78
Biochimica et Biophysics @ Elsevier/North-Holland
Acta, 530 (1978) 78-90 Biomedical Press
BBA 57208
TRIACYLGLYCEROL SYNTHESIS IN LIPID PARTICLES YEAST (SACCHAROMYCES CEREVZSZAE)
FROM BARER’S
KIRSTEN CHRISTIANSEN Department of Biochemistry C, University DK-2200 Copenhagen N (Denmark)
of Copenhagen,
Panum Institute,
Blegdamsvej
3,
(Received January 23rd, 1978)
Summary Triacylglycerol synthesis has been studied in a lipid particle preparation of baker’s yeast (S’accharomyces cereuisiue), and compared with the synthesis in other subcellular fractions. Fatty acid-CoA ligase (AMP) (EC 6.2.1.3) activity and sn-glycerol 3-phosphate acyltransferase activity (EC 2.3.1 .15) were present in all the subcellular fractions tested but the highest specific activities of both enzymes were observed with the lipid particle fraction. The products of the glycerol 3-phosphate acylation indicate that triacylglycerol synthesis proceeds through the phosphatidic acid pathway. However, only a small and nearly constant amount of lysophosphatidic acid was found with the lipid particle fraction while the other subcellular fraction produced lysophosphatidic and phosphatidic acid with a more pronounced precursor/product relationship. Triacylglycerol synthesis from endogenous diacylglycerol present in the lipid particle was also demonstrated.
Introduction Lipid particles have been described as a common structure in the yeast cell [l]. However, little is known about the biochemical basis for the formation and development of these intracellular particles in the course of cellular growth. In our laboratory, lipid particles have been isolated from yeast cells in the stationary phase of growth [2]. The isolated lipid particles are composed of 95% lipid and 5% protein. The lipid fraction of the particles consists of triacylglycerols (45-50%) and sterol esters (45-50%) together with minor amounts of phospholipids and free fatty acids [2]. Also about 2% diacylglycerol, but no monoacylglycerol is present in the lipid particle preparation. The endoplasmic reticulum is considered the site for triacylglycerol syn-
79
thesis, at least in mammalian cells. However, enzymes necessary for triacylglycerol synthesis have been reported also to be associated with triacylglycerolcontaining particles, e.g. in the so-called fat fraction of plant cells such as castor bean seeds [3] and Crambe seeds [ 41. In mammalian cells, the membranebound lipid particles of beef heart [5] and the lipid micelles of rat adipose tissue [6] have been reported to contain enzymes necessary for triacylglycerol synthesis. The purpose of the present report has been to study whether triacylglycerol-synthesizing enzymes also are associated with lipid particles in yeast cells. Materials and Methods Chemicals. Unlabelled lipids were purchased from Nu Chek Prep., Elysian, Minn., U.S.A. and from Serdary Research Laboratories, London, Canada. [1-“C]Oleic acid (spec. act. 54 Ci/mol) was purchased from The Radiochemical Centre, Amersham, England. sn-[ 2-3H] Glycerol 3-phosphate was prepared enzymatically from sn-[ 2-3H] glycerol (The Radiochemical Centre, Amersham, England) and purified as descibed by Chang and Kennedy [ 71, with the modifications proposed by Davidson and Stanacev [8]. The final product had a specific activity of 8.3 Ci/mol sn-glycerol 3-phosphate and was diluted appropriately with rat-glycerol 3-phosphate before use in enzyme assays. ATP (disodiurn) and coenzyme A were from Boehringer, Mannheim, W. Germany. Baker’s yeast (Saccharomyces cereukiae) was obtained by the courtesy of De Danske Spritfabrikker A/S, Copenhagen. Preparation of subcellular fractions. The yeast cells were washed and homogenized as previously described [2,9]. Whole cells, nuclei and cell debris were removed by centrifugation at 1000 X g for 10 min. The cytoplasmic extract containing cytosol plus intact and disrupted organelles [lo] was further centrifuged at 20 000 X g for 15 min, after which crude mitochondria were collected as a pellet and crude lipid particles as a top layer. The intermediate supernatant was centrifuged at 48 000 X g for 20 min, and at 85 000 X g for 90 min, whereby, respectively, the intermediate fraction and the microsomal fraction were obtained. The supernatant from the last centrifugation was designated the soluble supernatant. All stages were performed at 4°C. The mitochondrial fraction was extensively purified and only the heavy mitochondrial fraction was used in this study [5]. The purity of this fraction was evaluated by electron microscopy as described earlier [9]. The crude preparation of lipid particles was purified by flotation as described [2], except that 0.25 M sucrose was used instead of the mannitol medium. The purity of this fraction was also evaluated by electron microscopy [2]. The intermediate and the microsomal fractions were not purified further. The intermediate fraction appeared in the electron micrographs as a mixture of the membranous material of the cell including some intact mitochondria. The microsomal fraction appeared to consist mostly of polysomes with minor amounts of membranous material. The protein concentration of the fractions was measured by the method of Lowry et al. [ll] ,with crystalline bovine serum albumin as standard. For the lipid particles a modification for lipid-rich material was used [12]. RNA was
80
measured by the orcinol method as described by Schneider [13]. Marker enzyme activities were measured at 27”C, cytochrome oxidase activity as described by de Duve et al. [lo], NA~PH-cytochrome c reductase as described by Sottocasa et al. [14], except that rotenone was excluded from the assay and the subcellular fraction was diluted in Nonidet P-40 [ 151. The diacylglycerol of the total lipid extract [ 161 was isolated by preparative thin-layer chromatography on silica gel using light petroleum (b.p. 60-7O”C)/ diethyl ether/acetic acid (70 : 30 : 1, v/v). The appropriate band was scraped off the plate, and the diacylglycerol isolated by extraction with 2 X 3 ml chloroform/methanol (1 : 2, v/v). After evaporation of the solvent, the diacylglycerol content was determined after enzymatic hydrolysis and determination of glycerol as described by Wahlefeld [ 171, Dioleylglycerol in known amounts was chromatographed and analyzed in the same way as the unknown samples. Assay procedures for enzymes invoZued in tria~y~~lycero~ synthesis. Optimum conditions with respect to the cofactors and the substrates glycerol 3phosphate and the fatty acid (complexed to albumin in the ratio 4 : 1) for the sn-glycerol 3-phosphate acyltransferase activity with the lipid particle preparation as enzyme source were established in preliminary experiments. There was an absolute requirement for ATP and CoA and a 3-fold increase with 15 mM MgClz. Maximal activity was obtained at 5 mM sn-glycerol 3-phosphate and 0.4 mM fatty acid, respectively. ~-Mercaptoethanol was added routinely in order to protect SW-groups but had no influence on the measured enzymic activities. The following basic incubation system was used: 100 mM Tris * HCl (pH 7.4), 15 mM ATP, 15 mM MgC12, 1 mM CoA, 8 mM fl-mercaptoethanol and 0.4 mM oleic acid bound to 0.1 mM defatted bovine serum albumin (fraction V, Sigma) 1181. When added the concentration of glycerol 3-phosphate was 5 mM. The reactions were carried out in a shaking waterbath at 27°C. Acyl-CoA synthetase: The conversion of 0.4 gmol [ l-‘4C]oleate (spec. act. 0.64 Ci/mol) to [l-‘4C]oleyl-CoA was measured in a total reaction volume of 0.25 ml of the basic incubation system. The reaction was started by addition of protein (lo-40 pg), carried out for 4 min and stopped by addition of 1 ml Dole reagent [19] 0.35 ml water and 0.6 ml n-heptane. The extraction and scintillation counting of the product, acyl-CoA, was carried out as described by Lloyd-Davies and Brindley [20]. The specific activity was determined with two different concentrations of protein. The oleyl-CoA was identified by co-chromatography with authentic oleyl-CoA [Zl]. sn-Glycerol 3-phosphate acylation: The biosynthesis of tria~ylglycerol was determined by measuring the rate of incorporation of sn-[2-3H]glycerol 3-phosphate (spec. act. 0.10 Ci/mol) into lipids in a total reaction volume of 1 ml. The reaction was started by addition of 0.1 -0.3 mg protein and carried out for different time intervals or for 60 min. The reaction was terminated by adding 1.5 ml butanol followed by 2 ml water. The lipid was extracted as described by Daae [ 221. The butanol was removed under reduced pressure and the lipid dissolved in a known amount of chloroform. Aliquots were taken for counting total radioactivity and for chromatography. Neutral lipids were analyzed on silica gel-loaded paper (I.T.L.C. type SA from Gelman Instruments Co., Mich., U.S.A.) in light petroleum (b.p. 60-70”C)/diethyl ether/acetic acid (80 : 20 : 1, v/v or 65 : 45 : 1, v/v). Phospholipids were analyzed on silica gel F,,, pIates
81
in the two-dimensional thin-layer chromatography system described by Brotherus and Renkonen [23], after modifying the solvent system used in the first direction to chloroform/methanol/7 M ammonia (65 : 20 : 3.5, v/v). The lipid classes were detected by iodine and identified by comparing their Rf values with those of standards. The spots were either cut from paper or scraped from the plates and the radioactivity determined as described [21]. Oleate esterification with added glycerol 3-phosphate as acyl acceptor: Oleate esterification was measured by incubating 0.4 E.rmol [1-r4C]oleic acid (spec. act. 0.64 Ci/mol) with 5 pmol glycerol 3-phosphate in a total reaction volume of 1 ml. The reaction was started by addition of 0.1-0.3 mg protein, and carried out for different time intervals or 60 min. The reaction was stopped by addition of 20 ml chlorofo~/methanol (2 : 1, v/v) and the extraction carried out as recommended for incubations were both labelled oxoesters and thioesters are formed [21]. The separation and determination of radioactivity of individual lipids was as described above. Oleate esterification with endogenous acyl acceptor: [1-‘“C]Oleate was incubated under exactly the same conditions as stated above except that no glycerol 3-phosphate was added. The extraction of the incubation mixture and the radioactivity determ~ation was as described for incubations with labelled fatty acids [ 211. Hydrolysis of labelled triacylglycerol by pancreatic lipase. The triacylglycerol fraction from the 14C-labelled extracts was isolated by preparative thinlayer chromato~aphy on 0.5 mm silica gel G plates. The triacylglycerolcontaining band was localized and the compound extracted from the silica gel with 3 X 5 ml of diethyl ether. The diethyl ether was evaporated and the triacylglycerol treated with pancreatic lipase at 37°C for 5 min as described by Mattson and Volpenhein [ 241. The unhydrolyzed substrate and the hydrolysis products were separated by chromatography on silica gel-loaded glass fiber paper type SA from Gelman Products, in the solvent system light petroleum (b.p. 60-70”C)/diethyl ether/acetic acid (65 : 45 : 1, v/v). The bands were detected by IZ vapor and the radioactivity determined as described [21]. “C-labelling of endogenous 1,2-diacylglycerol in lipid particles and its use as substrate for 1,2-diacylglyceroltransferase. Purified lipid particles were incubated in the assay system for [ 14C]oleic acid acylation with added glycerol 3-phosphate as acyl acceptor as described above. The reaction was stopped by mixing the incubation mixture with an equal volume of ice-cold 50% (w/v) sucrose. The mixture was overlayered with ice-cold 0.25 M sucrose and centrifuged at 27 000 X g for 15 mm at 4°C. The top layer of labelled lipid particles was isolated by freezing the content of the centrifuge tube up to 1 cm below the lipid layer. The resuspended particles contained the labelled particle-bound diacylglycerol together with some phosphatidic acid and triacylglycerol (Table III). The labelled diacylglycerol was used as a substrate for studying diacylglycerol acyltransferase activity by incubating the prelabelled particles with unlabelled fatty acid, ATP, CoA, MgClz and P-mercaptoethanol in the concentrations stated for various time intervals.
82
Results Characterization of the lipid particle fraction The subcellular fractions were characterized both by electron microscopy and specific activities of marker enzymes. The results of Table I indicate that the lipid particles were contaminated with mitochondrial inner fragments to an extent of 6%. NADPH-cytochrome c reductase has been used as a marker enzyme for the microsomal fraction of baker’s yeast [25]. In our hands the specific activities of this enzyme in the different subcellular fractions did not show an exclusively microsomal origin, in agreement with the results from Saccharomyces Carkbergensis [ 261, Tetrahymena pyriformis [ 271, and from rat liver [28] and pig heart [29]. Thus, the low NADPH-cytochrome c reductase activity seen in the lipid particle fraction may be due to a small contamination probably with both endoplasmic reticulum and outer mitochondrial membranes. RNA could not be detected in the lipid particle fraction. The results obtained for marker enzymes and RNA content corroborate the results obtained with electron microscopy [2]. Thus, the lipid particle preparation appeared as a relatively pure fraction of particles with a homogeneous core surrounded with a thin layer of more heavily stained material, without any indications of a trilaminar membrane structure. Some heavily stained material is seen close to the particle surface or separate from the particles, which may be of either mitochondrial or microsomal origin, but whole mitochondria or ribosomes are never seen, Among the subcellular fractions tested the lipid particle fraction had the highest specific activities of the enzymes involved in lipid synthesis. An acyl-CoA synthetase activity of 142 nmol per mg of protein per min was obtained with the lipid particle fraction. This activity was 10 times higher than the activity obtained with any of the other subcellular fractions. Triacylglycerol synthesis from sn-glycerol3-phosphate occurred in the lipid particle fraction with a specific activity of 62 nmol glycerol 3-phosphate incorporated per mg protein per h, an activity which was six times higher than obtained with the other subcellular fractions. These results indicate that these enzymes are present in the lipid particle fraction not as a result of contamination with other subcellular components, but as a result of a specific attachment of these enzymes to the lipid particles. Also the specific activity of the acyl-CoA synthetase increase with purification of the lipid particles, while the triacylglycerol content remained constant. The following comparable results were obtained by three consetiutive purifications by the flotation method 121: 23, 125, 150 nmol per mg protein per min (acyl-CoA synthetase activity) and 30,25,26 mg (triacylglycerol content). Furthermore, when [‘4C]oleate was used as the label for triacylglycerol synthesis in a system where no glycerol 3-phosphate was added, the lipid particles had a specific activity for triacylglycerol synthesis of 317 nmol fatty acid incorporated per mg protein per h. This activity is 20-40 times that of the other subcellular fractions and thus indicates a difference in triacylglycerol synthesis between the lipid particles and the other subcellular fractions. The incorporation of labelled precursors into individual lipids The time course for sn-[ 2-3H]glycerol 3-phosphate acylation
into individual
are the
OF
157
190
Microsomal
Lipid
2980
f
*
43
21
+ 128
? 266
(6)
(6)
(7).
(5)
4.5
22.7
28.1
25.1
* 0.5
t 2.8
f 0.3
f 3.5
(5)
(3)
(3)
(4)
protein
not
458
126
41* (3)
detectable
r 39
(3)
6 (3)
protein)
f 16
(nmol/mg
per min)
(nmol/mg
protein
(pg/mg
per min)
RNA
c
NADPHsyto-
FRACTIONS
chrome
of experiments.
SUBCELLULAR
number
Cytochrome
617
particles
CONTENT
in parentheses
RNA
oxidase
+ S.E.;
AND
Intermediate
Mitochondrial
Fraction
mean
ACTIVITIES
values
The
I
ENZYME
TABLE OF
(nmol/mg
142
14.0
13.6
10.3 1.7
2.9
0.9
_+ 12
r
k
t
per min)
(5)
(3)
(3)
(3)
protein
YEAST
synthetase
Awl-CoA
BAKER’S
synthesis
8.0
62
9.5
11.0
? 6
.t 3.2
? 3.8
f 0.6
(3)
(10)
(4)
(4)
8.0
317
12.6
1.43
?
3.0
1.6
(12)
(5)
(4)
(4)
protein 2.5
t 29
r
f
asterifica-
per h)
endogenous acceptor
with (mnol/mg
acyl per h)
acylation protein
tion
[14C101eate
by
3-phosphate (nmol/mg
sn-[3HlGlycerol
Triacylglycerol
10" 30 L5 60
90
120 In!"
Fig. 1. Time course of sn-[2-3Hlglycerol 3-phosphate acylation with different subcellular fractions of baker’s yeast. In A. 0.28 mg lipid particle protein; in B, 0.15 mg mitochondrial protein: and in C, 0.22 mg microsomal protein was used as enzyme source. Lyso-PA, lysophosphatidic acid; PA, phosphatidic acid; DG, diacylglycerol and TG, triacylglycerol.
lipids of the lipid particle fraction is shown in Fig. 1A. The labelling of the products proceeds in a manner which is compatible with the Kennedy pathway [30] for the biosynthesis of triacylglycerol. It is noteworthy that the amount of lysophosphatidic acid was very small and nearly constant during the time of incubation. This was in contrast to the other subcellul~ fractions (Figs. IB and 1C) where the amount of lysophosphatidic acid exceeded or was equal to that of phosphatidic acid.
1000
Fig. 2. Time course of [l-14Cloleate esterification, (B) with endogenous acyl acceptor by lipid particles was in A 0.30 mg, and in B 0.24 mg.
(A) with glycerol 3-phosphate as acyl acceptor, and of baker’s yeast. The amount of lipid particle protein
When the label was placed in the fatty acid moiety instead of in the glycerol moiety, the reaction gave the same labelled products plus acyl-CoA (Fig. 2A). However, the labelling of the triacylglycerol fraction now equalled the diacylglycerol fraction in contrast to what was obtained with the sn-[3H]glycerol 3phosphate acylation. This observation may indicate that the triacylglycerol fraction has been labelled by other means besides by de novo synthesis. This observation is confirmed in experiments, where [l-14C]oleic acid was incubated without any exogenous acyl acceptor. As shown in Fig. 2B, the only labelled products were acyl-CoA, triacylglycerol and a small amount of sterol ester. The activity of this acylation reaction was high, higher than the rate of triacylglycerol formation through the phosphatidic acid pathway. The observed triacylglycerol formation from [ 14C] oleate with glycerol 3-phosphate as acyl acceptor (350 nmol fatty acid incorporated per mg protein per h, Fig. 2A), agrees with the sum of the triacylglycerol synthesis by glycerol 3-phosphate acylation (50 X 3 nmol fatty acid incorporated per mg protein per h, Fig. 1A) and the [ 14C]oleate esterification without added glycerol 3-phosphate (250 nmol fatty acid incorporated per mg protein per h, Fig. 2B). Nature of the endogenous [l -14C]oleate
acyl acceptor
in the synthesis
of triacylglycerol
from
Pancreatic hydrolysis of the triacylglycerol fraction synthesized de novo through the phosphatidic acid pathway should give monoacylglycerol (MG), diacylglycerol (DG) and free fatty acid (FFA) as labelled products, and the amount of label in the free fatty acid should follow the equation FFA = 2 X MG + DG/2 if the fatty acids in all three positions were labelled equally. Lipase treatment of the triacylglycerol fraction from experiments where labelled oleic acid was incubated with unlabelled glycerol 3-phosphate gave as labelled hydrolysis products free fatty acid, monoacylglycerol and diacylglycerol (Table
TABLE
II
LABELLED PRODUCTS GLYCEROL FRACTION 3-PHOSPHATE ADDED BAKER’S YEAST
OBTAINED BY PANCREATIC LIPASE TREATMENT OF THE TRIACYLSYNTHESIZED BY [14C]OLEATE ACYLATION WITH EITHER GLYCEROL OR WITH ENDOGENOUS ACYL ACCEPTOR BY LIPID PARTICLES OF
acylation with added glycerol 3-phosphate and with The triacylglycerol fractions from [ 14Cloleate endogenous acyl acceptor were isolated from the incubation mixture by preparative thin-layer chromatography. The triacylglycerols (approx. 1 qnol) and a commercial glycerol (tri[14C]oleate (3.5 @mol. spec. act. 34 * lo3 cpm/~mol) were each mixed with 900 ~1 Tris/buffer, pH 8. 20 ~1 1% sodium taurocholate and 50 ~1 45% CaClZ . 2H20. The mixture was shaken vigorously on-a Vortex mixer before 2 units of lipase (Sigma, Type II from hog pancreas) was added. The hydrolysis was done at 37°C for 10 min. The reaction was stopped by adding 500 ~1 cont. HCl/H20 (1 : 1, v/v). The hydrolysis were extracted with 2 X 3 ml diethyl and represent
ether.
the
Triacylglycerol
The ether phases were washed
average
source
Glyceryl (tri[14C]oleate) [14ClOleate + glycerol 3-phosphate [14C101eate + endogenous acyl acceptor
of duplicate
with 2 ml H20.
The values are expressed
as cpm.
experiments.
Diacylglycerol
Monoacylglycerol
Free fatty acid
13 230 8 260
9130 2140
28 510 18 220
4 200
-
9 200
86
II). However, the distribution of label was not in accordance with the formula. This supports the suggestion of an additional pathway for ~~~acylgly~e~o~ synthesis which would give rise to a mixture of molecular species. Hydrolysis of the triacylglycerol fraction formed from labelled oleic acid and the endogenous acyl acceptor gave only labelled diacylglycerol and free fatty acid, bot not labelled monoacylglycerol, as would be expected if the acceptor was either 1,2diacylglycerol or 2-monoacylglycerol. Formation of triacylglycerol from the latter and labelled fatty acid should lead to formation, albeit transient, of labelled dia~ylgly~erol and this was never observed even at short incubation times (Fig. ZB). 1,2-Diacylglycerol therefore seems to be the most likely endogenous acyl acceptor in the lipid particles. Monoacylglycerol could not be detected in extracts of lipid particles, but diacylglycerol was present in the amount of 1.6 t 0.3% (mean I S.E. from five determinations) of total lipid. Still, the amount of tria~ylglycerol synthesized from endogenous diacylglycerol, calculated from data of [l”C]oleate esterification (41, 51 and 22 nmol fatty acid incorporated in three experiments) was 3-O---60% greater than the decrease in the amount of diacylglycerol measured chemically (28, 32 and 15 nmol diacylglycerol, used, respectively). An increase in the amount of diacylgly~erol by addition of 1.2-dioleylglycerol in Tween-80 or ethanol 1311 failed to enhance incorporation of labelled fatty acid in triacylglycerol. This may reflect either competitior between exogenous and endogenous diacylglycerols or %imore appropriate fatt;i acid composition of the endogenous diacylglycerol. Experiments with labelled diacylglycerol were not performed. Instead, utilization of endogenous labelled ~,~dia~ylglycerol as substrate for dia~ylgiycerol a~yltr~sferase present in the lipid particles was studied (Table III). Incubation of the labelled lipid particles with unlabelled fatty acid and the cofactors, ATP, CoA, MgClz and fi-mercaptoethanol, leads to an increase in Iabelled triacylglycerol. The decrease in Iabelled diacylglycerol and phosphatidic acid was proportional to the increase in activity of the triacylglycerol fraction, suggesting that the prelabelled 1,2-diacyl-
TABLE III UTILIZATION OF ENDOGENOWS 14C-LABELLED DIACYLGLYCEROL IN LIPID PARTICLES SUBSTRATE FQR DIACYLGLYCEROL ACYLTRANSFERASE ACTIVITY Lipid particles (2.5 mg protein),
75 pmol ATP, 5 firno CoA, 75 pmol hIgCl~, 2 pmol [I-14CloIeic
AS A acid
complexed to 8 &mol defatted albumin, 40 &rnol ~-mer~aptoethanol and 50 @maI glycerol 3-phoshate were incubated at 27°C for 1 h. The labelled lipid particles were purified from the incubation mixture by centrifugation as described under Materials and Methods. The 14&-labelled particles were incubated with 2 gmol unlabelled oleic acid. 75 &mof ATP, 5 pmol CoA, 75 pmol MgCl2 and 40 pmol P-mercaptoethanol at 27°C for different time intervals. The values are expressed as percentage (9) of tatal label and change (A) in percentage label and represent the average of duplicate experiments. Time (Inin)
0
15 30 60
Phosphatidic acid ____ w A
Diacylglyceroi
Triacylgiycerol
Free fatty acid -
Sterol ester
%
A
%
A
%
A
%
A
32.4 28.2 25.3 22.7
43.7 44.0 41.8 37.8
+o.a -1.9 -5.9
20.4 23.4 27.7 33.3
+3.0 +7.3 +12.9
0.8 1.9 2.6 4.0
+1.1 rI.8 i-3.2
2.7 2.6 2.6 2.5
-0.1 -0.1 -9.2
-4.2 -7.1 -9.7
87
glycerol was acting as a substrate for diacylglycerol acyltransferase. The labelled diacylglycerol pool was evidently being replenished from labelled phosphatidic acid through the action of a phosphatidate phosphohydrolase which is present in the lipid particle preparation. A steady increase of label in the free fatty acid fraction during the incubation suggests that the lipid particle preparation also contains lipase activity. Role of phosphatidate
phosphohydrolase
It has been inferred from studies of triacylglycerol synthesis in in vitro preparations of rat liver that phosphatidate phosphohydrolase may be rate limiting [ 32,331. A similar role of the phosphohydrolase may be suspected in the yeast system. Incubations were carried out with additions of NaF and CaCl,, which are known inhibitors of the liver phosphohydrolase [33,34]. Further incubations were done with added supernatant which has a stimulating effect with liver systems [ 35-391. As shown in Table IV addition of NaF (10 mM) and CaClz in the concentration range of l-5 mM inhibited the neutral lipid formation from glycerol 3phosphate. At the highest CaCl, concentration (5 mM) the phosphatidic acid synthesis was also inhibited. Addition of a 100 000 X g particle-free supernatant markedly increased the formation of neutral lipids from glycerol 3-phosphate. The supernatant alone does not synthesize neutral lipids, but does synthesize the phosphorylated intermediates with a specific activity of 56 nmol glycerol 3-phosphate incorporated per mg protein per h. When the soluble supernatant was added to lipid particles a nearly 4-fold increase in the neutral lipid to phospholipid ratio was observed. The addition of boiled supernatant slightly stimulated the formation of all products of the phosphatidic acid pathway, probably as a result of addition of a more appropriate mixture of fatty acids for the different acyltransferases involved in triacylglycerol synthesis [ 371.
TABLE
IV
EFFECTS
OF
ACYLATION
F-,
Ca2+,
BY
LIPID
The
values
the
average
of duplicate
DG.
diacylglycerol;
acid;
are
expressed
AND
THE
PARTICLES as nmol experiments. TG,
SOLUBLE OF
sn-glycerol PL,
SUPERNATANT
BAKER’S
3-phosphate
phospholipid
triacylglycerol;
NL,
ON
s~-[~H]GLYCEROL
3-PHOSPHATE
YEAST converted which neutral
per
for more lipid,
which
mg
than
protein 90%
is the
per
represents sum
of
triacylglycerol. PL
DG
system
533
135
(10
461
Conditions Complete + NaF Complete
mM)
system
793
8 112
TG
NL/PL
40
0.21
2
0.02
56
0.21
+ CaClZ
(1 mM)
860
71
41
0.14
+ CaClZ
(2 mM)
825
47
26
0.09
+ CaCl2
(5 mM)
652
19
Complete
system
+ soluble + boiled
supernatant supernatant
(3 mg) (3 mg)
a
0.04 0.44
397
147
28
145
244
41
1.68
433
173
36
0.48
h. and
represent
phosphattidic
diacylglycerol
and
88
Discussion Baker’s yeast has been used extensively for the study of fatty acid synthesis [40]. Phospholipid synthesis has been studied in cell-free particulate fractions [25,41-431, but triacylglycerol synthesis and its regulation in yeast cells has attracted little interest. The present report describes triacylglycerol synthesis by sn-[ 3H]glycerol 3-phosphate acylation and [ 14C]oleate esterification in subcellular fractions of baker’s yeast. Specifically, it has been the main object to establish whether lipid particles were able to synthesize the triacylglycerols which constitute one of their main components. The results of this study show that lipid particles of baker’s yeast contain an active system for the synthesis of triacylglycerol as also shown from other cells [3-61. Compared to the other subcellular fractions the lipid particle fraction has the lowest activities of the marker enzymes, cytochrome oxidase and while it has the highest activities of the NADPH-cytochrome c reductase, enzymes involved in lipid synthesis, acyl CoA synthetase, and triacylglycerol synthesis by glycerol 3-phosphate acylation and oleate esterification. Therefore, the enzymes involved in triacylglycerol synthesis associated with the lipid particles cannot be due to contamination only, but represent a specific attachment of these enzymes to the lipid particles. Also the flotation method used for isolation and purification of the lipid particles minimizes contamination. Finally the specific activity of the acyl-CoA synthetase increases with purification, while the triacylglycerol content of the lipid particle fraction remains constant. The time course of glycerol 3-phosphate acylation indicates that the labelled phosphorylated intermediates in triacylglycerol synthesis obtained with the lipid particle fraction differ from the other subcellular fractions. Thus, phosphatidic acid is the main intermediate and only a small amount od lysophosphatidic acid is formed with the lipid particle fraction, whereas the mitochondrial fraction produces mainly lysophosphatidic acid as the intermediate, and the microsomal fraction produces equal amounts of lysophosphatidic acid and phosphatidic acid. A number of suggestions may be offered in explanation of this difference in activity and in the amounts of phosphorylated intermediates observed between lipid particles and the other subcellular fractions. The differences may be due to the higher activity of acyl-CoA synthetase of the lipid particles. Effects caused by differences in lysophosphatidic acid acyltransferase activity or in phospholipase activities cannot be excluded, but have not been examined. The effects of fluoride, CaCl, and the soluble supernatant on triacylglycerol formation through the phosphatidic acid pathway are in agreement with what has been found in rat liver, where F- and Ca2+ have been reported to inhibit the phosphatidate phosphohydrolase [ 33,341. Our experiments indicate that the action of these ions in yeast cell is at the level of phosphatidate phosphohydrolase. Addition of the soluble supernatant increased the formation of neutral lipid from glycerol 3-phosphate in rat liver mitochondrial and microsomal fractions [35-391. The same effect of the soluble supernatant was observed with lipid particles of yeast cell and suggests that the phosphatidate phosphohydrolase activity may be rate limiting in neutral lipid formation, and
89
that the soluble supernatant contains this enzyme as in mammalian systems. With the lipid particle fraction as enzyme source, triacylglycerol is also formed by acylation of an endogenous acyl acceptor, probably diacylglycerol. The acylation reaction proceeds even under circumstances where a de novo synthesis of triacylglycerol occurs, and the rate of triacylglycerol synthesis is higher by the acylation reaction than by de novo synthesis. The lipid particle fraction contains, in contrast to the other subcellular fractions, diacylglycerol in significant amounts This may explain the difference in the activity between the lipid particles and the other subcellul~ fractions. The acylation reaction was not stimulated by exogenous l,Zdioleylglycerol, but endogenous labelled diacylglycerol could act as a substrate for diacylglycerol acyltransferase. Triacylglycerol synthesis from an endogenous acyl acceptor has also been reported by Johnston and Paltauf [44] to occur in an in vitro study in S. curlsbergensis. An ATP-dependent formation of 14C-labelled t~acylglycerol from [14CJoleate was observed regardless of the presence of added acyl acceptors. However, the pathway for triacylglycerol formation was not established. The amount of triacylglycerol formed from endogenous diacylglycerol, calculated from data [ 14C]oleate esterification is usually greater than the decrease in the amount of diacylglycerol measured chemically. This means that if diacylglycerol is the proposed acyl acceptor, the amount of dia~ylglycerol must be replenished from another reaction, probably by the action of triacylglycerol lipase. Diacylglycerol is in fact an intermediate in the lipolytic degradation of triacylglycerol by the mitochondrial lipase of baker’s yeast [45]. The amount of protein associated the crude lipid particle fraction constitutes only 0.3% of the total protein of a cytoplasmic extract. In comparison the other crude subcellul~ fractions the mitochondrial, the intermediate, the microsomal and the supernatant contribute 19,10, 3 and 63% respectively, tp the total protein of a cytoplasmic extract. The amount of protein associated with triacylglycerol containing particles in vivo is difficult to evaluate, since only 15-20% of the total triacylglycerol of the cytoplasmic extract is recovered in the lipid particle prep~ation. Based on studies of plant and different animal tissue [46,47--491, we assume that a part of the remaining 8O--85% of triacylglycerol is located in cisternae, in vesicles attached to the endoplasmic reticulum, and in minute lipid particles associated the cell organelles. Triacylglycerol in these locations will not float in our centrifugation procedure, and therefore is not isolated with the lipid particles. However, the present study shows that lipid particles (0.3-l pm in diameter) of baker’s yeast can synthesize triacylglycerol de novo, and can introduce fatty acids by acyltransferases into preformed glycerolipid acceptors in the lipid particle. Thus, triacylglycerol can be synthesized and its fatty acid composition changed at the site where they were are stored without the need for transport. Acknowledgement This investigation was supported by the Danish Science Research Foundation grant no. 511-1838. 6 thank Dr. P.K. Jensen and Dr. B. Jensen for valuable comments on the manuscript, and Miss Birthe Nystr$m for skilful technical assistance.
90
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Acta, 530 (1978) 78-90 Biomedical Press
BBA 57208
TRIACYLGLYCEROL SYNTHESIS IN LIPID PARTICLES YEAST (SACCHAROMYCES CEREVZSZAE)
FROM BARER’S
KIRSTEN CHRISTIANSEN Department of Biochemistry C, University DK-2200 Copenhagen N (Denmark)
of Copenhagen,
Panum Institute,
Blegdamsvej
3,
(Received January 23rd, 1978)
Summary Triacylglycerol synthesis has been studied in a lipid particle preparation of baker’s yeast (S’accharomyces cereuisiue), and compared with the synthesis in other subcellular fractions. Fatty acid-CoA ligase (AMP) (EC 6.2.1.3) activity and sn-glycerol 3-phosphate acyltransferase activity (EC 2.3.1 .15) were present in all the subcellular fractions tested but the highest specific activities of both enzymes were observed with the lipid particle fraction. The products of the glycerol 3-phosphate acylation indicate that triacylglycerol synthesis proceeds through the phosphatidic acid pathway. However, only a small and nearly constant amount of lysophosphatidic acid was found with the lipid particle fraction while the other subcellular fraction produced lysophosphatidic and phosphatidic acid with a more pronounced precursor/product relationship. Triacylglycerol synthesis from endogenous diacylglycerol present in the lipid particle was also demonstrated.
Introduction Lipid particles have been described as a common structure in the yeast cell [l]. However, little is known about the biochemical basis for the formation and development of these intracellular particles in the course of cellular growth. In our laboratory, lipid particles have been isolated from yeast cells in the stationary phase of growth [2]. The isolated lipid particles are composed of 95% lipid and 5% protein. The lipid fraction of the particles consists of triacylglycerols (45-50%) and sterol esters (45-50%) together with minor amounts of phospholipids and free fatty acids [2]. Also about 2% diacylglycerol, but no monoacylglycerol is present in the lipid particle preparation. The endoplasmic reticulum is considered the site for triacylglycerol syn-
79
thesis, at least in mammalian cells. However, enzymes necessary for triacylglycerol synthesis have been reported also to be associated with triacylglycerolcontaining particles, e.g. in the so-called fat fraction of plant cells such as castor bean seeds [3] and Crambe seeds [ 41. In mammalian cells, the membranebound lipid particles of beef heart [5] and the lipid micelles of rat adipose tissue [6] have been reported to contain enzymes necessary for triacylglycerol synthesis. The purpose of the present report has been to study whether triacylglycerol-synthesizing enzymes also are associated with lipid particles in yeast cells. Materials and Methods Chemicals. Unlabelled lipids were purchased from Nu Chek Prep., Elysian, Minn., U.S.A. and from Serdary Research Laboratories, London, Canada. [1-“C]Oleic acid (spec. act. 54 Ci/mol) was purchased from The Radiochemical Centre, Amersham, England. sn-[ 2-3H] Glycerol 3-phosphate was prepared enzymatically from sn-[ 2-3H] glycerol (The Radiochemical Centre, Amersham, England) and purified as descibed by Chang and Kennedy [ 71, with the modifications proposed by Davidson and Stanacev [8]. The final product had a specific activity of 8.3 Ci/mol sn-glycerol 3-phosphate and was diluted appropriately with rat-glycerol 3-phosphate before use in enzyme assays. ATP (disodiurn) and coenzyme A were from Boehringer, Mannheim, W. Germany. Baker’s yeast (Saccharomyces cereukiae) was obtained by the courtesy of De Danske Spritfabrikker A/S, Copenhagen. Preparation of subcellular fractions. The yeast cells were washed and homogenized as previously described [2,9]. Whole cells, nuclei and cell debris were removed by centrifugation at 1000 X g for 10 min. The cytoplasmic extract containing cytosol plus intact and disrupted organelles [lo] was further centrifuged at 20 000 X g for 15 min, after which crude mitochondria were collected as a pellet and crude lipid particles as a top layer. The intermediate supernatant was centrifuged at 48 000 X g for 20 min, and at 85 000 X g for 90 min, whereby, respectively, the intermediate fraction and the microsomal fraction were obtained. The supernatant from the last centrifugation was designated the soluble supernatant. All stages were performed at 4°C. The mitochondrial fraction was extensively purified and only the heavy mitochondrial fraction was used in this study [5]. The purity of this fraction was evaluated by electron microscopy as described earlier [9]. The crude preparation of lipid particles was purified by flotation as described [2], except that 0.25 M sucrose was used instead of the mannitol medium. The purity of this fraction was also evaluated by electron microscopy [2]. The intermediate and the microsomal fractions were not purified further. The intermediate fraction appeared in the electron micrographs as a mixture of the membranous material of the cell including some intact mitochondria. The microsomal fraction appeared to consist mostly of polysomes with minor amounts of membranous material. The protein concentration of the fractions was measured by the method of Lowry et al. [ll] ,with crystalline bovine serum albumin as standard. For the lipid particles a modification for lipid-rich material was used [12]. RNA was
80
measured by the orcinol method as described by Schneider [13]. Marker enzyme activities were measured at 27”C, cytochrome oxidase activity as described by de Duve et al. [lo], NA~PH-cytochrome c reductase as described by Sottocasa et al. [14], except that rotenone was excluded from the assay and the subcellular fraction was diluted in Nonidet P-40 [ 151. The diacylglycerol of the total lipid extract [ 161 was isolated by preparative thin-layer chromatography on silica gel using light petroleum (b.p. 60-7O”C)/ diethyl ether/acetic acid (70 : 30 : 1, v/v). The appropriate band was scraped off the plate, and the diacylglycerol isolated by extraction with 2 X 3 ml chloroform/methanol (1 : 2, v/v). After evaporation of the solvent, the diacylglycerol content was determined after enzymatic hydrolysis and determination of glycerol as described by Wahlefeld [ 171, Dioleylglycerol in known amounts was chromatographed and analyzed in the same way as the unknown samples. Assay procedures for enzymes invoZued in tria~y~~lycero~ synthesis. Optimum conditions with respect to the cofactors and the substrates glycerol 3phosphate and the fatty acid (complexed to albumin in the ratio 4 : 1) for the sn-glycerol 3-phosphate acyltransferase activity with the lipid particle preparation as enzyme source were established in preliminary experiments. There was an absolute requirement for ATP and CoA and a 3-fold increase with 15 mM MgClz. Maximal activity was obtained at 5 mM sn-glycerol 3-phosphate and 0.4 mM fatty acid, respectively. ~-Mercaptoethanol was added routinely in order to protect SW-groups but had no influence on the measured enzymic activities. The following basic incubation system was used: 100 mM Tris * HCl (pH 7.4), 15 mM ATP, 15 mM MgC12, 1 mM CoA, 8 mM fl-mercaptoethanol and 0.4 mM oleic acid bound to 0.1 mM defatted bovine serum albumin (fraction V, Sigma) 1181. When added the concentration of glycerol 3-phosphate was 5 mM. The reactions were carried out in a shaking waterbath at 27°C. Acyl-CoA synthetase: The conversion of 0.4 gmol [ l-‘4C]oleate (spec. act. 0.64 Ci/mol) to [l-‘4C]oleyl-CoA was measured in a total reaction volume of 0.25 ml of the basic incubation system. The reaction was started by addition of protein (lo-40 pg), carried out for 4 min and stopped by addition of 1 ml Dole reagent [19] 0.35 ml water and 0.6 ml n-heptane. The extraction and scintillation counting of the product, acyl-CoA, was carried out as described by Lloyd-Davies and Brindley [20]. The specific activity was determined with two different concentrations of protein. The oleyl-CoA was identified by co-chromatography with authentic oleyl-CoA [Zl]. sn-Glycerol 3-phosphate acylation: The biosynthesis of tria~ylglycerol was determined by measuring the rate of incorporation of sn-[2-3H]glycerol 3-phosphate (spec. act. 0.10 Ci/mol) into lipids in a total reaction volume of 1 ml. The reaction was started by addition of 0.1 -0.3 mg protein and carried out for different time intervals or for 60 min. The reaction was terminated by adding 1.5 ml butanol followed by 2 ml water. The lipid was extracted as described by Daae [ 221. The butanol was removed under reduced pressure and the lipid dissolved in a known amount of chloroform. Aliquots were taken for counting total radioactivity and for chromatography. Neutral lipids were analyzed on silica gel-loaded paper (I.T.L.C. type SA from Gelman Instruments Co., Mich., U.S.A.) in light petroleum (b.p. 60-70”C)/diethyl ether/acetic acid (80 : 20 : 1, v/v or 65 : 45 : 1, v/v). Phospholipids were analyzed on silica gel F,,, pIates
81
in the two-dimensional thin-layer chromatography system described by Brotherus and Renkonen [23], after modifying the solvent system used in the first direction to chloroform/methanol/7 M ammonia (65 : 20 : 3.5, v/v). The lipid classes were detected by iodine and identified by comparing their Rf values with those of standards. The spots were either cut from paper or scraped from the plates and the radioactivity determined as described [21]. Oleate esterification with added glycerol 3-phosphate as acyl acceptor: Oleate esterification was measured by incubating 0.4 E.rmol [1-r4C]oleic acid (spec. act. 0.64 Ci/mol) with 5 pmol glycerol 3-phosphate in a total reaction volume of 1 ml. The reaction was started by addition of 0.1-0.3 mg protein, and carried out for different time intervals or 60 min. The reaction was stopped by addition of 20 ml chlorofo~/methanol (2 : 1, v/v) and the extraction carried out as recommended for incubations were both labelled oxoesters and thioesters are formed [21]. The separation and determination of radioactivity of individual lipids was as described above. Oleate esterification with endogenous acyl acceptor: [1-‘“C]Oleate was incubated under exactly the same conditions as stated above except that no glycerol 3-phosphate was added. The extraction of the incubation mixture and the radioactivity determ~ation was as described for incubations with labelled fatty acids [ 211. Hydrolysis of labelled triacylglycerol by pancreatic lipase. The triacylglycerol fraction from the 14C-labelled extracts was isolated by preparative thinlayer chromato~aphy on 0.5 mm silica gel G plates. The triacylglycerolcontaining band was localized and the compound extracted from the silica gel with 3 X 5 ml of diethyl ether. The diethyl ether was evaporated and the triacylglycerol treated with pancreatic lipase at 37°C for 5 min as described by Mattson and Volpenhein [ 241. The unhydrolyzed substrate and the hydrolysis products were separated by chromatography on silica gel-loaded glass fiber paper type SA from Gelman Products, in the solvent system light petroleum (b.p. 60-70”C)/diethyl ether/acetic acid (65 : 45 : 1, v/v). The bands were detected by IZ vapor and the radioactivity determined as described [21]. “C-labelling of endogenous 1,2-diacylglycerol in lipid particles and its use as substrate for 1,2-diacylglyceroltransferase. Purified lipid particles were incubated in the assay system for [ 14C]oleic acid acylation with added glycerol 3-phosphate as acyl acceptor as described above. The reaction was stopped by mixing the incubation mixture with an equal volume of ice-cold 50% (w/v) sucrose. The mixture was overlayered with ice-cold 0.25 M sucrose and centrifuged at 27 000 X g for 15 mm at 4°C. The top layer of labelled lipid particles was isolated by freezing the content of the centrifuge tube up to 1 cm below the lipid layer. The resuspended particles contained the labelled particle-bound diacylglycerol together with some phosphatidic acid and triacylglycerol (Table III). The labelled diacylglycerol was used as a substrate for studying diacylglycerol acyltransferase activity by incubating the prelabelled particles with unlabelled fatty acid, ATP, CoA, MgClz and P-mercaptoethanol in the concentrations stated for various time intervals.
82
Results Characterization of the lipid particle fraction The subcellular fractions were characterized both by electron microscopy and specific activities of marker enzymes. The results of Table I indicate that the lipid particles were contaminated with mitochondrial inner fragments to an extent of 6%. NADPH-cytochrome c reductase has been used as a marker enzyme for the microsomal fraction of baker’s yeast [25]. In our hands the specific activities of this enzyme in the different subcellular fractions did not show an exclusively microsomal origin, in agreement with the results from Saccharomyces Carkbergensis [ 261, Tetrahymena pyriformis [ 271, and from rat liver [28] and pig heart [29]. Thus, the low NADPH-cytochrome c reductase activity seen in the lipid particle fraction may be due to a small contamination probably with both endoplasmic reticulum and outer mitochondrial membranes. RNA could not be detected in the lipid particle fraction. The results obtained for marker enzymes and RNA content corroborate the results obtained with electron microscopy [2]. Thus, the lipid particle preparation appeared as a relatively pure fraction of particles with a homogeneous core surrounded with a thin layer of more heavily stained material, without any indications of a trilaminar membrane structure. Some heavily stained material is seen close to the particle surface or separate from the particles, which may be of either mitochondrial or microsomal origin, but whole mitochondria or ribosomes are never seen, Among the subcellular fractions tested the lipid particle fraction had the highest specific activities of the enzymes involved in lipid synthesis. An acyl-CoA synthetase activity of 142 nmol per mg of protein per min was obtained with the lipid particle fraction. This activity was 10 times higher than the activity obtained with any of the other subcellular fractions. Triacylglycerol synthesis from sn-glycerol3-phosphate occurred in the lipid particle fraction with a specific activity of 62 nmol glycerol 3-phosphate incorporated per mg protein per h, an activity which was six times higher than obtained with the other subcellular fractions. These results indicate that these enzymes are present in the lipid particle fraction not as a result of contamination with other subcellular components, but as a result of a specific attachment of these enzymes to the lipid particles. Also the specific activity of the acyl-CoA synthetase increase with purification of the lipid particles, while the triacylglycerol content remained constant. The following comparable results were obtained by three consetiutive purifications by the flotation method 121: 23, 125, 150 nmol per mg protein per min (acyl-CoA synthetase activity) and 30,25,26 mg (triacylglycerol content). Furthermore, when [‘4C]oleate was used as the label for triacylglycerol synthesis in a system where no glycerol 3-phosphate was added, the lipid particles had a specific activity for triacylglycerol synthesis of 317 nmol fatty acid incorporated per mg protein per h. This activity is 20-40 times that of the other subcellular fractions and thus indicates a difference in triacylglycerol synthesis between the lipid particles and the other subcellular fractions. The incorporation of labelled precursors into individual lipids The time course for sn-[ 2-3H]glycerol 3-phosphate acylation
into individual
are the
OF
157
190
Microsomal
Lipid
2980
f
*
43
21
+ 128
? 266
(6)
(6)
(7).
(5)
4.5
22.7
28.1
25.1
* 0.5
t 2.8
f 0.3
f 3.5
(5)
(3)
(3)
(4)
protein
not
458
126
41* (3)
detectable
r 39
(3)
6 (3)
protein)
f 16
(nmol/mg
per min)
(nmol/mg
protein
(pg/mg
per min)
RNA
c
NADPHsyto-
FRACTIONS
chrome
of experiments.
SUBCELLULAR
number
Cytochrome
617
particles
CONTENT
in parentheses
RNA
oxidase
+ S.E.;
AND
Intermediate
Mitochondrial
Fraction
mean
ACTIVITIES
values
The
I
ENZYME
TABLE OF
(nmol/mg
142
14.0
13.6
10.3 1.7
2.9
0.9
_+ 12
r
k
t
per min)
(5)
(3)
(3)
(3)
protein
YEAST
synthetase
Awl-CoA
BAKER’S
synthesis
8.0
62
9.5
11.0
? 6
.t 3.2
? 3.8
f 0.6
(3)
(10)
(4)
(4)
8.0
317
12.6
1.43
?
3.0
1.6
(12)
(5)
(4)
(4)
protein 2.5
t 29
r
f
asterifica-
per h)
endogenous acceptor
with (mnol/mg
acyl per h)
acylation protein
tion
[14C101eate
by
3-phosphate (nmol/mg
sn-[3HlGlycerol
Triacylglycerol
10" 30 L5 60
90
120 In!"
Fig. 1. Time course of sn-[2-3Hlglycerol 3-phosphate acylation with different subcellular fractions of baker’s yeast. In A. 0.28 mg lipid particle protein; in B, 0.15 mg mitochondrial protein: and in C, 0.22 mg microsomal protein was used as enzyme source. Lyso-PA, lysophosphatidic acid; PA, phosphatidic acid; DG, diacylglycerol and TG, triacylglycerol.
lipids of the lipid particle fraction is shown in Fig. 1A. The labelling of the products proceeds in a manner which is compatible with the Kennedy pathway [30] for the biosynthesis of triacylglycerol. It is noteworthy that the amount of lysophosphatidic acid was very small and nearly constant during the time of incubation. This was in contrast to the other subcellul~ fractions (Figs. IB and 1C) where the amount of lysophosphatidic acid exceeded or was equal to that of phosphatidic acid.
1000
Fig. 2. Time course of [l-14Cloleate esterification, (B) with endogenous acyl acceptor by lipid particles was in A 0.30 mg, and in B 0.24 mg.
(A) with glycerol 3-phosphate as acyl acceptor, and of baker’s yeast. The amount of lipid particle protein
When the label was placed in the fatty acid moiety instead of in the glycerol moiety, the reaction gave the same labelled products plus acyl-CoA (Fig. 2A). However, the labelling of the triacylglycerol fraction now equalled the diacylglycerol fraction in contrast to what was obtained with the sn-[3H]glycerol 3phosphate acylation. This observation may indicate that the triacylglycerol fraction has been labelled by other means besides by de novo synthesis. This observation is confirmed in experiments, where [l-14C]oleic acid was incubated without any exogenous acyl acceptor. As shown in Fig. 2B, the only labelled products were acyl-CoA, triacylglycerol and a small amount of sterol ester. The activity of this acylation reaction was high, higher than the rate of triacylglycerol formation through the phosphatidic acid pathway. The observed triacylglycerol formation from [ 14C] oleate with glycerol 3-phosphate as acyl acceptor (350 nmol fatty acid incorporated per mg protein per h, Fig. 2A), agrees with the sum of the triacylglycerol synthesis by glycerol 3-phosphate acylation (50 X 3 nmol fatty acid incorporated per mg protein per h, Fig. 1A) and the [ 14C]oleate esterification without added glycerol 3-phosphate (250 nmol fatty acid incorporated per mg protein per h, Fig. 2B). Nature of the endogenous [l -14C]oleate
acyl acceptor
in the synthesis
of triacylglycerol
from
Pancreatic hydrolysis of the triacylglycerol fraction synthesized de novo through the phosphatidic acid pathway should give monoacylglycerol (MG), diacylglycerol (DG) and free fatty acid (FFA) as labelled products, and the amount of label in the free fatty acid should follow the equation FFA = 2 X MG + DG/2 if the fatty acids in all three positions were labelled equally. Lipase treatment of the triacylglycerol fraction from experiments where labelled oleic acid was incubated with unlabelled glycerol 3-phosphate gave as labelled hydrolysis products free fatty acid, monoacylglycerol and diacylglycerol (Table
TABLE
II
LABELLED PRODUCTS GLYCEROL FRACTION 3-PHOSPHATE ADDED BAKER’S YEAST
OBTAINED BY PANCREATIC LIPASE TREATMENT OF THE TRIACYLSYNTHESIZED BY [14C]OLEATE ACYLATION WITH EITHER GLYCEROL OR WITH ENDOGENOUS ACYL ACCEPTOR BY LIPID PARTICLES OF
acylation with added glycerol 3-phosphate and with The triacylglycerol fractions from [ 14Cloleate endogenous acyl acceptor were isolated from the incubation mixture by preparative thin-layer chromatography. The triacylglycerols (approx. 1 qnol) and a commercial glycerol (tri[14C]oleate (3.5 @mol. spec. act. 34 * lo3 cpm/~mol) were each mixed with 900 ~1 Tris/buffer, pH 8. 20 ~1 1% sodium taurocholate and 50 ~1 45% CaClZ . 2H20. The mixture was shaken vigorously on-a Vortex mixer before 2 units of lipase (Sigma, Type II from hog pancreas) was added. The hydrolysis was done at 37°C for 10 min. The reaction was stopped by adding 500 ~1 cont. HCl/H20 (1 : 1, v/v). The hydrolysis were extracted with 2 X 3 ml diethyl and represent
ether.
the
Triacylglycerol
The ether phases were washed
average
source
Glyceryl (tri[14C]oleate) [14ClOleate + glycerol 3-phosphate [14C101eate + endogenous acyl acceptor
of duplicate
with 2 ml H20.
The values are expressed
as cpm.
experiments.
Diacylglycerol
Monoacylglycerol
Free fatty acid
13 230 8 260
9130 2140
28 510 18 220
4 200
-
9 200
86
II). However, the distribution of label was not in accordance with the formula. This supports the suggestion of an additional pathway for ~~~acylgly~e~o~ synthesis which would give rise to a mixture of molecular species. Hydrolysis of the triacylglycerol fraction formed from labelled oleic acid and the endogenous acyl acceptor gave only labelled diacylglycerol and free fatty acid, bot not labelled monoacylglycerol, as would be expected if the acceptor was either 1,2diacylglycerol or 2-monoacylglycerol. Formation of triacylglycerol from the latter and labelled fatty acid should lead to formation, albeit transient, of labelled dia~ylgly~erol and this was never observed even at short incubation times (Fig. ZB). 1,2-Diacylglycerol therefore seems to be the most likely endogenous acyl acceptor in the lipid particles. Monoacylglycerol could not be detected in extracts of lipid particles, but diacylglycerol was present in the amount of 1.6 t 0.3% (mean I S.E. from five determinations) of total lipid. Still, the amount of tria~ylglycerol synthesized from endogenous diacylglycerol, calculated from data of [l”C]oleate esterification (41, 51 and 22 nmol fatty acid incorporated in three experiments) was 3-O---60% greater than the decrease in the amount of diacylglycerol measured chemically (28, 32 and 15 nmol diacylglycerol, used, respectively). An increase in the amount of diacylgly~erol by addition of 1.2-dioleylglycerol in Tween-80 or ethanol 1311 failed to enhance incorporation of labelled fatty acid in triacylglycerol. This may reflect either competitior between exogenous and endogenous diacylglycerols or %imore appropriate fatt;i acid composition of the endogenous diacylglycerol. Experiments with labelled diacylglycerol were not performed. Instead, utilization of endogenous labelled ~,~dia~ylglycerol as substrate for dia~ylgiycerol a~yltr~sferase present in the lipid particles was studied (Table III). Incubation of the labelled lipid particles with unlabelled fatty acid and the cofactors, ATP, CoA, MgClz and fi-mercaptoethanol, leads to an increase in Iabelled triacylglycerol. The decrease in Iabelled diacylglycerol and phosphatidic acid was proportional to the increase in activity of the triacylglycerol fraction, suggesting that the prelabelled 1,2-diacyl-
TABLE III UTILIZATION OF ENDOGENOWS 14C-LABELLED DIACYLGLYCEROL IN LIPID PARTICLES SUBSTRATE FQR DIACYLGLYCEROL ACYLTRANSFERASE ACTIVITY Lipid particles (2.5 mg protein),
75 pmol ATP, 5 firno CoA, 75 pmol hIgCl~, 2 pmol [I-14CloIeic
AS A acid
complexed to 8 &mol defatted albumin, 40 &rnol ~-mer~aptoethanol and 50 @maI glycerol 3-phoshate were incubated at 27°C for 1 h. The labelled lipid particles were purified from the incubation mixture by centrifugation as described under Materials and Methods. The 14&-labelled particles were incubated with 2 gmol unlabelled oleic acid. 75 &mof ATP, 5 pmol CoA, 75 pmol MgCl2 and 40 pmol P-mercaptoethanol at 27°C for different time intervals. The values are expressed as percentage (9) of tatal label and change (A) in percentage label and represent the average of duplicate experiments. Time (Inin)
0
15 30 60
Phosphatidic acid ____ w A
Diacylglyceroi
Triacylgiycerol
Free fatty acid -
Sterol ester
%
A
%
A
%
A
%
A
32.4 28.2 25.3 22.7
43.7 44.0 41.8 37.8
+o.a -1.9 -5.9
20.4 23.4 27.7 33.3
+3.0 +7.3 +12.9
0.8 1.9 2.6 4.0
+1.1 rI.8 i-3.2
2.7 2.6 2.6 2.5
-0.1 -0.1 -9.2
-4.2 -7.1 -9.7
87
glycerol was acting as a substrate for diacylglycerol acyltransferase. The labelled diacylglycerol pool was evidently being replenished from labelled phosphatidic acid through the action of a phosphatidate phosphohydrolase which is present in the lipid particle preparation. A steady increase of label in the free fatty acid fraction during the incubation suggests that the lipid particle preparation also contains lipase activity. Role of phosphatidate
phosphohydrolase
It has been inferred from studies of triacylglycerol synthesis in in vitro preparations of rat liver that phosphatidate phosphohydrolase may be rate limiting [ 32,331. A similar role of the phosphohydrolase may be suspected in the yeast system. Incubations were carried out with additions of NaF and CaCl,, which are known inhibitors of the liver phosphohydrolase [33,34]. Further incubations were done with added supernatant which has a stimulating effect with liver systems [ 35-391. As shown in Table IV addition of NaF (10 mM) and CaClz in the concentration range of l-5 mM inhibited the neutral lipid formation from glycerol 3phosphate. At the highest CaCl, concentration (5 mM) the phosphatidic acid synthesis was also inhibited. Addition of a 100 000 X g particle-free supernatant markedly increased the formation of neutral lipids from glycerol 3-phosphate. The supernatant alone does not synthesize neutral lipids, but does synthesize the phosphorylated intermediates with a specific activity of 56 nmol glycerol 3-phosphate incorporated per mg protein per h. When the soluble supernatant was added to lipid particles a nearly 4-fold increase in the neutral lipid to phospholipid ratio was observed. The addition of boiled supernatant slightly stimulated the formation of all products of the phosphatidic acid pathway, probably as a result of addition of a more appropriate mixture of fatty acids for the different acyltransferases involved in triacylglycerol synthesis [ 371.
TABLE
IV
EFFECTS
OF
ACYLATION
F-,
Ca2+,
BY
LIPID
The
values
the
average
of duplicate
DG.
diacylglycerol;
acid;
are
expressed
AND
THE
PARTICLES as nmol experiments. TG,
SOLUBLE OF
sn-glycerol PL,
SUPERNATANT
BAKER’S
3-phosphate
phospholipid
triacylglycerol;
NL,
ON
s~-[~H]GLYCEROL
3-PHOSPHATE
YEAST converted which neutral
per
for more lipid,
which
mg
than
protein 90%
is the
per
represents sum
of
triacylglycerol. PL
DG
system
533
135
(10
461
Conditions Complete + NaF Complete
mM)
system
793
8 112
TG
NL/PL
40
0.21
2
0.02
56
0.21
+ CaClZ
(1 mM)
860
71
41
0.14
+ CaClZ
(2 mM)
825
47
26
0.09
+ CaCl2
(5 mM)
652
19
Complete
system
+ soluble + boiled
supernatant supernatant
(3 mg) (3 mg)
a
0.04 0.44
397
147
28
145
244
41
1.68
433
173
36
0.48
h. and
represent
phosphattidic
diacylglycerol
and
88
Discussion Baker’s yeast has been used extensively for the study of fatty acid synthesis [40]. Phospholipid synthesis has been studied in cell-free particulate fractions [25,41-431, but triacylglycerol synthesis and its regulation in yeast cells has attracted little interest. The present report describes triacylglycerol synthesis by sn-[ 3H]glycerol 3-phosphate acylation and [ 14C]oleate esterification in subcellular fractions of baker’s yeast. Specifically, it has been the main object to establish whether lipid particles were able to synthesize the triacylglycerols which constitute one of their main components. The results of this study show that lipid particles of baker’s yeast contain an active system for the synthesis of triacylglycerol as also shown from other cells [3-61. Compared to the other subcellular fractions the lipid particle fraction has the lowest activities of the marker enzymes, cytochrome oxidase and while it has the highest activities of the NADPH-cytochrome c reductase, enzymes involved in lipid synthesis, acyl CoA synthetase, and triacylglycerol synthesis by glycerol 3-phosphate acylation and oleate esterification. Therefore, the enzymes involved in triacylglycerol synthesis associated with the lipid particles cannot be due to contamination only, but represent a specific attachment of these enzymes to the lipid particles. Also the flotation method used for isolation and purification of the lipid particles minimizes contamination. Finally the specific activity of the acyl-CoA synthetase increases with purification, while the triacylglycerol content of the lipid particle fraction remains constant. The time course of glycerol 3-phosphate acylation indicates that the labelled phosphorylated intermediates in triacylglycerol synthesis obtained with the lipid particle fraction differ from the other subcellular fractions. Thus, phosphatidic acid is the main intermediate and only a small amount od lysophosphatidic acid is formed with the lipid particle fraction, whereas the mitochondrial fraction produces mainly lysophosphatidic acid as the intermediate, and the microsomal fraction produces equal amounts of lysophosphatidic acid and phosphatidic acid. A number of suggestions may be offered in explanation of this difference in activity and in the amounts of phosphorylated intermediates observed between lipid particles and the other subcellular fractions. The differences may be due to the higher activity of acyl-CoA synthetase of the lipid particles. Effects caused by differences in lysophosphatidic acid acyltransferase activity or in phospholipase activities cannot be excluded, but have not been examined. The effects of fluoride, CaCl, and the soluble supernatant on triacylglycerol formation through the phosphatidic acid pathway are in agreement with what has been found in rat liver, where F- and Ca2+ have been reported to inhibit the phosphatidate phosphohydrolase [ 33,341. Our experiments indicate that the action of these ions in yeast cell is at the level of phosphatidate phosphohydrolase. Addition of the soluble supernatant increased the formation of neutral lipid from glycerol 3-phosphate in rat liver mitochondrial and microsomal fractions [35-391. The same effect of the soluble supernatant was observed with lipid particles of yeast cell and suggests that the phosphatidate phosphohydrolase activity may be rate limiting in neutral lipid formation, and
89
that the soluble supernatant contains this enzyme as in mammalian systems. With the lipid particle fraction as enzyme source, triacylglycerol is also formed by acylation of an endogenous acyl acceptor, probably diacylglycerol. The acylation reaction proceeds even under circumstances where a de novo synthesis of triacylglycerol occurs, and the rate of triacylglycerol synthesis is higher by the acylation reaction than by de novo synthesis. The lipid particle fraction contains, in contrast to the other subcellular fractions, diacylglycerol in significant amounts This may explain the difference in the activity between the lipid particles and the other subcellul~ fractions. The acylation reaction was not stimulated by exogenous l,Zdioleylglycerol, but endogenous labelled diacylglycerol could act as a substrate for diacylglycerol acyltransferase. Triacylglycerol synthesis from an endogenous acyl acceptor has also been reported by Johnston and Paltauf [44] to occur in an in vitro study in S. curlsbergensis. An ATP-dependent formation of 14C-labelled t~acylglycerol from [14CJoleate was observed regardless of the presence of added acyl acceptors. However, the pathway for triacylglycerol formation was not established. The amount of triacylglycerol formed from endogenous diacylglycerol, calculated from data [ 14C]oleate esterification is usually greater than the decrease in the amount of diacylglycerol measured chemically. This means that if diacylglycerol is the proposed acyl acceptor, the amount of dia~ylglycerol must be replenished from another reaction, probably by the action of triacylglycerol lipase. Diacylglycerol is in fact an intermediate in the lipolytic degradation of triacylglycerol by the mitochondrial lipase of baker’s yeast [45]. The amount of protein associated the crude lipid particle fraction constitutes only 0.3% of the total protein of a cytoplasmic extract. In comparison the other crude subcellul~ fractions the mitochondrial, the intermediate, the microsomal and the supernatant contribute 19,10, 3 and 63% respectively, tp the total protein of a cytoplasmic extract. The amount of protein associated with triacylglycerol containing particles in vivo is difficult to evaluate, since only 15-20% of the total triacylglycerol of the cytoplasmic extract is recovered in the lipid particle prep~ation. Based on studies of plant and different animal tissue [46,47--491, we assume that a part of the remaining 8O--85% of triacylglycerol is located in cisternae, in vesicles attached to the endoplasmic reticulum, and in minute lipid particles associated the cell organelles. Triacylglycerol in these locations will not float in our centrifugation procedure, and therefore is not isolated with the lipid particles. However, the present study shows that lipid particles (0.3-l pm in diameter) of baker’s yeast can synthesize triacylglycerol de novo, and can introduce fatty acids by acyltransferases into preformed glycerolipid acceptors in the lipid particle. Thus, triacylglycerol can be synthesized and its fatty acid composition changed at the site where they were are stored without the need for transport. Acknowledgement This investigation was supported by the Danish Science Research Foundation grant no. 511-1838. 6 thank Dr. P.K. Jensen and Dr. B. Jensen for valuable comments on the manuscript, and Miss Birthe Nystr$m for skilful technical assistance.
90
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