From the Institute of Molecular Medicine and
§ Department of Internal Medicine, National Taiwan
University Hospital, College of Medicine, National Taiwan University, 7 Chung-Shan South Road, Taipei 100, Taiwan
Received for publication, December 30, 2002, and in revised form, February 19, 2003
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ABSTRACT |
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Acetyl-CoA hydrolase (Ach1p), catalyzing the
hydrolysis of acetyl-CoA, is presumably involved in regulating
intracellular acetyl-CoA or CoASH pools; however, its intracellular
functions and distribution remain to be established. Using
site-directed mutagenesis analysis, we demonstrated that the enzymatic
activity of Ach1p is dependent upon its putative acetyl-CoA binding
sites. The ach1 mutant causes a growth defect in acetate
but not in other non-fermentable carbon sources, suggesting that Ach1p
is not involved in mitochondrial biogenesis. Overexpression of Ach1p,
but not constructs containing acetyl-CoA binding site mutations, in
ach1-1 complemented the defect of acetate
utilization. By subcellular fractionation, most of the Ach1p in yeast
was distributed with mitochondria and little Ach1p in the cytoplasm. By
immunofluorescence microscopy, we show that Ach1p and acetyl-CoA
binding site-mutated constructs, but not its N-terminal deleted
construct, are localized in mitochondria. Moreover, the onset of
pseudohyphal development in homozygote ach1-1
diploids was abolished. We infer that Ach1p may be involved in a novel
acetyl-CoA biogenesis and/or acetate utilization in mitochondria and
thereby indirectly affect pseudohyphal development in yeast.
The concentration of acetyl-CoA in cells is primarily
regulated by its rate of synthesis and its utilization in various
metabolic pathways. In the yeast Saccharomyces cerevisiae,
biosynthesis of acetyl-CoA is mainly achieved by the acetyl-CoA
synthetase reaction, whereas oxidative decarboxylation by the
mitochondrial pyruvate dehydrogenase complex appears to be of minor
importance (reviewed in Ref. 1). Even under glycolytic growth
conditions, S. cerevisiae converts pyruvate into acetate,
catalyzed by the subsequent action of pyruvate decarboxylase,
acetaldehyde dehydrogenase, and acetyl-CoA synthetase (2). In the
presence of a fermentable carbon source, acetyl-CoA may be mainly used
as a precursor of fatty acid and sterol biosynthesis. On the other
hand, an additional pool of acetyl-CoA is required for the glyoxylate
cycle (citrate synthase and malate synthase reactions) when cells grow
with a non-fermentable substrate such as ethanol or acetate.
Acetyl-CoA hydrolase, catalyzing the hydrolysis of acetyl-CoA, was
first identified in the pig heart (3), and subsequently the enzyme has
been found in many mammalian tissues (4-10). During the purification
of yeast N It has been shown that the expression of acetyl-CoA hydrolase
(ACH1) from S. cerevisiae is glucose-repressible
(15) and subjected to cAMP-dependent repression (16). The
function of Ach1p1 in
vivo is still speculative. Previously, we have shown that the
ability of ach1 mutants to grow on acetate is impaired (17). ACH1 is highly homologous to the aarC gene of
Acetobacter aceti (18) and the Neurospora crassa
gene acu8 (19, 20). An acu-8 mutant strain,
characterized as acetate non-utilizing, shows strong growth inhibition
by acetate but will use it as a carbon source at low concentrations
(20). The acu-8 mutant was also shown to be deficient in
acetyl-CoA hydrolase and to accumulate acetyl-CoA when supplied with
acetate. As in Saccharomyces, the Neurospora enzyme is acetate-inducible. The arrC-defective mutant also
showed an inability to assimilate acetic acid (18). However, in all three organisms, disruption of these genes yields strains that grow
normally on ethanol (17, 18, 20). Possibly, the acetyl-CoA balance
during growth on acetate is disturbed in such mutants. Whether or not
acetyl-CoA hydrolase is involved in regulating the endogenous pool(s)
of acetyl-CoA remains to be established. In this study, we took an
initial step to characterize the biochemical property of Ach1p in
vivo and determine its subcellular localization. We demonstrate
that the enzymatic activity of Ach1p is dependent upon its putative
nucleotide (CoA) binding sites and show that Ach1p is a mitochondrial
enzyme. In addition, we provide initial evidence that Ach1p is involved
in development of pseudohyphae but not in mitochondrial biogenesis.
Strains, Media, and Microbiological Techniques--
Yeast
culture media were prepared as described by Sherman et al.
(21). YPD contained 1% Bacto-yeast extract, 2% Bacto-peptone, and 2%
glucose. SD contained 0.7% Difco yeast nitrogen base (without amino
acids) and 2% glucose. Nutrients essential for auxotrophic strains
were supplied at specified concentrations. For comparison of Ach1p
expression in different carbon sources, synthetic media containing 5%
glucose, 2% galactose, 2% glycerol, and 2% potassium acetate were
used. Yeast cells were transformed by the lithium acetate method (22).
Plasmids were constructed by standard protocols as described by
Sambrook et al. (23). Yeast strains YPH250 (MATa ade2, his3, leu2, lys2,
trp1, ura3-52), YPH252 (MAT Expression and Purification of Recombinant Proteins and
Polyclonal Antibody Production--
The open reading frame of
ACH1 was obtained by PCR, by the use of primers that
incorporated unique NcoI and BamHI sites at the
initiating methionine and 6 bp downstream of the stop codon, respectively. For the His-tagged Ach1p, a DNA fragment containing the
ACH1 coding region was generated by amplifying of yeast
genomic DNA with sequence-specific primers. The PCR product was
purified and ligated to the expression vector pET15b (Novagen),
yielding pET15bACH1. The His-tagged fusion protein was synthesized in
BL21(DE3) Escherichia coli and purified on
nickel-nitrilotriacetic acid resin (Qiagen, Chatsworth, CA) as
described (25). Denatured, purified recombinant Ach1p isolated from an
SDS-PAGE gel was used as antigen for raising polyclonal antibodies in
rabbits essentially as described (25).
Preparation of Crude Yeast Lysates and Assay of Acetyl-CoA
Hydrolase Activity--
Crude yeast lysates were prepared, and
acetyl-CoA hydrolase activity was determined by radioactive assay, as
described previously (15). One unit of activity is defined as the
amount of enzyme that hydrolyzes 1 nmol of
[1-14C]acetyl-CoA in 1 min.
Western Blot Analysis and Immunofluorescence
Microscopy--
Yeast total proteins were prepared and subjected to
Western blot analysis as described previously (26). Cells were prepared for immunofluorescence staining as described by Huang et al.
(25). Alexa 594- or Alexa 488-conjugated anti-IgG antibodies (Molecular Probes, Eugene, OR) were used as secondary antibodies. H33258 was
diluted in mounting solution for nucleic acid staining. Fluorescence microscopy was performed with a Nikon Microphot SA microscope.
The monoclonal anti-yeast mitochondria porin antibodies were
purchased from Molecular Probes. The monoclonal anti-yeast ribosomal protein Rpl3p antibody (also called anti-TCM1 antibody) was a gift of
Dr. J. Warner (Albert Einstein College of Medicine, Bronx, NY), and the
polyclonal anti-Kar2p antibody was a gift from Dr. M. Rose (Princeton
University, Princeton, NJ). Polyclonal anti-porin antibody was diluted
1:20,000 for Western blot analysis and 1:5000 for immunofluorescence
staining. The polyclonal antibodies against yeast Arf1p were generated
by the use of recombinant proteins from our laboratory (26).
Subcellular Fractionation--
Yeasts grown in selective minimal
medium or YPD medium were harvested by centrifugation and washed once
with 10 mM NaN3, before Lyticase digestion of
cell walls in a solution of 1.2 M sorbitol and 100 mM potassium phosphate, pH 6.5. Spheroplasts were suspended in buffer containing 0.1 M sorbitol, 20 mM
HEPES (pH 7.4), 50 mM potassium acetate, and 1 mM EDTA with protease inhibitors and disrupted on ice with
20 strokes in a Dounce homogenizer. The lysate was centrifuged
(450 × g) to remove debris and unbroken cells. Cleared
lysate (0.8 ml) was loaded on top of a manually generated six-step
sucrose gradient (0.7 ml each of 60, 50, 40, 30, 20, and 10% sucrose
in lysis buffer), which was then centrifuged at 170,000 × g for 3 h in a Beckman SW55 rotor at 4 °C. Proteins in samples (100 µl) of fractions, collected manually from the top,
were precipitated with 10% trichloroacetic acid, separated by
SDS-PAGE, and analyzed by immunoblotting. Diluted antibodies against
mitochondrial porin (1:500) (Molecular Probes), Kar2 (1:1000), Emp47
(1:5000), and Arf1p (1:5000) (26) were used to identify organelles.
Nycodenz Gradients--
The medium formula, cell treatments, and
fractionated centrifugation were performed as described (27-30) and
with some modifications. Wild-type yeast cells were cultured in 50 ml
of synthetic medium containing 2% glucose at 30 °C with shaking
overnight. Then the overnight culture was harvested and transferred
into 100 ml of oleic acid-containing medium (0.3% yeast extract, 0.3%
peptone, 0.1% oleic acid, 0.2% Tween 40, and 0.5% potassium
phosphate) to induce the formation of peroxisomes. After 24 h, the
yeast cells were harvested and washed once with 0.1 M
potassium phosphate buffer (pH 6.3). Then cells were suspended in 3 ml
of spheroplast solution (1.2 M sorbitol, 0.1 M
potassium phosphate, pH 6.3), 150 µl of Isolation and Fractionation of Mitochondria--
Yeast was grown
in YPGal to early stationary phase. Mitochondria were isolated as
described previously (31). Mitochondria were resuspended in lysis
buffer to give an approximate final concentration of 10 mg of
protein/ml. To isolate the mitochondrial intermembrane space, a
suspension of mitochondria (10-20 mg of protein/ml in 0.6 M sorbitol, 10 mM Tris, pH 7.4) was diluted with 5 volumes of 10 mM Tris, pH 7.4, to a final sorbitol
concentration of 0.1 M. The suspension was incubated at
4 °C with gentle rocking for 20 min. The "shocked" mitochondria
were sedimented at 20,000 rpm in a Beckman SW55 Ti rotor for 20 min.
The supernatant contains the contents of the intermembrane space; then
proteins were precipitated by 10% trichloroacetic acid and analyzed by
Western blotting. To isolate mitochondrial membrane and matrix, shocked
mitochondria were resuspended in 10 mM Tris, pH 7.4, to a
protein concentration of about 2 mg/ml with five strokes in a Dounce
homogenizer and left on ice to allow further swelling of the
mitochondrial matrix space. After 5 min, "shrinking buffer"
(one-third of the suspension volume) containing 1.8 M
sucrose, 8 mM ATP, 8 mM MgC12,
adjusted to pH 7.4 with KOH, was added. The suspension was mixed
carefully by three strokes in the Dounce homogenizer and left on ice.
After 5 min, the suspension was exposed to ultrasonic irradiation for 3 × 5 s on ice. Total mitochondrial membranes were
sedimented for 60 min at 35,000 rpm in a Beckman SW55 Ti rotor at
4 °C. The supernatant represents the matrix fraction; then proteins
were precipitated by 10% trichloroacetic acid and analyzed by Western blotting.
Identification of Endogenous Ach1p--
To characterize the
ACH1 gene product, we prepared a rabbit antiserum against an
E. coli synthesized recombinant full-length His-tagged Ach1p
fusion protein. Among total cellular proteins, antibodies prepared
against Ach1p reacted only with a protein of ~64 kDa, the expected
size for Ach1p (Fig. 1). This protein was
not detected in an ach1 mutant (Fig. 1) or by the preimmune serum (not shown). Immunoblotting with this antiserum detected nanogram
amounts of Ach1p (data not shown) as well as various mutant forms of
Ach1p (Fig. 1A). As in previous RNA blot analysis (15),
Ach1p was subjected to glucose-dependent repression (Fig. 1B).
Putative Nucleotide (CoA) Binding Site Is Required for Ach1p
Activity--
A data base search showed significant homologies among
Ach1p and other CoA-transferases, including Schizosaccharomyces
pombe ACH1 (SpACH1; 64% identity), N. crassa Acu8
(NcAcu8; 57% identity), E. coli ACH1-like
(EcCat1, GenBankTM accession U28377; 38% identity),
A. aceti AarC (AarC; 39% identity), Clostridium
kluyveri CAT1 (CkCat1, succinyl-CoA:coenzyme A transferase; 37%
identity), and C. kluyveri CAT2 (CkCat2, butyryl-CoA-acetate Coenzyme A; 20% identity). The amino acid sequence of the CoA (ADP)
binding site (GXGXX(G/A)) was reported
from an analysis of the known three-dimensional structures of ADP
binding Subcellular Localization of Endogenous Ach1p--
To study the
subcellular distribution of Ach1p, the total yeast
spheroplast-homogenized lysate was fractionated by 30-60% discontinuous sucrose gradient centrifugation. As shown in Fig. 3A, distribution of most of
Ach1p was similar to that of the mitochondrial protein porin, although
little Ach1p was found in cytoplasmic fractions. To determine further
whether Ach1p may also be present in peroxisomes, a homogenate of
oleate-grown cells was first subjected to differential centrifugation
to obtain an organellar pellet. This material was further fractionated
by density gradient centrifugation on Nycodenz. Fig. 3B
shows good resolution between mitochondria (porin marker) and
peroxisomes (thiolase marker), and the distribution of Ach1p was
similar to that of the mitochondrial protein porin. By
immunofluorescence microscopy, endogenous Ach1p, similar to porin,
appeared to be localized to mitochondria (Fig. 3C). Yeast mitochondria can form branched networks distributed evenly around the
circumference of the cell in the peripheral cytoplasm. Abnormal mitochondrial morphology was not seen in ach1 mutant yeast.
To further localize the Ach1p within the purified mitochondria, we analyzed the intermembrane space, matrix, and membrane fractions (Fig.
4). Ach1p cofractionated with Mge1p but
not with cytochrome oxidase subunit IV and porin, indicating that it is
localized to the mitochondrial matrix.
N terminus but Not Putative Nucleotide (CoA) Binding Sites
of Ach1p Are Required for Ach1p Mitochondrial Localization--
Most
proteins targeted to the mitochondrial matrix contain a cleavable
N-terminal presequence with basic and hydroxylated amino acids
interspersed throughout their length (35, 36). Although the N terminus
of Ach1p contains no typical matrix-targeting sequence, we suspected
that deletion of the N-terminal domain from Ach1p might interfere with
its mitochondrial localization. After expression in the ach1
mutant, Ach1pdN, lacking 64 amino acids at the N terminus, was
recovered in the least dense fractions of the lysate (data not shown).
In cells, most of the Ach1pdN mutant was in the cytoplasmic region with
a punctate distribution (Fig. 5), and the
mitochondrial morphology was similar to that in wild-type cells (Fig.
5). In addition, fusion between the N terminus (64 amino acids) of
Ach1p and GFP protein failed to be imported into mitochondria (data not
shown).
We further tested whether overexpression of Ach1p or its mutant
constructs (Ach1pES and Ach1pSS) in yeast might cause dominant-negative effects on mitochondrial morphology. By immunofluorescence microscopy, Ach1pES and Ach1pSS, like overexpressed Ach1p, were present in some
tubular or spherical structures that also stained with anti-porin antibody (Fig. 5). Thus, N terminus, but not putative Co-A binding sites of Ach1p, was required for Ach1p mitochondrial localization.
Ach1p Is Not Required for Mitochondrial Biogenesis--
Because
most of Ach1p is localized in the mitochondria, we next examined
whether Ach1p can affect mitochondrial function. Yeast, when
cultured in glycerol medium, requires mature active mitochondria for
oxidative metabolism and growth. Yeast grown in glucose initially does
not need active, mature mitochondria, and the mitochondria are not well
developed before being switched from anaerobic glucose fermentation to
aerobic ethanol oxidation. The wild-type strain, ach1
strain, and ach1 strains overexpressing Ach1p and its
deleted or mutated constructs (Ach1pES, Ach1pSS, and Ach1pdN) were
cultured overnight in synthetic medium containing glucose. Cells were
harvested, suspended in double distilled H2O, subjected to
serial dilution, and dropped onto glucose, acetate, glycerol,
succinate, and ethanol media plates. Fig.
6 shows that all strains grew on the
glucose, glycerol, succinate, and ethanol media plates; however, the
ach1 mutant and the Ach1pES, Ach1pSS, and Ach1pdN expression
strains did not grow in acetate medium plates. Moreover, the
Ach1p-overexpressing cells grew as well as the wild-type yeast on
acetate plates. These results suggested that Ach1p is not involved in
cellular events in mitochondrial biogenesis, which is required for
cells to grow in non-fermentable carbon sources.
Ach1p Is Required for Pseudohyphal Formation--
Diploid cells of
the yeast S. cerevisiae undergo pseudohyphal differentiation
in response to nutrient limitation (37). We have characterized the
connection between acetyl-CoA changes and pseudohyphal growth.
Wild-type, ach1/ach1 mutant, and ach1/ach1-overexpressing Ach1p
yeast cells were grown in low ammonium sulfate (SLAD; 50 µM) medium to characterize their pseudohyphal
differentiation. Importantly, we found that cells lacking the Ach1p
were completely defective in pseudohyphal differentiation, whereas
ach1/ach1-overexpressing Ach1p restores pseudohyphal growth (Fig.
7). However, expression of Ach1p deleted
or mutated constructs (Ach1pES, Ach1pSS, and Ach1pdN) failed to restore
pseudohyphal growth (data not shown).
In this study, we show that putative conserved nucleotide
(CoA) binding sites and N terminus of Ach1p require its enzyme
activity. Our data also show that Ach1p is localized to the
mitochondria, and the N terminus of Ach1p is required for its
localization. Finally, we show that Ach1p is not involved in
mitochondrial biogenesis but may be involved in pseudohyphal differentiation.
The amino acid sequences of Ach1p and homologous
CoA-transferases contain two conserved CoA (ADP) binding sites
(GXGXX(G/A)) from heterodimeric CoA transferases
(Fig. 2) (9, 33). We determined whether these putative nucleotide (CoA)
binding sites are required for Ach1p activity. Enzyme assays confirmed
that Ach1pSS and Ach1pES expressed in the ach1 mutant
contained little detectable acetyl-CoA hydrolyzing activity. We also
showed that utilization of acetate as carbon source by the
ach1 mutant is impaired, and overexpression of Ach1pES, or
Ach1pSS, cannot restore this activity. Our data indicate that one or
more putative nucleotide (CoA) binding sites are required for Ach1p
enzymatic activity.
The subcellular localization of a protein is an important
characteristic with functional implications. Our data show that most of
Ach1p is localized to the mitochondria, although a little Ach1p was
also found in small punctate form distributed in the cytoplasm. The
majority of mitochondrial matrix-targeting signals are cleaved upon
import into the mitochondria (36). The matrix-located MTF1 protein in
yeast, which is a transcription-stimulating factor (38), is an
exceptional case. This protein lacks a recognizable matrix-targeting
sequence, and its import is reported to be independent of outer
membrane receptors. How specificity of targeting is achieved in this
case and whether there is an entirely separate pathway for importing
this protein remain to be clarified. We demonstrate that N-terminal but
not putative CoA binding sites of Ach1p are required for localization
to mitochondria. Thus, translocation of Ach1p from the cytoplasm to the
mitochondrial matrix may, like that of MTF1, require that the
N-terminal sequence lacks a recognizable matrix-targeting signal. A
recent report described that Ach1p has two different protein spots by
two-dimensional gel electrophoresis (39). These two Ach1ps have the
same relative molecular weight but differ in their pI, suggesting that
Ach1p might be modified post-translationally. However, we attempted,
but failed, to confirm that there are two different protein spots of
Ach1p by two-dimensional gel
electrophoresis.2 Carnitine
acetyltransferase (CAT) is known to be present in mitochondria and peroxisomes of oleate-grown S. cerevisiae, and both
proteins are encoded by the same gene, YCAT (27). We also
speculated whether Ach1p in oleate-grown cells might have a different
subcellular localization. Our data showed that the majority of Ach1p is
present in mitochondria but not in peroxisomes. Thus, we concluded that Ach1p is a mitochondrial enzyme and may execute its physiologic function in the matrix space.
Contemporary knowledge of the structure and function of
acetyl-CoA hydrolases (i.e. cytosolic (8, 40) and
mitochondrial (5)) is incomplete. Ach1p resembles the rat mitochondrial
acetyl-CoA hydrolase, is not affected by ADP or ATP, and is inhibited
by Diploid cells of the yeast S. cerevisiae undergo
pseudohyphal differentiation in response to nutrient limitation (37).
Studies on the way in which nitrogen and carbon starvation induce
invasive and filamentous growth suggest multiple regulatory points in
each pathway and cross-talk between the pathways (41). Changing any single component may rearrange metabolic fluxes in a manner that is
difficult to predict. New approaches to metabolic system modeling and
design are likely to contribute to identifying the particular components that signal invasion and filamentation and offer predictions for how to regulate the intracellular activities of those components. Our study showed that Ach1p was required for pseudohyphal formation, suggesting a physiologic connection between acetyl-CoA changes and
pseudohyphal growth.
In recent studies, sequence alignment of genes with similar regulation
patterns revealed a putative regulatory promoter element (CCWTTSRNCCG)
for the glyoxylate cycle (42). This specific element was present in
seven genes, including CIT2 (citrate synthase in peroxisomal
matrix), ICL1 (isocitrate lyase), MLS1 (malate
synthase in peroxisomal matrix), MDH2 (malate dehydrogenase
in peroxisomal matrix), CAT2 (carnitine acetyltransferase in
peroxisomal matrix and mitochondria), ACR1
(succinate-fumarate transporter in mitochondrial inner membrane), and
ACH1, which were derepressed on ethanol or acetate.
Consistent with this observation, three glyoxylate cycle genes,
ICL1, MLS1, and MDH2, showed the same
regulation pattern as ACH1 (42). In addition,
acu-8 mycelium exhibited no significant flux through the
glyoxylate cycle 10 h after transfer to acetate. Thus, it is
reasonable to speculate that Ach1p may be involved in the glyoxylate
cycle. Recently, Lorenz and Fink (43) showed that live S. cerevisiae cells isolated from the phagolysosome are induced for
genes of the glyoxylate cycle. These findings in fungi, in conjunction
with reports that isocitrate lyase is both up-regulated and required
for the virulence of Mycobacterium tuberculosis, demonstrate
the wide ranging significance of the glyoxylate cycle in microbial
pathogenesis. It will be interesting to learn whether Ach1p, similar to
genes involved in the glyoxylate cycle, can be induced in phagolysosomes.
The role of acetyl-CoA hydrolases catalyzing the scission of the high
energy thioester bond acetyl-CoA with no apparent metabolic advantage
represents a biochemical conundrum. Because acetyl-CoA hydrolase
is highly expressed in yeast when media contain acetate, we suspect
that, under conditions when acetate is used as the main carbon source,
a large amount of acetyl-CoA is generated but not effectively
incorporated into the trichloroacetic acid or glyoxylate cycle. Such an
excess of acetyl-CoA could lead to autoacetylation of proteins, as well
as to the generation of toxic ketone bodies or other noxious
metabolites. It is possible, albeit not yet established, that the
intracellular level of acetyl-CoA could be regulated at a "safe"
level by hydrolysis of excessive acetyl-CoA by acetyl-CoA hydrolase.
Another interpretation is that the ACH1 enzyme, in vivo, is
regulated by its associated factor, which can alter the Ach1p enzyme to
be an acetyltransferase. It has been shown that the distinction between
acyltransferases and thioesterases is quite narrow (44). Moreover,
acetyltransferases, in the absence of the acetyl acceptor, can transfer
the acetyl group from acetyl-CoA to water and act as hydrolases
in vitro. Interestingly, a recent proteomic analysis showed
that exposure of S. cerevisiae to sorbic acid in YEPD
medium, pH 4.5, resulted in the up-regulation of 10 proteins, including
Ach1p (45), suggesting that the induction of Ach1p may confer
resistance to the inhibitory effects of sorbic acid. Furthermore, Ach1p
was indicated to have high homologies (42% identity and 62%
similarity) to C. kluyveri CAT1 (CkCat1,
succinyl-CoA:coenzyme A transferase) and was suggested to be a
succinyl-CoA:CoA transferase. However, the specific enzymatic activity
of C. kluyveri CAT1 in recombinant E. coli clones
was very low (9). We attempted, but failed, to prove that Ach1p has such enzymatic activity.2
In conclusion, this study has confirmed that putative
conserved nucleotide (CoA) binding sites of Ach1p are required for its enzyme activity in vivo. We also demonstrated that Ach1p is
a mitochondrial enzyme, although its potential function in the
glyoxylate cycle needs to be investigated further. We also showed that
Ach1p is not involved in mitochondrial biogenesis, and our data suggest that the metabolism of acetyl-CoA by Ach1p is involved indirectly in
pseudohyphal differentiation. Although yeast can use acetate or ethanol
as carbon source by converting them to acetyl-CoA in the metabolic
pathway, it will be interesting to know how ach1 mutants
could impair acetate but not ethanol utilization. The exact physiologic
role of this mitochondrial Ach1p needs to be investigated further.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-acetyltransferase, an endogenous "inhibitor" of acetyltransferase was purified and shown to be acetyl-CoA hydrolase (11, 12). Acetyl-CoA hydrolase also inhibits purified rat brain pyruvate carboxylase (13) and
[acyl-carrier-protein]acetyltransferase (14).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ade2,
his3, leu2, lys2, trp1,
ura3-52), and INVSc1 (MAT
his3-1,
leu2, trp1-289, ura3-52) were used in this
study. For creating the yeast expression vector encoding Ach1p, the
sequence encoding yeast ACH1 was amplified by PCR and
inserted into the SstI-XbaI site of the yeast
expression vector pVT101U, a 2-µ-based expression plasmid containing
the ADH1 promoter (24). ACH1 constructs containing truncations of the
N-terminal region (Ach1pdN, deletion of 1-64 amino acid residues), the
first mutation of the putative CoA binding site (Ach1pSS, substitution
of amino acids Gly-277 and Gly-279 by Ser-277 and Ser-279), and the
second mutation of the putative CoA binding site (Ach1pES, substitution
of amino acids Gly-393 and Gly-395 by Glu-393 and Ser-395) were made.
All mutations were generated by PCR-based mutagenesis. The sequences of
the resulting constructs were verified by sequencing.
-mercaptoethanol was
added, and the cells were kept at room temperature for 15 min with
gentle rocking. After spinning down and resuspending in 3 ml of fresh
spheroplast solution containing 30 µl of
-mercaptoethanol, 1,000 units of lyticase were added. The cells were mixed gently and incubated
at 30 °C for generation of spheroplasts. The spheroplasts were spun
down, washed once in ice-cold spheroplast solution, and finally
suspended in lysis buffer (0.6 M sorbitol, 5 mM
MES, pH 6.0, 1 mM KCl, and 0.5 mM EDTA). The
spheroplasts were lysed by passage through a 26-gauge needle 20 times
and incubated on ice for 30 min. Unbroken cells and nuclei were removed
by centrifugation at 1,500 × g for 5 min. The
supernatant was centrifuged at 15,000 rpm for 15 min. The crude
organelle pellet, consisting mainly of mitochondria and peroxisomes,
was gently resuspended in lysis buffer and centrifuged at low speed
(600 × g) to remove larger aggregates. The organelle suspension was loaded on a 14-36% Nycodenz gradient to further fractionate mitochondria and peroxisomes. The gradient was centrifuged at 32,500 rpm, 4 °C, for 3.5 h. After centrifugation, the
sample was divided into 14 fractions from 14 to 36% Nycodenz
gradients; then proteins were precipitated by 10% trichloroacetic acid
and analyzed by Western blotting.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Western blot analysis and
enzyme activity of Ach1p and its mutants. As shown in
A, wild-type (WT) yeast, achl mutant,
and achl mutant with expressed Ach1p, Ach1pdN, Ach1pES, and
Ach1pSS were cultured with synthetic medium containing 2% glucose.
Acetyl-CoA hydrolase activity was determined by radioactive assay, as
described previously (15). One unit of activity is defined as the
amount of enzyme that hydrolyzes 1 nmol of
[1-14C]acetyl-CoA in 1 min. As shown in B,
wild-type yeast was cultured with synthetic medium containing 5%
glucose, 2% galactose, 2% glycerol, or 2% potassium acetate. Total
proteins (~20 µg/lane) were separated by SDS-PAGE, stained with
Coomassie Blue (upper panels), or subjected to Western blot
analysis (lower panels). Ach1p were identified with specific
antibody (1:5000). The yeast Arf1p was used as internal control.
Positions of protein standards are indicated on the
left.
-
-
-folds (32). Fig. 2
shows that the conserved CoA (ADP) binding site (GXGXX(G/A)) from heterodimeric CoA transferases
are present in the amino acid sequences of Ach1p and homologous
CoA-transferases (33, 34). To determine whether these putative
nucleotide (CoA) binding sites are required for Ach1p activity, we
generated two mutants, Ach1pSS and Ach1pES, by site-directed
mutagenesis (as described under "Materials and Methods"). Wild-type
Ach1p, Ach1pSS, and Ach1pES were expressed in the ach1
mutant, and their expression was confirmed by Western blotting with
anti-Ach1p antibody (Fig. 1A). Enzyme assays of protein
extracts from these strains confirmed that Ach1pSS and Ach1pES
expressed in the ach1 mutant contained little (<5%)
detectable acetyl-CoA hydrolyzing activity, whereas the untransformed
wild type had normal enzyme activity (Fig. 1A). In addition,
in comparison with wild-type yeast, overexpressing Ach1p in the
ach1 mutant had ~3.8-fold of activity. Because of the low
solubility of E. coli producing Ach1p recombinant proteins, we failed to isolate recombinant Ach1p and its mutant constructs to
assess their enzymatic activity. Previously, we have shown that the
ability of ach1 mutants to grow on acetate is impaired (17).
We next tested whether overexpressed Ach1p and its mutant constructs
have biological function in ach1 mutant yeast. Specific growth rates of tested strains (wild-type strain, ach1
strain, ach1 strains overexpressing Ach1p, Ach1pES, and
Ach1pSS) were obtained by the growth of cells in the synthetic
medium containing acetate, and A600
values were determined at specific time intervals. Overexpressing Ach1p
in ach1 mutant can grow on acetate medium; however, the
ability of the ach1 mutant and strains overexpressing Ach1pES and Ach1pSS to grow on acetate is impaired (data not shown). These data suggest that Ach1pES and Ach1pSS lose their biological activity in vivo.
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Fig. 2.
Aligment of the homologous regions of the
Ach1p and other related Co-A transferases. ScAch1,
S. cerevisiae ACH1; SpAch1, S. pombe
ACH1; NcAcu8, N. crassa Acu8; EcCat1,
E. coli ACH1-like (GenBankTM accession number
U28377); AarC, A. aceti AarC; CkCat1,
C. kluyveri CAT1 (succinyl-CoA:coenzyme A transferase);
CkCat2, C. kluyveri CAT2 (butyryl-CoA-acetate
Coenzyme A). Arrows indicate the site-directed mutagenesis
of the of Co-A binding sites to generate ACH1ES and ACH1SS
mutants.
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Fig. 3.
Ach1p is localized to mitochondria.
A, sucrose gradient centrifugation analyses of Ach1p.
Wild-type yeast cell lysate was subjected to centrifugation in a
discontinuous 30-60% sucrose gradient. Fractions were collected, and
proteins were separated by SDS-PAGE followed by immunoblot analysis by
the use of antibodies against proteins indicated on the left
of each panel. Kar2p, endoplasmic
reticulum protein; Porin, mitochondrial protein;
Arf1p, cytoplasmic protein. B, subcellular
distribution of Ach1p in oleate-grown S. cerevisiae. The
organelle pellet (P15) of oleate-grown cells was prepared by
differential centrifugation and then analyzed by Nycodenz-gradient
centrifugation. Fractions 1-14 correspond to the 14-36% Nycodenz
gradients. The Ach1 distribution was detected by a polyclonal anti-Ach1
antibody at a 1:5000 dilution. The locations of mitochondria and
peroxisomes were detected by anti-mitochondrial porin antibody and
anti-thiolase antibody at 1:1000 and 1:2000 dilutions,
respectively. C, immunofluorescence staining of Ach1p.
Wild-type (WT) and achl mutant were fixed with
formaldehyde; spheroplasts were prepared and reacted with anti-Achlp
antibody (1:1000) and anti-mitochondrial porin antibody (1:50) followed
by secondary antibodies. Nucleic acids were stained with H33258.
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Fig. 4.
Ach1p is localized to
mitochondrial matrix. The mitochondria of yeast were
subfractionated as described under "Materials and Methods." Protein
samples of submitochondrial fractions were subjected to SDS-PAGE,
transferred to polyvinylidene difluoride membrane, and probed with
specific antisera to detect the following proteins: membrane markers
(Porin and cytochrome oxidase IV), matrix marker
(Mge1p), and Ach1p protein. Mem, mitochondrial
membrane proteins; IMS, mitochondrial intermembrane space;
Matx, mitochondrial matrix; MW, molecular weight
markers.
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Fig. 5.
The N terminus, but not putative CoA binding
sites of Ach1p, is required for localization to mitochondria. The
yeast achl mutant cells were transformed with pVTl0lU-ACHl,
pVTl0lU-ACHldN, pVTl0lU-ACH1ES, and pVTl0lU-ACH1SS. Cells were fixed
with formaldehyde; spheroplasts were prepared and reacted with
anti-Achlp antibody (1:1000) and anti-mitochondrial porin antibody
(1:50) followed by secondary antibodies. Nucleic acids were stained
with H33258.
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Fig. 6.
Ach1p is required for acetate utilization but
not for mitochondrial biogenesis. Various Ach1p-expressing cells
were grown and prepared as described under "Materials and Methods."
10-fold serial dilutions of cell suspensions were spotted on synthetic
media containing glucose, acetate, glycerol, succinate, or ethanol as
carbon source. Cells were incubated at 30 °C for 3 days.
WT, wild type.
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Fig. 7.
Ach1p is required for pseudohyphal
development. Diploid wild-type (WT)
(ACH1/ACH1), achl mutant
(ach1/ach1), and Ach1p-overexpressing
(ach1/ach1: pVTl0lU-ACHl) cells were
grown in low ammonium sulfate (SLAD; 50 µM) medium. Cells
were spotted on microscope slides and photographed at ×400
magnification.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
NADH (9, 12). The Km of the Ach1p is similar
to the mitochondrial acetyl-CoA hydrolase from hamster brown fat (6,
12). In addition, the pH optima for Ach1p and rat brain mitochondrial
acetyl-CoA hydrolase are identical (pH ~8) (5). Our data demonstrate
that Ach1p is localized to mitochondria, consistent with the previous
finding that the biochemical properties of Ach1p is similar to those of
mitochondrial acetyl-CoA hydrolases.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. T. Langer and M. Rose for providing us with antibodies. We thank Chih-Hsin Chen for preparing the anti-Ach1p antibody.
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FOOTNOTES |
---|
* This work was supported by grants from the Program for Promoting Academic Excellence of University (Grant EDU-89-FA01-1-4) and from the Yung-Shin Biomedical Research Funds (Grant YSP-86-019) (to F.-J. S. L.).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.: 886-2-2312-3456 (ext. 5730); Fax: 886-2-2395-7801; E-mail: fangjen@ha.mc.ntu.edu.tw.
Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M213268200
2 L.-M. Buu, Y.-C. Chen, and F.-J. S. Lee, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Ach1p, acetyl-CoA hydrolase; MES, morpholine-ethanesulfonic acid; CAT, carnitine acetyltransferase.
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