ARCHIVES
OF
BIOCHEMISTRY
Acyltransferase
KAZUYO
AND
183, 281-289
BIOPHYSICS
Systems Involved Saccharomyces YAMADA,
(19771
in Phospholipid cerevisiae
HARUMI OKUYAMA,’ HIROH IKEZAWA
Metabolism
,YOSHIKO
in
ENDO,
AND
Faculty
of Pharmaceutical
Sciences,
Nagoya
City
Received
Crn~vcrsity, March
3-l
Tanabedori,
Mizuho-ku,
Nagoya,
Japan
7, 1977
Membrane preparations from Saccharo7nyce.s cerevisrae OC-2 catalyzed the acylation of glycerophosphate, l-acyl and 2-acyl isomers of monoacylglycerophosphate, and l-acyl and 2-acyl isomers of monoacylglycerylphosphorylcholine. The acyl-CoA:glycerophosphate acyltransferase system (EC 2.3.1.15) showed a broad specificity for acyl-CoAs when the maximal velocities were compared under optimized conditions. The acylCoA:2-acylglycerophosphate acyltransferase activity was much lower than the l-acylglycerophosphate acyltransferase activity. Although the 1-acylglycerophosphate acyltransferase system utilized saturated and unsaturated acyl-CoAs at comparable rates, the acylations at the l- and 2-positions were relatively more selective for palmitate and oleate, respectively, when assayed in the presence of palmitoyl-CoA, oleoyl-CoA, lacylglycerophosphate, and 2-acylglycerophosphate. The acyl-CoA:l-acylglyceryl-phosphorylcholine acyltransferase system (EC 2.3.1.23) was relatively more specific for unsaturated acyl-CoAs, while the acyl-CoA:2-acylglycerylphosphorylcholine acyltransferase system (EC 2.3.1.23) utilized both palmitoyl-CoA and oleoyl-CoA at a comparable rate. Although various acyltransferase systems showed a different degree of specificity for acyl-CoAs, the positional distribution of fatty acids in the phospholipid molecules could not be explained simply by the observed specificities. Zymolyase, P-1,3-glucanase from Arthrobacter luteus, was used successfully for the protoplast formation. Subcellular fractionation of the protoplast revealed that these acyltransferase activities were localized mainly in the microsomal fraction. However, the glycerophosphate and l-acylglycerophosphate acyltranferase activities in the mitochondrial fraction could not be explained by the contamination of microsomes in this fraction. These observations are apparently inconsistent with a current concept that the mitochondrial fraction is the major site of phospholipid synthesis m yeast
In yeast, the fatty acid patterns of membrane phospholipids as well as the phospholipid composition were significantly different between the aerobically and anaerobically grown cells (l-3). The changes in the phospholipid composition have also been studied in relation to the biogenesis of mitochondria (4). Mutants defective in the syntheses of saturated and unsaturated fatty acids provided excellent systems in which to study the importance of the fatty acyl moiety of phospholipids in the maintenance of proper membrane fluidity (5-7). However, the mechanisms determining the fatty acid pattern of mem’ To whom
correspondence
should
brane phospholipids in yeast have not been fully elucidated. Kuhn and Lynen showed that sn-glycerol3-phosphate (glycerophosphate) was acylated by a yeast membrane preparation to form monoacylsn-glycerol 3-phosphate (monoacyl-GP)” and that acyl-CoA was the obligatory acyl donor (8). Steiner and Lester revealed the major biosynthetic routes of phospholipids, which were similar to those in animal cells (9). In this paper, we describe some features of the acyltransferase systems involved in y Abbreviations used: acyl-GP, acyl-sn-glycerol phosphate: acyl-GPC. acyl-sn-glycerol Y-phosphorylcholine.
be addressed. 281
Copyright All
rights
0 1977 by Academic Press of reproduction in any fzrr.
Inc. reserved.
ISSN
0003-9861
3-
282
YAMADA
diacyl-sn-glycerol3-phosphate (diacyl-GP) synthesis and the presence of acyltransferase systems for the reacylations of monoacyl-sn-glycerylphosphorylcholine (monoacyl-GPC) (10) in a eucaryotic microorganism, Saccharomyces cerevisiae OC-2. MATERIALS
AND
METHODS
I l-‘ACIPalmitic acid and [9,10-:1Hloleic acid were purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo). CoA and NADPH were obtained from Kyowa Hakko Co. (Tokyo). Bovine serum albumin, fraction V (fatty acid-poor), was a product of Miles Laboratories, Inc. (Kankakee, Illinois). Antimycin A and cytochrome c (horse heart) were the products of Boehringer Mannheim (Germany). Zymolyase (/% 1,3-glucanase from Arthrobacter luteus) was obtained from Kirin Brewery Co. Ltd. (Tokyo). AcylCoAs were prepared by a modification of Seubert’s procedure (11) as described previously (12). [2“HIGlycerol 3-phosphate was prepared from [Z“Hlglycerol (Daiichi Pure Chemicals Co. Ltd., Tokyo) according to the method of Chang and Kennedy (13). Preparations of 1-acyl-GP and isomeric monoacyl-GPC were described elsewhere (14, 15). 2-AcylGP was prepared by the hydrolysis of diacyl-GP with Rhizopus lipase (Seikagaku Kogyo Co. Ltd., Tokyo) as described elsewhere (16). The isomeric purity was at least 85%. These monoacylglycerophospholipids were derived from egg yolk phosphatidylcholine or beef heart choline plasmalogen and retained the original fatty acids at the l-position (mainly palmitate and stearate) or the a-position (mainly oleate and linoleate). Preparation ofyeast membranes. A yeast, Saccharomyces cerecisiae OC-2 (kindly supplied by Dr. E. Ichishima, Tokyo Noko University, Tokyo) was grown aerobically at 30°C in a medium containing 1% yeast extract (Kyokuto Seiyaku Co., Tokyo), 1% polypeptone (Daigo Eiyo Co., Osaka) and 0.8% galactose (pH 5.6). Cells were harvested at the midexponential phase. For the characterization of the acyltransferase systems, the membrane preparations were obtained routinely as follows. Five grams of wet cells were ground with about 100 g of Al,O:, (Katayama Chemicals Co., Ltd., Osaka) and then 50 ml of 2 mM EDTA-0.3 M mannitol (pH 7.5) was added to the mixture. After centrifugation at 6OOg for 10 min, the cell pellets and Al,O,, were ground again with an additional 100 g of Al,Ozi and the homogenate was extracted with 50 ml of the same solution. The combined extracts were centrifuged at 1500g for 10 min. The centrifugations of the supernatant were further carried out at 15,OOOg for 15 min, 28,000g for 30 min, and finally at 100,OOOg for 60 min to obtain the high-speed pellet fraction. The pellet was suspended in a small volume of 2 mM EDTA-0.3 M mannitol (pH 7.5) (5-15 mg of protein/
ET
AL
ml). The suspension was divided into several tubes and stored at -70°C. The acyltransferase activities were not lost significantly after 2 months of storage under these conditions. Since the final high-speed pellet had the highest specific activities for oleoylCoA:l-acyl-GP and oleoyl-CoA:l-acyl-GPC acyltransfer reactions, this fraction was used in most of experiments unless otherwise described. For the determination of the subcellular distribution of the acyltransferase systems, the cell walls were disrupted enzymatically by using Zymolyase (17). Cells harvested at mid-exponential growth phase were washed with water and immediately subjected to protoplast formation. Typically, 25 g of wet cells was suspended in 125 ml of 20 mM Tris-HCl (pH 7.5) containing 0.65 M sorbitol and 5 rnM EDTA (SET buffer). Then 1.25 ml of cysteamine and 75 mg of Zymolyase were added successively. The incubation was carried out at 30°C. Each 50 ~1 of the incubation mixture was diluted with 5 ml of cold water and the turbidity at 600 nm was followed. The incubation was terminated when the turbidity decreased to lo-20% of the initial value. The protoplast thus formed was sedimented by centrifugation at 27,000g for 10 min. The cell pellet was suspended in 125 ml of SET buffer and homogenized by using a glass homogenizer with a Teflon pestle. The homogenate was centrifuged at 600g for 5 min and the sedimented unbroken cells were suspended in 65 ml of SET buffer. The homogenization and centrifugation were repeated. The combined cell extracts were centrifuged at 3000g for 5 min and the pellets were discarded. The supernatant was centrifuged at 17,000g for 10 min to obtain the crude mitochondrial fraction. This fraction was resuspended in SET buffer and sedimented again by centrifugation at 17,OOOg for 10 min to obtain the mitochondrial fraction. Further centrifugations of the supernatant at 20,OOOg for 20 min, 48,000g for 30 min, and 100,OOOg for 90 min yielded the intermediate, microsomes 1, and microsomes 2 fractions, respectively. These pellets were suspended in a small volume of SET buffer and stored at -70°C. Assay systems. The acyl-CoA:glycerophosphate acyltransferase activity was determined by following the incorporation of [“Hlglycerophosphate into the lipid fraction as described previously (18). The assay mixture contained 3 rnM glycerophosphate (1000 cpminmol), 20 PM acyl-CoA, 0.2 mgiml of albumin, 0.2 M KCl, and 0.1-0.4 mg/ml of enzyme protein in 0.1 M Tris-HCl (pH 7.2). The incubations were carried out at 23-25°C for 1 min. The acylations of monoacyl-GP and monoacylGPC were routinely followed spectrophotometrically (19). The assay mixture contained 50 FM l-acyl-GP or 100 pM 1-acyl-GPC, lo-20 KM acyl-CoA, 1 mM 5,5’-dithiobis-(2-nitrobenzoic acid), and 0.05-0.3 mg/ ml of enzyme protein in 0.08 M Tris-HCl (pH 7.5). Reactions were started by adding acyl-CoA after 1
ACYLTRANSFERASE
SYSTEMS
min of preincubation. Experiments with labeled acyl-CoAs were carried out without 5,5’-dithiobis(2-nitrobenzoic acid). Antimycin-insensitive NADPH:cytochrome c reductase was measured spectrophotometrically (20, 21). One milliliter,of the incubation mixture contained 30 pmol of phosphate buffer (pH 7.4), 70 nmol of cytochrome c, 2 Fmol of KCN, 5 ~1 of an ethanolic solution of antimycin A 12 mgiml), and the enzyme protein. The reactions were started by adding 0.6 smol of NADPH. The increases in the absorbance at 550 nm were recorded continuously. Succinate dehydrogenase was measured according to the method of King (22). Protein was determined by the method of Lowry et al. (23) with bovine serum albumin as a standard. All figures presented are the averages of at least two separate determinations. Positional distribution of fatty acids ln glycerophospholipids. Lipids were extracted according to the method of Jacovcic et al. (2) from the cells grown aerobically at 30°C and harvested at the late exponential growth phase. Phospholipids were separated by Silica Gel H thin-layer chromatography with chloroform:methanol:l4% NH,OH (65:40:4, v/v/v) as a solvent. Individual phospholipid was extracted from the silica gel with chloroform:methanol (1:2, vi v) and then with chloroform:methanol:3% NH,OH (6:5:1, v/v/v). Solvents were evaporated with a rotary evaporator and the residues dissolved in a small volume of chloroform:methanol (1:2, v/vi were applied to a Silica Gel H thin-layer chromatogram with chloroform:methanol:water (65:25:4, v/v/v) as a solvent to obtain chromatographically pure ethanolamine phospholipid, phosphatidyl choline, and phosphatidyl inositol. The ethanolamine phospho-
TABLE
IN
283
S. CEREVISIAE
lipid fraction includes phosphatidyl ethanolamine and N-methylated phosphatidyl ethanolamine (91. About 2 pmol of ethanolamine phospholipid in 1 ml of diethyl ether was incubated at 37°C with 1 ml of phospholipase A, solution containing 5 mgiml of Crotaku adamanteus venom in 4 mM CaCIZ-O.I M Tris-HCl \pH 7.5). The hydrolysis was complete in 1 h. Phosphatidyl choline was hydrolyzed with 0.1 ml of enzyme solution in 0.5 h, and phosphatidyl inosito1 was hydrolyzed with 1 ml of enzyme solution in 2 h. The hydrolysis products were separated by Silica Gel H thin-layer chromatography with chloroform:methanol:water (65:25:4, v/v/vi as a solvent. The liberated fatty acids and the fatty acids from the lysophospholipids were analyzed as methyl esters by a gas-liquid chromatograph with 20% EGSSX on Gas Chrom W (Hitachi KGL 2Ai. RESULTS
Glycerophosphate
Acyltransferase
System
The effect of varying acceptor concentration on the glycerophosphate acyltransferase system is shown in Table I. The K,,, value for glycerophosphate obtained by a double reciprocal plot was in a range of 1.5 to 1.8 mM, which was lower than the reported value (10 mM) (8). MgCl, was added to the glycerophosphate acyltransferase system of Escherichia coli (24). However, no appreciable stimulatory effect was observed in the yeast system. KCl, which was shown to be stimulatory in an Escherichia coli system (18), also stimulated the I
EFFECT OF SUBSTRATE CONCENTRATIONS ON ACYLTRANSFERASE SYSTEM" Percentage
activity
Glycerophosphate acyltransferase, glycerophosphate concentration (mMi
100-95 80 60 40 20 Concentrations
for typical
assays
l-Acyl-GP 1-Acyl-GP concentration (p*M)
2.6-4.3’ 1.9, 4.9 1.2 0.6 0.3
30-100 13 8 6 3
3
50
acyltransferaseb OleoylCoA concentration (PM) 5-15 3. 22 1.6 0.8 0.4. 10
l-AcylGPC
acyltransferase”
1-AcylGPC concentration (@I) 60-200 35 18 7 5 100
OleoylCoA concentration (PM) 8-40 6 4 2.5 1.0 20
n Figures were taken from the best fit to the curves obtained and were arranged sothat one can reproduce the original curves from the percentage activity at the given substrate concentrations. D High-speed pellet was used as the enzyme source. The palmitoyl-CoA:glycerophosphate, oleoyl-CoA:lacyl-GP, and oleoyl-CoA:l-acyl-GPC acyl transfer reactions showed 12, 45, and 15 nmoliminimg of protein at 25”C, respectively. ’ Glycerophosphate concentrations of 2.6-4.3 mM gave 100-951; of the maximum activity.
284
YAMADA
acylation of glycerophosphate about 2.5fold at a concentration of 0.2 M (Fig. 1). When the time course of the acylation of glycerophosphate was followed in the presence of palmitoyl-CoA or oleoyl-CoA, a significant amount of monoacyl-GP was detected with palmitoyl-CoA as acyl donor, whereas the amount of monoacyl-GP was very low with oleoyl-CoA. Attempts to accumulate monoacyl-GP with oleoyl-CoA have been unsuccessful. The reactions in the presence of 30 PM acyl-CoA were linear for about 5 min at protein concentrations varying between 0.05 and 0.4 mglml. The specificity for acyl-CoAs of the glycerophosphate acyltransferase system was examined at 3 acyl-CoA concentrations (Table II). Saturated acyl-CoAs as well as unsaturated acyl-CoAs were transferred at comparable rates. Acyl-GP
Acyltransferase
System
The 1-acyl-GP acyltransferase system was saturated at much lower concentrations of acyl donor and acyl acceptor than those described for the glycerophosphate acyltransferase system (Tables I and II). In a typical assay system, the reactions were linear for about 2 min with protein concentrations varying between 0.05 and
$T
AL TABLE
II
SPECIFICITY OF GLYCEROPHOSPHATE ACYLTRANSFERASE~
Acyl-CoA
14:o 16:0 18:O cis-A”-16:1 cis-Ay-18:1 cis-A”-18:l trans.A!‘-18:1
Acyl transfer rates (nmol/min/ mg of protein) at acyl-CoA concentrations (PM) of 10
20
30
5.0 13.5 5.2 11.0 8.5 7.7 7.8
8.6 16.4 9.7 19.0 13.3 10.R 7.7
7.2 18.0 6.6 20.0 12.3 8.2 6.5
a The incubation mixture consisted of 0.4 mgiml of enzyme protein, 3 mM 1”Hlglycerophosphate (1000 cpminmol), 0.2 M KCl, 0.2 mg/ml of bovine serum albumin, and the indicated amounts of acyl-CoA in 0.1 M Tris-HCl (pH 7.2). The combined microsomes 1 and 2 fractions obtained after Zymolyase treatment were used as the enzyme source. Incorporation of glycerophosphate into total lipid fraction was followed. The control value for zero-time incubation (0.2 nmoliminimg of protein) has been subtracted to give net acyl transfer rates. TABLE
III
SPECIFICITIES OF I-ACYL-GP AND ACYLTRANSFERASES FOR VARIOUS
Acyl-CoA
Acyl transfer rates minimg of protein) 1 -Acyl-GP
16:0 18:O cis-A!‘-16:1 cis-A!‘-18:l cis-A”-18:l trans-~!‘-18:1
15 F -2 .E 5 E
10
cis-~!‘.“-1!3:2 5 0
0'
KCI,M 0
25
Mg Cl,
50
75
100
, mM
FIG. 1. Effects of KC1 ( - 0 - i and MgCl, (- 0 - 1 on the glycerophosphate acyltransferase system. The basic incubation mixture consisted of 3 mM 1”Hlglycerophosphate (1000 cpminmol), 30 @M acylCoA, 0.2 mg/ml of enzyme protein (high-speed pellet), and 0.1 M Tris-HCl (pH 7.2). When the effect of MgCl, was examined, 0.2 M KC1 was present in the incubation mixture.
l-ACYL-GPC ACYL-CoAs”
25.6 13.4 51.3 41.1 27.6 32.6 34.1
(1.91 (1.61 (3.11 (2.71 (3.1 I (2.91 (4.51
(nmoli with 1-AcylGPC 5.5 4.9
24.0 11.8 6.2 15.2 7.0
” High-speed pellet (0.3 mg proteinimli was used in the system described in the text. Reactions were continuously followed spectrophotometrically. The molecular extinction coefficient of 13,600 was used for the reaction product of CoA and 5,5’-dithiobis-(Znitrobenzoic acid) (19). Figures are averages of two separate determinations. Control values without acceptor, which are shown in parentheses, have been subtracted to give net acyl transfer rates.
0.3 mglml. The enzyme system showed a broad pH optimum between 7.0 and 9.0. The specificity for acyl-CoAs of the l-acylGP acyltransferase system is shown in Table III. This acyltransferase system
ACYLTRANSFERASE
SYSTEMS
showed very broad specificity for acylCoAs as described for the glycerophosphate acyltransferase system. PalmitoylCoA as well as oleoyl-CoA was incorporated at a relatively high rate. These enzyme systems do not seem to discriminate strictly the cis and trans configurations or the position of double bond in the fatty acyl moiety. When 2-acyl-GP was the acceptor, the rate of CoA liberation was much lower than with 1-acyl-GP. Furthermore, most of the radioactivity incorporated was found at the 2-position of diacyl-GP. This phenomenon, which had been observed with rat liver microsomes or E. coli preparations (14, 181, was understood to indicate that the contaminating 1-acyl-GP in the 2acyl-GP preparation was preferentially acylated under the conditions. The 2-acyl-GP acyltransferase activities could be measured more accurately by using labeled acyl-CoA and correcting the radioactivity incorporated into the individual position of diacyl-GP. The 2-acyl-GP acyltransferase activities thus determined were 2.8 and 1.6 nmol/min/mg of protein with palmitoylCoA and oleoyl-CoA, respectively, which were much lower than the 1-acyl-GP acyltransferase activities (Table III). In the next experiments, the relative acyl transfer rates were measured in the presence of saturating amounts of four substrates, [‘JClpalmitoyl-CoA, [:‘H]oleoyl-CoA, 1-acyl-GP, and 2-acyl-GP (Table IV). With the four donor:acceptor comTABLE ACYLATION
OF A MIXTURE
2-ACYL-GP
IN THE
PRESENCE
PALMITOYL-COA
Fatty
acids
OF 1-ACYL-GP
OF A MIXTURE
AND
position 0.9 2.5 0.4
AND (I:11
OF
OLEOYL-COA”
Incorporation (nmol/min/mg C-l
:‘H-l&I ’ ‘C-16:0 “H,‘tC
IV (1:l)
of fatty acids of protein) into the C-2 position 23.1 9.0 2.6
’ The incubation mixture contained 40 nmol of lacyl-GP, 40 nmol of 2-acyl-GP. 10 nmol of [:xH]oleoylCoA (7000 cpminmol), 10 nmol of I”C]palmitoyl-CoA (8000 cpminmol), and 0.1 mg of enzyme protein (high-speed pellet) in 1 ml of 0.09 M Tris-HCl (pH 7.5). Values are averages of two separate determinations.
IN
285
S. CEREVISIAE
binations, the acyl transfer rates determined under the conditions were in the following order: oleoyl-CoA:l-akyl-GP > palmitoyl-CoA:l-acyl-GP > palmitoylCoA:2-acyl-GP > oleoyl-CoA:2-acyl-GP. The acylation of 2-acyl-GP (C-l position) with palmitoyl-CoA and oleoyl-CoA was less than ‘1sof that determined for l-acylGP (C-2 position). The ratios of oleate to palmitate incorporated were approximately 112.5 and 2.6 for the acylations at the l- and the 2-positions of isomeric monoacyl-GP, respectively. The selectivity for oleate at t,he 2-position and that for palmitate at the l-position observed under the conditions are significantly higher than those expected from the ratios of the maximal velocities with oleoyl-CoA and palmitoyl-CoA shown in Table III. Acyl-GPC
Acyltransferase
System
The effect of substrate concentration on the 1-acyl-GPC acyltransferase is summarized in Table I. The reactions were linear for about 2 min at protein concentrations varying between 0.05 and 0.3 mg/ml. The enzyme showed a broad pH optimum within the range of 7.0 to 9.0. Under these conditions, the 1-acyl-GPC acyltransferase system showed the specificity shown in Table III. Palmitoleoyl-CoA gave the highest rate among the acyl-CoAs examined. When the major fatty acids of the phospholipids (16:0, 16:1, and 18:l) were compared, saturated fatty acyl-CoA showed a relatively lower maximal velocity than unsaturated acyl-CoAs. The specificity for various acyl-CoAs of the 1-acyl-GPC acyltransferase system was not significantly different at pH 7.3 and 8.5 (data not shown). 2-Acyl-GPC was examined as an acceptor of the acyltransferase system (Fig. 2). The enzyme system became saturated at 15 PM acyl-CoA. Palmitoyl-CoA appeared to be less effective as an acyl donor than oleoyl-CoA in the acylation of 2-acyl-GPC, although palmitate was transferred into 2acyl-GPC at a higher rate than into l-acylGPC. Rat liver microsomes showed 34 and 11 nmol/min/mg of protein for the palmitoyl-CoA:2-acyl-GPC and oleoyl-CoA:lacyl-GPC acyl transfer reactions, respectively, as reported previously (12, 15).
286
YAMADA
Positional Distribution Phospholipids
of Fatty
Acids
in
The content of saturated fatty acids in the phospholipids increased in the order of ethanolamine phospholipid, phosphatidyl choline, and phosphatidyl inositol (Table VI. The stearate content also increased in this order, although palmitate was the major saturated fatty acid in these phospholipids. In general, the 2-position was occupied mainly with unsaturated fatty acids while the saturated fatty acids were localized in the l-position of the phospholipids. Among the unsaturated fatty acids, more palmitoleate (16:l) was found at the l-positions of ethanolamine phospholipid and phosphatidyl choline while phosphatidylinositol contained a greater amount of this fatty acid at the 2-position. Oleate was
15.
--
0'
50 100 Z-acyl-GPC , p M
0
FIG. 2. 2-Acyl-GPC acyltransferase activity. The activities for palmitoyl-CoA ( - 0 - 0 ~ i or oleoylCoA (-0-O-i are shown as a function of the acceptor concentrations. The incubation mixture consisted of 0.2 mgiml of enzyme protein (high speed pellet), 20 PM acyl-CoA. 0.18 M Tris-HCl (pH 8.91, and the indicated concentrations of Z-acyl-GPC.
ET
the major fatty acid at the 2-position of all the phospholipids examined. Thus, the localization of the individual fatty acid in the phospholipid molecules is a typical one found in rat liver phospholipids, except palmitoleate (16:l) which was found mainly at the l-position or at the 2-position depending upon the phospholipid classes. Other minor phospholipids such as cardiolipin and phosphatidyl serine have not been examined. Subcellular uses
DISTRIBUTION
OF FATTY
Ethanolamine
ACIDS
IN
Phosphatidyl
FROM
S. cereuisiae
CELLS
Total
C-l
c-2
Total
- C-l
c-2
Total
9” 41 tr.’
14 75 tr.
18:l
41
3 25 tr. 66
11 54 3 21
23 53 9 12
4 36 1 55
47 15 10 23
” Only the major components are listed. * Figures are expressed as percentage of fatty ” Trace amount.
acids
of intact
GROWN
Phosphatidyl
choline
16:0 16:l 18:0
I
of Acyltransfer-
V
PHOSPHOLIPIDS
phospholipid
Distribution
Subcellular membrane fractions were obtained from the cells treated with Zymolyase as described under Materials and Methods. The recovery of protein and the acyltransferase activities in these subcellular fractions are shown together with the distributions of some marker enzymes in Table VI. From the distribution of marker enzymes, the pellets obtained by centrifugations at 17,000g for 10 min, 20,OOOgfor 20 min, 48,000g for 30 min, and 100,OOOg for 90 min were tentatively designated as mitochondria, intermediate, microsomes 1, and microsomes 2 fractions, respectively (25). The distribution of the 1-acyl-GPC acyltransferase system roughly paralleled the antimycin-insensitive NADPH:cytochrome c reductase, while the glycerophosphate and 1-acyl-GP acyltransferase activities in the mitochondrial fraction could not be explained by the contamination of microsomes in this fraction. Thus, the acyltransferase systems involved in diacyl-GP synthesis as well as the one involved in the reacylation system (acylCoA:lysophospho!ipid acyltransferase sys-
TABLE POSITIONAL
AL
lipids
or of the indicated
AT 30°C
inositol C-l 51 I 23 9 positions.
c-2 3 24 1 70
ACYLTRANSFERASE
SYSTEMS TABLE
SUBCELLULAR
Protein*
Fraction”
Mitochondrial Intermediate Microsomal
1
64 53 30
Microsomal
2
20
Glycerophosphate acyltransferase, palmitoylCoA’ 1.0” 10.0 17.6 9.9
DISTRIBUTION
I-Acyl-GP acyltransferase, oleoylCoA’ 10 71 173 57
IN
287
S. CEREVZSIAE
VI OF ACYLTRANSFERASES
l-Acyl-GPC acyltransferase, oleoylCoA’ 2” 47 96 36
~~~~~
NADPH:cytochrome c reductase”
~~_~~~~~~-~~~
Succinate dehydrogenase”
4” 58 156 52
U Subcellular fractions obtained from the Zymolyase-treated cells were used (see text). h Milligrams of protein from 25 g of wet cells. r The amounts of enzyme protein for the acyl transfer assays were varied in a range described to obtain the initial velocities. “The l-ml incubation mixture contained 0.1 to 0.2 mg ofenzyme protein. Antimycin-insensitive * nmol/min/mg of protein.
tern) are localized mainly in the microsomal fraction, but diacyl-GP synthesizing activity is also present in the mitochondrial fraction although the activity in this fraction is much lower. DISCUSSION
Both saturated and unsaturated fatty acids were found at the l- and 2-positions of phospholipids. The ratio of unsaturated fatty acids to saturated fatty acids ranged from 0.2 to 4.5 at the l-position and from 16 to 24 at the 2-position of the phospholipids (Table V). The specificities of the l-acylGP and l-acyl-GPC acyltransferase systems for various acyl-CoAs (as compared with the respective maximal velocities) were much lower than those expected from the fatty acid composition at the 2-position of phospholipids. The specificities for acylCoAs of the glycerophosphate, 2-acyl-GP, and 2-acyl-GPC acyltransferase systems do not directly explain the fatty acid composition at the l-position of phospholipids, either. It might be that other enzyme systems which have not been examined play more important roles in the selective positioning of various fatty acids in the phospholipid molecules. However, the dihydroxyacetone phosphate acyltransferase system, the presence of which was reported in Saccharomyces carlsbergensis (26), has been shown to have quite similar specificity for acyl-CoAs to that of the glycerophosphate acyltransferase system in rat liver (27), and evidence was presented recently to suggest that the acylations of
128’ 24 4 2
in the text activity.
dihydroxyacetone phosphate and glycerophosphate were catalyzed by the same enzyme in rat fat cells (28,. Other factors such as the availability of acyl donors and the acceptor concentrations (29-31) must also be taken into account to correlate these acyltransferase systems with the fatty acid patterns of the membrane lipids. The activities for the reacylation system (e.g., acylation of monoacyl-GPC) were comparable to those for the de nouo synthetic system (acylation of glycerophosphate and monoacyl-GP) in S. cerevisiae as in rat liver cells, while E. coli membranes with relatively high de nouo synthetic activity showed very low reacylation activity (31). In a protozoan, Tetrahymena pyriformis, the activity for the acylation of 1-acyl-GPC was comparable to the de nouo synthetic activity, whereas the 2-acyl-GPC acyltransferase activity was very low (32). Thus, the relative importance of various acyltransferase systems in membrane lipid synthesis may be different in different kinds of cells. In rat liver, the major site of phospholipid synthesis is in the microsomal fraction. The acyltransferase systems involved in phospholipid synthesis are found mainly in the microsomes (33), although glycerophosphate acyltransferase is also present in mitochondria. In Tetrahymena, acyltransferase activities in the mitochondrial fraction are also very low as reported earlier (32). However, the mitochondrial fraction from yeast cells had been characterized as the major site of phospholipid
288
YAMADA
synthesis (25, 34). In S. carlsbergensis, the glycerophosphate acyltransferase system was reported to be equally distributed between the mitochondria and the mitochondria-free supernatant fractions, although the obtained subcellular fractions were not sufficiently characterized (26). The present experiments revealed that the acyltransferase systems are localized mainly in the microsomal fraction, although very low glycerophosphate and 1-acyl-GP acyltransferase activities are also in the mitochondrial fraction. Preliminary experiments showed that the promitochondrial fraction (35) from the cells grown anaerobically also had the l-acyl-GP acyltransferase activity, although the specific activity was much lower. These observations are apparently inconsistent with a current concept that the mitochondrial fraction is the major site of phospholipid synthesis in yeast (34). Further investigations are necessary to evaluate the relative contributions of the mitochondrial and microsomal fractions to the phospholipid synthesis in yeast. Recently, the effectiveness of various fatty acids in supporting growth and respiration in S. cereuisiae was determined by Walenga and Lands (36, 37). It would be interesting to see whether the specificities of these acyltransferase systems can be correlated with those observations. ACKNOWLEDGMENTS The authors are indebted to Dr. Takashi Institute of Applied Microbiology, University kyo, Tokyo, Japan for his helpful advices subcellular fractionation of yeast membranes
Miura, of Toin the
REFERENCES 1. ERWIN, J. A. (1973) in Lipids and Biomembranes of Eukaryotic Microorganisms (Erwin, J. A., ed.1, p. 42, Academic Press, New York. 2. JAKOVCIC, S., GETZ, G. S., RABINOWITZ, M., JAKOB, H., AND SWIFT, H. (1971) J. Cell BiOl. 48, 490. 3. MEYER, F., AND BLOCH, K. (1963) J. Biol. Chem. 238, 2654. 4. GETZ, G. S. (1972) 1~ Membrane Molecular Biology (Fox, C. F., and Keith, A., eds.1 p. 386, Sinauer Associate, Stanford. 5. KEITH, A. D., RESNICK, M. R., ANDHALEY, A. B. (19691 J. Bacterial. 98. 415. 6. KEITH, A. D. (19731 in Lipids and Biomem-
ET
7. 8. 9. 10.
11. 12.
13. 14. 15.
16.
17. 18. 19.
AL. ‘_ branes of Eukaryotic Microorganisms (Erwm, J. A., ed.1, p. 259, Academic Press, New York. SCHWEIZER, S., AND BOLLINC, H. (19701 Proc,. Nat. Acad. SC;. USA 67, 660. KUHN, N. J., AND LYNEN, F. (1972)Bzochcm. .I. 94, 240. STEINER, M. R., AND LESTER. R. L. (1972) B&him. Biophys. Acta 260, 222. HILL, E. E., AND LANDS, W. E. M. (1970) in Lipid Metabolism (Wakil, S. tJ.. ed.1, p. 185, Academic Press, New York. SEUBERT, W. (1960) Biochem. Prep. 7, 80. OKUYAMA, H.. LANDS, W. E. M., CHRISTIE, W. W., AND GUNSTONE, F. D. (19691 J. Biol. Chem. 244, 6514. CHANG, Y., AND KENNEDY, E. P. (19671J. Lipid Res. 8, 447. OKUYAMA, H., EIBL, H., AND LANDS, W. E. M. (1971) Biochim. Biophys. Acta 248, 263. OKUYAMA, H., LANDS, W. E. M., GUNSTONE, F. D., AND BARVE, J. A. (19721 Biochematry 11. 4392. ISHIHARA, H., OKUYAMA, H., IKEZAWA, H., AND TEJIMA, S. (1975lBiochzm. Biophys. Acta 388, 413. KANEKO, T., KITAMKJRA, K.. AND YAMAMOTO, Y. (1973) J. Agw. Binl. Chem. 37, 2295. OKUYAMA, H., AND WAKIL, S. J. (1973) J. Biol. Chem. 248, 5197. LANDS, W. E. M., AND HART, P. (1965) J. Btol. Chum. 240, 1905.
20. SCHATZ, phys. 21. YOSHIDA,
G., AND
Acta
KLIMA,
d. (1964)
Bmchim,
BLO-
81. 448. KUMAOKA,
H.. ANDSATO, R. (19741 75, 1201. KING, T. E. (1963) J. Biol. Chem. 238, 4037. LOWRY, 0. H., ROSEBROUGH, N. J., FARR. A. L., AND RANDALL. R. J. (1951 i J. Biot. Chem. 193, 265. RAY, T. K., CRONAN, J. E., JR.. MAVIS, R. D., AND VAGELOS. P. R. (1970) J. Biol. Chem. 21.5, 6442. COBON, G. S., CROWFOOT, P. D., AND LINNANE, A. W. (1974) Biochena. .I. 141, 265. JOHNSTON, J. M., AND PALTAUF, F. (19701 B&-him. Biophys. Acta 218, 431. HAJRA, A. K. (1968lJ. Biol. Chem. 243, 3458. SCHLOSSMAN, D. M., AND BELL, R. M. (19761 -J. Biol. Chem. 251, 5738. OKUYAMA, H., AND LANDS. W. E. M. (19721 J. Biol. Chem. 217, 1414. OKUYAMA, H., YAMADA, K.. AND TKEZAWA, H. (1974) J. Biol. Chem. 250, 1710. OKUYAMA, H., YAMADA, K., IKEZAWA, H., AND WAKIL, S. J. (197615. Biol. Chum 251, 2487. OKUYAMA, H., YAMADA, K., KAMEYAMA, Y., Y..
cl. Biochem.
22. 23.
24.
25. 26. 27. 28. 29. 30. 31. 32.
IKEZAWA,
H..
Y. (1977lArch.
FUKUSHIMA,
Brochem.
H.
Biophvs.
AND
NOZAWA.
178, 319.
ACYLTRANSFERASE
SYSTEMS
H., HILL, E. E., AND LANDS, W. E. M. (1969)Eur.J.Biochem. 9, 250. 34. MANCNALL, D., AND GETZ, G. S. (1973) in Lipids and Biomembranes of Eukaryotic Microorganisms (Erwin, J. A., ed.1, p. 146, Academic Press, New York. 33.
EIBL,
35.
IN
289
S. CEREVISIAE
CRIDDLE,
R. S., AND
8, 322. 36. WALENGA. R. Bml. (‘hem. 37. WALENGA. R. Rid Chem.
SCHATZ,
G. (1969)Bioc~hrm
istry
W.,
AND
LANDS.
250, 9121. W.. AND LANDS. 2.50. 9130
W. E. .M. (19751./
W. E. M. I 197% ,/.
OF
BIOCHEMISTRY
Acyltransferase
KAZUYO
AND
183, 281-289
BIOPHYSICS
Systems Involved Saccharomyces YAMADA,
(19771
in Phospholipid cerevisiae
HARUMI OKUYAMA,’ HIROH IKEZAWA
Metabolism
,YOSHIKO
in
ENDO,
AND
Faculty
of Pharmaceutical
Sciences,
Nagoya
City
Received
Crn~vcrsity, March
3-l
Tanabedori,
Mizuho-ku,
Nagoya,
Japan
7, 1977
Membrane preparations from Saccharo7nyce.s cerevisrae OC-2 catalyzed the acylation of glycerophosphate, l-acyl and 2-acyl isomers of monoacylglycerophosphate, and l-acyl and 2-acyl isomers of monoacylglycerylphosphorylcholine. The acyl-CoA:glycerophosphate acyltransferase system (EC 2.3.1.15) showed a broad specificity for acyl-CoAs when the maximal velocities were compared under optimized conditions. The acylCoA:2-acylglycerophosphate acyltransferase activity was much lower than the l-acylglycerophosphate acyltransferase activity. Although the 1-acylglycerophosphate acyltransferase system utilized saturated and unsaturated acyl-CoAs at comparable rates, the acylations at the l- and 2-positions were relatively more selective for palmitate and oleate, respectively, when assayed in the presence of palmitoyl-CoA, oleoyl-CoA, lacylglycerophosphate, and 2-acylglycerophosphate. The acyl-CoA:l-acylglyceryl-phosphorylcholine acyltransferase system (EC 2.3.1.23) was relatively more specific for unsaturated acyl-CoAs, while the acyl-CoA:2-acylglycerylphosphorylcholine acyltransferase system (EC 2.3.1.23) utilized both palmitoyl-CoA and oleoyl-CoA at a comparable rate. Although various acyltransferase systems showed a different degree of specificity for acyl-CoAs, the positional distribution of fatty acids in the phospholipid molecules could not be explained simply by the observed specificities. Zymolyase, P-1,3-glucanase from Arthrobacter luteus, was used successfully for the protoplast formation. Subcellular fractionation of the protoplast revealed that these acyltransferase activities were localized mainly in the microsomal fraction. However, the glycerophosphate and l-acylglycerophosphate acyltranferase activities in the mitochondrial fraction could not be explained by the contamination of microsomes in this fraction. These observations are apparently inconsistent with a current concept that the mitochondrial fraction is the major site of phospholipid synthesis m yeast
In yeast, the fatty acid patterns of membrane phospholipids as well as the phospholipid composition were significantly different between the aerobically and anaerobically grown cells (l-3). The changes in the phospholipid composition have also been studied in relation to the biogenesis of mitochondria (4). Mutants defective in the syntheses of saturated and unsaturated fatty acids provided excellent systems in which to study the importance of the fatty acyl moiety of phospholipids in the maintenance of proper membrane fluidity (5-7). However, the mechanisms determining the fatty acid pattern of mem’ To whom
correspondence
should
brane phospholipids in yeast have not been fully elucidated. Kuhn and Lynen showed that sn-glycerol3-phosphate (glycerophosphate) was acylated by a yeast membrane preparation to form monoacylsn-glycerol 3-phosphate (monoacyl-GP)” and that acyl-CoA was the obligatory acyl donor (8). Steiner and Lester revealed the major biosynthetic routes of phospholipids, which were similar to those in animal cells (9). In this paper, we describe some features of the acyltransferase systems involved in y Abbreviations used: acyl-GP, acyl-sn-glycerol phosphate: acyl-GPC. acyl-sn-glycerol Y-phosphorylcholine.
be addressed. 281
Copyright All
rights
0 1977 by Academic Press of reproduction in any fzrr.
Inc. reserved.
ISSN
0003-9861
3-
282
YAMADA
diacyl-sn-glycerol3-phosphate (diacyl-GP) synthesis and the presence of acyltransferase systems for the reacylations of monoacyl-sn-glycerylphosphorylcholine (monoacyl-GPC) (10) in a eucaryotic microorganism, Saccharomyces cerevisiae OC-2. MATERIALS
AND
METHODS
I l-‘ACIPalmitic acid and [9,10-:1Hloleic acid were purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo). CoA and NADPH were obtained from Kyowa Hakko Co. (Tokyo). Bovine serum albumin, fraction V (fatty acid-poor), was a product of Miles Laboratories, Inc. (Kankakee, Illinois). Antimycin A and cytochrome c (horse heart) were the products of Boehringer Mannheim (Germany). Zymolyase (/% 1,3-glucanase from Arthrobacter luteus) was obtained from Kirin Brewery Co. Ltd. (Tokyo). AcylCoAs were prepared by a modification of Seubert’s procedure (11) as described previously (12). [2“HIGlycerol 3-phosphate was prepared from [Z“Hlglycerol (Daiichi Pure Chemicals Co. Ltd., Tokyo) according to the method of Chang and Kennedy (13). Preparations of 1-acyl-GP and isomeric monoacyl-GPC were described elsewhere (14, 15). 2-AcylGP was prepared by the hydrolysis of diacyl-GP with Rhizopus lipase (Seikagaku Kogyo Co. Ltd., Tokyo) as described elsewhere (16). The isomeric purity was at least 85%. These monoacylglycerophospholipids were derived from egg yolk phosphatidylcholine or beef heart choline plasmalogen and retained the original fatty acids at the l-position (mainly palmitate and stearate) or the a-position (mainly oleate and linoleate). Preparation ofyeast membranes. A yeast, Saccharomyces cerecisiae OC-2 (kindly supplied by Dr. E. Ichishima, Tokyo Noko University, Tokyo) was grown aerobically at 30°C in a medium containing 1% yeast extract (Kyokuto Seiyaku Co., Tokyo), 1% polypeptone (Daigo Eiyo Co., Osaka) and 0.8% galactose (pH 5.6). Cells were harvested at the midexponential phase. For the characterization of the acyltransferase systems, the membrane preparations were obtained routinely as follows. Five grams of wet cells were ground with about 100 g of Al,O:, (Katayama Chemicals Co., Ltd., Osaka) and then 50 ml of 2 mM EDTA-0.3 M mannitol (pH 7.5) was added to the mixture. After centrifugation at 6OOg for 10 min, the cell pellets and Al,O,, were ground again with an additional 100 g of Al,Ozi and the homogenate was extracted with 50 ml of the same solution. The combined extracts were centrifuged at 1500g for 10 min. The centrifugations of the supernatant were further carried out at 15,OOOg for 15 min, 28,000g for 30 min, and finally at 100,OOOg for 60 min to obtain the high-speed pellet fraction. The pellet was suspended in a small volume of 2 mM EDTA-0.3 M mannitol (pH 7.5) (5-15 mg of protein/
ET
AL
ml). The suspension was divided into several tubes and stored at -70°C. The acyltransferase activities were not lost significantly after 2 months of storage under these conditions. Since the final high-speed pellet had the highest specific activities for oleoylCoA:l-acyl-GP and oleoyl-CoA:l-acyl-GPC acyltransfer reactions, this fraction was used in most of experiments unless otherwise described. For the determination of the subcellular distribution of the acyltransferase systems, the cell walls were disrupted enzymatically by using Zymolyase (17). Cells harvested at mid-exponential growth phase were washed with water and immediately subjected to protoplast formation. Typically, 25 g of wet cells was suspended in 125 ml of 20 mM Tris-HCl (pH 7.5) containing 0.65 M sorbitol and 5 rnM EDTA (SET buffer). Then 1.25 ml of cysteamine and 75 mg of Zymolyase were added successively. The incubation was carried out at 30°C. Each 50 ~1 of the incubation mixture was diluted with 5 ml of cold water and the turbidity at 600 nm was followed. The incubation was terminated when the turbidity decreased to lo-20% of the initial value. The protoplast thus formed was sedimented by centrifugation at 27,000g for 10 min. The cell pellet was suspended in 125 ml of SET buffer and homogenized by using a glass homogenizer with a Teflon pestle. The homogenate was centrifuged at 600g for 5 min and the sedimented unbroken cells were suspended in 65 ml of SET buffer. The homogenization and centrifugation were repeated. The combined cell extracts were centrifuged at 3000g for 5 min and the pellets were discarded. The supernatant was centrifuged at 17,000g for 10 min to obtain the crude mitochondrial fraction. This fraction was resuspended in SET buffer and sedimented again by centrifugation at 17,OOOg for 10 min to obtain the mitochondrial fraction. Further centrifugations of the supernatant at 20,OOOg for 20 min, 48,000g for 30 min, and 100,OOOg for 90 min yielded the intermediate, microsomes 1, and microsomes 2 fractions, respectively. These pellets were suspended in a small volume of SET buffer and stored at -70°C. Assay systems. The acyl-CoA:glycerophosphate acyltransferase activity was determined by following the incorporation of [“Hlglycerophosphate into the lipid fraction as described previously (18). The assay mixture contained 3 rnM glycerophosphate (1000 cpminmol), 20 PM acyl-CoA, 0.2 mgiml of albumin, 0.2 M KCl, and 0.1-0.4 mg/ml of enzyme protein in 0.1 M Tris-HCl (pH 7.2). The incubations were carried out at 23-25°C for 1 min. The acylations of monoacyl-GP and monoacylGPC were routinely followed spectrophotometrically (19). The assay mixture contained 50 FM l-acyl-GP or 100 pM 1-acyl-GPC, lo-20 KM acyl-CoA, 1 mM 5,5’-dithiobis-(2-nitrobenzoic acid), and 0.05-0.3 mg/ ml of enzyme protein in 0.08 M Tris-HCl (pH 7.5). Reactions were started by adding acyl-CoA after 1
ACYLTRANSFERASE
SYSTEMS
min of preincubation. Experiments with labeled acyl-CoAs were carried out without 5,5’-dithiobis(2-nitrobenzoic acid). Antimycin-insensitive NADPH:cytochrome c reductase was measured spectrophotometrically (20, 21). One milliliter,of the incubation mixture contained 30 pmol of phosphate buffer (pH 7.4), 70 nmol of cytochrome c, 2 Fmol of KCN, 5 ~1 of an ethanolic solution of antimycin A 12 mgiml), and the enzyme protein. The reactions were started by adding 0.6 smol of NADPH. The increases in the absorbance at 550 nm were recorded continuously. Succinate dehydrogenase was measured according to the method of King (22). Protein was determined by the method of Lowry et al. (23) with bovine serum albumin as a standard. All figures presented are the averages of at least two separate determinations. Positional distribution of fatty acids ln glycerophospholipids. Lipids were extracted according to the method of Jacovcic et al. (2) from the cells grown aerobically at 30°C and harvested at the late exponential growth phase. Phospholipids were separated by Silica Gel H thin-layer chromatography with chloroform:methanol:l4% NH,OH (65:40:4, v/v/v) as a solvent. Individual phospholipid was extracted from the silica gel with chloroform:methanol (1:2, vi v) and then with chloroform:methanol:3% NH,OH (6:5:1, v/v/v). Solvents were evaporated with a rotary evaporator and the residues dissolved in a small volume of chloroform:methanol (1:2, v/vi were applied to a Silica Gel H thin-layer chromatogram with chloroform:methanol:water (65:25:4, v/v/v) as a solvent to obtain chromatographically pure ethanolamine phospholipid, phosphatidyl choline, and phosphatidyl inositol. The ethanolamine phospho-
TABLE
IN
283
S. CEREVISIAE
lipid fraction includes phosphatidyl ethanolamine and N-methylated phosphatidyl ethanolamine (91. About 2 pmol of ethanolamine phospholipid in 1 ml of diethyl ether was incubated at 37°C with 1 ml of phospholipase A, solution containing 5 mgiml of Crotaku adamanteus venom in 4 mM CaCIZ-O.I M Tris-HCl \pH 7.5). The hydrolysis was complete in 1 h. Phosphatidyl choline was hydrolyzed with 0.1 ml of enzyme solution in 0.5 h, and phosphatidyl inosito1 was hydrolyzed with 1 ml of enzyme solution in 2 h. The hydrolysis products were separated by Silica Gel H thin-layer chromatography with chloroform:methanol:water (65:25:4, v/v/vi as a solvent. The liberated fatty acids and the fatty acids from the lysophospholipids were analyzed as methyl esters by a gas-liquid chromatograph with 20% EGSSX on Gas Chrom W (Hitachi KGL 2Ai. RESULTS
Glycerophosphate
Acyltransferase
System
The effect of varying acceptor concentration on the glycerophosphate acyltransferase system is shown in Table I. The K,,, value for glycerophosphate obtained by a double reciprocal plot was in a range of 1.5 to 1.8 mM, which was lower than the reported value (10 mM) (8). MgCl, was added to the glycerophosphate acyltransferase system of Escherichia coli (24). However, no appreciable stimulatory effect was observed in the yeast system. KCl, which was shown to be stimulatory in an Escherichia coli system (18), also stimulated the I
EFFECT OF SUBSTRATE CONCENTRATIONS ON ACYLTRANSFERASE SYSTEM" Percentage
activity
Glycerophosphate acyltransferase, glycerophosphate concentration (mMi
100-95 80 60 40 20 Concentrations
for typical
assays
l-Acyl-GP 1-Acyl-GP concentration (p*M)
2.6-4.3’ 1.9, 4.9 1.2 0.6 0.3
30-100 13 8 6 3
3
50
acyltransferaseb OleoylCoA concentration (PM) 5-15 3. 22 1.6 0.8 0.4. 10
l-AcylGPC
acyltransferase”
1-AcylGPC concentration (@I) 60-200 35 18 7 5 100
OleoylCoA concentration (PM) 8-40 6 4 2.5 1.0 20
n Figures were taken from the best fit to the curves obtained and were arranged sothat one can reproduce the original curves from the percentage activity at the given substrate concentrations. D High-speed pellet was used as the enzyme source. The palmitoyl-CoA:glycerophosphate, oleoyl-CoA:lacyl-GP, and oleoyl-CoA:l-acyl-GPC acyl transfer reactions showed 12, 45, and 15 nmoliminimg of protein at 25”C, respectively. ’ Glycerophosphate concentrations of 2.6-4.3 mM gave 100-951; of the maximum activity.
284
YAMADA
acylation of glycerophosphate about 2.5fold at a concentration of 0.2 M (Fig. 1). When the time course of the acylation of glycerophosphate was followed in the presence of palmitoyl-CoA or oleoyl-CoA, a significant amount of monoacyl-GP was detected with palmitoyl-CoA as acyl donor, whereas the amount of monoacyl-GP was very low with oleoyl-CoA. Attempts to accumulate monoacyl-GP with oleoyl-CoA have been unsuccessful. The reactions in the presence of 30 PM acyl-CoA were linear for about 5 min at protein concentrations varying between 0.05 and 0.4 mglml. The specificity for acyl-CoAs of the glycerophosphate acyltransferase system was examined at 3 acyl-CoA concentrations (Table II). Saturated acyl-CoAs as well as unsaturated acyl-CoAs were transferred at comparable rates. Acyl-GP
Acyltransferase
System
The 1-acyl-GP acyltransferase system was saturated at much lower concentrations of acyl donor and acyl acceptor than those described for the glycerophosphate acyltransferase system (Tables I and II). In a typical assay system, the reactions were linear for about 2 min with protein concentrations varying between 0.05 and
$T
AL TABLE
II
SPECIFICITY OF GLYCEROPHOSPHATE ACYLTRANSFERASE~
Acyl-CoA
14:o 16:0 18:O cis-A”-16:1 cis-Ay-18:1 cis-A”-18:l trans.A!‘-18:1
Acyl transfer rates (nmol/min/ mg of protein) at acyl-CoA concentrations (PM) of 10
20
30
5.0 13.5 5.2 11.0 8.5 7.7 7.8
8.6 16.4 9.7 19.0 13.3 10.R 7.7
7.2 18.0 6.6 20.0 12.3 8.2 6.5
a The incubation mixture consisted of 0.4 mgiml of enzyme protein, 3 mM 1”Hlglycerophosphate (1000 cpminmol), 0.2 M KCl, 0.2 mg/ml of bovine serum albumin, and the indicated amounts of acyl-CoA in 0.1 M Tris-HCl (pH 7.2). The combined microsomes 1 and 2 fractions obtained after Zymolyase treatment were used as the enzyme source. Incorporation of glycerophosphate into total lipid fraction was followed. The control value for zero-time incubation (0.2 nmoliminimg of protein) has been subtracted to give net acyl transfer rates. TABLE
III
SPECIFICITIES OF I-ACYL-GP AND ACYLTRANSFERASES FOR VARIOUS
Acyl-CoA
Acyl transfer rates minimg of protein) 1 -Acyl-GP
16:0 18:O cis-A!‘-16:1 cis-A!‘-18:l cis-A”-18:l trans-~!‘-18:1
15 F -2 .E 5 E
10
cis-~!‘.“-1!3:2 5 0
0'
KCI,M 0
25
Mg Cl,
50
75
100
, mM
FIG. 1. Effects of KC1 ( - 0 - i and MgCl, (- 0 - 1 on the glycerophosphate acyltransferase system. The basic incubation mixture consisted of 3 mM 1”Hlglycerophosphate (1000 cpminmol), 30 @M acylCoA, 0.2 mg/ml of enzyme protein (high-speed pellet), and 0.1 M Tris-HCl (pH 7.2). When the effect of MgCl, was examined, 0.2 M KC1 was present in the incubation mixture.
l-ACYL-GPC ACYL-CoAs”
25.6 13.4 51.3 41.1 27.6 32.6 34.1
(1.91 (1.61 (3.11 (2.71 (3.1 I (2.91 (4.51
(nmoli with 1-AcylGPC 5.5 4.9
24.0 11.8 6.2 15.2 7.0
” High-speed pellet (0.3 mg proteinimli was used in the system described in the text. Reactions were continuously followed spectrophotometrically. The molecular extinction coefficient of 13,600 was used for the reaction product of CoA and 5,5’-dithiobis-(Znitrobenzoic acid) (19). Figures are averages of two separate determinations. Control values without acceptor, which are shown in parentheses, have been subtracted to give net acyl transfer rates.
0.3 mglml. The enzyme system showed a broad pH optimum between 7.0 and 9.0. The specificity for acyl-CoAs of the l-acylGP acyltransferase system is shown in Table III. This acyltransferase system
ACYLTRANSFERASE
SYSTEMS
showed very broad specificity for acylCoAs as described for the glycerophosphate acyltransferase system. PalmitoylCoA as well as oleoyl-CoA was incorporated at a relatively high rate. These enzyme systems do not seem to discriminate strictly the cis and trans configurations or the position of double bond in the fatty acyl moiety. When 2-acyl-GP was the acceptor, the rate of CoA liberation was much lower than with 1-acyl-GP. Furthermore, most of the radioactivity incorporated was found at the 2-position of diacyl-GP. This phenomenon, which had been observed with rat liver microsomes or E. coli preparations (14, 181, was understood to indicate that the contaminating 1-acyl-GP in the 2acyl-GP preparation was preferentially acylated under the conditions. The 2-acyl-GP acyltransferase activities could be measured more accurately by using labeled acyl-CoA and correcting the radioactivity incorporated into the individual position of diacyl-GP. The 2-acyl-GP acyltransferase activities thus determined were 2.8 and 1.6 nmol/min/mg of protein with palmitoylCoA and oleoyl-CoA, respectively, which were much lower than the 1-acyl-GP acyltransferase activities (Table III). In the next experiments, the relative acyl transfer rates were measured in the presence of saturating amounts of four substrates, [‘JClpalmitoyl-CoA, [:‘H]oleoyl-CoA, 1-acyl-GP, and 2-acyl-GP (Table IV). With the four donor:acceptor comTABLE ACYLATION
OF A MIXTURE
2-ACYL-GP
IN THE
PRESENCE
PALMITOYL-COA
Fatty
acids
OF 1-ACYL-GP
OF A MIXTURE
AND
position 0.9 2.5 0.4
AND (I:11
OF
OLEOYL-COA”
Incorporation (nmol/min/mg C-l
:‘H-l&I ’ ‘C-16:0 “H,‘tC
IV (1:l)
of fatty acids of protein) into the C-2 position 23.1 9.0 2.6
’ The incubation mixture contained 40 nmol of lacyl-GP, 40 nmol of 2-acyl-GP. 10 nmol of [:xH]oleoylCoA (7000 cpminmol), 10 nmol of I”C]palmitoyl-CoA (8000 cpminmol), and 0.1 mg of enzyme protein (high-speed pellet) in 1 ml of 0.09 M Tris-HCl (pH 7.5). Values are averages of two separate determinations.
IN
285
S. CEREVISIAE
binations, the acyl transfer rates determined under the conditions were in the following order: oleoyl-CoA:l-akyl-GP > palmitoyl-CoA:l-acyl-GP > palmitoylCoA:2-acyl-GP > oleoyl-CoA:2-acyl-GP. The acylation of 2-acyl-GP (C-l position) with palmitoyl-CoA and oleoyl-CoA was less than ‘1sof that determined for l-acylGP (C-2 position). The ratios of oleate to palmitate incorporated were approximately 112.5 and 2.6 for the acylations at the l- and the 2-positions of isomeric monoacyl-GP, respectively. The selectivity for oleate at t,he 2-position and that for palmitate at the l-position observed under the conditions are significantly higher than those expected from the ratios of the maximal velocities with oleoyl-CoA and palmitoyl-CoA shown in Table III. Acyl-GPC
Acyltransferase
System
The effect of substrate concentration on the 1-acyl-GPC acyltransferase is summarized in Table I. The reactions were linear for about 2 min at protein concentrations varying between 0.05 and 0.3 mg/ml. The enzyme showed a broad pH optimum within the range of 7.0 to 9.0. Under these conditions, the 1-acyl-GPC acyltransferase system showed the specificity shown in Table III. Palmitoleoyl-CoA gave the highest rate among the acyl-CoAs examined. When the major fatty acids of the phospholipids (16:0, 16:1, and 18:l) were compared, saturated fatty acyl-CoA showed a relatively lower maximal velocity than unsaturated acyl-CoAs. The specificity for various acyl-CoAs of the 1-acyl-GPC acyltransferase system was not significantly different at pH 7.3 and 8.5 (data not shown). 2-Acyl-GPC was examined as an acceptor of the acyltransferase system (Fig. 2). The enzyme system became saturated at 15 PM acyl-CoA. Palmitoyl-CoA appeared to be less effective as an acyl donor than oleoyl-CoA in the acylation of 2-acyl-GPC, although palmitate was transferred into 2acyl-GPC at a higher rate than into l-acylGPC. Rat liver microsomes showed 34 and 11 nmol/min/mg of protein for the palmitoyl-CoA:2-acyl-GPC and oleoyl-CoA:lacyl-GPC acyl transfer reactions, respectively, as reported previously (12, 15).
286
YAMADA
Positional Distribution Phospholipids
of Fatty
Acids
in
The content of saturated fatty acids in the phospholipids increased in the order of ethanolamine phospholipid, phosphatidyl choline, and phosphatidyl inositol (Table VI. The stearate content also increased in this order, although palmitate was the major saturated fatty acid in these phospholipids. In general, the 2-position was occupied mainly with unsaturated fatty acids while the saturated fatty acids were localized in the l-position of the phospholipids. Among the unsaturated fatty acids, more palmitoleate (16:l) was found at the l-positions of ethanolamine phospholipid and phosphatidyl choline while phosphatidylinositol contained a greater amount of this fatty acid at the 2-position. Oleate was
15.
--
0'
50 100 Z-acyl-GPC , p M
0
FIG. 2. 2-Acyl-GPC acyltransferase activity. The activities for palmitoyl-CoA ( - 0 - 0 ~ i or oleoylCoA (-0-O-i are shown as a function of the acceptor concentrations. The incubation mixture consisted of 0.2 mgiml of enzyme protein (high speed pellet), 20 PM acyl-CoA. 0.18 M Tris-HCl (pH 8.91, and the indicated concentrations of Z-acyl-GPC.
ET
the major fatty acid at the 2-position of all the phospholipids examined. Thus, the localization of the individual fatty acid in the phospholipid molecules is a typical one found in rat liver phospholipids, except palmitoleate (16:l) which was found mainly at the l-position or at the 2-position depending upon the phospholipid classes. Other minor phospholipids such as cardiolipin and phosphatidyl serine have not been examined. Subcellular uses
DISTRIBUTION
OF FATTY
Ethanolamine
ACIDS
IN
Phosphatidyl
FROM
S. cereuisiae
CELLS
Total
C-l
c-2
Total
- C-l
c-2
Total
9” 41 tr.’
14 75 tr.
18:l
41
3 25 tr. 66
11 54 3 21
23 53 9 12
4 36 1 55
47 15 10 23
” Only the major components are listed. * Figures are expressed as percentage of fatty ” Trace amount.
acids
of intact
GROWN
Phosphatidyl
choline
16:0 16:l 18:0
I
of Acyltransfer-
V
PHOSPHOLIPIDS
phospholipid
Distribution
Subcellular membrane fractions were obtained from the cells treated with Zymolyase as described under Materials and Methods. The recovery of protein and the acyltransferase activities in these subcellular fractions are shown together with the distributions of some marker enzymes in Table VI. From the distribution of marker enzymes, the pellets obtained by centrifugations at 17,000g for 10 min, 20,OOOgfor 20 min, 48,000g for 30 min, and 100,OOOg for 90 min were tentatively designated as mitochondria, intermediate, microsomes 1, and microsomes 2 fractions, respectively (25). The distribution of the 1-acyl-GPC acyltransferase system roughly paralleled the antimycin-insensitive NADPH:cytochrome c reductase, while the glycerophosphate and 1-acyl-GP acyltransferase activities in the mitochondrial fraction could not be explained by the contamination of microsomes in this fraction. Thus, the acyltransferase systems involved in diacyl-GP synthesis as well as the one involved in the reacylation system (acylCoA:lysophospho!ipid acyltransferase sys-
TABLE POSITIONAL
AL
lipids
or of the indicated
AT 30°C
inositol C-l 51 I 23 9 positions.
c-2 3 24 1 70
ACYLTRANSFERASE
SYSTEMS TABLE
SUBCELLULAR
Protein*
Fraction”
Mitochondrial Intermediate Microsomal
1
64 53 30
Microsomal
2
20
Glycerophosphate acyltransferase, palmitoylCoA’ 1.0” 10.0 17.6 9.9
DISTRIBUTION
I-Acyl-GP acyltransferase, oleoylCoA’ 10 71 173 57
IN
287
S. CEREVZSIAE
VI OF ACYLTRANSFERASES
l-Acyl-GPC acyltransferase, oleoylCoA’ 2” 47 96 36
~~~~~
NADPH:cytochrome c reductase”
~~_~~~~~~-~~~
Succinate dehydrogenase”
4” 58 156 52
U Subcellular fractions obtained from the Zymolyase-treated cells were used (see text). h Milligrams of protein from 25 g of wet cells. r The amounts of enzyme protein for the acyl transfer assays were varied in a range described to obtain the initial velocities. “The l-ml incubation mixture contained 0.1 to 0.2 mg ofenzyme protein. Antimycin-insensitive * nmol/min/mg of protein.
tern) are localized mainly in the microsomal fraction, but diacyl-GP synthesizing activity is also present in the mitochondrial fraction although the activity in this fraction is much lower. DISCUSSION
Both saturated and unsaturated fatty acids were found at the l- and 2-positions of phospholipids. The ratio of unsaturated fatty acids to saturated fatty acids ranged from 0.2 to 4.5 at the l-position and from 16 to 24 at the 2-position of the phospholipids (Table V). The specificities of the l-acylGP and l-acyl-GPC acyltransferase systems for various acyl-CoAs (as compared with the respective maximal velocities) were much lower than those expected from the fatty acid composition at the 2-position of phospholipids. The specificities for acylCoAs of the glycerophosphate, 2-acyl-GP, and 2-acyl-GPC acyltransferase systems do not directly explain the fatty acid composition at the l-position of phospholipids, either. It might be that other enzyme systems which have not been examined play more important roles in the selective positioning of various fatty acids in the phospholipid molecules. However, the dihydroxyacetone phosphate acyltransferase system, the presence of which was reported in Saccharomyces carlsbergensis (26), has been shown to have quite similar specificity for acyl-CoAs to that of the glycerophosphate acyltransferase system in rat liver (27), and evidence was presented recently to suggest that the acylations of
128’ 24 4 2
in the text activity.
dihydroxyacetone phosphate and glycerophosphate were catalyzed by the same enzyme in rat fat cells (28,. Other factors such as the availability of acyl donors and the acceptor concentrations (29-31) must also be taken into account to correlate these acyltransferase systems with the fatty acid patterns of the membrane lipids. The activities for the reacylation system (e.g., acylation of monoacyl-GPC) were comparable to those for the de nouo synthetic system (acylation of glycerophosphate and monoacyl-GP) in S. cerevisiae as in rat liver cells, while E. coli membranes with relatively high de nouo synthetic activity showed very low reacylation activity (31). In a protozoan, Tetrahymena pyriformis, the activity for the acylation of 1-acyl-GPC was comparable to the de nouo synthetic activity, whereas the 2-acyl-GPC acyltransferase activity was very low (32). Thus, the relative importance of various acyltransferase systems in membrane lipid synthesis may be different in different kinds of cells. In rat liver, the major site of phospholipid synthesis is in the microsomal fraction. The acyltransferase systems involved in phospholipid synthesis are found mainly in the microsomes (33), although glycerophosphate acyltransferase is also present in mitochondria. In Tetrahymena, acyltransferase activities in the mitochondrial fraction are also very low as reported earlier (32). However, the mitochondrial fraction from yeast cells had been characterized as the major site of phospholipid
288
YAMADA
synthesis (25, 34). In S. carlsbergensis, the glycerophosphate acyltransferase system was reported to be equally distributed between the mitochondria and the mitochondria-free supernatant fractions, although the obtained subcellular fractions were not sufficiently characterized (26). The present experiments revealed that the acyltransferase systems are localized mainly in the microsomal fraction, although very low glycerophosphate and 1-acyl-GP acyltransferase activities are also in the mitochondrial fraction. Preliminary experiments showed that the promitochondrial fraction (35) from the cells grown anaerobically also had the l-acyl-GP acyltransferase activity, although the specific activity was much lower. These observations are apparently inconsistent with a current concept that the mitochondrial fraction is the major site of phospholipid synthesis in yeast (34). Further investigations are necessary to evaluate the relative contributions of the mitochondrial and microsomal fractions to the phospholipid synthesis in yeast. Recently, the effectiveness of various fatty acids in supporting growth and respiration in S. cereuisiae was determined by Walenga and Lands (36, 37). It would be interesting to see whether the specificities of these acyltransferase systems can be correlated with those observations. ACKNOWLEDGMENTS The authors are indebted to Dr. Takashi Institute of Applied Microbiology, University kyo, Tokyo, Japan for his helpful advices subcellular fractionation of yeast membranes
Miura, of Toin the
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W. E. M. I 197% ,/.
