From the Departments of Biological Chemistry and
¶ Pediatrics, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205 and the § Department of
Physiological Chemistry, Ruhr-Universitat Bochum,
44780 Bochum, Germany
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ABSTRACT |
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A computer-based screen of the
Saccharomyces cerevisiae genome identified
YJR019C as a candidate oleate-induced gene.
YJR019C mRNA levels were increased significantly during
growth on fatty acids, suggesting that it may play a role in fatty acid
metabolism. The YJR019C product is highly similar to tesB,
a bacterial acyl-CoA thioesterase, and carries a tripeptide sequence,
alanine-lysine-phenylalanineCOOH, that closely resembles
the consensus sequence for type-1 peroxisomal targeting signals.
YJR019C directed green fluorescence protein to peroxisomes, and
biochemical studies revealed that YJR019C is an abundant component of
purified yeast peroxisomes. Disruption of the YJR019C gene
caused a significant decrease in total cellular thioesterase activity,
and recombinant YJR019C was found to exhibit intrinsic acyl-CoA
thioesterase activity of 6 units/mg. YJR019C also shared significant
sequence similarity with hTE, a human thioesterase that was previously
identified because of its interaction with human immunodeficiency
virus-Nef in the yeast two-hybrid assay. We report here that hTE is
also a peroxisomal protein, demonstrating that thioesterase activity is
a conserved feature of peroxisomes. We propose that YJR019C
and hTE be renamed as yeast and human PTE1 to
reflect the fact that they encode peroxisomal thioesterases. The
physical segregation of yeast and human PTE1 from the cytosolic fatty
acid synthase suggests that these enzymes are unlikely to play a role
in formation of fatty acids. Instead, the observation that PTE1
contributes to growth on fatty acids implicates this thioesterase in
fatty acid oxidation.
Acyl-CoA thioesterases catalyze the hydrolysis of acyl-CoA
molecules to free fatty acids and CoA. This enzymatic activity is an
intrinsic component of animal fatty acid synthetase and in this context
serves to terminate chain elongation (1). Additional thioesterases may
associate noncovalently with fatty acid synthetase to modify the length
of fatty acids produced by fatty acid synthetase in certain tissues.
For instance, a mammary gland-specific thioesterase has been shown to
direct the production of medium chain fatty acids during lactation (2).
Although these fatty acid synthetase-associated thioesterases
perform a relatively well understood role in cellular metabolism,
eukaryotic cells contain a number of additional thioesterase activities
with less well defined functions. The most notable of these are the
intraorganellar thioesterases of peroxisomes and mitochondria.
Both peroxisomal and mitochondrial acyl-CoA thioesterases have been
reported in mammalian cells (3-7). Peroxisomes and mitochondria do not
contain fatty acid synthetase, and their thioesterases would not be
expected to participate in the synthesis of fatty acids. In fact, the
mere presence of thioesterases in these organelles seems
counterproductive, because the oxidation of acyl-CoAs is a main
function of both peroxisomes and mitochondria. Although the molecular
cloning of mitochondrial fatty acyl-CoA thioesterase has been reported
recently (5), the structural genes for peroxisomal acyl-CoA
thioesterases remain to be identified. Here we report that
YJR019C is a novel fatty acid-induced gene of the yeast
Saccharomyces cerevisiae. This gene encodes a peroxisomal
protein, and both genetic and biochemical evidence demonstrate that the
YJR019C product is an acyl-CoA thioesterase. Loss of
YJR019C interferes with the ability of yeast to grow on
fatty acids, suggesting that this thioesterase plays an ancillary role
in fatty acid oxidation rather than fatty acid synthesis. Furthermore,
we report that this gene is conserved in humans and that the human form
of this gene also encodes a peroxisomal thioesterase. The implications of these results for the role of peroxisomal thioesterases are discussed.
Plasmids--
The YJR019C open reading frame
(ORF)1 was amplified from
S. cerevisiae genomic DNA using the primers
5'-GGGAGATCTATGAGTGCTTCCAAAATGGCG-3' and
5'-CCCGAGCTCAGGCTCCTCCCATTGCGAG-3'. The PCR product was
digested with BglII and SacI (sites underlined)
and cloned into the BglII and SacI sites of the
yeast GFP fusion vector
pGFP-X2 to make pGFP-YJR019C.
The YJR019C ORF was reamplified by PCR from S. cerevisiae genomic DNA using the primers
5'-CCCGTCGACCATGAGTGCTTCCAAAATGGCCATG-3' and
5'-CCCGCGGCCGCTCAGAACTTGGCTCGAATGTCTCG-3'. This PCR
product was digested with SalI and NotI (sites
underlined) and cloned into the SalI and NotI
sites of pMBP, a modified form of the pMALc2 expression vector (New
England Biolabs) to make pJMJ19.
The entire hTE ORF was amplified by PCR from human muscle
cDNA using the primers
5'-CCCGGATCCGGCTCGAGCATGTCGTCCCCGCAGGCCCCAGAAG-3' and 5'-CCCTCTAGAGCGGCCGCCTCTGGCTAAAGCTTGCTCTCTG-3'. A form
of hTE lacking the final two codons of the ORF
(hTE Strains and Media--
All bacterial manipulations were
performed in the Escherichia coli strain DH10B (8). The
yeast strains FY86 (9), BY4733 (10), SKQ2N (11), and BY4733,
pex3 Northern Blot Analysis--
BY4733 cells were maintained in
midlog phase growth for 24-48 h in YPOLT or YPE medium. Two-liter
cultures of cells were then harvested at an A600
of 1.0 and RNA was extracted using standard procedures (12).
Poly(A)+ RNA was purified using Dynabeads according to the
manufacturer's directions (Dynal, Inc.). 0.5 µg of
poly(A)+ RNA was loaded per lane, separated by denaturing
agarose gel electrophoresis, and transferred to nylon membranes.
Filters were prepared for hybridization and hybridized with
radiolabeled DNA fragments using standard protocols (12).
Localization of GFP-YJR019C--
The plasmid pGFP-YJR019C was
used to transform the strain FY86 and its pex3 derivative to
uracil prototrophy. The resulting strains were grown for 24 h in
minimal medium lacking uracil. Cells were washed once in minimal medium
and then transferred to YPOLT and grown for an additional 24 h.
Cells were harvested and attached to cover glasses coated with
poly-L-lysine. The subcellular distribution of GFP-YJR019C
was determined by confocal fluorescence and phase contrast microscopy.
Purification and Amino Acid Sequencing of the YJR019C Gene
Product--
Preparation of high salt-extracted peroxisomal proteins
from oleic acid-induced SKQ2N cells and their further separation by reverse phase HPLC was performed as described by Erdmann and Blobel (11). HPLC fractions were examined by SDS-PAGE. Fractions 26-28 were
pooled, separated on a 12% polyacrylamide gel, and electrophoretically transferred to a polyvinylidene difluoride membrane. Protein bands were
visualized with 0.1% Amido Black in 10% acetic acid and subjected to
amino-terminal sequence analysis (13). The amino-terminal 12 amino
acids of a 40-kDa protein in these fractions corresponded precisely to
the amino-terminal 12 amino acids of the deduced YJR019C sequence.
Growth Curves--
Wild-type BY4733 cells, pex8 cells
(BY4733, pex8 Expression and Purification of Recombinant Proteins--
50-ml
cultures of DH10B cells containing either the pMBP-hTE Whole Cell Lysates and Enzyme Assays--
For preparations of
whole cell lysates, BY4733 cells or their yjr019c derivative
were grown for 24 h in YPD, washed once with water, and
transferred to YPOLT for an additional 24 h. Cells were incubated
in reducing buffer (50 mM KPi, pH 7.4, 1 mM EDTA, and 10 mM 2-mercaptoethanol) for 20 min at 20 °C. The cells were then converted to spheroplasts in
KPi-sorbitol buffer (50 mM KPi, pH
7.4, and 1.2 M sorbitol) using 0.1 unit of Zymolase (ICN
Biomedical) per A600 equivalent of cells for 30 min at 30 °C. Spheroplasts were pelleted and resuspended in 40 ml of
Dounce buffer (5 mM 4-morpholineethanesulfonic acid, pH
6.0, 0.6 M sorbitol, 1 mM KCl, 0.5 mM EDTA, and 0.1% ethanol) supplemented with protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride and 25 µg/ml each aprotinin and leupeptin). The spheroplasts were then
homogenized by 10 passes in a tight-fitting Dounce homogenizer
(Wheaton) followed by brief high intensity sonication. A cleared lysate
was generated by centrifugation at 3000 × g for 10 min
at 4 °C. Total protein concentration in each lysate was determined
by Bio-Rad protein assay (New England Biolabs). 18.1 µg of total
protein from each lysate were added to the assay mixture (100 µM n-decanoyl-CoA, 0.1 mM
5,5'-dithiobis(2-nitrobenzoic acid), 50 mM KPi,
pH 8.0) and the absorbance at 412 nm (A412) was
read at 5-s second intervals for 5 min.
For assays of purified proteins, 0.75 µg of the two recombinant MBP
fusion proteins were added to 50 mM KPi, pH
8.0, containing a mixture of 0.1 mM
5,5'dithiobis(2-nitrobenzoic acid), 20 µg/ml bovine serum albumin,
and five different concentrations (2, 5, 20, 50, and 100 µM) of n-decanoyl-CoA. The
A412 was monitored at 5-s intervals for 2 min.
Recombinant MBP-YJR019C was assayed at 30 °C, whereas recombinant
MBP-hTE was assayed at 37 °C. One unit of thioesterase activity is
defined as the amount of enzyme required to hydrolyze 1 µmol of
substrate/min.
Transfections, Indirect Immunofluorescence, Antibodies, and
Fluorescence Microscopy--
Indirect immunofluorescence studies were
done in transformed human skin fibroblasts (GM5756-T) or the human
hepatoblastoma cell line HepG2. The cell line PBD100 was derived from a
Zellweger syndrome patient, has inactivating mutations in
PEX10, and has been described previously (14). Cell lines
were cultured and transfected as described (15). After electroporation,
cells were resuspended in complete medium, transferred onto cover
glasses, and incubated at 37 °C for 2 days. The cells were fixed,
permeabilized, and processed for indirect immunofluorescence as
described (15). Permeabilization was normally performed for 5 min with
1% Triton X-100, which permeabilizes both plasma and peroxisome
membranes. For selective permeabilization of only the plasma membrane,
the cells were incubated for 5 min in 25 µg/ml digitonin instead of Triton X-100. Rabbit antibodies to hTE were generated against purified
MBP-hTE Immunoblot and Subcellular Fractionation--
For immunoblot
experiments, anti-hTE antibodies were affinity purified using
MBP-hTE YJR019C Is an Oleate-induced Gene--
A screen for potential
fatty acid-regulated genes in yeast identified a consensus oleate
response element (CGGN3TNAN(6-13)(C/G)CG) in
the YJR019C promoter region (Fig.
1).2 To test whether
YJR019C is regulated by fatty acids, we determined the
relative abundance of YJR019C mRNA in yeast grown in
medium containing either oleic acid or ethanol. Both oleic acid and
ethanol are converted to acetyl-CoA before use in further metabolic or biosynthetic pathways. Therefore, any increases in mRNA levels in
yeast grown on oleic acid versus those grown on ethanol
should reflect changes specific to fatty acid metabolism.
Polyadenylated RNA was extracted from log phase yeast grown in either
oleic acid medium (YPOLT) or ethanol medium (YPE) and analyzed by
Northern blot with the YJR019C gene as probe (Fig.
2). Although a significant amount of
YJR019C mRNA was present in ethanol-grown cells, the level of YJR019C mRNA was increased greatly in
oleate-grown cells. The promoter region of YEL020C lacks
ORE-like elements, and its mRNA was present at similar levels in
cells grown in either oleic acid or ethanol medium.
The YJR019C Gene Encodes a Peroxisomal Protein--
Growth
of yeast on fatty acids requires a wide array of peroxisomal
proteins. Because YJR019C was induced by fatty acids and encodes a protein with a PTS1-like sequence at its carboxyl terminus (alanine-lysine-phenylalanineCOOH), we tested whether the
YJR019C gene product might be located in peroxisomes.
Previous studies have established that GFP can be targeted to
peroxisomes by fusion to peroxisomal proteins (18). We therefore fused
the GFP ORF to the amino-terminal end of the YJR019C ORF in
an oleate-inducible expression vector and introduced this vector into
the wild-type yeast strain FY86. The distribution of the GFP-YJR019C
fusion protein was assessed by confocal and phase contrast microscopy (Fig. 3A). GFP fluorescence
was present in discrete punctate structures typical of S. cerevisiae peroxisomes. To determine whether these structures were
in fact peroxisomes, we next examined the distribution of this fusion
protein in a pex3
The identification of YJR019C as a peroxisomal protein was also
accomplished by a direct biochemical approach. In an independent attempt to identify the constituents of peroxisomes, highly purified peroxisomes were isolated from yeast grown in oleic acid medium. Soluble matrix proteins were removed by hypotonic lysis of the peroxisomes, and membrane-associated proteins were released by high
salt wash of the peroxisomal membrane. The proteins released by high
salt extraction were then separated by reverse phase HPLC as described
previously (11). HPLC fractions were separated further by SDS-PAGE,
transferred to a membrane, and visualized by Amido Black staining.
Individual bands were then subjected to amino-terminal sequence
analysis. The amino-terminal sequence of an ~40-kDa protein, which
eluted in fractions 26-28, corresponded to the deduced amino
terminus of YJR019C (Fig. 4). These
results demonstrated that YJR019C is an abundant membrane-associated
component of yeast peroxisomes.
YJR019C Encodes an Acyl-CoA Thioesterase and Contributes to Growth
on Fatty Acids--
Because YJR019C mRNA was highly
induced by oleic acid and encoded a peroxisomal protein, we next tested
whether YJR019C might have a role in growth on fatty acids.
A yjr019c
The deduced product of YJR019C shared significant amino acid
similarity with tesB, a bacterial acyl-CoA thioesterase (20). Disruption of YJR019C resulted in a loss of ~80% of the
total n-decanoyl-CoA esterase activity in yeast cells (Fig.
5B). To test directly whether YJR019C is an acyl-CoA
thioesterase, we expressed and purified YJR019C in fusion with
maltose-binding protein. MBP-YJR019C had significant acyl-CoA
thioesterase activity using n-decanoyl-CoA as substrate,
exhibiting a specific activity of 6.1 units/mg at 30 °C.
The Human Acyl-CoA Thioesterase hTE Is Homologous to
YJR019C--
The amino acid sequence of YJR019C was used to
scan the data base of expressed sequence tags for any human homologues
of this yeast thioesterase. Multiple human expressed sequence tags
were identified, all of which corresponded to a single, previously characterized gene, hTE (21, 22). The deduced amino acid
sequence of hTE shows 26.3% identity to YJR019C and, like
its yeast counterpart, contains a PTS1 motif
(serine-lysine-leucineCOOH) at its carboxyl terminus
(Fig. 6). Although hTE has been shown
previously to have acyl-CoA thioesterase activity, we tested its
activity as an MBP fusion for direct comparison with YJR019C. MBP-hTE
displayed a specific activity of 12 units/mg hTE at 37 °C, an
activity similar to that of MBP-YJR019C.
The hTE Gene Encodes a Peroxisomal Protein--
Given that the
hTE gene product is similar to YJR019C and has a canonical
PTS1 sequence, we tested whether hTE encoded a peroxisomal protein. To address this issue, we first modified the hTE
ORF to include a 10-amino acid myc tag at its 5' end. A plasmid
designed to express this myc-hTE fusion,
pcDNA3-Nmyc-hTE, was introduced into human skin
fibroblasts. Indirect immunofluorescence experiments revealed that
Nmyc-hTE was localized to peroxisomes (Fig.
7A), as determined by
colocalization with the peroxisomal marker protein PMP70 (Fig.
7B). To assess whether Nmyc-hTE was imported into the
peroxisome lumen, we repeated these experiments under differential permeabilization conditions. Skin fibroblasts expressing
pcDNA3-Nmyc-hTE were permeabilized with a limiting
amount of digitonin, which permeabilizes the plasma
membrane but does not permeabilize the peroxisomal membrane. Indirect
immunofluorescent labeling of Nmyc-hTE in digitonin-permeabilized cells
showed only background cytosolic staining, even though PMP70-containing
peroxisomes were readily detected (Fig. 7, C and
D). The PMP70 antibodies that were used in these experiments
recognize the cytosolic domain of this peroxisomal membrane protein.
These results demonstrate that Nmyc-hTE is translocated into the
peroxisome lumen.
Zellweger syndrome is caused by defects in genes that are required for
peroxisome biogenesis, and cells from Zellweger syndrome patients
display defects in peroxisomal matrix protein import (23). As an
independent test of whether hTE is a peroxisomal protein, we expressed
Nmyc-hTE in the Zellweger syndrome cell line PBD100. This cell line is
homozygous for an inactivating mutation in PEX10 and is
unable to import peroxisomal matrix proteins, although it does contain
numerous peroxisomes and imports peroxisomal membrane proteins normally
(14). Nmyc-hTE accumulated in the cytosol of PBD100 cells, as would be
expected for a peroxisomal matrix protein, although intact peroxisomes
were detected using antibodies to PMP70 (Fig. 7, E and
F).
Previous studies identified hTE as a protein that interacts with
HIV-Nef (21, 22). Given that there is no evidence that HIV-Nef is
peroxisomal, we tested whether endogenously synthesized hTE was located
in peroxisomes using antibodies raised against a bacterially
synthesized version of hTE. The immune sera recognize peroxisomes, as
evident from their colocalization with the peroxisomal marker protein
catalase (Fig. 8, A and
B). In contrast, the preimmune sera do not recognize
peroxisomes (Fig. 8, C and D).
The distribution of this mammalian thioesterase was also assessed by
subcellular fractionation experiments. We first generated affinity-purified anti-hTE antibodies and tested whether they recognized the product of the hTE gene. Rabbit reticulocyte
lysates were used to synthesize either Nmyc-hTE or an unrelated
myc-tagged protein, and the two lysates were then examined by
immunoblot. Affinity purified anti-hTE antibodies recognized a protein
of the correct molecular mass only in the lysate in which Nmyc-hTE was
synthesized (Fig. 9A).
Anti-myc polyclonal antibodies confirmed expression of both proteins
(data not shown). Next, a postnuclear supernatant was prepared from rat
liver and separated by Nycodenz density gradient centrifugation.
Fractions across the gradient were assayed for peroxisomal and
mitochondrial markers as well as by immunoblot with the affinity
purified anti-hTE antibodies (Fig. 9B). These antibodies
detected a 36-kDa protein in rat liver peroxisomes, as well as in
cytosolic fractions at the top of the gradient. Peroxisomes will
rupture during homogenization and centrifugation, releasing peroxisomal
matrix proteins to the cytosol. Peroxisome rupture in this experiment
is confirmed by the presence of significant catalase activity in the
fractions at the top of the gradient. Although the amount of
cytoplasmic thioesterase may be partly explained by peroxisome rupture,
we cannot rule out the possibility that a small pool of thioesterase
exists in the cytoplasm at steady state. These results support the
hypothesis that hTE is a peroxisomal protein but do not rule out the
possibility that some hTE may be cytosolic and available to interact
with HIV-Nef.
In this report we have demonstrated that YJR019C is an
oleate-regulated gene that encodes a novel peroxisomal protein.
Furthermore, we find that YJR019C is required for the majority of
acyl-CoA thioesterase activity in yeast cells and show that recombinant YJR019C displays significant acyl-CoA thioesterase activity in vitro. The physical segregation of peroxisomal YJR019C from
cytosolic fatty acid synthetase, the enzyme required for fatty acid
synthesis, provides compelling evidence that the peroxisomal
thioesterase is not involved in fatty acid synthesis. Our finding that
loss of YJR019C results in impaired growth on fatty acids
implicates this gene in fatty acid oxidation, a process that is
exclusively peroxisomal in yeast. The fact that the human homologue of
YJR019C, the previously identified hTE gene, also
encodes a peroxisomal thioesterase suggests that there may be a
conserved role for this enzyme in peroxisomal fatty acid oxidation. We
propose that YJR019C and human hTE both be
renamed PTE1 to reflect the peroxisomal distribution and
acyl-CoA thioesterase activity of their gene products.
Fatty acids must be esterified with CoA before their This indirect model for PTE function has the potential to explain the
fact that loss of PTE1 impairs growth of yeast on fatty acids, but we
readily admit that there is not yet any independent corroborating
evidence in its favor. As such, it should be viewed with skepticism. At
the same time, it is worthwhile to consider this model as a possible
explanation for the role of mammalian PTE1. If CoASH depletion does
serve to inhibit peroxisomal fatty acid An alternative role for mammalian peroxisomal thioesterases has also
been suggested (7). Peroxisomal The identification of PTE1 in both yeast and human cells raises the
question of whether they correspond to previously described peroxisomal
enzymes. This is clearly not the case for yeast PTE1, because this
report is the first description of this enzyme activity in yeast
peroxisomes. However, there have been several reports of peroxisomal
thioesterases in mammalian cells (4, 7). A purification of rat liver
peroxisomal thioesterases revealed that myristoyl-CoA thioesterase
activity eluted in two peaks, one of ~35 kDa and one of larger size
(4). The product of human PTE1 has a deduced Mr
of 36 kDa and may represent the human homologue of the smaller rat
peroxisomal thioesterase. A second study found peroxisomal thioesterase
activity to be present in a broad peak of ~40 kDa (7). However,
subsequent fractionation of this sample led to the purification of a
single thioesterase of ~46 kDa. Interestingly, the putative cytosolic
thioesterase 1 contains a PTS1-like sequence at its carboxyl terminus,
proline-lysine-isoleucineCOOH, and has a deduced
Mr of 46 kDa (30). A further analysis of
cytosolic thioesterase 1 distribution may resolve the question of
whether it actually represents a second, 46-kDa thioesterase of peroxisomes.
The localization of human PTE1 (hTE) to the peroxisome lumen was
observed for an overexpressed, epitope-tagged version of HsPTE1 as well as for the endogenously synthesized,
wild-type HsPTE1 of human fibroblasts. Thus, there is little
doubt that this protein is predominantly peroxisomal. However, it is
also true that human PTE1 (hTE) was first identified because of its interaction with HIV-Nef in the yeast two-hybrid system (21, 22). There
is no indication that Nef is associated with peroxisomes, nor is there
any known role for peroxisomes in the life cycle of HIV. Nevertheless,
the interaction between PTE1 (hTE) and HIV-Nef was also detected in
lysates of CEM cells that stably expressed HIV-Nef. Although the
PTE1-Nef interaction might have occurred after the formation of the
cell-free lysate in these experiments, it is also quite possible that
small amounts of PTE1 are present in the cytosol. Our fractionation
data cannot rule out the possibility that some PTE1 may be cytoplasmic
at steady state and available for interaction with HIV-Nef. Also, it is
possible that HIV-Nef may bind PTE1 before its import into peroxisomes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
KL) was amplified using the oligonucleotide
5'-CCCTCTAGAGCGGCCGCCTCTGGCTAAAGCTAGCTCTCTG-3' in
conjunction with the first hTE primer above. These
oligonucleotides append BamHI and XhoI sites to
the 5' end and XbaI and NotI sites to the 3' end
of the ORF. The PCR product was digested with BamHI and
XbaI and cloned into the corresponding sites in pMALc2. The resulting plasmids were denoted pMBP-hTE for the complete
hTE ORF and pMBP-hTE
KL for the truncated form. The
sequences of all ORFs in pMBP and pMALc2 were confirmed by automated
fluorescent sequencing. The BamHI-XbaI fragment
from pMBP-hTE was excised and transferred to the mammalian expression
vector pcDNA3-Nmyc, a modified version of pcDNA3 (Invitrogen).
This plasmid contains the sequence
5'-AAGCTTACCATGGCGGAGCAGAAGCTGATCTCCGAGGAGGACCTGCTGGGATCC-3' between the HindIII and BamHI sites of pcDNA3
and is designed to express proteins in fusion with an amino-terminal
myc epitope.
::HIS32 have been
described. The yeast strain BY4733,
yjr019c
::HIS3 was generated by
PCR-mediated, one-step gene replacement as described by Baker-Brachmann
et al. (10) using the oligonucleotides YJR019Cko.5 (5'-AGTATCCACCATGAGCAAGACAAGATAAGACAAGATTGCATAAAAAGATTGTACTGAGAGTGCAC-3') and YJR019Cko.3
(5'-ATAAGAATATATATGTATGTGTTTATACGTGGGAGGGAATTGTCCCTGTGCGGTATTTCACACCG-3'). Disruption of YJR019C was confirmed by PCR analysis of HIS3+
clones. For routine culture, yeast strains were grown on either rich
medium (YPD: 1% yeast extract, 2% bacto-peptone, and 2% dextrose) or synthetic medium (SC: 0.17% yeast nitrogen base, 0.5%
(NH4)2SO4, 2% dextrose,
supplemented with amino acids and nucleotides appropriate for
each experiment). For analysis of RNA levels, growth on ethanol was in
YPE (1% yeast extract, 2% bacto-peptone, and 2% ethanol) and growth
on oleic acid was in YPOLT (1% yeast extract, 2% bacto-peptone, 0.02% Tween 40, and 0.2% oleic acid). For growth curves, growth on
oleic acid was in SYOLT (0.5% yeast extract, 0.5%
(NH4)2SO4, 0.17% yeast nitrogen
base, 0.02% Tween 40, and 0.2% oleic acid).
::HIS3), and
yjr019c cells (BY4733,
yjr019c
::HIS3) were maintained in
midlog phase for 2 days in YPD or selective medium. Cells were washed with water and used to inoculate 30 ml of SYOLT medium at an initial A600 of 0.001. Growth was assessed by removing
1-ml aliquots, washing the cells twice with water, and measuring the
A600. Aliquots were removed at 24-h intervals
for 168 h.
KL or the
pMBP-YJR019C plasmid were grown overnight at 37 °C in Luria broth
supplemented with 100 µg/ml ampicillin. 40 ml of this culture were
diluted into 1 liter of 2YT media (12) supplemented with 0.2% glucose
and 100 µg/ml ampicillin. This culture was grown at 37 °C
until the A600 reached 0.4, at which time
isopropyl-1-thio-
-D-galactopyranoside was added to 1 mM to induce protein expression. The induced culture was
incubated at 37 °C for 4 h for cells containing pMBP-hTE
KL or 30 °C for 15 h for cells containing pMBP-YJR019C. Cells were harvested, washed with Luria broth, and resuspended in 25 ml of amylose
column buffer (20 mM Tris-HCl, pH 7.5, 200 mM
NaCl, 1 mM EDTA, and 10 mM 2-mercaptoethanol).
The cell suspension was incubated on ice with 0.4 mg/ml lysozyme for 20 min. After this incubation the cells were frozen in liquid
N2, thawed at 37 °C, and sonicated briefly at high
intensity. This cycle of freezing, thawing, and sonication was repeated
twice for a total of three cycles. A cleared lysate was generated by
centrifugation at 17,500 × g for 20 min. Proteins were
purified by one-step affinity chromatography using a 10-ml
amylose-agarose (New England Biolabs) column according to the
manufacturer's instructions. Fractions were analyzed by SDS-PAGE, and
those containing highly purified (>90% purity) MBP-YJR019C (Mr
83,000) or MBP-hTE
KL
(Mr
79,000) were pooled and precipitated with 0.4 g/ml (NH4)2SO4. Aliquots
of the purified fusion proteins were stored at
70 C until needed.
KL. Immune serum was used directly for immunofluorescence experiments. Rabbit anti-PMP70 antibodies were obtained from Dave Valle
and Gerardo Jimenez-Sanchez. The anti-myc mouse monoclonal antibody,
anti-catalase sheep monoclonal antibody, and fluorescent secondary
antibodies were obtained from commercial sources.
KL covalently linked to CNBr-activated Sepharose resin
(Amersham Pharmacia Biotech). The protein-coupled column was prepared
and used according to the manufacturer's specifications. Immune serum
was diluted 1:15 in TBST (25 mM Tris-HCl, pH 7.5, 140 mM NaCl, and 0.1% Tween 20) and passed over the column.
The column was washed with 30 bed volumes of TBST, and the purified anti-hTE antibodies were eluted with 100 mM glycine, pH
2.5, neutralized with 1 M Tris-HCl, and precipitated with
0.4 g/ml (NH4)2SO4 for storage.
Anti-myc polyclonal antibodies were obtained from Santa Cruz
Biotechnology. Expression of proteins in rabbit reticulocyte lysates
(Promega) was according to the manufacturer's instructions, using the pcDNA3-Nmyc plasmid containing either the hTE
open reading frame or that of an unrelated peroxisomal enzyme.
Subcellular fractionation of rat liver was as described by Mihalik
(16). Immunoblots were performed as described by Crane et
al. (17).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and predicted protein sequence of
the YJR019C gene. The open reading frame is shown with
500 bp of flanking sequence at both the 5' and 3' ends. A consensus
oleate response element (underlined) is present from
nucleotides 130 to
108 relative to the first nucleotide of the
YJR019C open reading frame. The 1050-bp open reading frame,
which terminates in the atypical type 1 peroxisomal-targeting sequence
alanine-lysine-phenylalanineCOOH (underlined),
is predicted to encode a basic protein (pI, 8.82) with a mass of 40.2 kDa.
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Fig. 2.
YJR019C is an oleate-induced gene.
Northern blots containing 0.5 µg of poly(A)+ RNA/lane
were probed with radiolabeled fragments of the YJR019C
(upper panel) and YEL020C (lower
panel) genes. Lane 1, 0.5 µg of poly(A)+
RNA from log phase ethanol-grown cells. Lane 2, 0.5 µg of
poly(A)+ RNA from log phase oleate-grown cells.
derivative of FY86 (PEX3 is required for peroxisomal matrix protein import; Ref. 19). In the
pex3 strain, GFP fluorescence was found throughout the
cytoplasm (Fig. 3B), a distribution that is expected for a
peroxisomal matrix protein.
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Fig. 3.
YJR019C targets GFP to peroxisomes in a
PEX3-dependent manner. YJR019C with a GFP
tag fused to its amino terminus was expressed in wild-type yeast cells
(A) and a pex3 mutant (B). The
distribution of GFP-YJR019C was examined by confocal phase contrast
(left panels) and fluorescence (right panels)
microscopy. Scale bar, 5 µm.
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Fig. 4.
Preparative chromatographic separation of
high salt extract of peroxisome membranes for microsequence
analysis. Highly purified peroxisomes were disrupted by hypotonic
lysis, and soluble matrix proteins were removed by low salt extraction.
Membrane-associated proteins were released by high salt extraction and
separated by reverse phase HPLC. Fractions 26-47 were further
separated by SDS-PAGE and visualized by Coomassie Blue staining. The
position of the YJR019C gene product is indicated by an
arrowhead. The amount per lane corresponds to 5% of the
total fraction. Molecular mass standards are indicated on the
left.
derivative of BY4733 was created, and its
growth on oleic acid medium was compared with that of the wild-type
BY4733 strain and a pex8 derivative of BY4733. The
yjr019c cells exhibited a partial growth defect, growing to
53% of the final wild-type density, whereas the pex8 strain
showed the typical pex phenotype, growing to ~20% of
wild-type density (Fig. 5A).
Thus, YJR019C appears to play an ancillary role in growth on
fatty acids.
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Fig. 5.
YJR019C is an acyl-CoA thioesterase involved
in fatty acid metabolism. A, the growth of wild-type
(open circle), yjr019c (filled
circle), and pex8
(open triangle) cells
on oleic acid was monitored spectrophotometrically over 168 h. The
yjr019c
cells grew to 53% of the final wild-type
density. B, lysates were prepared from oleate-grown
wild-type (WT) and yjr019c
cells. Equal
amounts of protein from each lysate were assayed for acyl-CoA
thioesterase activity. The lysate of the wild-type strain had a
specific activity of 1.8 × 10
2 unit/mg, whereas the
lysate of yjr019c
strain had a specific activity of
4.0 × 10
3 unit/mg.
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Fig. 6.
Amino acid alignment of YJR019C and hTE
proteins. Sequence alignment was performed using DNASTAR (Madison,
WI) and the PAM 250 matrix. Identical residues are
boxed.
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Fig. 7.
hTE is a peroxisomal matrix protein.
Human skin fibroblasts expressing Nmyc-hTE were processed for double
indirect immunofluorescence by fixing cells and permeabilizing with 1%
Triton X-100. The distribution of Nmyc-hTE was examined using anti-myc
(A) and anti-PMP70 (B) antibodies. Additional
cells from the same set expressing Nmyc-hTE were permeabilized with 25 µg/ml digitonin and examined again using anti-myc (C) and
anti-PMP70 (D) antibodies. The distribution of Nmyc-hTE was
also examined in the PEX10-deficient cell line PBD100, again
by double indirect immunofluorescence using anti-myc (E) and
anti-PMP70 (F) antibodies. Scale bar, 25 µm.
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Fig. 8.
Endogenously synthesized hTE colocalizes with
the peroxisomal matrix enzyme catalase. HepG2 cells were processed
for double indirect immunofluorescence using anti-hTE antiserum
(A) or preimmune serum (C) and anti-catalase
(B and D) antibodies. Scale bar, 25 µm.
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Fig. 9.
Anti-hTE antibodies recognize a 36-kDa
protein in peroxisomal and cytoplasmic subcellular fractions.
A, rabbit reticulocyte lysate was used to synthesize
Nmyc-hTE (lane 1) or an unrelated peroxisomal protein
(lane 2). Equal amounts of each lysate were separated by
SDS-PAGE and analyzed by immunoblot with affinity-purified anti-hTE
antibodies. B, postnuclear supernatant from rat liver was
fractionated by Nycodenz density centrifugation. Equal amounts of
fractions were assayed for catalase (dark bars) and
succinate dehydrogenase (light bars) activity. Equal amounts
of fractions were also analyzed by immunoblot using affinity-purified
anti-hTE antibodies. The bar graph shows the relative amounts of the
peroxisomal and mitochondrial marker enzyme activities in each
fraction. The lower panels show the distribution of the
~36 kDa rat protein recognized by the anti-hTE antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
oxidation.
Thus, it is somewhat paradoxical that a peroxisomal acyl-CoA thioesterase is required for normal growth of yeast on fatty acids. In
fact, our current understanding of peroxisomal fatty acid oxidation in
yeast provides no direct role for thioesterase activity in fatty acid
oxidation or growth on fatty acids. Therefore, it is important to
consider possible indirect roles for PTE1 in fatty acid oxidation.
Previous studies have established that the peroxisome membrane is
impermeable to small molecules such as NAD and NADP (24, 25). As a
result, loss of enzymes involved in NAD regeneration lead to loss of
NAD-dependent activities within the peroxisome, including
fatty acid oxidation. If the peroxisome membrane is also impermeable to
CoASH, then the intraperoxisomal free CoASH pool may also be dependent
on constant regeneration. Previous studies in yeast have established
that CoASH regeneration would occur via complete oxidation of acyl-CoAs
to acetyl-CoA, followed by transfer of acetate to carnitine and release
of CoASH in a reaction catalyzed by CAT1 (26). However, if CoA were
appended to poorly metabolized or nonmetabolizable fatty acids, CoASH
levels would be expected to fall as more and more CoA was incorporated into these metabolic sinks. In this scenario, a peroxisomal
thioesterase that acted primarily at high acyl-CoA and/or CoASH levels
might serve a stimulatory role in fatty acid oxidation by providing an
alternative mechanism for generating the free CoASH that is necessary
for fatty acid
oxidation. As for the fate of the fatty acids that
may be released, these would be free to equilibrate with cellular and
extracellular pools, reducing their concentration in the peroxisome.
oxidation in yeast, we
might also expect CoASH depletion to pose problems for fatty acid
oxidation in mammalian peroxisomes and mitochondria. In fact, a similar
hypothesis has been proposed to explain the function of rat
mitochondrial thioesterase (6).
oxidation in mammalian cells
differs from the yeast system in that it does not oxidize fatty acids
completely. Instead, it transfers medium chain fatty acids out of the
peroxisome as acyl-carnitine (27). These are then exported to
mitochondria where their oxidation is completed. This facet of
mammalian peroxisomal fatty acid oxidation led to the hypothesis that
peroxisomal thioesterases may serve to regulate the chain length at
which fatty acids are exported (7). However, there are three flaws with
this model. First, mammalian peroxisomal carnitine acyltransferase
accepts fatty acids in the form of CoA esters, not as free acids (28).
Thus, it is unclear how a thioesterase would contribute to
acyl-carnitine export. Second, the chain length specificities of
mammalian peroxisomal
-oxidation enzymes are thought to be
sufficient to control the extent to which fatty acyl-CoAs are shortened
in peroxisomes (7, 27). Third, this model cannot explain the presence
of a thioesterase in yeast peroxisomes, because they oxidize fatty
acids to completion (29).
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ACKNOWLEDGEMENTS |
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We thank Jianwu Bai and James Morrell for technical assistance and Brian Geisbrecht, Katie Sacksteder, and Stephanie Mihalik for assistance with subcellular fractionation experiments.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK45787 (to S. J. G.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AFI24264 and AFI24265.
To whom correspondence should be addressed: Dept. of
Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3085; Fax: 410-955-0215; E-mail: Stephen.Gould{at}qmail.bs.jhu.edu.
2 M. T. Geraghty, D. Bassett, J. C. Morrell, G. J. Gatto, Jr., J. Bai, B. V. Geisbrecht, P. Hieter, and S. J. Gould, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: ORF, open reading frame; PCR, polymerase chain reaction; GFP, green fluorescent protein; MBP, maltose-binding protein; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; HIV, human immunodeficiency virus.
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REFERENCES |
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