* Division of Cellular and Molecular Medicine and Department of Biology, University of California at San Diego, La Jolla,
California 92093-0322
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Abstract |
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Abstract. Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris,
we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing
a peroxisomal targeting signal type II (PTS2). Like its
Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor. The pex7 mutant was not impaired for growth on methanol,
indicating that there are no PTS2-containing enzymes
involved in peroxisomal methanol metabolism. In contrast, pex7
cells failed to grow on oleate, but growth
on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1.
Because the subcellular location and mechanism of action of this protein are controversial, we used various
methods to demonstrate that Pex7p is both cytosolic
and intraperoxisomal. This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing
proteins from the cytosol to the peroxisomes. In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata.
The corresponding PpPex7p mutant proteins were stably expressed in P. pastoris, but they failed to complement the pex7
mutant and were impaired in binding
to the PTS2 sequence.
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Introduction |
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THE sorting of proteins to distinct subcellular compartments is achieved by the coordinated action of
organelle-specific targeting signals and receptors.
Studies on protein targeting across biological membranes
have uncovered two general paradigms for the action of
targeting signal receptors. In certain cases, such as the
SecA/B-dependent secretion of proteins across the bacterial inner membrane and the signal recognition particle
(SRP)1-dependent insertion of proteins across ER membranes or transport across the nuclear pore, the targeting
signal is recognized by a mobile receptor that typically recognizes the signal in the cytosol and then shuttles the
cargo to the target organelle (for review see Schatz and
Dobberstein, 1996; Görlich and Mattaj, 1996
). These mobile receptors cycle either between the cytosol and the organelle membrane, as seen for SecA/B and SRP, or they cycle into and out of the organelle, as observed for importin and transportin. In other instances, such as mitochondrial (Schatz and Dobberstein, 1996
) and chloroplast import (Schnell, 1995
), the receptors are located on the
target membrane (membrane-associated receptors), where
they bind the targeting signal but their mobility is believed
to be limited to the plane of the membrane.
Peroxisomal matrix proteins are synthesized on free
polyribosomes and are posttranslationally imported into
the peroxisome (for review see Lazarow and Fujiki, 1985).
Sorting to peroxisomes requires the presence of a peroxisomal targeting signal (PTS). Most peroxisomal matrix
proteins contain a PTS1 sequence that consists of the
COOH-terminal tripeptide SKL or other variants (Gould
et al., 1987
, 1989
).
A second peroxisomal targeting signal (PTS2) was first
identified in the NH2 terminus of rat peroxisomal thiolase
(Osumi et al., 1991; Swinkels et al., 1991
) and has been
identified in several other proteins since (for review see
Elgersma and Tabak, 1996
). Alignments and site-directed
mutagenesis of these proteins suggest a consensus PTS2
sequence of R/K-L/V/I-X5-H/Q-L/A (Gietl et al., 1994
;
Glover et al., 1994
a; Tsukamoto et al., 1994
).
Additional evidence for at least two pathways for peroxisomal protein import was provided by cloning of the
PTS1 and the PTS2 receptors. The PTS1 receptor, Pex5p,
has seven tetratricopeptide repeats (TPRs) in the COOH-terminal half of the protein, but surprisingly, it lacks a hydrophobic domain that could anchor the protein in the
peroxisomal membrane (McCollum et al., 1993; Van der
Leij et al., 1993
; Dodt et al., 1995
; Fransen et al., 1995
; Nuttley et al., 1995
; Szilard et al., 1995
; Van der Klei et al., 1995
; Wiemer et al., 1995
). There has been much debate about
the localization of Pex5p, but current evidence suggests
that in most organisms, at least a fraction of this protein is
cytosolic (Dodt et al., 1995
; Van der Klei et al., 1995
; Wiemer et al., 1995
; Elgersma et al., 1996a
; Gould et al., 1996
),
while the rest is peroxisome associated. This led to the
proposal that Pex5p functions as a mobile receptor, shuttling between the cytosol and the peroxisome. This model
is further supported by the characterization of the SH3 domain-containing peroxisomal membrane protein, Pex13p,
and another protein, Pex14p, which serve as docking proteins for Pex5p (Elgersma et al., 1996a
; Erdmann and Blobel, 1996
; Gould et al., 1996
; Albertini et al., 1997
). Furthermore, kinetic evidence for Pex5p as a mobile receptor
has been uncovered recently (Dodt and Gould, 1996
).
The Saccharomyces cerevisiae (Sc) pex7 mutant is characterized by the mislocalization of thiolase, whereas import of other proteins analyzed is unaffected. A phenotype
analogous to that of the pex7 mutant has also been described for a human patient cell line (Motley et al., 1994;
Slawecki et al., 1995
). Cloning of the yeast, and more recently the human, PEX7 genes led to the identification of
the PTS2 receptor (Pex7p; Marzioch et al., 1994
; Zhang
and Lazarow, 1995
; Braverman et al., 1997
; Motley et al., 1997
; Purdue et al., 1997
). Pex7p belongs to the WD-40 repeat (
-transducin) family, which is characterized by the
presence of a 43-amino acid repeat domain. Like TPRs,
the WD repeats are probably involved in multiple protein-protein interactions (Komachi et al., 1994
). Interestingly, a functional relationship between proteins belonging
to the TPR and the
-transducin (WD-40) families has
been described (for review see Goebl and Yanagida, 1991
;
Van der Voorn and Ploegh, 1992
). Although it is not yet
clear whether Pex5p and Pex7p interact directly, evidence
exists that they share a common import machinery. In humans, it has been shown that a certain cell line that is defective in the PEX5 gene is not only deficient in the import
of PTS1-containing proteins, but is also deficient in the import of PTS2-containing proteins (Dodt et al., 1995
; Wiemer et al., 1995
). Moreover, a peroxin (Pex) has been identified (ScPex14p) that is required for both import
pathways and which interacts with both Pex5p and Pex7p
(Albertini et al., 1997
).
The location of Pex7p is not clear. An HA-tagged Pex7p
(Pex7p-HA3) was found to be entirely intraperoxisomal in
S. cerevisiae, whereas a Myc-tagged (overexpressed)
ScPex7p (Myc-Pex7p) was predominantly cytosolic (Marzioch et al., 1994; Zhang and Lazarow, 1995
). Human Myc-Pex7p expressed from the strong cytomegalovirus (CMV)
promoter was found predominantly in the cytosol, and no
peroxisome-associated Pex7p was detected (Braverman et
al., 1997
). Because antibodies suitable for studying endogenous Pex7p are lacking, it is not clear which of these
tagged or overexpressed proteins reflects its real location.
The model for the action of Pex7p as a receptor has remained elusive because of uncertainties regarding the precise subcellular location of this protein. The cytosolic localization of epitope-tagged yeast or human Pex7p has
been cited as evidence for a mobile receptor, shuttling between the cytosol and the peroxisome (Marzioch et al.,
1994; Rehling et al., 1996
; Braverman et al., 1997
). In contrast, the presence of an intraperoxisomal receptor that
acts from within the organelle to pull proteins in would be
novel in comparison with the existing paradigms for targeting signal receptors described earlier (Zhang and Lazarow, 1995
, 1996
).
The finding that the PTS2 import pathway may not be
induced in methanol-grown Hansenula polymorpha (Faber et al., 1994) suggests that this pathway may not be required for growth on methanol. This may explain why despite elaborate screening procedures, mutants deficient in
the PTS2 import pathway have not yet been described in
methylotrophic yeasts, since the primary screening was
performed by selecting mutants that were unable to grow
on methanol (Cregg et al., 1990
; Gould et al., 1992
; Liu et al.,
1992
). To obtain more insight into the mechanism of import of PTS2-containing proteins in Pichia pastoris, we
have used a new screening procedure that is more selective for the PTS2 import pathway. We have isolated the P. pastoris (Pp) PEX7 gene, studied the function and localization of endogenous PpPex7p, and examined the use of
the PTS2-dependent import pathway upon growth of P. pastoris on different carbon sources. Furthermore, because recent studies have identified several mutations in
human Pex7p that are responsible for the disease rhizomelic chondrodysplasia punctata (RCDP; Braverman
et al., 1997
; Motley et al., 1997
; Purdue et al., 1997
), we
used the corresponding mutations in PpPex7p to provide
insights regarding why the mutant proteins were nonfunctional.
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Materials and Methods |
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Strains and Media
P. pastoris strains used in this study were as follows: PPY12 (arg4, his4; parental wild-type strain); pex7.1 (his4, pex7, ARG4::pTW84 (PTS2-GFP
(S65T)/GAPDH; PTS2-BLE/GAPDH)); pex7 (his4, pex7::PpARG4);
and fox3
(his4, fox3::PpARG4). S. cerevisiae strains used in this study
were as follows: BJ1991 (MAT
, leu2, trp1, ura3-251, prb1-1122, pep4-3)
(wild-type strain); pex7 (pex7, ura3-251, leu2, his3); and L40 (MATa,
his3
200, trp1-901, leu2-3,112, ade2, LYS2::(lexAop)4-HIS3, URA3::(lexAop)8-lacZ (for two-hybrid assays). Escherichia coli strains used in this
study were as follows: DH5
(recA, hsdR, supE, endA, gyrA96, thi-1,
relA1, lacZ; for cloning procedures), and SG13009 (nals, strs, rifs, lac
,
ara
, gal
, mtl
, F
, recA
; for protein expression).
Minimal methanol contained the following: 0.25% (NH4)2SO4, 0.02%
MgSO4, 0.39% NaH2PO4, 0.05% yeast extract, 0.1% (vol/vol) Vishniac
minerals mix, 0.1% vitamins, 20 mg/ml amino acids as needed, and 0.5%
methanol as carbon source. Minimal oleate medium contained 0.2% oleate and 0.02% Tween 40 as carbon source. Minimal oleate plates were
made according to Elgersma et al. (1993). Other media used in this study
were described by Wiemer et al. (1996)
.
Isolation of pex Mutants
A culture of P. pastoris PPY12+pTW84 (PTS2-GFP(S65T)/GAPDH,
PTS2-BLE/GAPDH)) cells was grown for 15 h on rich glucose medium (YPD), diluted 12-fold in 600 ml YPD, and grown for an additional 8 h.
Cells were pelleted and washed twice with water and once with 100 mM
sodium citrate, pH 5.5. The cells were then resuspended in 150 ml 100 mM
Na-citrate, pH 5.5, and 25-ml batches were treated with increasing concentrations (0-2%) N-methyl-N-nitro-nitrosoguanidine (NTG) for 30 min.
Mutagenized cells were washed three times with 100 mM sodium citrate,
pH 5.5, resuspended in 20 ml YPD, and recovered for 2 h. Small aliquots
were used to determine the survival rate, and the remainder was frozen in
glycerol at 80°C. Cells with an NTG survival of 4% were used to inoculate 50 ml YPD and were grown for 24 h. These cells were pelleted by centrifugation at 2,500 g, washed with water, and transferred to 300 ml rich
oleate medium (YPO) and induced for 15 h. These cells were collected, resuspended in 250 ml YPO, aliquoted in five portions of 50 ml, and induced in oleate medium for an additional 2 h. Increasing amounts of phleomycin were added to these cultures (to concentrations of 0-100 µg/ml), and
the cells were incubated for 3 h at 30°C. The phleomycin-treated cells were
washed twice with water and resuspended in YPD to recover for 2 h at
30°C. Cells were diluted and plated on YPD to determine the survival rate.
Colonies obtained from the batch showing a 5% survival rate were tested for
growth on minimal oleate and methanol medium, and they were analyzed for
PTS2-green fluorescent protein (GFP) import by fluorescence microscopy.
Cloning of the PpPEX7 and PpPAT1 Genes
A genomic library was transformed into pex7.1 cells. Three different plasmids (pM1, pM2, and pM3), which restored growth on oleate, were isolated. Subclone analysis revealed that pM1 contained ~1 kb of the 5 end
of a gene (PpPAT1) that is able to suppress the pex7.1 phenotype. pM2
and pM3 had an overlapping fragment of 2.8 kb. This 2.8-kb insert contained the PpPEX7 gene, which was sequenced on both strands. The
PpPEX7 sequence data have been submitted to the GenBank database
under accession number AF021797.
Construction of Plasmids Expressing PpPex7p
A 2.6-kb genomic fragment containing the PEX7 gene was cloned in a three-fragment ligation, as a SalI-EcoRI and EcoRI-BamHI fragment in a SalI/BamHI-digested pUC18, resulting in pM19. The PEX7 gene knockout was made by cloning the PpARG4 gene as a blunt fragment in the blunted NsiI sites (86 bp upstream of the ATG and 28 bp downstream of the stop codon of PEX7) of pM19. The polylinker of pUC18 was modified by ligating adaptors P43 and P44 (see Table I) in the EcoRI and BamHI sites so that these sites were destroyed and the BglII, KpnI, EcoRV, NsiI, and XbaI sites were introduced (pM24). The PEX7 gene was amplified by PCR using primers P33 and P34, thereby introducing a KpnI site immediately upstream of the ATG, and was cloned as a KpnI-NsiI fragment in pM24. To eliminate PCR errors, the BamHI-Nsi fragment was replaced by the BamHI-NsiI fragment of the genomic clone, and the remainder of the PEX7 gene was sequenced. The PEX7 gene on this plasmid (pM27) was used for further manipulations.
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For NH tagging, a linker encoding the NH tag (primers P26/P28) was ligated in the EcoRI site of pUC18, resulting in pEL210. PEX7 was cloned as a KpnI-NsiI fragment from pM27 in the KpnI/PstI sites of pEL210, resulting in pM22. The NH-tagged PEX7 was cloned downstream of the ACO1 promoter in the BglII/EcoRI-digested pTW72 (a pHILD2-based plasmid in which the alcohol oxidase promoter was replaced by the ACO1 promoter), using the BglII/HindIII fragment from pM22 and a EcoRI/ HindIII adaptor (P49/P50), resulting in plasmid pM39. The nontagged gene was cloned downstream of the ACO1 promoter by replacing the BglII/AvrII fragment from pM39 (encoding NH-PEX7) by the BglII/XbaI fragment from pM27 (encoding PEX7), resulting in pM70. The ACO1 promoter was replaced by the endogenous PEX7 promoter by replacing the NdeI/BamHI fragment from pM39, containing the ACO1 promoter, with the genomic XhoI/BamHI fragment and the NdeI/XhoI adapter P122/P123, resulting in pM78.
To introduce the C347ter mutation, we ligated linker P124/P125 in an
EcoRI/SalI-digested pM27, resulting in pM75, and sequenced the inserted
linker. To introduce the G249V and A248R point mutations in PEX7, we
designed primers that introduced the desired mutation and a unique restriction site at the location of the mutation. The 5 part of the gene was
amplified by PCR with primers P32 (internal primer just upstream of the
BamHI site) and P128 (G249V) or P126 (A248R). The 3
part of the gene
was amplified by PCR with primers P35 (a primer just downstream of
PEX7) and P129 (G249V) or P127 (A248R). The PCR fragments corresponding to the 5
segments of PEX7 were digested with BamHI and PstI
(G249V) or BamHI and XbaI (A248R), and the PCR fragments corresponding to the 3
segments of PEX7 were digested with PstI and EcoRI
(G249V) or XbaI and EcoRI (A248R). These fragments were ligated in a
three-fragment ligation into the BamHI/EcoRI-digested pM27, resulting
in pM76 (G249V) and pM77 (A248R). The BamHI/EcoRI inserts were
sequenced. The mutated PEX7 genes were cloned as BamHI/SalI fragments, downstream of the ACO1 promoter in a BamHI/XhoI-digested
pM70, resulting in pM85 (C347ter), pM86 (G249V), and pM87 (A248R).
The mutated PEX7 genes were cloned as BamHI/SalI fragments downstream of the PEX7 promoter in a BamHI/XhoI-digested pM78, resulting
in pM82 (C347ter), pM83 (A248R), and pM84 (G249V).
PpFox3-SKL was amplified by PCR using primers based on the sequence of the PpFOX3 gene so that a BamHI site was introduced directly
upstream of the ATG, and an SKL sequence and an XbaI site were added
at the 3 end of the gene. The PCR product was digested with BamHI and
XbaI and was cloned downstream of the ACO1 promoter between the
BglII/SpeI sites of pM39.
For the yeast two-hybrid analysis, plasmids were generated that expressed appropriate protein fusions to the DNA binding (DB) domain of
LexA, and the activation domain (AD) of VP16. The LexA(DB)-ScFox3p (DB-ScFox3p) fusion protein was made by cloning ScFOX3 as a BamHI/ XbaI fragment in pBTM116B, resulting in pM35. pBTM116 (Bartel et al.,
1993) was modified by introducing a polylinker in all three open reading
frames, resulting in pBTM116A, B, and C. The DB-ScPTS2 fusion protein
was made by digesting pM35 with NcoI/SalI, followed by a filling-in reaction using Klenow polymerase and religation, thereby deleting ScFOX3
downstream of the PTS2 (pM52). The DB-ScFox3p
PTS2 fusion protein
was made by digesting pM35 with NotI/NcoI, followed by a filling-in reaction using Klenow polymerase and religation, thereby deleting the PTS2
signal of ScFOX3 (pM51). The AD-PpPex7p fusion protein was made by
cloning PpPEX7 as a BglII/SalI fragment from pM27 in the BamHI/SalI-digested pVP16C, resulting in pM34. pVP16 (Hollenberg et al., 1995
) was modified by introducing a polylinker in all three open reading frames, resulting in pVP16A, B, and C. The RCDP mutations were introduced in
the two-hybrid vector as BamHI/SalI fragments into a BamHI/SalI-digested
pM34, resulting in pM79 (Pex7p(G249V)), pM80 (Pex7p(A248R)), and
pM81 (Pex7p(C347ter)).
Raising Antibodies against Pex7p
To raise antibodies against Pex7p, we cloned the DNA encoding amino acids 1-143 of Pex7p as a KpnI/HpaI fragment from pM27 into pQE30 (QIAGEN Inc., Chatsworth, CA), which was first digested with HindIII, blunted with Klenow polymerase, and subsequently digested with KpnI. The (His)6-tagged protein was expressed in E. coli SG13009 and purified under denaturing conditions on Ni2+-NTA beads according to the manufacturer's manual (QIAGEN Inc.). The protein was further purified by SDS-PAGE, visualized with 0.25 M KCl, 1 mM DTT, and subsequently excised and eluted from the gel in elution buffer (50 mM Tris, pH 8.0, 0.1% SDS, 0.1 mM EDTA, 5 mM DTT, 0.15 M NaCl). This purified protein was used to immunize rabbits.
The antibody was purified using a pex7 depletion column and, subsequently, a Pex7p affinity column. The depletion column was made by coupling overnight ~20 mg of proteins from a cell-free pex7
lysate to 0.6 g of
cyanogen bromide-activated Sepharose 4B (Sigma Immunochemicals, St.
Louis, MO) in coupling buffer (0.1 M NaHCO3, 0.5 M NaCl). After blocking the column for 2 h with blocking buffer (1 M ethanolamine, 0.1 M
NaHCO3, 0.5 M NaCl, pH 8.0), the column was washed extensively, alternating between coupling buffer and low pH wash buffer (20 mM sodium
acetate, 0.5% [vol/vol] acetic acid, 0.5 M NaCl, pH 3.5) and subsequently
with PBS, followed by 0.2 M glycine, 1 mM EGTA, pH 2.4, followed by
PBS. After overnight incubation with 10 ml of serum, the serum was
drained off and collected together with the first 3 ml of PBS wash. This
purified serum was used for further purification on an affinity column. This was done essentially in the same manner as the depletion column, except that 2 mg of purified Pex7p was bound to the column, and after binding the serum to the column, it was washed extensively with PBS, low pH
wash buffer (0.3% [vol/vol] acetic acid, 50 mM sodium acetate, 1 M NaCl,
pH 4.5), high pH wash (0.1 M NaHCO3, 1 M NaCl, pH 9.0), and again
with PBS. The antibodies were eluted with 15 ml of 0.2 M glycine, 1 mM
EGTA, pH 2.4, and immediately neutralized to pH 7.0 with 1 M Tris base.
Finally, the antibodies were concentrated to ~1 ml using a Centriprep 30 (Amicon, Beverly, MA). The antibody was used at a 1:10,000 dilution for
Western blotting.
Subcellular Fractionation and Nycodenz Gradients
Subcellular fractionations of oleate-grown cells were essentially performed as described by Van der Leij et al. (1992), in the presence of protease inhibitors (1 mM PMSF, 0.2 mg/ml NaF, and 2 µg/ml of chymostatin,
leupeptin, antipain, and pepstatin). 6 ml of a 1,000 g postnuclear supernatant was layered on a continuous 16-35% Nycodenz gradient (30 ml), with
a 4-ml cushion of 42% Nycodenz dissolved in 5 mM MES, pH 6.0, 1 mM
EDTA, 1 mM KCl, and 8.5% sucrose. The sealed tubes were centrifuged
for 2.5 h in a vertical rotor at 29,000 g at 4°C. Afterwards, fractions were
collected and analyzed by SDS-PAGE and Western blotting.
Microscopy Analysis
Fluorescence microscopy for GFP analysis was done as described by
Monosov et al. (1996). Fluorescence microscopy of semithin sections and
immuno-EM analysis of ultrathin sections was done as follows: P. pastoris
cells were grown to log phase and fixed in either periodate-lysine-paraformaldehyde (75 mM phosphate buffer containing 2% formaldehyde, 70 mM lysine, and 10 mM sodium periodate, pH 6.2) for 6 h, or in
4% paraformaldehyde contained in 100 mM phosphate buffer, pH 7.4, for
18-24 h at 4°C. The cells were washed, pelleted, and embedded in 1% ultralow temperature gelling agarose, and were trimmed into 1-mm3 blocks.
The cells were cryoprotected in 2.3 M sucrose containing 20% polyvinyl
pyrollidone for 1 h, mounted on aluminum cryopins, and frozen in liquid
nitrogen. Ultrathin cryosections were then cut on a Reichert Ultracut E
microtome (Leica Inc., Heerbrugg, Switzerland) equipped with an FC4
cryostage, and sections were collected onto 300 mesh, Formvar/carbon-coated nickel grids and immunolabeled as follows. Grids were washed
with several drops of PBS containing 5% FCS and 0.01 M glycine, pH 7.4. The grids were blocked in 10% FCS and incubated for 16 h with
NH antibodies in PBS containing ~10 µg/ml specific antibody. After washing the grids in PBS, they were incubated for 2 h with a 50× diluted goat anti-rabbit 5-nm gold conjugate (Amersham Life Sciences, Arlington Heights,
IL). The grids were washed several times with PBS, subsequently with water, and then the grids were embedded in a mixture containing 2.7% polyvinyl alcohol (mol wt = 10,000), 0.2% methyl cellulose (400 cP), and 0.2%
uranyl acetate. The sections were observed and photographed on a
1200EX II TEM microscope (Jeol Ltd., Tokyo, Japan).
Miscellaneous
Digitonin titrations were essentially performed as described by Zhang and
Lazarow (1995), with modifications as described in Elgersma et al. (1996a)
.
For TCA lysates, cells (50 ml of culture OD600 = 0.7) were pelleted by centrifugation, washed, and collected in a 2-ml Eppendorf tube. After the addition of 500 µl of 10% TCA and ~200 µl of glass beads, the cells were broken by vigorous vortexing for 30 min at 4°C. Cell debris was pelleted by centrifugation for 30 s at 6,000 rpm, and the supernatant was further centrifuged for 20 min at 12,000 rpm at 4°C in a microcentrifuge. The pellet was washed once with acetone, dried, and resuspended in Laemmli sample buffer for SDS-PAGE.
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Results |
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Isolation of PTS2 Import-deficient Mutants
The negative screening procedure for isolating P. pastoris
pex mutants that has been used thus far (Gould et al.,
1992; Liu et al., 1992
) has not resulted in the isolation of
mutants that are specifically disturbed in the PTS2 import
pathway. Therefore, we developed a positive selection
procedure that would enrich for such mutants. In S. cerevisiae, such a scheme has been developed, which is based on
the ability of peroxisomes to import the bleomycin resistance protein (BLE) fused to the PTS1 (Elgersma et al.,
1993
). In wild-type cells, BLE-PTS1 is imported into peroxisomes, thereby sequestering the BLE protein from its
toxic ligand, phleomycin. Peroxisomal import mutants
(pex) are unable to import BLE-PTS1 into their peroxisomes, which allows the protein to bind phleomycin, thereby preventing its toxicity. We modified this procedure by fusing the BLE protein to the PTS2. Since a PTS2
sequence of an endogenous P. pastoris protein had not
been identified, we tested the PTS2 sequence of S. cerevisiae thiolase (Fox3p) in P. pastoris. We therefore made a
fusion of the first 17 amino acids of thiolase (containing
the PTS2 signal) to GFP (PTS2-GFP). When expressed in
oleate-grown P. pastoris, we observed a punctate GFP expression pattern, suggesting that PTS2-GFP was imported
into the peroxisomes and that the ScPTS2 is functional in
P. pastoris (Fig. 1 E). We also observed some cytosolic labeling, however, suggesting that PTS2-GFP import was
not as efficient as GFP-SKL import (Fig. 1, E versus C).
|
A yeast strain (PPY12 + pTW84) that expressed PTS2-GFP was made, and the BLE fused to the PTS2 sequence
(PTS2-BLE) under the control of the glyceraldehyde
3-phosphate dehydrogenase promoter (GAPDH) from a
plasmid integrated in the genome. These cells were mutagenized, induced on oleate, and treated with phleomycin. We expected that PTS2 import mutants would be able
to grow on methanol because none of the known enzymes
involved in methanol metabolism contain a PTS2 import
signal. Conversely, because thiolase is required for growth
on oleate, we expected that a PTS2 import-deficient mutant would be unable to grow on oleate. Therefore, colonies that had survived the phleomycin selection were
screened for their ability to grow on methanol but not on
oleate. Two mutants (fox3.1 and pex7.1) clearly fulfilled
this criterion (shown for pex7 in a growth curve in Fig. 2,
A and B). Inspection of PTS2-GFP import revealed, however, that one of these mutants (fox3.1) was unaffected in
PTS2 protein import. Cloning of the complementing gene
revealed that this mutant was deficient in the thiolase gene itself (Koller, A., and S. Subramani, unpublished results).
|
pex7.1 Cells Are Deficient in PTS2 Import Only
The second mutant (pex7.1) was not only specifically disturbed in its growth on oleate (Fig. 2, A and B), but was also unable to import PTS2-GFP (Fig. 1 F), whereas import of GFP-SKL was normal (Fig. 1 D). This suggested a general deficiency of pex7.1 cells in the import of PTS2-containing proteins. The pex7.1 mutant was therefore further characterized by biochemical subcellular fractionation. In wild-type cells, we observed an equal distribution of thiolase between the organellar pellet fraction and the cytosolic supernatant fraction. As expected, thiolase was exclusively present (27,000 g) in the cytosolic supernatant fraction of pex7.1, providing additional evidence that the import of PTS2-containing proteins was disturbed (Fig. 2 C). Targeting of the membrane protein Pex3p was normal, as was the import of acyl CoA oxidase (Aco1p), because these proteins were predominantly present (27,000 g) in the organellar pellet fraction. Furthermore, the import of presumed PTS1-containing proteins such as catalase (Cta1p) and trifunctional enzyme (Tfe1p) was unaffected, suggesting that the import deficiency of pex7.1 is specific for PTS2-containing proteins only.
Cloning of pex7.1 Complementing Genes
The pex7.1 mutant was complemented for growth on oleate medium using a genomic library. Two plasmids (pM1
and pM2) with nonoverlapping inserts restored growth on
oleate, although growth was better when using plasmid
pM2 (Fig. 2 D). Further analysis of plasmid pM1 revealed
that complementation of the oleate-minus phenotype was
caused by a truncated protein (amino acids 1-342) with
highest homology to ScPat1p, a peroxisomal ABC transporter (Hettema et al., 1996). Interestingly, it has been reported that the peroxisomal ABC transporter Pmp70 can
suppress the peroxisomal protein import deficiency of a
mammalian pex2-deficient cell line (Gartner et al., 1994
).
Therefore, we tested whether plasmid pM1 was able to restore the PTS2-GFP import in the pex7.1 mutant. Microscopy analysis did not reveal any restoration of import of
PTS2-GFP in these cells, suggesting that this protein did
not act as a suppressor of the PTS2 import deficiency (data
not shown). It has been recently demonstrated that
ScPat1p and ScPat2p are involved in the transport of (activated) long-chain fatty acids across the peroxisomal membrane (Hettema et al., 1996
). We therefore speculate that
the truncated PpPat1p somehow disturbed the normal functioning of the Pat1p/Pat2p complex, thereby allowing
leakage of the partially completed
oxidation product (3-oxoacyl-CoA) into the cytosol, allowing cytosolic thiolase
to complete the
oxidation in this pex7.1 mutant.
The second clone (pM2) was not only able to restore growth on oleate of the pex7.1 mutant, but it also corrected the import deficiency of PTS2-GFP (data not shown). Further analysis revealed that the complementing gene encoded a protein of 376 amino acids with a calculated molecular mass of 42.4 kD. The protein contains six WD-40 repeats and has 43% sequence identity with ScPex7p (Fig. 3). Evidence that we cloned a true orthologue of ScPex7p was obtained by expressing the gene in S. cerevisiae. It was able to complement the growth defect of S. cerevisiae pex7 (Fig. 4 A); hence, the gene is designated PpPEX7.
|
|
Pex7p Interacts with the PTS2
Pex7p from S. cerevisiae has been shown to interact with
the PTS2 sequence in multiple ways (Rehling et al., 1996;
Zhang and Lazarow, 1996
). Because PpPex7p complements the Scpex7 mutant, we expected PpPex7p to interact with the PTS2 sequence. This was tested using the
yeast two-hybrid system (Fields and Song, 1989
). Therefore, we fused Pex7p to the LexA activation domain (AD-PpPex7p) and Sc-thiolase to the LexA DNA-binding domain (DB-ScFox3p). These plasmids were coexpressed in
S. cerevisiae L40 cells with either an empty control plasmid
or with each other. High induction of the HIS3 gene and
-galactosidase was obtained when AD-PpPex7p and DB-ScFox3p were coexpressed, whereas no induction was observed when AD-PpPex7p or DB-ScFox3p was coexpressed
with a control plasmid (shown for HIS3 expression in Fig.
4 B). This indicates that indeed Pex7p interacts with Fox3p
in vivo. To test whether this interaction was dependent upon
the presence of the PTS2, we made a construct from which
the 17 NH2-terminal amino acids of thiolase were removed (DB-ScFox3p
PTS2). No induction of the HIS3 gene and
-galactosidase was observed when DB-ScFox3p
PTS2 was
coexpressed with AD-PpPex7p, indicating that the interaction of PpPex7p with thiolase was PTS2 dependent. To
test whether the PTS2 alone would be sufficient to bind
to PpPex7p, we fused the first 17 amino acids of ScFox3p to
the LexA DNA-binding domain (DB-ScPTS2). When
DB-ScPTS2 was coexpressed with AD-PpPex7p, we found
again a strong induction of the HIS3 gene and
-galactosidase (shown for HIS3 expression in Fig. 4 B). These results
indicate that the ScPTS2 sequence alone is sufficient for interaction with PpPex7p in vivo, supporting a receptor-like
function for this protein.
Fox3p-SKL Is Efficiently Imported into Peroxisomes of
pex7 Cells
To study the function of Pex7p, we made a PEX7 deletion
mutant (pex7) in which the entire open reading frame
was replaced by the PpARG4 gene. Subcellular fractionation of this mutant showed the same phenotype as was
observed for the pex7.1 mutant: a specific import deficiency of the PTS2-containing enzyme thiolase (shown later in Figs. 5 B and 6, B and D).
|
|
Like the original pex7.1 mutant, pex7 did grow on
methanol medium, suggesting that peroxisomal import of
PTS2-containing enzymes is not necessary for growth on
methanol (Fig. 2 A). Conversely, pex7
did not grow on
oleate, indicating that import of at least one PTS2-containing protein is required for growth on this medium (Fig. 2
B). We investigated whether the growth defect on oleate was caused solely by the mistargeting of thiolase. Therefore, a construct (Fox3p-SKL) expressing P. pastoris
Fox3p (Koller, A., S. Subramani, unpublished results)
fused to the PTS1 sequence (SKL) under the control of
the ACO1 promoter was integrated in the genome of
pex7
and fox3
cells. This protein restored growth on
oleate plates to nearly wild-type levels when expressed in Ppfox3
, indicating that Fox3p-SKL is active and imported into peroxisomes (Fig. 5 A). Expression of Fox3p-SKL in pex7
only partially restored the growth on oleate
plates. This suggests that the inability to import thiolase is
not the only reason why pex7
cells do not grow on oleate,
but that other PTS2-containing proteins are also required
for growth on this medium (Fig. 5 A).
The import efficiency of Fox3p-SKL was studied by subcellular fractionation (Fig. 5 B). We observed a considerable amount of endogenous thiolase in the cytosolic fraction in the control (untransformed wild-type) cells, whereas
thiolase was exclusively cytosolic in untransformed pex7
cells. The control enzyme, Aco1p, was predominantly
present in the organellar pellet fractions (Fig. 5 B). These
results suggest that thiolase easily leaks out of peroxisomes during subcellular fractionation. Alternatively, the PTS2 import pathway in P. pastoris is not very efficient.
This is supported by the observation that PTS2-GFP is
also largely cytosolic and only partially peroxisomal (Fig.
1, E and G), although we cannot rule out that this localization is caused by the chimeric protein itself. Interestingly,
Fox3p-SKL was fully imported into peroxisomes of fox3
cells, with no protein detectable in the cytosolic fraction.
In pex7
cells expressing Fox3p-SKL, thiolase was equally
distributed between the pellet and supernatant fractions (Fig. 5 B). From the small difference in molecular weight
between Fox3p-SKL and endogenous Fox3p, we conclude
that Fox3p-SKL is fully imported into these cells via its
PTS1 sequence, whereas endogenous thiolase is almost exclusively cytosolic, as in the untransformed pex7
control
cells (Fig. 5 B). These results suggest that the PTS2 import
pathway in P. pastoris may be less efficient than the PTS1
import pathway (see Discussion).
Antibodies against Pex7p
The lack of useful antibodies against endogenous Pex7p
has greatly hampered the localization analysis of this protein in S. cerevisiae and in human cells. We therefore
wanted to raise antibodies against PpPex7p to analyze its
localization. We expressed the full-length Pex7p in E. coli
fused to a (His)6 tag, but the expression of this protein was
very poor. We were unable to increase its expression by
fusing it to dihydrofolate reductase, nor could we increase
the expression by fusing three different truncated versions
of Pex7p to dihydrofolate reductase. Good expression in
E. coli was obtained for a truncated protein corresponding to amino acids 1-143 fused to the (His)6 epitope. This purified protein, (His)6-Pex7p(143), was used to immunize
rabbits. The antiserum that was obtained recognized several proteins in the wild-type strain and in the pex7
strains (Fig. 6 A, lanes 1 and 2). Because this could indicate that a (nonfunctional) pseudogene of PEX7 might be
present, we checked this possibility with PCR and low
stringency Southern blotting, but did not find any evidence for the existence of such a gene (data not shown). We purified the antiserum with an immunodepletion column, using a lysate obtained from pex7
cells. The flow-through
of this column was used to purify the antiserum on an affinity column using the coupled(His)6-Pex7p(143) protein.
The eluted antibody recognized two prominent bands in lysates of wild-type cells (Fig. 6, A, lane 4). The signal obtained from the lower molecular weight (cytosolic) protein
was present at the same intensity in pex7
cells (Fig. 6,
lane 3) and could be eliminated by using more stringent
washing conditions (see figures below). The higher molecular weight band was largely reduced in pex7
cells, indicating that this signal is from Pex7p (Fig. 6 A, lane 3 versus
lane 4). This could be confirmed by overexpressing Pex7p
from the acyl-CoA oxidase promoter (Fig. 6, lane 5). A 25-fold dilution of this lysate (compared to a wild-type and
pex7
lysate) gave only one signal at the expected molecular weight (Fig. 6 A, lane 6). Because there is still a weak
signal present at this molecular weight in a pex7
lysate
(from a mitochondrial protein, see below), in our subsequent analyses, we included the pex7
control and separated the peroxisomes from mitochondria where necessary.
PpPex7p Is Distributed between the Peroxisomes and the Cytosol
The intracellular location of Pex7p was determined by
subcellular fractionation. Wild-type and pex7 cells were
fractionated, and the organellar pellet and the cytosolic supernatant were analyzed. Aco1p, Pex3p, and thiolase
(Fox3p) were present in the organellar (27,000 g) pellet
fraction of wild-type cells, although varying portions of
some of these proteins were also present in the cytosolic (27,000 g) supernatant fraction, possibly because of leakage (Fig. 6 B). A similar distribution was observed for
pex7
cells, with the exception of thiolase, which was exclusively present in the cytosolic fraction, reflecting the
PTS2 import deficiency of pex7
(Fig. 6 B). Pex7p was
present in both the organellar pellet (varying from 10 to
30%) and the cytosol fraction of wild-type cells, suggesting that about a quarter of the protein is associated with peroxisomes. In Fig. 6 B, the mitochondrial protein that
sometimes cross-reacts with the anti-Pex7p antibodies (see
Fig. 6, A and D) would not affect our conclusion that
Pex7p is partially found in the cytosol because (a) we did
not observe the cross-reacting band in this particular experiment (see H and P fractions of control pex7
cells),
and (b) the cross-reacting mitochondrial band was never found in the supernatant fraction during subcellular fractionation. The protein band found in the cytosolic fraction
can therefore be only Pex7p.
To determine whether the pelletable Pex7p was indeed
associated with peroxisomes, we used a postnuclear supernatant for further analysis on a Nycodenz gradient. A portion of Pex7p comigrated with the peroxisomal marker enzymes Aco1p and Fox3p, suggesting that at least some
Pex7p is associated with peroxisomes (Fig. 6 C). We also
found significant amounts of Pex7p-cross-reacting material at the top of the gradient (fractions 14-20), representing mitochondrial and cytosolic proteins. In these gradients, the mitochondrial protein (Fig. 6 D, fractions 14-18)
that cross-reacted with the antibody against Pex7p was
never found at the top of the gradient in fractions 19 and
20, where cytosolic proteins would be found. The presence
of Pex7p in fractions 19 and 20 of the gradient from wild-type cells (Fig. 6 D) shows that some Pex7p is indeed cytosolic. Because of the cross-reacting protein present in the
mitochondrial fractions (see fractions 14-18 in the pex7
control gradient, Fig. 6 D), however, it is impossible to estimate the exact amount of Pex7p present in the cytosol in
this experiment.
To study the localization of Pex7p by an independent
biochemical method, we selectively permeabilized spheroplasts of wild-type cells, pex7 and pex3
(a mutant disturbed in the import of PTS1- and PTS2-containing proteins; Wiemer et al., 1996
) by digitonin, and we followed
the subsequent leakage of Pex7p and control proteins from the cells. In this procedure, cytosolic enzymes are released at low concentrations of digitonin, whereas peroxisomal matrix enzymes are released at much higher concentrations of digitonin (Zhang and Lazarow, 1995
). As
can be seen in Fig. 7, the cytosolic enzyme glucose-6 phosphate dehydrogenase (G6PDH) was completely released
at low concentrations (50 µg/ml) of digitonin. This was also observed for catalase in pex3
, as well as thiolase in
the pex7
and pex3
strain, which reflects the peroxisomal
import deficiency of these mutants. In contrast, thiolase
and catalase were released at significantly higher concentrations of digitonin in wild-type cells, starting at 150 µg/
ml digitonin, and release was not complete until 300-400
µg/ml digitonin was added. The membrane protein Pex3p
could be completely released at only very high concentrations of digitonin (>600 µg/ml; Fig. 7). Using the
Pex7p
antibody, we observed release of the mitochondrial
Pex7p-cross-reacting protein in pex7
cells at digitonin
concentrations of
400 µg/ml. In wild-type cells, a substantial amount of Pex7p was already released before release of peroxisomal matrix proteins took place (before
150 µg/ml digitonin). In contrast to the cytosolic markers, however, release was not complete until 300-400 µg/ml
digitonin, which is similar to what was observed for the release of Cta1p and Fox3p. In pex3
cells, Pex7p was completely released at 50 µg/ml. These results confirm that
Pex7p is distributed between the peroxisome and the cytosol. Furthermore, since complete release of Pex7p is observed at digitonin concentrations <400 µg/ml, these results also suggest that some Pex7p is present in the lumen
of peroxisomes.
|
Overexpressed PpPex7p Is Primarily Located in the Cytosol
If Pex7p functions solely as an intraperoxisomal receptor,
it probably has a PTS that directs it efficiently to the peroxisomal matrix. Some experiments in S. cerevisiae suggest
that this is indeed the case (Zhang and Lazarow, 1996). To
test whether PpPex7p can direct a reporter protein to the
peroxisome, we fused PpPex7p to the peroxisomal malate
dehydrogenase of S. cerevisiae, which lacks its PTS1 (NH-Mdh3p
PTS1). It has previously been shown that NH-Mdh3p
PTS1 is cytosolic and that it can not complement
the growth defect on oleate of S. cerevisiae mdh3
cells
(Elgersma et al., 1996b
). When PpPex7p was fused to NH-Mdh3p
PTS1 (NH-Mdh3p-PpPex7p) and expressed in S. cerevisiae mdh3
cells under the control of the ScCTA1
promoter, we observed some complementation of the mdh3
mutant (data not shown). This suggests that PpPex7p does
indeed have a signal that can direct it to the peroxisomal
matrix.
To study the effect of overexpressing and/or epitope-tagging Pex7p, the expression level of Pex7p was increased
by expressing it under the control of the ACO1 promoter.
Western blot analysis revealed that this resulted in an
~25-fold higher expression level (see Fig. 6 A). The overexpressed protein fully complemented the growth deficiency of pex7 cells (Fig. 8 A). Full complementation was
also observed when we epitope tagged Pex7p by fusing it
to the NH tag (Elgersma et al., 1996b
) and expressed it under the control of the ACO1 promoter (NH-Pex7p/ACO1)
(Fig. 8 A). Neither tagging nor overexpression affected its
ability to restore the peroxisomal import of thiolase in
pex7
cells, as shown by subcellular fractionation (Fig. 8
B). Interestingly, we found on average >90% of overexpressed Pex7p in the cytosol (Fig. 8 B). Comparison of the
absolute amount present in the organellar pellet revealed that the amount of pelletable Pex7p was only increased
5-10-fold (Fig. 8 C) compared to untransformed cells.
These experiments suggest that targeting of PpPex7p to
the peroxisomal matrix is not as efficient as the PTS1 or
PTS2 import pathway, and that an unknown component is
limiting the import of Pex7p.
|
The localization of overexpressed NH-Pex7p was also
addressed by fluorescence microscopy of semithin sections
of yeast cells labeled with the NH antibody. This antibody
and epitope tag have previously been shown to be very
suitable for immuno-EM analysis (Elgersma et al., 1996b).
Analysis of control cells expressing NH-tagged malate dehydrogenase of S. cerevisiae (NH-Mdh3p) under the control of the ACO1 promoter showed a punctate labeling, indicating efficient import of NH-Mdh3p into peroxisomes
(Fig. 9, A and B). In contrast, cells expressing NH-Pex7p
showed only limited punctate staining and a high cytosolic
staining (Fig. 9, C and D). This supports the biochemical
data that a large amount of overexpressed Pex7p is cytosolic. To verify the nature of the punctate staining, we used
the cells expressing NH-Mdh3p or NH-Pex7p for immuno-EM analysis. This revealed that a portion of NH-Pex7p is
indeed present in the peroxisomal matrix (Fig. 10, B-D).
These results confirm the predominantly cytosolic and
partially intraperoxisomal location of overexpressed NH-Pex7p.
|
|
Human PEX7 Mutations Affect the Ability of PpPex7p to Bind PTS2
The human PEX7 gene has recently been cloned and
shown to be responsible for RCDP (Braverman et al.,
1997; Motley et al., 1997
; Purdue et al., 1997
). The most
frequently occurring mutations were the L292ter and
A218V mutations. Five patients were genetic compounds
of the L292ter as the first allele and a G217R mutation as
the second allele (Braverman et al., 1997
). Expression
studies showed that the L292ter and A218V mutations impaired Pex7p function, but the functional consequence of
the G217R mutation was undetermined. To test whether
the mutated proteins were nonfunctional because of instability, mistargeting, or failure to bind the PTS2 sequence, we made the PpPEX7 equivalents of the RCDP G217R,
A218V, and L292ter mutations (A248R, G249V, and
C347ter, respectively, as indicated in Fig. 3). The mutated
PpPex7p proteins were expressed under the control of the
PEX7 and ACO1 promoters, and they were tested for the
ability to correct the growth deficiency of pex7
on oleate. As shown in Fig. 11 A, all mutated proteins were unable to
restore the growth defect on oleate. When (over)expressed under the control of the ACO1 promoter, we observed some oleate usage by Pex7p(G249V), as judged by
the formation of a halo on an oleate plate (data not
shown). This correlates with the finding that patients with this corresponding allele have a phenotype milder than in
classical RCDP (Braverman et al., 1997
).
|
We made lysates of pex7 cells expressing the mutated
Pex7p. As shown in Fig. 11 B, all proteins were expressed
stably, suggesting that the deficiency is not caused by
protein instability. We then tested whether the mutated
proteins were able to bind the PTS2 sequence, using the
two-hybrid system as described above. Interestingly, all
mutated proteins had lost the ability to bind the PTS2 sequence (Fig. 11 C). This suggests that the mutations lead
to impaired PTS2 binding.
To test whether the mutations affected the peroxisomal
targeting of Pex7p, we expressed the mutated proteins in
pex7 cells and compared their targeting to that observed
for wild-type Pex7p by Nycodenz gradient analysis. We
did not observe significant differences between the distribution of wild-type or mutated proteins (data not shown).
This indicates that peroxisomal targeting of the mutated
Pex7p proteins is not affected. Moreover, this suggests that binding of the PTS2 sequence is not a prerequisite for
targeting Pex7p to the peroxisome.
![]() |
Discussion |
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PpPex7p Is a Receptor for PTS2 Protein Import
Using a new screening procedure devised to yield mutants
specifically compromised in the PTS2 import pathway, we
have obtained a P. pastoris pex7 mutant, cloned the
PpPEX7 gene, and shown in several ways that PpPex7p is
the homologue of ScPex7p, the PTS2 receptor for protein
import (Rehling et al., 1996, Zhang and Lazarow, 1996
):
(a) PpPex7p has high sequence identity with ScPex7
(43%), (b) PpPEX7 complements the ScPex7 mutant, (c)
Pppex7
is specifically disturbed in PTS2 protein import,
and (d) PpPex7p interacts with the PTS2 sequence in the
two-hybrid system. The observation that Pppex7
is unaffected in its growth on methanol medium suggests that
there are no PTS2-containing enzymes or peroxins required for growth on this medium. Furthermore, it explains why screening procedures for pex mutants, based on
their inability to grow on methanol, did not result in the
isolation of this mutant.
PpPex7p Functions Probably as a Mobile Receptor
The role of Pex7p in PTS2 import has been a matter of debate. Zhang and Lazarow (1995, 1996
) concluded that
ScPex7p is an intraperoxisomal receptor and functions by
pulling PTS2 proteins inside, analogous to the role of
mitochondrial HSP70. Although they found significant
amounts of a triple-tagged Pex7p (Pex7p-HA3) in the cytosol upon fractionation, they observed in digitonin experiments a strikingly similar pattern of latency of Pex7p-HA3 compared to peroxisomal matrix proteins. Therefore, they
concluded that the cytosolic pool found in fractionation
studies was probably caused by leakage of Pex7p-HA3. In
contrast, others proposed that Pex7p functions as a mobile
cytosolic receptor because most of the (overexpressed and
tagged) Pex7p protein was found in the cytosol by biochemical fractionation (Marzioch et al., 1994
; Rehling et al.,
1996
) or by immunofluorescence (Braverman et al., 1997
).
We raised antibodies against the endogenous Pex7p and
studied its localization. Subcellular fractionation and digitonin experiments suggest that Pex7p is largely cytosolic
but that some of the protein is peroxisomal. Evidence that
the peroxisome-associated protein is present in the matrix
was deduced from digitonin experiments and immuno-EM
using NH-tagged Pex7p, which showed clear peroxisomal matrix labeling and no obvious membrane labeling. Interestingly, a 25-fold overexpression of Pex7p results in a
modest 5-10-fold increase in peroxisome-associated Pex7p
and in a large increase of cytosolic Pex7p. This explains
why others found an almost exclusively cytosolic localization for Pex7p when they studied the localization of overexpressed Pex7p (Marzioch et al., 1994; Rehling et al., 1996
; Braverman et al., 1997
).
Our analyses indicate that most of Pex7p is cytosolic
(varying from 70 to 90%), suggesting a role in the cytosol
where it binds to PTS2-containing proteins and delivers
them at the peroxisomal membrane. The identification of
Pex14p as a putative peroxisomal docking protein for
Pex7p supports this model (Albertini et al., 1997). A similar model has also been proposed for the PTS1 receptor Pex5p, which binds to the peroxisomal membrane protein
Pex13p (Elgersma et al., 1996a
; Erdmann and Blobel,
1996
; Gould et al., 1996
).
In addition to the cytosolic pool of Pex7p, we found also a significant fraction of Pex7p in the peroxisomal matrix (varying from 10 to 30%). Therefore, it is possible that Pex7p is also involved in the subsequent translocation step. In that case, we expect that Pex7p has to get recycled to the cytosol because thiolase expression is at least 25-fold higher than Pex7p expression. The observation that the amount of intraperoxisomal Pex7p did not increase proportionally upon overexpression suggests that the Pex7p import signal is not very efficient. Therefore, it is conceivable that intraperoxisomal Pex7p does not have a role at all, but that some Pex7p enters the peroxisome coincidentally and remains in the peroxisomal matrix without any function. Further kinetic experiments are required to address this point.
Targeting of Pex7p to the Peroxisomal Matrix
The ability of Pex7p to enter the peroxisomes, albeit at
low efficiency, appears to be real. Some amounts of the
wild-type and overexpressed Pex7p were intraperoxisomal, as judged by digitonin permeabilization and immuno-EM (Figs. 7 and 10). Furthermore, NH-Mdh3p-PpPex7p was able to complement an mdh3 strain of S. cerevisiae
(data not shown). The mechanism involved in this targeting is unknown. Because protein unfolding is not essential
for the import of proteins into the peroxisomal matrix,
Pex7p might enter the matrix solely by association with
PTS2-containing proteins. This is unlikely, however, because the A248R, G249V, and C347ter mutants cannot interact with the PTS2 sequence and show the same subcellular location as wild-type Pex7p. Moreover, within experimental error, we did not observe a different distribution of Pex7p when we deleted the FOX3 gene or when we
raised its expression twofold by overexpressing it from the
ACO1 promoter. An alternative possibility is that Pex7p
has a new targeting signal or that it enters the peroxisome in association with some other protein. Such a targeting or
protein association signal might reside in the first 56 amino
acids of ScPex7p, as suggested by the experiments of
Zhang and Lazarow (1996)
.
Yeast Model System for Human Peroxisome Biogenesis Defects
The cloning of yeast PEX genes has tremendously facilitated the characterization of their human orthologues (for
review see Subramani, 1997). For instance, the human
PEX7 gene was cloned by screening databases using the
yeast PEX7 gene (Braverman et al., 1997
; Motley et al.,
1997
; Purdue et al., 1997
). Our studies show a new role for
the yeast model system: the rapid analysis of the effect of
the mutations found in the PEX genes of patients. We introduced the mutations found in the RCDP patients at the corresponding positions in the PpPEX7 gene and found
that this resulted in impaired growth on oleate. Furthermore, we found that the mutated Pex7p proteins in P. pastoris are stably expressed and not disturbed in peroxisomal
targeting, but that they are unable to bind to the PTS2 sequence. The application of a similar strategy to other human PEX genes involved in disease is likely to provide
useful insights regarding the effect of mutations.
Fox3p-SKL Only Partially Restores Growth on Oleate
When Expressed in pex7 Cells
Pppex7 did not grow on oleate plates, but this deficiency
could be partially suppressed by expressing Fox3p-SKL in
these cells. This indicates that the lack of thiolase import is
possibly the main, but certainly not the only, reason that
pex7
cells do not grow on oleate. Hence, we expect that
other PTS2-containing proteins are also required for oleate metabolism. Surprisingly, it appeared that Fox3p-SKL
was either imported or retained in peroxisomes much
more efficiently than endogenous thiolase. Moreover, it
should be noted that we did not observe significant import of endogenous thiolase in pex7
cells expressing Fox3p-SKL. This indicates that the formation and import of heterodimers of Fox3p-SKL with endogenous thiolase did not
take place, in contrast to what has been observed for S. cerevisiae cells expressing thiolase with and without a
PTS2 (Glover et al., 1994b
). This may suggest that the import of Fox3p-SKL via the PTS1 pathway is much faster than the import of endogenous thiolase via the PTS2 pathway, and that import of Fox3p-SKL takes place before the
folding and oligomerization of Fox3p-SKL is completed.
Further kinetic experiments, as conducted by Ruigrok et al.
(1996)
, are necessary to validate this hypothesis.
![]() |
Footnotes |
---|
Received for publication Received for publication 17 October 1997 and in revised form 18 December 1997..
We are grateful to Dr. A. Koller for providing us the PpFOX3 gene sequence, to Dr. H.F. Tabak for providing us thiolase antibodies and S. cerevisiae strains and plasmids, Dr. Peter van der Sluijs for providing the NH antibody, and Drs. P. Bartel and S. Fields for providing the two-hybrid plasmids. We thank Dr. K.-N. Faber for sharing his preliminary characterization of the pex7 mutant with us. ![]() |
References |
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