Download Developmental Analysis of a Putative ATP/ADP Carrier Protein

Plant Cell Physiol. 42(8): 835–841 (2001)
JSPP © 2001
Developmental Analysis of a Putative ATP/ADP Carrier Protein Localized on
Glyoxysomal Membranes During the Peroxisome Transition in Pumpkin
Cotyledons
Youichiro Fukao 1, 2, Yasuko Hayashi 3, Shoji Mano 1, Makoto Hayashi 1 and Mikio Nishimura 1, 2, 4
1
Departmnt of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan
Department of Molecular Biomechanics, School of Life Science, Graduate University for Advanced Studies, Okazaki, 444-8585 Japan
3
Departmnt of Environmental Science, Faculty of Science, Niigata University, Ikarashi, Niigata, 950-2181 Japan
2
;
oxidation cycle and the glyoxylate cycle, and leaf peroxisomes
contain enzymes of the photorespiration pathway that act in
concert with enzymes in chloroplasts and mitochondria (Tolbert
et al. 1968). Peroxisomes are transformed from glyoxysomes to
leaf peroxisomes in greening cotyledons of fatty seedlings, and
this transformation is controlled by light (Nishimura et al.
1993, Titus and Becker 1985). The reverse conversion from
leaf peroxisomes to glyoxysomes also occurs in senescing
cotyledons (De Bellis and Nishimura 1991). In contrast to the
matrix enzymes, how membrane proteins change during the
peroxisome transition is not well understood. To date only a
few peroxisomal membrane proteins (PMPs) have been characterized in higher plants, for example PMP31 (peroxisomal
ascorbate peroxidase), PMP28 and PMP22 (Yamaguchi et al.
1995a, Tugal et al. 1999).
In this paper, we report the presence of a novel PMP,
PMP38, in higher plant peroxisomes as an integral membrane
protein. The deduced amino acid sequence of the cDNA of
Arabidopsis thaliana has high similarity with those of Homo
sapiens (Hs) PMP34 and Candida boidinii (Cb) PMP47.
Developmental analysis revealed that the level of PMP38 was
similar to those of the fatty acid >-oxidation enzymes during
greening and senescence, suggesting that PMP38 functions in
concert with the fatty acid >-oxidation cycle.
In order to clarify the peroxisomal membrane proteins (PMPs), we characterized one of the major PMPs,
PMP38. The deduced amino acid sequence for its cDNA in
Arabidopsis thaliana contained polypeptides with 331 amino
acids and had high similarity with those of Homo sapiens
PMP34 and Candida boidinii PMP47 known as homologues
of mitochondrial ATP/ADP carrier protein. We expected
PMP38 to be localized on peroxisomal membranes, because
it had the membrane peroxisomal targeting signal. Cell
fractionation and immunocytochemical analysis using
pumpkin cotyledons revealed that PMP38 is localized on
peroxisomal membranes as an integral membrane protein.
The amount of PMP38 in pumpkin cotyledons increased
and reached the maximum protein level after 6 d in the
dark but decreased thereafter. Illumination of the seedlings
caused a significant decrease in the amount of the protein.
These results clearly showed that the membrane protein
PMP38 in glyoxysomes changes dramatically during the
transformation of glyoxysomes to leaf peroxisomes, as do
the other glyoxysomal enzymes, especially enzymes of the
fatty acid >-oxidation cycle, that are localized in the matrix
of glyoxysomes.
Key words: ATP/ADP carrier protein — Fatty acid >-oxidation
cycle — Glyoxysome — Integral membrane protein — The
peroxisome transition.
Results
Abbreviations: AAC, ATP/ADP carrier protein; mPTS, membrane peroxisomal targeting signal; PMP, peroxisomal membrane protein.
Deduced amino acid sequences of AtPMP38
To identify the major PMPs, we prepared the pumpkin
glyoxysomal membrane fraction and determined the internal
amino acid sequences of some major membrane proteins by ingel digestion (data not shown). By this procedure, we obtained
one internal amino acid sequence (IQELYDEAGI) from the
polypeptides with a molecular mass of about 38 kDa,
designated PMP38 from the molecular mass. This sequence
had high homology with the Arabidopsis putative peroxisomal
ATP/ADP carrier protein (AAC) using the BLAST algorithm
(Altschul et al. 1990). To characterize this protein, we obtained
EST clone (173G9T7) corresponding to the sequences of putative peroxisomal AAC and determined the sequences. As
shown in Fig. 1, the complete sequence of the cDNA of A.
Introduction
The peroxisome is a single membrane-bound organelle
present in eukaryotic cells (Lazarow and Fujiki 1985). In
higher plants, peroxisomes are classified into three functionally different types, namely glyoxysomes, leaf peroxisomes and
unspecialized peroxisomes (Beevers 1979, Huang et al. 1983).
Enzymatic components of peroxisomes have been characterized well. Glyoxysomes contain enzymes for the fatty acid >4
Corresponding author: E-mail, [email protected]; Fax, +81-564-55-7505.
835
836
Glyoxysomal ATP/ADP carrier protein
Fig. 2 Comparison of energy-transfer-protein signatures of
AtPMP38, HsPMP34 and CbPMP47. Energy-transfer-protein signature of CbPMP47 is single underlined and transmembrane domains are
double underlined according to the transmembrane domain of
AtPMP38; white letters indicate the corresponding conserved amino
acid residue. The alignment only shows the conserved energy-transferprotein signature. The sequence alignment was performed using the
CLUSTAL W program.
Fig. 1 Comparison of the deduced amino acid sequences with
AtPMP38 and HsPMP34 and CbPMP47. (A) The internal amino acid
sequence corresponding to the pumpkin mature protein, as determined
by Edman degradation, is single underlined, the putative mPTS motif
is double underlined and putative transmembrane domains are indicated by dotted underlines; white letters indicate corresponding conserved amino acid residue. The sequence alignment was performed
using the CLUSTAL W program. (B) Hydropathy plots of AtPMP38,
HsPMP34 and CbPMP47. Hydrophobic residues are depicted as positive and hydrophilic residues as negative. The putative transmembrane
domains are indicated by bars. At, Arabidopsis thaliana; Hs, Homo
sapience; Cb, Candida boidinii.
thaliana revealed an open reading frame encoding for a 331amino acid protein with a calculated molecular mass of
36,171 Da. The alignment of peroxisomal AAC homologues of
other organisms showed 25% identity to the HsPMP34 proteins, and 26% identity to the CbPMP47 proteins (Wylin et al.
1998, McCammon et al. 1990). However, although AtPMP38
showed low similarity (14% identity) to the putative mitochondrial AAC of A. thaliana that had no membrane peroxisomal
targeting signal (mPTS), AtPMP38 has the energy-transfer-
protein signature that is a conserved residue characteristic of
peroxisomal AAC homologues of other organisms, such as
HsPMP34 and CbPMP47, and mitochondrial AAC homologues
of Saccharomyces cerevisiae that is located predominantly at
the borders of the transmembrane domains as shown in Fig. 2
(Bairoch 1993). The hydropathy plots of AtPMP38, HsPMP34
and CbPMP47 (analyzed with the algorithm of von Heijne)
show that these proteins belong to the six-transmembrane
family of transport systems (Fig. 1), supporting that PMP38 is
a member of the AAC family.
AtPMP38 has a putative mPTS between putative transmembrane domains 4 and 5 (Fig 1A). This motif was determined on CbPMP47 sequences (Dyer et al. 1996). From this
result, AtPMP38 was expected to be localized on peroxisomal
membranes. We found this motif on other AtPMPs, although it
is unknown yet whether the motif of AtPMP38 actually functions as mPTS.
The genomic DNA sample digested with the restricted
enzymes, which had no site within the cDNA, was hybridized
to the AtPMP38 probe and gave one fragment. By contrast, the
genomic DNA digested with restricted enzymes, which had a
single site within the cDNA, gave two fragments (data not
shown). The results of Southern analysis of A. thaliana
genomic DNA probed with AtPMP38 suggested that AtPMP38
is a single-copy gene. The gene encoding AtPMP38 is located
on chromosome II within BAC T28M21 (Gen Bank accession
no. AAB95282).
Assessment of antibody raised against AtPMP38
As shown in Fig. 3, the antibody raised against AtPMP38
cross-reacted with only a single polypeptide of about 38 kDa in
the total homogenates from etiolated A. thaliana and pumpkin
Glyoxysomal ATP/ADP carrier protein
Fig. 3 Immuno-cross reactivity with antibody raised against
AtPMP38. Homogenates of etiolated cotyledons of Arabidopsis thaliana (lane 1), of pumpkin (lane 2) and peroxisomal membrane fraction from etiolated cotyledons of pumpkin (lane 3) were applied to
SDS-PAGE and subsequent immunoblotting. Molecular markers are
indicated in kDa on the right.
Fig. 4 Subcellular localization of PMP38 in etiolated pumpkin cotyledons. Extracts prepared from 5 d etiolated pumpkin cotyledons were
fractionated after sucrose density gradient centrifugation. (A) Immunological detection of PMP38. Each fraction (50 and 5 ml) was analyzed by immunoblotting using antibodies raised against AtPMP38 and
isocitrate lyase, respectively. (B) The distribution of COX and catalase was monitored by the determination of enzyme activity (COX,
closed triangle and catalase, closed circle). Open circle, sucrose concentration (w/w). ICL, isocitrate lyase; COX, cytochrome c oxidase.
837
Fig. 5 Immunoelectron microscopic analysis of an etiolated cotyledon of Arabidopsis thaliana with an antibody raised against the
AtPMP38. Arrowheads mark the gold particles. G, glyoxysome; O, oil
body. Bar = 500 nm.
cotyledons, and in the isolated peroxisomes, respectively, indicating that the antibody was monospecific. We concluded that
the antibody cross-reacted with not only AtPMP38 but also
PmPMP38, although it was indicated that the molecular mass
of PmPMP38 was slightly higher than that of AtPMP38.
Subcellular and suborganellar localizations of PMP38
To investigate the subcellular localization of the PMP38,
homogenates from 5 d etiolated pumpkin cotyledons were subjected to sucrose density gradient centrifugation. Fractions
obtained were analyzed using an immunoblot technique with
antibodies raised against the AtPMP38 and isocitrate lyase. Isocitrate lyase was used as a marker for glyoxysomal enzymes.
As shown in Fig. 4A, PMP38 and isocitrate lyase were present
together in fractions 23–25, as well as catalase activities (Fig.
4B). By contrast, no PMP38 was detected in fractions 11–15,
which corresponds to the activity of a mitochondrial marker
enzyme, cytochrome c oxidase. These results indicated that
PMP38 is localized in glyoxysomes, but not in mitochondria.
Fig. 5 shows an immunoelectron microscopic analysis of
PMP38 in the cells of 5 d etiolated cotyledons of A. thaliana.
The gold particles were detected in glyoxysomes, especially on
glyoxysomal membranes. The particles were not detectable in
any other organelles such as oil bodies. These results clearly
indicated that the PMP38 is exclusively localized on glyoxysomal membranes.
To investigate the localization of the PMP38 in glyoxysomes, we subfractionated glyoxysomes of pumpkin cotyledons
into four fractions as reported previously (Yamaguchi et al.
1995b). Most matrix proteins were recovered in the water(lane 2) and salt-soluble (lane 3) fractions as shown for catalase
(Fig. 6D). As shown in Fig. 6B, PMP38 was found in the
alkali-insoluble fraction (lane 5). PMP38 was hardly extracted
from glyoxysomes even with alkali solution. However, small
amounts of thiolase (a matrix protein) were found in the alkali-
838
Glyoxysomal ATP/ADP carrier protein
Fig. 6 SDS-PAGE and immunoblot analysis of AtPMP38 (B), peroxisomal ascorbate peroxidase (C) and thiolase (D). The various solubilized fractions of glyoxysomal membrane proteins were prepared
using pumpkin cotyledons as described in “Materials and Methods”
and subjected to SDS-PAGE, subsequent staining with Coomassie
Brilliant Blue (A) and immunoblotting (B, C and D). Lane 1, isolated
glyoxysomes; lane 2, the water-soluble fraction; lane 3, the salt-soluble fraction; lane 4, the alkali-soluble fraction and lane 5, the alkaliinsoluble fraction. Black arrow indicates the bands of AtPMP38. White
and gray arrows indicate the bands of thiolase and peroxisomal ascorbate peroxidase, respectively. Molecular markers are indicated in kDa
on the right.
soluble and insoluble fractions (lane 4 and 5), indicating that
both fractions were slightly contaminated with matrix proteins.
Peroxisomal ascorbate peroxidase, another peroxisomal membrane protein, was mainly detected in the alkali-soluble fraction (lane 4). These results indicated that PMP38 is a glyoxysomal integral membrane protein. This is also supported by the
hydropathy plot that shows six-transmembrane domains (Fig.
1B).
Developmental changes in the level of PMP38
Fig. 7 shows the changes in the level of PMP38 during the
post-germinative growth of the pumpkin seedlings. An immunoblot analysis of the cotyledons of the pumpkin seedlings
grown in the dark showed that PMP38, as well as peroxisomal
ascorbate peroxidase, glyoxysomal membrane proteins, and
short-chain acyl-CoA oxidase and isocitrate lyase, glyoxysomal matrix proteins, reached a maximum level 6 d after germination. These enzymes were still present in the etiolated cotyledons 9 d after germination, although their levels were gradually
decreased. After illumination of the seedlings, the amount of
these enzymes was markedly decreased, but PMP38 was still
present in greening cotyledons in a low amount like another
enzyme of the fatty acid >-oxidation cycle, the short-chain
acyl-CoA oxidase, and a related enzyme, peroxisomal ascor-
Fig. 7 Developmental changes in the level of proteins for PMP38.
Total homogenate prepared from pumpkin cotyledons (10–20 mg) was
subjected to SDS-PAGE, which was followed by immunoblotting with
antibodies raised against two peroxisomal membrane proteins,
AtPMP38 and peroxisomal ascorbate peroxidase (pAPX), two glyoxysomal enzymes, short-chain acyl-CoA oxidase (SACOX) and isocitrate lyase (ICL) and leaf-peroxisomal enzymes, hydroxypyruvate
reductase (HPR).
Fig. 8 Changes in levels of peroxisomal enzymes during senescence
in vitro. Green pumpkin cotyledons grown in a greenhouse for 20 d
were placed in a plastic case in darkness. Total protein from cotyledons kept in darkness for 0, 2, 4, 6 and 8 d was subjected to SDSPAGE (10 mg each), which was followed by immunoblotting with antibodies raised against two peroxisomal membrane proteins, AtPMP38
and peroxisomal ascorbate peroxidase (pAPX), two glyoxysomal
enzymes, short-chain acyl-CoA oxidase (SACOX) and isocitrate lyase
(ICL) and leaf-peroxisomal enzymes, hydroxypyruvate reductase
(HPR).
Glyoxysomal ATP/ADP carrier protein
bate peroxidase. These suggest that PMP38 is a glyoxysomal
protein and may function in the fatty acid >-oxidation cycle.
While hydroxypyruvate reductase, a matrix protein of leaf peroxisomes, was slightly detectable in pumpkin seedlings grown
in the dark, it was increased markedly after illumination of the
seedlings.
We also investigated the changes in the levels of PMP38
during in vivo senescence. A small amount of PMP38 protein
was present in cotyledons after 20 d in continuous light (0 d of
senescence), and not changed during further incubation in continuous darkness (2–8 d) (Fig. 8). The developmental pattern
was similar to those of peroxisomal ascorbate peroxidase and
short-chain acyl-CoA oxidase, whereas the amount of isocitrate lyase increased markedly and the amount of hydroxypyruvate reductase gradually decreased during senescence. These
results indicated that PMP38 plays a role during senescence as
well as germination, and may act in concert with short-chain
acyl-CoA oxidase and peroxisomal ascorbate peroxidase.
Discussion
PMP38 is a candidate of ATP/ADP carrier protein on
glyoxysomal membranes
AAC has been studied well in mitochondria and plastids.
Generally, the mitochondrial AAC exchanges ATP synthesized
in the matrix with ADP in the cytosol (Klingenberg 1989).
Since ATP-synthesis pathways have not been found in the peroxisomes, PMP38 seems to import ATP into peroxisomes.
PMP38 may play a role in glyoxysomal specific events,
because the developmental pattern is similar to those of glyoxysomal enzymes (Fig. 7). Two metabolic pathways are known
in glyoxysomes, namely glyoxylate cycle and fatty acid >oxidation cycle. The proteins of the fatty acid >-oxidation
enzymes, such as short-chain acyl-CoA oxidase, increase during the early stage of germination in continuous darkness,
reached the maximum level at 5–6 d like the developmental
pattern of PMP38, and then decrease after exposure to light.
After the completion of the peroxisome transition, proteins of
the fatty acid >-oxidation enzymes were still detectable in leaf
peroxisomes like the developmental pattern of PMP38,
whereas the proteins of glyoxylate cycle, such as isocitrate
lyase, disappeared (Fig. 7). They showed a similar pattern during senescence (Fig. 8). In fact, it was reported recently that the
PMP47-deleted strain of C. boidinii could not grow well on a
middle-chain fatty acid, laureate, but showed growth similar to
that of the wild-type strain when provided with lauroyl-CoA
(Nakagawa et al. 2000), suggesting that CbPMP47 is involved
in the transport of ATP required in the conversion of laureate to
lauroyl-CoA in peroxisomes.
Altogether, we conclude that PMP38 may be a transporter
involved in the transport of ATP that is required for the reaction
of fatty acyl-CoA synthesis and functions in concert with
enzymes participating in the fatty acid >-oxidation cycle. We
are now examining the activity of the expressed PMP38 as an
839
ATP/ADP carrier. We have been screening for PMP38
knockout mutants of A. thaliana and generating anti-sense
transgenic plants to clarify whether PMP38 is responsible for
the fatty acid >-oxidation cycle or not. Recently, we screened
for ped mutants with defects in the fatty acid >-oxidation cycle
(Hayashi et al. 1998, Hayashi et al. 2000). Since 2,4dichlorophenoxybutyric acid (2,4-DB) is metabolized to produce a herbicide, 2,4-D, by the action of the fatty acid >oxidation cycle in higher plants, wild-type plants cannot germinate in the presence of 2,4-DB (Wain and Wightman 1954).
However, ped mutants could germinate in the presence of 2,4DB, because these mutants are defective in the fatty acid >oxidation cycle. Therefore, if PMP38 is involved in the fatty
acid >-oxidation cycle, a mutant of PMP38 will be able to
germinate on a 2,4-DB plate. Further analysis of these mutants
will help us to clarify the functions of PMP38.
Change in levels of PMP38 during the peroxisome transition
The peroxisomal matrix enzymes can be classified into
three categories; (1) the glyoxysome-specific enzymes such as
malate synthase and citrate synthase, which are members of the
glyoxylate cycle; (2) the leaf peroxisome-specific enzymes
such as glycolate oxidase and hydroxypyruvate reductase,
which function in glycolate metabolism of photorespiration;
and (3) the enzymes present in glyoxysomes and leaf peroxisomes such as catalase and the enzymes of the fatty acid >oxidation cycle. In dark-grown cotyledons, the glyoxysomespecific enzymes increased with the germination and decreased
gradually after reaching the maximum level. By contrast, the
leaf peroxisome-specific enzymes were at a low level in darkgrown cotyledons. When the seedlings were illuminated, the
glyoxysome-specific enzymes decreased rapidly whereas the
leaf peroxisome-specific enzymes increased markedly. The
developmental changes of the enzymes present in both peroxisomes (glyoxysome and leaf peroxisome) were similar to those
of glyoxysome-specific enzymes. After exposure to light, the
glyoxysome-specific enzymes disappeared, whereas enzymes
present in both peroxisomes decreased but were still retained at
a low level after completion of the peroxisome transition from
glyoxysomes to leaf peroxisomes.
In this report, we showed that a membrane protein,
PMP38 increases in the dark and is retained at a low level during greening of pumpkin cotyledons, indicating that PMP38
belongs to the enzymes present in both peroxisomes like the
enzyme of the fatty acid >-oxidation cycle. We have previously shown that the membrane proteins of glyoxysomes, peroxisomal ascorbate peroxidase (PMP31) and PMP28, undergo
drastic changes during the peroxisome transition and PMP28
disappeared after completion of the peroxisome transition
whereas peroxisomal ascorbate peroxidase remained at a low
level like PMP38 (Yamaguchi et al. 1995b).
These results clearly show that membrane proteins of glyoxysomes are degraded to a considerable extent and may be
replaced by leaf peroxisomal membrane proteins during the
840
Glyoxysomal ATP/ADP carrier protein
transition as the matrix enzymes of glyoxysomes. Further characterization of leaf peroxisomal membrane proteins will provide us information how these membrane proteins are
exchanged during the transformation of glyoxysomes to leaf
peroxisomes.
Materials and Methods
Plant materials
Pumpkin (Cucurbita sp. cv. Kurokawa Amakuri) seeds were
soaked overnight, planted in moist rock fiber (66R; Nitto Bouseki,
Chiba, Japan) and allowed to germinate at 25°C in darkness. The etiolated cotyledons obtained from seedlings kept in darkness for 5 d after
germination were used for the preparation of glyoxysomes and those
kept in darkness for 1–9 d for the developmental analysis. Some seedlings were transferred to light after 5 d in darkness and the development of the green cotyledons was also analyzed. Seeds of A. thaliana
(L.) Heynh. Ecotype Columbia were germinated at 22°C in darkness.
Preparation of glyoxysomes
Glyoxysomes were isolated from etiolated pumpkin cotyledons.
Etiolated cotyledons (50 g FW) were harvested from pumpkin seedlings kept in darkness for 5 d after germination, and homogenized with
200 ml of grinding medium that contained 20 mM pyrophosphateHCl, pH 7.5, 1 mM EDTA and 0.3 M mannitol in a chilled Waring
blender for 3 s twice. The homogenate was squeezed through four layers of cheesecloth. The residue was homogenized similarly with
another 200 ml of grinding medium. The filtrates were combined and
were centrifuged at 1,500´g for 10 min to remove plastids and cell
debris. After centrifugation at 10,000´g for 20 min, the pellet was
resuspended in 150 ml of grinding medium, and was centrifuged similarly. The resultant pellet was then resuspended in 5 ml of buffer A
(10 mM HEPES-KOH, pH 7.2, 1 mM EDTA and 0.3 M mannitol) and
was subjected to centrifugation in Percoll. A 4 ml aliquot of the suspension was layered directly on top of 30 ml of a 28% (v/v) solution of
Percoll in 10 mM HEPES-KOH, pH 7.2, 1 mM EDTA and 0.3 M raffinose and was centrifuged at 40,000´g for 30 min with slow acceleration and deceleration. Glyoxysomes sedimented near the bottom of the
self-generated gradient were collected with a Pasteur pipette after centrifugation at 5,000´g for 10 min in 4 volumes of buffer A. The pellet
was suspended carefully in 1 ml of buffer A and was used as purified
glyoxysomes for subsequent analysis. All procedures were carried out
at 4°C.
Characterization of glyoxysomal membrane proteins
Purified glyoxysomes were pelleted again by centrifugation at
5,000´g for 10 min and were suspended in 200 ml of 10 mM HEPESKOH buffer, pH 7.2. After centrifugation at 100,000´g for 30 min, the
supernatant was analyzed as the water-soluble fraction. The pellet was
resuspended in 10 mM HEPES-KOH, pH 7.2, which contained 0.2 M
KCl and was centrifuged again at 100,000´g for 30 min to yield a saltsoluble fraction. The pellet was extracted with 0.1 M Na2CO3 by
means of a sodium carbonate procedure that removed peripheral membrane proteins (Fujiki et al. 1982). After centrifugation at 100,000´g
for 30 min, the supernatant was neutralized by the addition of 1 M HCl
and was analyzed as the alkali-soluble fraction. The pellet was resuspended in 100 mM HEPES-KOH, pH 7.2, and was used as the alkaliinsoluble fraction. All procedures were carried out at 4°C.
Determination of N-terminal amino acid sequences of internal peptides
of PmPMP38
SDS-PAGE (12.5% polyacrylamide) was performed by the
method of Laemmli (1970) for the separation of glyoxysomal membrane proteins. The separated proteins were then stained with Coomassie Brilliant Blue. The bands corresponding to PmPMP38 were cut out
from the blot, and incubated in 300 ml of a solution containing ammonium bicarbonate (pH 8.0) and 50% acetonitrile with shaking for
30 min twice. The supernatant was discarded. The gel was dried and
was then incubated in 10 ml of 0.2 M ammonium bicarbonate (pH 8.0)
for 5 min. The sample was digested with 10 ml of 0.1 mg ml–1 trypsin
solution (Wako, Osaka, Japan) in 20 ml of 0.2 M ammonium bicarbonate (pH 8.0) for 16 h at 37°C. The reaction was stopped by the addition of 4 ml of trifluoroacetic acid (TFA). The peptides were collected
by shaking in 300 ml of 0.1% TFA and 60% acetonitrile for 30 min
twice and subjected to SMART system (Pharmacia, Uppsala, Sweden)
to separate the peptides. The separated peptides were subjected to
automatic Edman degradation on a peptide sequencer (model 473A;
Applied Biosystems Inc., Foster City, CA, U.S.A.).
Sequencing of AtPMP38 EST clones
AtPMP38 EST clone (stock number 173G9T7) was obtained
from ABRC. These were derived from a lZip-Lox plasmid (GIBCO
BRL, MD, U.S.A.). The nucleotide sequences with the longest cDNA
insert, designated 173G9T7 were determined with an automatic DNA
sequencer (model 310; Perkin Elmer/Applied Biosystems) according
to the manufacturer’s instructions. The nucleotide and the deduced
amino acid sequences were analyzed using the Gene-Works program
(Intelli Genetics, Mountain View, CA, U.S.A.).
Preparation of a specific antibody raised against AtPMP38
The DNA fragment of AtPMP38, corresponding to amino acid
residues 26–108, was inserted into pET32a vector (Novagen, Madison, WI, U.S.A.). A fusion protein between the N-terminal region of
AtPMP38 and histidine tag was expressed in E. coli cells and was purified by column chromatography on Ni2+-resin. The purified protein in
1 ml of sterilized water was emulsified with an equal volume of Freund’s complete adjuvant (DIFCO, Detroit, MI, U.S.A.). The emulsion
was injected (about 0.4 mg of protein) on the back of a rabbit and then
was similarly given to the first injection. Blood was taken from an
auricular vein 7 d after the second booster injection. The serum was
used for immunoblotting and immunogold analysis.
Subcellular fractionation
Etiolated cotyledons of pumpkin 5 d after germination in darkness were homogenized in a Petri dish by chopping with a razor blade
for 5 min in 10 ml of a medium that contained 150 mM Tricine-KOH
(pH 7.5), 1 mM EDTA and 0.5 M sucrose. The homogenate was
passed through four layers of cheesecloth. The filtrate (3 ml) was
layered onto a sucrose gradient that contained a 1 ml cushion of 60%
(w/w) sucrose and an 11 ml linear sucrose gradient from 30 to 60%
without buffer. The gradient was centrifuged at 25,000´g for 3 h using
a SW 28-2 rotor in an ultracentrifuge (XL-90; Beckman, Fullerton,
CA, U.S.A.). After centrifugation, fractions (0.5 ml each) were collected with an automatic liquid fractionator (ALC-2L; Advantec,
Tokyo, Japan). All procedures were carried out at 4°C.
Ultrastructural analysis
For immunoelectron microscopy, etiolated cotyledons of A. thaliana 5 d after germination in darkness were frozen with a high pressure freezing machine (model HPM 010, Bal-Tec Inc., Balzers, FL,
U.S.A.) and the ice in the cells was substituted with acetone for 2 d at
–85°C. After being washed with acetone, cotyledons were embedded
in LR white resin. Sections were treated successively with 1% bovine
serum albumin (BSA) for 30 min and antibody raised against PMP38
rabbit IgG (1 : 100) for 10 h at 4°C. After being washed with phos-
Glyoxysomal ATP/ADP carrier protein
phate-buffered saline, the sections were treated with colloidal gold
conjugated protein A for 1 h at room temperature. After being washed
with 0.1% BSA-C (Aurion, Netherlands) and distilled water, sections
were stained with an aqueous solution of uranyl acetate and with lead
citrate. All sections were examined with a transmission electron microscope (model 1200EX; JEOL, Tokyo, Japan).
Immunoblot analysis
Pumpkin cotyledons kept under various conditions were homogenized with extraction buffer [20 mM Tris-HCl (pH 6.8), 2% SDS,
10% b-mercaptoethanol, 24% glycerol and a protein inhibitor cocktail
(Boehringer Mannheim)] and then the homogenates were heated at
95°C for 5 min. The samples were centrifuged at 15,000´g for 5 min,
and the protein in the supernatant was quantified using a protein assay
kit (Nippon Bio-Rad Laboratories, Tokyo, Japan) with bovine gamma
albumin as a standard. The samples were subjected to SDS-PAGE on a
12.5% polyacrylamide gel as described by Laemmli (1970). Then, an
immunoblot analysis was performed by the method of Towbin et al.
(1979). Immunologic reactions were detected by monitoring the activity of horseradish peroxidase (ECL system; Amersham). The antibody
raised against isocitrate lyase, peroxisomal ascorbate peroxidase, thiolase, short-chain acyl-CoA oxidase and hydroxypyruvate reductase
were prepared as described previously (Maeshima et al. 1988, Nito et
al. 2001, Kato et al. 1996, Hayashi et al. 1999).
Acknowledgements
We are grateful to M. Maeshima (Nagoya University) for kind
donation of the antibody raised against isocitrate lyase from castor
bean and N.-H. Chua (Rockefeller University) for the antibody raised
against hydroxypyruvate reductase from spinach. This work was supported in part by a grant from the Research for the Future Program of
the Japanese Society for the Promotion of Science (JSPSRFTF96L00407), and by a grant-in-aid for scientific research from the
Ministry of Education, Science, Sports and Culture of Japan
(12440231).
References
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic
local alignment search tool. J. Mol. Biol. 215: 403–410.
Bairoch, A. (1993) The PROSITE dictionary of sites and patterns in proteins, its
current status. Nucl. Acids Res. 21: 3097–3103.
Beevers, H. (1979) Microbodies in higher plants. Annu. Rev. Plant Physiol. 30:
133.
De Bellis, L. and Nishimura, M. (1991) Development of enzymes of the glyoxylate cycle during senescence of pumpkin cotyledons. Plant Cell Physiol. 32:
555–561.
Dyer, J.M., McNew, J.A. and Goodman, J.M. (1996) The sorting sequence of
the peroxisomal integral membrane protein PMP47 is contained within a
short hydrophilic loop. J. Cell Biol. 133: 269–280.
Fujiki, Y., Fowler, S., Siho, H., Hubbard, A.L. and Lazarow, P.B. (1982)
Polypeptide and phospolipid composition of the membrane of rat liver peroxisomes: comparison with endoplasmic reticulum and mitochondrial membranes. J. Cell Biol. 93: 103–110.
Hayashi, H., Bellis, L.D., Ciurli, A., Kondo, M., Hayashi, M. and Nishimura, M.
(1999) A novel acyl-CoA oxidase that can oxidase short-chain acyl-CoA in
plant peroxisomes. J. Biol. Chem. 274: 12715–12721.
Hayashi, M., Nito, K., Toriyama-Kato, K., Kondo, M., Yamaya, T. and
841
Nishimura, M. (2000) AtPex14 maintains peroxisomal functions by determining protein targeting to three kinds of plant peroxisomes. EMBO J. 19: 5701–
5710.
Hayashi, M., Toriyama, K., Kondo, M. and Nishimura, M. (1998) 2,4-dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects in glyoxysomal fatty acid >-oxidation. Plant Cell 10: 183–195.
Huang, A.H.C., Trelease, R.N. and Moore, T.S. (1983) Plant Peroxisomes. Academic Press, New York, pp. 89–94.
Kato, A., Hayashi, M., Takeuchi, Y. and Nishimura, M. (1996) cDNA cloning
and expression of a gene for 3-ketoacyl-CoA thiolase in pumpkin cotyledons. Plant Mol. Biol. 31: 843–852.
Klingenberg, M. (1989) Molecular aspects of adenine nucleotide carrier from
mitochondria. Arch. Biochem. Biophys. 270: 1–14.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227: 680–685.
Lazarow, P.B. and Fujiki, Y. (1985) Biogenesis of peroxisomes. Annu. Rev. Cell
Biol. 1: 489.
Maeshima, M., Yokoi, H. and Asahi, T. (1988) Evidence for no proteolytic
processing during transport of isocitrate lyase into glyoxysomes in castor
bean endosperm. Plant Cell Physiol. 29: 381–384.
McCammon, M.T., Dowds, C.A., Orth, K., Moomaw, C.R., Slaughter, C.A. and
Goodman, J.M. (1990) Sorting of peroxisomal membrane protein PMP47
from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265: 20098–20105.
Nakagawa, T., Imanaka, T., Morita, M., Ishiguro, K., Yurimoto, H., Yamashita,
A., Kato, N. and Sakai, Y. (2000) Peroxisomal membrane protein Pmp47 is
essential in the metabolism of middle-chain fatty acid in yeast peroxisomes
and is associated with peroxisome proliferation. J. Biol. Chem. 275: 3455–
3461.
Nishimura, M., Takeuchi, Y., Bellis, L.D. and Hara-Nishimura, I. (1993) Leaf
peroxisomes are directly transformed to glyoxysomes during senescence of
pumpkin cotyledons. Protoplasma 175: 131–137.
Nishimura, M., Yamaguchi, J., Mori, H., Akazawa, T. and Yokota, S. (1986)
Immunocytochemical analysis shows that glyoxysomes are directly transformed to leaf peroxisomes during greening of pumpkin cotyledon. Plant
Physiol. 81: 313–316.
Nito, K., Yamaguchi K., Kondo M., Hayashi M. and Nishimura M. (2001)
Pumpkin peroxisomal ascorbate peroxidase is localized on peroxisomal membranes and unknown membrane structures. Plant Cell Physiol. 42: 20–27.
Titus, D.E. and Becker, W.M. (1985) Investigation of the glyoxysome-peroxisome transition in germinating cucumber cotyledons using double-label
immunoelectron microscopy. J. Cell Biol. 101: 1288–1299.
Tolbert, N.E., Oeser, A., Kisaki, T., Hageman, R.H. and Yamazaki, R.K. (1968)
Peroxisomal from spinach leaves containing enzymes related to glycolate
metabolism. J. Biol. Chem. 243: 5179–5184.
Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some
applications. Proc. Natl. Acad. Sci. USA 76: 4350–4354.
Tugal, H.B., Pool, M. and Baker, A. (1999) Arabidopsis 22-kilodalton peroxisomal membrane protein. Nucleotide sequence analysis and biochemical characterization. Plant Physiol. 120: 309–320.
Wain, R.L. and Wightman, F. (1954) The growth-regulating activity of certain
M-substituted alkyl carboxylic acids in relation to their >-oxidation within the
plant. Proc. R. Soc. Lond. B Biol. Sci. 142: 525–536.
Wylin, T., Baes, M., Brees, C., Mannaerts, G.P., Fransen, M. and Veldhoven,
P.P.V. (1998) Identification and characterization of human PMP34, a protein
closely related to the peroxisomal integral membrane protein PMP47 of Candida boidinii. Eur. J. Biochem. 258: 332–338.
Yamaguchi, K., Mori, H. and Nishimura, M. (1995a) A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal membranes in pumpkin. Plant Cell Physiol. 36: 1157–1162.
Yamaguchi, K., Takeuchi, Y., Mori, H. and Nishimura, M. (1995b) Development
of microbody membrane proteins during the transformation of glyoxysomes
to leaf peroxisomes in pumpkin cotyledons. Plant Cell Physiol. 36: 455–464.
(Received March 7, 2001; Accepted May 25, 2001)