Download Lipid Characterization of an Enriched Plasma Membrane

Received for publication May 9, 1988
and in revised form September 8, 1988
Plant Physiol. (1989) 89, 970-976
0032-0889/89/89/0970/07/$01 .00/0
Lipid Characterization of an Enriched Plasma Membrane
Fraction of Dunaliella salina Grown in Media of Varying
Salinity1
Thomas C. Peeler, Martha B. Stephenson, Kregg J. Einspahr, and Guy A. Thompson, Jr.*
Department of Botany, University of Texas, Austin, Texas 78713
high salinity. It is generally accepted that the high NaCl
concentrations in which Dunaliella lives would disrupt membrane processes in the majority of organisms by inhibiting
enzyme activities and disordering membrane structure. Cytoplasmic enzymes isolated from Dunaliella cells are inhibited
by high concentrations of NaCl (7), indicating that the plasma
membrane of Dunaliella must not only remain functional
under the high external concentrations of NaCl, but also
maintain a permeability barrier against the high NaCl concentration outside of the cell. Indeed, internal Na+ concentrations of less than 100 mm have been reported in Dunaliella
cells grown in 0.5 to 4 M NaCl (16). The plasma membrane
must also enable the cell to sense changes in the surrounding
osmotic conditions so that it can adjust its internal glycerol
concentration to achieve osmotic equilibrium.
We have adapted an aqueous two-phase system (19) that
allows us to purify D. salina plasma membrane more quickly
and completely than was possible with earlier methods (28).
Utilizing this method, we obtained an enriched plasma membrane fraction from D. salina grown in our standard growth
medium containing 1.7 M NaCl, and from cells grown in
media containing one-half the amount of NaCl (0.85 M) and
twice the amount of NaCl (3.4 M). The enriched plasma
membrane fractions were then characterized as to lipid composition. Several significant differences were observed which
may be responsible for the tolerance of D. salina for a wide
range of osmotic conditions.
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 4,5bisphosphate 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.
MATERIALS AND METHODS
Cell Culture
Axenic cultures of Dunaliella salina (UTEX 1644) were
grown in synthetic medium under conditions previously described (22). The cultures were maintained at 30C in continuous light (100 gmol m-2s-'). Cell population density was
measured using a Coulter Counter model ZB.
Dunaliella salina is a single-celled alga that is extraordinarily tolerant to salt stress. It can withstand a wide variety of
NaCl concentrations (0.86 to 4.3 M) (1 1) and is one of the
few organisms that can survive in extreme environments such
as the Great Salt Lake. D. salina can also survive rapid
fluctations in external NaCl concentrations. The cell osmoregulates by rapidly increasing or decreasing the internal
concentration of glycerol to osmotically balance the external
concentration of NaCl (4, 7).
The plasma membrane of Dunaliella must therefore have
particular characteristics which allow the cell to tolerate such
Plasma Membrane Purification
Cells were harvested and fractionated as previously described (22, 23), except that the cell disruption buffer (400
mM mannitol, 2 mm EDTA, 1 mM MgCl2, 100 mm NaPO4
[pH 8.0] contained phosphate instead of Tris-HCl. After
disruption in the Parr bomb, the suspension of broken cells
was centrifuged at 2639g for 3.5 min to pellet chloroplasts
and cellular debris. The supernatant over the chloroplast pellet
'This study was supported in part by grants from the Robert A.
Welch Foundation (F-350), the National Science Foundation (DMB8506750), and the Texas Advanced Technology Research Program.
970
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PLASMA MEMBRANE COMPOSITION AND NaCI ACCLIMATION
was then added to a dextran-PEG aqueous two-phase system
to partition the plasma membrane away from the remainder
of the organelles. The final concentrations of each of the
components of the two-phase system were; 6.7% Dextran T
500 (w/w), 6.7% PEG 3350 (w/w), 178 mM mannitol, 0.89
mM EDTA, 0.44 mm MgCl2, 44.6 mM NaPO4 (pH 8.0). In
addition, approximately 30 mM NaCl was added so that the
two-phase system partitioned properly. The NaCl that remained with the pelleted cells (from the growth medium) was
also necessary for plasma membrane purification. Membranes
from cells washed in NaCl-free media did not partition properly. In a typical experiment, 1.5 x 109 cells were disrupted
in 40 mL of phosphate disruption buffer, and the subsequent
supernatant was divided between two separate tubes of the
dextran-PEG system. Each 17.5 mL aliquot of the supernatant
was added to 21.7 mL of the dextran-PEG system. The
complete two-phase system was mixed by shaking and centrifuged at 660g for 10 min in a Sorvall HB-4 swinging-bucket
rotor. The upper phase (PEG-rich) was removed and centrifuged at 150,000g for 1 h in a Beckman SW-41 swinging
bucket rotor. The resulting pellet was enriched in plasma
membrane. The remaining lower phase (dextran-rich) was
diluted with buffer and also centrifuged at 1 50,000g for 1 h.
This lower phase pellet was enriched in cellular membranes
other than the plasma membrane and chloroplasts. All steps
of this isolation procedure were carried out at 4°C.
Standard Assays for Fraction Characterization
Proteins were measured using the bicinchoninic acid protein assay (Pierce) (29). Lipid phosphorus was quantified using
the method of Bartlett (3). Chl was measured according to
Arnon (2). Nucleic acid was precipitated with 2.5 vol ethanol,
and the concentration determined at A260 nm (24).
ATPase activity was measured at 37°C by the method of
Sandstrom et al. (27). The sensitivity of the ATPase activity
to vanadate was assayed by adding 0.5 mM Na3VO4 to inhibit
the activity. Cyt c oxidase and antimycin A-insensitive
NAD(P)H Cyt c reductase were assayed according to Hodges
and Leonard (14). Antimycin A (1.0 nmol) was added to the
reaction mixture for measuring antimycin A-insensitive
NAD(P)H Cyt c reductase. The values reported are from a
single representative experiment.
Samples utilized for gel electrophoresis were boiled in SDS
sample buffer containing 50 mM Tris (pH 6.8), 1% SDS, and
2% f3-mercaptoethanol, and electrophoresed on a discontinous Tris-glycine-buffered polyacrylamide gel system using a
17.5% resolving gel and a 3% stacker (18).
Lipid Extraction and Characterization
Lipids were extracted from cell fractions using the
procedure of Bligh and Dyer (5). Plasma membrane lipids
were separated by two-dimensional TLC on precoated
silica gel plates (Kieselgel 60 F254, Merck), using acetone/
benzene/methanol/H20 (91:30:20:8, v/v/v/v) for the first
dimension, and chloroform/acetone/methanol/acetic acid/
H20 (50:20:10:10:5, v/v/v/v/v) for the second dimension.
Following development, lipid spots were located under ultraviolet light after spraying with primulin. Individual lipids were
971
identified by their TLC mobility, by specific spray reagents,
and by comparison with authentic standards. After the addition of a known amount of heptadecanoic acid as an internal
standard, lipid spots were scraped from the TLC plate. Fatty
acid methyl esters were made from the lipids by adding 14%
BF3 in methanol (Sigma) and heating for 10 min at 100CC in
a sealed ampule. The fatty acid methyl esters were extracted
in hexane and quantified by GC on a Varian model 3700 gas
chromatograph using the procedures of Lynch and Thompson
(23). Concentrations of the individual phospholipids were
calculated by comparison with the internal standard. Polar
lipid analyses were performed 5 times for 1.7 M plasma
membranes, and 2 times each for 0.85 and 3.4 M plasma
membranes. The values reported are from a single representative experiment.
Sterol Characterization
Lipids were extracted following the procedure of Bligh and
Dyer (5). Whole cell and chloroplast extracts were concentrated and separated by TLC using Silica Gel H in a solvent
system consisting of petroleum ether/diethyl ether/acetic acid
(70:30:1, v/v/v). After drying the plate under N2, the free
sterol-containing areas were immediately scraped into conical
centrifuge tubes, and sterols were eluted two times with 5 mL
of chloroform/methanol (2:1, v/v). The supernatants were
combined and concentrated to dryness under N2. To remove
any traces of nonsterol lipids, the residue was saponified by
adding 5 mL of 1 N KOH in 90% methanol, 10% H20 and
incubating the mixture under N2 and in darkness for 3 h at
25°C. After adding water, the nonsaponifiable products were
extracted with hexane, and the washed hexane extracts were
concentrated to dryness and redissolved in chloroform/methanol 6:1 (v/v) for storage in darkness under N2 at -20°C.
For use as a standard, ergosterol peroxide was prepared
from ergosterol following the procedure of Gunatilaka et al.
(12). The peroxide was purified by TLC, using Silica Gel H
in a solvent system consisting of benzene/diethyl ether/
ethanol/acetic acid (50:40:2:0.2, v/v/v/v). Elution from silica
gel and preparation for HPLC analysis was performed in the
same manner as described for the sterols.
Individual sterols and sterol peroxides were resolved by
HPLC, using a Waters model 510 dual piston pump equipped
with a Waters model U6K universal injector and a C18, 25
cm x 4.6 mm, 5 gm Rainin Microsorb ODS reverse phase
column. The mobile phase consisted of methanol/H20 (98:2,
v/v). Samples were prepared for analysis following the procedure of Bulder et al. (8). After adding an appropriate
amount of internal standard (fucosterol), samples were injected in 50 gL of isopropanol. The identity of individual
sterols was based upon comparison with authentic standards
using GC, HPLC, and GC-MS. Because the amounts of lipid
obtained from the aqueous two-phase separations were very
small, these were injected directly onto the HPLC column
without TLC purification or saponification. Sterols separated
by HPLC were quantified with a Tracor Instruments 945
Flame Ionization LC Detector. Sterol fractions were analyzed
three times for each of the plasma membrane and whole cell
samples. The values reported are from a single representative
experiment.
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Plant Physiol. Vol. 89,1989
PEELER ET AL.
972
Acidic Extraction
The acidic lipid extraction procedure used to quantify PIP2
and PIP2 is described in Einspahr et al. (10).
RESULTS
Purification of Plasma Membrane
A standard cell fractionation procedure was used to break
D. salina cells and pellet the chloroplasts and other cellular
debris from the remainder of the cell membranes. The membranes in the supernatant over the chloroplast pellet were
partitioned directly, without first pelleting them, by a dextranPEG two-phase system. After testing a number of different
concentrations, 6.7% dextran and 6.7% PEG were found to
partition mainly plasma membrane into the upper phase of
the two-phase system, as judged by the criteria described
below. The concentrations ofphosphate buffer and NaCl were
also of critical importance to the success of the plasma membrane enrichment. Phosphate ions partition preferentially into
the lower (dextran) phase, causing the lower phase to become
negatively charged, and the upper phase to be positively
charged (19). This difference in charge is believed to be
responsible for the selective partitioning of plasma membrane
into the upper phase. Other ions, such as Nae and Cl-, also
influence the charge differential between the two phases.
Following partitioning of membranes between the two phases,
the membranes of each phase were collected by centrifugation
at higher speeds. The optimized procedure for obtaining an
enriched plasma membrane fraction from D. salina took
approximately 2.5 h. Three membrane pellets were subsequently used for analysis: the initial chloroplast pellet, the
upper phase pellet (plasma membrane), and the lower phase
pellet (mitochondria, endoplasmic reticulum, and other remaining membranes).
Assay of Purity of the Isolated Plasma Membrane
The isolation procedure and characterization of the plasma
membrane fraction was first performed on cells grown in
standard growth medium containing 1.7 M NaCl. Analysis of
the three major fractions obtained in this isolation procedure
(Table I) showed that the plasma membrane fraction accounted for approximately 1% of the total membrane protein
in the sample, was free of detectable chlorophyll, and had a
relatively low nucleic acid/protein ratio compared with the
other fractions.
Enzyme activities characteristic for specific organelles and
membranes were measured in each of the three fractions
(Table II). The plasma membrane fraction was enriched in an
ATPase activity that was stimulated by K+ and inhibited by
vanadate. This type of ATPase activity is considered a specific
marker for plasma membrane in higher plants (14). Thus the
2 Abbreviations: PIP, phosphatidylinositol 4-phosphate; PIP2,
phosphatidylinositol 4,5-bisphosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PI, phosphatidylinositol; DGTS, diacylglyceryltrimethylhomoserine; SL, sulfoquinovosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol;
DGDG, digalactosyldiacylglycerol.
Table 1. Characterization of the Three Membrane Fractions Isolated
from D. salina
Membrane protein (,gg/
1 06 cells)
Percent of total membrane protein
Lipid P (1gmol)/protein
(mg)
Chl (Ag)/protein (mg)
Nucleic acid (mg)/protein
(mg)
aND = not detected.
Chloroplast
Plasma
Membrane
Lower phase
Membranes
0.4
3.5
28.2
1.2
10.9
87.8
Membranes
0.28
0.15
0.07
ND"
0.07
0.03
0.31
0.86
0.15
Table II. Marker Enzyme Activities in the Three Membrane
Fractions Isolated from D. salina
Plasma
Lower phase Chloroplast
Membrane Membranes Membranes
Arnol/mg
protein/min
ATPase
Basal
+K+
K+ + vanadate
Cyt c oxidase
NAD(P)H Cyt c reductase
a ND = not detected.
0.61
0.97
0.28
NDa
0.039
0.29
0.38
0.32
0.14
0.017
0.1
0.11
0.1
0.08
ND
plasma membrane of D. salina was selectively partitioned to
the upper phase during the two-phase isolation procedure.
Cyt c oxidase activity was absent in the plasma membrane
fraction, demonstrating that mitochondrial contamination
was insignificant. NAD(P)H-Cyt c reductase activity has been
considered characteristic of ER in many plant species (15).
This enzyme activity was present in the plasma membrane
fraction, indicating that there may have been some ER contamination of the plasma membrane preparation. However,
the majority of the activity remained in the supernatants of
both the upper and lower phases, suggesting that this activity
may be soluble and not be specific to ER in D. salina. In
addition, two recent reviews (20, 25) have noted that
NAD(P)H-reductases are often associated with higher plant
plasma membranes purified by aqueous two-phase systems,
and are not reliable markers for ER.
Proteins of the three membrane fractions were resolved by
gel electrophoresis. Characteristic protein patterns are apparent in the Coomassie blue-stained gels (Fig. 1). The proteins
present in the plasma membrane fraction indicate a number
of bands unique to that fraction, as well as some bands that
are common to other fractions. The identity of specific proteins was not determined.
Lipid Characterization of the Plasma Membrane
Fractions
An overall comparison of the amounts of polar lipids and
fatty acids present in the plasma membrane of cells grown in
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PLASMA MEMBRANE COMPOSITION AND NaCI ACCLIMATION
ab
.88
.i.
.iii
_1 1
1 46
-
.I
.p~
14
A.W
Figure 1. SDS-PAGE of the proteins from the three fractions isolated
from D. salina. Each sample lane contains approximately 100 'gg of
protein. Lane a, protein standards (kilodaltons); b, plasma membrane;
c, lower phase membranes; d, chloroplast membranes.
0.85, 1.7, and 3.4 M NaCl is shown in Table III. The plasma
membrane polar lipids from Bligh and Dyer extractions were
quantified by scraping individual lipid classes from two-dimensional TLC plates and analyzing fatty acid methyl esters
following the addition of an internal standard. PE was the
phospholipid present in the highest amounts at each NaCl
concentration, followed by PC, PG, and PI. DGTS and SL
were the only polar lipids in the plasma membrane fraction
that did not contain phosphorus. DGTS was present as a
major component of the plasma membrane at each NaCl
concentration, and was more concentrated in cells grown in
3.4 M NaCl than in 0.85 M or our standard 1.7 M medium.
The concentration of SL was relatively low at all NaCl
concentrations.
The fatty acid compositions of the polar lipids extracted by
the Bligh and Dyer procedure are also shown in Table III.
There was a large amount of 16:0 present in all of the polar
lipids of the plasma membrane. PE contained a large percentage of 14:2, accounting for all that was present in the
plasma membrane. PG was responsible for all of the 18:2
(A6,9) found in the plasma membrane fraction. At 3.4 M
NaCl, there was a decrease in 18:3 and an increase in 18:2
(A9,12) (DGTS) and 18:1 (PC), compared with plasma membranes from cells grown at the lower NaCl concentrations.
Phospholipids were also quantified by extracting lipids from
973
membrane fractions by an acidic extraction procedure which
maximizes the recovery of charged inositol phospholipids. In
these experiments 1.7 M cells were labeled with 32PO4 for 48
h in order to achieve isotopic equilibrium. PIP and PIP2 were
present in the plasma membrane fraction in significant quantities, accounting for 5.2% and 1.5%, respectively, ofthe total
phospholipid, which amounted to a 6-fold (PIP) and a 7-fold
(PIP2) enrichment of the polyphosphoinositides over the
amounts found in whole cell lipids (10). In addition, a slightly
higher percentage of PI was recovered by this method than
by the Bligh and Dyer procedure. The relative amounts of
PE, PC, and PG were similar in the two extraction procedures.
Analysis of the neutral lipid components of plasma membrane fractions purified from 0.85, 1.7, and 3.4 M cells revealed sterol/phospholipid molar ratios of approximately 1.7.
The principal sterols, 7-dehydroporiferasterol and ergosterol
(Table IV), were enriched in the plasma membrane fractions
compared to whole cell values (Table V). The relative amount
of individual sterols in the plasma membrane fraction
changed only slightly over the range of NaCl conditions, in
contrast to whole cell sterols, where the proportions of individual sterols varied with changes in NaCl growth conditions.
The mol% of 7-dehydroporiferasterol increased from 25% of
the total in 0.85 M cells to 45% of the total in 3.4 M cells.
The percentage of whole cell sterol peroxides changed in an
opposite fashion, decreasing from 44% of the total in 0.85 M
to 17% of the total in 3.4 M cells. Ergosterol peroxide was
identified by comparison to a known standard. We believe
that a second peak, with a retention time slightly longer than
that of ergosterol peroxide, was 7-dehydroporiferasterol peroxide, although no standard could be obtained for comparison. Unlike previous findings (28), we did not find sterol
peroxides to be present in significant amounts in plasma
membrane preparations. In contrast, sterol peroxides accounted for 30 mol% of the sterols plus sterol peroxides
extracted from whole 1.7 M cells. A preferential enrichment
of sterol peroxide in the lower phase of the two-phase system
was apparent following TLC oflipids extracted from the upper
phase (plasma membrane-enriched) and the lower phase (remaining nonchloroplast membranes).
DISCUSSION
Purification and General Characterization of the Plasma
Membrane
The aqueous two-phase system for purifying plasma membranes described here has several advantages over previous
methods for purifying plasma membrane. It is relatively fast,
requiring 2.5 h for enrichment of D. salina plasma membranes, as compared to approximately 6 h for isolation by a
previous method not using a dextran-PEG system (28). We
were able to reduce the time required for purification by not
pelleting the non-chloroplast membrane fraction before applying it to the two-phase system. Although this increased the
volume of the two-phase system necessary to partition the
plasma membranes from the other microsomal components,
it did not affect the quality of the purification.
The two-phase method that we used partitioned plasma
membrane into the upper (PEG) phase, as measured by
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Plant Physiol. Vol. 89,1989
PEELER ET AL.
974
Table Ill. Polar Lipid and Fatty Acid Composition of D. salina Plasma Membranes Grown at 3.4, 1.7, and 0.85 M NaCI
Percent of
L
N
14-2
14-1*
Polar Lipid
NaCI
Concn
total
16-0
18:1
59.9
64.1
65.3
32.9
32.0
5.8
6.4
8.8
12.6
4.0
39.3
52.9
50.1
52.8
33.0
38.2
47.8
45.9
48.6
53.4
70.8
61.4
64.0
5.3
7.9
7.2
9.5
42.6
34.6
47.1
6.5
3.4
3.8
11.3
12.4
16.7
18:1
18:2
All
A9
18:2
A9,12
18:3
A6,9
18:3
A9,12,15
A6,9,12
8.1
5.6
2.2
2.3
3.7
34.8
36.8
1.0
0.7
1.1
10.8
17.2
0.4
0.8
0.8
4.4
4.1
33.4
14.5
3.4
wt %
mol %
M
27.9
32.3
3.2
3.4
24.0
42.4
2.1
1.7
41.2
3.7
16.4
0.85
3.4
12.6
PC
17.8
1.7
0.85
12.6
13.4
PG
3.4
1.7
10.7
0.85
17.0
Pi
3.4
6.2
1.7
6.3
0.85
4.9
3.4
26.8
DGTS
1.7
16.0
0.85
19.4
3.4
SL
6.9
1.7
7.1
4.9
0.85
a
A blank space indicates the fatty acid was not detected.
PE
39.2
42.7
37.6
20.1
20.6
4.3
6.6
4.4
2.1
29.1
21.9
11.3
15.3
2.8
8.7
20.2
9.2
10.8
11.6
16.1
8.7
15.4
7.7
6.5
5.2
Table IV. Sterol Composition of Plasma Membranes Isolated from D. salina Grown at 0.85, 1.7, and
3.4 M NaCI
1.7 M
3.4 M
Sterol
0.85 M
7-Dehydroporiferasterol
Ergosterol
moP/O
Sterol (m/ol)/
MOO/O
1.07
0.58
65
35
1.08
0.62
64
36
P (p.MOl)
1.05
0.70
mo/O
3.4 M
Sterol (mo/)/
Table V. Sterol Composition of D. salina Whole Cells
0.85 M
Sterol
P (4smol)
1.7 M
Sterol (m/)ol)/
mo/0
P
(AMI)P (uml)
Sterol (Pmo)/
7-Dehydroporiferasterol
Ergosterol
f3-Sitosterol
Peroxides (total)
Unknown
Sterol (Aroly
Sterol (m/ol)/
P (pfMOl)
0.08
0.05
0.04
0.14
0.01
25
16
12
44
3
several different assays. The enrichment of a K+-stimulated,
vanadate-inhibited ATPase in the plasma membrane fraction
demonstrates that the plasma membrane partitioned preferentially into the upper phase. The complete absence of Chl in
the plasma membrane fraction indicates that no chloroplast
contamination was present. Mitochondrial contamination
was ruled out be the absence of Cyt c oxidase activity in the
plasma membrane pellet. Endoplasmic reticulum may have
been present, as reflected by the presence of NAD(P)H Cyt c
reductase activity in the plasma membrane fraction. However,
the unusual distribution of the total NAD(P)H Cyt c reductase
activity in the plasma membrane fraction. However, the
unusual distribution of the total NAD(P)H Cyt c reductase
activity, that is, with the majority of the activity in the soluble
0.07
0.05
0.03
0.07
0.01
P
30
22
13
30
4
(pumol)
0.15
0.07
0.04
0.06
0.02
moP
60
40
mO/
ml
45
20
12
17
6
fractions, suggests that this activity may not be a specific
marker for the presence of ER in this species. In fact,
NAD(P)H Cyt c reductase has been reported to be associated
with the plasma membrane (20, 25). Some nucleic acids were
present in the plasma membrane fraction, which could have
resulted from ribosomes on contaminating RER. However,
the amount of nucleic acid was small compared to the total
present in all fractions, suggesting that any contamination by
RER was minor.
Phospholipids accounted for a 34.6 mol% of the total
plasma membrane fraction lipids in D. salina grown in 1.7 M
NaCl. PE was the most abundant phospholipid, followed by
PC and PG. The fact that PC is not the prevalent phospholipid
here, as it is in the plasma membrane of a number of other
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PLASMA MEMBRANE COMPOSITION AND NaCI ACCLIMATION
species (21, 26), may be related to the presence of the unusual
lipid DGTS in large amounts. DGTS, although not a phospholipid, is, like PC, a zwitterionic compound and therefore
may fulfill certain roles ordinarily played by PC. The inositol
phospholipids (PI, PIP, and PIP2) were present, with PIP and
PIP2 being highly enriched in the plasma membrane, as they
are in the plasma membrane of several animal cell types (1),
and in fusogenic carrot cells (30). Inositol phospholipids are
involved in signal transduction in a number of cell types, and
levels of inositol phospholipids respond dramatically to osmotic shock in D. salina (9, 10).
SL was also a component of the plasma membrane fraction.
However, its presence in the plasma membrane in even small
amounts is unusual, because it is normally associated with
the chloroplast. Its presence in the plasma membrane fraction
is not likely to be simply the result of chloroplast contamination, however, since the other characteristic chloroplast
galactolipids MGDG and DGDG were not detectable in the
plasma membrane fraction. This implies that SL is a genuine
component of the plasma membrane.
With the exception of PC and DGTS, which were quite
similar in their fatty acid compositions, the major plasma
membrane fraction polar lipids each had a characteristic fatty
acid pattern. The fatty acid patterns for each lipid class in the
plasma membrane strongly resembled the distributions reported for D. salina microsomes, although the relative proportions of the lipid classes in the two membrane types
differed (22, 23). The plasma membrane fraction SL fatty
acid pattern resembled that reported for chloroplast SL (22).
Sterols were present in the plasma membrane fraction (55%
of the total lipid) entirely in the form of free sterols. The
composition of the plasma membrane sterols was quite different from that found in higher plants, in agreement with
previous characterizations of D. salina sterols (28). Higher
plant plasma membranes have also been found to contain
modified sterols, such as acylated steryl glucosides and steryl
glucosides (21, 26), but these lipids appeared to be completely
absent from even heavily loaded TLC plates of D. salina
lipids. Likewise, there was no evidence for the presence of
cerebrosides in the D. salina plasma membrane fraction.
An earlier paper by Sheffer et al. (28) reported the lipid
composition of a D. salina plasma membrane fraction purified by a different method. They ruptured D. salina cells
under hypoosmotic conditions, and used a sucrose density
gradient to separate plasma membranes from other organelles
and membranes. There are several differences between their
results and those reported here. Sheffer et al. found more PC
than PE in their plasma membrane fraction (which was the
reverse of our results), and they found DGTS to be the most
prevalent polar lipid, accounting for 23.5% of the total lipid
(compared to 7.9% in our plasma membrane fraction). They
also found a small amount of MGDG and DGDG (6.0% of
the total lipid), which was not present in our plasma membrane fraction. These galactolipids may indicate that there
was some chloroplast contamination in their plasma membrane preparation. The most striking difference between the
two plasma membrane preparations concerns the presence of
sterol peroxides. Sheffer et al. found that sterol peroxides were
21.8% of the total plasma membrane lipid, while free sterols
975
were only 4.6% of the lipid present. In our membrane fraction
free sterols accounted for 55% of the total, and sterol peroxides
were not present. Perhaps the more rapid isolation procedure
that we employed reduced the percentage of plasma membrane sterols that were oxidized to sterol peroxides. Sterols
such as ergosterol and 7-dehydroporiferasterol, which contain
A5,7 unsaturation, are very prone to peroxidation, as confirmed by our observation that authentic ergosterol was rapidly converted to ergosterol peroxide by refluxing in ethanol
in the presence of light and a trace of eosin ( 12).
A recent review of Dunaliella has called into question some
of the species designations used in various laboratories (1 1).
The possibility exists that even though we both have reported
on the plasma membrane composition of D. salina, we may
indeed be using two different species of Dunaliella. Perhaps
this could explain the differences between our results and
those reported by Sheffer et al.
Plasma Membrane Lipid Changes in Response to
Varying Salinity
Polar lipid changes in plasma membranes from cells grown
in various NaCl concentrations were relatively minor. The
most notable change was the 10% increase in the concentration of DGTS in 3.4 M plasma membranes compared to 1.7
M cells, with a concomitant loss of PE in the 3.4 M plasma
membranes. The capacity to regulate DGTS concentration in
the plasma membrane may play a role in the ability of D.
salina to grow in a variety of salinities.
The fatty acid compositions of the polar lipids also showed
small but significant changes in response to NaCl concentration. There was a reduction in 18:3 in both PC and DGTS in
plasma membranes from cells grown at 3.4 M NaCl, with an
increase in 18:2 in DGTS and 18:1 in PC. Similar changes
have been seen in higher plant species in response to high
concentrations of NaCl ( 13). Increases in the degree of saturation of membrane-associated fatty acids have been hypothesized to make the membrane less permeable to NaCl (17).
Although the sterol composition of whole cells changed
with increasing NaCl concentration, the sterol composition
of the plasma membrane fraction changed only slightly. The
plasma membrane fraction contained a high proportion of
sterol (55% of the total lipid), which may be responsible for
some of the unusual permeability characteristics of D. salina
plasma membranes. Membrane permeability is affected by
the proportion of sterol in the lipid bilayer (6).
In conclusion, the lipid composition of D. salina plasma
membranes remained relatively constant in cells grown at
varying NaCl concentrations. There were several significant
differences, however, including an increase in the amount of
DGTS at 3.4 M NaCl, and a decrease in the degree of fatty
acid unsaturation in prevalent phospholipids at 3.4 M NaCl.
The relative stability of the plasma membrane composition
under a wide range of osmoticums may enable D. salina to
adapt so successfully to sudden changes in the external NaCl
concentration, because major changes in lipid composition
are not necessary to maintain plasma membrane integrity
after osmotic shock.
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PEELER ET AL.
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