From the Institut für Physiologische Chemie,
Ruhr-Universität Bochum, D-44780 Bochum, Germany and
the European Molecular Biological Laboratory (EMBL)
Heidelberg, Meyerhofstr. 1, D-69012 Heidelberg, Germany
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ABSTRACT |
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Import of matrix proteins into peroxisomes
requires two targeting signal-specific import receptors, Pex5p and
Pex7p, and their binding partners at the peroxisomal membrane, Pex13p
and Pex14p. Several constructs of human PEX5 have been overexpressed
and purified by affinity chromatography in order to determine
functionally important interactions and provide initial structural
information. Sizing chromatography and electron microscopy suggest that
the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetramers. Surface plasmon resonance analysis indicates that PEX5
binds to the N-terminal fragment of PEX14-(1-78) with a very high
affinity in the low nanomolar range. Stable complexes between recombinant PEX14-(1-78) and both the full-length and truncated versions of PEX5 were formed in vitro. Analysis of these
complexes revealed that PEX5 possesses multiple binding sites for
PEX14, which appear to be distributed throughout its N-terminal half. Coincidentally, this part of the molecule is also responsible for
oligomerization, whereas the C-terminal half with its seven tetratricopeptide repeats has been reported to bind PTS1-proteins. A
pentapeptide motif that is reiterated seven times in PEX5 is proposed
as a determinant for the interaction with PEX14.
Proteins residing inside organelles must be translocated across
lipid bilayers to reach their final destination. It has been shown for
some compartments that protein translocation across hydrophobic
membranes occurs through proteinaceous complexes, which are
evolutionary conserved (1). However, the initial notion that the
translocation machineries and their mechanisms are also conserved among
different organelles has turned out to be an oversimplification
(2).
Combined classical and molecular genetic analyses of protein import
into peroxisomes in lower and higher eukaryotes have identified a large
number of genes (PEX genes), the protein products (peroxins) of which play essential roles in the different steps of transport from
the cytoplasm to the peroxisomal matrix (for recent reviews, see Refs.
3-6). In a number of cases it has been possible to link mutations in
human orthologues to peroxisomal disorders, most being fatal (7,
8).
Import of peroxisomal matrix proteins (for recent reviews, see Refs.
3-6) depends on two well defined targeting signals, termed PTS1 and
PTS2.1 Two import receptors,
Pex5p and Pex7p, have been identified which specifically bind PTS1 and
PTS2, respectively. Both receptor proteins contain repetitive sequence
motifs, each belonging to established structural families. Pex5p
possesses seven tetratricopeptide repeats (TPRs) (9-11), while Pex7p
has six WD-40 motifs (12). Pex13p and Pex14p, membrane-bound peroxins,
have been demonstrated to bind the two PTS receptors. Both are
components of a recently reported complex network of interacting
peroxins (4, 8, 13, 14). Pex13p binds the PTS1 receptor Pex5p with its
cytoplasmic SH3 domain (15-17). Pex14p interacts with both
PTS-dependent receptors (14, 18, 19). Therefore, it was
proposed that Pex14p may represent the point of convergence of both
PTS-dependent import pathways (14). The functional
importance of Pex14p for peroxisome biogenesis is further supported by
its interaction with Pex13p and with an additional membrane bound
peroxin Pex17p (13).
An understanding of peroxisomal protein import at the molecular level
requires knowledge about the structure of the peroxins and
conformational changes resulting from their interactions. Herein, we
report initial biochemical and biophysical studies of the human PTS1
receptor PEX5, overexpressed in Escherichia coli, purified
to homogeneity, and its interaction with PEX14.
Construction of His6-PEX5 and GST-PEX14-(1-78)
Expressing Vectors--
Cloning experiments were performed with
E. coli strain DH5
DNA fragments encoding full-length PEX5L, PEX5S, and the C-terminal
fragments PEX5L-(214-639) and PEX5L-(323-639) were digested with
NcoI/BglII. The resulting fragments, which also
contained additional 218 base pairs from the 3'-noncoding region and an NcoI/NotI fragment corresponding to
PEX5L-(1-251), were subcloned into expression plasmids kindly provided
by G. Stier (EMBL, Heidelberg). These plasmids were derived from pET9d
(Novagen) by replacing the unique NcoI site with a DNA
fragment encoding a hexahistidinyl (His6) tag and a TEV
(tobacco etch virus) protease cleavage site and containing several
unique endonuclease recognition sites. The PEX5 coding fragments were
ligated with NcoI/BamHI or
NcoI/NotI digested vectors, thus, fusing a
peptide with the sequence MKHHHHHHPMSDYDIPTTENLYFQGAM to the N termini
of the PEX5 proteins.
A DNA fragment encoding GST-PEX14-(1-78) was amplified by polymerase
chain reaction using
pGEX-PEX14-(1-134)3 as a
template and the primers
5'-GCAGTGGTCTCTCATGTCCCCTATACTAGGTT-3' (sense,
BsaI recognition site underlined) and
5'-CCAAGCTTAGTCGACCGAAGGCTCATCGGCAGC-3' (antisense,
HindIII recognition site underlined), digested with BsaI (creating a NcoI-compatible 5'-overhang) and
HindIII and subcloned into
NcoI/HindIII digested pET21d plasmid (Novagen).
The DNA constructs were verified by restriction analysis and partial
DNA sequence analysis using an ABI automated sequencer (Applied
Biosystems). A nucleotide exchange was found in the coding region for
PEX5L-(1-251), resulting in an amino acid substitution of glutamic
acid to aspartic acid at position 33.
Expression and Purification of Recombinant
Proteins--
Expression of His6-tagged PEX5 forms and
GST-PEX14-(1-78) was carried out in E. coli strain
BL21(DE3). Fresh transformants were grown in Luria Broth medium
supplemented with 30 mg/liter kanamycin or 100 mg/liter ampicillin.
Cells were induced in the mid-log phase with 0.4 mM
isopropyl-
Protease cleavage was performed at 37 °C for 4 h. Thrombin
(Serva) was used at a concentration of 3 NIH units/mg of purified GST-PEX14-(1-78), whereas 0.1 NIH units of TEV protease (Life Technologies, Inc.)/mg of PEX5 proteins effectively cleaved off the
His6 tag. The thrombin activity was inactivated with 1 mM phenylmethylsulfonyl fluoride for 15 min at
37 °C.
A Mono Q ion exchange column (HR5/5, Pharmacia) equilibrated with 20 mM Tris-HCl, pH 8.0, 1 mM DTT was used to
remove proteases and fusion parts and to concentrate the recombinant
proteins. For that purpose PEX14-(1-78) was eluted with a 20-ml linear
gradient between 0 and 120 mM NaCl, whereas for all
recombinant PEX5 proteins, 20-ml linear gradients from 150 to 800 mM NaCl were applied.
Protein Analysis--
Size exclusion chromatography was
performed on a Superose 6 or a Superose 12 column HR 10/30 (Pharmacia)
with buffer A at a flow rate of 0.5 ml/min at 20 °C. Molecular mass
standards were thyroglobulin (669 kDa), apoferritin (443 kDa), amylase
(200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and glutathione S-transferase (GST, 52 kDa),
respectively. Protein concentrations were determined according to
Bradford (21) using bovine serum albumin as a standard. SDS-denatured
proteins were separated by standard PAGE (22) or Tricine-PAGE according
to the method of Schägger and von Jagow (23) and visualized by Coomassie Blue or silver stain (24).
Electron Microscopy--
Protein solutions were adjusted to
concentrations of 0.1-1 mg/ml and applied onto glow discharged 400 mesh carbon-coated grids for 1 min. Grids were flashed with 150 µl of
buffer and stained with a drop of 1% (w/v) aqueous uranyl acetate for
30 s. Excess stain was removed by flash washing. The blotted and
air-dried grids were examined in a Philips 400T Transmission Electron
Microscope operating at 80 kV.
Surface Plasmon Resonance--
Studies on the interaction
between the PEX5 proteins and GST-PEX14-(1-78) were performed by
surface plasmon resonance spectroscopy using a BIAcore 2000 instrument
(BIAcore AB). Here one binding partner, referred to as the ligand, is
immobilized on a sensor chip, and the interaction with an interactant
in free solution, the analyte, is detected. Changes in the mass
concentration on the sensor surface are proportional to changes in the
refractive index of the sensor surface. This refractive index change is
monitored by using the physical phenomenon of surface plasmon resonance and expressed in response units (RU), whereas 1000 RU correspond to a
change in surface concentration of about 1 ng of
protein/mm2 (25). Anti-GST antibodies (Code number
BR-1002-23, BIAcore AB) were coupled to the surface to a total response
of 3000 RU by standard amine coupling following the manufacturers'
instructions. Purified GST-PEX14-(1-78) at a concentration of 0.09 mg/ml was captured to an immobilization level of about 400 RU to a CM5
sensor chips via anti-GST antibodies. All interaction experiments were carried out in a buffer containing 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl at a constant flow rate of 30 µl/min.
PEX5L, PEX5S, PEX5L-(1-251),and PEX5L-(214-639) were injected in
varying concentrations from 500 to 1 nM. Unspecific binding
was subtracted using an unmodified sensor surface for each
concentration individually. Bulk refractive index changes due to buffer
variations were subtracted by using runs without protein. After each
interaction cycle the surfaces were regenerated by either two
injections (1 min each) with 10 mM glycine-HCl, pH 2.2, or
one injection with the glycine solution followed by one injection with
0.05% SDS. Association and dissociation phase were both monitored for
5 min.
The resulting data were evaluated with the BIAevaluation software
version 3.0 (BIAcore AB). The dissociation rate constants were
determined separately from the binding curves obtained for the highest
concentration of the respective PEX5 protein. The data from 5 to
60 s after injection stop were fitted to Equation 1.
Recombinant Human PEX5 Exists as a Homotetramer--
It has been
reported previously that the PTS1 receptor Pex5p of yeasts and higher
eukaryotes can bind to the two peroxins Pex13p (15-17) and/or Pex14p
(14, 18, 19) at the cytoplasmic side of the peroxisomal membrane. In
higher eukaryotes two forms of PEX5 have been reported which differ in
an insertion of 37 amino acids as a result of alternative splicing (20,
26, 27). To gain insight into the structural basis of the interactions of Pex5p with other peroxins, large quantities of homogeneous protein
were required. For this purpose we created His6-tagged versions of both, the long and the short form of human PEX5 (Fig. 1, PEX5L and PEX5S), and expressed them
in E. coli by using a T7 promotor-based expression system. A
TEV protease cleavage site was introduced between the His6
tag and the PEX5 sequence, allowing the removal of the affinity tag.
Expression and purity were analyzed by SDS-PAGE.
With both recombinant PEX5 forms we obtained high levels of expression
(Fig. 2A, lanes 1 and 2). During SDS-PAGE both proteins did not migrate with
their calculated molecular masses of 67 kDa (PEX5S) and 71 kDa (PEX5L),
respectively, but with an apparent molecular mass of 80 kDa, as had
been described previously for PEX5S (27, 28). In our studies truncated
versions of PEX5 were also used. A similar difference between empirical
(45 kDa) and calculated molecular mass (28 kDa) was observed for an
N-terminal fragment but not for C-terminal fragments (Fig. 2),
suggesting that the aberrant migration is due to the high density of
negatively charged residues (about 20%) within the N-terminal half.
Both proteins, PEX5L and PEX5S, were virtually completely soluble and could be purified to close homogeneity in one step using Ni-NTA affinity chromatography (Fig. 2A, lanes 6 and
7). The His6 tag was efficiently cleaved off by
TEV protease. The protease, degradation products, and minor
contaminants were removed by anion exchange chromatography. Typically
20 mg of pure protein could be obtained from one liter bacterial
culture. Crystallization experiments have been initiated.
Size exclusion chromatography indicated a molecular mass of about 270 kDa for both PEX5 forms (Fig. 3),
suggesting a tetramer of identical subunits. The tetrameric assembly of
PEX5 was supported by results from electron microscopy. Electron
micrographs of negatively stained recombinant PEX5S preparations show
numerous particles with a diameter of about 13 nm (Fig.
4A). These particles have the
shape of a square with a central stain filled cavity and appear to
consist of four subunits. It is important to note that the recombinant
PEX5 also tends to form filamentous aggregates, which were detected in
the same micrographs. It is noteworthy that their number and size is
increased in low salt environment (Fig. 4B), suggesting that
their formation is governed by electrostatic interactions. Similar
aggregates consisting of more than 100 molecules have been described
for another TPR containing protein (9).
Oligomerization of PEX5 Is Mediated by Its N-terminal Half and Does
Not Require the TPR Region--
TPR domains are known to be involved
in protein-protein interactions. For example the binding of the PTS1 to
its receptor Pex5p is mediated by the TPRs (27, 29, 30). TPRs are also believed to interact with each other forming inter- and/or
intramolecular contacts (10, 31). In order to map the determinants
responsible for tetramerization and aggregation of PEX5, three
truncated His6-tagged versions, PEX5L-(1-251),
PEX5L-(214-639), and PEX5L-(323-639) (Fig. 1), were expressed in
E. coli (Fig. 2A, lanes 3-5),
purified by Ni-NTA affinity chromatography (Fig. 2A,
lanes 8-10), and subjected to size exclusion chromatography.
While the first two proteins were soluble the third one comprising
little more than the seven TPR motifs was insoluble and could only be
purified under denaturing conditions. Thus, approximately 100 amino
acids, which distinguish PEX5L-(214-639) and PEX5L-(323-639), are
sufficient to prevent aggregation and render the former protein soluble.
Gel filtration analyses suggested an apparent molecular mass of 105 kDa
for PEX5L-(1-251), whereas PEX5L-(214-639) behaved like a protein of
45 kDa (Fig. 3). These results are consistent with a tetrameric
structure for PEX5L-(1-251), which contains only the N-terminal half
of PEX5, and a monomeric structure for PEX5L-(214-639) comprising the
C-terminal two-thirds of PEX5.
Electron micrographs of negatively stained PEX5L-(1-251) showed
particles very similar in shape but smaller in size to those found in
preparations of full-length PEX5 (data not shown). However, in contrast
to full-length PEX5 no filamentous aggregates were detected.
A comparison of the properties determined for the four soluble
PEX5 proteins, PEX5L, PEX5S, PEX5L-(1-251), and PEX5L-(214-639), revealed that the presence of the segment between amino acid 214 and 251 does not correspond to the ability to form tetramers. Thus,
this structural property is dependent on a determinant located within
the first 213 amino acids. Aggregation in contrast seems to be due to
the seven TPR motifs.
PEX5 and PEX14-(1-78) Form a Complex in Vitro--
In
Saccharomyces cerevisiae interaction of Pex5p and Pex14p was
indicated previously by the two-hybrid method leading to the suggestion
that Pex14p could be a candidate for the docking protein of the PTS1
receptor at the peroxisomal membrane (14, 18). Deletion analysis mapped
the binding site for ScPex5p to the first 58 amino acid residues of
ScPex14p.4 Recently, the
human orthologue of ScPex14p was identified and also was shown to
interact with PEX5 (19). Sequence comparison of all available Pex14p
orthologues suggests that the first 58 amino acid residues of ScPex14p
correspond to the first 78 amino acid residues of human PEX14 (Fig.
5). Thus, we used a fusion protein
composed of GST, a thrombin cleavage site, and the first 78 amino acid
residues of human PEX14 to demonstrate the direct binding of PEX5 and
PEX14 in vitro. The fusion protein was expressed in E. coli and could be purified in high amounts (Fig. 2B).
Lysates of cells expressing either GST-PEX14-(1-78) or
His6-PEX5L in similar amounts were mixed and divided into
two parts. One aliquot was run through a glutathione-Sepharose column,
while the other was subjected to Ni-NTA chromatography. As seen in Fig.
6A, in both cases the
identical complex consisting of His6-PEX5L and
GST-PEX14-(1-78) could be specifically eluted by glutathione and
imidazole, respectively. The binding of PEX5 to the N-terminal fragment
of PEX14 was specific, since expression of GST alone instead of
GST-PEX14-(1-78) did not recruit His6-PEX5L to the
glutathione column (data not shown). We conclude that PEX5 directly
binds PEX14. This conclusion was supported by another in
vitro binding experiment with purified GST-PEX14-(1-78) and
His6-PEX5L. The two proteins were mixed in a molar ratio of
1:6 (PEX5L: GST-PEX14-(1-78)) and subsequently analyzed by sizing
chromatography. Fig. 6B shows that the mixture of the two
proteins contained an early eluting species, which is not detectable
when each protein was analyzed individually. This high molecular weight
complex consisted of GST-PEX14-(1-78) and His6-PEX5 as
confirmed by SDS-PAGE analysis (Fig. 6A, lane 4).
The formation of stable complexes indicated a high affinity binding
between PEX5 and PEX14-(1-78). The kinetics of interaction between
PEX5 and the peroxisomal docking protein PEX14 were analyzed using
surface plasmon resonance spectroscopy. Both recombinant PEX5 isoforms,
PEX5L and PEX5S, bind to the immobilized GST-PEX14-(1-78) with a fast
association rate, whereas the dissociation rate is very slow (Fig.
7). The calculated equilibrium binding
constants are in the low nanomolar range (Table
I). Data were also analyzed by steady
state analysis (not shown) and found to be in good agreement with those
obtained by evaluating the apparent rate constants. Vice versa
experiments with His6-tagged PEX5 immobilized on a Ni-NTA
sensor chip surface as ligand and GST-PEX14-(1-78) as analyte confirmed the high affinity binding (data not shown).
PEX5 Possesses Multiple Binding Sites for PEX14-(1-78)--
In
order to determine whether the GST part of GST-PEX14-(1-78) affects
the binding with PEX5, the fusion protein was cleaved with thrombin to
obtain a 10-kDa fragment PEX14-(1-78) (Fig. 2B). This
allowed in vitro binding experiments with four different forms of PEX5: the long form PEX5L, the short form PEX5S, the N-terminal fragment PEX5L-(1-251), and the C-terminal fragment PEX5L-(214-639). For this purpose the components were mixed and analyzed by sizing chromatography. Fig. 8
summarizes the findings. In all four binding experiments PEX14-(1-78)
was present in excess. Comparison of the elution profiles obtained with
the individual PEX5 proteins in the presence or absence of
PEX14-(1-78) revealed that the PEX5 proteins eluted earlier, when
PEX14-(1-78) was present, indicating the formation of complexes.
SDS-PAGE analyses of the corresponding peak fractions confirmed the
presence of both PEX14-(1-78) and the respective PEX5 protein, whereas
the unbound PEX14-(1-78) was found as a late peak in all elution
profiles. This is shown for PEX5L in Fig. 8.
One surprising result was that all soluble forms of PEX5 were able to
interact with PEX14-(1-78), suggesting more than one binding site.
This is consistent with the findings from surface plasmon resonance
analysis that both truncated versions PEX5L-(1-251) and
PEX5L-(214-639) bind to GST-PEX14-(1-78) with similar affinity as the
long and the short form of PEX5 (Fig. 7 and Table I).
Using the apparent molecular masses of PEX14-(1-78), of the different
PEX5 proteins, and of their complexes, we estimated the subunit
composition of the complexes (Table II).
This led to interesting and unexpected results. The tetrameric long and short forms of PEX5 seem to bind seven and six PEX14-(1-78) fragments per subunit, respectively, suggesting that the 37-amino acid insertion (amino acids 215-251) of the long form contains one binding site. The
calculated number of binding sites was confirmed by analysis of the
complexes formed by the two truncated PEX5 proteins. The tetrameric
N-terminal fragment PEX5L-(1-251) seems to bind four fragments of
PEX14-(1-78) per subunit, whereas the C-terminal fragment
PEX5L-(214-639) binds three or four fragments. As the overlapping
region of these two forms of PEX5 contained the insertion (amino acids
215-251) with one putative binding site, their presumed binding sites
add up to six or seven.
In order to initiate a structural analysis of the import machinery
for peroxisomal matrix proteins, we prepared recombinant human PEX5
(PTS1 receptor) and examined its binding to human PEX14 (binding
partner at the peroxisomal membrane) in vitro. In these studies we used an N-terminal fragment of PEX14 comprising only the
first 78 amino acid residues rather than the full-length protein as the
binding site for the PTS1 receptor of S. cerevisiae had been
mapped to the corresponding fragment of
ScPex14p.5 Human PEX5, as all
other members of the steadily growing Pex5 protein family, is composed
of two distinctly different parts, the highly conserved C-terminal half
comprising seven TPR motifs and the N-terminal half, which possesses
only a few amino acids that are strictly conserved among all members
(Fig. 9). While the TPR region was shown
to mediate the binding to PTS1-containing proteins (27, 29, 30), a
specific function was not yet assigned to the N-terminal half. In this
study we present evidence that the N-terminal half of PEX5 binds
PEX14.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
. The cDNA fragments coding for
PEX5L, PEX5S, and the truncated versions PEX5L-(1-251) and
PEX5L-(323-639) were from pcDNA3 (Invitrogen) derived
plasmids.2 A polymerase chain
reaction product corresponding to PEX5L-(214-639) was amplified from
pGD106 (20) using the sense primer
5'-ATTGTCGACCATGGAGTTCCTGAAATTC-3' containing a
NcoI site (underlined) and a vector-specific antisense primer corresponding to the Sp6 promotor region
(5'-TATTTAGGTGACACTATAG-3').
-D-thiogalactopyranoside and grown for another
4-6 h at a temperature of 37 °C. Cells were harvested by
centrifugation and were stored at
80 °C. Cell pellets were thawed
in buffer A containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1 mM dithiothreitol (DTT) and
broken with a French pressure cell (Aminco) in one or two passages.
Cell debris and other insoluble material were removed by centrifugation
(44,000 × g, 45 min). The supernatant containing the
soluble proteins was loaded directly on either a Ni-NTA-agarose
(Qiagen) or a glutathione-Sepharose 4B (Pharmacia) column equilibrated
with buffer A. PEX5L-(323-639) was purified from inclusion bodies on
Ni-NTA-agarose columns under denaturing conditions as described in the
QIAexpress system instructions (Qiagen). PEX5 proteins were eluted from
Ni-NTA-agarose with 60 mM imidazole. For GST-PEX14-(1-78)
bound to glutathione-Sepharose a single-step elution with 10 mM glutathione was performed. All affinity purification
steps were carried out at 4 °C and monitored by SDS-PAGE analysis.
Association rate constants were calculated with the previously
obtained dissociation rate constants and respective concentrations of
analytes according to the pseudo first order model A + B = AB
using Equation 2 in global fit analysis.
(Eq. 1)
The equilibrium binding constants were calculated from the
respective rate constants according to Equation 3.
(Eq. 2)
In these equations the following abbreviations are used:
R0 is the response at the initial time
t0, R(t) is the relative response at a given time t, Rmax is
the maximum analyte binding capacity, C is the molar
concentration of the analyte, ka and
kd are the association and dissociation rate
constants, respectively, and KD is the equilibrium
binding constant.
(Eq. 3)
RESULTS
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Fig. 1.
Schematic representation of human PEX5
constructs. The two isoforms, PEX5L and PEX5S, and truncated
versions of PEX5L were expressed in E. coli as fusion
proteins with an N-terminal His6 tag. Numbers represent the
amino acid residues of the long form of human PEX5.
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Fig. 2.
Heterologous expression and purification of
recombinant PEX5 proteins and PEX14-(1-78). Proteins were
expressed in E. coli and purified as described under
"Experimental procedures," analyzed by 12% SDS-PAGE, and stained
with Coomassie Blue. A, total cell lysates (lanes
1-5) and Ni-NTA-agarose eluate fractions (lanes 6-10)
demonstrating the high expression level and affinity purification of
His6-tagged versions of PEX5L (lanes 1 and
6), PEX5S (lanes 2 and 7),
PEX5L-(1-251) (lanes 3 and 8), PEX5L-(214-639)
(lanes 4 and 9), PEX5L-(323-639) (lanes
5 and 10). B, the fragment PEX14-(1-78) was
obtained by overexpressing GST-PEX14-(1-78) in E. coli
(lane 1), affinity purification on glutathione-Sepharose
column (lane 2), digestion with thrombin (lane
3), and removal of the GST-fusion part by anion exchange
chromatography (lane 4).
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Fig. 3.
Size estimation of recombinant PEX5 forms by
gel filtration on Superose 6. The calibration curve was determined
using the standards: apoferritin (443 kDa) (1); amylase (200 kDa) (2); alcohol dehydrogenase (150 kDa) (3);
bovine serum albumin (66 kDa) (4); and glutathione
S-transferase (52 kDa) (5). The void volume,
V0, of the column was determined with blue
dextran. Each calibration point is the result of at least two
independent measurements in the same buffer containing 20 mM Tris, pH 8.0, 150 mM NaCl, and 1 mM DTT. VE, elution volume.
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Fig. 4.
Electron microscopy of recombinant human
PEX5. Survey view of negatively stained PEX5S preparations.
A, purified PEX5S (0.9 mg/ml) in 20 mM Tris, pH
8.0, 150 mM NaCl, and 1 mM DTT. Typical
tetramers are marked by arrowheads. Two of them are shown as
insets at higher magnification. B, purified PEX5S
after dialysis against the same buffer but without NaCl. Whereas the
number of tetramers is decreased, the number and size of filamentous
aggregates is increased. The bar represents 100 nm.
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Fig. 5.
Sequence alignment of the N-terminal region
of known (Hs, Sc, Hp) and putative
(Sp, An, Ce, Dm,
Os) Pex14p orthologues. Black boxes
indicate identical amino acid residues in at least six out of eight
sequences. The sequences were found by searching SWISSPROT, TREMBL, and
EST data bases using the BLAST (2.0.4) program. Hs,
Homo sapiens (EMBL AF045186); Sc, S. cerevisiae (SwissProt P53112); Hp, Hansenula
polymorpha (SwissProt P78723); Sp,
Schizosaccharomyces pombe (SwissProt e1287778);
An, Aspergillus nidulans (EMBL AA966699);
Ce, Caenorhabditis elegans (SwissProt Q93930);
Dm, Drosophila melanogaster (EMBL AA950716);
Os, Oryza sativa (EMBL D22333).
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Fig. 6.
Isolation of in vitro formed
complexes between His6-PEX5L and
GST-PEX14-(1-78). A, SDS-PAGE analysis of
complexes isolated by glutathione (lane 2) and Ni-NTA
(lane 3) affinity chromatography and by size exclusion
chromatography (lane 4). Proteins were separated in 12%
polyacrylamide gels and visualized with Coomassie Blue. The formation
of complexes was carried out in 20 mM Tris, pH 8.0, 150 mM NaCl, and 1 mM DTT. For the affinity
purification of the complexes by glutathione-Sepharose and by
Ni-NTA-agarose, total cell lysates of E. coli expressing
His6-PEX5L or GST-PEX14-(1-78) were mixed (lane
1) and incubated for 1 h at 4 °C with gentle shaking. Size
exclusion chromatography on a Superose 6 column was performed with a
mixture of both affinity purified proteins incubated for 5 min at room
temperature. B shows the gel filtration chromatograms
obtained for purified His6-PEX5L and GST-PEX14-(1-78) and
for the mixture of both proteins.
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Fig. 7.
Binding of the soluble PEX5 proteins to
GST-PEX14-(1-78). The binding curves were obtained by surface
plasmon resonance analyses. GST-PEX14-(1-78) was immobilized to an
anti-GST sensor chip surface. Association and dissociation of the
His6-tagged proteins, PEX5L, PEX5S, PEX5L-(1-251), and
PEX5L-(214-639) were both monitored for 5 min at a flow rate of 30 µl/min. Concentrations used were 1.5, 3, 6, 12, 25, 50, and 100 nM for PEX5L; 3, 6, 12, 25, 50, and 100 nM for
PEX5S; 1, 2, 4, 8, 18, and 70 nM for PEX5L-(1-251); and
15, 31, 62, 125, 250, and 500 nM for
PEX5L-(214-639).
Rate and equilibrium binding constants of the interaction of
immobilized GST-PEX14-(1-78) and PEX5 proteins
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Fig. 8.
Size exclusion chromatography with
full-length and truncated PEX5 proteins in absence and presence of
isolated PEX14-(1-78) fragment. The His6-tagged
proteins PEX5L, PEX5S, PEX5L-(1-251), and PEX5L-(214-639) were
affinity-purified and loaded separately (dashed lines) or in
complex with isolated PEX14-(1-78) fragment (solid lines)
on a Superose 6 column equilibrated with a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 1 mM -mercaptoethanol. The complexes were formed by mixing
protein solutions in the same buffer and incubating for 5 min at room
temperature. Excess of PEX14-(1-78) (10 kDa) is indicated by the
amount of unbound protein represented by the late peak. Optical
density, OD, of the column effluent was monitored at 280 nm.
Arrows indicate the positions of globular proteins used as
molecular mass standards (see "Experimental Procedures"). The
insets in the upper panel show SDS-PAGE analysis
of two peak fractions. Proteins were separated by Tricine-SDS-PAGE and
visualized by silver stain.
Summary of the results of molecular mass analysis of PEX5 proteins in
absence and presence of isolated PEX14-(1-78) fragment
DISCUSSION
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Fig. 9.
Occurrence of WXXXF/Y motifs
within the PEX5 protein family. Protein sequences of known and
presumed Pex5p orthologues were aligned using the ClustalW (1.74)
program. Identical amino acid residues in six or more out of eight
sequences are shaded red. WXXXF/Y-motifs are
indicated by yellow boxes. Hs, H. sapiens (SwissProt P50542); Dm, D. melanogaster (SwissProt O46085); Hp, H. polymorpha (SwissProt Q01495); Pp, Pichia
pastoris (SwissProt P33292); Yl, Yarrowia
lipolytica (SwissProt Q99144); Sc, S. cerevisiae (SwissProt P35056); Ce, C. elegans (SwissProt Q18426); Cl, C. lanatus
(EMBL AF068690).
Recombinant human PEX5 is a soluble protein (Fig. 2) with a tetrameric structure, as judged by sizing chromatography (Fig. 3) and by electron microscopy (Fig. 4). However, it also forms aggregates. As the C-terminal fragment (amino acids 214-639) alone, in contrast to the N-terminal fragment (amino acids 1-251), behaves as a monomer the ability to form tetramers appears to be a property of the first 213 amino acids. Conversely, the present data suggest that aggregation could be caused by the TPR motifs. A C-terminal fragment (amino acids 323-639), which consists virtually of nothing else but the TPR motifs is completely insoluble, while the N-terminal fragment (amino acids 1-251) does not aggregate.
A striking property of the recombinant PEX5 is the occurrence of
multiple (at least six or seven per subunit) binding sites for the
PEX14 fragment which seem to bind with very high affinity. Interestingly, sequence analysis of PEX5L reveals seven pentapeptide repeats within the segment of amino acids 115-315. These repeats are
characterized by the consensus WXXXF/Y. Six of them have
been reported to exist in the short form of human PEX5 (27). An
alignment of the known Pex5 proteins shows that these repeats are found in each but the number and spacings within the N-terminal half of the
molecules differ (Fig. 9). For example, Pex5p of water melon
(Citrullus lanatus) possesses nine of these motifs whereas Pex5p of S. cerevisiae contains only two. Sequence analysis
of these repeats in all of the orthologues revealed that the first position between the conserved aromatic amino acid residues can be any
amino acid, whereas there is a strong preference for hydrophilic residues at the second position and the third position is occupied in
35 out of 36 motifs by aspartic acid, glutamic acid, or glutamine. Secondary structure prediction (32) for all known Pex5 proteins revealed that the WXXXF/Y motifs are parts of amphipathic
-helices, suggesting that the strictly conserved aromatic residues
of tryptophan, phenylalanine, or tyrosine are positioned to the same
side of the helix. As the number and distribution of the repeats in
human PEX5 closely matches the estimated number of binding sites for the PEX14 fragment in the various PEX5 forms (Table II), it is tempting
to speculate that the repeats might provide the structural basis for
this interaction.
It seems unlikely that all the binding sites found for the PEX14-(1-78) fragment in vitro in the absence of any other possible interacting protein (peroxins and/or cargoproteins) are occupied in vivo by full-length PEX14 simultaneously. It is very difficult to envisage how a protein of this size, and especially with all its predicted partners, could sterically fit into seven binding sites within a segment of about 200 amino acid residues.
This then raises the question which function the observed multiple binding sites of PEX5 for PEX14 could have in vivo. One explanation could be that the number of binding sites increases the probability of binding between the two partners without the necessity that they all have to be occupied. In the case that the individual binding sites turn out to have different binding constants, it is conceivable that PEX14 first interacts with low affinity binding sites and is then transferred to the high affinity one(s). To address this possibility additional fragments and mutant forms of PEX5, as well as full-length PEX14, have to be tested.
Taken together, the fact that recombinant PEX5 can be obtained in high
amounts and successfully used for in vitro binding studies,
including surface plasmon resonance analysis, now opens up new ways to
study the structural basis of interactions between essential components
of peroxisomal protein import. Studies including ternary complexes are
in progress.
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ACKNOWLEDGEMENTS |
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We thank Uschi Dorpmund for her excellent technical assistance, Gunther Stier for the supply of plasmids, Garnet Will for providing PEX14 cDNA, and Dr. John Williams for critical reading the manuscript. We are most grateful to all members of the Structural Biology Program, EMBL (Heidelberg) for encouragement and helpful discussions throughout the course of this work.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB394, C4, and B4).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.
§ To whom correspondence should be addressed: Institut für Physiologische Chemie, Ruhr-Universität Bochum, D-44780 Bochum, Germany. Tel.: 49-234-700-2430; Fax: 49-234-709-4279; E-mail: wolf-h.kunau{at}ruhr-uni-bochum.de.
2 G. Dodt, D. Warren, T. Yahraus, M. Soukupova, E. Becker, P. Rehling, and S. J. Gould, manuscript in preparation.
3 Will, G., Soukupova, M., Hong, X., Erdmann, K. S., Kiel, J. A. K. W., Dodt, G., Kunau, W.-H., and Erdmann, R., in press.
4 K. Niederhoff and W.-H. Kunau, manuscript in preparation.
5 K. Niederhoff and W.-H. Kunau, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: PTS, peroxisomal targeting signal; TPR, tetratricopeptide repeat; GST, glutathione S-transferase; DTT, dithiothreitol; TEV, tobacco etch virus; PAGE, polyacrylamide gel electrophoresis; RU, response units; Ni-NTA, nickel-nitrilotriacetic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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