Lipid Characterization of an Enriched Plasma Membrane Fraction of Dunaliella salina Grown in Media of Varying Salinity

Abstract

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.

Salt Induction of Fatty Acid Elongase and Membrane Lipid Modifications in the Extreme Halotolerant Alga Dunaliella salina

“…In this instance, increased desaturation of thylakoid fatty acids was causally linked to enhanced salt tolerance (Somerville, 1995). In D. salina, increased desaturation was reported in polar lipid fractions from plasma membranes of D. salina grown in high compared with low salinity (Peeler et al, 1989). Another analysis (Al-Hasan et al, 1987) detected a salt-related increase in the relative proportion of linolenic acid (18:3) in the total lipids of another D. salina strain.…”

“…Centrifugation of fraction 1 through a glycerol gradient yielded purified plasma membranes (fraction 2). Analyses of fractions 1 and 2 (Table I) for the microsomal marker NADPH cytochrome c reductase (Peeler et al, 1989) and for chlorophyll, a thylakoid membrane marker, indicated that fraction 1, but not fraction 2, contained microsomal membranes and that both fractions were largely free of thylakoid membranes. Together with the detection of the D. salina plasma membrane proteins Dca (Fisher et al, 1996) and TTf (Fisher et al, 1997) in both fractions 1 and 2 (data not shown), these results led to the conclusion that fraction 1 contained both microsomes and plasma membranes, whereas fraction 2 contained microsome-free plasma membranes.…”

“…A comparison of the Cell growth and isolation of fractions 1 and 2 were as described in "Materials and Methods." Assay of antimycin A-insensitive NADPH CytC reductase and unit definition were as described (Peeler et al, 1989) course of change in Kcs activity and transcript accumulation indicates both reach a maximum within 12 to 27 h after the salt shock. To determine whether kcs induction reflected a salt-specific or general osmotic response, the level of the kcs transcript was determined in cells subjected to osmotic upshock generated by added glycerol rather than NaCl.…”

Diacylglycerol Metabolism in the Green Alga Dunaliella salina under Osmotic Stress

“…Analysis of these components and the product of partial hydrolysis, DAG, by GC revealed that approximately half of the TAG contained 16:0 at the sn-2 position. TAG associated with the chloroplasts, perhaps as contami- The FA composition of the major D. salina plasma membrane phospholipids has been determined (24). Each of these phospholipid classes has a distinctive FA pattern that should produce characteristic DAG upon hydrolysis by phospholipase C. Two quantitatively minor but functionally important plasma membrane lipids, PIP and PIP2, have not been analyzed previously.…”

“…I Abbreviations: DAG, diacylglycerols; PIP, phosphatidylinositol 4-monophosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; 921 MATERIALS AND METHODS Chemicals [9,10-3H]Myristic acid (22.4 Ci/mmol) and [9,10-3H] (24). In order to reduce the likelihood of lipid hydrolysis during the course of cell fractionation, the upper (polyethyleneglycol) phase, containing plasma membrane vesicles, and the lower (dextran) phase, containing the endoplasmic reticulum, mitochondria, and other membraneous organelles (microsome-enriched fraction), recovered following the aqueous two-phase separation were sometimes subjected to lipid extraction immediately rather than being routinely centrifuged at high speed to sediment membranes.…”