleton dynamics. Previous results have demonstrated the accumulation of cargo with Bgl2p in the internal un-secreted pool for cdc42-6, as well for sec6-4, and rho3-V51 [START_REF] Adamo | Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud[END_REF][START_REF] Adamo | The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity[END_REF] . A modified version of the Bgl2p-HA secretion assay suitable for the detection of disturbances in the early stages of the cell cycle 67 also revealed a secretion defect in the psi1Δ strain [START_REF] Doignon | Requirement of phosphoinositides containing stearic acid to control cell polarity[END_REF] . Thus, we analysed genetic interactions between PSI1 and genes involved in vesicle docking during exocytosis. The genetic interactions we have demonstrated between PSI1 and CDC42 or RHO3, encoding two key proteins involved in the late steps of polarised exocytosis, are consistent with previous results based on the Bgl2p secretion assay, showing an accumulation of Bgl2p-HA in an endocellular fraction at the early bud stage in the psi1∆ mutant. Furthermore, a mild synergic effect was highlighted between PSI1 and a thermosensitive allele of SEC62, a gene encoding a member of the translocon complex involved in the early steps of trafficking. This is in agreement with previous results highlighting a link between the early and late steps of trafficking, more precisely between members of the exocyst (Sec4p, Sec8p and Sec15p) and Seb1p, a subunit of the translocon [START_REF] Guo | The exocyst meets the translocon: a regulatory circuit for secretion and protein synthesis?[END_REF][START_REF] Toikkanen | The β Subunit of the Sec61p Endoplasmic Reticulum Translocon Interacts with the Exocyst Complex in Saccharomyces cerevisiae[END_REF] .

In a yeast strain carrying a PSI1 deletion, the decrease in fatty acyl chains with stearic acid in phosphoinositides generates strong perturbations of cell polarity [START_REF] Doignon | Requirement of phosphoinositides containing stearic acid to control cell polarity[END_REF] . The cell polarity is also highly disturbed in the Caenorhabditis elegans acl-10 mutant (an orthologous gene of PSI1), since the percentage of cells with abnormal seam orientation during cell division is strongly increased. In the triple mutant for the three orthologous genes acl-8, acl-9 and acl-10, which encode lysophosphatidylinositol acyltransferases, an abnormal seam orientation was also observed. In agreement with our results, a suppressor effect of this phenotype was revealed in a quadruple mutant disrupted for acl-8, acl-9, acl-10 and the ArfGEF mon-2, belonging to the Sec7 family and involved in retrograde trafficking [START_REF] Imae | Intracellular phospholipase A1 and acyltransferase, which are involved in Caenorhabditis elegans stem cell divisions, determine the sn-1 fatty acyl chain of phosphatidylinositol[END_REF] . In addition, physical and functional links between SEC7 and PIK1 (involved in PI(4)P synthesis to coordinate Arf activation) were previously highlighted [START_REF] Gloor | Interaction between Sec7p and Pik1p: the first clue for the regulation of a coincidence detection signal[END_REF] . Our results demonstrated a similar genetic link between SEC7 (coding also for an ArfGEF) and PSI1, which is involved in phosphoinositide synthesis.

In S. cerevisiae, two pools of PI(4)P are synthesised in the Golgi apparatus and plasma membrane by two essential type III PI 4-kinases, Pik1p and Stt4p, respectively [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF][START_REF] Wera | Phosphoinositides in yeast: genetically tractable signalling[END_REF] . The pool of PI(4)P specifically synthesised by Stt4p is essential for recruiting both the Cdc42p GTPase module itself [START_REF] Meca | Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions[END_REF] and Cla4p, an activator downstream of Cdc42p involved in septin hourglass formation [START_REF] Wild | The p21-activated protein kinase-related kinase Cla4 is a coincidence detector of signaling by Cdc42 and phosphatidylinositol 4phosphate[END_REF][START_REF] Versele | Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4[END_REF] . PSI1 overexpression induced the overproduction of PIP (36:0), and in the stt4-4ts mutant at 30°C, the amount of this molecular species grows up to 15% to the level observed at permissive temperatures. In comparison, PIP (36:0) production increases by only 9% in the stt4-4ts mutant (without PSI1 overexpression) when grown at 30°C. It was also shown that the bulk of the PI(4,5)P2 produced at the plasma membrane by the PI(4)P-kinase Mss4p is dependent on Stt4p activity [START_REF] Strahl | Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae[END_REF] . By recruiting two subunits of the exocyst, Sec3p and Exo70p, in cooperation with Cdc42p 7,39,45 , Rho1p 53 and Rho3p 54 , PI(4,5)P2 is required for secretory vesicle docking and fusion with the plasma membrane [START_REF] Liu | Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells[END_REF] . In our work, using a PI(4,5)P2 biosensor, we observed that, in the stt4 mutant, a pool of PI(4,5)P2 was present at the plasma membrane, but excluded from the growth area at the non-permissive temperature. This mislocalisation of PI(4,5)P2 in the stt4-4ts mutant has been previously described at a non-permissive temperature in yeast during shmoo formation [START_REF] Garrenton | Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5-bisphosphate distribution is required for MAPK signaling[END_REF] . Unlike mammalian cells [START_REF] Nakatsu | PtdIns4P synthesis by PI4KIIIalpha at the plasma membrane and its impact on plasma membrane identity[END_REF] , we did not find an intracellular compartment containing PI(4,5)P2 in stt4-4ts yeast at a non-permissive temperature. The Cdc42p GTPase module, including the guanine exchange factor Cdc24p, the scaffold Bem1p and the kinase Cla4p, bind to the PI(4)P, PI(4,5)P2 and PS (phosphatidylserine) pools in the plasma membrane. Together, these components make it possible for Bem1p and Cdc24p to localise to the membrane and for Cdc42p to be activated by GTP [START_REF] Meca | Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions[END_REF][START_REF] Rapali | Scaffold-mediated gating of Cdc42 signalling flux[END_REF] . The constitutive overexpression of PSI1 should rescue the lethality of stt4-4ts by maintaining a pool of PI(4)P with stearic acid PIP (36:0) at the plasma membrane (Figure 5B). This pool may be required for the function of this module, a lack of PIPs containing stearic acid being detrimental to the late steps of secretion due to either a slight decrease in the activity of Cdc42p or/and its reduced localization to the growth area. Moreover, the high proportion of PIP (36:0) may induce the greater localisation of PI(4,5)P2 to the growth zone (Figure 4), suppressing the effect of the stt4-4ts mutation to some degree.

Looking at the sedimentation profiles of Exo70p and Sec3p using OptiPrep™ density gradients, we observed intriguing differences between the psi1∆/psi1∆ strain and the control (Figure 6). Sedimentation of cell extracts using the gradient exhibited a shift in the psi1∆/psi1∆ strain compared to the control for both Rho1p and Cdc42p, but not for Rho3p (Figure 6). Interestingly, in contrast with Rho3p, the C-terminal domains of Rho1p 79 and Cdc42p [START_REF] Meca | Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions[END_REF][START_REF] Sartorel | Phosphatidylserine and GTPase activation control Cdc42 nanoclustering to counter dissipative diffusion[END_REF] contain a polybasic region enriched in positively charged amino acids (lysine) that interacts with phosphoinositides and PS.

Sec3p and a pool of Exo70p are located at polarised growth sites independent of the secretory pathway, which uses actin cables [START_REF] Boyd | Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p[END_REF] . These subunits bind to the inner leaflet of the plasma membrane at PIP2 clusters [START_REF] Pleskot | Membrane targeting of the yeast exocyst complex[END_REF] . Exo70p binds to the GTP-activated forms of Cdc42p and Rho3p [START_REF] Wu | The Exo70 subunit of the exocyst is an effector for both Cdc42 and Rho3 function in polarized exocytosis[END_REF] , while Sec3p binds to the activated forms of Cdc42p [START_REF] Zhang | Membrane association and functional regulation of Sec3 by phospholipids and Cdc42[END_REF][START_REF] Zhang | Cdc42 interacts with the exocyst and regulates polarized secretion[END_REF] and Rho1p [START_REF] Guo | Spatial regulation of the exocyst complex by Rho1 GTPase[END_REF] . Exo70p and Sec3p act in concert to mediate the anchorage of secretory vesicles to the plasma membrane via other exocyst subunits [START_REF] He | Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane[END_REF][START_REF] Guo | Spatial regulation of the exocyst complex by Rho1 GTPase[END_REF] . In the mss4 strain (mutated for PI(4,5)P2 kinase) Sec3p and Exo70p are mislocalised, emphasising the role of PI(4,5)P2 in polarised growth [START_REF] Yakir-Tamang | A phosphatidylinositol-transfer protein and phosphatidylinositol-4-phosphate 5-kinase control Cdc42 to regulate the actin cytoskeleton and secretory pathway in yeast[END_REF][START_REF] He | Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane[END_REF] . In contrast with the mss4 temperature-sensitive mutant, our work shows that the deficiency of phosphoinositides with stearic acid does not disturb the localisation of either Exo70p or Sec3p; however, we did observe a slight modification of the subcellular localisation of Cdc42p at the bud tip (13% less compared to the wild-type strain). In the rho3-V51 strain, the interaction of Rho3p with Exo70p was decreased [START_REF] Wu | The Exo70 subunit of the exocyst is an effector for both Cdc42 and Rho3 function in polarized exocytosis[END_REF] and a marked decrease in the interaction between Cdc42p with Exo70p was shown in the cdc42-6 strain in vitro [START_REF] Wu | The Exo70 subunit of the exocyst is an effector for both Cdc42 and Rho3 function in polarized exocytosis[END_REF] . Interactions of Rho3p with Exo70p and Cdc42p with Sec3p are not essential for growth at the permissive temperature [START_REF] He | Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane[END_REF][START_REF] Zhang | Membrane association and functional regulation of Sec3 by phospholipids and Cdc42[END_REF] , but the thermosensitive mutations cdc42-6 or rho3-V51 display severe secretion and growth defects in non-permissive conditions, as well as a synthetic lethal phenotype in the double mutant [START_REF] Adamo | Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud[END_REF] .

In this paper, we used a BiFC approach to demonstrate the role of phosphoinositides with stearic acid in optimising the interactions of GTPases with the two exocyst subunits, both using FACS analysis and confocal microscopy. In addition, we also demonstrated a synergistic effect of psi1∆ associated with rho3-V51, as well as synthetic lethality at intermediate temperatures in the double mutant carrying the psi1∆ and cdc42-6 mutations. These results reflect the disorganisation of Rho GTPase biology in yeast lacking stearic acid in their phosphoinositides.

Although GTP hydrolysis of Cdc42p and Rho3p is not required for their role in trafficking [START_REF] Roumanie | Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex[END_REF] , we showed that the lack of phosphoinositides with stearic acid in a psi1-deficient strain decreased the ratio of GTP/GDP forms for both Rho1p and Rho3p, and perhaps for Cdc42p . This consequently disturbs the role of Sec3p and Exo70p in secretion by decreasing their interaction with their Rho GTPase partners. Hence, the modification of the PI(4,5)P2 environment modulates the activation state of Rho, disturbing the positioning of these proteins in the membrane, which may influence their ability to interact with the exocyst [START_REF] Pleskot | Membrane targeting of the yeast exocyst complex[END_REF] . These interactions between Rho GTPases and exocyst members take place in their GTP-bound state and should induce an allosteric conformational switch to an activated state [START_REF] Roumanie | Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex[END_REF][START_REF] Rossi | Exocyst structural changes associated with activation of tethering downstream of Rho/Cdc42 GTPases[END_REF] . The cycle between GTP-state and GDP-state is regulated by the RhoGEFs and their RhoGAPs partners. Some RhoGEFs (Tus1p, Rom1/2p and Cdc24p) and RhoGAPs (Bem2/3p, Rgd1/2p and Rga1/2p) for Rho1p, Rho3p and Cdc42p, respectively, and the scaffold protein Bem1 for Cdc42p activation, contain specific domains which bind phosphoinositides: PH, F-BAR, PX and basic cluster motifs [START_REF] Meca | Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions[END_REF][START_REF] Mukherjee | Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes[END_REF][START_REF] Saito | Transbilayer phospholipid flipping regulates Cdc42p signaling during polarized cell growth via Rga GTPase-activating proteins[END_REF][START_REF] Prouzet-Mauleon | Phosphoinositides affect both the cellular distribution and activity of the F-BAR-containing RhoGAP Rgd1p in yeast[END_REF][START_REF] Schmelzle | Yeast protein kinases and the RHO1 exchange factor TUS1 are novel components of the cell integrity pathway in yeast[END_REF][START_REF] Ozaki | Rom1p and Rom2p are GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP binding protein in Saccharomyces cerevisiae[END_REF][START_REF] Roumanie | Functional characterization of the Bag7, Lrg1 and Rgd2 RhoGAP proteins from Saccharomyces cerevisiae[END_REF][START_REF] Martinez | Structural evidence of a phosphoinositide-binding site in the Rgd1-RhoGAP domain[END_REF] . In this way, they may control Rho activity through the action of phosphoinositides.

For the first time, our work demonstrates that the qualitative modification of the acyl chain of phosphoinositides modifies the activity of at least two Rho GTPases involved in the management of exocyst proteins Exo70 and Sec3, consequently controlling the activation of the exocyst and intracellular trafficking. The comparison of architectural modelling of the exocyst complex in wild reference strains [START_REF] Mei | Cryo-EM structure of the exocyst complex[END_REF][START_REF] Heider | Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex[END_REF][START_REF] Donovan | Tracking individual secretory vesicles during exocytosis reveals an ordered and regulated process[END_REF] and in cells lacking stearic acid in their phosphoinositides would be crucial to accurately determine the role of phosphoinositides and Rho GTPases in controlling the assembly and functioning of the exocyst complex.

We speculate that the importance of phosphoinositide acyl chains in the interactions between small G proteins and members of the exocyst may be partially or totally shared by other organisms, specifically in filamentous fungi, plants or mammals. For instance, in Candida albicans, pathogenicity is related to its ability to induce hyphae. This morphological switch requires the action of Rho GTPases and a steep gradient of PI(4,5)P2 generated from the tip of the hyphal filament . In this pathogenic yeast, CaSec3 and CaExo70 localise at the hyphae tip, spatially separated from a specific structure present during hyphal growth: the Spitzenkörper [START_REF] Jones | Spitzenkorper, exocyst, and polarisome components in Candida albicans hyphae show different patterns of localization and have distinct dynamic properties[END_REF] . Furthermore, previous work showed that plant plasma membrane SEC3, a protein in the pollen tube, is co-localised with PIP2 via its PH domain [START_REF] Bloch | Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth[END_REF] and can indirectly recruit ROP (Rho Of Plant) GTPases [START_REF] Guan | Signaling in pollen tube growth: crosstalk, feedback, and missing links[END_REF][START_REF] Lavy | A Novel ROP/RAC effector links cell polarity, rootmeristem maintenance, and vesicle trafficking[END_REF] . In animal cells, EXOC1 (Sec3) and EXOC7 (Exo70) proteins bind to PI(4,5)P2 [START_REF] Liu | Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells[END_REF][START_REF] Li | Ezrin/Exocyst complex regulates mucin 5AC secretion induced by neutrophil elastase in human airway epithelial cells[END_REF][START_REF] Xiong | An association between type Igamma PI4P 5-kinase and Exo70 directs E-cadherin clustering and epithelial polarization[END_REF] and the exocyst complex plays a role mainly during trafficking [START_REF] Liu | Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells[END_REF][START_REF] Li | Ezrin/Exocyst complex regulates mucin 5AC secretion induced by neutrophil elastase in human airway epithelial cells[END_REF][START_REF] Xiong | An association between type Igamma PI4P 5-kinase and Exo70 directs E-cadherin clustering and epithelial polarization[END_REF] . The interactions between Rho-GTPases and the exocyst complex also regulate the transport of Glut4 (the glucose transporter) to the plasma membrane after stimulation by insulin [START_REF] Inoue | The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin[END_REF] . Phagocytosis requires an interaction between Cdc42 and the exocyst complex [START_REF] Mohammadi | Cdc42 interacts with the exocyst complex to promote phagocytosis[END_REF] and interaction of TC10 with Exo70 is essential for membrane expansion in developing hippocampal neurons and axon regeneration [START_REF] Dupraz | The TC10-Exo70 complex is essential for membrane expansion and axonal specification in developing neurons[END_REF] .

Materials and methods

Yeast strains and media

The S. cerevisiae strains used in this study are listed in Supplementary Table 1. Standard techniques for culturing the yeast were used and the composition of rich (YPD) and synthetic (SC) media for yeast cultures have been reported elsewhere [START_REF] Rose | A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae[END_REF] . Yeast strains were usually grown at 30°C, except for thermosensitive mutants when grown at the indicated permissive temperatures. Double-mutants were generated by inserting a PSI1 deletion cassette carrying the G418 R marker, or by crossing the conditional sec pathway mutants with psi1Δ. Double mutants were selected for G418 R and thermosensitivity or cryosensitivity. For crossing with mutants from the Euroscarf collection, double-mutants were isolated from Non-Parental Ditype (NPD) asci.

Plasmid constructs

For PSI1 overexpression, a BamHI-NotI fragment corresponding to the PSI1 ORF was inserted under the control of the tetO promoter in pCM189 [START_REF] Gari | A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae[END_REF] . pRS314-GFP-PH PLCδ1 dimers were constructed by inserting a KpnI-SacII fragment from pRS406-2xGFP-PH PLCδ1 44 into the pRS314 vector 100 . 3xHA-RHO constructions under the control of the MET25 promoter were generated using a plasmid derived from pUG36 101 , where GFP was replaced by 3xHA [START_REF] Claret | Evidence for functional links between the Rgd1-Rho3 RhoGAP-GTPase module and Tos2, a protein involved in polarized growth in Saccharomyces cerevisiae[END_REF] . The bacterial plasmids for the production of GST-C21RBD (rhotekin RBD) and GST-CRIB were generously gifted by Dr Pilar Perez 102 and Dr Robert Arkowitz, respectively 66 .

Yeast strains constructs

Exo70p and Sec3p were tagged at C-terminal part with 6xHA at their chromosomal loci using toolbox 103 . Rho proteins used for OptiPrep™ Floatation Assay were tagged at their N-terminal part with a 6xHA using the pOM13 vector allowing N-terminal tagging of proteins expressed by own promoters at their native loci 104 . Strains used for pull-down assay carried plasmid constructs with 3xHA-RHO genes transcribed by the MET25 promoter to conserve a chromosomal allele for RHO genes, this is particularly important for strains producing the constitive GTP forms of Rho.

Determination of cell lethality

After overnight growth, OD600 was measured and dead cells were visualised 10 min after mixing 30 µl of culture with 30 µl of methylene blue staining solution. Living cells reduce the dye to a colourless state, but in dead cells the dye stays oxidized and blue. Cell mortality was quantified by the ratio of blue-coloured cells over the total number of cells by counting at least 400 cells [START_REF] De Bettignies | RGD1 genetically interacts with MID2 and SLG1, encoding two putative sensors for cell integrity signalling in Saccharomyces cerevisiae[END_REF]105 .

Analysis of phosphoinositide molecular species

Yeast cells were cultured in 100 mL of SC media lacking uracil at 25°C, up to a cell density of 0.5 OD600. Then, the cells were re-inoculated immediately in pre-warmed media and divided into two separate flasks of equal volume: one was maintained for one hour at 25°C (permissive temperature) and the other was shifted to 30°C (intermediate temperature) for one hour. The protocol used was derived from Clark 106 and was described in previous work [START_REF] Doignon | Requirement of phosphoinositides containing stearic acid to control cell polarity[END_REF] . Briefly, after lipid extraction, phosphoinositides were extracted and derived with trimethylsilyl-diazomethane (TMS-diazomethane, Sigma-Aldrich, St Louis, MO). Reverse-phase separation was carried out on a Jupiter C4 50×1 mm column using 5 µm particles (Phenomenex) and then molecular species analysis was performed using a mass spectrometer (QTRAP ® 5500, ABSciex) coupled to a LC (Liquid Chromatography) system (Ultimate 3000, Dionex).

Determination of global PIP and PIP2 amounts by [ 14 C] acetate labelling

For logarithmic growth phase labelling, 50 mL samples of both stt4-4ts mutants (either overexpressing PSI1 or not) were grown at 25°C in SC medium lacking uracil and labelled with [1-14 C]acetate (1 µCi/mL, specific activity 50.5 Ci/mol) for an additional 15 h of steady-state lipid labelling (10 generations) 107 . During the log phase, cells were re-inoculated immediately in pre-warmed media and divided into two separate flasks of equal volume: one was maintained for one hour at 25°C (permissive temperature) and the other was shifted to 30°C (intermediate temperature) for one hour. Quantitative analysis of phosphoinositides was carried out according to the method used in a previous paper [START_REF] Doignon | Requirement of phosphoinositides containing stearic acid to control cell polarity[END_REF] .

OptiPrep™ Floatation Assay

OptiPrep™ floatation was performed as described previously 108,109 using strains with trafficking landmarks tagged using 6xHA integrated into the native locus. Cells were lysed using glass beads in TNE (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 5 mM EDTA), then the lysate was cleared by centrifugation at 500 g for 5 min at 4°C. The cleared lysates were adjusted to 40% OptiPrep™ (Nycomed, Oslo, Norway) with the stock OptiPrep™ solution (60%) to a final volume of 1.35 mL, then deposited at the bottom of 5 mL centrifugation tubes, covered by a 2.16 mL cushion of 30% OptiPrep™ prepared with TNE and 490 µL of TNE. Tubes were centrifuged overnight at 100,000 g using a TH660 rotor. Fractions were collected from the top of the gradient and subjected to Western blot analysis.

Membrane fluidity assessment

Membrane fluidity assessment was determined by Laurdan spectroscopy from intact cells using a protocol described previously [START_REF] Ishmayana | Further investigation of relationships between membrane fluidity and ethanol tolerance in Saccharomyces cerevisiae[END_REF] . Cells were cultured in YPD media, to an OD600 nm of 0.4, then 1 ml of each cell culture was pelleted and resuspended at identical concentrations in 1 ml of phosphate buffer, before the addition of Laurdan (prepared in ethanol to a final concentration of 5 µM) and incubation for 1 h at 30°C in the dark. Aliquots of 200 µl of the cells were centrifuged 2 min, resuspended in 50 µl of phosphate buffer and analysed using a CLARIOstar spectrofluorimeter (BMG LABTECH). Fluorescence emission spectra were recorded between 400 nm and 600 nm with excitation at 380 nm. The emission spectrum for each cell sample was corrected by subtraction of the background fluorescence from the unlabelled cell suspension. The The fluorescence of GFP or Venus were observed at 500-600 nm, with the maximum intensities of the emission spectra at 510 and 530 nm respectively, after excitation of the cells with a laser at 488 nm.

BiFC approaches

Cdc42p, Rho1p and Rho3p were tagged at their N-terminals with VC using a one-step PCR cassette from the pFA6a-His3MX6-pCET1-VC vector [START_REF] Sung | Bimolecular fluorescence complementation analysis system for in vivo detection of protein-protein interaction in Saccharomyces cerevisiae[END_REF] integrated using homologous recombination in BY4742 (Mat α) and psi1∆ (Mat α). Exo70p and Sec3p were tagged at their Cterminals with VN using a PCR cassette from the pFA6a-VN-His3MX6 vector and integrated using BY4741 (Mat a) and psi1∆IVc (Mat a). Different combinations were generated by mating haploid and diploid cells with VC and VN tags, selected on SC lacking histidine, lysine and methionine. The BiFC signal was only detected in the growth area for each of the four combinations and scored for quantitative data.

BiFC fluorescence was also quantified by flow cytometry as previously reported 110 , using a CyFlow Space (PARTEC). Fluorescence intensity was measured in exponential phase cultures in SC medium, which were then harvested and resuspended in phosphate buffered saline at approximately 50,000 cells per mL. Cells were excited at 488 nm, and fluorescence detected through a 530 nm band pass filter. 50% of the cell population was gated to avoid fluorescence from small molecules. Some autofluorescence was observed, mainly originating from riboflavin 111 . This was measured in both the psi1∆ background and the control strain without Venus and subtracted from the fluorescence intensities detected for each diploid strain comprising the two domains (VC and VN) of the fluorochrome. Calibration was performed by measuring the fluorescence intensity signal for 20,000 cells.

RT-qPCR

A RNeasy-PlusMini Kit (Qiagen) was used for RNA purification. rDNase I was used to remove contaminating DNA (DNA-Free Kit; Invitrogen), and cDNA was obtained using a SuperScript™ II Reverse Transcriptase kit (Invitrogen). Real-time quantitative PCR reactions were performed in a CFX96 Real-Time System (Bio-Rad) using the GoTaq qPCR Master mix (Promega). The software CFX Manager (v.3.1; Bio-Rad) was used for data acquisition and analysis. The Ct method was used to normalise transcript abundance with the references mRNA ALG9 (Mannosyltransferase), TAF10 (TFIID and SAGA complexes), and UBC6 (Ubiquitinconjugating) validated for optimal quantitative expression analysis 112 .

Determination of Rho activation by Rho-GTP pull-down assay

The degree of Rho activation was determined in vivo by the amount of GTP-bound Rho, revealed by a pull-down assay performed according to a previously described protocol [START_REF] Claret | Evidence for functional links between the Rgd1-Rho3 RhoGAP-GTPase module and Tos2, a protein involved in polarized growth in Saccharomyces cerevisiae[END_REF] . The wild-type and psi1∆ strains were each transformed with one of pU009-3xHA-RHO1, pU009-3xHA-RHO3 or pU009-3xHA-CDC42, producing the wild-type 3xHA-Rho1p, 3xHA-Rho3p and 3xHA-Cdc42p strains, respectively. The wild-type strain was also transformed with one or other of the plasmids pU009-3xHA-RHO1 Q68L -GTP, pU009-3xHA-RHO3 Q74L -GTP or pU009-3xHA-CDC42 Q61L -GTP, expressing constitutively activated Rho1p, Rho3p and Cdc42p. Cells were cultured in SC lacking uracil up to a density of 0.4 OD600nm, then moved into prewarmed SC lacking uracil and methionine and grown up to a density of approximately 2 OD600nm before harvesting and lysing the cells using glass beads as described previously [START_REF] Claret | Evidence for functional links between the Rgd1-Rho3 RhoGAP-GTPase module and Tos2, a protein involved in polarized growth in Saccharomyces cerevisiae[END_REF] . Then, GTP-bound Rho1p and Rho3p were pulled down from the cell extracts by binding to GST-C21RBD (rhotekin RBD), previously obtained and purified from E. coli [START_REF] Onishi | Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis[END_REF][START_REF] Claret | Evidence for functional links between the Rgd1-Rho3 RhoGAP-GTPase module and Tos2, a protein involved in polarized growth in Saccharomyces cerevisiae[END_REF] . Cdc42-GTP was pulled down using the CRIB domain from the Ste20 protein [START_REF] Barale | Cdc42p GDP/GTP cycling is necessary for efficient cell fusion during yeast mating[END_REF] . Anti-HA 12CA5 monoclonal antibodies were used for Western blot analysis. The amount of GTP-bound Rho pulled down compared to total Rho levels for each sample was based on band intensity quantification. Densitometric analysis was performed using the free ImageJ software after BIO-RAD ChemiDoc™ acquisitions. Pgk1p was used as a loading control for total Rho and was measured using an anti-Pgk1 monoclonal antibody 22C5D8 (Thermo Fisher Scientific). GST-CRIB or GST-RDB were quantified after staining the blotted membranes with Red Ponceau [START_REF] Onishi | Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis[END_REF] .

  GP value was calculated according to: GP = 𝐼𝐼440-𝐼𝐼490 𝐼𝐼440+𝐼𝐼490 , where I440 and I490 represent the fluorescence intensity emitted at 440 nm and 490 nm respectively, as described by Ishmayana[START_REF] Ishmayana | Further investigation of relationships between membrane fluidity and ethanol tolerance in Saccharomyces cerevisiae[END_REF] .4.9. Fluorescence microscopyCells were grown in YPD or SC lacking the appropriate metabolites. Aliquots of exponentially growing yeast cells (0.2 to 0.4 OD600 units) were centrifuged, washed once with phosphatebuffered saline solution and the pellet was resuspended in DAKO Fluorescence Mounting Medium (DAKO S3023; 25% [v/v] PBS and 75% [v/v] glycerol). Confocal microscopy was performed using a Zeiss LSM 880 laser microscope (1 Airy pinhole) with a 63× oil objective.

FIGURE 1

 1 FIGURE 1 Deletion of PSI1 shows genetic interactions with some mutants involved in secretory

FIGURE 2 PSI1

 2 FIGURE 2 PSI1 constitutive overexpression suppresses growth defect of cdc42-6 mutant.

FIGURE 3 PSI1

 3 FIGURE 3 PSI1 constitutive overexpression suppresses the growth defect of the stt4-4ts mutant.Exponentially growing cells with the empty vector (pCM189) or constitutively overexpressing

FIGURE 4 PSI1

 4 FIGURE 4 PSI1 constitutive overexpression suppresses the growth defect of the stt4-4ts mutant

FIGURE 5

 5 FIGURE 5 Effect of PSI1 constitutive overexpression in stt4-4ts mutant, on the molecular fatty

FIGURE 6

 6 FIGURE 6 Specific cell polarity landmarks change their membrane environment in cell

FIGURE 7

 7 FIGURE 7The specific interactions between Rho GTPases and subunits of the exocyst are

FIGURE 8

 8 FIGURE 8The lack of stearic acid in phosphoinositides greatly reduces the activation level of

ACKNOWLEDGEMENTS

The heat-sensitive strains AAY104, SEY6210 and AAY202 were generously provided by Chris Stefan (University of California, San Diego) and strain CTY1568 was obtained from Dr Vytas Bankaitis. We thank Dr Randy Schekman for the gift of the sec mutants, Dr Patrick Bernnwald for cdc42-6 and rho3-V51, Dr Wei Guo for SEC3-GFP and EXO70-GFP, Dr Isabelle Sagot for the gift of the vectors for BiFC and for helpful discussions, Dr Derek McCusker for the gift of the GFP-CDC42 construction, Dr Tim Levine for the plasmids with biosensors to visualise PI(4,5)P2 in vivo, Dr Robert Arkowitz for the gift of the plasmid carrying the CRIB domain from Ste20p and Dr Pilar Perez for the plasmid producing rhotekin RBD. Lipidomic analyses were performed at the Toulouse INSERM Metatoul-Lipidomique and at Bordeaux Metabolome Facility-MetaboHUB (ANR-INBS-0011). Imaging was performed at the Bordeaux Imaging Center, part of the France BioImaging national infrastructure, with technical help from Brigitte Batailler and Lysiane Brocard. FACS analyses were performed with the help of Dr Catherine Cheniclet.

All authors declare that there is no conflict of interest