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Plant Physiol. (1999) 120: 309-320
Arabidopsis 22-Kilodalton Peroxisomal Membrane
Protein.
Nucleotide Sequence Analysis and
Biochemical
Characterization1
H. Bülent Tugal,
Martin Pool2, and
Alison Baker*
Centre for Plant Sciences, Leeds Institute for Plant Biotechnology
and Agriculture, University of Leeds, Leeds LS2 9JT, United Kingdom
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ABSTRACT |
We
sequenced and characterized PMP22 (22-kD
peroxisomal membrane protein) from
Arabidopsis, which shares 28% to 30% amino acid identity and 55% to
57% similarity to two related mammalian peroxisomal membrane proteins,
PMP22 and Mpv17. Subcellular fractionation studies confirmed that the
Arabidopsis PMP22 is a genuine peroxisomal membrane protein.
Biochemical analyses established that the Arabidopsis PMP22 is an
integral membrane protein that is completely embedded in the lipid
bilayer. In vitro import assays demonstrated that the protein is
inserted into the membrane posttranslationally in the absence of ATP,
but that ATP stimulates the assembly into the native state. Arabidopsis
PMP22 is expressed in all organs of the mature plant and in
tissue-cultured cells. Expression of PMP22 is not associated with a
specific peroxisome type, as it is detected in seeds and throughout
postgerminative growth as cotyledon peroxisomes undergo conversion from
glyoxysomes to leaf-type peroxisomes. Although PMP22 shows increased
accumulation during the growth of young seedlings, its expression is
not stimulated by light.
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INTRODUCTION |
The peroxisome is a single membrane-bound organelle present in
almost all eukaryotic cells (Lazarow and Fujiki, 1985 ). Peroxisomes possess different metabolic functions depending not only on the type of
tissue in which they are found, but also on the metabolic and
developmental state of the organism (Tolbert, 1981). A common function
of all peroxisomes is their ability to degrade hydrogen peroxide,
thereby allowing the co-compartmentalization of metabolic pathways that
produce this toxic by-product. In plants specialized peroxisomes
(glyoxysomes) in cotyledons or endosperm tissues perform  -oxidation
and undergo the glyoxylate cycle to convert carbon from stored fatty
acids to carbohydrate during germination. In photosynthetic tissues
peroxisomes are involved in the salvage of glycolate produced by
photorespiration (Canvin and Salon, 1997). Such functional diversity in
a single compartment is possible because the composition of peroxisomes
can be modified through the uptake and assembly of the required
proteins and enzymes.
The peroxisome membrane forms the interface between the organelle and
the rest of the cell (Mullen and Trelease, 1996). It mediates not only
the transport of metabolites, but also the import of proteins that
maintain or modify the identity and function of the organelle. It also
almost certainly plays a role in organelle movement and division. The
peroxisomal membrane (especially that of plants) is one of the least
characterized, mainly because of the difficulties in isolating pure
membranes that are free from contamination by other cellular membranes.
The use of yeast mutants that are defective in peroxisomal function
circumvents this difficulty and has resulted in the identification of
PMPs such as Pex2p (Per6) (Waterham et al., 1996), Pex3p (Pas3p)
(Höhfeld et al., 1991), Pex9p (Pay2p) (Eitzen et al., 1995),
Pex10p (Per8p) (Tan et al., 1995), Pex13p (Elgersma et al., 1996;
Erdman and Blöbel, 1996; Gould et al., 1996), and Pex15p
(Elgersma et al., 1997), all of which are required for peroxisome
biogenesis, although their precise functions remain unknown.
Other proteins identified by reverse genetics in yeasts include Pex11p
(Pmp27) (Erdman and Blöbel, 1995; Marshall et al., 1995), which
is implicated in the control of peroxisome size; Pmp47 (McCammon et
al., 1994), a putative solute transporter; and Pat1p and Pat2p (Hettema
et al., 1996), which are involved in fatty acid transport into
peroxisomes. Studies addressing the molecular basis of human genetic
disorders that result in peroxisomal dysfunction have also yielded the
identification of human PMPs such as PAF-1 (Pex2p) (Tsukamoto et al.,
1991) and the adrenoleukodystrophy gene product (Mosser et al.,
1993; Lombard-Platet et al., 1996). In other mammals the related PMPs,
PMP22 and Mpv17, were identified by biochemical (Fujiki et al.,
1982; Kaldi et al., 1993) and genetic (Weiher et al., 1990)
studies. Mpv17 was implicated in the production of reactive oxygen
species (Zwacka et al., 1994).
Plant homologs of these proteins have not yet been
identified. In the absence of a straightforward genetic approach, most work done to date to has tried to characterize plant PMPs by searching for activities that copurify with peroxisomal membranes. Some have also
attempted to purify and compare peroxisomal membranes from different
plant tissues to analyze their polypeptide compositions (Corpas et
al., 1995). Among the activities present in plant peroxisome membranes are those of alkaline lipase (Maeshima and Beevers, 1985),
NADH:Cyt c reductase, NADH:ferricyanide reductase (Hicks and
Donaldson, 1982; Fang et al., 1987; Luster and Donaldson, 1987;
Struglics et al., 1993), NADH:ascorbate free-radical reductase (Bowditch and Donaldson, 1990), Mn-superoxide dismutase (Sandalio and
del Rio, 1988), and an 18-kD Cyt b5
(López-Huertas et al., 1997).
Most of these activities are postulated to be involved in redox
reactions associated with the reoxidation of NADH, or in defense against potentially harmful reactive oxygen species such as hydrogen peroxide and superoxide radicals produced in the matrix and membranes. In peroxisomes from pea leaves, there are two sites of superoxide generation, one in the organelle matrix, in which the generating system
was identified as xanthine oxidase, and another in the peroxisomal
membrane, which is dependent on NADH (del Río et al., 1992).
Recently, the integral PMPs of pea leaf peroxisomes were identified
using SDS-PAGE by López-Huertas et al. (1995); three of these
membrane polypeptides with molecular masses of 18, 29, and 32 kD have
also been characterized and demonstrated to be responsible for
superoxide radical generation (López-Huertas et al., 1996, 1997).
However, in plants we lack genetic information on
oxygen-radical-producing PMPs. With the exception of a 31-kD membrane-associated peroxisomal ascorbate peroxidase (Yamaguchi et al.,
1995; Bunkelmann and Trelease, 1996), the genes encoding these
enzymic activities have not yet been identified.
Advances in the Arabidopsis Genome Project are providing a vital link
between genetic and biochemical approaches in plants. To further our
understanding of how plant peroxisomes are assembled, we sought to
identify and characterize a PMP. We were able to use the data from the
Arabidopsis sequencing project to identify a 22-kD integral membrane
protein that is related to the mammalian PMP22/Mpv17 family of PMPs. In
this paper we describe the characterization of this 22-kD Arabidopsis
PMP.
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MATERIALS AND METHODS |
Materials
The Arabidopsis clone TAY089 (accession no. Z18516), derived from
a cDNA library prepared from cycling cells of a cell-suspension culture
of the Columbia ecotype, was obtained from the Arabidopsis Biological
Research Center (Ohio State University, Columbus). We purchased all
chemicals, unless otherwise specified, from either Sigma or BDH (Poole,
Dorset, UK). DNA sequences were determined by automated sequencing
using the DNA sequencer (model 373A-XL, Applied Biosystems) at the
University of Cambridge Department of Biochemistry (Cambridge, UK).
Plasmid Constructs and Other Molecular Biological Procedures
All molecular biological procedures were performed as described by
Sambrook et al. (1989) unless otherwise specified. The 0.85-kb cDNA
insert of clone TAY089 flanked by EcoRI restriction enzyme
sites was subcloned into the same site in pBluescript SK (Stratagene)
(E10:SK). For in vitro transcription of the open reading frame, the
insert was excised from E10:SK with SalI and XbaI
and subcloned into pBluescript KS (Stratagene) to permit transcription
from the T7 promoter. For the production of recombinant hexahistidine-PMP22, the PMP22 open reading frame was cloned into a
pET16b expression vector (hexahistidine-PMP22:pET16b) to create an
in-frame N-terminal hexahistidine tag. Genomic DNA from Arabidopsis was
extracted from leaves using a DNA-extraction kit (PhytoPure, Scotlabs,
Woburn, MA); and 10-µg aliquots at a concentration of 0.1 µg
µL 1 were digested with the required
restriction enzymes for 14 h. The restriction fragments were
separated by agarose-gel electrophoresis and transferred to Hybond-N
membranes (Amersham) by capillary blotting as described by Sambrook et
al. (1989). The PMP22 open reading frame randomly labeled with
[32P] -dCTP 3000 Ci
mmol1 (Prime-It II, Stratagene) was used to
probe PMP22, which contained restriction fragments in the presence of
6× SSC, 5× Denhardt's solution, 0.1% SDS, and 250 µg
mL1 denatured and fragmented herring-sperm DNA
for 16 h at 65°C. The membranes were washed with 2× SSC and
0.1% (v/v) SDS for 10 min at 65°C, followed by two washes of 0.1×
SSC and 0.1% (v/v) SDS for 20 min at 65°C. After the membranes were
washed, they were autoradiographed.
Recombinant Hexahistidine-PMP22 Production and
Purification
The construct hexahistidine-PMP22:pET16b was transformed into
Escherichia coli strain NovaBlue DE3 (Novagen, Madison, WI). Recombinant hexahistidine-PMP22 production in exponentially growing cells was induced by 500 µM
isopropylthio--galactoside over 4 h at 37°C, and the protein
was purified by its affinity to nickel-agarose (Qiagen, Chatsworth, CA)
under denaturing conditions according to the manufacturer's
recommendations.
Plant Material and Growth Conditions
Arabidopsis plants were germinated and grown in compost:sand
(10:1, v/v) at 18°C with 8 h light d1
for the first 4 weeks, then at 20°C with 16 h light
d1. Suspension cultures of Arabidopsis
(obtained from Dr. Paul Knox, Leeds Institute for Plant Biotechnology
and Agriculture) were maintained at 20°C in 1× Murashige and Skoog
medium, pH 5.7, with 3% (w/v) Suc, 0.05 mg L1
kinetin, and 0.5 mg L1 NAA under dark and
sterile conditions with constant agitation. The cells were subcultured
once every 7 d by transferring 0.1× final culture volume into
fresh medium as described above. For the germination time-course
experiment, 0.1-g batches of Arabidopsis seeds were sterilized in 10%
(v/v) domestic bleach for 30 min and washed 10 times with 1 mL of
sterile distilled water. The seeds were imbibed for 1 h at room
temperature in the final sterile distilled water wash and placed onto
plates containing 0.2% (w/v) Phytogel (Sigma) and 0.5× Murashige and
Skoog medium, pH 5.7.
Protein Isolation and Subcellular Fractionation of Plant
Material
Glyoxysomes from sunflower cotyledons 3 d postimbibition were
prepared according to the method described by Horng et al. (1995). For
the isolation of organelles from dark-cultured Arabidopsis suspension-cultured cells, the cells were harvested by centrifugation at 1000g for 5 min at 4°C and resuspended in ice-cold 50 mM Mes-KOH, pH 6.0, 0.5 M
Suc, 10 mM KCl, and 1 mM
EDTA at a pellet-to-buffer ratio of 1:5. The cell clumps were loosened
by homogenization (10 strokes) of a loose-fitting Teflon/glass
homogenizer. The cell suspension was initially ruptured by five strokes
of a 3-mL glass/glass homogenizer (no. 2 clearance, Jencons Scientific, Bridgeville, PA), followed by 20 strokes of a 1-mL glass/glass homogenizer (no. 2 clearance, Jencons Scientific).
Postnuclear supernatant was obtained by pelleting the cellular debris
at 1,000g for 10 min at 4°C. For some experiments, a mixed-organelle pellet fraction was obtained by centrifuging the postnuclear supernatant at 20,000g for 30 min at 4°C.
Membranes were prepared from the mixed-organelle pellet by resuspending them by homogenization first in 20 mM Hepes-KOH,
pH 7.5, then with the addition of NaCl to a final concentration of 250 mM. A membrane pellet was obtained by
centrifuging the organelle lysate at 100,000g for 30 min at
4°C. Continuous Suc gradients of subcellular organelles were
performed in 13-mL SW40 tubes (Beckman). Gradients ranged from 0.7 to
2.1 M Suc dissolved in 50 mM Mes-KOH, pH 6.0, 10 mM
KCl, and 1 mM EDTA. Typically, 5 to 10 mg (1 mL)
of postnuclear supernatant was centrifuged at 200,000g for
12 h at 4°C, and the gradients were fractionated into 12 1-mL
aliquots from their dense Suc end. Total protein extracts of plant
material were obtained by freezing approximately 1 to 5 g of the
required tissue in liquid nitrogen and grinding it into a smooth
powder. This powder was immediately resuspended in 10 mL of ice-cold
TCA (10%, v/v). Proteins were removed from this mixture for analysis
by centrifuging aliquots at 14,000g for 15 min at 4°C.
Arabidopsis rosettes (20 g) were chopped with razor blades in 50 mL of
chilled 50 mM Mes-KOH, pH 6.0, 0.5 M Suc, 10 mM KCl, and 1 mM EDTA on ice for 20 min and
filtered through four layers of muslin to produce a homogenate
fraction. This was centrifuged at 1,000g for 10 min at 4°C
to produce a pellet and supernatant fraction. The supernatant fraction
was centrifuged at 25,000g for 30 min at 4°C to produce a
25,000g pellet and a supernatant fraction.
Treatments of Subcellular Fractions
Organelles at a protein concentration of 1 mg
mL1 were washed with 0.1 M
Na2CO3 (pH 11.0) as
described by Fujiki and Lazaraow (1982). Fractionation of organelles
with Triton X-114 was performed as described by Pryde and Phillips
(1986). In protease-digestion experiments, intact organelles or
their ruptured and salt-washed membranes (prepared as described above)
at a protein concentration of 1 mg mL1 were
incubated in the presence of the required concentration of thermolysin
in 50 mM Mes-KOH, pH 6.0, and 10 mM KCl for 30 min at 4°C. Enzymatic activities of catalase and fumarase were measured as described by Cooper and Beevers (1969), and the activity of
NADH:Cyt c reductase was measured as described by Gomez and Chrispeels (1994). Protein estimations were performed using BCA reagent
(Pierce) according to the manufacturer's recommendations, with BSA as
the standard.
Electrophoretic Methods
Protein composition was analyzed in 15% (w/v) polyacrylamide gels
(unless otherwise specified) as described by Laemmli (1970). For
immunodetection, proteins separated by SDS-PAGE were transferred electrophoretically onto 0.45-µm nitrocellulose membranes (Schleicher & Schuell) in the presence of 20 mM
Na2HPO4, 0.02% (w/v) SDS, and 20% (v/v) methanol using a semidry blotter (model 2117-250 Novablot, LKB-Pharmacia) for 2 h at 0.8 mA
cm2.
Immunological Methods
Nitrocellulose membranes for immunoblotting were blocked for
nonspecific immunoreactivity initially with 1× TBS (20 mM
Tris-HCl, pH 7.4, and 150 mM NaCl), 0.5% (v/v) Tween 20, and 0.1% (w/v) NaN3 containing 10 mg
mL1 BSA overnight and then with 1× TBS, 0.5%
(v/v) Tween 20, and 0.1% (w/v) NaN3 containing
10% (w/v) defatted dried milk (Sainsbury's, London) for 6 to 8 h. Antibodies were diluted in 1× TBS, 0.05% (v/v) Tween 20, 0.1%
(w/v) NaN3, and 10 mg mL1
BSA and incubated with the blots for 16 h. Nonspecifically bound antibodies were removed with eight 15-min washes using 1× TBS and
0.1% (v/v) Tween 20. Specifically bound IgGs were detected after
incubation with anti-rabbit antibodies conjugated with horseradish peroxidase (Sigma) using enhanced chemiluminescence (Amersham).
Anti-PMP22 specific antibodies were affinity purified by incubating the
antisera at a dilution of 1:100 with a blocked nitrocellulose strip
(0.5 × 8 cm) containing the region of migration of
electrophoretically separated, purified hexahistidine-PMP22 (250 µg).
Following incubation for 16 h, the strip was washed with eight
15-min washes of 1× TBS and 0.1% (v/v) Tween 20. The PMP22-specific
antibodies were eluted with 0.1 M Gly, pH 2.8, for 1 min
and immediately neutralized. For immunoblots, affinity-purified
anti-PMP22 antibodies were used at a dilution of 1:10,000 with respect
to the original antisera. Anti-glycolate oxidase antisera (Volokita and
Somerville, 1989) was affinity purified by the same procedure
using spinach glycolate oxidase (Sigma) and used at a dilution of
1:10,000 with respect to the original antiserum. The anti-isocitrate
lyase antiserum, previously characterized by Martin and Northcote
(1982), was used at a dilution of 1:100,000.
In Vitro Import Reactions
In vitro import assays were performed as described by Behari and
Baker (1993) with modifications according to Horng et al. (1995).
Glyoxysomes were isolated from cotyledons of sunflower 3 d
postgermination by separation of a postnuclear supernatant in a
Nycodenz step gradient. Glyoxysomes harvested from the gradient were
diluted and concentrated by centrifugation before resuspension in 25 mM Mes-KOH, pH 6.0, 0.5 M Suc, 10 mM KCl, and 1 mM MgCl2 at
a protein concentration of 1.0 to 1.5 mg mL1.
Organelle integrity was assessed by the latency of malate synthase activity. Radiolabeled PMP22 was prepared by in vitro transcription and
translation in wheat germ lysate, as previously described by Behari and
Baker (1993). Import assays contained 200 µg of glyoxysome fraction,
15 µL of translation product, 2.4 mM ATP, 0.26 mg
mL1 creatine kinase, 32 mM creatine
phosphate, 0.34 mg mL1 cold Met, 25 mM Mes-KOH, pH 6.0, 0.5 M Suc, 10 mM KCl, and 1 mM MgCl2 in
a final volume of 200 µL. For carbonate extraction the incubations
were carried out in 400 µL and contained 400 µg of glyoxysomes and
30 µL of translation. For minus-ATP controls, ATP and the
ATP-regenerating system were omitted and both glyoxysomes and
translation product were pretreated with apyrase. Import reactions were
incubated for 15 min at 26°C and terminated by chilling on ice.
Protease-treated reactions were incubated with 0.1 mg
mL1 (final concentration) thermolysin on ice
for 30 min. EDTA was added to 25 mM final concentration and
the organelles were re-isolated by centrifugation through a 0.7 M Suc cushion. Samples to be Triton-treated were
re-isolated through a Suc cushion, and the organelle pellet was
resuspended in 25 mM Mes-KOH, pH 6.0, 0.5 M
Suc, 10 mM KCl, 1 mM
MgCl2, 250 mM NaCl, 1% (v/v) Triton
X-100, and 0.1 mg mL1 thermolysin and digested
for 30 min on ice. Organelle pellets were subjected to SDS-PAGE and
radioactivity was detected by exposure to a phosphor-imaging cassette.
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RESULTS |
Identification and Characterization of Arabidopsis PMP22 cDNA
To identify putative PMPs we screened sequence databases generated
by the Arabidopsis Sequencing Project for genes similar to known PMPs
from other organisms using the BLAST algorithm (Altschul et al., 1990).
With this procedure we identified the cDNA TAY089 (accession no.
Z18516) encoding a 0.85-kb sequence with similarity to the 5 end of
mammalian PMP22 genes. The complete sequence of the cDNA, shown in
Figure 1A, revealed an open reading frame encoding for a 190-amino acid protein with a calculated molecular mass
of 21,687 D. As shown in Table I, the
alignment of all known PMP22 polypeptides reveals two distinct but
related mammalian families, the Mpv17s and the PMP22s, with the
Arabidopsis sequence showing 55% amino acid sequence similarity (30%
identity) to the mouse and human Mpv17 proteins, and 57% sequence
similarity (28% identity) to the rat and mouse PMP22 proteins. Regions
of identity at amino acids 77, 78 (-GP-), 151, 155, 156, 157, and 160 (-N-xxx-VPL-xx-R-) of the Arabidopsis polypeptide appear to be
signatures of all subgroups of the PMP22 family (Fig. 1A). Another
region of the Arabidopsis polypeptide, from amino acids 91 to 109 and
encompassing the di-Lys at positions 92 and 93, shows weak similarity
to the PMP-targeting signal postulated by Dyer et al. (1996) and
Elgersma et al. (1997). Further structural similarities between the
Arabidopsis polypeptide and the mammalian PMP22 sequences (especially
Mpv17) are apparent on comparison of their hydropathy profiles, as
shown in Figure 1B. We therefore refer to this protein as Arabidopsis PMP22.
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| Figure 1.
A, Clustal W alignment of the deduced amino acid
sequences of Arabidopsis PMP22, mouse Mpv17 (P19258), human Mpv17
(P39210), mouse PMP22 (P42925), and rat PMP22 (Q07066). The thick
underlining indicates the conserved regions mentioned in the text. B,
Arabidopsis PMP22, mouse Mpv17, and rat PMP22 according to the
algorithm of Kyte and Doolittle (1982) with an amino acid window of 19. Hydrophobic residues are depicted as positive and hydrophilic residues
as negative.
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Table I.
Amino acid sequence comparison of PMP22 related
proteins
Amino acid sequence similarities and identities between Mpv17 and PMP22
proteins from Arabidopsis and mammals. Data derived from BESTFIT
(Genetics Computer Group) by pairwise comparison of polypeptide
sequences.
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Figure 2 shows the results of Southern
analysis of Arabidopsis genomic DNA probed with PMP22. The result
suggests that the cDNA is the product of a single-copy gene, as genomic
DNA samples digested with the restriction enzymes
HindIII, EcoRI, and PstI, each
contained a single fragment that hybridized to the Arabidopsis PMP22
probe sequence at high stringency (Fig. 2, lanes 1, 2, and 3). DNA
digested with PvuII, which has a single site within the cDNA, gave two fragments of 6.0 and 1.7 kb (indicated by arrows in Fig.
2, lane 5). The gene encoding PMP22 is located on chromosome IV at 19.3 centimorgans within BAC T26N6 (accession no. AF076243). The start codon
is located at 50,654 and the stop codon at 49,343 in the BAC sequence.
The gene consists of seven exons: exon 1, 50,654 to 50,576; exon 2, 50,351 to 50,255; exon 3, 50,153 to 50,047; exon 4, 49,963 to 49,888;
exon 5, 49,801 to 49,735; exon 6, 49,636 to 49,550; and exon 7, 49,451 to 49,393.
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| Figure 2.
Southern blot showing 10 µg of Arabidopsis
genomic DNA digested with HindIII (lane 1),
EcoRI (lane 2), PstI (lane 3),
XbaI (lane 4), and PvuII (lane 5) probed
with the [32P]PMP22 open reading frame, washed under
stringent conditions, and detected by autoradiography.
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Arabidopsis PMP22 Is Ubiquitously Expressed
The Arabidopsis PMP22 open reading frame was cloned into the
pET16b expression vector with an N-terminal hexahistidine tag and
expressed in E. coli. The resulting fusion protein, which has a calculated molecular mass of 24,172 D (due to the presence of 6 His residues and 18 additional residues at the amino terminus of the
protein, which were introduced as a result of cloning the PMP22 open
reading frame into the pET16b expression vector) was purified
on nickel-agarose under denaturing conditions and used to produce
rabbit polyclonal antibodies. The anti-PMP22 antisera was
affinity purified using hexahistidine-Arabidopsis PMP22 (see ``Materials and Methods''). Because the PMP22 cDNA clone was isolated
from a cell-suspension culture library, we tested the antibody using a
total protein extract from a dark-grown Arabidopsis cell-suspension
culture (Fig. 3A, lane 1) and purified
recombinant hexahistidine PMP22 as a positive control (Fig. 3A, lane
2). The affinity-purified antibodies bound to a 22-kD protein (in good
agreement with the calculated molecular mass of 21,687 D) and
to hexahistidine-PMP22, which, as expected, migrated at approximately
24 kD.
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| Figure 3.
A, Immunoblot showing 150 µg of total protein
extract from dark-grown Arabidopsis cell-suspension culture (lane 1)
and 0.25 µg of recombinant purified hexahistidine-PMP22 (lane 2)
probed with affinity-purified anti-PMP22 antibodies and detected by
enhanced chemiluminescence. B, Equal amounts of protein (150 µg)
extracted from various organs of Arabidopsis plants and from dark-grown
tissue-cultured cells were separated by SDS-PAGE and probed with
affinity-purified anti-PMP22 antibodies. Lane 1, Dark-grown
tissue-culture cells; lane 2, flowers; lane 3, siliques; lane 4, stems;
lane 5, upper leaves; lane 6, basal leaves; and lane 7, roots.
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To determine whether PMP22 was expressed in whole plants, equal amounts
of total protein from organs of 12-week-old flowering Arabidopsis
plants (see ``Materials and Methods'' for growth conditions) were
immunoblotted with the affinity-purified anti-PMP22 antibody. Figure 3B
shows that PMP22 is present in all Arabidopsis tissues, with the
highest enrichment in flowers and green siliques. There appeared to be a tissue-dependent difference in the electrophoretic mobility of PMP22, with a larger isoform detected in flowers, stems,
and roots. Although the anti-PMP22 was affinity purified
using recombinant hexahistidine-PMP22 and was monospecific when tested
against whole-cell extracts of suspension-cultured cells (Fig. 3, lanes
1), there was an additional and persistent cross-reaction with a
protein of approximately 70 kD in green tissues and roots (Fig. 3B,
lanes 4-7) that was absent in flowers and siliques (Fig. 3B, lanes 2 and 3). Because it was not possible to remove the cross-reaction with
this protein by affinity purification of the anti-PMP22 IgGs, it may
share epitopes with PMP22.
Localization of PMP22 to Peroxisomes by Subcellular Fractionation
and Suc-Density-Gradient Centrifugation
Arabidopsis rosette leaves were fractionated by differential
centrifugation. A homogenate fraction was centrifuged to produce a
1,000g pellet and supernatant. The 1,000g
supernatant was further centrifuged to produce a 25,000g
pellet and supernatant. The 1,000g pellet would be expected
to be enriched in nuclei and chloroplasts, whereas the
25,000g pellet would be expected to be enriched in mitochondria and peroxisomes. The 25,000g supernatant
contained soluble proteins and light membranes. Equal amounts of
protein (120 µg) from each fraction were separated by SDS-PAGE and
subjected to blotting with affinity-purified anti-PMP22 and antibodies
against the 23-kD subunit of the PSII oxygen-evolving complex (a
peripheral membrane protein of the thylakoid membrane). Figure
4A shows that the 70-kD protein is
detectable in the homogenate and greatly enriched in the
1,000g pellet. A trace was detectable in the
25,000g supernatant, but nothing was detectable in the
25,000g pellet. Because the 70-kD protein behaved as a
soluble protein (data not shown), a low level of the antigen in the
25,000g supernatant due to the release of soluble proteins
from ruptured organelles was to be expected. The 23-kD protein (Fig.
4B) and chlorophyll (Fig. 4C) showed a similar distribution, being
predominantly in the 1,000g pellet. However, both of these
markers are associated with the thylakoid membrane, so they were also
present at a low level in the 25,000g pellet due to the
presence of fragments from the thylakoid membrane released from
ruptured chloroplasts. In contrast, PMP22 could not be detected in the
homogenate, in the 1000g supernatant, or in the pellet
(because of its low abundance), but was readily detectable in the
25,000g pellet (Fig. 4A). The specific activity of catalase
(a peroxisomal marker) showed a similar distribution (Fig. 4C), being
most enriched in the 25,000g pellet. These results are
consistent with a peroxisomal localization for PMP22 but not for the
70-kD protein, which may be a soluble plastidial protein.
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| Figure 4.
Subcellular localization of Arabidopsis PMP22 in
leaf tissue by differential centrifugation. Arabidopsis leaves were
fractionated as described in ``Materials and Methods''. Equal amounts
of protein (120 µg) were separated by SDS-PAGE, transferred to
nitrocellulose, and probed with affinity-purified antibodies against
PMP22 (A) or the 23-kD protein of the PSII oxygen-evolving complex (B).
C, Catalase activity, a peroxisomal matrix marker (shaded bars) and
chlorophyll (white bars) were measured in each fraction. Catalase
recovery was 102% and chlorophyll recovery was 61%. Lane 1, Homogenate; lane 2, 1,000g supernatant; lane 3, 1,000g pellet; lane 4, 25,000g
supernatant; lane 5, 25,000g pellet.
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To provide more definitive evidence for the location of PMP22,
organelles were separated by Suc-density-gradient centrifugation. Because of the size and abundance of chloroplasts it was not possible to obtain clean separation between chloroplast and peroxisomal fractions in Suc gradients from leaf tissue. Therefore,
suspension-cultured cells were chosen for the Suc-gradient experiments
because sufficient quantities of material could readily be obtained.
PMP22 is abundant in these cells, which also lack the 70-kD
cross-reacting protein. A postnuclear supernatant prepared from
dark-grown tissue-cultured cells was separated in a 0.7 to 2.1 M continuous Suc gradient. The gradient was fractionated
and the distribution of organelles was determined by the enzymatic
activities of the marker enzymes catalase (for peroxisomes), fumarase
(for mitochondria), and NADH:Cyt c reductase (for the ER),
as well as with the immunoreactivity of isocitrate lyase. Figure
5 is an immunoblot of equal volume loadings for each fraction, and shows that Arabidopsis PMP22 was localized mainly in fractions 3 and 4 (1.80 and 1.65 M Suc), where the peroxisomal markers catalase
and isocitrate lyase were also detected. The fairly broad distribution
of these markers is typical for peroxisomal proteins and reflects the
heterogeneity of the peroxisomal compartment (Lüers et al.,
1993). Fumarase and NADH:Cyt c reductase were localized in
fractions 4 and 5 (1.5 and 1.35 M Suc,
respectively).
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| Figure 5.
Subcellular localization of PMP22 in tissue
culture cells by Suc-density-gradient centrifugation. A postnuclear
supernatant prepared from dark-grown Arabidopsis suspension-cultured
cells was separated on a 0.7 to 2.1 M continuous
Suc-density gradient. Fractions were assayed for catalase (a
peroxisomal marker), fumarase (a mitochondrial marker), NADH:Cyt
c reductase (an ER marker), and protein. The recoveries
of the enzyme activities relative to the postnuclear supernatant were:
catalase, 76%; fumarase, 96%; and NADH Cyt c
reductase, 91%. Equal volumes of the Suc-gradient fractions were
separated by SDS-PAGE and probed with affinity-purified anti-PMP22
antibodies and anti-castor bean isocitrate lyase antibodies.
|
|
Membrane Localization of Arabidopsis PMP22
The presence of significant hydrophobic regions in the Arabidopsis
PMP22 and its sequence similarity to other PMP22s that are known to be
integral membrane proteins indicated its probable membrane association.
To determine the nature of this membrane association, a
20,000g organelle pellet obtained from dark-cultured Arabidopsis suspension-cultured cells enriched in PMP22 and isocitrate lyase was treated with 0.1 M
Na2CO3 (pH 11.0) to lyse
the organelles and to remove peripheral and lumenal proteins. As shown
in Figure 6A, Arabidopsis PMP22 remained
in the membrane pellet fraction, whereas isocitrate lyase, which is a
peroxisomal matrix protein, was extracted into the supernatant
(Fig. 6A).
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| Figure 6.
A, PMP22 is resistant to extraction by
Na2CO3 and partitions in the detergent phase of
Triton X-114. A 20,000g organelle pellet (P) and the
corresponding supernatant (S) were prepared from dark-grown
tissue-cultured cells and analyzed for PMP22 and isocitrate lyase (ICL)
by SDS-PAGE and immunoblotting (20,000g). The
20,000g pellet fraction enriched in PMP22 was treated
with 0.1 M Na2CO3 (pH 11.0) and
centrifuged at 100,000g to produce a membrane pellet and
supernatant fraction. A postnuclear supernatant prepared from
dark-grown tissue-cultured cells was fractionated using 2% (v/v)
Triton X-114 phase separation, and the various fractions were analyzed
by SDS-PAGE and immunoblotting with antibodies against isocitrate lyase
and affinity-purified anti-PMP22 antibodies. Lane 1, Postnuclear
supernatant; lane 2, Triton X-114-insoluble pellet; lane 3, detergent
phase; lane 4, aqueous phase; lane 5, "glycoprotein"-rich pellet;
and lane 6, postaqueous supernatant. In all lanes 150 µg of protein
was analyzed. B, Accessibility of Arabidopsis PMP22 to protease. The
peroxisome-enriched 20,000g pellet fraction was
subjected to hypotonic lysis followed by a wash with 250 mM
NaCl. Salt-washed membranes (250 µg) were incubated with the protease
thermolysin at the indicated concentrations (see ``Materials and Methods''). After the incubation the protease was inhibited and the
membrane-bound and protease-solubilized peptides were separated by
ultracentrifugation. Triton X-100 was included in a duplicate
incubation containing 100 µg mL1 thermolysin, which was
not subjected to ultracentrifugation before analysis by SDS-PAGE and
immunoblotting. All samples were analyzed by SDS-PAGE and
immunoblotting with affinity-purified anti-PMP22 antibodies.
|
|
Fractionation of biological membranes using the phase-separation
properties of the nonionic detergent Triton X-114 provides a method of
determining the hydrophobic nature of membrane-associated proteins
(Bordier, 1981; Pryde and Phillips, 1986). Although all membrane-associated proteins are extracted by Triton X-114, only integral membrane proteins with significant hydrophobic regions partition into a "detergent" phase; those that are peripherally associated and/or glycosylated are enriched in the "aqueous" phase. A postnuclear supernatant prepared from dark-grown Arabidopsis suspension-cultured cells was treated with Triton X-114, and the resulting detergent-soluble fraction was phase-separated according to
the method of Pryde and Phillips (1986). Arabidopsis PMP22 was observed
to partition only into the Triton X-114 detergent phase (Fig. 6A), as
expected for an integral membrane protein and unlike the soluble matrix
protein isocitrate lyase, which partitioned into the aqueous phase.
To investigate the topology and the extent of integration of
Arabidopsis PMP22 in the peroxisomal membrane, both intact organelles (not shown) and lysed membranes derived from them were treated with the
protease thermolysin at a range of concentrations. Following protease
treatment, membrane-bound and protease-solubilized peptides were
separated by ultracentrifugation and analyzed for Arabidopsis PMP22
immunoreactivity. Figure 6B shows that Arabidopsis PMP22 remained
completely protease resistant even when lysed membranes prewashed with
250 mM NaCl were treated with high protease concentrations (Fig. 6B). Protease resistance is dependent on the presence of a
phospholipid bilayer, because the inclusion of 1% (v/v) Triton X-100
during the protease treatment resulted in the complete digestion of
Arabidopsis PMP22. Similar results were obtained with the protease trysin (data not shown).
Integration of Arabidopsis PMP22 into Peroxisomes in Vitro
As an independent method of determining the intracellular location
of Arabidopsis PMP22, the specificity of its targeting and insertion
into peroxisomes in vitro was addressed. Transcripts were translated in
vitro with wheat germ lysate in the presence of
[35S]-L-Met and incubated with
isolated intact sunflower glyoxysomes, a stage-specific type of
peroxisome whose purity and integrity was established by Behari and
Baker (1993) and Horng et al. (1995). As established in the
previous section, native Arabidopsis PMP22 is deeply buried in the
membrane so that it is resistant to proteolysis by thermolysin and
cannot be extracted with 0.1 M
Na2CO3, pH 11.0. This
provided the means of assessing the correct insertion of PMP22 into
peroxisomal membranes in vitro.
After the incubation of intact peroxisomes with radiolabeled, in
vitro-translated Arabidopsis PMP22 in the presence of ATP and
an ATP-regeneration system, the peroxisomes were incubated with
thermolysin and then re-isolated through a 0.7 M Suc
cushion. To differentiate between correctly integrated and possible
membrane-associated protease-resistant aggregates of PMP22, the
re-isolated peroxisomes were washed with 0.1 M
Na2CO3 (pH 11.0). As shown
in Figure 7, most of the radiolabeled
Arabidopsis PMP22 bound to peroxisomes (Fig. 7, lane 2), and was
resistant to carbonate washing (Fig. 7, lane 10). Protease treatment
removed surface-bound PMP22 and left intact those molecules that were
correctly inserted (Fig. 7, lane 3). These molecules were also
resistant to carbonate extraction (Fig. 7, lane 12). Insertion was
dependent on the presence of the peroxisome membrane, because
pretreatment with 1% (v/v) Triton X-100 prior to protease treatment
had abolished protease resistance (Fig. 7, lane 4).
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| Figure 7.
Insertion of Arabidopsis PMP22 into isolated
peroxisomes. Radiolabeled PMP22 was prepared by in vitro transcription
and translation in wheat germ lysate and incubated with peroxisomes
isolated from 3-d postimbibition sunflower cotyledons (see ``Materials and Methods''). Lane 1, 40% of the translation product added to the
other reactions; lane 2, import reaction carried out in the presence of
the ATP-regeneration system; lane 3, import in the presence of the
ATP-regeneration system followed by protease treatment; lane 4, same as
lane 3 but in buffer containing 1% Triton X-100 and 250 mM
NaCl before the addition of protease; lane 5, import in the absence of
ATP and the ATP-regenerating system; lane 6, import in the absence of
ATP and the ATP-regenerating system followed by treatment with
protease; lane 7, same as lane 3 but no peroxisomes were added to the
import assay; lane 8, same as lane 2 but glyoxysomes were replaced with
50 µg of washed red blood cells from calf ascites; lane 9, same as
lane 8 but treated with protease; lanes 10 to 15, Na2CO3 (pH 11.0) treated pellets and
supernatants derived from import reactions (lane 10 is the pellet and
lane 11 the supernatant of the import reaction in lane 2, lane 12 is
the pellet and lane 13 the supernatant of the import reaction in lane
3; and lane 14 is the pellet and lane 15 the supernatant of the import
reaction in lane 5).
|
|
If peroxisomes were omitted from the import assay, (Fig. 7, lane 7) or
replaced with washed red blood cells from calf ascites (Fig. 7, lanes 8 and 9), Arabidopsis PMP22 was not re-isolated through the 0.7 M Suc cushion, demonstrating that the re-isolation and
insertion is dependent upon the presence of the correct membrane. Red
blood cells were used as an "irrelevant membrane" control in these
experiments; because of the fragility and heterogeneity of peroxisomes
it is extremely difficulty to prepare other organelles that are totally
free from peroxisome membrane contamination (Fig. 5; Behari and Baker,
1993).
When organelles and the translation product were pretreated with
apyrase to remove any nucleoside triphosphates and the ATP-regeneration system was omitted from the import reaction, the level of insertion was
substantially reduced (Fig. 7, compare lanes 3 and 6). In the absence
of ATP, slightly more PMP22 bound to peroxisomes (Fig. 7, compare lanes
2 and 5), and it was also resistant to carbonate extraction (Fig. 7,
lane 14).
Developmental Expression of PMP22 in Arabidopsis Seedlings
To begin to understand the function of Arabidopsis PMP22, we
investigated its expression pattern in seedlings and in
suspension-cultured cells. During germination glyoxysomes are important
organelles, taking part in the mobilization of stored lipids for the
provision of the carbohydrate required for postgerminative growth. By
the time stored lipid reserves begin to diminish, the cotyledons break out of the soil and become photosynthetic. As this occurs, peroxisomes change metabolic function from fatty acid oxidation to salvaging glycolate, a by-product of photorespiration. As a consequence, the
levels of isocitrate lyase decrease and the levels of glycolate oxidase
increase. To investigate whether PMP22 is involved in such
stage-specific roles, the levels of PMP22 in Arabidopsis seedlings of
different ages were compared with levels of isocitrate lyase and
glycolate oxidase. Total protein was extracted from equal numbers of
Arabidopsis seedlings at different time points after imbibition and
then immunoblotted with affinity-purified anti-PMP22,
affinity-purified anti-glycolate oxidase, and anti-isocitrate lyase antibodies. Figure 8A shows that
PMP22, which is present at low levels in seeds, progressively
accumulated on a per-seedling basis to reach a steady level by 48 h postimbibition. The level of isocitrate lyase was, as expected,
temporally dependent, with maximum protein levels at 48 h. The
level of glycolate oxidase increased steadily from 36 h on, as did
the level of chlorophyll (data not shown). Greening was observed
between 48 and 72 h by measuring total chlorophyll levels.
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| Figure 8.
A, Expression of PMP22 during seedling
development. Total protein was extracted from an equal number (based on
dry weight at sowing) of Arabidopsis seedlings at the indicated number
of hours postimbibition. Protein samples were separated by SDS-PAGE and
probed with affinity-purified anti-PMP22 antibodies, anti-castor bean
isocitrate lyase antibodies (ICL), or affinity-purified anti-spinach
glycolate oxidase antibodies (GO). B, Effects of light on expression of
PMP22 in tissue cultures. Arabidopsis suspension-cultured cells were
grown in either continuous light (lane 1) or total dark (lane
2), subcultured, and cultured for a further 5 d under the
same conditions. Total protein was extracted from these samples and
subjected to SDS-PAGE and immunoblotting with affinity-purified
anti-PMP22 antibodies.
|
|
To establish whether PMP22 expression was light dependent, total
protein was extracted from the same growth stage (5 d postsubculture) of constant-dark- and constant-light-cultured Arabidopsis
suspension-cultured cells (these cells turned green upon exposure to
light). Immunoblotting equivalent amounts of total protein (compared on
the basis of equal packed-cell volume) revealed that the levels of
PMP22 were the same in both dark and light conditions (Fig. 8B).
| |
DISCUSSION |
We have identified and characterized at the molecular level a
plant PMP, Arabidopsis PMP22, which is approximately 55% similar (30%
identical) to its mammalian counterparts, which form two subgroups,
Mpv17 and PMP22. Although Mpv17 from human and mouse are 92% identical
at the amino acid level, and rat and mouse PMP22 are 95% identical,
the identity between Mpv17 and PMP22 from mouse is only 28%. Thus, the
two different subgroups from the same organism are no more similar to
each other than they are to Arabidopsis PMP22. However, comparison of
the hydropathy profiles shows that Arabidopsis PMP22 is more similar to
Mpv17 than to PMP22. The Arabidopsis PMP22 is 190 amino acids long and
is the product of a single-copy gene.
Arabidopsis PMP22 was localized to peroxisomes by subcellular
fractionation and immunoblotting and by co-sedimenting in Suc gradients
with catalase and the peroxisomal marker protein isocitrate lyase. As
predicted by its hydropathy plot, Arabidopsis PMP22 behaves as an
integral PMP: it is not extracted by
Na2CO3 (pH 11.0) and it
fractionates into the Triton X-114 detergent phase. Despite the
prediction by the hydropathy profile that significant hydrophilic
domains flank the transmembrane regions, these were not accessible to
proteases such as thermolysin and trypsin even if salt-washed membranes
were treated with high concentrations of protease. This implies that
Arabidopsis PMP22 is either deeply buried in the membrane or that it
may form homo- or hetero-complexes in the membrane such that the
hydrophilic domains are not accessible to protease. Protease protection
by the membrane was completely abolished in the presence of Triton
X-100.
The protease resistance of native Arabidopsis PMP22 and its
inextractability with
Na2CO3 (pH 11.0) allowed us
to investigate the manner of its insertion into the peroxisomal
membrane. As established for PMP70 (Imanaka et al., 1996), PAF-1, and
rat PMP22 (Just and Diestelkötter, 1996), Arabidopsis PMP22
inserted into peroxisomal membranes posttranslationally. There was no
binding or insertion observed if glyoxysomes were replaced by an
irrelevant membrane (red blood cells), demonstrating that insertion was
not due to nonspecific partitioning of a hydrophobic protein into a
membrane. We observed that in contrast to results obtained with rat
PMP22, but similar to results with PMP70 and PAF-1, the insertion of
Arabidopsis PMP22 into peroxisomal membranes was stimulated by the
presence of 2.4 mM ATP with an ATP-regeneration system.
In the absence of ATP and the ATP-regeneration system, binding of
Arabidopsis PMP22 to the surface of glyoxysomes was not affected, and
this binding was carbonate-insensitive, suggesting partial insertion
into the bilayer. However, the level of the native protease-resistant
conformation was reduced by 50%. This suggests that after
binding/insertion of Arabidopsis PMP22 to and/or into the peroxisomal
membrane by an ATP-independent mechanism, ATP is required at the
membrane for rearrangement into the native conformation. However,
because this process was not completely abolished in the absence of
ATP, it may also suggest that the membrane protein insertion/assembly
machinery of the isolated peroxisomes could remain "primed" for the
import of membrane proteins, e.g. with ATP tightly bound. These results
are in contrast to those obtained for the matrix proteins isocitrate
lyase and glycolate oxidase, in which import into the matrix is
dependent upon added ATP (Behari and Baker, 1993; Horng et al.,
1995).
The expression of Arabidopsis PMP22 is not light dependent, because it
is expressed at similar levels in both dark- and light-grown suspension-cultured cells. After imbibition the levels of PMP22 per
seedling steadily increased to a constant level by the onset of
greening. Thus, the requirement for PMP22 was not associated with a
specific peroxisome type and may represent a universal component of the
peroxisome membrane. The steady-state level of PMP22 differs between
tissues it is most abundant in open flowers and siliques, with lesser
amounts in stems, leaves, and roots. We observed a tissue-specific
alteration in the mobility of PMP22 (<0.5 kD), with a larger isoform
detected in flowers, stems, and roots. We are currently investigating
the molecular basis of this heterogeneity.
The mouse Mpv17 protein has been shown to generate reactive oxygen
species (Zwacka et al., 1994), but it is not known if this is its
principal function or if it is a by-product of some other reaction
catalyzed by the protein. Although, like Arabidopsis PMP22, Mpv17
appears to be expressed in all tissues, its inactivation by retroviral
insertion principally affects the kidney, leading to glomerosclerosis
and nephrotic syndrome (Weiher et al., 1990). Plant peroxisomes are
significant generators of active oxygen species in both the membranes
and the matrix; they contain defense mechanisms in the form of
catalase, superoxide dismutase, and ascorbate peroxidase. It will be
interesting to discover if the loss of PMP22 has phenotypic effects on
any particular organ, or if it results in altered sensitivity to growth
conditions that stimulate free radical production in plant peroxisomes.
Such information may help to shed light on the function of PMP22 in
both mammals and plants.
| |
FOOTNOTES |
1
This work was supported by the Biotechnology and
Biology Research Council, UK (grant no. 24/C06847), and by the
Leverhulme Trust, UK (grant no. F/122/AW to A.B.).
2
Present address: Zentrum Molecular Biology
Heidelberg, Im Neuenheimerfeld 282, D69120 Heidelberg, Germany.
*
Corresponding author; e-mail a.baker{at}leeds.ac.uk; fax
44-113-233-3144.
Received October 8, 1998;
accepted January 18, 1999.
| |
ABBREVIATIONS |
Abbreviation:
PMP, peroxisomal membrane protein.
| |
ACKNOWLEDGMENTS |
We thank Dr Claude Kaplan for identifying the TAY089 expressed
sequence tag as a possible PMP22 ortholog and the Arabidopsis Biological Resource Center for dispatching the expressed sequence tag
clone. We are grateful to Prof. Colin Robinson (University of Warwick,
UK) for antibodies to the 23-kD subunit of PSII and to Prof. L.A. del
Río and Dr. L.M. Sandalio for helpful comments on the text.
The accession number for the Arabidopsis PMP22 sequence reported here
is AJ006053.
| |
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Top
Abstract
Introduction