Isolated fromFusogenic Carrot Cells - NCBI

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May 11, 1987 - phosphate and phosphatidylinositol 4,5-bisphosphate,play a piv- otal role in signal ... addition, DAG can activate protein kinase C (14). Evidence has been ... d intervals (3) and were used 3 or 4 d after transfer. Cells were.

Plant Physiol. (1987) 85, 389-392 0032-0889/87/85/0389/04/$0 1.00/0

Polyphosphoinositides Are Present in Plasma Membranes Isolated from Fusogenic Carrot Cells' Received for publication February 9, 1987 and in revised form May 11, 1987

JEFFERY J. WHEELER AND WENDY F. Boss* Department of Botany, North Carolina State University, Raleigh, North Carolina 27695 ABSTRACT

Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-12-3Hlinositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografmn density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in I3Hlinositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2). An additional

membranes of fusogenic carrot cells (Daucus carota L.), prelabeled with myo-[2-3H]inositol, were isolated by aqueous polymer two-phase partitioning (10) and discontinuous Renografin density gradients (5), and the [3H]inositol-labeled lipids were analyzed by TLC. The plasma membrane fraction was enriched in both PIP and PIP2. In addition, lysophosphatidylinositol monophosphate (LPIP) was found predominantly in the plasma membrane-rich fraction isolated from the fusogenic carrot cells.

I3H]inositol-labeled lipid, lysophosphatidylinositol monophosphate,

which migrated between PIP and PIP2 on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitochrondrial-rich fraction.


Fusogenic carrot cells were maintained by serial transfer at 7 d intervals (3) and were used 3 or 4 d after transfer. Cells were radiolabeled overnight (12 h) with myo-[2-3H]inositol (188.5 kBq/0.3 g fresh weight of cells; 622 gBq/mmol). While the amount of [3H] in the inositol lipids increased during the 12 h labeling, when lipid extracts of cells labeled 2 h and 12 h were compared, no newly labeled lipids were observed. This indicated that nonspecific labeling of lipids with [3H] was not occurring The polyphosphoinositides, phosphatidylinositol 4-mono- during the time course of these experiments. The 12 h labeling phosphate and phosphatidylinositol 4,5-bisphosphate, play a piv- was used to achieve optimal incorporation. EGTA (2 mM final otal role in signal transduction in many animal systems (2). In concentration) was added to the suspension medium 20 min response to an agonist, a phosphatidylinositol 4,5-bisphosphate- prior to harvesting the cells. The cells were collected on filter specific phosphodiesterase is activated, which hydrolyzes phos- paper and homogenized with a 15 ml ground glass homogenizer phatidylinositol 4,5-bisphosphate to yield DAG2 and inositol in a homogenizing medium consisting of 50 mM Tris, 95 mM 1,4,5-trisphosphate (IP3). Both DAG and IP3 function as second LiCl, 2 mM EGTA, 10 mM KCI, 1.0 mM EDTA, 0.2 mM MgCl2, messengers. IP3 mobilizes Ca2" from nonmitochondrial intracel- and 8% sucrose (pH 7.5). The homogenate was centrifuged for lular stores (25) thus initiating a cascade of calcium-regulated 4 min (1,OOOg), and the resulting supernatant was centrifuged processes affecting cell division, differentiation, and secretion. In for 45 min at 40,000g. The final pellet was resuspended in 0.5 addition, DAG can activate protein kinase C (14). ml distilled H20 before adding 0.5 g of the resuspended memEvidence has been found for the PI cycle in plant cells. PIP branes to a 4 g aqueous polymer two-phase system consisting of and PIP2 have been identified in extracts from carrot cells (Dau- 6.3% w/w polymer concentration in 5 mm phosphate buffer (pH cus carota L.) grown in suspension culture (4) and in the tropical 7.5) and 0.25 M sucrose. The system was inverted 70 times at legume, Samanea saman (13). Membranes isolated from wheat 4°C. The polymers used were Dextran T-500 (Pharmacia) (12% phosphorylate phosphatidylinositol and phosphatidylinositol 4- hydration assumed) and PEG 3350 (Fisher Sci. Co.). The twomonophosphate to form PIP and PIP2 (19). In addition, IP3 has phase system was centrifuged for 10 min (600g) in a clinical been shown to stimulate calcium efflux both in vitro (6, 21) and centrifuge (swinging bucket rotor) at 4°C to quicken the separain vivo (18) in plant cells and there is in vitro evidence for a C- tion; and upper, interface and lower fractions were collected type protein kinase (15, 20). separately, diluted in 15 ml homogenizing medium, and centriIf phosphatidylinositol 4-monophosphate and phosphatidyli- fuged for 45 min (40,000g). The pellets were washed by resusnositol 4,5-bisphosphate are important in plant cells for signal pending in 5 ml homogenizing medium and centrifuging for 20 transduction from external stimuli, then they must be associated min (40,000g). The washed pellets were resuspended in 1 ml of with the plasma membrane. To test this hypothesis, the plasma homogenizing medium and lipids were extracted according to the procedure of Boss and Massel (4). The lipids were dried in 'Supported by the National Science Foundation (DCB-85028 13 AO 1) vacuo, reconstituted in CHC13:methanol (3:1) and spotted on and a United States Army Research Office fellowship to J. J. W. (DAA silica gel LK5 thin-layer plates (Whatman) which had been LU3-86-G-0035 P- 1). Paper No. 10929 of the Journal Series of the North soaked in 1% potassium oxalate for 80 s and dried at 10°C for Carolina Agricultural Research Service, NC 27695-760 1. 1.5 h. The plates were run in CHC13:methanol: 15 N NH40H:H20 2 Abbreviations: DAG, diacylglycerol; PI, phosphatidylinositol; LPI, (90:90:7:22) for 2 h, allowed to air dry, and the radiolabeled lysophosphatidylinositol; PIP, phosphatidylinositol monophosphate; lipids were quantitated with a Bioscan System 500 Imaging LPIP, lysophosphatidylinositol monophosphate; PIP2, phosphatidylinos- Scanner. The lipids were visualized with iodine and identified by itol bisphosphate; IP3, inositol trisphosphate. comigration with commercial standards (Sigma) except for ly389



sophosphatidylinositol 4-monophosphate which was synthesized from phosphatidylinositol 4-monophosphate from bovine brain according to the following modification of the method of Kates (9). The phosphatidylinositol 4-monophosphate (0.2 mg in CHCl3:methanol, 3:1) was dried to a film under nitrogen in a 15 ml screw-cap culture tube. Ethyl ether:methanol (0.5 ml of a 98:2 solution) and 0.1 ml of 100 mm Tris buffer (pH 8.9),

Plant Physiol. Vol. 85, 1987

lipids in the lower fraction. PIP was less than 3% of the inositollabeled lipid in the lower fraction and there was only a trace of


An additional [3H]inositol-labeled lipid, putatively LPIP, was found predominantly in the plasma membrane-rich fraction (Table I; Fig 1, a and b). This lipid incorporated 132PPJ as well as [2-3H]inositol, [2-3H]glycerol, [1-'4C]myristate, and [1-'4C]stearcontaining 0.89 mg CaCl2- 2H20 and 0.2 mg phospholipase A2 ate (data not shown) and was completely hydrolyzed in mild from Crotalus durissus terrificus venom, were added to the base indicating that it was a glycerolinositol phospholipid and phosphatidylinositol 4-monophosphate, and vortexed vigorously not a sphingolipid. The [3H]inositol-labeled peak was in fact two for 30 s. After 4 h, the reaction mixture was dried in vacuo. The peaks which were not well resolved with the Bioscan but which lipids were reconstituted in CHCl3:methanol (3:1) and chromat- were readily resolved on autoradiographs. The two spots are typical of a lysolipid indicating the fatty acid could either be on ographed as described above. The presence of mitochondria was assayed by Cyt c oxidase the one or two position of the glycerol backbone. Since LPIP activity (5). Endoplasmic reticula were assayed by NADH-de- with the lower Rf was the predominant isomer detected and since pendent Cyt c reductase (5). Due to the inhibitory effects of the the two isomers were not always well resolved with the Bioscan, PEG and Dextran on the Cyt c oxidase and reductase assays and the two peaks were combined and denoted LPIP for quantitative the loss of enzyme activity upon pelleting when two-phase par- analysis. In the solvent system used, the putative LPIP had a titioning was used, an aliquot from each fraction was diluted 8- slightly lower Rf than the lysophosphatidylinositol 4-monophosfold with Tris buffer (pH 7.5) before assaying. Vanadate-sensitive phate synthesized from brain phosphatidylinositol 4-monophosK+/ATPase was used as the marker enzyme for plasma mem- phate; how,ever, mild base hydrolysis of [3H]inositol-labeled PIP brane (7) and latent IDPase was used for the Golgi apparatus from carrot cells gave two spots which comigrate with the puta(17). Protein was quantified according to Lowry et al. ( 11) with tive LPIP. Taken in toto, these data suggested that the previously BSA as a standard. Pi was determined according to Taussky and unidentified [3Hlinositol-labeled lipid (4) was LPJP. The increase in the percentage of LPIP in the upper fractions Shorr (26). Discontinuous Renografin density gradients were utilized ac- (Table I; Fig 1), was not due to increased lysis of PIP during cording to the method of Boss and Ruesink (5). Labeling with membrane isolation since if the counts in the upper and lower myo-[2-3H]inositol was done in the same manner as for aqueous fractions were added together, the lysolipid as percent total polymer two-phase partitioning. The lipids were extracted and inositol lipid was the same (within the limits of error) in the isolated membranes (4.3 ± 1.0%) as the total cell extract (4.7 ± run on thin-layer plates as described above. 1.3%). Thus, the enrichment reported represents a true change in the distribution of the inositol-labeled phospholipids. RESULTS To further substantiate the location of the polyphosphoinosiUsing the aqueous polymer two-phase partitioning technique, tides, membranes were isolated on Renografin density gradients it was possible to obtain a fraction which was enriched in plasma (Table II; Fig. 2, a and b). Others have reported that isolation of membrane based on marker enzyme assays (Table I). Greater plasma membrane from plant cells by two-phase partitioning is than 90% of the K+/ATPase activity was inhibited by vanadate typically superior to isolation of plasma membrane on sucrose in the plasma membrane-rich fraction. This compared to about density gradients (1, 8, 12). We also found this to be true for 60% vanadate inhibition in the mitochondrial and microsomal discontinuous Renografin density gradients. With the Renografin phase. In addition, the total ATPase activity was 2- to 4-fold density gradients, the distribution of PIP and PIP2 in several of higher in the absence of vanadate in the upper, plasma mem- the gradient fractions reflected the distribution of the plasma brane-rich fraction. membrane based on marker enzymes (5) and was typical of the When cells were prelabeled with [3H]inositol, the plasma mem- purity of gradient isolated membranes. brane fraction was enriched in (3HJinositol-labeled PIP and PIP2. When expressed as percent total [3H]inositol-labeled lipid reDISCUSSION covered, PIP and PIP2 were about 4- to 7-fold higher in the In this paper, we have demonstrated that [3Hlinositol-labeled plasma membrane (upper) fraction than in the lower fraction (Table I). PI and LPI were the predominant inositol-labeled PIP and PIP2 are located in the plasma membrane of wild carrot Table I. A Comparison of the Distribution of fHJInositol-Labeled Lipid from Each Fraction ofAqueous Polymer Two-Phase Partitioning and from Whole Cells Marker enzyme activity is given for the isolated membranes. The data are the means ± SD from three separate experiments. Marker Enzymes Activity per mg Protein [3H]Inositol-Labeled Lipids as Percent Total [3H]Inositol Recovered

Phospholipid PI

+ Microsomes (lowner) (lower)(lwr

Plasma membrane (upper)

+ Microsomes mitochondria

Whole cells

Marker enzymes

Plasma membrane (upper)

54.0 ± 4.5

73.5 ± 1.6

76.0 ± 6.0

K+/ATPase (ihmol Pi



4.5 ± 0.5

18.6 ± 1.4

7.4 ± 1.6


14.2 ± 1.5

2.7 ± 0.3

7.0 ± 3.4

min-') Percent inhibition by vanadate Cyt c oxidase (AA


18.9 ± 3.3

2.0 ± 0.6

4.7 ± 1.3

min-') Cyt c reductase (AA min-')


1.3 ± 0.1

0.3 ± 0.1

0.58 ± 0.3

Latent IDPase (Mmol Pi min-')


0.045 56











0 ~-.



- 4. 00








a JS




0 6 _1 2_0







-2000 Ta

0 1



1~~~~6 20

CENTIMETERS FIG. 1. Thin layer analysis of [3H]inositol-labeled lipids extracted from membranes of fusogenic cells isolated by aqueous polymer twophase partitioning; a, upper phase, plasma membrane-rich fraction; b, lower phase, microsomal + mitochondrial-rich fraction. Phosphatidylinositol (PI); lysophosphatidylinositol (LPI); phosphatidylinositol monophosphate (PIP); lysophosphatidylinositol monophosphate (LPIP); phosphatidylinositol -bisphosphate (PIP2). Note the enrichment of PIP, LPIP, and PIP2 in the plasma membrane-rich fraction and the high ratio of PIP to LPI in the plasma membrane-rich fraction versus that found in the microsomal + mitochondrial-rich fraction. (Boxes underneath figures indicate regions of integration.) Table II. A Comparison of the Distribution of[3HlInositol-Labeled Lipids from Each Fraction of Discontinuous Renografin Density

Gradients The experiment was repeated three times and the trends were consistent. Representative data are from one experiment. [3H]Inositol-Labeled Lipids as Percent Total [3H]Inositol Recovered LPIP LPI PIP PI PIP2 Fraction ER-rich

Plasma membranerich Mitochondria-rich







o Cl 4000


Pi ~~~~~~LPI



82.7 90.2 87.3

10.5 4.3 3.0

0.8 1.0 1.4

1.9 3.3

0.1 0.2 0.2







cells grown in suspension culture. These data, obtained from in vivo labeling of the membrane lipids, are in agreement with the in vitro data of Sandelius and Sommarin (19) who showed that PI and PIP kinase activities were associated with the plasma membrane of wheat cells. Phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate must be located on the plasma membrane to function in signal transduction from external stimuli. This does not preclude, however, that they may also be associated





FIG. 2. Thin layer analysis of [3H]inositol-labeled lipids extracted from membranes of fusogenic cells isolated on discontinuous Renografin density gradients: a, plasma membrane-rich fraction; b, mitochondrialrich fraction. Note the enrichment of PIP, LPIP, and PIP2 in the plasma membrane-rich fraction. (Legend and analysis as per Fig. 1.)

with other cellular membranes. Phosphatidylinositol 4-monophosphate and/or phosphatidylinositol 4,5-bisphosphate have been reported to be associated with the plasma membrane (22, 23), lysosomes (22), and nuclear membrane (16, 24) in animal systems. With the carrot cells, nuclei were discarded along with cell wall debris, starch granules, and unbroken cells in the IOOOg pellet. Thus, one cannot eliminate the possibility for PIP and PIP2 association with the carrot nuclear membrane. It is clear, however, that the microsomal or mitochondrial membranes of the carrot cells are not enriched in PIP, LPIP, and PIP2 but are enriched in LPI. In fact, in these cells, the ratio PIP/LPI can be used as measure of the purity of the plasma membrane fraction. In summary, phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate, potential key regulators in signal transduction, are associated with the plasma membrane of fusogenic carrot cells grown in suspension culture. In addition, the plasma membranes isolated from these fusogenic cells are enriched in LPIP but not LPI. While the enrichment in LPIP appears to correlate with the fusion potential of the cells (JJ Wheeler, unpublished data), LPIP has been found in nonfusogenic carrot cells grown in suspension culture. Since the percentage of inositol-labeled LPIP is equal to or greater than the percentage of PIP2 and since LPIP comigrates with PIP2 in some solvents or when the chromatograms are not completely eluted using the solvents reported, care should be taken to resolve these lipids when studying plant phosphoinositides. Acknowledgments-The authors would like to acknowledge the assistance of Dr. Anna Sandelius in optimizing the aqueous two-phase system for isolating plasma

membrane from the fusogenic carrot cells and Mara Massel for her technical assistance.

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tion and tumour promotion. Nature 308: 693-698 15. OLAH Z, Z Kiss 1986 Occurrence of lipid and phorbol ester activated protein kinase in wheat cells. Fed Eur Biochem Soc Lett 195: 33-37 16. RAVAL PJ, D ALLAN 1985 Ca2l-induced polyphosphoinositide breakdown due to phosphomonoesterase activity in chicken erythrocytes. Biochem J 231: 179-183 17. RAY PM, TL SHININGER, MM RAY 1969 Isolation of,-glucan synthetase particles from plant cells and identification with Golgi membranes. Proc Natl Acad Sci 64: 605-612 18. RINCON M, WF Boss 1987 Myo-inositol trisphosphate mobilizes calcium from fusogenic carrot (Daucus carota L.) protoplasts. Plant Physiol 83: 395-398 19. SANDELIUS AS, M SOMMARIN 1986 Phosphorylation of phosphatidylinositols in isolated plant membranes. Fed Eur Biochem Soc Lett 201: 282-286 20. SCHAFER A, F BYGRAVE, S MATZENAUER AND D MARME 1985 Identification of a calcium- and phospholipid-dependent protein kinase in plant tissue. Fed Eur Biochem Soc Lett 187: 25-28 21. SCHUMAKER KS, H SZE 1987 Inositol 1,4,5-trisphosphate releases Ca2l from vacuolar membrane vesicles of oat roots. J Biol Chem 262: 3944-3946 22. SEYFRED MA, WW WELLS 1984 Subcellular incorporation of 32P into phosphoinositides and other phospholipids in isolated hepatocytes. J Biol Chem 259: 7659-7665 23. SMITH CD, CC Cox, R SNYDERMAN 1986 Receptor-coupled activation of phosphoinositide-specific phospholipase C by an N protein. Science 232: 97-100 24. SMITH CD, WW WELLS 1983 Phosphorylation of rat liver nuclear envelopes II. Characterization of in vitro lipid phosphorylation. J Biol Chem 258: 9368-9373 25. STREB H, RF IRVINE, MJ BERRIDGE, I SCHULTZ 1983 Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol1,4,5-trisphosphate. Nature 306: 67-69 26. TAUSSKY HH, E SHORR 1953 A microcolorimetric method for determination of inorganic phosphorus. J Biol Chem 202: 675-685

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