Supplemental Figure S1

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1 Supplemental Figure S1 GC-MS profile of total FA of BY-2 purified PM IS: h14 IS: 17: Glycerolipids 16: GIPCs 18:1,2,3 18: h22 h24 Sterols GluCER h16: 2: h2 22: h21 c23 24: h23 h25 h26 Supplemental Figure S1. Typical GC-MS spectrogram of total FAMES and sterols extracted from BY-2 cell PM. Two internal standards (IS) were added to quantify GIPC: 2-hydroxylated 14 carbon atom fatty acid (h14), and heptadecanoic acid (17:).

2 nmoles FAMES / mg protein nmoles FAMES / mg protein Mol. % Mol. % A. Supplemental Figure S2 Tobacco leaves Total Microsome PM BY-2 cells Total Microsome PM Tobacco leaves PM BY-2 cells PM LCFA VLCFA hvlcfa LCFA VLCFA hvlcfa Supplemental Figure S2. Fatty acid content of tissue, microsomal, plasma membrane (PM) and Detergent- Insoluble Membranes () fractions from tobacco leaves or BY-2 cell culture. A. Fatty Acids were released from biological samples by acid methanolysis; the resulting FAMEs were subsequently derivatized with BSTFA before GC/MS analysis. LCFA: Long Chain Fatty Acid with 16, 18 or 2 carbon atoms, VLCFA: Very Long Chain Fatty Acid with 22 to 26 carbon atoms, hvlcfa, 2-hydroxylated Very Long Chain Fatty Acid with 22 to 26 carbon atoms. The data are expressed as the mean of three independent experiments ± SEs (Garssen et al. 27). B. Sum of LCFA: Long Chain Fatty Acid with 16, 18 or 2 carbon atoms, VLCFA: Very Long Chain Fatty Acid with 22 to 26 carbon atoms, hvlcfa, 2-hydroxylated Very Long Chain Fatty Acid with 22 to 26 carbon atoms expressed as nmol of FAMES per mg of proteins. The data are expressed as the mean of three independent experiments ± SEs (Garssen et al. 27).

3 Mol. % Supplemental Figure S3 Tobacco leaves Sucrose gradient 3% w/w 35% w/w 4% w/w 48% w/w DSM DSM LCFA VLCFA hvlcfa Supplemental Figure S3. Fatty acid content of Detergent-Insoluble Membranes () vs. Detergent-Soluble Membranes (DSM) from tobacco leaf purified PM. Fatty Acids were released by acid methanolysis; the resulting FAMEs were subsequently derivatized with BSTFA before GC/MS analysis. LCFA: Long Chain Fatty Acid with 16, 18 or 2 carbon atoms, VLCFA: Very Long Chain Fatty Acid with 22 to 26 carbon atoms, hvlcfa, 2-hydroxylated Very Long Chain Fatty Acid with 22 to 26 carbon atoms. The data are expressed as the mean of three independent experiments ± SEs (Garssen et al. 27).

4 % total µg FAMES / 1µg protein µg FAMES / 1µg protein Supplemental Figure S4 FA content of ASG extracted from leaf PM : 18: 2: 22: 24: h16: h22: h24: FA and sterols content of ASG extracted from BY-2 PM : 18: 2: 22: 24: Campesterol Stigmasterol Sitosterol (h)vlcfa not detectable SG PM Leaf SG BY-2 ASG PM Leaf ASG BY-2 Supplemental Figure S4. Fatty acid and sterol content of purified Acyl Steryl Glucosides (ASG) extracted from tobacco leaves or BY-2 cell culture. ASG was purified by HP-TLC (Lefebvre et al., 27), scratched from the silica and submitted to either acid methanolysis for FAMES analysis, or to saponification for sterol analysis. After TMS derivatizasion, FAMES and sterols were quantified by GC-MS. The data are expressed as the mean of three independent experiments ± SEs (Garssen et al. 27).

5 µg FAMES / 1mg protein µg FAMES / 1µg protein Supplemental Figure S5 FA content of Glucer Tobacco leaves BY-2 cells Supplemental Figure S5. Fatty acid content of purified glucosyl ceramide (glucer) extracted from tobacco leaves or BY-2 cell culture and purified by TLC. GluCER was purified by HP-TLC (Lefebvre et al., 27), scratched from the silica and submitted to acid methanolysis for FAMES analysis, After BSTFA derivatizasion, FAMES were quantified by GC-MS. The data are expressed as the mean of three independent experiments ± SEs (Garssen et al. 27).

6 nmoles LCB / mg proteins LCB content (mol % of total LCB) nmoles LCB / mg proteins LCB content (mol% of total LCB) Supplemental Figure S6 A LCB present in GIPCs extracted from leaves and BY-2 6 Leaf GIPC 5 BY2 GIPC B LCB content of PM and extracted from tobacco leaves C LCB content of PM and s: Tobacco leaves PM BY-2 cells PM Supplemental Figure S6. LCB content of GIPCs, plasma membrane (PM) and Detergent-Insoluble Membranes () from tobacco leaves or BY-2 cell culture. A, LCBs were isolated by hydrolysis from GIPCs purified from leaf and BY-2 cells, converted to their fatty aldehydes by peroxydation and separated by GC, as described (Cacas et al., 212a); B- C, LCBs content were determined in PM and purified from tobacco leaves and BY-2 cells. Abbreviations are as follow: Peak nomenclature in the key is systematically based upon the 2-amino-acyl backbone of the LCB. t18:: 2- aminooctadecane-1,2,4-triol (trivial name phytosphingosine); t18:1(8z): (Z)-2-aminooctadec-8-ene-1,2,4-triol, (trivial name (8Z)-phytosphingenine); d18:1(4e): (E)-2-aminooctadec-4-ene-1,2-diol (trivial name sphingosine); d18:2(4e/8z,e): (E,Z)-2- aminooctadeca-4,8-dienine-1,2-diol (trivial name (4E,8Z)-sphingadienine); d18:: 2-aminooctadecane-1,2-diol (trivial name sphinganine).

7 Figure S7 supplemental informations GIPCs extracted from BY-2 cells serie A: Hex(R1)-HexA-Ins-P-(LCB-FA) R1=NH 2 R1=NHCOCH 3 serie B: Hex(R1)-HexA-Ins(Hex)-P-(LCB-FA) R1=NH 2 R1=NHCOCH 3

8 serie C: Hex(R1)-HexA-Ins(Hex-Pen)-P-(LCB- FA) R1=NH 2 R1=NHCOCH 3 serie D: Hex-Hex(R1)-HexA-Ins(Hex-Pen)-P-(LCB-FA) R1=NH 2 R1=NHCOCH 3

9 serie E: Hex 2 -Hex(R1)-HexA-Ins(Hex-Pen)-P-(LCB-FA) R1=NH 2 R1=NHCOCH 3 Supplemental Figure S7. MALDI-MS analysis of GIPC extracts from BY-2 cells. Spectra were acquired in the negative ion mode using 2,6-dihydroxyacetophenone (DHA) as a matrix. GIPCs are grouped in series according to their number of saccharide units, from two sugars (series A) to six (series E).

10 Supplemental Figure S8 MALDI-TOF of GIPCs purified from Tobacco leaf PM and PM Supplemental Figure S8. MALDI-MS analysis of GIPC extracts purified from PM and s extracted from tobacco leaves. Spectra were acquired in the negative ion mode using 2,6-dihydroxyacetophenone (DHA) as a matrix.

FA content (µg) Figure S9 supplemental informations Extraction of 1µg of BY-2 cell PM 1 75 FA content (µg) (h)vlcfa content (µg) 5 25 Supplemental Figure S14.

11 FA content (µg) Figure S9 supplemental informations Extraction of 1µg of BY-2 cell PM 1 75 FA content (µg) (h)vlcfa content (µg) 5 25 Supplemental Figure S14. Determination of lipid-to-protein ratio in plant PM. A, 1 µg of BY-2 cell purified PM were extracted by protocol #1 (see experimental procedures) using chloroform/methanol/hcl, the aqueous phase were re-extracted by buranol-1. Histograms shows the total FA content recovered in each solvent fractions expressed as the mean ± SD of three independent experiments, compare with the direct transesterification of 1 µg of BY-2 cell purified PM.

12 Fa content (µg/mg of dry weight) Figure S1 A-B supplemental informations A Aqueous phase (protein/s ugars ) Organic phase LIPIDS Optional: extraction by butanol-1 Organic phase LIPIDS Aqueous phase (protein/sug ars ) Optional: extraction by butanol-1 Organic phase (LIPIDS/prot ein/sugars ) Insoluble pellet Optional: extraction by butanol-1 Extraction 1: CHCl3/MeOH 2:1 Extraction 2: MTBE/MeOH Extraction 3: Markham B Total FA content of grape cell culture FA VLCFA hvlcfa total h26: h25: h24: h23: C24: h22: C23: C22: h2: C2 C18: C18:3-1 C18:2 C16: % mol of FA in grape cell culture

% total FA content Figure S1C-D supplemental informations C 1 9 8 7 6 5 4 3 2 1 TOTAL C/M 2/1 MTBE MARKHAM C/M 2/1 MTBE MARKHAM FA hvlcfa VLCFA + Extraction by butanol-1 of the aqueous phase

Supplemental Figure S1. Fatty acid content of total lipids from grape cell culture.

13 % total FA content Figure S1C-D supplemental informations C TOTAL C/M 2/1 MTBE MARKHAM C/M 2/1 MTBE MARKHAM FA hvlcfa VLCFA + Extraction by butanol-1 of the aqueous phase Treatment by hot isopropanoll (no second extraction by butanol-1) D TLC Control of glycerolipid extraction Without hot isopropanol + hot isopropanol PA-- C/M 2:1 MTBE Markham C/M 2:1 MTBE Markham Supplemental Figure S1. Fatty acid content of total lipids from grape cell culture. A, Fatty Acids were released from biological samples by acid methanolysis; the resulting FAMEs were subsequently derivatized with BSTFA before GC/MS analysis. LCFA: Long Chain Fatty Acid with 16, 18 or 2 carbon atoms, VLCFA: Very Long Chain Fatty Acid with 22 to 26 carbon atoms, hvlcfa, 2-hydroxylated Very Long Chain Fatty Acid with 22 to 26 carbon atoms. The data are expressed as the mean of three independent experiments ± SEs (Garssen et al.); B, Lipids from grape cell culture were extracted with or without preliminary hot isopropanol treatment. Polar lipis are further separated by HP-TLC by the solvent migration described in (Vitiello and Zanetta, 1978). Abbreviations are as described in Fig. 3; C, Rationale for the three lipid extraction protocols; D, Fatty acid analyses of lyophilized grape cell culture by the three lipid extraction protocols with or without hot isopropanol pre-treatment, compare with the direct transesterification of grape cell culture (TOTAL, left). The data are expressed as means of three independent experiments ± SD.

1274. 1396. 145. 132. 1687.8 1715.8 249.3 283.2 2111.2 2153.1 2183.1 2286.8 2314.7 2447.4 2476.4 1422. 1789.7 1869.5 }series B }series D 221.4 1979.4 1951.4 Intens. [a.u.] 1819.6 1847.6 1422. 145. 1493.

$12 13-----------------------------------------2.8 25 mm Ac Ammo 1123_fraction 4 du 11128_feuilles tabac :K2 MS Raw 21--------------------------------------27 Fractions.6.4.2 GIPCs Series A.$ PSL1-- PSL2-- GIPC series B-F B 5 mm Ac Ammo 1 mm Ac Ammo 25 mm Ac Ammo 28----------------------------------34 GIPC BY2 Test for the purity of BY-2 cell GIPCs of different series }

PSL1-- PSL2-- GIPC series B-F B 5 mm Ac Ammo 1 mm Ac Ammo 25 mm Ac Ammo 28----------------------------------34 GIPC BY2 Test for the purity of BY-2 cell GIPCs of different series }

$35----------------------- -----------------------------43 44-----------------------49 Fractions } series E } series F 1123_fraction ABC du 11128_feuilles tabac :K22 MS Raw.75.5 A{.25.$

14 }series B }series D Intens. [a.u.] Intens. [a.u.] Supplemental Figure S11 A Purification on DEAE column of BY-2 GIPCs Phospholipids + glucer+ Sterols mm Ac Ammo 5 mm Ac Ammo x1 4 1 mm Ac Ammo GIPC BY mm Ac Ammo 1123_fraction 4 du 11128_feuilles tabac :K2 MS Raw Fractions GIPCs Series A. PSL1-- PSL2-- GIPC series B-F B 5 mm Ac Ammo 1 mm Ac Ammo 25 mm Ac Ammo GIPC BY2 Test for the purity of BY-2 cell GIPCs of different series } glycerolipids/sterols x Fractions } series E } series F 1123_fraction ABC du 11128_feuilles tabac :K22 MS Raw.75.5 A{.25. B- D- E- -B -D -E } -F MALDI-MS: Fractions series B-F m/z Supplemental Figure S1. Purification of GIPCs from BY-2 cells by DEAE chromatography. A, Total BY-2 cell GIPC were separated by DEAE. The different fractions were eluted with increasing amount of ammonium acetate: fractions 9-49, see material and methods. The purification process was monitored by on HP-TLC. Lipids were visualized by spraying plates with primuline. GIPC BY2 are the starting material used as control standards for HP-TLC; B, Purified and dialyzed fractions (GIPC series A and B-F) were check for purifty by HP-TLC and MALDI-MS. Note the absence of residual glycerolipids and sterols, and the strong enrichment of series A in one hand (middle) and series B-F in the other hand.

ΔDO 45nm Figure S12 supplemental informations Specificity of antibodies to polyglycosylated GIPC tested by ELISA 2.5 2. 1.5 1..5. BSA PM BSA PM BSA PM BSA PM without antibodies pre-immun serum #46 1/5 Serum final immunsation #46 1/5 Purified antibodies #46 1/1 Antibodies to Polyglycosylated GIPC Supplemental Figure S11.

15 ΔDO 45nm Figure S12 supplemental informations Specificity of antibodies to polyglycosylated GIPC tested by ELISA BSA PM BSA PM BSA PM BSA PM without antibodies pre-immun serum #46 1/5 Serum final immunsation #46 1/5 Purified antibodies #46 1/1 Antibodies to Polyglycosylated GIPC Supplemental Figure S11. Test by ELISA of the specificity of antibodies against polyglycosylated GIPCs. ELISA were performed using BSA as negative control. Data represent mean value of four technical replicates. Vertical bars indicate standard error of the mean.

Figure S13 supplemental informations Antibodies against polyglycosylated GIPCs : test of cross reactivity toward

Purified polyclonal antibody to polyglycosylated GIPCs (rabbit #46) 1/1; secondary mouse antibody 1/15, B.

Cross reactivity of antibodies against polyglycosylated GIPCs.

manufacturers s instructions (Echelon Bioscience, USA; http://www.echelon-inc.com).

and further with horseradish peroxydase-conjugated secondary anti mouse antibody (dilution 1/15, for 1h at RT);

16 Figure S13 supplemental informations Antibodies against polyglycosylated GIPCs : test of cross reactivity toward other lipids on PIP strips A. Purified polyclonal antibody to polyglycosylated GIPCs (rabbit #46) 1/1; secondary mouse antibody 1/15, B. NEGATIVE CONTROLS: Preimmune serum Anti GIPC ABC (rabbit #46) ; secondary mouse antibody 1/15, secondary mouse antibody alone 1/15, C. POSITIVE CONTROL: Monoclonal antibody to PtdIns (4,5)P 2 1/1, ; secondary mouse 1/1, Supplemental Figure S12. Cross reactivity of antibodies against polyglycosylated GIPCs. A, The cross reactivity of antibodies against polyglycosylated GIPCs was performed on PIP strips according to manufacturers s instructions (Echelon Bioscience, USA; Membranes were first incubated with rabbit antibodies against polyglycosylated GIPCs (dilution 1/1 for 1h at RT) and further with horseradish peroxydase-conjugated secondary anti mouse antibody (dilution 1/15, for 1h at RT); B, A negative control with preimmume serum, or without primary antibodies is shown at the bottom panel; C, Positive control is performed with antibodies against PI4,5P2 (Antibodies against native PI4,5P2 from bovine spinal cord ( designs.com); dilution 1/1, for 1h at RT). Note that these antibodies are 1-fold more diluted than antibodies against polyglycosylated GIPCs.

Figure S14 supplemental informations Immunogold labeling controls A Immunogold

PM with pre-immun serum 2 nm 2 nm 2 nm C Positive control : Immunogold labeling on

, 29) 1 nm Supplemental Figure S13. Immumogold labeling controls of PM vesicles.

for immunization of polyglycosylated GIPCs with the preimmune serum of rabbit used

17 Figure S14 supplemental informations Immunogold labeling controls A Immunogold labeling on purified PM without primary antibody B Immunogold labeling on purified PM with pre-immun serum 2 nm 2 nm 2 nm C Positive control : Immunogold labeling on PM vesicles with antibodies to PMA (Raffaele et al., 29) 1 nm Supplemental Figure S13. Immumogold labeling controls of PM vesicles. A, omission of the primary antibody; B, use of the pre-immune serum of rabbit used for immunization of polyglycosylated GIPCs with the preimmune serum of rabbit used for immunization of polyglycosylated GIPCs; C, with antibodies against the proton pump ATPase PMA as used in (Raffaele et al., 29)

Hiroya Hidaka *1), Masaki Takiwaki 2), Mine Yamashita 2), Shinya Otsuki 1), Kenji Kawasaki 3), Mitsutoshi Sugano 3) and Takayuki Honda 4)

Mild acid hydrolysis of sphingolipids yields lysosphingolipids: a matrix-assisted laser desorption and ionization time-of-flight mass spectrometry study Hiroya Hidaka *1), Masaki Takiwaki 2), Mine Yamashita