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INTRODUCTION |
After synthesis in the cytosol, many proteins are sorted to the
various organelles characteristic of a eukaryotic cell. For the import
of matrix proteins into the peroxisome, two different pathways have
been identified (for reviews, see Refs. 1-3). Most peroxisomal matrix
proteins contain a peroxisomal targeting signal type 1 (PTS1)1 consisting of three
amino acids at the extreme carboxyl terminus. A consensus sequence has
been defined as (S/C/A)(K/R/H)(L/M) (4, 5). Only a few peroxisomal
proteins contain a peroxisomal targeting signal type 2 (PTS2). The
consensus sequence for PTS2 is
(R/K)(L/V/I)X5(H/Q)(L/A), and it is located in
the N-terminal part of a protein (6-8).
The first step in the import process is the recognition of the
targeting signal by the receptor protein. PTS1 is recognized by Pex5p
(peroxin-5 protein)
(9-13), and PTS2 by Pex7p (14, 15). After binding PTS1-containing
proteins in the cytosol, the Pex5p-cargo complex docks at the
peroxisomal membrane. Several proteins are thought to be part of the
docking complex, e.g. the integral peroxisomal membrane
protein Pex13p (16-20) and the two peroxisomal membrane-associated
proteins Pex14p (21-25) and Pex17p (26). Not much is known about the
actual translocation step across the peroxisomal membrane.
Deletion studies have shown that the seven (or eight, depending on the
organism) tetratricopeptide repeats (TPR) in the C-terminal part of
Pex5p are important and also sufficient for the binding of PTS1
proteins (27, 28). To investigate in more detail how Pex5p binds
PTS1-containing proteins, we have now used a different approach. A
library of pex5 mutants was created by random mutagenesis of
Saccharomyces cerevisiae PEX5. The yeast two-hybrid system was used to select pex5 mutants that had lost the capacity
to bind the PTS1-containing protein Mdh3p (malate
dehydrogenase-3 protein) from S. cerevisiae. In a separate
screen, pex5 mutants were selected that had gained
interaction with a mutant PTS1 protein (Mdh3-SEL). We also derived a
structural model for the TPR motifs of Pex5p based on the crystal
structure of the three TPR motifs in protein phosphatase-5 (PP5) (29).
By relating the mutations found in Pex5p to this structural model, we
were able to explain why certain mutations affected the interaction
with Mdh3p. On the basis of orthologous sequence alignments, additional
mutations were made in strongly conserved amino acids in Pex5p by
site-directed mutagenesis. The selected mutants, together with the
structural model, allowed us to put forward a proposal as to how
PTS1-containing proteins are selected by Pex5p.
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EXPERIMENTAL PROCEDURES |
Strains and Culture Conditions--
The yeast strains
used in this study were S. cerevisiae BJ1991
(MAT
, leu2, trp1, ura3-251, prb1-1122, pep4-3,
gal2), BJ1991pex5
(MAT
, pex5::LEU2, leu2, trp1, ura3-251, prb1-1122, pep4-3,
gal2), HF7c (MATa, ura352,
his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4-542,
gal80-538,
LYS2::GAL1UAS-GAl1TATA-HIS3, URA3::GAL417-mers(×3)-CyC1TATA-lacZ), and PCY2 (MAT
,
gal4,
gal80, URA3::GAL1-lacZ, lys2-801,
his3-
200, trp1-
63, leu2, ade2-101).
The Escherichia coli strain DH5
(recA, hsdR,
supE, endA, gyrA96, thi-1, relA1, lacZ) was used for all transformations and plasmid isolations. Yeast transformations were
carried out as described (30). Transformants were selected and
grown on minimal medium containing 0.67% yeast nitrogen base without
amino acids (Difco), 2% glucose, and amino acids as needed.
Cloning Procedures--
Standard techniques for DNA
manipulations were used (31). The construct pAN1 was made by cloning
the complete open reading frame of PEX5 between
EcoRI and HindIII in the multiple cloning site of
pUC19. Some restriction sites in the multiple cloning site were
deleted, and some additional sites were introduced: EcoRI
and BamHI at the 5'-end of PEX5 and
PstI, SphI, SpeI, SphI, and
HindIII at the 3'-end of PEX5. Two additional
restriction sites were introduced in PEX5 by silent
mutations: a XbaI site at position 1140 by mutating the
codon for leucine 381 from CTG to CTA and a SalI site at
position 1356 by mutating the codon for leucine 452 from TTA to TTG and
the codon for serine 453 from AGC to TCG. The plasmid encoding the
Gal4 activation domain fusion with PEX5 (pAN4) was
constructed by cloning PEX5 from pAN1
EcoRI-SpeI in the two-hybrid vector pPC86 (32).
The plasmid encoding the Gal4 DNA-binding domain fusion with
GFP-SKL (pAN25) was constructed by PCR on GFP containing the S65T
mutation with primers p330 and p331. The PCR product was cloned into
EcoRI-PstI in pAN1, resulting in pAN21.
Subsequently, the two complementary oligonucleotides p332 and p333
were ligated between the PstI and SpeI sites of pAN21, resulting in pAN22. The insert from pAN22 was cloned into SalI-SpeI in the multiple cloning site of the
two-hybrid vector pPC97 (32). The amino acid sequence of the extreme
carboxyl terminus reads GMDELYLQGGGSKL. Gal4 DNA-binding domain fusions with the Pex13p SH3 domain (pGB17) and Pex14p (pGB47) have been described before (19). pPR6/56 encodes a fusion protein of the Gal4
DNA-binding domain with Pex8p (amino acids 19-589) (33). pDBMDH3
was made by PCR on pEL102 (34). The PCR product was cloned into
SalI-SpeI in the multiple cloning site of pPC97
(32). pDBMDH3-SEL was made by PCR on pMDH3-SEL (34). The PCR product was cloned into SalI-SpeI in the multiple cloning
site of pPC97. The glutathione S-transferase (GST)-Pex5p
fusion protein (pGST-Pex5p) has been described before (19). A GST
fusion with the Pex5p-N393A mutant was made by site-directed
mutagenesis (see below). A maltose-binding protein (MBP) fusion with
Mdh3p (pAN60) was made by digestion of pEL102 with BamHI and
cloning the fragment into pMal-c2 (New England Biolabs Inc.). An MBP
fusion with Mdh3
SKL (pAN56) was made by PCR on pEL102 with
primers p325 and p326. The PCR product was cloned into
BamHI-HindIII in pMal-c2. The oligonucleotides used were p325 (AAGGATCCATGGTCAAAGTCGCAATTCTTG), p326
(AAAAAGCTTCAAGAGTCTAGGATGAAACTCTTG), p330
(CGGAATTCTGTCGACTGGATCCATGAGTAAAGGAGAAGAACTTTTC), p331
(CCCAAGCTTGCATGCCTGCAGGTATAGTTCATCCATGCCATGTG), p332
(GGGTGGTGGTTCCAAGCTATGA), and p333 (CTAGTCATAGCTTGGAACCACCACCCTGCA).
Construction of the pex5 Mutant Libraries--
The
PEX5 gene spanning 1839 base pairs was randomly mutagenized
by error-prone PCR. We used Taq DNA polymerase lacking the 3'
5' proofreading activity (35). pAN1 was used as a template under
standard reaction conditions (10 mM Tris-HCl (pH 7), 50 mM KCl, 1.5 mM MgCl2, 0.8 mM dNTPs, and 0.03 units/µl Taq polymerase) (36) using the M13/pUC primers (6 ng/µl) 1224 and 1233 (New England
Biolabs Inc.).
The mutation frequency was determined by sequencing 23 randomly picked
pex5 clones at 1.8 × 10
3 per
nucleotide synthesized, resulting in 3.3 mutations per pex5 clone synthesized. The mutagenized pex5 was split into two
parts using the XbaI site at position 1140. In this way, two
sublibraries of pex5 mutants were made (see Fig. 1). The
C-mutant pex5 library was made by replacing the
3'-XbaI-PstI fragment of the wild-type PEX5 sequence (nucleotides 1141-1839) in plasmid pAN1 with
the corresponding mutagenized pex5 sequence. This
mutagenized region of PEX5 includes the motifs TPR3-7
(amino acids 381-612). The N-mutant pex5 library was made
from the same PCR product as the C-mutant pex5 library by
replacing the 5'-BamHI-XbaI fragment of the
wild-type PEX5 region (nucleotides 1-1140) in plasmid pAN1 with the corresponding mutagenized pex5 sequence. This
includes the motifs TPR1 and TPR2. For screening in the two-hybrid
system (37), both libraries were subcloned in plasmid pPC86 (using EcoRI-SpeI), leading to a fusion of the
pex5 mutants with the Gal4 transcription activation domain.
For in vivo complementation studies, both libraries were
subcloned (BamHI-PstI) behind the PEX5
promoter in plasmid pEL91.
Two-hybrid Screen with the C-mutant and N-mutant pex5
Libraries--
Yeast strain HF7c expressing pDBMDH3 was transformed
with the C-mutant pex5 library fused to the Gal4
transcription activation domain; 20,000 double transformants were
selected on 2% glucose/leu
trp
plates.
These transformants were replica-plated onto 2%
glucose/leu
trp
his
plates
containing 25 mM 3-amino-1,2,4-triazole (3-AT) to test for
the interaction between the PTS1 protein Mdh3p and the pex5 mutant library. About 300 transformants (1.5%) were selected that were
not able to grow on plates without histidine. Total protein was
isolated from 72 of the 300 transformants for Western blotting using
anti-Pex5p antibodies; 28 of the 72 transformants expressed a
full-length Gal4 transcription activation domain-Pex5p fusion. The 28 plasmids coding for full-length Pex5p were rescued and retransformed to HF7c for two-hybrid analysis. Nine of the 28 mutants
displayed interaction with Mdh3p, indicating a first-round false-negative mutant. The remaining 19 clones were sequenced to
determine the sites of the mutations. Five mutants contained more than
two amino acid substitutions and were not further analyzed. The
N-mutant pex5 library was screened for loss of interaction with Mdh3p in the same way as described for the C-mutant
pex5 library. The percentage of mutants that could not grow
was much larger for the N-mutant pex5 library than for the
C-mutant pex5 library. Of the 3000 transformants, 100 were
not able to grow. However, only 8 of the 100 selected mutants produced
the full-length fusion protein, and from these mutants, the plasmid was
rescued. After retransformation, only one of these mutants
(pex5.42) still showed no interaction with Mdh3p.
Suppressor Analysis--
Yeast strain HF7c expressing
pDBMDH3-SEL was transformed with either the C-mutant or N-mutant
pex5 library, and in each case, 100,000 transformants were
made and plated onto 2% glucose/leu
trp
his
plates containing 25 mM
3-amino-1,2,4-triazole. Transformants that were able to grow on these
plates and thus contained pex5 mutants that had gained an
interaction with Mdh3-SEL were selected. One pex5 suppressor
mutant (pex5.sup2) was isolated from the N-mutant pex5 library, and three pex5 suppressor mutants
(pex5.sup1, pex5.sup3, and pex5.sup21)
were isolated from the C-mutant pex5 library. These mutants
were sequenced to determine the sites of the mutations.
In Vitro Binding Assay--
The GST and MBP fusion proteins were
expressed and isolated as described elsewhere (19, 20). For the
in vitro binding experiments, 250 µl of cleared lysate
containing the MBP fusion protein was first bound to an amylose resin
column; and subsequently, the purified GST fusion protein (100 µg)
was passed over the column. After washing, bound proteins were eluted
with maltose and analyzed by SDS-polyacrylamide gel electrophoresis.
The fusion proteins used in these experiments were MBP-Mdh3p,
MBP-Mdh3
SKL, GST-Pex5p, and GST-Pex5p-N393A.
Modeling of the Pex5p TPR Domain--
The small amino acids at
positions 8, 10, and 27 of the TPR motifs of Pex5p were aligned with
the small amino acids of the PP5 TPR motifs. By using this optimized
alignment, it was possible to model the Pex5p TPR domain based on the
PP5 TPR crystal structure (29). For the modeling procedure, the Pex5p
TPR domain region was split into two parts, TPR1-3 and TPR5-7, thus
excluding TPR4. Alignments and initial optimized template PP5 and model
Pex5p overlays were generated using the Swiss-PdbViewer/SWISS-MODEL interface (38). Subsequent models were checked, refined, and energy-minimized using WHAT IF (39) and the Biotech Validation Suite in combination with the Swiss-PdbViewer. Models show
backbone root mean square deviations from the PP5 template of
0.4-0.8 Å. The final total energies were
4600 kJ/mol for TPR1-3
and
5900 kJ/mol for TPR5-7.
Site-directed Mutagenesis--
Site-directed mutations
were introduced with the QuickChange site-directed mutagenesis kit
(Stratagene) using pAN4 as a template. The oligonucleotides used were
N325A (GCTGCCTACTGATGGAAGCCGGAGCCAAATTGAGCG), N360A
(GGTCTAGTACAAACCCAGGCTGAAAAAGAGTTGAACGGC), E363A
(CCCAGAATGAAAAAGCGTTGAACGGCATAAGCGC), I389D
(GAGGCAATGAAAACTTTAGCGGACAGTTATATAAACGAAGG), N393A
(CTTTAGCGATAAGTTATATAGCCGAAGGTTATGATATGAGCGCC), N393G
(CTTTAGCGATAAGTTATATAGGCGAAGGTTATGATATGAGCGCC), E394A
(GCGATAAGTTATATAAACGCAGGTTATGATATGAGCGCC), N503A
(GGGGCTTCATTGGCCGCTTCCAATAGATCAGAGG), S504A
(GCTTCATTGGCCAATGCCAATAGATCAGAGGAAGC), N505A
(GGCTTCATTGGCCAATTCCGCTAGATCAGAGGAAGC), L531A
(GTTAGAGCTCGCTATAATGCGGCGGTATCATCCATGAATATAGGC), and N537A
(GGCGGTATCATCCATGGCTATAGGCTGTTTCAAAGAAGC). The mutations that were introduced using these primers are underlined. For each primer listed, also the complementary primer was used.
-Galactosidase activity was determined as described (40,
41).
 |
RESULTS |
Isolation of pex5 Mutants Disturbed in PTS1 Recognition--
To
investigate which parts of Pex5p are responsible for contacting
PTS1-containing proteins, mutations were introduced randomly into the
S. cerevisiae PEX5 gene by PCR amplification (see
"Experimental Procedures"). For technical reasons, the mutant
library was divided into two halves (Fig.
1). The N-terminal part of the mutant
library was linked to the wild-type C-terminal part, and the C-terminal part of the mutant library was linked to the wild-type N-terminal part.
Both libraries were used for a yeast two-hybrid interaction screen to
select mutants that had lost the capacity to interact with yeast Mdh3p,
a PTS1 (SKL)-containing peroxisomal matrix protein (see "Experimental
Procedures"). Each of the selected mutants was analyzed by Western
blotting to determine whether full-length Pex5p was still produced at
normal levels.

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Fig. 1.
Construction of the pex5
mutant libraries. The XbaI restriction site was
used to swap wild-type PEX5 sequences for randomly
mutagenized pex5 sequences.
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From the C-mutant pex5 library, 14 pex5 mutants
were selected, whereas from the N-mutant pex5 library, only
one mutant (pex5.42) was selected that expressed full-length
Pex5p. (Table I). These numbers confirm
previous results that the C-terminal TPR domain is responsible for PTS1
recognition (11, 27, 28).
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Table I
pex5 mutants that have lost the interaction with Mdh3p
Randomly mutagenized pex5 libraries were screened for
pex5 mutants that had lost the interaction with the PTS1
protein Mdh3p in the two-hybrid system. The sites of the mutations were
determined by sequencing. For the underlined substitutions, it is
indicated in which TPR motif the mutations are located.
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None of the mutations we found gave rise to a gross structural
alteration or instability of Pex5p since the two-hybrid interactions of
these mutants with known partner peroxins such as Pex8p (33), Pex13p
(16-20), and Pex14p (21-23) were unaffected, and proteins were
expressed at wild-type levels (data not shown). The pex5 mutants, when expressed in vivo under the control of the
PEX5 promoter, were unable to complement the oleate
non-utilizer (onu) phenotype of a pex5
strain,
indicating that they had also lost their function in vivo
(data not shown). The sites of the mutations in these pex5
mutants were determined, and we found that for every mutant, there was
at least one mutation located in a TPR motif (Table I). This indicated
that these motifs are important for the interaction with PTS1 proteins,
i.e. in TPR2, TPR3, and TPR5-7. No mutations were found in
TPR1 and TPR4, suggesting that these motifs do not contribute to PTS1
interaction. One residue, asparagine 393 in TPR3, was found to be
mutated in five different clones (pex5.38,
pex5.45, pex5.46, pex5.70, and
pex5.97), suggesting an important role for this residue in
PTS1 recognition.
In Vitro Binding Studies--
Previous studies of Pex5 proteins in
different species have shown that the interaction between Pex5p and
PTS1-containing proteins is direct (10, 12, 28). To determine whether
the interaction between S. cerevisiae Pex5p and Mdh3p is
direct and dependent on PTS1, an in vitro binding assay was
carried out. The genes encoding Pex5p and Mdh3p were fused in frame to
DNA sequences encoding GST and MBP, respectively, and the chimeric
genes were expressed in E. coli. As a control, mutant Mdh3p
lacking its PTS1 (Mdh3
SKL) was fused to MBP and expressed in
E. coli. GST-Pex5p was purified on a glutathione-Sepharose
column, and the purified fusion protein was tested for its ability to
bind to a column with immobilized MBP-Mdh3p and MBP-Mdh3
SKL,
respectively. Fig. 2 shows that GST-Pex5p
interacted with MBP-Mdh3p, but not with MBP-Mdh3
SKL. Furthermore,
GST alone was not retained on the MBP-Mdh3p column (data not shown).
These data indicate that the interaction between Pex5p and Mdh3p is
direct and is dependent on PTS1 of the latter protein. To determine
whether asparagine 393, found to be mutated in five different clones,
is also important for direct interaction with Mdh3p, an in
vitro binding experiment was performed with the Pex5p-N393D
mutant. GST-Pex5p-N393D did not interact with Mdh3p because it was not
retained on a column with immobilized MBP-Mdh3p. These data underscore
the important role of asparagine 393 in TPR3 of Pex5p for PTS1
interaction.

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Fig. 2.
In vitro binding experiments of
Pex5p and Mdh3p. Purified GST-Pex5p (100 µg) or GST-Pex5p-N393D
(100 µg) was passed over an amylose column loaded with 250 µl of
cleared lysate containing either MBP-Mdh3p or MBP-Mdh3 SKL. After
washing, the columns were eluted with 20 mM maltose, and
the proteins in the elution fractions were separated by
SDS-polyacrylamide gel electrophoresis and revealed by staining with
Coomassie Blue. WT, wild-type.
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Isolation of pex5 Suppressor Mutants--
To identify additional
residues in Pex5p involved in PTS1 recognition, we carried out a
positive two-hybrid screen. Instead of screening for pex5
mutants that had lost the interaction with a PTS1 protein, we
screened for pex5 mutants that gained interaction with the
PTS1 mutant Mdh3-SEL. Four suppressor mutants were selected in this
positive two-hybrid screen (Table
II). Remarkably, one mutant
(pex5.sup2) contained a glutamic acid-to-lysine substitution at position 361 in TPR2, a mutation that is exactly the opposite of the
mutation introduced in PTS1, i.e. lysine to glutamic acid. This suppressor mutant specifically suppressed the PTS1 mutation because it did not interact with Mdh3p from which PTS1 had been deleted
(Mdh3
SKL). The other suppressor mutants still showed a weak, but
detectable interaction with Mdh3
SKL (see "Discussion"). It is
noteworthy that all suppressors were still able to bind to Mdh3p with
wild-type PTS1 (Mdh3-SKL), indicating that the mutations had no gross
structural effects on the protein.
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Table II
pex5 suppressor mutants restore the interaction with a PTS1 mutant
Two-hybrid interaction was measured in the two-hybrid reporter strain
HF7c by growth on glucose plates lacking histidine and containing 25 mM 3-AT. +, growth was observed after 4 days; , no growth
was observed after 4 days; +*, when double-transformed cells were first
grown on glucose plates containing histidine and later were transferred
to glucose plates lacking histidine (and containing 25 mM
3-AT), growth was observed on these his plates. When cells
were directly plated after the transformation onto glucose plates
without histidine (and containing 25 mM 3-AT), no growth
was detectable.
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Modeling of the Pex5p TPR Domain--
The mutations that altered
PTS1 binding were not clustered in a small region of Pex5p as expected
on the basis of the small PTS1 ligand comprising only three amino
acids. Instead, the mutations were distributed over the entire TPR
domain and were present in most TPR motifs, except TPR1 and TPR4 (Fig.
3 and Table I). To understand why the
mutations led to a loss of Mdh3p interaction, we used the known
three-dimensional structure of another TPR protein, PP5 (29). The
crystal structure of PP5 shows that individual TPR motifs consist of
two
-helices,
-helix A and
-helix B, which are antiparallel.
The small hydrophobic amino acids at positions 8, 20, and 27 are
important for packing these
-helices close together. Most of the TPR
motifs of Pex5p also contain these small hydrophobic amino acids such
as glycine and alanine at positions 8, 20, and 27 (Fig. 3). A common
feature in many TPR motifs is a proline at position 32. This proline at
the end of
-helix B probably supports a turn in the structure (42),
leading to an antiparallel arrangement of
-helix A relative to
-helix B of the previous TPR. Analyzing the primary amino acid
sequence of the seven TPR motifs of Pex5p for these features indicated
that TPR4 differed from the other six TPR motifs. The amino acid
sequence that should form the fourth TPR motif should be 34 amino
acids, instead of the 42 amino acids found in between TPR3 and TPR5. In
addition, there are no small amino acids found at positions 8, 20, and
27, and also proline 32 is not present. We suggest therefore that TPR4
is not a true TPR motif, but may rather function as a flexible hinge
that connects two clusters of three TPR motifs. This may explain how
two TPR subdomains can interact with the small ligand. However, to
avoid possible confusion, we continued the numbering from TPR1 to
TPR7.

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Fig. 3.
Sequence alignment of Pex5p TPR motifs.
Aligned are TPR1-3 and TPR5-7 from S. cerevisiae
(Sc), H. polymorpha (Hp), P. pastoris (Pp), and H. sapiens
(Hs). The part of the TPR motif that forms an -helix or a
loop (based on PP5) is indicated by arrows. The
stars mark the small amino acids at positions 8, 20, and 27 of a TPR motif. These amino acids were aligned with the small amino
acids of the TPR motifs of PP5. The hatched arrows indicate
the positions where mutations were found that affect the packing of the
-helices such that PTS1 recognition is affected as a secondary
effect. The black arrows indicate the positions involved in
PTS1 binding. The white arrows indicate the mutant positions
found in the suppressor screen with Mdh3-SEL.
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It was possible to model the Pex5p TPR domain based on the PP5 TPR
crystal structure by using an optimized alignment (see "Experimental
Procedures"). Structural models were made of Pex5p TPR1-3 (Fig.
4, A and B) and
Pex5p TPR5-7 (C and D). Relating the
loss-of-interaction mutations to the derived structural model of Pex5p
allowed us to separate them into two groups as follows.

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Fig. 4.
Structural model of the TPR motifs of
Pex5p. A, ribbon model of TPR1-3. Each TPR
motif consists of two -helices connected by a short intra-repeat
loop. Side chains of amino acids involved in PTS1 interaction are
indicated. B, space-filling model of TPR1-3. Indicated in
blue are the side chains of the amino acids that are
involved in PTS1 recognition. Glutamic acids and isoleucine are shown
in dark blue, and asparagines are shown in
light blue. Ile389 and Asn393 are
located on one side of the small TPR groove, and Glu361 and
Glu363 are on the other side. The general TPR groove is
indicated. C, ribbon model of TPR5-7 with the side chain of
Arg526 indicated. D, space-filling model of
TPR5, 6 and 7. Indicated in blue is Arg526,
sticking out into the general TPR groove. Also indicated in
blue are Asn503 and Ser534, where
suppressor mutations were found.
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The first group of mutants contains an amino acid substitution that
probably results in a small local change in the structure of a TPR
domain. These mutations are located in
-helix A or B, where they may
interfere with correct packing of the
-helix in the structure. This
can arise by a small amino acid changing into a more bulky one. An
example is the G354C mutation in pex5.42; this glycine is
present at position 8 of the second TPR motif. Another example is the
G498E mutation in pex5.74; here the small amino acid at
position 8 of TPR6 is mutated. The same effect is probably achieved
when certain amino acids are replaced by a proline. These mutations are
also localized in the
-helices of the TPRs. Mutants belonging to
this group are pex5.14 (containing the L531P mutation in
-helix A of TPR7), pex5.48 (containing the L518P mutation
in
-helix B of TPR6), pex5.30 (containing the L465P mutation in
-helix A of TPR5), and pex5.79 (containing
the L404P mutation in
-helix B of TPR3). The introduction of a
proline will probably disturb the continuity of an
-helix, and this
may lead to (local) misfolding of a TPR motif. To test this hypothesis, we changed, by site-directed mutagenesis, the prolines in
pex5.14, pex5.30, and pex5.79 to
alanines, a residue that is accepted in an
-helix. Two-hybrid
analysis revealed that in all cases, the alanine substitution restored
PTS1 interaction (data not shown). This indicated that the prolines at
these positions give a disturbance of the TPR structure and that the
mutated residues (leucines in the
-helices of TPR3, TPR5, and TPR7)
are not directly involved in binding of PTS1-containing proteins.
The second group consists of mutants that contain an amino acid
substitution located in the small intra-repeat loop that connects
-helix A with
-helix B within a TPR motif (Fig. 4A).
Here, several mutants contain a substitution of the same residue in the
small loop of TPR3. Asparagine 393 was found to be mutated to aspartic acid (N393D) in pex5.38 and pex5.45, to tyrosine
(N393Y) in pex5.46 and pex5.97, and to serine
(N393S) in pex5.70. Unlike the
-helices, these loops are
somewhat projecting outwards from the folded TPR structure (Fig.
4A). The loop of TPR3 might therefore be a position for
direct contact with PTS1. This is in line with the substitution of
asparagine 393 with alanine, which resulted in a loss of Mdh3p interaction in the two-hybrid system (Table
III). Additional evidence that the
intra-repeat loops are directly involved in PTS1 binding came from the
screen for pex5 mutants that gained interaction with the
PTS1 mutant Mdh3-SEL (Table II). This screen identified glutamic acid
361 in loop 2 as being directly involved in contacting PTS1.
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Table III
Quantification of two-hybrid interactions for Pex5p mutants made by
site-directed mutagenesis
Two-hybrid interaction between the Pex5p mutants and either Mdh3p or
GFP-SKL was quantitated in the two-hybrid reporter strain PCY2 by
measuring -galactosidase activity. Indicated is the average of two
independent measurements with the range in parentheses. ONPG,
o-nitrophenyl -D-galactopyranoside.
|
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Site-directed Mutagenesis of the Pex5p TPR Motifs--
The results
from the two different screens for pex5 mutants described
above indicated that the intra-repeat loops of TPR2, TPR3, and TPR6,
which connect
-helices A and B within a TPR motif, are important for
the interaction with PTS1 proteins. A sequence alignment of the Pex5p
TPR motifs from S. cerevisiae, Hansenula polymorpha, Pichia pastoris, and Homo
sapiens showed that besides residues in the
-helices, also
certain residues in the loops are well conserved (Fig. 3). The sequence
conservation of amino acids in the intra-repeat loops is not a general
feature of TPRs in other proteins. However, in the intra-repeat loops
of Pex5p TPRs, with the exception of the loop of TPR5, some very
well conserved asparagines are present, next to other conserved residues.
To further investigate the importance of the loops of the different TPR
motifs in Pex5p, we mutated the conserved residues and studied the
effect on PTS1 protein binding. In every TPR loop, except that in TPR5,
at least one amino acid was mutated (Fig. 3 and Table III). The
conserved asparagine 360 (loop 2), asparagine 393 (loop 3), asparagine
503 (loop 6), asparagine 505 (loop 6), and asparagine 537 (loop 7)
residues were all mutated to alanines. Glutamic acids 361 and 363 (both
in loop 2) and glutamic acid 394 (loop 3) were also mutated, as were
the nonconserved asparagine 325 (loop 1) and serine 504 (loop 6)
residues. These pex5 mutants were still able to interact
with Pex13p and Pex14p, indicating that the Pex5 protein is still at
least partially functional (data not shown).
We tested the pex5 mutants in the two-hybrid system for
interaction with Mdh3p and an artificial PTS1 protein (GFP) extended with the PTS1 SKL sequence at its carboxyl terminus (GFP-SKL). Mutations in the loops of TPR1 and TPR7 did not influence the binding
of either protein (Table III). This indicated that amino acids in these
loops do not directly participate in the binding of PTS1 proteins or
that loss of a weak interaction is insufficient to evoke a phenotype.
Mutations in the loops of TPR2 and TPR3 did have an effect on PTS1
protein binding and some of the mutants showed differences in
interactions with Mdh3p and GFP-SKL (Table III). The mutations N360A
and E394A in the loops of TPR2 and TPR3, respectively, resulted
in complete loss of GFP-SKL binding, but interaction with Mdh3p was
still present. Similarly, the suppressor mutant E361K and the
site-directed mutant E363A in the loop of TPR2 showed complete loss of
GFP-SKL binding, whereas interaction with Mdh3p was only 2-3-fold reduced.
Mutations in the loop of TPR6 (N503A, S504A, and N505A) did not disturb
binding of either PTS1 protein. However, it should be noted that in our
pex5 suppressor screen, asparagine 503 was found to be
mutated twice, suggesting that the loop of TPR6 contributes to the
interaction with PTS1.
A much stronger phenotype was found when asparagine 393 in the loop of
TPR3 was mutated either to alanine or to glycine. In both cases, we
found a complete loss of interaction with GFP-SKL and Mdh3p. This
position was also found to be mutated several times in the screen for
pex5 mutants with no Mdh3p interaction.
The structural model of Pex5p TPR1-3 (Fig. 4A) suggests
that the intra-repeat loops of TPR2 and TPR3 are localized close
together and that the intra-repeat loop of TPR1 is farther away. A
groove similar to that found in the structure of the TPR of PP5 (29) is
present in Pex5p TPR1-3 (Fig. 4B). For PP5, Das et
al. (29) postulated that this is the binding groove for target
proteins. In our model, besides the general TPR groove, there is a
smaller second groove in the area where the intra-repeat loops of TPR2 and TPR3 come together (Fig. 4B). There is a high sequence
conservation in this area, and close to the residues of the loops of
TPR2 and TPR3 is isoleucine 389, located in
-helix A of TPR3 (Fig.
4, A and B). Because of its conservation among
species and its close position to the residues of the loops of TPR2 and
TPR3, we decided to mutate this hydrophobic residue to aspartic acid.
The interaction of this Pex5p-I389D mutant with Mdh3p and GFP-SKL in
the two-hybrid system was completely lost (Table III).
The structural model for TPR5-7 (Fig. 4D) shows that the
strictly conserved arginine 526, located in
-helix A of TPR7,
projects outwards from the TPR groove. This might indicate that this
amino acid is important for the interaction with target proteins. In line with this suggestion, arginine 526 was found to be mutated to
glycine in the screen for pex5 mutants that had lost the
interaction with Mdh3p (Table I). This mutant (pex5.98),
however, contained a second mutation (S504P) in the loop of TPR6. To
investigate the contribution of each residue to PTS1 interaction,
single alanine mutants were generated by site-directed mutagenesis. The
R526A mutation resulted in a complete loss of interaction with both Mdh3p and GFP-SKL, whereas the S504A mutation had no effect on the
interaction with either PTS1 protein (Table III). These results indicate that arginine 526 might also be involved in the interaction with PTS1 proteins. Such an interaction might be possible due to the
flexible hinge region discussed before, which could allow the TPR
subdomains to come together.
 |
DISCUSSION |
We carried out a structure-function analysis of the PTS1 receptor
Pex5p to obtain insight into how recognition of PTS1 proteins destined
for import into peroxisomes is accomplished. To this end, mutations in
Pex5p were isolated that affected the binding of the peroxisomal matrix
protein Mdh3p in a yeast two-hybrid trap. Two types of mutants were
isolated: loss-of-interaction mutants and suppressor mutants,
i.e. mutants that gained interaction with Mdh3p containing a
mutation in its PTS1. The pex5 mutants were all located in
the C-terminal half of Pex5p containing six TPRs. Rather surprisingly,
they did not cluster in a particular region within the TPR domain. To
be able to interpret the location of the pex5 mutations in
relation to its structure, we derived a homology model of the TPR
domain of Pex5p based on the crystal structure of the three TPRs from
PP5 (29). Mapping of the mutations onto this structural model showed
that some of the loss-of-interaction mutations consisted of amino acid
substitutions with prolines or bulky amino acids in the
-helices of
TPRs. These mutations are predicted to disrupt the regular packing of
the TPR helices such that PTS1 protein recognition is affected as a
secondary effect. Indeed, we showed that changing a mutational proline
in an
-helix to alanine rescued Mdh3p recognition. Several
inactivating mutations in Pex5 proteins of different species have been
reported in the literature (43, 44). These mutations were found to involve substitutions of glutamic acid residues (a bulky amino acid)
for glycine residues located at position 8 of helix A in TPRs. Our
modeling studies suggest that the stacking of the TPR helices might be
compromised in these mutant Pex5 proteins.
The other loss-of-interaction mutations and most suppressor mutations
were located in the short hairpin loops of TPR2, TPR3, and TPR6 that
connect helices A and B (Fig. 4). These loops are somewhat exposed from
the folded TPR structure and probably form the direct contact site for
PTS1 proteins. In support of this, we found that changing a disabling
mutation in the loop of TPR3 to alanine did not restore PTS1 protein
recognition. Apparently, a much more critical property is involved
here, related to the side chain of the original amino acid, which would
be in line with direct interaction with Mdh3p. Additional site-directed
mutagenesis of conserved residues in intra-repeat loops underscored the
essential role of the loops of TPR2 and TPR3 in PTS1 interaction.
Interestingly, some of these mutants showed a differential effect when
tested in the two-hybrid trap against Mdh3p and GFP-SKL: interaction with Mdh3p remained or was slightly reduced, but interaction with GFP-SKL was completely lost. One possible explanation for this differential effect is that Mdh3p, an authentic peroxisomal matrix protein of yeast, contains, in addition to its PTS1, other sequences (so-called accessory sequences) that contribute to Pex5p binding. Most
likely, a heterologous, non-peroxisomal protein like GFP does not
contain such additional sites that can interact with Pex5p. Therefore,
it is completely dependent on the added PTS1 for the interaction with
Pex5p. The presence of amino acid sequences outside PTS1 that might
contribute to receptor recognition has been suggested before (34, 41,
45, 46).
A number of TPR structures have now been described that have
contributed significantly to our understanding of how TPR domains interact with their targets. In addition to the x-ray structure of the
isolated TPR domain of PP5 (29), two complex structures have been
recently published (47, 48). The complexes between the adaptor protein
Hop and peptides derived from either Hsp70 or Hsp90 showed that the
peptides bind to a groove formed on the helix A face of the TPR domain
(the general binding groove; see also Fig. 4). This general binding
groove for peptides in TPR domains had been predicted by Das et
al. (29) based on the isolated PP5 structure. Interestingly, the
second complex structure of the small GTPase Rac bound to the TPR
domain of p67phox revealed a novel mode of interaction
involving only the loop regions connecting TPR motifs. Our data now
show the importance of the intra-repeat loops of TPRs in target
recognition, suggesting yet another structural variation of TPR
motif-mediated protein-protein interaction.
Our Pex5p modeling studies suggest that the TPR domain does not
form a tandem array of seven TPRs, but rather two distinct clusters of
three TPR motifs (TPR1-3 and TPR5-7) that are connected by a
(flexible) linker of 42 amino acids (TPR4). Given the relative small
size of PTS1 (three amino acids) and the distribution of mutations
affecting PTS1 binding over both TPR clusters, it is tempting to
speculate that the two clusters of TPRs are localized close together in
space forming a single binding site for PTS1. The absence of a crystal
structure of Pex5p prevents the description of the interaction of the
TPRs with the PTS1 amino acids at the molecular level. However, based
on our mutational analysis and the homology model, some predictions can
be made. Very striking is the negatively charged patch in TPR1-3
formed by strictly conserved glutamic acid residues in intra-repeat
loops 2 and 3 (Fig. 4, A and B). These residues
might be involved in binding the positively charged amino acid at
position
2 of PTS1 via electrostatic interactions. In Mdh3p and
GFP-SKL, residue
2 is lysine, but other positively charged amino
acids like arginine and histidine can also be found at this position
(4, 5). This notion is supported by the charge-shift suppressor
mutation E361K in the loop of TPR2, which was isolated in a screen with
Mdh3p containing a negatively charged residue (glutamic acid) at
position
2. Close to the negatively charged residues in the loops of
TPR2 and TPR3, two conserved residues are located that are essential
for PTS1 binding: asparagine 393, found to be mutated in several
independent clones in the loss-of-interaction screen, and isoleucine
389 (valine in human PEX5). An asparagine residue can be
involved in different types of interactions because its side chain is
able to both donate and accept hydrogen bonds. In particular,
interactions of asparagine side chains with the backbone of short
peptides have been well documented (49, 50). Isoleucine 389 might be
important for contacting the hydrophobic side chain of leucine at
position
1. Other residues that can be found at this position in PTS1
signals are either large hydrophobic (methionine) or aromatic
(phenylalanine) amino acids.
The exact contribution of TPR5-7 to PTS1 binding cannot be easily
extracted from our data. However, while our work was in progress, a
speculative model for the interaction of only TPR5-7 of human PEX5
with PTS1 was published (51). This model, which is based solely on
homology modeling and orthologous sequence information, highlights the
importance of four strictly conserved asparagine residues in the A
helices of TPR6 and TPR7. These asparagine residues are predicted to
recognize the backbone of PTS1. One of these asparagine residues
(Asn503) (Fig. 4, C and D) was found
to be mutated in two independent clones (pex5.sup3 and
pex5.sup21) in our suppressor screen. Remarkably, the
phenotype of these suppressors (and the S534L suppressor) was different
from that of the previously mentioned charge-shift suppressor E361K.
Whereas the E361K mutant showed no interaction with Mdh3p without its
PTS1 (Mdh3
SKL), the other suppressors still displayed (a weak)
binding to Mdh3
SKL. This phenotype might be related to the possible
role of asparagine 503 (and serine 534) in peptide backbone recognition
(51). Finally, the model of Gatto et al. (51) predicts a
role for an absolutely conserved arginine residue in helix A of TPR7
(Arg526 in yeast Pex5p) in binding the carboxylate oxygens
of the PTS1 C terminus. Our experimental data support this prediction
since substitution of arginine 526 with alanine or glycine completely abrogated PTS1 interaction (Tables I and III).
The work described above demonstrates that by combining homology
modeling and mutational analysis, we were able to put forward a
possible model as to how PTS1 is recognized by the TPR domain of Pex5p.
Further refinement of this model requires the crystal structure of the
Pex5p TPR domain in complex with a PTS1 protein.