Canut, H.

Deswarte, Corinne

Fallot, J.

Peyrebrune, Sandrine

Roustan, J.-P.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 33, 99-100 (1994) Research Note Purification of plasma membrane vesicles from Vitis vinifera cv. Gamay suspension cells by free-flow electrophoresis CoRINE DESWARTE1), SANDRINE PEYREBRUNE1), H. CANUT'), J.-P. RousTAN1) and J. FALLOT1) Introduction: Eutypiosis, due to the ascomycete fungus Eutypa lata, affects a number of vineyards in the world. We have demonstrated that the pathogenic agent synthesises a toxin, 4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benz-aldehyde (RENAUD et al. 1989), named eutypine, which participates in the pathogenic action of Eutypa lata (TEY-RULH et al. 1991, PHILIPPE et al. 1992). The aim of our research is to determine the mechanism of action of eutypine. Its primary target can be located on the plasma membrane or into the cells. In both cases, an interaction between the toxin and the plasma membrane must necessarily occur. Also, we have developed an efficient method to obtain highly purified plasma mem-brane vesicles from Vitis vinifera cells, using free-flow electrophoresis. Material and methods: M e m b r a n e p r e p a r-a t i o n : The microsomal membranes were prepared from an 8-day-old cell Suspension of Vitis vinifera cv. Gamay (AMBID et al. 1983). The cells were harvested by filtration and 15 g fresh weight were homogenized using a biender (type 534, Moulinex, France) in 10 m1 of a medium containing 0.4 M sucrose, 2 mM EDTA, 0.1 % bovine serum albumin, 5 mM DTT, 1 % PVP and 50 mM MES (KOH) pH 5.5. The homogenatewas centrifuged at 1000 g for 5 min and the supematant was centrifuged for 10 min at 10000 g. Then, the supernatant was centrifuged for 30 min at 45000 g. The resu1ting pellet was resuspended in EC buffer (see below) and again centrifuged for 30 min at 45000 g. The final pellet (microsomal membranes) was then resuspended in EC buffer to give a protein concen-tration of about 5 mg · mi-1• The protein concentration was determined by the method of SMITH et al. ( 1985). Free- flow electrophoresis: The electrophoresis equipment was a Vap-22 continuous free-flow electrophoresis unit (Bender and Hobein, Munich). The e1ectrophoresis chamber (EC) buffer contained 5 mM KCl, 0.25 M sucrose, 10 mM triethanolamine, 10 mM acetic acid (pH 7.4) and the electrode buffer contained 100 mM triethanolamine and 100 mM acetic acid (pH 7.4). The microsomal membranes were injected at 1 m1· h-1 with an EC buffer flow of 3 ml · fraction· 1 • h- 1• Separations were performed with a constant current of 110 mA (about 1050 V) at 4 °C. The distribution of membranes in each 1) INP-ENSAT, 145 Av. de Muret, F-31076 Toulouse Cedex, France. 2) Univ. P. Sabatier, URA CNRS 1457, 118 Rte de Narbonne, F-31062 Toulouse Cedex, France. fraction was monitored by absorbance at 280 nm. The membranes were collected from pooled fractions by centrifugation for 30 min at 45000 g. E n z y m e a s s a y s : The activities of cytochrome (Cyt)-c oxidase, and NADPH cytochrome-c reductase were determined as reported by SANDEuus et al. (1986). The activity of pyrophosphatase (PPiase) was detefrnined by the method of WALKER and LEIGH (1981) and the re1ease of inorganic phosphate (Pi) was estimated by the procedure of AMES (1966). E I e c t r o n m i c r o s c o p y : Membrane fractions were fixed in 2% glutaraldehyde in 100 mM K-phosphate buffer (pH 7.2) for 5 min at room temperature then kept at 4 oc for 2 h. Sampies were post-fixed overnight with buffered osmium tetroxide (1 %) at 4 °C, dehydrated in an ethanol series and embedded in Epon. Thin sections were post-stained with uranyl acetate I Iead citrate (REYNOLDS 1963) and viewed with an electron microscope PHIUPS EM 301. Results and Discussion: Separation of microsomal membranes from Vitis vinifera cv. Gamay cells was realised by a free-flow e1ectrophoresis procedure that separates membranes according to their surface charge. A typica1 3-peak distribution profile for membrane vesicles (O.D. 280 nm) is shown in Fig. 1. Similar profiles have been obtained for other plant species studied, i.e. soybean hypocotyls (SANDELIUS et al. 1986), Catharanthus roseus cell suspension (CANUT et al. 1991) or wheat leaves (EGGER et al. 1992). The most electronegative fraction (fraction A) consisted of tonoplast and the least (fraction E) consisted of plasma membrane. The fraction C consisted of mixed membranes. To determine the membrane composition of Vitis vinifera microsomal preparation, fractions of each peak were pooled and marker enzyme activities were deter-mined. The mitochondrial marker, cytochrome-c oxydase, was recovered only in fractions A and C : 0.01 and 0.04 OD· min-1 • mg Prot·I, respectively. For the endop1asmic reticulum marker, NADPH Cyt-c reductase, only traces 400 350 300 ~ 250 ~ 200 0 0 150 100 50 0 -0 0 10 15 I I I :E 0 20 25 30 35 40 Fraction number Fig. 1: Absorbance at 280 nm of fractions obtained by free-flow electrophoretic separation of total microsomes from Vitis vinifera cells. The regions marked A, C and E represent pooled fractions. 100 CoRrNE DESWARTE, SANDRINE PEYREBRUNE, H. CANUT, J.-P. RousTAN and J. FALLOT were detected in the three fractions; it can be noted that the major activity (0.21 OD · min-1 · mg Prot-l) was elimi-nated in the 10000 g pellet during the microsome pre-paration. The PPiase activity, characteristic of vacuolar membranes, was mostly associated to fraction A (71.5 nmol Pi ·min-I· mg prot-I, 60% total activity). A low Ievel ofthis tonoplast marker contaminated fraction E (13 .9 nmol Pi· min-I· mg prot·I, 15% oftotal activity). These results demonstrate that the tonoplast is essen-tially recovered in fraction A and that fraction C contained I arger proportion of endomembranes. It also appeared that Fig. 2: Electron micrograph of isolated plasma membrane vesicles (fraction E) after free-flow electrophoresis of a microsomal preparation from Vitis vinifera cells. Bar = 0.1 f.Lm. plasmalemic fraction E presented very Iow contamination by tonoplast and endomembranes. Electron microscopy of this fraction E confirmed its plasma membrane composition (Fig. 2). Indeed, 95 % of the vesicles showed the typical three membrane Ieaflets (dark-light-dark pattern), only visible in the plasma membrane (SANDELIUS et al. 1986). In conclusion, highly purified plasma membrane vesi-cles can be rapidly obtained from Vitis vinifera cv. Gamay cells using a free-flow electrophoresis procedure, allowing eutypine plasma membrane receptor research or trans-membrane eutypine transport studies. This work is supported by EEC program «Lutte contre l'eutypiose», n°8001-CT-91.205, section DG VI FIU3. We wish to thank Dr. WINTERTON for reviewing the English manuscript. AMBID, C.; MOISSEF, M.; FALLOT, J.; 1983: Interconversion des aldehydes et des alcools monoterpeniques dans les cellules de raisin Museal cultivees in vitm. Physiol. Veg. 21 , 82-92. AMES, B. N.; 1966: Assay of inorganic phosphate. total phosphate and phosphatase. Method. Enzymol. 8, 115-118. CANUT, H.; BAUDRACCO. S.;CABANE, M.; BoUDET,A. M.; MARIGO, G.; 1991 : Preparation of sealed tonoplast and plasma membrane vesicles from Catharanthus roseus (L.) G. Don. cells by free-flow electrophoresis. Planta 184, 448-456. EGGER, A.; MoRRE, D. J.; SELLDEN, G.; SANDELIUS, A. S.; 1992: Isolation of plasma membrane fractions from green leaves by free-flow electro-phoresis or two-phase partition alone or in combination. Physiol. Plant. 84, 121-133. PHILIPPE, I. ; FALLOT, J.; PETITPREZ, M.; DARGENT, R.; 1992: Effets de I' eutypiose sur des feuilles de Vitis vinifera cv. Cabemet Sauvignon. Etude cytologique. Vitis 31, 45-53. RENAUD, J. M.; TsoUPRAS, G.; TABACCHI, R.; 1989: Biologically active natural acetylenic compounds from Eutypa lata (Pers.: F.) Tul. Helv. Chim. Acta 72, 929-932. REYNOLDS, B. S.; 1963: The use of Iead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell. Biol. 17,208-212. SANDELIUS, A. S.; PENEL, C. ; AUDERSET. G.; BRIGHTMAN, A. ; MILLARD, M.; MoRRE, D. J.; 1986: Isolation of highly purified fractions of plasma membrane and tonoplast from the same homogenate of soybean hypocotyls by free-flow electrophoresis. Plant Physiol. 81, 177-185. SMITH, P. K.; KROHN, R. I.: HERMANSON, G. T.; MALLIA, A. K.; GARTNER, F. H.; PROVENZANO, M. D.; FIJIMOTO. E. K.: GoEKE, N. M.; ÜLSON, B. J.; BLENK, D. C.; 1985: Measurement of protein using bicinchoninic acid. Anal. Biochem. 15, 76-85. TEv-RuLH, P.; PHILIPPE, I.: RENAUD, J. M.; TsouPRAS, G.; DE ANGELIS, P.; f ALLOT, J.; TABACCHI, R. ; 1991 : Eutypine, a phytotoxin produced by Eutypa lata the causal agent of dying-arm disease of grapevine. Phytochemistry 30, 471-473. WALKER, R. R. ; LEIGH, R. A.; 1981: Mg2+.dependent cation-stimulated inorganic pyrophosphatase associated with vacuoles from storage roots of red beet. Planta 153, 150-155. 

Purification of plasma membrane vesicles from Vitis vinifera cv. Gamay suspension cells by free electrophoresis

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Purification of plasma membrane vesicles from Vitis vinifera cv. Gamay suspension cells by free electrophoresis

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