From the Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan
Received for publication, December 26, 2000
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ABSTRACT |
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Candida boidinii Pmp20
(CbPmp20), a protein associated with the inner side of peroxisomal
membrane, belongs to a recently identified protein family of
antioxidant enzymes, the peroxiredoxins, which contain one cysteine
residue. Pmp20 homologs containing the putative peroxisome targeting
signal type 1 have also been identified in mammals and lower
eukaryotes. However, the physiological function of these Pmp20 family
proteins has been unclear. In this study, we investigated the
biochemical and physiological functions of recombinant CbPmp20 protein
in methanol-induced peroxisomes of C. boidinii using the
PMP20-deleted strain of C. boidinii
(pmp20 The peroxisome is a ubiquitous organelle found in most eukaryotic
cells in which various kinds of oxidative metabolism occur through at
least one H2O2-generating peroxisomal oxidase
(1, 2). These oxidative metabolisms yield reactive oxygen species (ROS),1 which can cause
damage to all cellular constituents, i.e. nucleic acids,
proteins, lipids, etc. In mammalian cells, treatment with peroxisome
proliferators or constitutive expression of peroxisomal oxidases
(acyl-CoA oxidase and urate oxidase) were shown to result in neoplastic
transformation (3), and these and other studies indicate that ROS
generation within peroxisomes is related to carcinogenesis.
The peroxisomal matrix and membranes are assumed to be exposed to a
high level of ROS. Therefore, as in other ROS-generating organelles,
such as mitochondria (4), peroxisomes are assumed to have defensive
enzyme activities against ROS toxicity (5). One such activity is
peroxisomal catalase, which decomposes H2O2. It
is distributed from lower to higher organisms, and as such, it is also
used as a marker enzyme for peroxisomes. In mammalian peroxisomes, two
other antioxidant enzymes are also present, i.e. superoxide
dismutase (6-8) and glutathione peroxidase (GPX) (9).
Pmp20 (20-kDa peroxisomal membrane protein) was initially
identified as a peroxisomal membrane protein of unknown function in the
methylotrophic yeast Candida boidinii (10). Recently, Pmp20
family proteins have been suggested to detoxify ROS within human and
murine peroxisomes (11, 12). Subsequently, putative Pmp20 homologs,
having a putative peroxisome targeting signal type 1 (PTS1) at their C
terminus, were found to be widely distributed in various eukaryotic
cells from yeasts and mammalian cells, e.g. Saccharomyces cerevisiae (ScPmp20; also designated as Ahp1p
or type II thioredoxin peroxidase (TPX)) (13, 14), Lipomyces kononenkoae (15), human (HsPmp20), and mouse (MmPmp20) (11).
Immunocytochemical and biochemical studies have clearly demonstrated
that C. boidinii Pmp20 (CbPmp20) is a peroxisomal
membrane-associated protein present also in the peroxisome matrix (16,
17). However, the physiological significance and function of this and
other Pmp20 family proteins seem to be more complex than initially
expected. For example, a green fluorescent protein (GFP)-ScPmp20 fusion protein was localized to mitochondria despite the presence of a
putative PTS1 sequence (18). Depletion of Pmp20 in S. cerevisiae did not affect its growth on oleate medium where normal
peroxisomal metabolism is necessary for its
growth.2 Although the binding
of HsPmp20 to the PTS1 receptor Pex5p was shown, up to 50% of
epitope-tagged HsPmp20 was also detected in the cytosolic fraction of
HeLa cells (11). More recently, a long form of HsPmp20, designated as
AOEB166, having a mitochondrial sorting signal at its N terminus and
peroxisome targeting signal at its C-terminus, showed bimodal
distribution of AOEB166 in these organelles (12).
Recently, the Pmp20 family proteins were recognized from their primary
amino acid sequence to belong to a new antioxidant family, the
peroxiredoxins (Prx) (19). HsPmp20 and ScPmp20 exhibited antioxidant
activity in vitro (11, 14), and both MmPmp20 (20) and
ScPmp20 (21) were reported to have TPX activity. However, the presence
of thioredoxin molecule within peroxisomes has not been demonstrated.
Furthermore, bimodal localization of Pmp20 proteins in peroxisomes and
mitochondria has made it difficult for us to investigate the
physiological function of Pmp20 proteins in relation to the ROS
protection system within peroxisomes.
To answer these questions, we initiated a study of the biochemical and
physiological functions of CbPmp20. The rationale for using CbPmp20 in
the methylotrophic yeast C. boidinii are as follows: 1)
Because CbPmp20 is specifically induced in methanol-containing medium
and is exclusively localized within peroxisomes, we can specify the
function of CbPmp20 within methanol-induced peroxisomes (17, 22). 2)
The molecular genetics (23, 24) and organelle fractionation techniques
(25, 26) have been well established with C. boidinii.
Through this study we have demonstrated that the Pmp20-antioxidant
system indeed functions within peroxisomes as GPX and that the
antioxidant function of CbPmp20 is biochemically and physiologically
distinct from that of peroxisomal catalase.
Microorganisms and Growth Conditions--
C. boidinii
strain S2 was the original haploidal strain for construction of the
genomic library. C. boidinii strain TK62 (ura3) (24) was used as the host for transformation. C. boidinii
strain GC (27), which was derived from strain TK62 via gene conversion with URA3 fragment (24), was routinely used as the control
wild type strain. C. boidinii strain
cta1 Cloning of Pmp20-encoding Gene from C. boidinii Strain
S2--
Two PCR primers, PMP20N1 and PMP20R1 (Table
I), were designed based on the conserved
DNA sequences between CbPmp20A and CbPmp20B (10), and synthesized.
These primers were used to amplify an approximately 500-base pair
fragment that encoded a partial Pmp20 sequence from C. boidinii strain S2. This PCR-amplified fragment was gel purified,
32P-labeled according to the method of Feinberg and
Vogelstein (29), and used for further cloning experiments. A pool of
BamHI-digested genomic DNA of ~3.8 kb was gel purified and
ligated into the BamHI site of pBluescript II KS+
(Strategene, San Diego, CA). E. coli transformants were
transferred onto a Biodyne nylon membrane (Pall Bio Support, New York,
NY). After lysis of the bacteria, the liberated DNA was bound to the
nylon membrane, and these blots were then used for colony hybridization
under high stringency hybridization conditions using Church-Gilbert
buffer (1% bovine serum albumin, 1 mM EDTA, 0.25 M NaCl, 0.25 M Na3PO4,
pH 7.2, 7% SDS) (30). Hybridization was performed at 65 °C
overnight, and then the membranes were washed six times in 0.2× SSC
(1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate) at the same temperature. Five clones that showed strong
positive signals were found to harbor a reactive 3.8-kb
BamHI fragment. The nested deletion mutants were derived as
previously described (31), and the entire PMP20 gene was sequenced in both directions using a 7-deaza sequencing kit (Thermo Sequence fluorescent labeled primer cycle sequencing kit) from Amersham
Pharmacia Biotech and DNA sequencer model DSQ-2000L from SHIMADZU Co.
Ltd. (Kyoto, Japan).
Construction of the Disruption Cassette and One-step Gene
Disruption of PMP20--
pMP200, carrying 3.8-kb BamHI
fragment harboring the C. boidinii PMP20 gene, was digested
with BglII and StyI to delete a 1.7-kb fragment
that included 5' half of PMP20 gene. The remaining fragment
was gel purified, blunted with T4 DNA polymerase, and ligated to the
SacI-XhoI fragment of pSPR (C. boidinii
URA3 gene having repeated sequence at the 5'- and 3'-franking
regions) (23). The ligation reaction generated the C. boidinii
PMP20 disruption vector pD20SPR. After propagation of pD20SPR in
E. coli, the insert DNA fragment was liberated following
HindIII digestion and was used to transform C. boidinii TK62 (ura3) to uracil prototrophy. The
disruption of PMP20 was confirmed by genomic Southern
analysis with HindIII-digested DNA from a Ura+
transformant, using the 1.0-kb StyI-HindIII
fragment from pMP200 as the probe. The PMP20 disruptant
strain pmp20 Expression of CbPmp20 and Its Derivatives in C. boidinii--
NotI sites were added to both ends of the
PMP20 coding sequence by PCR using two primers NOT20N and
20CNOT (Table I) and PMP20 DNA as the template. To replace
the conserved Cys53 residue of CbPmp20 with serine, at
first, two fragments were PCR-amplified using either set of primers,
primers NOT20N and C53S-R, or, primers C53S-F and C53S-R (Table I).
Each fragment was purified and used as the templates of the second PCR,
where primers NOT20N and 20CNOT were used. Each of the wild type or C53S-mutagenized PMP20-fragments amplified was introduced
into the C. boidinii expression vector pNoteI (32), and the
constructed plasmids were named pNot20 and pNot20C53S, respectively.
pNoteI harbored the C. boidinii AOD1 promoter and terminator
sequences with a unique NotI site to insert coding sequences
for expression (32). pNot20- Preparation and Purification of His6-CbPmp20
Proteins--
CbPmp20 or CbPmp20 C53S-coding protein was expressed in
E. coli BL21(DE3) (Novagen, Madison, WI) as a
His6 fusion protein in pRSET A (Invitrogen, Carlsbad, CA)
containing six histidine residues at their N terminus. pNot20 and
pNot20C53S was each digested with NotI, and the insert DNAs
was blunt-ended and ligated to the blunt-ended BamHI site of
pRSET A, yielding pRSET20 and pRSET20C53S, respectively. The proper
in-frame integration was confirmed by DNA sequencing.
A single colony was inoculated in 100 ml of LB medium containing
ampicillin (50 µg/ml) and incubated at 28 °C with reciprocal shaking until the OD610 reached ~0.6. The recombinant
protein production was induced by adding isopropyl
Enzyme Assays--
GPX activity was determined by measuring
consumption of NADPH in the presence of GSH (Wako, Osaka, Japan) and
GSH reductase (Oriental Yeast, Tokyo, Japan) using cumene
hydroperoxide, tert-butyl hydroperoxide, and hydrogen
peroxide as substrates. The standard reaction mixture (1 ml) was 20 µg of purified His6-tagged CbPmp20, 70 µM
NADPH, 0.1 mM GSH, and 0.4 units/ml GSH reductase in 50 mM Tris-HCl, pH 7.5. The reaction was carried out at
30 °C and was started by adding peroxide on a double beam
spectrophotometer UV 2200-A (Shimadzu Co. Ltd., Kyoto, Japan). In the
thioredoxin system, 4 µM thioredoxin (Oriental Yeast,
Tokyo, Japan) and 0.4 units/ml thioredoxin reductase (Oriental Yeast,
Tokyo, Japan) were substituted for GSH and GSH reductase. Enzyme
activity was expressed as µmol of NADPH oxidized/min/mg protein. The
amount of GSH and GS-SG was determined according to Tietze (35). GSH reductase (GR) activity was determined by the method of Casalone et al. (36). Catalase and cytochrome c oxidase
activities were determined as described previously (37, 38). Protein
quantitation was performed using the method of Bradford (39) with a
protein assay kit (Bio-Rad) using bovine serum albumin as the standard.
Protein Methods and Antibody--
Standard 9% Laemmli gels (40)
with a separating gel of pH 9.2 were used. Immunoblotting was performed
by the method of Towbin et al. (41) using the ECL detection
kit (Amersham Pharmacia Biotech). The VA9 monoclonal anti-Pmp20
antibody and anti-alcohol oxidase were kindly provided by Dr. J. M. Goodman (University of Texas Southwestern Medical Center, Dallas, TX).
O2 Uptake--
O2 dissolved in aqueous
solution was measured polarographically at 37 °C in 50 mM Hepes-NaOH, pH 7.4, using a Clark electrode placed in a
water-jacketed cell assembly from Rank Brothers Ltd. (model 0646). The
calibration of the O2 monitor was performed as described
before (42). O2 uptake was initiated by the addition of the
reducing agent (ascorbate or DTT), unless specifically noted. The
profile of DTT oxidation is comprised of a lag phase of several
minutes, followed by a rapid O2 uptake step when the maximal rate is reached. The maximum rate was determined from the slope
of the steepest, nearly linear segment of the O2 uptake curve.
H2O2 Concentration--
Fluorescent
measurement was employed in the horseradish peroxidase-catalyzed
oxidation of homocanillic acid by H2O2 (43, 44). Because thiols are substrates for horseradish peroxidase (45),
sulfhydryl groups were alkylated with N-ethylmaleimide, prior to the determination of H2O2
concentration. Calibration curves were generated with known amounts of
H2O2.
Organelle Fractionation--
Wild type C. boidinii
cells were grown on YPMGy medium overnight, spheroplasted with
Zymolyase 100T (Seikagaku Co., Tokyo Japan), and osmotically lysed
according to the method of Goodman et al. (46), as described
previously (26). Unlysed cells, nuclei, and other cell debris were
removed carefully from the lysate by centrifugation at 1,000 × g at 4 °C for 10 min. The supernatant was subjected to
centrifugation at 20,000 × g at 4 °C for 20 min to
obtain a crude pellet containing mainly peroxisomes and mitochondria.
To prepare a continuous Nycodenz (Sigma) gradient solution, a step
gradient of 10.6 ml (1.3 ml of 60% Nycodenz, 2 ml of 50% Nycodenz, 4 ml of 35% Nycodenz, and 3.3 ml of 28% Nycodenz (w/v)), was frozen
once in liquid nitrogen, and then thawed. Then, the organellar
suspension was layered on top of the 10.6-ml gradient, and the samples
were spun at 100,000 × g for 2 h at 4 °C in a VTi 65.1 vertical rotor (Beckman Instruments, Inc., Palo Alto, CA). The
gradients were fractionated from the bottom.
Fluorescence Microscopy--
Cells representing GFP fluorescence
were placed on a microscope slide and examined under a Carl Zeiss
Axioplan 2 Fluorescence Microscope (Oberkochen, Germany), and set at
the fluorescein isothiocyanate channel. Images were acquired using a
CCD camera (Carl Zeiss ZVS-47DE) and a CG7 Frame Grabber (Scion Corp.,
Frederick, MD).
Electron Microscopy--
C. boidinii strain
pmp20 GenBank Accession Number--
The nucleotide sequence of
PMP20 gene published here has been submitted to GenBank and
is available under accession number AB055180.
Gene Structure of CbPmp20 in C. boidinii Strain S2--
From the
deduced encoded amino acid sequence of the gene cloned from strain S2
of C. boidinii, CbPmp20 is a protein of 167 amino acids with
a molecular mass of 18,083 Da. Fig. 1
shows a multiple alignment of several Pmp20 family proteins. All family members contain a putative PTS1 sequence at their C- terminus. The
amino acid identity of CbPmp20 based on deduced amino acid sequences
with CbPmp20B, CbPmp20A, HsPmp20, MmPmp20, and ScPmp20 was 99.4%
(C. boidinii ATCC32195), 96.4% (C. boidinii
ATCC32195), 25.7% (human), 23.4% (mouse), and 18.6% (S. cerevisiae), respectively. BLAST analysis revealed that these
Pmp20 family proteins exhibited significant sequence similarities with
other Prx family proteins, especially in the region around
Cys53 of CbPmp20. The most unique feature of the C. boidinii Pmp20 proteins is that CbPmp20 has only one cysteine
residue, whereas mammalian and S. cerevisiae Pmp20s have
more than two cysteine residues. Therefore, CbPmp20 was classified as
1-Cys Prx, whereas other Pmp20 proteins belonged to 2-Cys Prx (48-50).
From genomic Southern analysis (data not shown) and gene disruption
analysis (see below), C. boidinii strain S2 was found to
contain a sole gene encoding for CbPmp20. On the other hand, C. boidinii strain ATCC32195 contained two genes, CbPMP20A
and CbPMP20B (10).
Antioxidant Activity of His6-tagged CbPmp20--
Prx
family proteins are known to exert their antioxidant activity through
an active site cysteine residue resulting in the formation of a
homodimeric form by disulfide linkage through the cysteine residues
(13, 50). To study the biochemical characteristics of CbPmp20, a
His6-tagged version of CbPmp20 (His6-CbPmp20)
and His6-CbPmp20 C53S in which the cysteine 53 residue
corresponding to CbPmp20 was replaced by serine were overproduced in
E. coli under the control of the T7 promoter. The resistance
to tert-butyl hydroperoxide of E. coli cells
producing His6-CbPmp20 or His6-CbPmp20 C53S was
then determined (Fig. 2A). A
filter paper containing tert-butyl hydroperoxide was placed
on a lawn of E. coli cells, and growth inhibition was
evaluated by the size of the clear zone surrounding the filter paper.
The cells producing His6-CbPmp20 became more resistant to
tert-butyl hydroperoxide than the cells producing
His6-CbPmp20 C53S (Fig. 2A). This indicated that
His6-CbPmp20 exhibited its anti-oxidant activity in
E. coli and that the Cys-53 residue of CbPmp20 is necessary
for its anti-oxidant activity. Similarly, overproduction of ScPmp20
(Ahp1p) in S. cerevisiae was reported to result in yeast
resistance to alkyl hydroperoxides in a
thioredoxin-dependent manner (13). However, overproduction of CbPmp20 in S. cerevisiae did not show such a resistance,
even though a considerable amount of CbPmp20-protein was detected by Western analysis (data not shown).
His6-CbPmp20 and His6-CbPmp20 C53S were
purified to homogeneity judged on SDS-PAGE through
nickel-nitrilotriacetic acid column as described under "Experimental
Procedures" (Fig. 2B). His6-CbPmp20 prepared
in E. coli showed two bands corresponding to the monomeric and dimeric size under the nonreducing conditions (Fig. 2B,
lane 1). Upon the reduction by 10 mM DTT, the
upper band disappeared (Fig. 2B, lane 2).
Therefore, we assumed that CbPmp20 dimerized through disulfide linkage
between a single cysteine residue corresponding to Cys53 of
the monomeric form. Indeed, His6-CbPmp20 C53S was present only as a a monomeric form in E. coli cell-free extract
under nonreducing conditions (data not shown). The
His6-CbPmp20 C53S protein was also purified to homogeneity
via the same procedure for His6-CbPmp20 and used for
further analyses (Fig. 2B, lane 3).
The thiol specificity of His6-CbPmp20 in antioxidant
activity was studied by monitoring the consumption of O2
using a metal-catalyzed oxidation system (51). This oxidation system is
comprised of Fe3+, O2, and electron donors.
When DTT was used as an electron donor, O2 was consumed
slowly in several minutes (lag phase) (Fig. 2C). During this
lag phase, H2O2 accumulated, and propagation of
radical chain reaction lead to very rapid consumption of O2
(Fig. 2C, no addition). The rapid O2 consumption
was prevented by His6-CbPmp20 (Fig. 2C). The
lack of this inhibitory effect of His6-CbPmp20 added at 16 min (Fig. 2C) indicated that CbPmp20 prevented the accumulation of H2O2 at the lag phase. In the
ascorbate system, the lag phase of O2 consumption is
shorter than the DTT system, and His6-CbPmp20 did not
inhibit the O2 consumption (Fig. 2D) (51).
Therefore, purified His6-CbPmp20 prevented the
O2 consumption using a thiol-metal-catalyzed oxidation
system (DTT/Fe3+/O2) (Fig. 2B) but
did not prevent the O2 consumption in a
non-thiol-metal-catalyzed oxidation system
(ascorbate/Fe3+/O2) (Fig. 2D). On
the other hand, catalase prevented the O2 consumption in
both systems (Fig. 2, C and D). These results
indicated that His6-CbPmp20 does carry thiol-specific
peroxidase activity. Furthermore, because purified
His6-CbPmp20 C53S could not prevent O2 uptake (Fig. 2C), Cys53 of CbPmp20 is considered to be
essential for thiol peroxidase activity.
Glutathione Peroxidase Activity of
His6-CbPmp20--
Some thiol-specific antioxidants which
reduce ROS have been shown to be themselves reduced by electron donors,
e.g. glutathione, thioredoxin, and tryparedoxin, to recycle
oxidized thiol groups (19, 52). CbPmp20 belongs to the 1-Cys Prx
subfamily, which has been suggested to use glutathione as an electron
donor (19). Therefore, we tested glutathione-dependent
peroxidase activity of purified His6-CbPmp20. The rate of
cumene hydroperoxide reduction catalyzed by purified
His6-CbPmp20 was measured by monitoring the decrease in
A340 (attributable to the oxidation of NADPH) in
a glutathione-dependent system. In the presence of GSH, GR, and NADPH, His6-CbPmp20 reduced cumene hydroperoxide
efficiently (Fig. 3, column
1). Only background activity was detected when either CbPmp20
(column 2) or GSH (column 3) or GR (column
4) was omitted from the reaction. When His6-CbPmp20
was replaced with His6-CbPmp20 C53S (column 5),
the GPX activity was lost. Our biochemical results established
His6-CbPmp20 as a functional GPX. On the other hand,
thioredoxin has recently been identified as a biochemical hydrogen
donor for HsPmp20 and ScPmp20 (12, 13, 20). We assessed the effect of
thioredoxin on the peroxidase activity of purified
His6-CbPmp20. In this system, however, CbPmp20 did not
exhibit a thioredoxin-dependent peroxidase activity (Fig. 3, column 6).
As shown in Fig. 2B, the dimeric form of
His6-CbPmp20 was converted to the monomeric form upon
reduction by 10 mM DTT. Because His6-CbPmp20
could use GSH as an electron donor, we asked whether a dimeric form of
His6-CbPmp20 could be reduced by GSH (Fig.
4A). We found that GSH indeed
reduced the oxidized dimeric form of His6-CbPmp20 protein
into the reduced monomeric form and that the reduction was in
dose-dependent manner. The physiological level of GSH, ~2
mM, was sufficient to complete reaction (Fig. 4B).
Next, we measured the initial rates of NADPH oxidation at various
concentrations of cumene hydroperoxide, tert-butyl
hydroperoxide, and H2O2 (Table
II). Double-reciprocal plots of GPX
activity versus substrate concentration were linear for
these peroxides (data not shown). Because GPX activity did not show
saturation with GSH, apparent kinetic constants were determined at 0.1 mM GSH (Table II) (53). Although the apparent
Vmax measured with each of the three peroxides
was similar: 80.0 µmol/min/mg protein for cumene hydroperoxide, 75.8 µmol/min/mg protein for tert-butyl hydroperoxide, and 71.4 µmol/min/mg protein for H2O2, the apparent Km values for the alkyl hydroperoxides were lower
than that for H2O2: 0.562 mM for
cumene hydroperoxide, 0.952 mM for tert-butyl
hydroperoxide, and 2.86 mM for
H2O2.
CbPmp20 Is Necessary for the Methylotrophic Growth of C. boidinii--
CbPmp20 is specifically induced when C. boidinii cells are grown in methanol medium (16, 22, 34). To study
the physiological function of CbPmp20 during methylotrophic growth,
gene disruption of CbPMP20 was carried out as described in
Fig. 5A, and the growth of the
disrupted strain (strain pmp20
The pmp20
The growth defect of the pmp20 Peroxisomal Targeting of CbPmp20 and Complementation of the Growth
Defect in Strain pmp20
Next, three C-terminal amino acids, -AKL, were deleted from
CbPmp20 (CbPmp20
The localization of CbPmp20 and CbPmp20
As demonstrated with the biochemical in vitro experiment, an
antioxidant activity or GPX activity was executed through the Cys53 residue of CbPmp20. Therefore, CbPmp20 C53S was
introduced in pmp20 Glutathione System in Peroxisomes--
Our results demonstrated
that His6-CbPmp20 had GPX activity in vitro and
that CbPmp20 exerted its physiological activity within peroxisomes.
Glutathione is an abundant cellular thiol compound that has been
implicated in many cellular processes, but its existence within
peroxisomes has not previously been established. We detected a dimeric
oxidized form of His6-CbPmp20 in E. coli, the
formation of which was stimulated by addition of tert-butyl
hydroperoxide into the culture medium (data not shown). However, the
dimeric oxidized form of CbPmp20 could not be detected in C. boidinii cells even when the cells were exposed to highly
ROS-stressed conditions, e.g. in the cta1
Next, we asked whether peroxisomes contain GPX (and/or TPX), and the
other components of the GPX system, i.e. GR and glutathione. The highly purified peroxisomal fractions (Fig. 8B, fraction
4 from the rCbPmp20 strain; fraction 3 from the rCbPmp20
Fig. 8C shows the glutathione and protein concentration
throughout the fractionated fractions from the rCbPmp20 strain.
Glutathione showed a small peak around the peroxisomal fraction
(fraction 4) in proportion to the amount of protein (Fig.
8C). Glutathione in fraction 4 was found to be in the
reduced form (GSH) (0.950 µg/mg protein), and the oxidized form of
glutathione (GS-SG) was less than the detectable level (0.05 µg/mg
protein). Therefore, GSH is found to be present in peroxisomes at least
at the physiological level.
Morphology of Peroxisomes in C. boidinii pmp20 Three antioxidant enzymes, i.e. catalase, CuZn
superoxide dismutase, and GPX, were described previously as antioxidant
enzymes in mammalian peroxisomes and were assumed to play important
roles in the detoxification of ROS. In contrast to higher organisms, catalase was until recently the only known anti-oxidant enzyme in yeast
peroxisomes. Recently described Pmp20 family members are novel
candidate peroxisomal anti-oxidant proteins. Thus far, the anti-oxidant
activity of ScPmp20 and mammalian Pmp20 has been demonstrated only
in vitro, and their in vivo function has been unclear. In this study, we have characterized the biochemical and
physiological function of CbPmp20 in the methylotrophic yeast C. boidinii, taking advantage of its well characterized molecular genetics and cell fractionation technique and its methylotrophic growth.
The most important fact revealed in this study is that CbPmp20
performs its physiological anti-oxidant function within peroxisomes as
a GPX, which can use alkyl hydroperoxides and
H2O2 as substrates. Interestingly, the
knock-out defect caused by CbPmp20 depletion was much more severe than
that induced by depletion of peroxisomal catalase. Moreover, the
viability of pmp20 Although other Pmp20 family members (HsPmp20, ScPmp20) contain more
than two cysteine residues and belong to the 2-Cys Prx subfamily,
CbPmp20 is unique among Pmp20 family proteins in having only one
conserved cysteine, 1-Cys Prx. Many 2-Cys Prxs are known as TPXs, and
the catalytic model involving 2-Cys residues for TPX activity has also
been postulated (50, 60). In contrast, the role of GSH as the reductant
for 1-Cys Prx protein has been controversial (49, 53, 61). In this
study, we have shown that 1-Cys Prx CbPmp20 can utilize GSH as a
reductant for both the catalytic reaction and monomerization from
oxidized dimeric form in vitro. In contrast, yeast
thioredoxin did not replace GSH, which fits with the model requiring
both Cys residues for the catalysis of thioredoxin reductase (50).
However, because a physiological level of GSH was detected in a
peroxisomal fraction and a dimeric form of CbPmp20 was not detected in
C. boidinii, GSH may be a native reductant for CbPmp20
within peroxisomes.
The presence of GPX and glutathione within peroxisomes has raised
another question regarding whether the glutathione regeneration system
is present in peroxisomes. However, we could not detect GR activity in
the peroxisomal fraction of C. boidinii. Because of the
fragile nature of the isolated peroxisomes, early studies led to the
hypothesis that peroxisomal membrane are freely permeable to compounds
of low molecular weight (62). However, recent studies indicated that
some metabolites are unable to permeate the peroxisomal membrane and
that there are transporters for low molecular weight compounds (26, 63,
64). Likewise, there may be some glutathione transport system in
peroxisome membrane to import GSH and to export GS-SG. In methanol
metabolism, formaldehyde produced by peroxisomal alcohol oxidase should
be hydrated spontaneously to form S-hydroxymethyl glutathione, being a substrate for cytosolic
glutathione-dependent formaldehyde dehydrogenase (65).
Therefore, S-hydroxymethyl glutathione might be formed in
peroxisomes and then exported to cytosol via the glutathione transport
system in peroxisomal membrane.
Among Pmp20 family members, CbPmp20 has another unique feature,
i.e. CbPmp20 is exclusively localized in peroxisomes,
whereas AOEB166 (a long form of HsPmp20) and ScPmp20 seem to show
bimodal distribution between peroxisomes and mitochondria (12, 18). CbPmp20 may have evolved in a unique pathway such that the Pmp20 molecule specifically protects the peroxisome membrane from ROS generated during the methanol metabolism. In fact, CbPmp20 was induced
specifically in methanol medium but not in other peroxisome-inducing carbon sources, e.g. oleate or D-alanine (22).
Although oleate- and D-alanine metabolism also generate
H2O2 within peroxisomes through acyl-CoA
oxidase and D-amino acid oxidase, respectively, CbPmp20 is
not necessary for their metabolism. Why then does methanol-metabolism specifically require Pmp20? Methanol-induced peroxisomes harbor a high
amount of FAD protein alcohol oxidase, which can amount to nearly 40%
of the total soluble proteins (66), whereas intracellular protein
contents of acyl-CoA oxidase and D-amino acid oxidase were
low (less than 1%) (34). Alcohol oxidase contains FAD in both quinone
and semiquinone forms (67), and these quinone cofactors could be
released from the alcohol oxidase protein during the methylotrophic
growth (68). These released quinone compounds may catalyze radical
chain reaction together with generated H2O2 and
induce highly oxidative conditions within peroxisomes as described above (57, 58). Therefore, C. boidinii may have retained
Pmp20 molecule during the evolution to protect against these highly oxidative conditions in the methanol metabolism. In contrast, ScPmp20
might have lost its physiological function in peroxisomes during the
evolution, because S. cerevisiae can grow on oleate medium
without ScPmp20.
Our experiments, using highly purified peroxisomes, indicate that
methanol-induced peroxisomes of C. boidinii contain a GPX activity catalyzed by CbPmp20 (Fig. 9). Singh and Shichi (61) reported
that peroxisomes purified from rat liver had a GPX activity and the
peroxisomal fraction showed a cross-reacting band around 20 kDa with
antibody raised against cytosolic GPX (1-Cys Prx). In human cells,
HsPmp20 or another Pmp20 family protein may have peroxisomal GPX
activity, which will play an antioxidant role similar to CbPmp20.
In summary, ROS in methanol-induced peroxisomes of C. boidinii is scavenged by two anti-oxidant enzymes: CbPmp20 and
peroxisomal catalase. Our results show that ROS generated in
peroxisomes could cause severe injury that leads to cell death, and
these ROS species are eliminated by CbPmp20 having a GPX activity at
the peroxisomal membrane surface. This is the first report that
clarifies the physiological role of Pmp20 family proteins as the
anti-oxidant system within a ROS generating organelle, the peroxisome.
strain). The His6-tagged CbPmp20
fusion protein was found to have glutathione peroxidase activity
in vitro toward alkyl hydroperoxides and
H2O2. Catalytic activity and dimerization of
His6-CbPmp20 depended on the only cysteine residue
corresponding to Cys53. The pmp20
strain was
found to have lost growth ability on methanol as a carbon and energy
source. The pmp20
growth defect was rescued by CbPmp20,
but neither CbPmp20 lacking the peroxisome targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the
growth defect. Interestingly, the pmp20
strain had a
more severe growth defect than the cta1
strain, which
lacks catalase, another antioxidant enzyme within the peroxisome.
During incubation of these strains in methanol medium, the
cta1
strain accumulated H2O2,
whereas the pmp20
strain did not. Therefore, it is
speculated to be the main function of CbPmp20 is to decompose reactive
oxygen species generated at peroxisomal membrane surface, e.g. lipid hydroperoxides, rather than to decompose
H2O2. In addition, we detected a physiological
level of reduced glutathione in peroxisomal fraction of C. boidinii. These results may indicate a physiological role for
CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive
oxygen species.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which is the disruptant of the
peroxisomal catalase-encoding gene (CTA1), will be described elsewhere.3 Synthetic MI
medium was used as the basal medium on which C. boidinii was
cultivated (26). One or more of the following was used as the carbon
source in each experiment: 1% (w/v) glucose, 1% (v/v) methanol. The
initial pH of the medium was adjusted to 6.0. The complex YP medium
containing 2% bactopeptone and 1% yeast extract (Difco Laboratories,
Detroit, MI) was also used as the basal medium in some experiments. YPD
contained 2% glucose, and YPMGy contained 0.5% methanol and 0.5%
glycerol as the carbon source(s). The C. boidinii strains
were incubated aerobically at 28 °C under reciprocal shaking, and
the growth of the yeast was followed by measuring the
OD610. Escherichia coli DH5
(28) was
routinely used for plasmid propagation. LB medium (0.5% yeast extract,
1% bactopeptone, and 1% NaCl) (28) was used for bacterial expression
of the recombinant CbPmp20s.
Oligonucleotide primers used in this study
, obtained with pD20SPR, was reverted to
uracil auxotrophy, yielding strain pmp20
ura3,
using our previously described procedure (23).
AKL, in which the C-terminal 3 amino
acids of CbPmp20 were deleted, was constructed in the same way with the use of primers NOT-20-N and 20dAKL-C (Table I). These constructed plasmids were linearized by BamHI and introduced into
C. boidinii strain pmp20
ura3 by
the modified lithium acetate method (33). The expression plasmid,
pGFP-AKL containing GFP tagged with the Pmp20 C-terminal -AKL sequence
(34), was also introduced into strain pmp20
ura3, yielding strain GFP-AKL/pmp20
. Strain
GFP-AKL/wt (34) was used as the control wild type strain.
-D-thiogalactopyranoside to a 1 mM final
concentration and cultured further for 5 h. The cells were
harvested by centrifugation at 5000 × g for 5 min at 4 °C, resuspended in 25 ml of cold 50 mM Tris-HCl, pH
7.5, and recentrifuged. The cell pellet was resuspended in 25 ml of the same buffer, frozen and thawed six times, and sonicated for 30 s
three times. Solubilized proteins were recovered by centrifugation at
15,000 × g for 15 min. The obtained protein-containing
supernatant was applied on the nickel-nitrilotriacetic acid-agarose
column (Qiagen, Chatsworth, CA), and proteins were purified through a protein nondenaturation procedure as recommended by the manufacturer. Purified proteins were dialyzed against 50 mM Tris-HCl, pH
7.5, and subjected to the biochemical analyses.
cells were incubated in MI medium containing 0.5%
yeast extract for 10 h. Cells were fixed with 2% glutaraldehyde
and postfixed sequentially with 1.5% KMnO4 and 1.5%
uranyl acetate as described previously (47). The material was
dehydrated in a graded acetone series and then embedded in Spurr resin
(Polysciences, Inc., Warrington, PA). Ultrathin sections were prepared
by using a diamond knife for cutting and observed under an electron
microscope (JEOL model C100).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequence alignment of CbPmp20 with
other Pmp20 family proteins. The cysteine residues
responsible for catalysis in Prx is boxed with a solid
line. The putative PTS1 sequences at the C terminus are
boxed with a dashed line. The residues with
asterisks represent residues conserved in all sequences, and
those with dots represent conserved similar amino
acids.
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Fig. 2.
Purified His6-CbPmp20 showed a
thiol-specific antioxidant activity. A, effects of
His6-CbPmp20 overexpression in E. coli on the
growth inhibition exerted by tert-butyl hydroperoxide. Discs
containing either (left panel) 5 µl or (right
panel) 10 µl of 500 mM tert-butyl
hydroperoxide were placed on lawns of E. coli expressing
His6-CbPmp20 or His6-CbPmp20 C53S.
B, SDS-polyacrylamide gel electrophoresis analysis of
His6-CbPmp20 and His6-CbPmp20 C53S purified
from the E. coli cell-free extract through
nickel-nitrilotriacetic acid column. The recombinant protein was eluted
with 100 mM imidazole. The recombinant protein (5 µg/lane) was loaded to each lane and visualized by Coomassie Blue
staining. Lanes 1 and 2,
His6-CbPmp20; lane 3, His6-CbPmp20
C53S (without DTT). The upper band of purified
His6-CbPmp20 (lane 1) disappeared upon the
reduction in the presence of 10 mM DTT (lane 2).
These recombinant proteins were used for antioxidant activity assays.
C, antioxidant activity of His6-CbPmp20 in the
metal-catalyzed oxidation system containing 5 mM DTT. 1 µM His6-CbPmp20 (closed circle), 1 µM His6-CbPmp20 C53S (closed
triangle), or 0.1 µM catalase (open
triangle) was added prior to the initiation of the reaction.
Closed diamond, His6-CbPmp20 was added at 16 min
(arrow) after the reaction had been started. Open
circle, no addition. The basal reaction mixture contained 5 µM Fe3+ and 1 mM EDTA in 50 mM Hepes-NaOH buffer, pH 7.4, and the reaction was
performed at 37 °C. D, antioxidant activity of
His6-CbPmp20 in the metal-catalyzed oxidation system
containing 10 mM ascorbate. Symbols are the same as in
C.
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Fig. 3.
GSH-dependent peroxidase activity
of purified His6-CbPmp20. Assays were performed with
the purified His6-CbPmp20 (20 µg) in the presence of the
GSH system (columns 1-4) or the thioredoxin system
(column 6) using 1 mM cumene hydroperoxide as
the substrate. Column 5, His6-CbPmp20 was
replaced by His6-CbPmp20 C53S in the complete GSH system.
Experiments were done in triplicates as described under "Experimental
Procedures," and the error bars indicate standard
deviations.
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Fig. 4.
Cys53-dependent dimerization of
His6-CbPmp20 and regeneration of monomeric form by
GSH. A, His6-CbPmp20 purified from E. coli were in a dimeric and monomeric form (lane 1). The
protein (~5 µg for each lane) was visualized by Coomassie Blue
staining. The addition of 10 mM DTT (lane 2) or
10 mM GSH (lane 3) or the C53S mutation
(lane 4) abolished dimer formation. B, purified
His6-CbPmp20 (5 µg of protein) was treated under
indicated concentrations of GSH, and the protein was visualized by
Coomassie Blue staining.
GPX activity of His6-CbPmp20 with several substrates
) on methanol was compared with both the wild type strain, and the cta1
strain, in
which the peroxisomal catalase-encoding gene was
disrupted.3 Proper gene disruption and subsequent excision
of the URA3 sequence were confirmed by Southern analysis
with HindIII-digested genomic DNAs from the transformant,
using the 1.0-kb StyI-HindIII fragment from
pMP200 as the probe (Fig. 5B). The 3.0-kb hybridizing band in the host strain (Fig. 5B, lane 1) shifted to
6.0-kb in the pmp20
strain (Fig. 5B,
lane 2), and this 6.0-kb band shifted to 2.4-kb upon the
regeneration of uracil auxotrophy (Fig. 5B, lane
3). In addition, immunoblotting with an anti-Pmp20 antibody confirmed the loss of the signal in the pmp20
strain
(data not shown).
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Fig. 5.
One-step disruption of the PMP20
gene in C. boidinii genome. A,
physical map of the cloned fragment and disruption strategy.
Arrows show the direction of the coding sequences. The
shaded boxes at both ends of URA3 show repeated
sequences for homologous recombination to remove the URA3
gene after gene disruption (23). B, genomic Southern
analysis of HindIII-digested total DNAs (3 µg each) from
the host strain TK62 (lane 1), the pmp20
strain (lane 2), and the pmp20
ura3
strain (lane 3), probed with the 32P-labeled
1.0-kb StyI-HindIII fragment, including the
3'-flanking region of PMP20.
strain lost the capacity for methylotropic
growth (Fig. 6A), but it could
grow normally in other peroxisome-inducing carbon sources, such as
oleate or D-alanine (data not shown). On the other hand,
the cta1
strain could grow on methanol, although its
growth rate was retarded (Fig. 6A).
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Fig. 6.
Growth and viability of the C. boidinii
pmp20 and cta1
strains during the incubation in the methanol
medium. A, growth on methanol. Open circle,
the wild type strain; open triangle, the pmp20
strain; closed circle, the cta1
strain.
B, viability. At the incubation time in the methanol medium
indicated, 5 µl of the cell suspension was plated onto YPD medium
plate. C, H2O2 concentration in the
incubation medium. Symbols are the same as in A.
strain, which was more
severe than that of the cta1
strain, was also
demonstrated by another experiment. During the incubation of each
strain in methanol medium, the viability on YPD medium-plate (Fig.
6B) and the H2O2 concentration in
the medium (Fig. 6C) were determined. A considerable amount of H2O2 was accumulated with the cell
suspension of the cta1
strain, whereas the
H2O2 in the medium was not detected for the pmp20
strain. Nevertheless, the viability of the
pmp20
strain decreased drastically, whereas that of the
cta1
strain was not affected (Fig. 6B). These
results show that H2O2 generated during the
methylotrophic growth is mainly decomposed by peroxisomal catalase and
also suggest that CbPmp20 is more involved in decomposition of ROS
which is more toxic than H2O2. The in
vitro activity of His6-CbPmp20 toward
alkylhydroperoxides and the association of CbPmp20 with the inner side
of peroxisomal membrane lead us to speculate that CbPmp20 reduces and
detoxifies lipidperoxides generated in peroxisomal membrane. However,
the peroxisomal location of Pmp20 family proteins has not been
previously demonstrated to be necessary for their physiological
function. To obtain evidence that CbPmp20 indeed executes its
physiological antioxidant function within peroxisomes, we asked whether
peroxisomal targeting of CbPmp20 is necessary to complement the growth
defect of strain pmp20
.
--
Pmp20 family proteins contain PTS1
sequences at their C-terminal ends (11, 12, 16-18). At first, we
introduced GFP tagged with the C-terminal PTS1 of CbPmp20, -AKL, to
visualize the peroxisomes in strain pmp20
. Previous
studies showed that the C-terminal -AKL sequence was sufficient for
targeting cytosolic GFP to peroxisomes (34). As shown in Fig.
7A, transport of GFP-AKL to
peroxisomes was normal in the pmp20
strain.
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Fig. 7.
Peroxisomal protein transport and the growth
defect of the C. boidinii pmp20 strain
on methanol medium. A, GFP-AKL fluorescence in the wild
type and pmp20
strains. B, growth of the
pmp20
strains on methanol medium expressing various
versions of CbPmp20 protein. Open circle, the rCbPmp20
strain; closed triangle, the rCbPmp20
AKL strain; open
triangle, the rCbPmp20 C53S strain; closed circle, the
host pmp20
strain.
AKL), and CbPmp20 and CbPmp20
AKL were expressed in the pmp20
strain to determine whether the peroxisomal
localization of CbPmp20 was necessary to complement the growth defect
of the pmp20
strain. Whereas CbPmp20 could complement the
growth defect of the pmp20
strain comparable with the
level of the wild type strain, the Pmp20 protein without PTS1
sequence, CbPmp20
AKL, could not (Fig. 7B).
AKL was analyzed by
subjecting each strain (the rCbPmp20 strain and the rCbPmp20
AKL strain, respectively) to differential centrifugation, which separated the intracellular components into a cytosolic supernatant (S) and an
organelle-pellet fraction (P) consisting mainly of peroxisomes and
mitochondria (Fig. 8A). More
than 80% of CbPmp20 and peroxisomal alcohol oxidase was detected in
the organelle-pellet fraction (Fig. 8A), and CbPmp20 protein
colocalized with a peroxisomal marker protein catalase on Nycodenz
gradient (Fig. 8B). On the other hand, CbPmp20
AKL was
found in the cytosolic supernatant fraction (Fig. 8A).
Therefore, deletion of the C-terminal -AKL sequence from CbPmp20 lead
to mistargeting of CbPmp20 from peroxisomes to the cytosol.
Furthermore, because rCbPmp20
AKL strain showed a 9.8-fold higher
level of cytosolic GPX activity (7.90 units/mg protein) when compared
with the rCbPmp20 strain (0.808 unit/mg protein), CbPmp20
AKL is
considered to be present in the cytosol in an enzymatically active
form.
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Fig. 8.
The C-terminal -AKL in CbPmp20 is necessary
for peroxisomal transport. A, methanol-induced cells
were lysed by osmotic shock, and cell debris was removed by
centrifugation at 500 × g. The resultant supernatant
was separated into pellettable fraction (P) at 20000 × g including peroxisomes and mitochondria and its supernatant
(S) and dissolved in the same volume of the buffer. The same
aliquots were analyzed by Western analysis using anti-alcohol oxidase
or anti-CbPmp20. B, the organellar pellet fraction after
centrifugation at 20,000 × g was further fractionated
on Nycodenz equilibrium density gradient centrifugation. The relative
activity 100% for catalase (open circle) corresponds to
2210 units/ml for the rCbPmp20 strain and 1430 units/ml for the
rCbPmp20 AKL strain, and that for cytochrome c oxidase
(closed circle) corresponds to 0.297 unit/ml for the
rCbPmp20 strain and 0.104 unit/ml for the rCbPmp20
AKL strain.
Peroxisomal localization of CbPmp20 was confirmed on Western blot using
anti-Pmp20 monoclonal antibody with the rCbPmp20
strain
C, peroxisomal fraction contained glutathione. Open
square, protein amount; bars, glutathione amount.
(the rCbPmp20 C53S strain) to see
whether this anti-oxidant activity CbPmp20 was necessary for its
physiological function. As expected, CbPmp20 C53S could not complement
the growth of C. boidinii pmp20
on methanol (Fig.
7B), although CbPmp20 C53S was targeted to peroxisomes (data
not shown). These growth complementation and subcellular fractionation
experiments demonstrate that CbPmp20 exerts its physiological function
through its antioxidant activity specifically within peroxisomes.
strain or cells grown under high methanol concentrations (data not
shown). Therefore, we speculated that a dimeric form of CbPmp20 is
rapidly re-reduced into its monomeric form via reduced glutathione
present within peroxisomes of C. boidinii. In addition,
His6-tag might alter the conformational change of CbPmp20
and hinder the TPX activity of CbPmp20.
AKL strain), which contained no mitochondrial contamination as judged by cytochrome c oxidase activity, were tested for further analyses. As
expected, the purified peroxisomal fraction from the rCbPmp20 strain
exhibited both GPX activity (3.78 units/mg protein) and catalase
activity (2080 units/mg protein), but neither TPX nor GR activity could be detected. On the other hand, the purified peroxisomal fraction from
the rCbPmp20
AKL strain contained a catalase activity (1460 units/mg
protein) bud did not contain a detectable GPX activity. These results
show that peroxisomal GPX is CbPmp20 and that CbPmp20 with or without
His6-tag sequence (cf. Fig. 3) did not have TPX activity.
Cells--
Recently, Schrader et al. (54) reported that ROS
could induced a tubular form of peroxisomes in HepG2 cells. To
determine whether ROS generation caused by CbPmp20-depletion induced
abnormal peroxisomal morphology, methanol-induced cells of strain
pmp20
were observed under electron microscopy. Electron
microscopic observation confirmed normal morphology of peroxisomes in
pmp20
cells (Fig. 9).
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Fig. 9.
Peroxisomal morphology is normal in the
pmp20 strain. Subcellular EM
morphology of KMnO4-fixed pmp20
cells
incubated in the methanol medium for 12 h. P,
peroxisome; N, nucleus; M, mitochondrion;
V, vacuole. Bar, 1 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells decreased during the incubation
in methanol medium, whereas that of the cta1
strain did
not, even though a considerable level of H2O2
accumulation was observed with the cta1
strain. During
methylotrophic growth, the majority of H2O2
generated by alcohol oxidase may be mainly decomposed by catalase
within peroxisomes. However, because the affinity of catalase toward
H2O2 is low (Km for
H2O2, 25 mM) (55), a trace amount
of H2O2 will not be eliminated by catalase
alone. Therefore, CbPmp20 having higher affinity against H2O2 (Km for
H2O2, 2.86 mM; Table II) may also
have a role in eliminating H2O2 at lower
H2O2 concentrations. However, we think that
CbPmp20 has a more important role in decomposing lipid hydroperoxides.
A major peroxisomal enzyme alcohol oxidase contains FAD (or flavin
semiquinone) (56), and peroxisomal catalase contains heme as a cofactor
(55). When these cofactors or iron molecules are released from the
protein, they could catalyze formation of hydroxyl radicals from
H2O2 (57, 58), which then attack peroxisomal
membrane, resulting in the generation of lipid hydroperoxides. Accumulation of lipid hydroperoxides will further accelerate oxidative decomposition of membrane lipids through radical chain reactions (59).
Therefore, it may be reasonably speculated that CbPmp20 is present and
functions at the inner side of peroxisomal peripheral membranes. The
main physiological function of CbPmp20 is most likely the elimination
of lipid hydroperoxides generated in peroxisomal membranes so that
cells can maintain the integrity of peroxisomal membrane to avoid cell death.
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ACKNOWLEDGEMENTS |
---|
We are very grateful to Dr. Joel M. Goodman (University of Texas, South Western Medical Center, Dallas) for the generous gift of valuable reagents, Dr. Tokichi Miyakawa (Hiroshima University) for information on S. cerevisiae ahp1 knock-out phenotype. We acknowledge Dr. Tsuneo Imanaka (Toyama Medical and Pharmaceutical University) and Dr. Jun'ichi Mano (Kyoto University) for valuable advice, helpful discussion, and critical reading of the manuscript and Dr. Hirokazu Matsukawa (Oriental Yeast, Co. Ltd.) for the generous gift of yeast thioredoxine and thioredoxine reductase.
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FOOTNOTES |
---|
* This work was supported by a Ministry of Education, Science, Sports, and Culture of Japan grant-in-aid for scientific research and the research fund from Noda Institute for Scientific Research.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) AB055180.
To whom correspondence should be addressed. Tel.:
+81-75-753-6455; Fax: +81-75-753-6385; E-mail:
ysakai@kais.kyoto-u.ac.jp.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011661200
2 I. C. Farcasanu and T. Miyakawa, personal communication.
3 H. Horiguchi, H. Yurimoto, T.-K. Goh, T. Nakagawa, N. Kato, and Y. Sakai, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: ROS, reactive oxygen species; Prx, peroxiredoxin; GPX, glutathione peroxidase; GSH, reduced form of glutathione; GS-SG, oxidized form of glutathione; GR, GSH reductase; TPX, thioredoxin peroxidase; CbPmp20, C. boidinii Pmp20; HsPmp20, Homo sapiens Pmp20; MmPmp20, Mus musculus Pmp20; ScPmp20, S. cerevisiae Pmp20; GFP, green fluorescent protein; PTS, peroxisome targeting signal; PCR, polymerase chain reaction; kb, kilobase(s); DTT, dithiothreitol.
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