From the Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
Received for publication, October 19, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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Triacylglycerol is one of the major storage forms
of metabolic energy in eukaryotic cells. Biosynthesis of
triacylglycerol is known to occur in membranes. We report here the
isolation, purification, and characterization of a catalytically active
cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis
from Rhodotorula glutinis during exponential growth. The
complex was characterized and was found to contain lysophosphatidic
acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol
acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier
protein. The 10 S triacylglycerol biosynthetic complex rapidly
incorporates free fatty acids as well as fatty acyl-coenzyme A into
triacylglycerol and its biosynthetic intermediates. Lysophosphatidic
acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol
acyltransferase from the complex were microsequenced. Antibodies were
raised against the synthetic peptides corresponding to lysophosphatidic
acid acyltransferase and phosphatidic acid phosphatase sequences.
Immunoprecipitation and immunolocalization studies show the presence of
a cytosolic multienzyme complex for triacylglycerol biosynthesis.
Chemical cross-linking studies revealed that the 10 S multienzyme
complex was held together by protein-protein interactions. These
results demonstrate that the cytosol is one of the sites for
triacylglycerol biosynthesis in oleaginous yeast.
The de novo biosynthesis of triacylglycerol occurs by
the sequential acylation of glycerol-3-phosphate (1-3).
Glycerol-3-phosphate acyltransferase catalyzes the first step in
glycerolipid synthesis (4), generating lysophosphatidic acid
(LPA).1 Alternatively, LPA is
formed by acylation followed by reduction of dihydroxyacetone
phosphate. Dihydroxyacetone phosphate acyltransferase (5) and
acyl-dihydroxyacetone phosphate reductase (6, 7) catalyze the formation
of LPA from dihydroxyacetone phosphate, respectively. The acylation of
LPA is catalyzed by LPA acyltransferase to form phosphatidic acid (PA),
which is the branch point for the synthesis of diacylglycerol (DAG) and
phospholipids. PA phosphatase catalyzes the dephosphorylation of PA to
DAG that is an immediate precursor of triacylglycerol (TAG),
phosphatidylcholine, and phosphatidylethanolamine. DAG, an important
signal molecule leading to protein kinase C activation (8), can also be
derived from phospholipids by the action of phospholipase C (9). DAG
acyltransferase catalyzes the acylation of DAG, which is a committed
step in TAG biosynthesis. Recently, an acyl-CoA-independent enzyme for
TAG synthesis has been reported in plants and yeast cells that uses
phospholipid as acyl donor and DAG as acyl acceptor. This reaction is
catalyzed by phospholipid-DAG acyltransferase (10) The same reaction
can also be catalyzed by lecithin-cholesterol acyltransferase in yeast (11). All of the enzymes in these pathways are membrane-bound in
eukaryotic systems (1-4, 12, 13). In Saccharomyces
cerevisiae, mitochondrial membranes and endoplasmic reticulum have
been identified as the major sites for phospholipid and TAG synthesis
(3, 6, 14).
Rhodotorula glutinis, a pink budding yeast, accumulates
about 50% dry weight per cell as lipid (15). Due to the large
accumulation of TAG, the biosynthetic enzymes are active in R. glutinis. Here we present for the first time the isolation,
characterization, polypeptide composition, and immunolocalization of a
cytosolic multienzyme complex for TAG biosynthesis in oleaginous yeast
cells during exponential growth phase. This study provides direct
evidence for the cytosol to be one of the sites for TAG biosynthesis.
Understanding the lipid biosynthesis would enable one to genetically
engineer fungi and plants with desired fatty acid composition and the
altered oil content (16).
Materials--
R. glutinis (MTCC 1151) was obtained
from the Institute of Microbial Technology (Chandigarh, India).
[1-14C]Palmitoyl-CoA (51 mCi/mmol),
[1-oleoyl-9,10-3H]LPA ((50 Ci/mmol),
[glycerol-U-14C]PA (100 mCi/mmol),
[2-3H]G3P (12 Ci/mmol), [1-14C]fatty acid
(55 mCi/mmol), and [35S]protein labeling mix were
obtained from NEN Life Sciences. [1-14C]sodium acetate
(56.4 mCi/mmol) was from the Board of Radiation and Isotope Technology
(Mumbai, India). Superose 12 (10/30) FPLC column, gel filtration
molecular mass standards, and ampholines (pH 3-10) were from Amersham
Pharmacia Biotech. Protein assay reagents were obtained from Pierce.
Thin layer chromatography plates were from Merck. All other reagents
were obtained from Sigma.
Growth Conditions--
Yeast cells were grown in malt-yeast
extract medium (pH 7.0) containing 0.3% yeast extract, 0.5% peptone,
0.3% malt extract supplemented with 1% glucose with aeration at
30 °C. Cell density was determined by colony-forming units (one
A600 = 9 × 107 cells).
Nile Blue A Staining--
A smear of cells at various stages of
growth was prepared on a glass slide and heat-fixed. The slides were
immersed in a 1% aqueous solution of Nile blue A stain for 10 min at
55 °C. The slides were washed with water to remove the excess stain
followed by a 1-min wash in 8% acetic acid. The slides were then
rinsed in water, air-dried, and visualized under fluorescence
microscope (× 100 magnification).
Incorporation of [1-14C]Acetate into
TAG--
Yeast cells (8 × 107 cells/ml of
phosphate-buffered saline, pH 7.4) were labeled with 2.5 µCi of
[1-14C]acetate for 3 h. Cells were harvested by
centrifugation, and the cell pellet was washed with ice-cold
phosphate-buffered saline. To the pellet, 0.5 ml of 10% acetic acid in
isopropyl alcohol was added and boiled for 5 min. To the mixture, 1 ml
of hexane was added and vortexed thoroughly (17). The hexane layer was removed and concentrated, and the lipids were separated on silica gel G
thin layer plates developed with petroleum ether/diethyl ether/acetic
acid (70:30:1, v/v/v). Lipids were identified by their migration with
standards and then scraped from the plate and counted in liquid
scintillation counter.
Preparation of Subcellular Fractions--
Logarithmic phase
cells (21 h) were suspended in 10 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 100 µM
leupeptin, and 5% sucrose. The cells were lysed using glass beads
(0.45-0.6 µm) in the absence of detergent. Differential centrifugation was used to fractionate intracellular components. The
supernatant (10,000 × g) thus obtained was centrifuged
at 240,000 × g for 60 min to obtain the soluble
fraction (cytosol). The pellet was washed with the lysis buffer and
centrifuged again at 240,000 × g for 60 min to obtain
membranes. All the operations were carried out at 4 °C.
Enzyme Assays--
The assay mixtures consisted of all of the
components of lysis buffer except protease inhibitors: with labeled
acyl donor, 20 µM [1-14C]palmitoyl-CoA
(100,000 dpm), 5-25 µg of enzyme, and 0.1 mM G3P for G3P
acyltransferase or 50 µM LPA (1-oleoyl) for LPA
acyltransferases or 50 µM 1,2-diolein for DAG
acyltransferase in a total volume of 100 µl. The incubation was
carried out at 30 °C for 30 min and stopped by extracting lipids as
described above. Lipids were separated on silica-TLC plates using
petroleum ether/diethyl ether/acetic acid (70:30:1, v/v/v) and
chloroform/methanol/acetic acid/water (170:25:25:4, v/v/v/v) as the
solvent systems for separating neutral lipids and phospholipids,
respectively. The lipids were visualized by staining with iodine vapor,
and spots corresponding to LPA, PA, DAG, and TAG were scraped off for
measurement of radioactivity. In addition, acyltransferases were also
assayed using labeled acyl acceptors [2-3H]G3P (50 µM, 100,000 dpm) for G3P acyltransferase or
[1-oleoyl-9,10-3H]LPA (50 µM; 150,000 dpm)
for LPA acyltransferase along with 20 µM palmitoyl-CoA.
PA phosphatase activity was measured by monitoring the formation of DAG
from [glycerol-U-14C]PA-dipalmitoyl (1.1 × 105 dpm).
ACP and acyl-ACP synthetase assays were carried out as described (18).
The reaction mixture consisted of 0.1 M Tris-HCl (pH 8.0),
0.4 M LiCl, 5 mM MgCl2, 5 mM ATP, 0.2% Triton X-100, [1-14C]palmitate
(0.25 µCi) and the enzyme source. The reaction mixture was incubated
at 30 °C for 30 min. The amount of labeled acyl-ACP formed was
determined by spotting the reaction mixture on Whatman No. 3MM and
washing the filter paper with chloroform/methanol/acetic acid (3:6:1,
v/v/v) followed by liquid scintillation counting.
Size Exclusion Chromatography--
The soluble fraction was
concentrated (2 mg of protein) and was applied to a Superose 12 FPLC
column fitted with the Bio-Rad BioLogic low pressure chromatography
system. The column was preequilibrated with 10 mM Tris-HCl,
pH 7.5 containing 0.1 M NaCl, and the elution was with the
same buffer at a flow rate of 0.3 ml/min. Fractions (1 ml) were
collected and assayed for TAG biosynthetic enzyme activities (LPA
acyltransferase, PA phosphatase, and DAG acyltransferase).
Sucrose Density Gradient--
The soluble fraction (75 mg) or
the purified complex (25 µg) was layered onto a 10-30% linear
sucrose gradient containing 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 0.1 M NaCl. The tubes were centrifuged for 18 h at 200,000 × g (Beckman
SW 41 rotor), and fractions (1 ml) were collected and assayed for
enzyme activities.
Purification of TAG Biosynthetic Enzyme Complex--
All
operations were conducted at 4 °C except for the FPLC purification
step, which was conducted at ambient temperature. The soluble fraction
from the exponentially growing cells was used for the purification.
Cytosol was loaded on a 7% native polyacrylamide gel and
electrophoresed under constant current at 4 °C. After the run, the
resolving gel was progressively cut into 0.5-cm slices, and the protein
was eluted by finely crushing the gel pieces in 10 mM
Tris-HCl (pH 7.5) buffer containing 0.1 M NaCl, 5 mM MgCl2, and 5% sucrose and incubated
overnight at 4 °C. The gel-eluted protein was used for further studies.
SDS-polyacrylamide gel electrophoresis was performed based on the
method of Laemmli (19). Isoelectric focusing of proteins was performed
using a gradient of pH 3-10 according to the instructions of Amersham
Pharmacia Biotech. Protein was measured by the Bradford method (20).
Protein Sequencing--
The 10 S complex was resolved on a 15%
SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride
membrane. The five proteins were subjected to N terminus
microsequencing by the Edman method. The proteins that did not yield N
terminus sequences were subjected to tryptic digestion, and
microsequencing of the tryptic peptides was performed. Protein
sequencing was performed at the protein sequencing facility at
Rockefeller University.
Antisera Production--
Rabbits were immunized by subcutaneous
injection of 250 µg of purified ACP emulsified in Freund's complete
adjuvant. Three booster doses of 125 µg of protein emulsified in
Freund's incomplete adjuvant were administered at three weekly
intervals. Ten days after the last injection, blood was collected, and
serum was separated and stored at
The major peptide CY-ALELQADDFNK corresponding to LPA
acyltransferase and phosphatidic acid phosphatase peptides (major
peptide CY-NALTGLHMGGGK, and minor peptide C-YVEGARP) were conjugated to bovine serum albumin using
m-maleimidobenzoyl-N-hydroxysuccinimide ester
(21). The conjugated peptides (300 µg) were emulsified and injected
into rabbits. The antibody production, specificity, and titer were
analyzed by enzyme-linked immunosorbent assay (22).
Western Blotting--
Proteins were separated by either native
or SDS-polyacrylamide gel electrophoresis and transferred onto a
nitrocellulose membrane for immunoblotting as described (23).
Peptide-specific antibodies were used at a dilution of 1:600, ACP
antibody at 1:3000, and acyl-ACP synthetase antibody was at 1:400 in
0.1% bovine serum albumin in Tris-buffered saline containing 0.05%
Tween 20.
Protein Labeling and Immunoprecipitation--
Logarithmic phase
R. glutinis cells (107 cells/ml) were labeled
with 30 µCi/ml of [35S]protein labeling mix for 3 h at 32 °C. The labeled cells were lysed and centrifuged at
10,000 × g for 15 min to obtain the lysate. Primary
antibodies were incubated with lysate for 1 h, and the immunocomplex was sequestered with protein A beads. The bound proteins
were analyzed by gel electrophoresis followed by fluorography.
Cross-linking of Proteins--
Cross-linking was carried out as
described (24). In short, preparations of the purified complex (5 µg)
were mixed with disuccinimidyl suberate to a final concentration of 0.5 mM in a total volume of 50 µl. The cross-linking was
performed for 60 min at 4 °C. The reaction was stopped by the
addition of 5 µl of 0.25 M Tris-HCl, pH 7.5. The
cross-linked products were resolved by 6% SDS-PAGE and electroblotted
onto a nitrocellulose membrane. The membrane was probed with anti-ACP
and anti-LPA acyltransferase antibodies for the detection of the
cross-linked products.
Indirect Immunofluorescence--
Logarithmic phase (21 h)
R. glutinis cells were fixed with 4% paraformaldehyde for
20 min followed by 4% formaldehyde for 60 min. The fixed cells were
washed three times with 0.1 M phosphate buffer (pH 5.9) and
resuspended in 50 mg of lytic enzyme with 1.2 M sorbitol
for 6 h at 30 °C to obtain spheroplasts. The cells were washed,
resuspended (107 cells/ml), and plated on 12-mm coverslips,
which were pretreated with poly-L-lysine. The coverslips
smeared with R. glutinis spheroplasts were treated with
ice-cold methanol for 6 min and acetone for 30 s. The primary
antibodies were added at a dilution of 1:20 except for anti-ACP
antibody, which was used at a dilution of 1:200. Secondary antibody
fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate
conjugates were used for localization. The slides were viewed in a
confocal laser-scanning microscope (Leica TCS SP, Heidelberg, Germany)
to locate the TBC.
Growth and TAG Synthesis in R. glutinis--
The growth of the
oleaginous yeast cells was monitored by both
A600 and colony-forming units at 30 °C (Fig.
1, A and B). Both A600 and viable cell count increased
proportionally with time. Analysis of TAG profiles at various growth
periods indicated that TAG accumulation was found even at the early
logarithmic phase (Fig. 1C). Cells stained at various time
intervals with Nile blue A and viewed under a fluorescence microscope,
revealed that 21-h grown cells accumulate low amounts of TAG, whereas
stationary phase cells showed an intense Nile blue A staining (Fig.
1D), indicating large accumulation of TAG. At 21 h, TAG
is present as small oil droplets, which is evident by the fluorescent
staining, whereas in the stationary phase cells, TAG is present as
large oil bodies. To determine the rate of TAG biosynthesis in R. glutinis, cells were metabolically labeled with
[14C]acetate, and its incorporation into free fatty
acids, DAG, TAG, and PL was measured at various growth intervals (Fig.
1E). These results indicated that exponentially growing
(21-h) cultures are active in synthesizing TAG. Therefore, all
subsequent experiments were carried out on 21-h cultures.
Distribution of TAG Biosynthetic Activity--
We investigated TAG
formation in the exponentially growing R. glutinis cells and
found that both the soluble (240,000 × g supernatant)
and the particulate fractions were capable of synthesizing TAG. Table
I summarizes the enrichment of TAG
biosynthetic enzyme activities in the soluble (cytosol) fraction. Of
the various TAG biosynthetic enzymes assayed, the soluble fraction
exhibited high amounts (49-69% total activity) of LPA and DAG
acyltransferases and PA phosphatase activities as compared with the
corresponding enzymes in the particulate fraction (Table I). On the
other hand, a negligible amount of glycerol-3-phosphate acyltransferase
activity could be detected in the soluble fraction. The pattern of
distribution of the enzyme activities remained the same under different
lysis procedures such as French press and sonication (data not shown). In the 21-h grown culture, no discrete fraction of lipid body was
observed upon centrifugation, thus eliminating the possibility of
contamination of TAG biosynthetic enzymes from lipid particles. These
results indicated that an additional TAG biosynthetic pathway could
exist in the soluble fraction. LPA and DAG acyltransferases and PA
phosphatase activities are collectively represented as "triacylglycerol synthase" (TAG synthase).
To demonstrate that the soluble fraction had TAG synthase activity,
this fraction was loaded onto a gel exclusion column (Superose 12).
Most of the TAG synthase activity eluted between 158 and 200 kDa (Fig.
2A). TAG synthase activity
(5%) was also found in the void volume fraction that could be due to
the nonsedimentable membrane fragments and lipid particles generated
during the fractionation procedure. This experiment confirmed that the
TAG biosynthetic activity in R. glutinis is also present in
the cytosol.
The sedimentation value of the purified complex was estimated by
loading the cytosol onto a 10-30% linear sucrose gradient, and the
various fractions were analyzed for TAG synthase activity. The analysis
revealed that the LPA acyltransferase, PA phosphatase, and DAG
acyltransferase activities were associated with one fraction. The
sedimentation value of the active fraction was calculated to be 10 S
(Fig. 2B).
Purification of TAG Synthase--
TAG biosynthetic activity was
found to be high in the soluble fraction and this fraction was
electrophoresed on a 7% native polyacrylamide gel at 4 °C. To
determine the region of the gel corresponding to TAG synthase, the
eluted proteins were assayed for various enzyme activities. LPA
acyltransferase, PA phosphatase, and DAG acyltransferase were detected
in the same region of the gel (Fig. 3,
A and B). To identify the number of bands present in the active gel eluted fraction, the same fraction was
reloaded onto a native gel and a single band was visualized upon silver staining (Fig. 3C). An overall summary of the purification
procedure is shown in Table II. The
native polyacrylamide gel electrophoresis step was effective and
resulted in 469-, 426-, and 409-fold purification of LPA
acyltransferase, PA phosphatase, and DAG acyltransferase, respectively,
with a recovery of 39-56%. The ratio of acyltransferases to PA
phosphatase activity remained constant during purification. Upon
loading this fraction onto a Superose 12 column, the TAG synthase
activity eluted as a single peak with the native molecular size of 180 kDa. The active fraction from the gel filtration column contained LPA
acyltransferase, PA phosphatase, and DAG acyltransferase activities
(data not shown). These results suggested the possibility of a soluble
enzyme complex for TAG biosynthesis in R. glutinis.
Identification of a 10 S Multienzyme Complex--
We wanted to
examine if the enzymes were present as a multifunctional protein or
multienzyme complex. The gel-eluted active fraction containing TAG
synthase was resolved under denaturing and reducing conditions on a
polyacrylamide gel that showed five polypeptides upon silver staining
(Fig. 3D). The purified complex was subjected to isoelectric
focusing (IEF) followed by silver staining, and the profile
showed the presence of five polypeptides, of which four were basic (pI
>8.0) and one was acidic (pI 4.0) proteins (Fig. 3E). These
data, in conjunction with the native PAGE of the purified complex and
sucrose density gradient connote the presence of a 10 S multienzyme
complex for TAG biosynthesis in the cytosol of R. glutinis.
10 S Complex and Its Polypeptide Composition--
To identify the
nature of polypeptides in the complex, the purified 10 S multienzyme
complex was loaded onto a 12% SDS-polyacrylamide gel in the presence
of 0.1% SDS without boiling the sample and electrophoresed at 4 °C.
The gel was cut into 0.5-cm sections, and proteins were eluted from the
gel and assayed for TAG synthase activity. As shown in Figs.
4A and 5A, LPA
acyltransferase and PA phosphatase activities were predominantly found
at the 5th and 4th cm, respectively, and the yield was 7-10%. LPA
acyltransferase and PA phosphatase migrated separately with molecular
masses of 32 and 48 kDa, respectively. DAG acyltransferase activity
could not be localized in the gel. These results indicated that two of
the polypeptides in the 10 S TBC were LPA acyltransferase and PA
phosphatase. The electrophoresed proteins were blotted onto polyvinylidene difluoride membrane; the polypeptides corresponding to
molecular sizes of 32 and 48 kDa were excised and digested with
trypsin; and the tryptic peptide sequences were determined. The
internal sequences (major peptide XALELQADDFNK and minor
peptide XXVNNVXPGXIEQ) of LPA
acyltransferase (32 kDa) did not match with any known sequences in the
data base. Tryptic peptide sequences (major peptide NALTGLHMGGGK and
minor peptide YVEGARPXK) of PA phosphatase (48 kDa) showed
40-100% identity with Homo sapiens PA
phosphatase 2a and 2b isoforms and with Musculus domesticus kidney PA phosphatase.
To confirm that 10 S TBC contained LPA acyltransferase, immunoblots of
native and SDS-polyacrylamide gels of proteins from purified complex,
cytosol, and membranes were probed with polyclonal antibodies raised
against the major internal peptide of the cytosolic LPA
acyltransferase. The antibody recognized the complex in the native
immunoblot (Fig. 4B) and a single band of 32 kDa from the complex and the cytosol in the SDS-polyacrylamide gel immunoblot (Fig.
4B). The same antibody was used for probing the 10 mM CHAPS-solubilized R. glutinis microsomal
membranes and was found to recognize a polypeptide in SDS gel with a
molecular size of 28 kDa (Fig. 4B). Similarly, immunoblots
were carried out on purified complex, cytosol, and solubilized
membranes with polyclonal antibodies raised against two peptides of
cytosolic PA phosphatase (Fig. 5,
B and C). The antibodies for the major and minor
peptides of PA phosphatase recognized a single protein in the cytosol,
which had a molecular mass of 48 and 45 kDa in the microsomal
membranes.
To determine whether the five different proteins identified in the TBC
were held together by physical interactions, logarithmic phase-grown
R. glutinis cells were metabolically labeled with [35S]methionine followed by immunoprecipitation with
antibodies raised to the three peptides. All three peptide-specific
antisera, one to LPA acyltransferase and two to PA phosphatase,
specifically immunoprecipitated the TBC, while normal rabbit serum or
protein A-Sepharose could not immunoprecipitate the complex.
Resolution of the immunoprecipitate by SDS-PAGE followed by
fluorography exhibited five distinct bands corresponding to TBC (Figs.
4C and 5D). Importantly, the fluorograms of both
native and SDS-polyacrylamide gels were identical in all three cases.
The presence of LPA acyltransferase, PA phosphatase, and DAG
acyltransferase was further confirmed by assaying for their activities
in the immunoprecipitate (Table III).
DAG acyltransferase (56 kDa) polypeptide was microsequenced, and the
internal sequence (XLWAVVGAQPFGGARGS) showed 40-80%
identity to the known DAG acyltransferase sequences available in the
data base. The presence of DAG acyltransferase in the TBC was confirmed by assaying for its activity in the immunoprecipitate (Table III). DAG
acyltransferase was found to be the most labile enzyme of the 10 S
multienzyme TBC.
To study the formation of TAG, the purified complex was incubated with
either [14C]palmitic acid in the presence of ATP,
MgCl2, and LPA or [14C]palmitoyl-CoA in the
presence of LPA. Surprisingly, the rate of TAG synthesis was comparable
with palmitic acid or palmitoyl-CoA, indicating that the complex was
capable of activating the fatty acid. The TBC preferred unsaturated
long chain fatty acids over saturated short chain fatty acids. The
order of preference for free fatty acid as substrate by the TBC was as
follows: linoleic > oleic > stearic > palmitic > myristic acids (data not shown). During fatty acid synthesis,
activation of fatty acids was shown to be via the formation of acyl-ACP
in the cytosol (25). Fatty acyl-CoAs were the substrates for TAG
biosynthesis, and this activation was established in the microsomes. To
examine the nature of fatty acid activation by the TBC, ACP was
purified to homogeneity from R. glutinis as described (26),
and polyclonal antibodies were raised to the purified ACP. To ensure
that the 10 S complex contained ACP, immunoblots of native and
SDS-polyacrylamide gels of purified TBC were probed with antibodies to
ACP purified from R. glutinis. The antibodies recognized a
21-kDa protein under denaturing conditions and the complex under
native conditions. The membrane fraction was devoid of ACP (Fig.
6A).
Cross-linking of the 10 S Complex--
To rule out the possibility
of any lipid-protein interactions, the TBC was subjected to
cross-linking using a homobifunctional cross-linker, disuccinimidyl
suberate. Upon probing the cross-linked product with LPA
acyltransferase and ACP antibodies, it was observed that the
cross-linked product migrated at ~200 kDa (Fig. 6B), confirming that the complex was held together by protein-protein interactions and not by lipid-protein interactions.
Immunolocalization of TAG Biosynthetic Complex in the
Cytosol--
To determine the subcellular localization of the TBC,
R. glutinis spheroplasts were probed with anti-LPA
acyltransferase, anti-PA phosphatase major and minor peptide, and
anti-ACP antibodies for indirect immunofluorescence. The staining
pattern revealed the cytosolic nature of TBC (Fig.
7A). PA phosphatase major
peptide antibodies were found to have lower affinity than the
antibodies to the minor peptide. This was also evident upon
immunostaining. The staining pattern for LPA acyltransferase and PA
phosphatase was similar to ACP, which was used as the cytosolic marker.
These results confirmed the cytosolic nature of the TBC. We have
proposed a model for the TAG biosynthesis in R. glutinis
(Fig. 7B).
Where does triacylglycerol biosynthesis occur in oleaginous yeast?
Our data demonstrate that TAG biosynthesis occurs in both the cytosol
and the membrane. The cytosolic fraction has higher total activity than
the membranes (Table I), and that may account for the increased TAG
accumulation in vivo. While there is no direct evidence to
support the possibility that membranes are the only sites for TAG
synthesis, there is also no evidence for the absence of this
biosynthetic pathway in the cytosol. The presence of soluble enzymes
that provide important precursors for triacylglycerol biosynthesis is
well documented. A soluble G3P acyltransferase has been isolated from
cocoa seed (27). PA phosphatase, responsible for dephosphorylation of
PA, is located in the cytosol of S. cerevisiae (28, 29) and
higher plants (30). In developing rapeseed, the presence of a soluble
DAG biosynthetic activity has been demonstrated (31). LPA phosphatase
(32), DAG kinase (33, 34), inactive choline cytidyltransferase (35),
and active ethanolaminephosphate cytidyltransferase (36) have also been
found in the cytosol in animal systems.
The following observations reveal the presence of the cytosolic TAG
pathway in R. glutinis. First, subcellular distribution studies indicate that more than 60% of the total activity is
associated with the soluble fraction (Table I). Second, the enzymes
involved in TAG synthesis are included in the gel filtration column
(Superose 12) as well as 7% native polyacrylamide gel (Fig.
3A). Third, the cytosolic TAG biosynthetic enzymes are
immunolocalized with specific antibodies to LPA acyltransferase and PA
phosphatase (Figs. 4B and 5, B and C).
This leads to the question of how cytosolic TAG biosynthesizing enzymes
function in a hydrophilic environment. The most likely explanation is
that these enzymes exist as a complex for efficient substrate
channeling and to achieve increased accumulation of TAG.
Immunoprecipitation (Figs. 4C and 5D) and
immunolocalization (Fig. 7A) studies carried out with
antibodies raised against the enzymes of the TAG biosynthetic pathway
indicated that these enzymes are indeed present as a complex. Protein
cross-linking studies confirmed the presence of a multienzyme complex.
This also ruled out the possibility that the proteins of the complex
could be held together by lipid protein interactions in the cytosol of R. glutinis. The isoforms of TAG biosynthetic enzymes that
catalyze the same reactions in different subcellular locations may
provide independent regulation at the level of enzyme and gene
expression (37). The segregated pools of lipids may be generated in
different regions of the cell and used for different functions. For
example, PA phosphatase in the endoplasmic reticulum has been shown to be involved in TAG synthesis (38), whereas the same enzyme in the
plasma membrane is involved in signal transduction (39).
The unexpected finding of this study is that the TBC accepts free fatty
acids to form TAG, and the complex consists of ACP, acyl-ACP
synthetase, LPA acyltransferase, PA phosphatase, and DAG
acyltransferase. Acyl-ACP synthetase in prokaryotes has been implicated
in fatty acid and lysophospholipid metabolism but does not provide
acyl-ACP for other intracellular enzymes (40). This enzyme has not been
reported previously in any eukaryotic system.
The demonstration of a cytosolic pathway for TAG synthesis in
oleaginous yeast by sucrose gradient velocity sedimentation, purification, specific antibody interaction, immunoprecipitation, cross-linking, and immunolocalization raises several fundamental questions concerning the currently accepted view of assembly and biogenesis of oil bodies from the endoplasmic reticulum. One
possibility is that oil bodies are synthesized in the cytosol and that
the extent of assembly depends on the local concentration of the newly synthesized TAG (41). The molecular structure of oil bodies in yeast
may be similar to that of plant oil bodies (42, 43).
Finally, is the cytosolic pathway responsible for TAG
accumulation? Preliminary studies on the isolation of mutants
(defective in TAG accumulation) and characterization indicate that it
is the cytosolic pathway that is responsible for TAG accumulation in
oleaginous yeast.2 It may be
advantageous for the cell to produce TAG near the site of fatty acid
synthesis. Our results provide the first direct evidence for the
existence of a soluble 10 S multienzyme triacylglycerol biosynthetic
complex in oleaginous yeast. The isolation of a cytosolic multienzyme
TBC has significant implications in understanding the biosynthesis and
regulation of triacylglycerol accumulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Growth, cell viability, TAG synthesis, and
accumulation. A, exponentially growing cultures of
R. glutinis in malt-yeast extract medium were added to a
final concentration of 1% to fresh medium and incubated at 30 °C.
At regular time intervals, A600 was
measured. B, culture aliquots were taken at regular time
intervals and diluted, and the cell count was taken. Viable cells were
expressed as colony-forming units/ml. C, profile of TAG
accumulation in R. glutinis at the indicated time points was
determined. Cells were isolated at different growth times, and the cell
number was adjusted to 2.0 A600. Lipids were extracted and separated by silica-TLC using a neutral
lipid solvent system as described under "Experimental Procedures."
D, Nile blue A staining of R. glutinis.
Phase-contrast and fluorescence micrographs are shown of R. glutinis cells grown at the indicated growth time intervals.
E, metabolic labeling of yeast cells with
[14C]acetate and its incorporation into TAG, DAG, free
fatty acids, and PL at various time intervals was performed.
Incorporation and analysis of labeled lipids was carried out as
described under "Experimental Procedures."
Distribution of triacylglycerol biosynthetic enzyme activities in
soluble and particulate fractions of R. glutinis
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Fig. 2.
Identification of a soluble triacylglycerol
biosynthetic enzyme complex. A, cytosol was applied
onto a Superose 12 gel filtration column, and the elution profile of
the TAG biosynthetic enzyme activities was determined. B,
the cytosol was subjected to a 10-30% linear sucrose density gradient
centrifugation. LPA acyltransferase, PA phosphatase, and DAG
acyltransferase activities were estimated in 1-ml fractions and found
to be colocalized in both the gel filtration and density gradient
studies.
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Fig. 3.
TAG biosynthetic enzymes exist as a
multienzyme complex in the cytosol. A, native PAGE
(7%) profile of R. glutinis cytosol is indicated in
lane 1. The mobility of electrophoretic markers
is shown on the right (lane 2).
B, cytosol was electrophoresed on 7% native gel, and the
proteins were eluted from the gel pieces. LPA acyltransferase, PA
phosphatase, and DAG acyltransferase activities were measured from the
eluted proteins. The protein eluted from the 2nd cm of the native gel
showed highest TAG synthase activity. C, a single band was
observed upon silver staining when the active fraction containing the
TAG biosynthetic enzymes was re-electrophoresed on a 7% native
polyacrylamide gel. D, native PAGE-eluted active fraction
was analyzed by 12% SDS-PAGE, and five proteins were visualized upon
silver staining (lane 2), indicating the presence
of a multienzyme complex. E, the purity of the multienzyme
complex was confirmed by isoelectric focusing.
Purification of triacylglycerol biosynthetic enzymes from oleaginous
yeast
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Fig. 4.
LPA acyltransferase is a part of the 10 S TAG
biosynthetic complex. A, purified multienzyme complex
was treated with 0.1% SDS and 50 mM dithiothreitol and
electrophoresed on a 12% polyacrylamide gel. The resolving gel was
progressively cut into 0.5-cm slices, and the eluted protein (~20
µg) was assayed for TAG synthase. The 5th cm of the gel exhibited LPA
acyltransferase activity. The molecular size of LPA acyltransferase
(LPAAT) corresponded to 32 kDa. B, a synthetic peptide
corresponding to the sequence of the major peptide of LPA
acyltransferase was conjugated to bovine serum albumin for generation
of polyclonal antibodies. The antiserum was used to probe the TBC,
cytosol, and CHAPS-solubilized membranes after electrophoresis on
native/SDS-polyacrylamide gels and subsequent transfer to
nitrocellulose membrane. TBC and a 32-kDa protein were visualized in
immunoblots under native and denaturing conditions, respectively. This
antibody also reacted with a 28-kDa protein in the solubilized membrane
fraction (lane 4). C,
35S-labeled R. glutinis lysate was
immunoprecipitated with anti-LPA acyltransferase antibody. The
immunoprecipitate was analyzed on 7% native gel as well as 12%
SDS-polyacrylamide gel, and proteins were visualized by fluorography.
Native and SDS-gel fluorographs showed the presence of TBC and five
polypeptides corresponding to the proteins of the TBC, respectively.
Normal rabbit serum was used as the negative control in
immunoprecipitations.
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Fig. 5.
PA phosphatase is a part of the TBC.
A, purified multienzyme complex was treated with 0.1% SDS
and 50 mM dithiothreitol and electrophoresed on a 12%
polyacrylamide gel. The resolving gel was progressively cut into 0.5-cm
slices, and the eluted protein (~20 µg) was assayed for TAG
synthase. The 4th cm of the gel exhibited PA phosphatase activity whose
molecular size corresponded to 48 kDa. B and C,
peptides were synthesized based on the internal sequences of PA
phosphatase and polyclonal antibodies raised to these peptides. TBC,
cytosol, and 10 mM CHAPS-solubilized membrane fraction was
electrophoresed on native/SDS gels and transferred to nitrocellulose
membranes, which were probed with PA phosphatase antibodies. Both of
the antibodies recognized a 48-kDa protein under denaturing and TBC in
nondenaturing conditions. PA phosphatase was found to be present in the
membrane fraction as a 45-kDa protein (lane 4).
D, 35S-labeled R. glutinis lysate was
immunoprecipitated with anti-PA phosphatase (major and minor peptides)
antibodies. The immunoprecipitates were analyzed on 7% native gel as
well as 12% SDS-polyacrylamide gel, and proteins were visualized by
fluorography. Native and SDS-gel fluorographs showed the presence of TBC and five
polypeptides corresponding to the proteins of the TBC, respectively.
Anti-PAPase 1 represents the antiserum to the PA phosphatase major
peptide and anti-PAPase 2 to PA phosphatase minor peptide. Normal
rabbit serum was used as the negative control in
immunoprecipitations.
TAG biosynthesizing capacity of the immunoprecipitate
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Fig. 6.
ACP is a part of the 10 S complex.
A, the presence of ACP in the TBC and in the cytosol was
observed by probing with antibodies for ACP from R. glutinis. The membrane fraction was devoid of ACP (lane
4). B, TBC was cross-linked with DSS, and the
cross-linked product was analyzed by 6% SDS-PAGE followed by
visualization by probing with antibodies to LPA acyltransferase and
ACP. With both of the antibodies, an ~200-kDa cross-linked product
was visualized by Western blotting.
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Fig. 7.
Localization of the multienzyme TAG
biosynthetic complex in R. glutinis. A,
spheroplasts from logarithmic phase R. glutinis were probed
with anti-LPA acyltransferase, anti-PA phosphatase (major and minor
peptides), and anti-ACP antibodies and visualized as described under
"Experimental Procedures." Cytosolic staining was observed with all
of the antibodies. Normal rabbit serum was used as the negative
control. PC, phase-contrast image. B, a schematic
model shows the biosynthesis of TAG in R. glutinis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. P. N. Rangarajan for helpful discussion and support. We acknowledge the Division of Biological Sciences Confocal Microscopy facility for allowing us to carry out indirect immunofluorescence and the help of Dr. P. Sarla.
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FOOTNOTES |
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* This work was supported by the Department of Science and Technology, New Delhi, India.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 91-80-3092881;
Fax: 91-80-3602627; E-mail: lipid@biochem.iisc.ernet.in.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M009550200
2 A. Gangar, S. Raychaudhuri, and R. Rajasekharan, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: LPA, lysophosphatidic acid; ACP, acyl carrier protein; DAG, diacylglycerol; G3P, glycerol 3-phosphate; PA, phosphatidic acid; TAG, triacylglycerol; TBC, triacylglycerol biosynthetic complex; FPLC, fast protein liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PL, phospholipid; FFA, free fatty acids.
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REFERENCES |
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