From the Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
Received for publication, June 12, 2000, and in revised form, September 21, 2000
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ABSTRACT |
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Peroxins are proteins required for peroxisome
assembly. The cytosolic peroxin Pex20p binds directly to the
Peroxisomes are members of the microbody family of organelles and
are found in organisms from yeasts to humans and in most cell types.
They are the site of many important biochemical processes that vary
depending on the organism or cell type. Among their many metabolic
activities, peroxisomes perform the Peroxins are proteins required for peroxisome assembly. A subset of
peroxins is required for the targeting and import of peroxisomal matrix
proteins. The PTS receptors Pex5p and Pex7p bind PTS1- and
PTS2-containing proteins, respectively, and are necessary for targeting
these proteins to peroxisomes (reviewed in Refs. 2-4). There are
conflicting findings regarding the localization of these receptors in
the cell. Both receptors have been found in the cytosol, associated
with the peroxisomal membrane or in the peroxisomal matrix (reviewed in
Refs. 3 and 4). Pex5p and Pex7p have therefore been proposed to be
mobile receptors that bind cargo proteins in the cytosol, dock at the
peroxisomal membrane, enter the peroxisome, release their cargo in the
matrix, and recycle to the cytosol (2-4, 7, 8). Pex13p and Pex14p are
integral and peripheral peroxisomal membrane peroxins, respectively, necessary for PTS receptor docking (reviewed in Refs. 2-4). Since Pex13p and Pex14p interact with each other and with both PTS receptors, it has been suggested that although PTS1- and
PTS2-dependent targeting pathways are divergent, the import
pathways are convergent (9, 10). Pex8p is a peroxin associated with the
inside of the peroxisomal membrane, and, in its absence, PTS1- and
PTS2-containing proteins are mislocalized to the cytosol (11-14).
Recently, a physical interaction was detected between Pex8p and the
PTS1 receptor Pex5p in the yeast Saccharomyces cerevisiae,
and Pex8p was suggested to function in protein translocation across the
peroxisomal membrane following the docking of Pex5p-cargo complexes
(14).
Peroxisomal targeting signals and the peroxisomal import apparatus have
been strongly conserved during evolution, but interestingly some
divergence has been recognized. In certain mammalian systems, two
isoforms of Pex5p have been identified, a short isoform necessary for
PTS1 import and a long isoform necessary for the import of both PTS1-
and PTS2-containing proteins (15, 16). In Yarrowia lipolytica, the PTS2 receptor Pex7p has not been identified. In this yeast, Pex20p, a peroxin with sequence similarity to Pex5p, is
necessary for the dimerization and peroxisomal targeting of thiolase, a
PTS2-containing protein (17). Since Pex20p has been shown to be found
in both the cytosol and associated with the peroxisomal membrane, it
has been proposed to act as a cycling protein that picks up thiolase in
the cytosol, docks at the peroxisomal membrane, and then repeats the
circuit (17).
Here we report the identification and characterization of a direct
interaction between Pex8p and Pex20p. Pex20p is apparently not required
for the targeting of Pex8p to peroxisomes, because Pex8p is still
peroxisomal in cells of a PEX20 disruption strain. Instead,
we provide data that are consistent with Pex8p being directly involved
in the import of thiolase into peroxisomes at a stage following docking
of the Pex20p-thiolase complex at the peroxisomal membrane. Our data
also suggest that, like Pex7p and Pex5p, Pex20p may accompany its cargo
into peroxisomes during import.
Yeast Strains and Culture Conditions--
The yeast strains used
in this study are listed in Table I.
Media components were as follows: YEPD, 1% yeast extract, 2% peptone,
2% glucose; YEPA, 1% yeast extract, 2% peptone, 2% sodium acetate;
YPBO, 0.3% yeast extract, 0.5% peptone, 0.5%
K2HPO4, 0.5% KH2PO4,
1% Brij 35, 1% (w/v) oleic acid; YND, 1.34% yeast nitrogen base
without amino acids, 2% glucose; YNO, 1.34% yeast nitrogen base
without amino acids, 0.05% (w/v) Tween 40, 0.2% (w/v) oleic acid;
YNA, 1.34% yeast nitrogen base without amino acids, 2% sodium
acetate. Liquid YNO and YND media and YNO agar plates were supplemented
with 2× Complete Supplement Mixture (minus the appropriate amino acids
or nucleotides) (Bio 101, Vista, CA). YNA agar plates were supplemented
with leucine, uracil, and lysine, each at 50 µg/ml, as required.
Growth was at 30 °C. Strains and culture conditions for two-hybrid
analyses were as described by the manufacturer
(CLONTECH, Palo Alto, CA).
Two-hybrid Analyses--
Physical interactions between peroxins
were detected using the Matchmaker Two-Hybrid System
(CLONTECH). Chimeric genes were generated by
amplifying the open reading frames (ORFs) of PEX genes from
Y. lipolytica genomic DNA by the polymerase chain reaction (PCR) and ligating them in-frame and downstream of the DNA encoding the
transcription-activating domain (AD) and the DNA-binding domain (DB) of
the GAL4 transcriptional activator in the plasmids pGAD424 and pGBT9,
respectively (Table II). To generate
pGAD-P8
Cells of the S. cerevisiae strain SFY526 were
transformed simultaneously with a pGBT9-derived plasmid and a
pGAD424-derived plasmid. Transformants were grown on synthetic media
lacking tryptophan and leucine and tested for activation of the
integrated lacZ construct using Construction of Chimeric Genes and Isolation of Recombinant
Proteins--
Chimeric genes encoding maltose-binding protein (MBP)
fusions and glutathione S-transferase (GST) fusions were generated as described below. The PEX8 ORF was excised from pGAD-PEX8
(Table II) with EcoRI and BglII and ligated into
EcoRI/BamHI-digested pBluescript SKII(+)
(Stratagene, La Jolla, CA) to generate the plasmid pBS-PEX8. To
generate pMAL-PEX8 encoding MBP-Pex8p, PEX8 was excised from
pBS-PEX8 with EcoRI/XbaI and ligated into
EcoRI/XbaI-digested pMAL-c2 (New England Biolabs,
Beverly, MA). To generate pGEX-PEX20 encoding GST-Pex20p, pGBT-PEX20
(Table II) was digested with EcoRI, and the PEX20
ORF was ligated into EcoRI-digested pGEX-4T1 (Amersham Pharmacia Biotech).
The plasmids pGEX-4T1, pGEX-PEX20, pMAL-c2, and pMAL-PEX8 were
introduced into the Escherichia coli strain BLR (DE3)
(Novagen, Madison, WI). Induction and purification of GST fusion
proteins were performed according to the manufacturer's (Amersham
Pharmacia Biotech) specifications, except that expression of chimeric
genes was induced at 30 °C with 1 mM
isopropyl- In Vitro Binding Assay with Recombinant Proteins--
An
in vitro binding assay was performed using recombinant GST,
GST-Pex20p, MBP, and MBP-Pex8p purified as described above. Glutathione-Sepharose 4B (Amersham Pharmacia Biotech) was washed with
RW buffer (20 mM HEPES-KOH, pH 6.8, 150 mM
potassium acetate, 5 mM magnesium acetate, 0.1% (w/v)
Tween 20, 0.1% casamino acids, 1 µg/ml leupeptin, 1 µg/ml
pepstatin A, 1 µg/ml aprotinin, 2.5 µg/ml antipain, 0.21 mg/ml NaF,
0.1 mg/ml Pefabloc SC (Roche Molecular Biochemicals)). GST and
GST-Pex20p were tumbled end-over-end in batch with
glutathione-Sepharose 4B (2.5 µg of GST or GST-Pex20p and 5 µl of
packed beads per reaction) in 0.5 ml of RW buffer for 20 min at room
temperature. Beads were collected by centrifugation and washed three
times with 0.5 ml of RW buffer. Purified MBP or MBP-Pex8p (5 µg/reaction) was added to 100 µl of RW buffer containing GST or
GST-Pex20p linked to beads and tumbled end-over-end for 30 min at room
temperature. Beads were collected by centrifugation, and supernatants
were retained as the unbound fractions. The beads were resuspended in
0.5 ml of RW buffer and applied to spin filters (CytoSignal, Irvine,
CA), washed three times with 0.5 ml of RW buffer, and eluted with 50 µl of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
at room temperature. 50% of bound fractions and 25% of unbound
fractions were run on 9% polyacrylamide gels, and proteins were
detected by staining with Coomassie Brilliant Blue R250.
In Vitro Binding Assay with Yeast Lysate--
Aliquots of
glutathione-Sepharose 4B (10 µl of packed beads/reaction) were washed
four times with 1 ml of RX buffer (20 mM HEPES-KOH, pH 6.8, 150 mM potassium acetate, 2 mM magnesium
acetate, 0.5% (w/v) Triton X-100, 0.1% (w/v) casamino acids, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml aprotinin, 2.5 µg/ml antipain, 0.21 mg/ml NaF, 0.1 mg/ml Pefabloc SC) and
resuspended in 0.5 ml of RX buffer. 3 µg of GST or GST-Pex20p was
added to the beads, and reactions were tumbled end-over-end at room
temperature for 80 min. Beads were collected by centrifugation and
washed three times with 0.5 ml of RX buffer. YPBO-grown cells of the
PEX8-HAc strain (Table I) were collected by
centrifugation, washed three times with water, and washed once with RX
buffer. Cells were disrupted in RX buffer by agitation with glass
beads. The lysate was clarified by centrifugation at 20,000 × g in a microcentrifuge for 45 min at 4 °C. 120 µl of
the cleared supernatant (10 mg of protein/ml) or of RX buffer alone was
tumbled end-over-end in batch with glutathione-Sepharose-bound GST or
GST-Pex20p for 2 h at 4 °C. Beads were washed twice with 0.8 ml
of RX buffer, applied to spin filters, and washed four times with 0.5 ml of RX buffer. Bound proteins were eluted in 40 µl of SDS-PAGE
sample buffer according to the manufacturer's instructions. Proteins
in equal fractions of each sample were separated on 10% polyacrylamide
gels, transferred to nitrocellulose, and subjected to immunoblotting.
Epitope Tagging of Pex8p--
A modified PEX8 gene
encoding Pex8p tagged at its carboxyl terminus was made by inserting a
DNA fragment encoding two copies of the influenza virus hemagglutinin
(HA) epitope in frame and downstream of the PEX8 ORF. A
fragment containing the PEX8 ORF flanked by 2.0 and 1.2 kilobase pairs of genomic DNA at its 5'- and 3'-ends, respectively, was
excised from p50LD (13) with HindIII and ligated into the
HindIII site of pGEM7Zf(+) to make the plasmid pG7PEX8.
Next, a BglII site was introduced immediately upstream of
the stop codon of PEX8 using sequential PCR. Essentially, two regions of the PEX8 gene flanking the proposed
BglII site were amplified from pG7PEX8 using
oligonucleotides RP and 761 for the 5' region and oligonucleotides UP
and 760 for the 3' region (Table III). The two products were used as
templates for a second round of PCR with oligonucleotides UP and RP.
Since oligonucleotides 760 and 761 have BglII recognition
sequences and complementary 5'-ends, the second round of PCR generated
a single fragment containing PEX8 with a BglII
site immediately upstream of the stop codon. This product was digested
with HindIII and ligated into pGEM7Zf(+) to generate the
plasmid pG7-PEX8B. Next, a fragment with
BamHI/BglII termini encoding the peptide
DPLAMYPYDVPDYAAMYPYDVPDYAAMGKGE, which contains
two repeats of the HA epitope (underlined residues) (18), was ligated
in-frame into the BglII site of pG7PEX8B to generate plasmid
pG7PEX8-HAc. The region of pG7PEX8-HAc
amplified by PCR was confirmed to be correct by sequencing.
A fragment containing the modified PEX8 gene was excised
from pG7PEX8-HAc with HindIII and integrated
into the genome of the pex8-1 mutant strain (Table I) by
homologous recombination to replace the pex8-1 allele of
PEX8. The pex8-1 allele has a substitution
mutation in the PEX8 gene (A1924T), creating a premature
stop codon2 and cannot grow
on oleic acid medium (13). Transformants were selected for
reestablished growth on oleic acid medium (YNO agar) and characterized
by Southern blotting and electron microscopy. One strain,
PEX8-HAc, having the correct genotype and
wild-type morphology, was chosen for further study.
To make a PEX8-HAc expression plasmid,
PEX8-HAc was excised from
pG7PEX8-HAc with HindIII and ligated into the
HindIII site of the E. coli/Y. lipolytica shuttle vector pINA445 to make the plasmid
pPEX8-HAc encoding Pex8p-HAc. pINA445 contains
the Y. lipolytica LEU2 gene for positive selection of yeast
transformants and the Y. lipolytica ARS68 gene for
autonomous plasmid replication in Y. lipolytica cells
(19).
Coimmunoprecipitation--
Coimmunoprecipitation was performed
by immunoaffinity chromatography using protein A-Sepharose CL-4B
(Sigma). All steps were performed at 4 °C unless otherwise
specified. YPBO-grown PEX8-HAc cells were
collected by centrifugation, washed three times with water, and washed
once with RX buffer. Cells were resuspended in RX buffer and lysed by
disruption with glass beads. The total cell lysate was clarified by
centrifugation at 100,000 × g in a TLA120.2 rotor
(Beckman, Palo Alto, CA) for 15 min, diluted to a protein concentration
of 10 mg/ml with RX buffer, and divided into 500-µl aliquots. 4.5 µl of immune serum or 18 µl of preimmune serum (volumes of serum
containing equal numbers of IgG molecules) was added to each of two
aliquots, and reactions were tumbled end-over-end for 30 min. Protein
A-Sepharose, preblocked in RX buffer containing 8% bovine serum
albumin for 1 h, was pelleted by centrifugation and resuspended in
RX buffer to a final concentration of 20% (v/v). 50 µl of the
protein A-Sepharose suspension was added to each reaction, and
reactions were tumbled end-over-end for 40 min. Antibody complexes were
pelleted by centrifugation and resuspended in 0.5 ml of RX buffer.
Samples were then applied to spin filters and washed three times
with RX buffer, and antibody complexes were eluted in SDS-PAGE sample
buffer at room temperature. Proteins in 20% of each eluate, along with
10 µg of protein of the cell lysate, were separated by SDS-PAGE,
transferred to nitrocellulose, and immunoblotted.
Subcellular Fractionation--
Subcellular fractionation of
Y. lipolytica was performed as described (17, 20) using 5 mM MES, pH 5.5, 1 M sorbitol containing 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml aprotinin, 2.5 µg/ml antipain, 0.21 mg/ml NaF, 0.1 mg/ml Pefabloc SC as
homogenization buffer. Essentially, a postnuclear supernatant isolated
from YPBO-grown cells was fractionated by differential centrifugation
at 20,000 × g for 20 min into a pellet (20KgP)
enriched for peroxisomes and mitochondria and a supernatant (20KgS)
enriched for cytosol and high-speed pelletable organelles. The 20KgS
fraction was further subfractionated by centrifugation at 200,000 × g for 1 h to yield a pellet (200KgP) enriched for
high-speed pelletable organelles and a supernatant (200KgS) highly
enriched for cytosol. Peroxisomes were purified from the 20KgP fraction
by isopycnic centrifugation on a discontinuous sucrose gradient
(21).
Subfractionation of Peroxisomes and Protease Protection
Analysis--
To subfractionate peroxisomes, 1.6 ml of ice-cold Ti8
buffer (10 mM Tris-HCl, pH 8.0, 5 mM EDTA
containing 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml
aprotinin, 2.5 µg/ml antipain, 0.21 mg/ml NaF, 0.1 mg/ml Pefabloc SC,
0.2 mM benzamidine hydrochloride) was added to 160 µl of
20KgP fraction containing 240 µg of protein. Samples were incubated
on ice for 30 min with occasional gentle agitation to disrupt
peroxisomes. Half of each disrupted 20KgP (20KgP-D) was separated into
a supernatant (Ti8S) enriched for soluble proteins and a pellet (Ti8P)
enriched for membranes by centrifugation at 200,000 × g at 4 °C for 1 h. Proteins from equal fractions of
the 20KgP-D, Ti8S, and Ti8P were separated by SDS-PAGE, transferred to
nitrocellulose, and subjected to immunoblotting.
Protease protection analysis was performed on 20KgP fractions isolated
in the absence of protease inhibitors as described (22). 0-32 µg of
trypsin was combined with 60 µg of protein of the 20KgP fraction in
the absence or presence of 0.1% (w/v) Triton X-100 in a final volume
of 160 µl. Reactions were incubated on ice for 20 min and terminated
by precipitation with trichloroacetic acid. Proteins from equal
fractions of each reaction were separated by SDS-PAGE, transferred to
nitrocellulose, and subjected to immunoblotting.
Immunofluorescence Microscopy--
Strains were grown at
30 °C in YEPD overnight, transferred to YPBO, and grown for 9 h. Cells were fixed for 30 min at room temperature and processed for
immunofluorescence microscopy as described (23), except that
spheroplasts were prepared in 100 mM potassium phosphate,
pH 7.5, containing 1.2 M sorbitol, 40 µg/ml
Zymolyase-100T (ICN, Aurora, OH), 0.4% (v/v) 2-mercaptoethanol for 30 min at 30 °C with gentle agitation. Images were captured using a
Spot Cam digital fluorescence camera (Spot Diagnostic Instruments,
Sterling Heights, MI).
Antibodies--
Guinea pig and rabbit antibodies to Y. lipolytica Pex20p (17), rabbit antibodies to Y. lipolytica peroxisomal isocitrate lyase and to thiolase, guinea
pig antibodies to Y. lipolytica Pex2p (24), and rabbit
antibodies to Y. lipolytica Kar2p (25) have been described.
Mouse monoclonal antibody 12CA5, which recognizes 9 amino acid residues
of the influenza virus HA protein, was purchased from the Berkeley
Antibody Company (Richmond, CA). To raise antibodies to Y. lipolytica Pex6p, a DNA fragment with
EcoRI/HindIII termini encoding the first 452 amino acid residues of Pex6p was amplified from Y. lipolytica genomic DNA by PCR with oligonucleotides 424 and 425 (Table III). This fragment was ligated into the corresponding sites of
the vector pMAL-c2 in frame and downstream of the malE ORF
encoding MBP. Antibodies to the fusion protein were raised in rabbit
and guinea pig as described previously (26, 27). Rabbit antibodies to
Pex20p and guinea pig antibodies to Pex2p were preabsorbed with
permeabilized spheroplasts of the respective disruption strains prior
to use in immunofluorescence microscopy.
Miscellaneous--
Oligonucleotides were synthesized on an Oligo
1000M DNA Synthesizer (Beckman). Sequencing was performed with an ABI
Prism 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CA). DNA was amplified using Ready-To-Go PCR beads (Amersham Pharmacia Biotech). Preparation of yeast lysates by disruption with glass beads,
growth of E. coli, and manipulations of DNA were performed as described (28). Protein concentrations were determined using a
commercially available kit (Bio-Rad). SDS-PAGE was performed essentially as described (29). Proteins were transferred to nitrocellulose for immunoblotting using a wet transfer system, and
antigen-antibody complexes were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Detection of Y. lipolytica Peroxin Interactions with the Yeast
Two-hybrid System--
A limited yeast two-hybrid screen (30) was
performed to identify physical interactions between Y. lipolytica peroxins Pex1p, Pex2p, Pex5p, Pex6p, Pex8p, Pex9p,
Pex16p, and Pex20p. Others have used this methodology successfully to
detect interactions between peroxins (for examples, see Refs. 10 and
31-33). Chimeric genes were generated by ligating Y. lipolytica
PEX genes in frame and downstream of sequences encoding one of the
two functional domains (AD or DB) of the GAL4 transcriptional
activator. All possible combinations of plasmid pairs encoding AD and
DB fusion proteins were transformed into the S. cerevisiae
strain SFY526, and
To confirm the results of the filter assay, liquid
Lysates of strains transformed with constructs encoding DB-Pex8p or
DB-Pex20p showed high levels of The Interaction between Pex8p and Pex20p Is Direct and
Autonomous--
Some interactions between peroxins that have been
detected using the yeast two-hybrid system have been shown to be
indirect (10, 33). Therefore, it is possible that an endogenous
S. cerevisiae protein may have bridged the observed
interaction between Pex8p and Pex20p. To determine whether the
interaction between Pex8p and Pex20p is direct and autonomous, an
in vitro binding assay was performed. The ORFs encoding
Pex8p and Pex20p were fused to the 3'-ends of DNA sequences encoding
MBP and GST, respectively. The chimeric genes were expressed in
E. coli, and the fusion proteins were purified. GST and
GST-Pex20p were immobilized on glutathione-Sepharose beads and tested
for their ability to bind MBP or MBP-Pex8p. Proteins in bound and
unbound fractions were separated by SDS-PAGE and visualized by staining
with Coomassie Brilliant Blue (Fig.
3). MBP-Pex8p interacted with GST-Pex20p,
while GST or MBP alone did not interact with MBP-Pex8p or GST-Pex20p,
respectively. We conclude that the interaction between Pex8p and Pex20p
is direct and autonomous.
The Pex8p-Pex20p Interaction Is Specific--
An in
vitro binding experiment was performed to determine whether
GST-Pex20p could interact specifically with Pex8p tagged with the
hemagglutinin epitope, Pex8p-HAc, in a yeast lysate. GST
and GST-Pex20p were immobilized on glutathione-Sepharose beads. A total
cell lysate was prepared from PEX8-HAc, a strain
synthesizing Pex8p-HAc, and incubated with immobilized GST
or GST-Pex20p. The proteins bound to each column and the proteins of
the total cell lysate were separated by SDS-PAGE, transferred to
nitrocellulose, and analyzed by immunoblotting with various antibodies
(Fig. 4). Pex8p-HAc
interacted specifically with GST-Pex20p, while the peroxins Pex1p and
Pex6p did not. Interestingly, peroxisomal thiolase, which has been
shown to interact with Pex20p (17), did not bind GST-Pex20p (Fig. 4).
Since an interaction between thiolase and Pex20p also could not be
detected in the yeast two-hybrid system,2 it may be that
peptides fused to the amino terminus of Pex20p inhibit its interaction
with thiolase.
Pex8p-HAc and Pex20p Coimmunoprecipitate--
To
determine whether Pex20p was capable of interacting with
Pex8p-HAc in vivo, Pex20p was immunoprecipitated
under native conditions from a total lysate of cells grown in YPBO
medium, and coimmunoprecipitating proteins were analyzed by SDS-PAGE
and immunoblotting with various antibodies (Fig.
5). Although all proteins tested were
present in the total cell lysate, Pex8p-HAc
coimmunoprecipitated specifically with Pex20p but not with peroxisomal isocitrate lyase or the peroxin Pex6p. Therefore, Pex8p-HAc
interacts with Pex20p in vivo. As previously reported (17), the precursor form of thiolase also specifically coimmunoprecipitated with Pex20p.
Pex20p Is Not Required for Targeting Pex8p-HAc to
Peroxisomes--
Since Pex20p binds thiolase in the cytosol and is
necessary for its targeting to peroxisomes (17) and because Pex20p
interacts with Pex8p-HAc, we investigated whether Pex20p
might also bind Pex8p in the cytosol and act in targeting it to
peroxisomes. To test this hypothesis, Pex8p-HAc was
localized by subcellular fractionation of cells of the wild-type strain
E122, the PEX20 disruption strain
pex20KO, and the original PEX20 mutant strain
pex20-1 (Table I), each
transformed with the expression plasmid pPEX8-HAc. For each
strain, a postnuclear supernatant fraction was separated into
20KgS enriched for cytosol and high-speed pelletable organelles and 20KgP enriched for peroxisomes and mitochondria (17). The 20KgS
fraction was further divided into 200KgS highly enriched for cytosol
and 200KgP enriched for high-speed pelletable organelles, including
high-speed pelletable peroxisomes (17, 20). Proteins in equal portions
of each fraction were separated by SDS-PAGE and analyzed by
immunoblotting (Fig. 6).
Pex8p-HAc was present in the 20KgP fraction enriched for
peroxisomes and in the 20KgS and 200KgP fractions enriched for
high-speed pelletable peroxisomes but not in the highly enriched
cytosol fraction (200KgS) of cells of all strains. For all strains,
Pex8p-HAc cofractionated with peroxisomal marker proteins
in a sucrose density gradient (data not presented). Together, these
results suggest that Pex20p is not required for the targeting of Pex8p to peroxisomes. As expected, thiolase was preferentially localized to
peroxisome-enriched fractions of cells of the wild-type strain and to
cytosol-enriched fractions of cells of the PEX20 mutant strains.
Pex20p and a Small Amount of Thiolase Associate with Peroxisomes in
Cells of a PEX8 Disruption Strain--
Interaction between Pex20p and
Pex8p may occur at the level of the peroxisomal membrane during the
targeting or import of thiolase. Consistent with this scenario, only a
small fraction of thiolase is peroxisomal in a PEX8
disruption strain (13). To further elucidate the role of Pex8p in the
import of thiolase, the localization of Pex20p and the subperoxisomal
localization of peroxisome-associated thiolase were determined in cells
of the PEX8 disruption strain, pex8-KA (13)
(Table I). Double labeling immunofluorescence microscopy of YPBO-grown
cells demonstrated that as expected, Pex20p showed a diffuse
localization characteristic of a cytosolic protein (17), while the
peroxisomal membrane protein Pex2p and the peroxisomal matrix protein
thiolase showed a punctate pattern of localization characteristic of
peroxisomes in cells of the wild-type strain E122 (Fig.
7). Interestingly, in cells of the
pex8-KA strain, Pex20p colocalized with Pex2p to punctate
structures characteristic of peroxisomes, while as expected, thiolase
had a diffuse localization characteristic of a cytosolic location.
These results suggest that while Pex20p is predominantly cytosolic in
wild-type cells, as has been reported previously (17), a significant
fraction of Pex20p associates with peroxisomes in cells lacking
Pex8p.
Pex20p and thiolase were also localized by subcellular fractionation of
cells of the wild-type E122 and pex8-KA strains.
In E122 cells, Pex20p was found largely in the 20KgS
fraction enriched for cytosol and high-speed pelletable organelles,
while thiolase was primarily in the 20KgP fraction enriched for
peroxisomes, as expected (Fig.
8A). In pex8-KA
cells, by contrast, Pex20p was found primarily in the 20KgP fraction
and thiolase was found primarily in the 20KgS fraction, although a
small amount of thiolase fractionated to the 20KgP, since some thiolase
is peroxisomal in pex8-KA cells (13). The results of
subcellular fractionation are consistent with those of
immunofluorescence analysis.
The suborganellar locations of Pex20p and thiolase in wild-type cells
and in pex8-KA cells were now compared. The 20KgP fraction of wild-type and pex8-KA cells was disrupted by incubation
in dilute Tris buffer. The 20KgP-D was then separated by centrifugation into a pellet fraction (Ti8P) enriched for membranes and a supernatant fraction (Ti8S) enriched for soluble proteins. Proteins in equal portions of each fraction were separated by SDS-PAGE and analyzed by
immunoblotting (Fig. 8B). Pex20p localized primarily to the Ti8P fraction from both wild-type and pex8-KA cells along
with peroxisomal membrane protein Pex2p, whereas thiolase was primarily found in the Ti8S fraction from both strains. These data indicate that
in both wild-type and pex8-KA cells, Pex20p associates with the peroxisomal membrane. In addition, since thiolase was present in
the soluble Ti8S fraction derived from pex8-KA cells, either thiolase is in the matrix of peroxisomes in the absence of Pex8p or
membrane-associated thiolase is released during the extraction incubation.
In wild-type cells, Pex20p is localized to the outer surface of
peroxisomes, as demonstrated by its susceptibility in vitro to the action of external proteases (17). To determine whether in the
absence of Pex8p, peroxisome-associated Pex20p and thiolase are
protected from the action of proteases by the peroxisomal membrane,
protease protection analysis was performed on a 20KgP fraction isolated
from cells of the pex8-KA strain. Equal portions of the
20KgP fraction were incubated with increasing amounts of trypsin in the
absence or presence of the nonionic detergent, Triton X-100. The
proteins in equal portions of each digest were separated by SDS-PAGE
and analyzed by immunoblotting (Fig. 8C). Both Pex20p and
thiolase showed greater resistance to trypsin in the absence of
detergent than in its presence, suggesting that they are afforded
protection from the action of proteases by the peroxisomal membrane in
pex8-KA cells.
Pex20p is a peroxin that binds thiolase in the cytosol and is
necessary for its oligomerization and targeting to peroxisomes (17).
Pex8p is an intraperoxisomal peroxin required for the import of a
number of peroxisomal proteins, including thiolase (11-14). Using the
yeast two-hybrid system, we identified an interaction between Pex8p and
the amino terminus of Pex20p. The Pex8p-Pex20p interaction was
confirmed by the isolation of a complex containing Pex8p and Pex20p
from yeast lysates by coimmunoprecipitation and by in vitro
binding studies with recombinant proteins showing that this interaction
is specific, direct, and autonomous. Although Pex20p and Pex8p are in
different cellular compartments, two readily possible scenarios for
their interaction can be proposed. One scenario is that Pex20p binds
Pex8p in the cytosol and is required for the oligomerization and/or
peroxisomal targeting of Pex8p. The second scenario is that Pex8p
interacts with Pex20p at the peroxisomal membrane at a step in the
import of thiolase into peroxisomes. We localized Pex8p and Pex20p in
cells of PEX20 and PEX8 disruption strains,
respectively, to elucidate the mechanism of interaction of these two proteins.
Pex20p-dependent Targeting and/or Oligomerization of
Pex8p--
The possibility of a role for Pex20p in the targeting
and/or oligomerization of Pex8p is consistent with evidence that
suggests that Pex8p of the yeast Hansenula polymorpha may be
an oligomer (11). It is also compatible with the fact that Pex8p is
intraperoxisomal and in most yeasts contains PTS1 (12) or both PTS1 and
PTS2 motifs (11, 14). Since Pex8p shares PTS with peroxisomal matrix enzymes, it is conceivable that it may also be targeted to peroxisomes by the same peroxins involved in targeting these enzymes. Although such
an explanation is plausible, two lines of evidence suggest that Pex20p
is not necessary for the targeting and/or oligomerization of Pex8p.
First, assuming that mislocalized or nonoligomerized Pex8p molecules
are nonfunctional and that Pex20p is necessary for the oligomerization
and/or the targeting of Pex8p, then a PEX20 disruption
strain should present a phenotype that is as severe as or more severe
than that of a PEX8 disruption strain. However, this is not
the case, since several matrix proteins are mislocalized in
PEX8 disruption strains (11-14), while only thiolase is
mislocalized in a PEX20 disruption strain (17). Second, an epitope-tagged version of Pex8p was localized to subcellular fractions from cells of pex20 mutant strains enriched for peroxisomes.
Although these data strongly suggest that Pex20p is not required for
the targeting and/or oligomerization of Pex8p, we cannot rule out the
possibility that redundant systems exist for these functions, with only
one requiring Pex20p.
A Role for Pex8p in Pex20p-dependent Import of
Thiolase--
The alternative scenario that Pex8p and Pex20p interact
at the peroxisomal membrane in a step in the targeting or import of thiolase is consistent with previously published findings. Pex8p has
been shown to be associated with the peroxisomal membrane in three
different yeasts, including Y. lipolytica (12-14). It has
also been shown that in cells of a Y. lipolytica PEX8
disruption strain, thiolase is primarily cytosolic, although a small
amount of thiolase associates with peroxisomes (13). The localization of Pex20p and thiolase in cells of a PEX8 disruption strain
also points to a direct role for Pex8p in the
Pex20p-dependent import of thiolase. While a small amount
of total Pex20p has previously been shown to be associated with
peroxisomes in wild-type cells (17), we find that a large fraction of
Pex20p associates with peroxisomal membranes in cells devoid of Pex8p
relative to wild-type cells. These data point to a role for the
interaction between Pex8p and Pex20p in the import of thiolase at a
stage following the docking of Pex20p-thiolase complexes at the
peroxisomal membrane. The exact role of Pex8p in thiolase import is
unknown; however, since Pex20p has been shown to interact with Pex8p in
the absence thiolase (or any other protein), it is tempting to
speculate that Pex8p has a role either in dissociating thiolase from
Pex20p or in recycling Pex20p after dissociation. Future experiments
will be aimed at studying the interactions between Pex8p and Pex20p bound to thiolase.
Interestingly, we find that the peroxisome-associated Pex20p and
thiolase in cells of the PEX8 disruption strain are
protected from the action of external proteases. These data suggest
that in the absence of Pex8p, both Pex20p and thiolase are protected by
membranes. This, together with the fact that Pex20p directly interacts
with Pex8p, an intraperoxisomal peroxin, suggests that Pex20p could
enter peroxisomes with its cargo. This event may not be readily
detectable in wild-type cells (17), either because the steady-state
level of the population of intraperoxisomal Pex20p is very low or
because Pex20p may exit the peroxisome through a putative translocation
channel during fractionation of wild-type cells.
A Role for Pex8p in Pex5p-dependent Import of
PTS1-containing Proteins--
As mentioned previously, thiolase is
mislocalized in cells lacking Pex20p, while several matrix proteins,
including PTS1-containing proteins, are mislocalized in cells lacking
Pex8p (13). One possible reason for this more extensive mislocalization
of matrix proteins in pex8 mutant strains is that in these
strains, Pex20p becomes trapped in the peroxisomal membrane, thereby
clogging the translocation apparatus. Since the PTS1- and
PTS2-dependent import pathways are apparently convergent
(9, 10), this obstruction may prevent PTS1-containing proteins from
being imported into the peroxisome. Alternatively, Pex8p may be
directly involved in both Pex5p-dependent and
Pex20p-dependent import. Our identification of an
interaction between Pex8p and Pex5p in the yeast two-hybrid system is
consistent with this possibility. In addition, Pex8p and Pex5p of
S. cerevisiae have recently been shown to interact, even in
the absence of the PTS1 of Pex8p (14). Although Pex8p and Pex5p have
not been shown to interact directly, Pex8p and Pex5p may interact at
their amino termini, as we have shown that Pex8p and Pex20p interact at
their amino termini, and Pex20p and Pex5p show sequence similarity at
their amino termini (17).
-oxidation enzyme thiolase and is necessary for its dimerization and
peroxisomal targeting. The intraperoxisomal peroxin Pex8p has a role in
the import of peroxisomal matrix proteins, including thiolase. We report the results of yeast two-hybrid analyses with various peroxins of the yeast Yarrowia lipolytica and characterize more
fully the interaction between Pex8p and Pex20p. Coimmunoprecipitation
showed that Pex8p and Pex20p form a complex, while in vitro
binding studies demonstrated that the interaction between Pex8p and
Pex20p is specific, direct, and autonomous. Pex8p fractionates with
peroxisomes in cells of a PEX20 disruption strain,
indicating that Pex20p is not necessary for the targeting of Pex8p to
peroxisomes. In cells of a PEX8 disruption strain, thiolase
is mostly cytosolic, while Pex20p and a small amount of thiolase
associate with peroxisomes, suggesting the involvement of Pex8p in the
import of thiolase after docking of the Pex20p-thiolase complex to the
membrane. In the absence of Pex8p, peroxisomal thiolase and Pex20p are
protected from the action of externally added protease. This finding,
together with the fact that Pex8p is intraperoxisomal, suggests that
Pex20p may accompany thiolase into peroxisomes during import.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation of fatty acids, bile
acid synthesis, plasmalogen synthesis, cholesterol metabolism, and
methanol oxidation (1). Soluble enzymes and other proteins found in the
peroxisomal matrix are synthesized on polysomes free in the cytosol (1)
and are targeted to peroxisomes by cis-acting peroxisomal
targeting signals (PTS).1
Matrix proteins are targeted most commonly by a PTS1, a tripeptide of
the sequence SKL or a conserved variant thereof, located at the extreme
carboxyl terminus of proteins. Less commonly, matrix proteins are
targeted by a PTS2, a nonapeptide located near or at the amino terminus
of proteins and having the consensus sequence (R/K)(L/V/I)X5(H/Q)(L/A) (reviewed in Refs.
2-4). Interestingly, unlike other organelles in which proteins
traverse the membrane in an unfolded conformation, peroxisomes are
capable of importing oligomeric protein complexes (5, 6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yarrowia lipolytica strains used in this study
NC, a plasmid encoding AD-Pex8p-(12-644), a portion
of the PEX8 gene with flanking EcoRI sites was
amplified from genomic DNA with oligonucleotides 122-1 and 549 (Table
III) and ligated into
EcoRI-digested pGAD424. To generate pGAD-P8-(12-370)
encoding AD-Pex8p-(12-370), pGAD-P8
NC was digested with
EcoRI and BamHI, and the resulting 1080-bp
fragment was ligated into the corresponding sites of pGAD424. pGAD-P8-(164-644) encoding AD-Pex8p-(164-644) was generated by digesting pGAD-P8
NC with ApoI and EcoRI and
ligating the 1442-bp fragment into EcoRI-digested pGAD424.
pGAD-P20-(1-76) encoding AD-Pex20p-(1-76) was generated by excising a
BalI/SmaI fragment from pGBT-PEX20 (Table
II).
Generation of plasmids for two-hybrid system analyses
Oligonucleotides used in this study
-galactosidase filter and
liquid assays. Both assays were performed according to the
manufacturer's (CLONTECH) instructions except that
for liquid assays, yeast from 1 ml of culture was used, and cells were
broken by three freeze-thaw cycles at
70 °C, and for filter
assays, agar plates contained 100 µg/ml adenine to reduce the red
color of the yeast.
-D-thiogalactopyranoside for 2 h. Cells
were lysed with B-PER reagent (Pierce). Induction and purification of
MBP fusion proteins was performed according to the manufacturer's (New
England Biolabs) specifications. All purified proteins were dialyzed
against buffer (20 mM HEPES-KOH, pH 6.8, 150 mM
potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 20% (w/v) glycerol) and stored at
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase filter detection assays
were performed initially. Physical interaction between peroxins was
expected to bring the two domains of the GAL4 transcriptional activator
together and result in activation of transcription of the integrated
lacZ construct and detectable
-galactosidase activity in
the form of blue coloration of the yeast colony in the presence of the
substrate 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal). Potential interactions
were detected between Pex8p and Pex5p and between Pex1p and Pex6p using
the filter detection assay (data not shown).
-galactosidase
assays were performed. Cell lysates of strains synthesizing both Pex1p
and Pex6p fusion proteins showed much greater
-galactosidase activity than lysates of control strains synthesizing either one or the
other of the fusion proteins (Fig.
1A), demonstrating that Pex1p
and Pex6p interact physically. Lysates of strains synthesizing both
Pex8p and Pex5p fusion proteins also showed very high
-galactosidase activity, but so did the lysates of control strains synthesizing one or
the other of the fusion proteins (Fig. 1B). Using the
Smith-Satterthwaite test for statistical significance (34), it was
determined that the levels of
-galactosidase activity from strains
synthesizing both DB-Pex8p and AD-Pex5p or both DB-Pex5p and AD-Pex8p
were significantly higher than those of the corresponding control
strains at confidence levels of 95 and 90%, respectively, indicating
that Pex8p and Pex5p interact physically.
View larger version (25K):
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Fig. 1.
Identification of Pex1p-Pex6p and Pex8p-Pex5p
interactions using the yeast two-hybrid system. A comparison of
-galactosidase activities of strains doubly transformed with
plasmids encoding the designated fusion (x axes).
-Galactosidase activity is measured in arbitrary units
(U) as defined by the manufacturer
(CLONTECH). A, Pex1p and Pex6p
physically interact. Each column is the measure of the
average
-galactosidase activity of at least three individual
transformants. B, Pex8p and Pex5p physically interact. Each
column is the measure of the average
-galactosidase
activity of at least six individual transformants. The levels of
activity from strains synthesizing DB-Pex8p and AD-Pex5p and
synthesizing DB-Pex5p and AD-Pex8p are significantly different from
those of the corresponding control strains at confidence levels of 95 and 90%, respectively. Error bars represent S.D.
values.
-galactosidase activity, making it
difficult to identify an interaction between Pex20p and Pex8p.
Therefore, a plasmid encoding DB-Pex20p-(1-76), a fusion between the
DB domain and the amino-terminal 76 amino acid residues of Pex20p, was
generated. DB-Pex20p does not have intrinsic transcription activation
activity. Lysates of strains synthesizing both AD-Pex8p and
DB-Pex20p-(1-76) showed significantly higher
-galactosidase activity than did lysates of control strains synthesizing one or the
other of the fusion proteins (Fig. 2,
compare row 1 with rows 5 and 6),
demonstrating that Pex8p and Pex20p interact physically. To
characterize this interaction further, fusions to smaller domains of
Pex8p were assayed. DB-Pex20p-(1-76) still interacted with AD-Pex8p-(12-644), containing Pex8p truncated at both its amino and
carboxyl termini, but did not interact with AD domain fusions to more
extensive amino- or COOH-terminal truncations of Pex8p (Fig. 2, compare
row 2 to rows 3 and 4). Therefore,
Pex8p physically interacts with the amino terminus of Pex20p, and the
11 amino-terminal and 27 COOH-terminal amino acid residues of Pex8p are
dispensable for this interaction.
View larger version (38K):
[in a new window]
Fig. 2.
Analysis of the interaction of Pex8p with the
amino terminus of Pex20p in the yeast two-hybrid system.
SFY526 cells doubly transformed with chimeric genes encoding
AD (first column) and DB (second column) fusion
proteins were tested for -galactosidase activity. The Pex8p and
Pex20p portions of fusions are represented by shaded bars
and open bars, respectively. Numbers indicate the
amino acid residues at the amino and carboxyl termini of peroxins. The
activity of
-galactosidase obtained from the liquid assay
(third column) is the average of the activities of three
independent transformants ± S.D. For the filter assay of
-galactosidase activity, the color intensities of two representative
independent transformants for each strain are shown (last
column).
View larger version (52K):
[in a new window]
Fig. 3.
The Pex8p-Pex20p interaction is direct and
autonomous. Genes encoding MBP, MBP-Pex8p, GST, and GST-Pex20p
were expressed in E. coli, and the proteins were purified.
GST or GST-Pex20p was bound to glutathione-Sepharose 4B and then
incubated with MBP or MBP-Pex8p as indicated at the top.
Bound (left panel) and unbound (right panel)
proteins were separated by SDS-PAGE on a 9% polyacrylamide gel and
stained with Coomassie Brilliant Blue R-250. 25% of unbound fractions
and 50% of bound fractions were loaded. The migrations of molecular
mass standards are marked between the
panels. The standards are 175, 83, 62, 47.5, 32.5, and 25 kDa from top to bottom.
View larger version (62K):
[in a new window]
Fig. 4.
Pex8p-HAc interacts specifically
with GST-Pex20p. Equal amounts of GST or GST-Pex20p were
immobilized on glutathione-Sepharose 4B and incubated with a total cell
lysate of strain PEX8-HAc or with buffer as
indicated at the top. Columns were washed, and bound
proteins were eluted and separated by SDS-PAGE along with a fraction of
the total cell lysate. Proteins were transferred to nitrocellulose and
analyzed by immunoblotting with various antisera as indicated at the
right.
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Fig. 5.
Coimmunoprecipitation of
Pex8p-HAc with Pex20p. Pex20p was subjected to
immunoprecipitation with antiserum to Pex20p or with preimmune serum
under nondenaturing conditions from total lysates of YPBO-grown cells
of the PEX8-HAc strain. 10 µg of cell lysate
proteins and proteins in 20% of each immunoprecipitate (IP)
were separated by SDS-PAGE and subjected to immunoblotting with various
antisera (designated at right). The bottom
panel shows that both immunoprecipitation reactions
contained the same number of IgG heavy chain molecules.
View larger version (66K):
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Fig. 6.
Subcellular localization of
Pex8p-HAc in wild-type and pex20 mutant
strains. Immunoblot analysis is shown of subcellular
fractions of the wild-type strain E122, the PEX20
disruption strain pex20KO, and the PEX20 mutant
strain pex20-1, each transformed with the plasmid
pPEX8-HAc encoding Pex8p-HAc. The postnuclear
supernatant fraction of each strain was divided by centrifugation at
20,000 × g into a supernatant fraction
(20KgS) enriched for cytosol and high-speed pelletable
organelles and a pellet fraction (20KgP) enriched for
peroxisomes and mitochondria. The 20KgS fraction of each strain was
divided by centrifugation at 200,000 × g into
supernatant fraction (200KgS) highly enriched for cytosol
and pellet fraction (200KgP) enriched for high-speed
pelletable organelles, including high-speed pelletable
peroxisomes.
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Fig. 7.
Pex20p is peroxisomal in the PEX8
disruption strain, pex8-KA. The PEX8
disruption strain pex8-KA (top panels) and the
wild-type strain E122 (bottom panels) were grown
overnight in YEPD medium and transferred to YPBO medium and grown for
an additional 9 h. Cells of each strain were processed for
immunofluorescence microscopy. Cells were double-labeled with guinea
pig anti-Pex2p antibodies (left panels) and rabbit
anti-Pex20p antibodies (middle panels) or labeled with
guinea pig anti-thiolase antibodies (right panels). Guinea
pig primary antibodies were detected with rhodamine-conjugated donkey
anti-guinea pig IgG secondary antibodies. Rabbit primary antibodies
were detected with fluorescein-conjugated donkey anti-rabbit IgG
secondary antibodies.
View larger version (52K):
[in a new window]
Fig. 8.
Peroxisomal Pex20p from the PEX8
disruption strain is membrane-associated and protected from the
action of externally added proteases. A, immunoblot
analysis of subcellular fractions of cells of the wild-type strain
E122 and of the PEX8 disruption strain
pex8-KA. Proteins in equal portions of each subcellular
fraction were separated by SDS-PAGE and subjected to immunoblotting
with antibodies to Pex20p and thiolase. B, organelles in the
20KgP fraction were disrupted by incubation with dilute Tris, and
membranes and soluble proteins were separated by differential
centrifugation. Proteins in equal portions of the 20KgP-D, the membrane
pellet (Ti8P), and supernatant (Ti8S) containing
soluble proteins were processed for immunoblotting as described for
A. C, protease protection analysis. Organelles in
the 20KgP fraction from cells of the pex8-KA strain were
incubated with 0, 2, 4, 8, 16, or 32 µg of trypsin in the absence
( ) or presence (+) of 0.1% (v/v) Triton X-100 for 20 min on ice.
Samples were subjected to immunoblotting with antibodies to thiolase
and Pex20p.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by grant MT-9208 from the Medical Research Council of Canada (MRC) (to R. A. R.).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.
Recipient of an MRC studentship.
§ An MRC Senior Scientist and an International Research Scholar of the Howard Hughes Medical Institute. To whom all correspondence should be addressed: Dept. of Cell Biology, 5-14 Medical Sciences Bldg., University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-9868; Fax: 780-492-9278; E-mail: rick.rachubinski@ualberta.ca.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M005072200
2 J. J. Smith and R. A. Rachubinski, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PTS, peroxisomal targeting signal(s); 20KgP, 20,000 × g pellet enriched for peroxisomes and mitochondria; 20KgS, 20,000 × g supernatant enriched for cytosol and high-speed pelletable organelles; 200KgP, 200,000 × g pellet enriched for high-speed pelletable organelles; 200KgS, 200,000 × g supernatant highly enriched for cytosol; 20KgP-D, disrupted 20KgP; AD, transcription-activating domain of GAL4; DB, DNA-binding domain of GAL4; GST, glutathione S-transferase; HA, hemagglutinin; MBP, maltose-binding protein; ORF, open reading frame; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid.
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