From the Departments of Biological Chemistry and
§ Biophysics and Biophysical Chemistry, Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205
Received for publication, July 3, 2002, and in revised form, November 8, 2002
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ABSTRACT |
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Most peroxisomal enzymes are targeted to
peroxisomes by virtue of a type-1 peroxisomal targeting signal (PTS1)
at their extreme C terminus. PEX5 binds the PTS1 through its C-terminal
40-kDa tetratricopeptide repeat domain and is essential for import of PTS1-contining proteins into peroxisomes. Here we examined the PTS1-binding activity of purified, recombinant, full-length PEX5 using
a fluorescence anisotropy-based assay. Like its C-terminal fragment,
full-length tetrameric PEX5 exhibits high intrinsic affinity for the
PTS1, with a Kd of 35 nM for the
peptide lissamine-Tyr-Gln-Ser-Lys-Leu-COO Peroxisomes lack nucleic acids and import all of their protein
content (1). Peroxisomal matrix protein import is initiated when newly
synthesized peroxisomal enzymes are bound by the peroxisomal matrix
protein import receptors, PEX5 or PEX7 (2). These interactions usually
require the presence of peroxisomal targeting signals (PTS)1 in the enzymes. Two
classes of PTS are known, the PTS1 and PTS2, either of which is
sufficient to direct proteins into the peroxisome lumen (3, 4). Most
peroxisomal enzymes contain the PTS1, a 3-amino acid-long signal that
functions only when present at the extreme C terminus of a protein. The
canonical PTS1 is Ser-Lys-Leu-COO PEX5 binds the PTS1 and is thought to act as an import receptor,
directing PTS1-containing proteins into the import pathway (5). PEX5 is
a predominantly cytoplasmic, partly peroxisomal protein that appears to
shuttle between these compartments as it mediates the import of
PTS1-containing proteins (6, 7). The dynamic behavior of PEX5 suggests
a model of peroxisomal matrix protein import that involves
receptor-docking factors, translocation factors, and receptor-recycling
factors. Several other lines of evidence support this model, including
the identification of other peroxins (peroxisomal import factors) that
have specific roles in receptor docking, enzyme translocation, and
receptor recycling (8).
A clear understanding of the initial molecular events in peroxisomal
matrix protein import is likely to reveal important features of
subsequent steps in the process (9). Toward this end, we recently
determined the structure of a 40-kDa, C-terminal fragment of PEX5 bound
to a PTS1-containing peptide (10). PEX5 uses nearly its entire
C-terminal half to bind the PTS, with two triplets of the
tetratricopeptide repeat making all of the contacts with the
PTS1. This structure, combined with our biochemical analysis of
PEX5-PTS1 binding, established that this fragment of PEX5 acts as a
monomer and binds the PTS1 peptide with a 1:1 stoichiometry, displays
an apparent affinity (Kd) for the PTS1 peptide lissamine-Tyr-Gln-Ser-Lys-Leu-COO Although the C-terminal half of PEX5 has intrinsic PTS1-binding
activity, there is clearly a potential for accessory factors to promote
or disrupt the PEX5-PTS1 interaction. For example, Harano et
al. (12) reported that Hsp70 binds to PEX5 and enhances the
PEX5-PTS1 interaction. Furthermore, PEX5 appears to act catalytically, which implies the existence of a PEX5-PTS1 dissociation event and
factors that promote PEX5-PTS1 dissociation. PEX12 is an integral peroxisomal membrane protein that is essential for peroxisomal matrix
protein import and is one of only two peroxins known to interact with
the ligand-binding domain of PEX5 (13). The other is PEX8, a
peroxisomal membrane protein that terminates with a PTS1 even though it
does not use the PTS1 for targeting to peroxisomes (14-16). These
properties suggest that PEX12 and PEX8 might also be regulators of
PEX5-PTS1 binding. Although there are many possible mechanisms by which
a regulator may alter the PEX5-PTS1 interaction, the simplest is that
they bind to PEX5 and alter the affinity of PEX5 for the PTS1. Here we
characterized the interaction between full-length PEX5 and the PTS1.
Our results demonstrate that full-length, tetrameric PEX5 has high
intrinsic affinity for the PTS1. In addition, we show that Hsp70
and PEX12 have no effect on PEX5- PTS1 binding.
Proteins--
Human Hsp70 was purchased from StressGen. ATPase
activity was determined by phosphate release and measured by a standard
malachite green reaction (for protocol, see StressGen customer
support). To generate human PEX5L containing an N-terminal
hexahistidine tag, the PEX5L open reading frame (6, 17) was amplified
using oligonucleotides designed to place the sequence GTCGACC
immediately preceding the start codon and the sequence GCGGCCGC
downstream of the stop codon. The resulting amplification product was
cleaved with SalI and NotI and cloned into the
plasmid pBG90A, also known as pT7 (18), a derivative of pET28a
(Novagen). This plasmid was introduced into BL21/DE3 cells, and the
resulting kanamycin-resistant strain was induced to express
His6-PEX5L by the addition of 0.5 mM isopropyl
thiogalactoside at 25 °C. Following an overnight induction of the
culture, the cells were incubated with lysozyme (15 mg/ml for 15 min on
ice) and then broken by sonication in lysis buffer, pH 7.8 (20 mM NaH2PO4-NaOH, 0.5 M
NaCl, 5 mM NaF, 5 mM benzamidine). The cell
lysates were then clarified by centrifugation at 15,000 × g for 15 min and fractionated by nickel affinity
chromatography as follows. A 100-ml lysate was poured over a 3-ml bed
volume of nickel-nitrilotriacetic acid Superflow-agarose (Qiagen)
twice, washed in 50 ml of lysis buffer, pH 7.8, washed in 50 ml of
lysis buffer, pH 6.0, and finally washed in 25 ml of lysis buffer, pH 6.0 containing 100 mM imidazole. Purified PEX5 was then
eluted with 1 M imidazole in lysis buffer, pH 6.0. PEX5L
was concentrated by centrifugation in a Pall Macrosep 30K Omega
filtration apparatus for 120 min at 5000 × g.
The plasmid encoding MBP-LacZ, pMAL-c2, is available from New England
Biolabs. The plasmids encoding the maltose-binding protein (MBP) fusion
with LacZ and PEX12 (MBP-LacZ and MBP-PEX12, respectively) have been
previously described (13). To generate the MBP-PEX8 fusion, the
full-length Pichia pastoris PEX8 open reading frame was
amplified using oligonucleotides designed to place the sequence GTCGACC
immediately preceding the start codon and the sequence GCGGCCGC
downstream of the stop codon. The resulting amplification product was
cleaved with SalI and NotI and cloned into the
plasmid pJM539, a derivative of pMAL-c2 that is also known as
pMBPtev. This plasmid places the
SalI-NotI PEX8 fragment in frame with, and
immediately downstream of, the tobacco etch virus protease recognition
site, which lies downstream of the MBP open reading frame. The plasmids
encoding MBP-LacZ, MBP-PEX12, and MBP-PEX8 were used to transform DH10B
cells to ampicillin resistance, and the resulting strain was induced to
express the MBP fusions by the addition of 0.5 mM isopropyl
thiogalactoside followed by incubation of the culture at 25 °C
overnight. The cells were then collected, incubated with lysozyme, and
broken by sonication in a buffer of 20 mM Tris, pH 7.5, 200 mM NaCl, 1 mM EDTA, and 10 mM
2-mercaptoethanol. The cell lysates were then clarified by
centrifugation at 15,000 × g for 15 min, generating
crude lysates that were used for subsequent protein binding assays.
Gel Filtration of PEX5--
Purified recombinant PEX5L was
separated on an S300 gel filtration (Amersham Biosciences)
column at a flow rate of 0.8 ml/min at 4 °C in a solution of 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM NaF, and 5 mM benzamidine. Blue dextran
( Protein Pull-down Experiments--
Clarified lysates from
strains expressing MBP fusions were bound to an amylose resin and
washed extensively in binding buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 5 mM NaF, 10 mM
2-mercaptoethanol). The resulting protein-saturated resins were
incubated with purified PEX5 and washed extensively. Samples were
eluted with binding buffer containing 10 mM maltose,
separated by SDS-PAGE, and stained with Coomassie Blue.
PEX5-PTS1 Binding Assay--
To examine the PEX5-PTS1
interaction in real time and in solution we employed a fluorescence
anisotropy-based peptide-protein binding assay, essentially as
described by Gatto et al. (10). To use fluorescence
anisotropy for measuring PEX5-PTS1 binding events, we employed small
peptides (YQSKL or YQSEL) with a fluorophore (lissamine) attached to
their N terminus as described (9). Prior to anisotropy experiments, a
3.5-ml Spectrosil Far UV Quartz window fluorescence cuvette (Starna
Cells, Inc.) containing a magnetic stir bar was incubated overnight at
25 °C in 320 µg/ml gelatin in 10 mM HEPES, pH 7.5, and
100 mM NaCl to prevent protein or peptide from binding the
walls of the cuvette. Peptide was added to a concentration of 200 nM, and stability of the fluorescence signal was determined
by measuring the emission spectra of the fluorophore prior to and
following the experiment. Increasing amounts of recombinant PEX5 were
then titrated into the cuvette. At each protein concentration, the
lissamine was excited with polarized light (Horiba Jobin Yvon Spex
Fluorolog-3 fluorometer), and the fluorescence anisotropy was measured
by monitoring the lissamine emission intensity with the excitation and
emission polarizers configured in the L-format. The
excitation monochromator (at 568 nm) and the emission monochromator (at
588 nm) were set to a slit width of 2 and 4 nm, respectively. Each
measurement was made with a 5-s integration time with at least 10 min
of equilibration time between each addition of PEX5. A Neslab RTE-111
bath circulator was used to maintain constant temperature (25 °C).
To test the effects of Hsp70 on PEX5-PTS1 binding, the buffer was
supplemented with 5 mM MgSO4, human Hsp70 (15 ng/µl), ATP (10 mM), and ADP (10 mM).
Concentrations of PEX5 used in the fluorescence anisotropy experiments
are indicated in the figures. For the anisotropy experiment performed
with PEX12, MBP-PEX12 was added at 4-fold molar excess relative to the
PEX5 concentration used throughout the titration.
Data Analysis--
Determination of the binding affinity of PEX5
for the PTS1 requires that we first determine the fraction of peptide
bound (fB) for any given anisotropy value
(r). Since a change in fluorescence intensity was typically
observed upon ligand binding, this change must be accounted for in the
final calculation. A correction factor, Q, represents the
quantum yield ratio of the bound to the free form and is estimated by
the ratio of the intensities of the bound to the free fluorophore.
Thus using the following equation, we can determine the fraction bound.
Full-length PEX5 Binds the PTS1 Specifically and with High
Affinity--
For most peroxisomal proteins, the first step in import
is their interaction with PEX5 (2). We previously characterized the
interaction between a PTS1 peptide and the minimal domain of PEX5 that
still retains specificity for the PTS1, a C-terminal, 40-KDa monomeric
fragment of PEX5 (10). Full-length human PEX5 is nearly twice as long
(6, 17) and assembles into tetramers (19), and there is evidence of
interaction between the N-terminal and C-terminal halves of PEX5 (12).
Thus, the PTS1 binding characteristics of full-length PEX5 may be more
complex than the PTS1-binding properties of its C-terminal, monomeric,
ligand-binding domain. We purified a recombinant form of full-length
PEX5 containing an N-terminal hexahistidine tag from bacteria. For
these studies we used PEX5L, one of two major PEX5 isoforms expressed
in humans, which is 639 amino acids in length and can rescue all
defects of PEX5-deficient human cells (17). Next, we characterized the interaction between tagged, full-length PEX5 and a
fluorophore-containing, PTS1-containing peptide
(lissamine-Tyr-Gln-Ser-Lys-Leu-COO The PEX5-PTS1 Interaction Is Not Affected by ATP or
Hsp70--
Harano et al. (12) reported that Hsp70
facilitated the binding of full-length PEX5 to an ~16-kDa
PTS1-containing protein. Harano et al. also reported that
Hsp70 bound to both PEX5 and the PTS1-containing protein. These results
can be explained by two distinct hypotheses. One is that Hsp70 alters
the affinity of PEX5 for the PTS1. The other is that Hsp70 alters the
folding of the PTS1 protein and/or the presentation of its PTS1. To
distinguish between these hypotheses we examined the effect of Hsp70 on
the affinity of PEX5 for the PTS1. Using the same concentration of Hsp70 employed by Harano et al. (12), we found that Hsp70
had no effect on the PEX5-PTS1 interaction (Fig.
2A). The ability of Hsp70 to
facilitate protein folding is dependent upon ATP, and the above
experiment was performed in the presence of 10 mM ATP and 5 mM MgSO4. However, Harano et al.
(12) reported that the presence of ATP reduced the stimulatory effect
of Hsp70 on the interaction between PEX5 and the PTS1 protein used in
their studies, leading them to conclude that Hsp70-ADP has a greater stimulatory effect on the PEX5-PTS1 interaction than Hsp70-ATP. To
determine whether Hsp70 might facilitate PEX5-PTS1 interactions in the
presence of ADP, we compared the affinity of PEX5 for the PTS1 in the
presence of Hsp70 and either ATP or ADP. No difference was observed
(Fig. 2B) even though the Hsp70 used in the experiments retained significant ATPase activity (Fig. 2C).
Peroxisomal matrix protein import requires ATP (20), ATP depletion
causes significant changes in PEX5 distribution within the cell (7),
and ATP is an allosteric regulator of many proteins (21). To determine
whether ATP itself might affect the PEX5-PTS1 interaction, we performed
the PTS1 binding assay in the presence of ATP and found that it had no
effect (Fig. 2D).
PEX12 Has No Effect on PEX5-PTS1 Binding--
We previously
established that PEX12 is an integral peroxisomal membrane protein
required for peroxisomal matrix protein import (22) and that the
C-terminal, a 10-kDa RING finger domain of PEX12, interacts with the
ligand-binding domain of PEX5 (13). This interaction might reflect any
number of possible roles for PEX12, but one distinct possibility is
that PEX12 might alter the affinity of PEX5 for the PTS1. This is a
particularly attractive hypothesis because a point mutation in this
domain of PEX12 reduced its ability to bind PEX5 and resulted in a
pronounced increase in the amount of intraperoxisomal PEX5 (13), which
might result from a defect in PEX5-PTS1 dissociation.
To test the hypothesis that PEX12 might alter the affinity of PEX5 for
the PTS1, we expressed a protein that contained the 10-kDa PEX5-binding
domain of PEX12 fused to the C terminus of maltose-binding protein
(MBP-PEX12). Following its purification, we used the fluorescence
anisotropy assay to test whether MBP-PEX12 had any effect on PEX5-PTS1
interactions. PEX12 had no effect on PEX5-PTS1 interactions (Fig.
3A). This was not due to an
inability of MBP-PEX12 to bind PEX5, since we were able to specifically pull down PEX5 with MBP-PEX12 but not with MBP-LacZ during amylose affinity chromatography (Fig. 3B).
PEX8 has yet to be identified in humans, precluding our ability to test
whether it affects the interaction between human PEX5L and the PTS1.
However, it has been identified in numerous yeast species, including
P. pastoris (14-16). Therefore, we purified P. pastoris PEX5 and PEX8 proteins. As with human PEX5L, we expressed full-length P. pastoris PEX5 (5) with an N-terminal
hexahistidine tag and purified the protein by nickel affinity
chromatography. P. pastoris PEX8 was expressed as an MBP
fusion and purified by amylose affinity chromatography.
As we found for human PEX5L, P. pastoris PEX5 bound the PTS1
as measured by fluorescence anisotropy indicating that this is a
general property of PEX5 proteins (Fig.
4). The apparent affinity of P. pastoris PEX5 for this PTS1 peptide was ~250 nM,
approximately an order of magnitude higher than that for the human
protein. Addition of a purified MBP-PEX8 fusion protein had no effect
on PEX5-PTS1 binding (data not shown). However, we were unable to detect binding between P. pastoris PEX5 and PEX8, rendering
that result irrelevant to the question of whether the binding of PEX8 to PEX5 affects the affinity of PEX5 for the PTS1.
Most peroxisomal matrix proteins contain the PTS1, are bound by
PEX5 prior to their import, and are then released from PEX5 at some
point during their translocation. It is therefore likely that
peroxisomal protein import involves accessory factors that regulate the
affinity of PEX5 for the PTS1. Furthermore, full-length PEX5 is a
homooligomeric protein (19) that displays intramolecular interactions
(12), making it possible that PEX5-PTS1 interactions are regulated, in
part, by cooperativity in the binding reaction (12). The present study
extends our understanding of PEX5-PTS1 interactions by providing a
detailed molecular analysis of the interactions between
full-length PEX5 proteins and PTS1-containing peptides.
We have shown here that full-length forms of PEX5 from both humans and
the yeast P. pastoris have intrinsic PTS1 binding activity. These results eliminate the possibility that accessory factors are
required for PEX5-PTS1 binding. We have also established that full-length PEX5 binds PTS1 peptides specifically and with high affinity. Full-length PEX5 proteins bind the PTS1 peptide with an
affinity similar to that of the isolated ligand-binding domain of human
PEX5 (Kd of ~35 nM). The actual
avidity of PEX5 for PTS1-containing proteins is even higher since many
peroxisomal enzymes are oligomeric and oligomerize prior to import
(23-25), and multidentate interaction may occur between tetrameric
PEX5 and folded, oligomeric PTS1-containing proteins.
One disadvantage of studying the interactions between PEX5 and
PTS1-containing peptides is that it eliminates potentially important
PEX5-protein interactions that occur outside of the PTS1-binding
pocket. However, this is also an advantage, since it allows a
quantitative assessment of whether and how different factors affect the
affinity of PEX5 for just the PTS1. The power of this approach is
evident from our analysis the effects of Hsp70 on PEX5-PTS1 binding.
Harano et al. (12) established that Hsp70 promotes the
interaction of PEX5 with a PTS1-containing protein. However, Harano
et al. (12) did not determine whether this was due to an
effect on the affinity of PEX5 for the PTS1 or the accessibility of the
PTS1 on the PTS1-containing protein. Using our assay of PEX5-PTS1
peptide binding, we observed that PEX5 maintains the same affinity for
the PTS1 regardless of whether Hsp70 is present and regardless of
whether the reaction mixtures contain ATP or ADP. Taken together, our
results and those of Harano et al. (12) suggest that Hsp70
acts on the PTS1-containing protein, presumably to increase the
accessibility of its PTS1, which is subsequently bound by PEX5 in an
Hsp70-independent manner. This model is also consistent with the well
established role for Hsp70 proteins in the initial folding of many proteins.
The presumption that PEX5 acts catalytically predicts that the
dissociation of PEX5-PTS1 complexes is an important and perhaps critical step in peroxisomal matrix protein import. It is possible that
the PEX5-PTS1 interaction could be disrupted or inhibited by one or
more PEX5-binding proteins, which might stabilize a form of PEX5 unable
to bind the PTS1. Six peroxins are known to bind PEX5 (PEX7 (26, 27),
PEX8 (16), PEX10 (28), PEX12 (13), PEX13 (29-33), and PEX14 (11, 31,
34, 35)), and two of these, PEX12 and PEX8, known to interact with the
ligand-binding domain of PEX5. Here we found that a 4-fold molar excess
of PEX12 did not alter the affinity of PEX5 for the PTS1. Thus, if
PEX12 does regulate PEX5-PTS1 interactions in vivo, the
mechanism must involve additional factors not present in our in
vitro assay. Unfortunately, we were unable to assess the effect of
PEX8-PEX5 interactions on the affinity of PEX5 for the PTS1 because we
were unable to generate a form of soluble, recombinant PEX8 that would bind to purified PEX5. Although the present study has failed to identify a regulator of PEX5-PTS1 binding, it will be interesting to
see if genetic screens and selections might be sensitive to such an
activity and help us identify the factors that participate in this
critical step in peroxisomal matrix protein import.
. The
specificity of this interaction was demonstrated by the fact that PEX5
had no detectable affinity for a peptide in which the Lys was replaced
with Glu, a substitution that inactivates PTS1 signals in
vivo. Hsp70 has been found to regulate the affinity of PEX5 for a
PTS1-containing protein, but we found that the kinetics of PEX5-PTS1
binding was unaffected by Hsp70, Hsp70 plus ATP, or Hsp70 plus ADP. In
addition, we found that another protein known to interact with the
PTS1-binding domain of PEX5, the PEX12 zinc RING domain, also had no
discernable effect on PEX5-PTS1 binding kinetics. Taken together, these
results suggest that the initial step in peroxisomal protein import,
the recognition of enzymes by PEX5, is a relatively simple process and
that Hsp70 most probably stimulates this process by catalyzing the
folding of newly synthesized peroxisomal enzymes and/or enhancing the accessibility of their PTS1.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
, though conservative
variants of this motif can also direct proteins into the peroxisome.
While the vast majority of peroxisomal enzymes use the PTS1, a few use
the PTS2, a nonapeptide of the sequence RLX5HL, or a conservative variant that is
found at or near the N terminus of these proteins.
of ~20
nM, and binds the PTS1 in the absence of any cofactors (10).2
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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2 MDa), tetrameric
-galactosidase (465 kDa), and bovine
serum albumin (67 kDa) were used to calibrate the column. Fractions
were analyzed by SDS-PAGE and stained with Coomassie Blue to determine
the approximate molecular weight of recombinant PEX5.
To calculate Kd, the data were fit using
Kaleidagraph (Synergy Software).2
(Eq. 1)
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) using the
technique of fluorescence anisotropy (10). Plotting the amount of
peptide bound against PEX5 protein concentration reveals a rectangular
hyperbola showing that PEX5 displayed an apparent affinity
(Kd) for the PTS1 of 35 nM, which is similar to the Kd reported for the
interaction between the same peptide and the C-terminal
ligand-binding domain of PEX5 (10).2 A nearly identical
peptide carrying a very low efficiency PTS1 (lissamine-Tyr-Gln-Ser-Glu-Leu-COO
) showed no
affinity for Pex5 under identical conditions indicating that the
PEX5/PTS1 binding is specific (Fig.
1A). Interestingly, purified PEX5 behaves as a tetramer in vitro. When purified
PEX5L is separated by gel filtration chromatography, nearly half of the
protein migrates with a size of ~300 kDa, as expected for a
homotetramer of 70 kDa subunits, whereas the other half of the protein
migrates in the void volume and probably represents aggregated forms of
PEX5L that may not contribute to PTS1 binding (Fig. 1B).
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Fig. 1.
PEX5 binds a PTS1 peptide specifically and
with high affinity. A, binding of purified PEX5L to a
PTS1 peptide as measured by fluorescence anisotropy. The titration of
PEX5 into lissamine-labeled YQSKL ( ) generated a shift in lissamine
fluorescence anisotropy, while titration of PEX5 into lissamine-labeled
YQSEL (
) showed no increase in anisotropy. Here, a fraction of
peptide bound is plotted against PEX5 concentration. B, gel
filtration of purified PEX5. Approximately half of the PEX5 migrates in
the void volume, while most of the remaining PEX5 migrates with an
apparent molecular mass of ~300 kDa. Blue dextran,
-galactosidase,
and bovine serum albumin were used as molecular weight markers, and the
fractions containing these markers are overlined.
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[in a new window]
Fig. 2.
Hsp70 does not affect the interaction between
PEX5 and the PTS1. A, binding of lissamine-labeled
YQSKL at increasing concentrations of PEX5 in the presence ( ) or
absence (
) of 15 ng/µl of Hsp70, 10 mM ATP, and 5 mM MgSO4 is plotted as a function of the
concentration of PEX5 protein. B, fluorescence anisotropy of
PEX5-PTS1 binding performed in the presence of 15 ng/µl Hsp70 in the
presence of 10 mM ATP and 5 mM
MgSO4 (
) or in the presence of 10 mM ADP and
5 mM MgSO4 (
). C, ATPase activity
of Hsp70 protein compared with bovine serum albumin. Activity is
defined as the µM PO
) or absence (
) of 10 mM
ATP and 5 mM MgSO4.
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Fig. 3.
The PEX5 binding domain of PEX12 has no
effect on the affinity of PEX5 for the PTS1. A,
PEX5-PTS1 binding was measured in the presence ( ) or absence (
)
of a 4-fold molar excess of MBP-PEX12 over PEX5. B, the PEX5
binding domain of PEX12 binds purified PEX5 in vitro.
Bacterial cell lysates from cells expressing MBP-LacZ or MBP-PEX12 were
bound to amylose resin, incubated with buffer or buffer supplemented
with PEX5, followed by elution from the resin with maltose, separation
by SDS-PAGE and staining with Coomassie Blue.
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[in a new window]
Fig. 4.
P. pastoris PEX5 displays
intrinsic binding to the PTS1. Fluorescence anisotropy of
lissamine-labeled YQSKL peptide was determined at various
concentrations of recombinant P. pastoris PEX5, and fraction
of bound peptide was plotted versus PEX5 protein
concentration.
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ACKNOWLEDGEMENTS |
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We thank Gregory Gatto for the PTS1 peptide.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health (to S. J. G and J. M. B.).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: Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3085/3424; Fax: 410-955-0215; E-mail: sgould@jhmi.edu.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M206651200
2 G. Gatto, E. L. Maynard, A. L. Guerrrio, B. V. Geisbrecht, S. J. Gould, and J. M. Berg, manuscript submitted.
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ABBREVIATIONS |
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The abbreviations used are: PTS, peroxisomal targeting signals; MBP, maltose-binding protein.
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