We have developed a rapid procedure for isolating a fraction enriched in plasma membrane from DunalielIa salUna using an aqueous two-phase system (dextran/polyethylene glycol, 6.7%/ 6.7%). An enriched plasma membrane fraction, free of chloroplast and mitochondrial contamination, could be obtained in 2.5 hours. Plasma membrane proteins, which accounted for approximately 1% of the total membrane protein, contained a number of unique proteins compared with the other cell fractions, as shown by gel electrophoresis. The lipids of the plasma membrane fraction from 1.7 molar NaCI-grown cells were extracted and characterized. Phosphafidylethanolamine and phosphatidylcholine were the two most prevalent phospholipids, at 20.6% and 6.0% of the total lipid, respectively. In addition, inositol phospholipids were a significant component of the D. salina plasma membrane fraction.Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate accounted for 5.2% and 1.5% of the plasma membrane phospholipid, respectively. Diacylglyceryltrimethylhomoserine accounted for 7.9% of the plasma membrane total lipid. Free sterols were the major component of the plasma membrane fraction, at 55% of the total lipid, and consisted of ergosterol and 7-dehydroporiferasterol. Sterol peroxides were not present in the plasma membrane fraction. The lipid composition of enriched plasma membrane fractions from cells grown at 0.85 molar NaCI and 3.4 molar NaCI were compared with those grown at 1.7 molar NaCI. The concentration of diacylglyceryltrimethylhomoserine and the degree of plasma membrane fatty acid saturation increased in 3.4 molar plasma membranes. The relative concentration of sterols in the plasma membrane fraction was similar in all three NaCI concentrations tested.

ABSTRACT

Abstract. Hyperosmotic shock, induced by raising the NaCI concentration of Dunaliella salina medium from 1.71 to 3.42 M, elicited a rapid decrease of nearly onethird in whole cell volume and in the volume of intracellular organelles. The decrease in cell volume was accompanied by plasmalemma infolding without overall loss of surface area. This contrasts with the dramatic increase in plasmalemma surface area after hypoosmotic shock (Maeda, M., and G. A. Thompson. 1986. J. Cell Biol. 102:289-297). Although plasmalemma surface area remained constant after hyperosmotic shock, the nucleus, chloroplast, and mitochondria lost membrane surface area, apparently through membrane fusion with the endoplasmic reticulum. Thus the endoplasmic reticulum serves as a reservoir for excess membrane during hyperosmotic stress, reversing its role as membrane donor to the same organelles during hypoosmotically induced cell expansion. Hyperosmotic shock also induced rapid changes in phospholipid metabolism. The mass of phosphatidic acid dropped to 56% of control and that of phosphatidylinositol 4,5-bisphosphate rose to 130% of control within 4 min. Further analysis demonstrated that within 10 min after hyperosmotic shock, there was 2.5-fold increase in phosphatidylcholine turnover, a twofold increase in lysophosphatidylcholine mass, a four-fold increase in lysophosphatidate mass, and an elevation in free fatty acids to 124% of control, all observations suggesting activation of phospholipase A. The observed biophysical and biochemical phenomena are likely to be causally interrelated in providing mechanisms for successful accommodation to such severe osmotic extremes. MECHANISMS by which eukaryotic cells tolerate acute and chronic osmotic stress are not well understood. The unicellular alga Dunaliella salina possesses extremely effective mechanisms for tolerating such osmotic stress. It will grow in saline environments ranging from 0.5 to 5 M NaC1 and successfully accommodates rapid and drastic changes in extracellular osmolarity (Brown and Borowitzka, 1979).Previously, we have demonstrated that the response of D. salina to hypoosmotic shock involves rapid alterations in membrane morphology (Maeda and Thompson, 1986) and equally rapid changes in phospholipid metabolism (Einspahr et al., 1988). The increase in cell volume that follows rapid transfer (2-4 s) to hypotonic medium is accommodated by increased plasmalemmal surface area through rapid vesicle fusion. More recently, we have reported that the response of D. salina to hypoosmotic shock also involves the rapid breakdown of the polyphosphoinositides, phosphatidylinositol 4,5-bisphosphate (PIP2),I and phosphatidylinositol 4-monophosphate (PIP) through the apparent activation of a Manabu Maeda's present address is Department of Dermatology, Gifu University School of Medicine, Tsuk~asamachi 40, Gifu, Japan.1. Abbreviations used in this paper: Ch, chloroplast; ER, endoplasmic reticulum; Gol, Golgi bodies; IMP, intramembranous particles; lysoPA, lysophosphatidic acid; lysoPC, lysophosphat...

In comparison with other cell organelles, the Dunaliella salina plasma membrane was found to be highly enriched in phospholipase C activity toward exogenous [3H]

Recent investigations have confirmed the presence of the polyphosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP2), as well as inositol phospholipid-specific phospholipase C in higher plant and microalgal cells. In addition, it has been shown that stimulation of some photosynthetic cell types by environmental or hormonal challenge is accompanied by degradation of the polyphosphoinositides. The products of phospholipase C-catalyzed PIP2 hydrolysis, inositol 1,4,5-trisphosphate and diacylglycerol, appear to (24). In the limited space available, this short review will (a) highlight selected new developments during the past 2 years, (b) identify possible sources of confusion and areas in need of clarification, and (c) preview potentially significant research areas of the future.The basic process of transmembrane signaling via the inositol phospholipids as it is understood in nonphotosynthetic cells is outlined in Figure 1. Recent evidence suggesting an involvement ofthis signaling pathway in plants is summarized below. EVIDENCE FOR COMPONENTS OF THE SIGNALING PATHWAY IN PLANTS Stimulation by Extracellular SignalsDespite the expected participation of a plasma membraneassociated receptor in plant PIP2-mediated signal transduction, no such receptor has been identified. In