Characterization of the 70-kDa Peroxisomal Membrane Protein,
an ATP Binding Cassette Transporter*
Tsuneo
Imanaka
§,
Kazutoshi
Aihara
,
Tatsuya
Takano¶,
Atsushi
Yamashita
,
Ryuichiro
Sato**,
Yasuyuki
Suzuki
,
Sadaki
Yokota§§, and
Takashi
Osumi¶¶
From the
Department of Biological Chemistry, Faculty
of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical
University, 2630 Sugitani, Toyama 930-0194, ¶ Departments of
Microbiology and Molecular Pathology and
Hygienic Chemistry and
Nutrition, Faculty of Pharmaceutical Sciences, Teikyo University,
Sagamiko, Kanagawa 199-0145, ** Laboratory of Biochemistry and Molecular
Biology, Graduate School of Pharmaceutical Sciences, Osaka University,
Suita, Osaka 565-0871, 
Department of
Pediatrics, Gifu University School of Medicine, Tsukasa, Gifu 500-8076, §§ Biological Laboratory, Yamanashi Medical
University, Tamaho, Yamanashi 490-3898, and ¶¶ Department of
Life Science, Himeji Institute of Technology, Kamigori,
Hyogo 678-1297, Japan
 |
ABSTRACT |
The 70-kDa peroxisomal membrane protein (PMP70)
is one of the major components of rat liver peroxisomal membranes and
belongs to a superfamily of proteins known as ATP binding cassette
transporters. PMP70 is markedly induced by administration of
hypolipidemic agents in parallel with peroxisome proliferation and
induction of peroxisomal fatty acid
-oxidation enzymes. To
characterize the role of PMP70 in biogenesis and function of
peroxisomes, we transfected the cDNA of rat PMP70 into Chinese
hamster ovary cells and established cell lines stably expressing PMP70.
The content of PMP70 in the transfectants increased about 5-fold when
compared with the control cells. A subcellular fractionation study
showed that overexpressed PMP70 was enriched in peroxisomes. This
peroxisomal localization was confirmed by immunofluorescence and
immunoelectron microscopy. The number of immuno-gold particles
corresponding to PMP70 on peroxisomes increased markedly in the
transfectants, but the size and the number of peroxisomes were
essentially the same in both the transfectants and the control cells.
-Oxidation of palmitic acid increased about 2-3-fold in the
transfectants, whereas the oxidation of lignoceric acid decreased about
30-40%. When intact peroxisomes prepared from both the cell lines
were incubated with palmitoyl-CoA, oxidation was stimulated with ATP,
but the degree of the stimulation was higher in the transfectants than
in the control cells. Furthermore, we established three Chinese hamster ovary cell lines stably expressing mutant PMP70. In these cells,
-oxidation of palmitic acid decreased markedly. These results suggest that PMP70 is involved in metabolic transport of long chain
acyl-CoA across peroxisomal membranes and that increase of PMP70 is not
associated with proliferation of peroxisomes.
 |
INTRODUCTION |
Peroxisomes are organelles bounded by a single membrane that are
present in almost all eukaryotic cells. The peroxisomes are involved in
a variety of metabolic processes including peroxide-based respiration,
oxidative degradation of fatty acids and purine, and synthesis of
plasmalogen and bile acids (1). Early studies on rat liver peroxisomes
led to the concept that peroxisomes are freely permeable to compounds
of low molecular weight (2, 3). However, Van Roermund et al.
(4) suggested recently that some metabolites were unable to permeate
the membrane of peroxisomes in Saccharomyces cerevisiae. The
peroxisomal membrane in human fibroblasts has been also shown not to be
freely permeable to at least one of the substrates of
acyl-CoA:dihydroxyacetonephosphate acyltransferase, unless ATP is
present (5). Therefore, the exchange of metabolites between peroxisomes
and cytosol seems to require specific transporters.
The 70-kDa peroxisomal membrane protein
(PMP70)1 is a major component
of peroxisomal membranes and markedly induced by the administration of
hypolipidemic agents, and its induction parallels peroxisome
proliferation and induction of peroxisomal
-oxidation enzymes (6,
7). We cloned and sequenced rat PMP70 and found that it was an ATP
binding cassette (ABC) transporter (8). ABC transporters are a
superfamily of membrane-bound proteins whose structure and function
have been highly conserved from eubacteria to mammals and catalyze the
ATP-dependent transmembrane translocation of a wide variety
of substrates, including anti-tumor drugs and several kind of lipids
(9, 10). They are composed of two homologous halves, each containing
the following two parts: a domain containing six potential
transmembrane segments and an ATP binding domain, which contains a
particular type of nucleotide binding fold. PMP70 is half the size of
an ABC transporter and seems to function as a dimer.
To date, four peroxisomal ABC transporters have been identified in
mammalian peroxisomes: PMP70 (8, 11, 12), adrenoleukodystrophy protein
(ALDP) (13), ALDP-related protein (ALDRP) (14, 15), and PMP70-related
protein (P70R/PMP69) (16, 17). A defect in ALDP is known to be
responsible for the X chromosome-linked neurodegeneration disorder
adrenoleukodystrophy; this defect is an inborn error of peroxisomal
-oxidation of very long chain fatty acids (18, 19). Mutations in the
PMP70 gene have been identified in one complementation group (group 1)
of Zellweger syndrome, an inborn error of peroxisome biogenesis, and it
is suggested that PMP70 has an important role in peroxisome assembly (12). However, a recent study of complementation group 1 probands by
Southern blotting and single-strand conformation polymorphism (SSCP) of
reverse transcription-PCR fragments failed to detect PMP70 mutations
(20). Therefore, it is not clear whether PMP70 is associated with
biogenesis of peroxisomes. On the other hand, the fact that
inducibility of PMP70 parallels peroxisome proliferation and induction
of peroxisomal
-oxidation enzymes suggests that PMP70 participates
in proliferation of peroxisomal membranes and/or transport of
substrates for peroxisomal
-oxidation enzymes.
As an approach to determining the role of PMP70 in peroxisome assembly
and function, we isolated CHO cells stably overexpressing PMP70 and its
mutant forms. We found that PMP70 is involved in metabolic transport of
long chain acyl-CoA across the peroxisomal membrane and is not required
for proliferation of peroxisomes or peroxisome biogenesis.
 |
EXPERIMENTAL PROCEDURES |
Materials--
125I-Protein A (2.60-3.70 TBq/g),
[1-14C]palmitic acid (1.85 GBq/mmol),
[1-14C]lignoceric acid (1.92 GBq/mmol), and
[1-14C]palmitoyl-CoA (1.46-2.22GBq/mmol) were purchased
from ICN Biochemicals Inc. (Irvine, CA), Moravek Biochemicals Inc.
(Brea, CA), CEA (Gif-Sur-Yvette, France), and NEN Life Science
Products, respectively. Palmitoyl-CoA, coenzyme A, NAD+,
and dithiothreitol were obtained from Sigma. Preparation of antibodies
against the C-terminal 15 amino acids of rat PMP70 was as described in
(21). Anti-rat liver catalase antibodies were raised in guinea pigs
(22), and anti-rat liver acyl-CoA oxidase antibodies were raised in
rabbits (23). Other reagents were of analytical grade.
Construction of PMP70 Expression Vector--
The plasmid
pTZ18U/PMP70 in which the rat cDNA sequence encoding PMP70 was
cloned into the EcoRI and HindIII sites of pTZ18U was described in (21). From this cDNA, a fragment containing the
first 1971 bases of PMP70 was excised with EcoRI and
recloned into pBluescript KS(+) at the EcoRI site. This was
cut with HpaI and PstI, and the linear plasmid
containing the N-terminal 35% of PMP70 was purified by agarose gel
electrophoresis. A 1.3-kilobase fragment containing the C-terminal 65%
of PMP70 was then excised from pTZ18U/PMP70 with HapI
and EcoT22I and purified. The fragment was ligated into
HapI and PstI sites of the above plasmid,
encoding the N-terminal 35% of PMP70 and the construct designated
pBluescriptKS(+)/PMP70. The pBluescript KS(+)/PMP70 was cut with
XhoI and XbaI, and a 2.1-kilobase fragment
encoding the full-length of PMP70 was subcloned into XhoI
and XbaI sites of expression vector pME18S (24), which was
then designated as pME18S/PMP70.
Construction of Mutant cDNAs--
A mutant version of PMP70
containing mutations in EAA-like motif, designated as PMP70
(E277D/E278D), was constructed by asymmetric PCR using pME18S/PMP70 as
a template. Two oligonucleotides (the site of substitution is
underlined), with
5'-660AAGCCATTTTTAGACATAGTTTTGTA685-3' and
5'-875GGCAATTTCTTCACTATTAGTAGTAAGCCG846-3'
were used in the first step, and a 220-bp fragment was generated by
PCR. The fragment and an oligonucleotide,
5'-1075TGAAGGTGTCGCGGATGCGCCAGGTC1050-3' was
used in the second step, and a 400-base pair fragment generated by PCR
was purified and digested with HpaI and KpnI. The
fragment containing the mutations of E277D and E278D was ligated into
the HpaI-KpnI sites of pME18S/PMP70. Another
version containing a mutation in Walker A motif, designated as PMP70
(K479A), was constructed with QuikChangeTM site-directed
mutagenesis kit (Stratagene) using pME18S/PMP70 as a template. Two
oligonucleotides with substitution sites and a new restriction site of
1468NarI,
5'-1445CATTTGTGGTCCAAATGGCTGTGGCGCCAGCTCCCTCTTC1484-3'
and
5'-1484GAAGAGGGAGCTGGCGCCACAGCCATTTGGACCACAAATG1445-3'
were used. A deletion construct of PMP70 containing amino acids 1-575,
designated PMP70(1-575), was made as follows. A mutation was generated
by PCR with two oligonucleotides:
5'-660AAGCCATTTTTAGACATAGTTTTGTA685-3' and
5'-TTGGAAGCTTGAATTC-1763TCATTCTCCTCCGCTGAGT1745-3',
using pTZ18U/PMP70 as template. The sequence of
5'-TTGGAAGCTTGAATTC-1763TCA-3' contained
new restriction sites EcoRI and HindIII. The PCR-generated fragment of about 1.1-kilobases containing a termination codon and the new restriction sites was digested with
HindIII and inserted into HindIII site of
pTZ18U/PMP70. The plasmid was then digested with EcoRI and
inserted into EcoRI site of pME18S/PMP70. The mutation in
each construct was confirmed by DNA sequencing.
Transfection of PMP70 cDNAs and Selection of Cells
Overexpressing PMP70--
Expression plasmids were stably transfected
into CHO cells. CHO cells were cultured with Ham's F-12 medium (100 units/ml penicillin and 100 µg/ml streptomycin) and transfected with
5.0 µg of pME18S/PMP70 plus 0.5 µg of pSVneo, which had been mixed
with Transfectam® (Promega). The procedure was essentially the same as
described (25). Surviving isolated colonies were picked up by cylinder technique and subjected to immunoblot analysis of PMP70. Cells overexpressing PMP70 were further purified by repeating the limited dilution twice. The same procedure was carried out to obtain cells overexpressing mutant PMP70.
Subcellular Fractionation of CHO Cells--
CHO cells were grown
with Ham's F-12 medium containing 10%(v/v) fetal calf serum and cell
homogenate and a postnuclear supernatant fraction were prepared with
the same procedure described (21). Then a fraction combining both heavy
and light mitochondrial fractions was obtained by centrifugation at
16,000 × g for 20 min. This fraction (~10 mg of
protein in 0.5 ml) was further subjected to equilibrium density
centrifugation in a 10.6-ml linear sucrose gradient (1.10-1.20 g/ml)
in a Hitachi RP55VF rotor. The gradient rested on 1.0 ml of 1.25 g/ml
sucrose. All solutions contained 1 mM EDTA, 3 mM imidazole, and 0.1%(v/v) ethanol. Centrifugation was
carried out at 50,000 rpm (193,000 × g) for 90 min at
4 °C. Fractions of approximately 1.0 ml were collected in preweighed Eppendorf tubes, and the density of each fraction was determined by refractometry.
Immunofluorescence--
CHO cells were seeded on LAB-TEK®II
Chamber SlideTM System that mounted 4 chambers on a glass
slide (Nalge Nunc) in Ham's F-12 medium with 10%(v/v) fetal calf
serum. After 18-24 h, the medium was replaced by a serum-free medium,
and the culture was continued for an additional 15-18 h (22).
Immunostaining microscopy was performed as described (26).
Immunoelectron Microscopy--
CHO cells were fixed with
4%(w/v) paraformaldehyde, 0.2%(w/v) glutaraldehyde in 0.15 M cacodylate buffer, pH 7.4, for 1 h at room
temperature. After washing with phosphate-buffered saline, the cells
were dehydrated with graded dimethylformamide at
20 °C and
embedded in LR White. Polymerization of the resin was performed under
UV light at
20 °C for 24 h. Thin sections were cut with a
diamond knife equipped with a Ultramicrotome (Amersham Pharmacia Biotech). Catalase was visualized by combination of guinea pig anti-catalase with a 15-nm protein A-gold probe applied to one side of
the thin sections, and PMP70 was detected by rabbit anti-PMP70 antibody
with a 3-nm protein A-gold probe applied to another side of the sections.
-Oxidation Assay in Cultured Cells--
-Oxidation assay
was done essentially as described (27). The cells cultured in 6-well
flasks were preincubated for 1 h in Ham's F-12 medium without
fetal calf serum. Fatty acid oxidation reaction was initiated by adding
4 nmol of [1-14C]palmitic acid or 4 nmol of
[1-14C]lignoceric acid to the freshly prepared Ham's
F-12 medium without fetal calf serum. Radioactive palmitic and
lignoceric acid dissolved in ethanol was evaporated under a stream of
nitrogen and dissolved in a solution of 0.1 M Tris-HCl, pH
8.0, containing 10 mM
-cyclodextrin. After incubation
for 0-120 min, the dishes were placed on ice, 0.15 ml of 10%(w/v)
bovine serum albumin and 0.2 ml of 3 M perchloric acid were
added to the medium, and the preparation was incubated for 30 min on
ice. The medium was centrifuged, and the unreacted fatty acids in the
supernatant were removed by extraction 3 times with 3 ml of hexane. The
acid-soluble radioactivity such as in acetate and citrate in the medium
was measured by a scintillation counter.
-Oxidation Assay Using Isolated Peroxisomes--
Peroxisomal
palmitoyl-CoA oxidation was measured with some modification of the
methods in (28, 29). The oxidation was measured in a final volume of
100 µl of 10 µM [1-14C]palmitoyl-CoA, 2 mM MgCl2, 0.5 mM coenzyme A, 2 mM NAD+, 2 mM KCN, 2 mM
dithiothreitol, 0.1%(w/v) bovine serum albumin, 0.25 M
sucrose, 10 mM Tris-HCl, pH 7.4, with or without 2 mM ATP. In some experiments, peroxisomes were disrupted
with 0.1%(v/v) of Triton X-100. After incubation for 10 min at
37 °C, reactions were terminated by adding 700 µl of ice-cold
H20, 0.15 ml of 10% (w/v) bovine serum albumin, and 0.2 ml
of 3 M perchloric acid to the reaction mixture, and the
mixture was incubated for 30 min on ice. The mixture was centrifuged,
and the supernatant was extracted 3 times with 3 ml of hexane. The
acid-soluble radioactivity in the medium was measured by the
scintillation counter.
Other Methods--
Protein, catalase,
N-
-D-glucosaminidase, cytochrome c
oxidase, and NADPH cytochrome c reductase were assayed as
described previously (21). Immunoblotting was done by the method of
Small et al. (30).
 |
RESULTS |
Overexpression of PMP70 in CHO Cells--
The expression vector
pME18/PMP70 was co-transfected with pSVneo into CHO cells, and stable
transformants were selected based on Geneticin resistance. The clones
were examined for the expression of PMP70 by immunoblot analysis.
Several cell lines transfected with pME18S/PMP70 showed strong
expression of a 70-kDa protein corresponding to PMP70 (Fig.
1). Cells transfected with pSVneo alone
(Neo cells) or untransfected cells (Cont cells) showed weak expression
of PMP70. The amount of PMP70 increased about 5-fold in the cells
transfected with pME18S/PMP70 compared with Cont and Neo cells. To
examine whether the overexpression of PMP70 affects the amount of other
peroxisomal proteins in CHO cells, immunoblot analysis was carried out
using #19 and #31 cells. As shown in Fig.
2, amounts of catalase, acyl-CoA oxidase,
hydratase-dehydrogenase, and thiolase in these cells were essentially
the same as those in Cont and Neo cells.

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Fig. 1.
Overexpression of PMP70 in stable CHO cells
expressing the PMP70 cDNA. Cell homogenates prepared from
Geneticin-resistant and control CHO cells were subjected to immunoblot
analysis of PMP70. Rat Liver fraction,
post-mitochondrial supernatant fraction prepared from rat liver (50 µg of protein). Cont, wild type CHO cells (150 µg of
protein). Neo, CHO cells transfected with pSVneo (150 µg
of protein). #19~#55, CHO cells co-transfected with
pME18S/PMP70 and pSVneo (150 µg of protein). The amount of PMP70 was
quantitated by BAS 1500 imaging analyzer. The arrowhead
indicates the position of PMP70.
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Fig. 2.
Immunoblot analysis of several peroxisomal
enzymes in control CHO cells and CHO cells overexpressing PMP70.
Cell homogenates (150 µg of protein) prepared from wild type CHO
cells (Cont), the cells transfected with pSVneo
(Neo), and the cells co-transfected with pME18S/PMP70 and
pSVneo (#19 and #31) were analyzed with
anti-catalase (Cat) (A), anti-acyl-CoA oxidase
(AOx; a and b mean subunits a and b,
respectively) (B), anti-hydratase-dehydrogenase
(HD) (C), and anti-thiolase (D).
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Subcellular Localization of PMP70--
To investigate the roles of
PMP70 in biogenesis and function of peroxisomes, we first examined
whether PMP70 was enriched in peroxisomes in the cells overexpressing
PMP70. When isolated subcellular fractions such as mitochondrial, light
mitochondrial, microsomal, and cytosolic fractions were subjected to
immunoblot analysis, a band corresponding to PMP70 was enriched in the
light mitochondrial fraction in Cont, Neo, #19, and #31 cells (data not shown).
The subcellular distribution of PMP70 was further determined by
equilibrium density centrifugation. In Neo cells, peroxisomes were well
separated from mitochondria, endoplasmic reticulum, and lysosomes (Fig.
3A). The PMP70 was mainly
recovered in fractions 10-11 on the sucrose gradient, which
corresponded to the position of peroxisomes as shown by the catalase
distribution (Fig. 3B). For #19 cells, peroxisomes were also
separated from other organelles (Fig. 3A). The PMP70 was
mainly recovered in fractions 9-10, and the distribution of PMP70 was
similar to that of catalase (Fig. 3B). These results suggest
that overexpressed PMP70 is enriched in peroxisomes of CHO cells. The
distribution of PMP70 in #31 cells is essentially the same as that of
#19 cells (data not shown).

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Fig. 3.
Subcellular localization of PMP70 in Neo and
#19 cells. A, heavy and light mitochondrial fractions
from Neo and #19 cells were fractionated by equilibrium density
centrifugation on sucrose. The marker enzyme distributions are plotted
as described previously (21); the recoveries varied between 70 and
120%. Catalase, cytochrome c oxidase (Cyt Ox),
NADPH cytochrome c reductase (Cyt Red), and
N-acetyl- -D-glucosaminidase
(NAGase) were measured as marker enzymes of peroxisomes,
mitochondria, microsomes, and lysosomes, respectively. Distribution of
PMP70 was quantitated by BAS 1500 imaging analyzer. B,
immunoblot analysis of PMP70. An aliquot of each fraction from the top
(1) to the bottom (12) of the sucrose gradient
was analyzed by immunoblotting. One hundred µl of each fraction was
used in the case of Neo cells and 25 µl was used in that of #19
cells.
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Localization of PMP70 to peroxisomes in Neo, #19, and #31 cells was
also examined by immunofluorescence microscopy. As shown in Fig.
4, PMP70 exhibited a punctulate staining
pattern in Neo cells. This pattern was completely superimposable on the
distribution of catalase in the same cells (A and
B). PMP70 also exhibited a punctulate structure with high
intensity of fluorescence in #19 and #31 cells, and this pattern was
also superimposable on the distribution of catalase on the same cells,
although the intensity of cytosolic catalase increased (D
and F). We observed cytosolic catalase activity increased
slightly after digitonin permeabilization of these cells (data not
shown). In immunoelectron microscopy (Fig.
5), gold particles corresponding to PMP70
were observed on peroxisomes, and their number increased in #19 and #31
cells (B and C) as compared with Neo cells
(A). However the size of peroxisomes seems to be similar
between Neo, #19, and #31 cells.

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Fig. 4.
Immunofluorescence microscopy of Neo, #19,
and #31 cells. Double immunostaining of Neo cells (A
and B), #19 cells (C and D), and #31
cells (E and F). Distribution of PMP70 was
visualized with a rabbit anti-PMP70 antibody and a fluorescein
isothiocyanate-labeled secondary antibody (Chemicon) (A,
C, and E). Distribution of catalase was
visualized with a guinea pig anti-catalase antibody and a
rhodamine-labeled secondary antibody (Cappel) (B,
D, and F). Bar = 20 µm.
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Fig. 5.
Immunoelectron microscopy of Neo, #19, and
#31 cells. A, Neo cells; B, #19 cells;
C, #31 cells. A-C show double labeling for
catalase (large gold particles) and PMP70 (small gold particles). Large
gold particles are predominantly present in peroxisomes (P).
Note that dots of PMP70 on peroxisomal membranes increased in #19 and
31 cells, but the size of peroxisomes in Neo, #19, and 31 cells are
essentially the same. Magnification, ×62,000.
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-Oxidation of Fatty Acids--
We have investigated the
enzymatic
-oxidation activity of Cont, Neo, #19, and #31 cells using
radiolabeled [1-14C]palmitic and
[1-14C]lignoceric acids. 14C-Labeled
acid-soluble degradation products derived from both substrates
increased linearly up to 2 h in all the cells. The rate of
palmitic acid
-oxidation in #19 and #31 cells increased about
2-3-fold compared with control and Neo cells (Fig.
6A). On the other hand, the
-oxidation of lignoceric acid in these cells was reduced about
30-40% (Fig. 6B). However, when
-oxidation activities
of palmitic acid and lignoceric acid were measured with 2 mM KCN in the lysates, prepared by freezing and thawing or
treatment of 0.01% (v/v) Triton X-100, these activities were unchanged
and indistinguishable among these cells, Cont, #19, and #31 cells,
under those conditions where peroxisomes were disrupted (data not
shown).

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Fig. 6.
-Oxidation of
[1-14C]palmitic acid and [1-14C]lignoceric
acid in Neo, #19, and #31 cells. Cells in 3.5-cm dishes were
incubated with [1-14C]palmitic acid and
[1-14C]lignoceric acid for 30 min and 1 h, and
14C-labeled acid-soluble products were counted. The
-oxidation rates in Neo cells were taken as reference (100%).
A, rate of formation of acid-soluble products from
[1-14C]palmitic acid and -oxidation in Neo cells was
1.90 pmol/h/100 µg of protein. B, rate of formation of
acid-soluble products from [1-14C]lignoceric acid and
-oxidation in Neo cells was 4.80 pmol/h/100 µg of protein. Values
are the mean ±S.D. of three experiments.
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To evaluate peroxisomal
-oxidation, we measured
-oxidation of
palmitic acid in cultured CHO cells in the presence of
2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a potent
inhibitor of mitochondrial carnitine palmitoyltransferase I (27, 31).
However, POCA (50 µM) did not inhibit
-oxidation of
palmitic acid in any of the cells, although under the same conditions
palmitic acid oxidation was inhibited about 70 and 80% in rat hepatoma
H4IIE cells and human fibroblasts. In addition, when a combined
fraction of both heavy and light mitochondrial fractions prepared from
CHO cells was separated on sucrose gradient centrifugation, about 80%
of the activity was recovered in peroxisomal fraction, and it was not
inhibited with 2 mM KCN (data not shown). We show,
therefore, that peroxisomes contribute mainly to the formation of
acid-soluble labeled products in CHO cells under our experimental conditions.
ATP Stimulates Palmitoyl-CoA Oxidation in Light Mitochondrial
Fraction--
As palmitic acid is activated to become palmitoyl-CoA
outside of peroxisomes, we further examined whether overexpression of PMP70 on peroxisomal membranes stimulates
-oxidation of
palmitoyl-CoA using light mitochondrial fractions in the presence of
KCN. In some experiments, peroxisomes were disrupted with Triton X-100. As shown in Fig. 7, when
-oxidation
was measured in the disrupted peroxisomes, the activity was essentially
the same with or without ATP. On the other hand, if peroxisomes were
intact, ATP stimulated
-oxidation of palmitoyl-CoA, and the degree
of the stimulation in #19 cells was higher than that in Neo cells.
Latency of catalase in both cells was essentially the same (about 90%)
(data not shown). In addition, in our incubation, the rate of
hydrolysis of palmitoyl-CoA to palmitic acid and CoA was about 10% and
was not changed by ATP (data not shown). These results suggest that
PMP70 is involved in transport of long chain acyl-CoA across
peroxisomal membranes. It is unlikely that extensive hydrolysis of
palmitoyl-CoA during incubation decreased its concentration and that
the stimulative effect of ATP on the
-oxidation was because of
reactivation of palmitate.

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Fig. 7.
Effect of ATP on
-oxidation of [1-14C]palmitoyl-CoA in
light mitochondrial fraction prepared from Neo and #19 cells.
Light mitochondrial fractions containing intact or disrupted
peroxisomes were incubated with [1-14C]palmitoyl-CoA
in the absence or presence of ATP for 10 min at 37 °C, and
acid-soluble products were counted. Rates of
[1-14C]palmitoyl-CoA -oxidation are expressed as a
percentage of the rates observed in disrupted peroxisomes in the
presence of ATP. The activity in Neo cells and #19 cells was 2.07 nmol/h/mg of protein and 1.86 nmol/h/mg of protein, respectively.
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Mutation of Conserved Motifs in PMP70 Inhibited
-Oxidation of
Palmitic Acid--
ABC transporters possess highly conserved regions
such as an EAA-like motif and Walker A and B motifs. The EAA-like motif seems to be important for the function of ABC transporters (32, 33),
and Walker A and B motifs seem to be important for ATP binding and
hydrolysis (9, 34). As PMP70 is a half-sized ABC transporter and seems
to function as a dimer, mutation of one of the conserved regions might
cause loss of the PMP70 function. We chose to mutate the two glutamic
acids (Glu277 and Glu278) in the EAA-like motif
of PMP70 to aspartic acids (E277D/E278D), because aspartic acid
resulting from a change of the corresponding glutamic acids
(Glu291) in ALDP is known to be the site of mutation
causing adrenoleukodystrophy. Concerning the Walker A motif, we changed
the corresponding lysine (Lys479) to alanine, because it
was shown that mutation of the lysine abolishes or markedly reduces ATP
hydrolysis and inhibits the protein function of several ATPases (35,
36). In addition, we constructed PMP70 (1-575) minus 84 C-terminal
amino acids including the Walker B motif, because recently,
Gärtner et al. (12) suggested that the C-terminal
sequence of PMP70 is important for the assembly of PMP70.
The three mutant cDNAs were transfected into CHO cells, and stably
expressing cells were selected. In the cells overexpressing PMP70
(E277D/E278D and K479A), the amount of PMP70 increased about 2-3-fold
compared with that in Neo cells (Fig. 8,
A and B). In the cells overexpressing PMP70
(1-575), the band of 60 kDa corresponding to C-terminal-truncated
PMP70 was detected, and the amount of the protein was increased
~2-fold over that of wild type PMP70 in Neo cells (Fig.
8C). Furthermore, the mutant PMP70s was found to be enriched
in peroxisomes in E1, W10, and S15 cells (data not shown).

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Fig. 8.
Overexpression of mutant PMP70 in CHO cells
and the -oxidation of
[1-14C]palmitic acid and [1-14C]lignoceric
acid in the cells. A-C, immunoblot analysis of PMP70.
A, missense mutation of EEA-like motif (E1-E22).
Neo, CHO cells transfected with pSVneo. B,
missense mutation of Walker A motif (W5-W19). C, deletion
mutation of C-terminal sequence including the Walker B motif (S1-S15).
Cell homogenates (150 µg of protein) were subjected to immunoblot
analysis. In the case of C-terminal-truncated PMP70, antibody against
purified PMP70 from rat liver peroxisomes (37) was used. The
arrowheads indicate the position of PMP70 and
C-terminal-truncated PMP70, respectively. D-E, the
-oxidation of palmitic acid (D) or lignoceric acid
(E) in Neo and the mutant cells. Rates of fatty acid
-oxidation are expressed as a percentage of the rates observed in
Neo cells. The rate of formation of acid-soluble products from
[1-14C]palmitic acid and [1-14C]lignoceric
acid in Neo cells was 2.80 pmol/h/100 µg of protein and 4.50 pmol/h/100 µg of protein, respectively. Values are the mean ±S.D. of
three experiments.
|
|
When we investigated the
-oxidation of palmitic acid in the mutant
cells, we found the rate of
-oxidation markedly decreased compared
with that of Neo cells (Fig. 8D). In the case of E1 and E21
cells,
-oxidation of palmitic acid decreased almost 60-70%. These
results support PMP70 involvement in
-oxidation of long chain fatty
acids, and furthermore, EAA-like and Walker A motifs, as well as the
C-terminal segment including the Walker B motif, are essential to the
function of PMP70.
Interestingly, the rate of lignoceric acid
-oxidation decreased
slightly in all the mutant cells (Fig. 8E) and cells
overexpressing wild type PMP70 (Fig. 6B). It might be that
overexpression of mutant as well as wild type PMP70 inhibited the
function of ALDP, which is thought to be involved in
-oxidation of
very long chain fatty acids.
Overexpression of PMP70 Did Not Change the Number and Size of
Peroxisomes in CHO Cells--
Administration of hypolipidemic agents
in rats induces peroxisome proliferation (6, 7). For example, in the
above conditions where the number of peroxisomes was increased
~5-fold, the amount of PMP70 increased ~10-fold (37). This
observation suggested that PMP70 induces peroxisomal membranes and
increases the number of peroxisomes. To address this issue we compared
the number and the size of peroxisomes between Neo, #19, and #31 cells
by immunofluorescence and immunoelectron microscopy.
As shown in Fig. 9, a number of
punctulate structures with high intensity of fluorescence representing
acyl-CoA oxidase were detected in Neo, #19, and #31 cells. The number
of fluorescent dots in #19 and #31 cells was similar or slightly
decreased compared with that in Neo cells. In immunoelectron
micrographs, the number of small gold particles corresponding to PMP70
located on peroxisomes in #19 and #31 cells markedly increased compared
with that of Neo cells, but the size and the number of peroxisomes
seemed to be similar between Neo cells and #19 and #31 cells. An
increase in the size and number of peroxisomes as was observed in rat
liver on the administration of hypolipidemic agents did not occur.
Thus, it seems that overexpression of PMP70 is not associated with
proliferation of peroxisomes.

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|
Fig. 9.
Distribution of acyl-CoA oxidase in Neo, #19,
and #31 cells. Immunofluorescence staining of acyl-CoA oxidase
using rabbit anti-acyl-CoA oxidase and Cy2-labeled secondary antibody
(Amersham Pharmacia Biotech). A, Neo cells; B,
#19 cells; C, #31 cells. The number of peroxisomal particles
in #19 and #31 cells was the same as or slightly decreased compared
with that of Neo cells.
|
|
 |
DISCUSSION |
In this study, we overexpressed PMP70 in CHO cells and examined
roles of PMP70 in biogenesis and function of peroxisomes. First,
subcellular localization of PMP70 overexpressed in CHO cells was
investigated because information about peroxisomal localization is
essential to the characterization of PMP70. The following findings suggest that the overexpressed PMP70 is enriched in peroxisomes. First,
the distribution of overexpressed PMP70 in a sucrose gradient was
similar to that of catalase (Fig. 3). Second, the numerous dots
detected by immunofluorescent staining of PMP70 in #19 and #31 cells
were superimposable on those stained with anti-catalase antibody (Fig.
4). Third, the enrichment of immunogold particles corresponding PMP70
on peroxisomes was shown in #19 and #31 cells by immunoelectron
microscopy (Fig. 5). In light of these results, we further investigated
roles of PMP70 in biogenesis and function of peroxisomes.
Function of PMP70--
An early study demonstrated that ATP
stimulated overall
-oxidation activity in isolated rat liver
peroxisomes (29). In addition, it is well known that PMP70 is markedly
induced by administration of hypolipidemic agents in parallel with the
induction of peroxisomal fatty acid
-oxidation enzymes (6, 7, 37).
These observations suggest the presence of an ATP-dependent
acyl-CoA carrier in the peroxisomal membranes and PMP70 may be a
carrier of fatty acids or their CoA ester. If PMP70 is a transporter of
long chain fatty acids or their CoA ester, its overexpression might
stimulate the
-oxidation in peroxisomes. On the other hand, as PMP70
is a half-sized ABC transporter, mutant PMP70 expressed in the cells
might interact with wild type PMP70, resulting in the inhibition of
peroxisomal
-oxidation. In fact, the rate of
-oxidation of
palmitic acid in #19 and #31 cells increased ~2-3-fold (Fig. 6). On
the other hand, the rate of
-oxidation of palmitic acid was markedly
decreased by overexpression of all the mutant PMP70 (Fig. 8). It seems
unlikely that the increase of
-oxidation activity in #19 and #31
cells is because of the induction of peroxisomal
-oxidation enzymes or increase in the permeability of peroxisomal membranes because the
amount of peroxisomal
-oxidation enzymes was essentially the same in
Neo, #19, and #31 cells (Fig. 2), as was the latency of catalase in
isolated peroxisomes (data not shown). Furthermore, the rate of
-oxidation of lignoceric acid decreased in CHO cells overexpressing
wild type and mutant PMP70 (Figs. 6 and 8), supporting the idea that
increase in nonspecific permeability of fatty acids across peroxisomal
membranes did not occur.
Because the active site of long chain acyl-CoA synthetase is located
outside of peroxisomes (38), the long chain fatty acids seem to be
activated to change to acyl-CoA ester prior to
-oxidation in cells.
To define whether PMP70 is involved in the transport of long chain
acyl-CoA, we studied
-oxidation of palmitoyl-CoA using isolated
peroxisomes prepared from Cont, #19, and #31 cells. Intact peroxisomes
showed latency to palmitoyl-CoA oxidation. ATP stimulated the
-oxidation of palmitoyl-CoA when intact peroxisomes were used. The
degree of stimulation was higher in the CHO cells overexpressing PMP70
than that in the Neo cells (Fig. 7). The effect of ATP was lost when
the peroxisomal membranes were disrupted by the use of Triton X-100
(Fig. 7). Based on these results, we suggest that PMP70 functions as a
transporter of long chain acyl-CoA across peroxisomal membranes to
serve substrates for
-oxidation, and ATP facilitates the transport
of the substrate by PMP70.
Another important point addressed by the present study is the
functional domain of PMP70. All ABC transporters characterized so far
contain the consensus sequence for an ATP binding site (Walker motifs A
and B (34, 35)). Walker A is characterized by the simple motif
GXXGXGK(T/S), and the lysine as a source of the
positive charge is invariant. In the case of PMP70, mutation of this
lysine to alanine resulted in marked inhibition of the palmitic acid
-oxidation (Fig. 8D). The fact that the
lysine479 is critical for the PMP70 function suggests that
PMP70 possesses ATP binding and hydrolytic activity, which couples to
the transport of palmitoyl-CoA. EAA-like motif is a conserved sequence
located in the region between the fourth and fifth putative
membrane-spanning domains of ABC transporters. Studies of prokaryotic
ABC transporters suggest missense mutations, which alter conserved
residues, cause loss of transporter function (32-34). Our mutant
demonstrated that this motif is also critical for the PMP70 function.
Recently, two ABC transporters called Pat1p (Pxa2p) and Pat2p (Pxa1p)
were identified on peroxisomal membranes of S. cerevisiae (39-43). Disruption of PAT1 and PAT2 resulted in
impaired growth on oleic acid and reduced ability to oxidize long chain
fatty acids such as oleic acid. Furthermore, a possibility that pat2p is involved in the transport of long chain acyl-CoA was suggested using
digitonin-permeabilized yeast cells (44). Therefore, PMP70 seems to be
a counterpart of yeast peroxisomal ABC transporter of Pat1p or Pat2p.
However, the functional unit of PMP70 and yeast peroxisomal ABC
transporters Pat1p and Pat2p seems to be different. Disruption of PAT1
or PAT2 in yeast cells resulted in impaired growth in oleic acid
medium, suggesting that Pat1p and Pat2p function as heterodimers
(43-46). On the other hand, overexpression of PMP70 alone in CHO cells
induced
-oxidation of palmitic acid, suggesting that PMP70 can
function as homodimer.
Unexpectedly, overexpression of wild type as well as mutant PMP70
suppressed the
-oxidation of lignoceric acid. We don't know the
reason for this at present, but one possibility is that because of the
overexpression of PMP70, a part of PMP70 may associate with ALDP,
although the interaction might be weak and such a heterodimer might
reduce the transport activity of lignoceric acids. This possibility is
now under investigation.
Effect of Overexpression of PMP70 on Structure of
Peroxisomes--
Proliferation of peroxisomes parallels induction of
PMP70 (6, 7, 37). A subset of patients with Zellweger syndrome, a
peroxisome biogenesis disorder (complementation group 1), has defects
at the locus encoding PMP70 (12). This observation led us to examine
whether PMP70 is involved in proliferation and biogenesis of
peroxisomes. To answer this, we analyzed the number and the morphology
of peroxisomes by immunofluorescence and immunoelectron microscopy
using CHO cells overexpressing PMP70. The following results suggest
that PMP70 is not involved in such a process. First, the number of
punctulate structures corresponding to peroxisomes did not increase on
overexpression of PMP70 (Fig. 9). Second, the number of gold particles
against PMP70 located on peroxisomes was markedly increased by
overexpression of PMP70, but the size and the number of peroxisomes did
not increase (Fig. 5). Furthermore, CHO cells expressing C-terminal
truncated PMP70 as well as the cells possessing missense mutations of
EAA-like and Walker A motifs had morphologically normal peroxisomes
(data not shown). Therefore, PMP70 seems not to be necessary for the
biogenesis and proliferation of peroxisomes but to be required for the
normal function of peroxisomes. Recently, it was revealed that
peroxisomes in mammalian cells preferentially oxidized several kinds of
lipids, prostanoids such as prostaglandin E2,
F2
, and leukotriens (45, 46) and dicarboxylic acids
(47), trihydroxycholestanic acid (48), and phytanic acid (49, 50).
These substances must be transported into peroxisomes to be oxidized.
It would be interesting to know whether PMP70 is involved in the
transport of these substances.
In this study we found that PMP70 is involved in metabolic transport of
long chain fatty acyl-CoA across the peroxisomal membrane and is not
required for proliferation of peroxisomes or peroxisome biogenesis
using CHO cells stably overexpressing wild and mutant type PMP70. The
mechanism by which PMP70 transports long chain acyl-CoA will be the
subject of further research.
 |
FOOTNOTES |
*
This research was supported by the Ministry of Education,
Science, Sports, and Culture of Japan (grant-in-aid for scientific research on priority areas of ABC Proteins).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.: 81-764-34-2281 (ext. 2635); Fax: 81-764-34-4656; E-mail:
imanaka{at}ms.toyama-mpu.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
PMP70, 70-kDa
peroxisomal membrane protein;
ABC, ATP binding cassette;
ALDP, adrenoleukodystrophy protein;
PCR, polymerase chain reaction;
CHO, Chinese hamster ovary;
POCA, 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate.
 |
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