1

Recognition of a functional peroxisome type 1 target by

the dynamic import receptor Pex5p

Will A. Stanley1, Fabian V. Filipp2, Petri Kursula1, Nicole Schüller1, Ralf

Erdmann3, Wolfgang Schliebs3, Michael Sattler2, Matthias Wilmanns1*

1EMBL-Hamburg Outstation, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany

2Structural and Computational Biology Unit, EMBL-Heidelberg, Meyerhofstrasse 1,

69117 Heidelberg, Germany

3 Institute for Physiological Chemistry, Department of Systems Biology, Faculty of

Medicine, Ruhr University of Bochum, 44780 Bochum, Germany.

*Correspondence:

Email: wilmanns@embl-hamburg.de

Phone: +49-40-89902-110

Fax: +49-40-89902-149

Running Title: Target recognition by the import receptor Pex5p

* C) Manuscript

2

Summary

Peroxisomes require the translocation of folded and functional target proteins of

various sizes across the peroxisomal membrane. We have investigated the

structure and function of the principal import receptor Pex5p, which recognizes

targets bearing a C-terminal peroxisomal targeting signal type 1. Crystal

structures of the receptor in the presence and absence of a peroxisomal target,

sterol carrier protein 2, reveal major structural changes from an open, snail-like

conformation into a closed, circular conformation. These changes are caused by a

long loop C-terminal to the seven-fold repeated tetratricopeptide repeat segments.

Mutations in residues of this loop lead to defects in peroxisomal import in human

fibroblasts. The structure of the receptor/cargo complex demonstrates that the

primary receptor binding site of the cargo is structurally and topologically

autonomous, enabling the cargo to retain its native structure and function.

3

Introduction

Diverse machineries involved in translocating proteins across organellar membranes are

required to maintain the compartmentalization of biological processes within eukaryotic

cells (Kunau et al., 2001; Wickner and Schekman, 2005). Many components of

membrane receptors recognize specific targeting sequences in proteins destined for

translocation (Eichler and Irihimovitch, 2003). However, they differ in their

requirements for cargo folding/unfolding during the translocation process to retain the

functional integrity of the cargo. To date, the only well characterized system supporting

the translocation of folded protein targets is the nuclear import/export system by

karyopherins (Conti and Izaurralde, 2001; Matsuura and Stewart, 2004). The extent to

which other translocation systems may resemble karyopherin-mediated processes

remains elusive.

Peroxisomal import is one of the few transport processes that uses a translocon for the

purpose of trafficking folded and functional cargo proteins across membranes (Gould

and Collins, 2002; Holroyd and Erdmann, 2001; Lazarow, 2003; Schnell, 2000; van der

Klei and Veenhuis, 2002). To date, more than two dozen proteins involved in

trafficking cargo to the peroxisome—referred to as peroxins—have been identified and

partially characterized. No pore-like structure in the peroxisomal membrane has thus far

been observed, and the exact composition of the import translocon, possibly assembled

according to the size and type of import substrates, has yet to be established. The

majority of peroxisomal matrix proteins destined for translocation into peroxisomes

share the C-terminal type 1 peroxisomal targeting signal (PTS1) motif. It comprises an

obligatory C-terminal tripeptide, conforming to the consensus sequence -[S/A/C]-

[K/H/R]-[L/M]-CO2-, which is specifically recognized by the C-terminal segment of the

4

import receptor peroxin Pex5p. Human diseases leading to impaired fatty acid

metabolism, organ dysfunction, and neonatal mortality, such as Zellweger syndrome,

are often caused by mutations in the Pex5p receptor (Weller et al., 2003), rendering the

receptor an important target for biomedical research.

At the molecular level, these Pex5p translocation targets appear to be released into the

peroxisomal lumen by interactions between the receptor and other peroxins and by its

association with the peroxisomal membrane (Gouveia et al., 2003a; Holroyd and

Erdmann, 2001; Madrid et al., 2004). Cargo loading may also influence the oligomeric

state of Pex5p and its interactions with other peroxisomal membrane docking factors,

such as Pex14p (Madrid et al., 2004; Wang et al., 2003). Other evidence demonstrates

that the cargo-loaded Pex5p receptor may even shuttle across the peroxisomal

membrane (Dammai and Subramani, 2001). However, to date, questions still remain

regarding whether, or to what extent, the receptor, or parts of the receptor, physically

shuttle or just become accessible to the peroxisomal lumen (Erdmann and Schliebs,

2005; Kunau et al., 2001).

To investigate the molecular requirements of this dynamic receptor both for cargo

loading and release, we determined the structures of the PTS1-cargo binding region of

the Pex5p receptor in the presence and in the absence of a peroxisome translocation

target, sterol carrier protein 2 (SCP2). The target is bound to the receptor by two

separate binding sites—a C-terminal PTS1 motif and a topologically separate secondary

site—providing a rationale as to how the target remains folded and functional during

translocation. A comparative analysis of the two Pex5p receptor structures reveals major

conformational changes in the receptor upon cargo loading, which are generated by the

loose structural arrangement of the receptor tetratricopeptide repeat (TPR) segments and

5

by the intrinsic structural flexibility within some of these structural segments. Our data

reveal unexpected common principles governing the conformational changes observed

in the peroxisome Pex5p import receptor and karyopherins, providing insight into the

major determinants of protein import/export into the peroxisome.

6

Results

Choice of a functional receptor/cargo system

In order to unravel the molecular basis of PTS1-driven protein translocation via the

Pex5p receptor into peroxisomes, we searched for a suitable physiological target that

could serve as a model system. We selected sterol carrier protein 2 (SCP2), which

contains a canonical C-terminal PTS1 motif (Seedorf et al., 1998). Its structure has been

characterized previously, both by X-ray crystallography and NMR spectroscopy

(Choinowski et al., 2000; Garcia et al., 2000). The SCP2 gene is translated into two

protein products: SCPx, a 58 kDa fusion protein comprising an N-terminal thiolase

domain and a C-terminal SCP2 domain, and preSCP2, a protein with a molecular mass

of about 15 kDa, which is processed into its mature form (mSCP2) by proteolytic

cleavage of a 20-residue leader sequence after translocation into peroxisomes (Figure

1). SCP2 binds to the Pex5p receptor both under in vivo and in vitro conditions,

allowing structural investigation of the receptor/cargo complex.

We used NMR spectroscopy and isothermal titration microcalorimetry (ITC) to

determine the molecular requirements of SCP2 for receptor binding. For both the

translated (pre) and the processed mature (m) forms—preSCP2 and mSCP2,

respectively—the chemical shift perturbations in 1H,15N correlation spectra upon

binding to Pex5p(C) affect the same set of SCP2 residues (Supplement, Figures S1

and S2). Furthermore, chemical shift perturbation and line width analysis show that the

presequence remains highly dynamic and is not involved in receptor binding. The

binding affinities of preSCP2 and mSCP2 for the receptor, as measured by ITC, are

7

both in the order of 100 nM (Table 1), indicating that the presequence is tolerated and

does not affect the receptor interaction.

To determine whether SCP2 retains its function upon loading onto the Pex5p import

receptor, we used two different but complementary approaches. First, we investigated

the capacity of SCP2 to bind specific lipids required for metabolic processes inside

peroxisomes in the presence and in the absence of Pex5p(C). To locate the lipid binding

site with SCP2, we used spin-labeled paramagnetic doxyl stearate as a fatty acid

derivative (Garcia et al., 2000). We observed similar bleaching of NMR signals upon

binding of this fatty acid derivative to either free SCP2 or Pex5p(C)-bound SCP2,

indicating that the functional integrity of SCP2, in terms of lipid binding, is not

impaired upon receptor loading (Figure 2). We further used ITC to quantify the

thermodynamic parameters governing the receptor-cargo interaction in the presence and

in the absence of stearoyl-CoA, a physiological ligand of SCP2 (Frolov et al., 1996).

The data show that mSCP2 binds to the receptor with about the same Kd and 1:1

stoichiometry, regardless of whether it is loaded with stearoyl CoA (Table 1).

Structure of the cargo-loaded Pex5p receptor complex

We have determined the crystal structure of the C-terminal part of the Pex5p import

receptor (Pex5p(C), residues 315-639) in the presence of the mature form of the

peroxisomal matrix protein SCP2, mSCP2(21-143), at 2.3 Å resolution (Figures 1, 3A-

B, 4A-B, 5; Table 2). The X-ray data reveal the complete structure, except for the very

N-terminus (315-334) and one loop (441-453) of Pex5p(C). Most of its structure is

formed by seven consecutive TPR motifs, each consisting of a helix-turn-helix motif

(D'Andrea and Regan, 2003). The forth segment, which matches the established TPR

8

sequence signature, is in a distorted arrangement (Figure 5A) and is preceded by a

glycine-rich loop that is, in part, flexible. The C-terminus of the structure is folded into

a three-helical bundle of which the first two helices display TPR-like properties, both in

terms of sequence signature pattern and structure. The long loop connecting the seventh

TPR segment and the C-terminus (589-601), referred to as “7C-loop”, interacts with the

two helices from the TPR1 segment (Figure 5B). This loop and the distorted TPR4

segment link the two arch-shaped TPR motif triplets (1-3, 5-7), thus generating a

pseudo-circular structure of the cargo-loaded receptor with a tunnel in its center, which

is open to both faces of the ring-like structure (Figure 4B). The connecting segments

represent the most mobile regions of the cargo-loaded receptor (Supplement, Figure

S3A) and, therefore, can be regarded as hinges. The C-terminal helical bundle does not

participate in the circular arrangement (Figures 3A-B and 5A-B).

The structure of the receptor-bound mSCP2 resembles that of free SCP2 (Choinowski et

al., 2000), except the C-terminus that bears the PTS1 motif (Supplement, Figure S3C).

In the Pex5p(C) receptor complex, the ten C-terminal residues (134-143) of mSCP2

adopt an extended conformation, pointing away from its core domain. However, unlike

the structure of free SCP2, wherein both termini are flexible, in the receptor-bound

SCP2 structure, the C-terminus becomes the most rigid part of the overall structure

while its N-terminus remains mobile (Supplement, Figures 1C and S3B-C). The most

C-terminal AKL motif (141-143) of mSCP2 binds within the central hole of the ring-

like structure of the receptor. It is involved in specific interactions with four asparagines

(N415, N526, N534, N561) that are located on the N-terminal helices of TPR segments

4, 5, 6, and 7. These residues are conserved (Figures 1, 5A-B), indicating that PTS1

binding is a general property of the Pex5p receptor. In contrast to the most C-terminal

9

region, the preceding residues (134-140) interact with the receptor by van der Waals

forces only.

The structure of the receptor/cargo complex also reveals a second interaction site of

about 500 Å2 that is formed by the C-terminal helical bundle of Pex5p(C) and a surface

patch of SCP2, covering parts of helices 1 and 3 (Figures 1, 4A-B, 5B). ITC binding

data using the PTS1 peptide motif only reveal that its binding affinity to Pex5p(C) is

reduced (Kd = 664 nM) compared with the entire protein cargo (Table 1). Thus, the data

indicate that there is a notable contribution by the secondary interface in SCP2 loading

onto the Pex5p receptor. However, none of the residues of either the Pex5p receptor or

SCP2 involved in these interactions are invariant (Figure 1). These findings suggest

that, in contrast to the PTS1-mediated cargo/receptor interactions, the formation of the

secondary SCP2/Pex5p(C) interface is specific and may not be conserved taxonomally.

Since PTS1 targets are generally unrelated in terms of structure and function, with the

exception of the C-terminal PTS1-receptor recognition motif, our findings suggest that

the involvement of secondary binding sites may serve as a determinant for the sorting of

folded import substrates.

Our NMR spectroscopy data on mSCP2 in the presence and in the absence of the

receptor correlate with the crystallographic analysis. The largest chemical shift

perturbations are found for the backbone amides of the C-terminus (136-143)

(Supplement, Figure S1B), coinciding with the structural alterations observed by

comparing the structures of unbound SCP2 and Pex5p(C)-bound SCP2 (Figure 5B;

Supplement, Figure 3C). NMR relaxation measurements indicate that the PTS1

backbone, which is flexible and disordered in free SCP2, rigidifies upon binding to

10

Pex5p(C) (Supplement, Figure 1C). Significant chemical shift changes have also been

detected for residues in the secondary binding site.

Taken together, our biochemical and structural data suggest bipartite binding of the

SCP2 cargo to the Pex5p receptor. The PTS1 binding site at the most C-terminal region

is structurally and topologically well separated from the functional SCP2 core domain.

As such, the data provide a rationale for our observations on a mechanism of receptor

recognition that does not interfere with the function of SCP2 as lipid binding protein.

Our data, however, do not support previous hypotheses proposing that binding of lipid

substrates to members of the SCP2 family may enhance the exposure of the PTS1 motif,

thereby driving ligand-dependent translocation (Choinowski et al., 2000; Garcia et al.,

2000; Lensink et al., 2002).

Structure of the unliganded Pex5p receptor

Protein translocation into peroxisomes requires a delicate balance between the binding

and release of cargo proteins to and from the appropriate import receptor. To investigate

the molecular parameters that govern cargo release, we have also determined the

structure of the import receptor Pex5(C) in the absence of a cargo at 2.5 Å resolution

(Figures 3C, 4C; Table 2). We have found a crystal form that contains four Pex5p(C)

molecules per asymmetric unit. For two of these, the entire sequence is well defined in

electron density, except for a gap from part of the TPR4 segment. In the other two

Pex5p(C) molecules, the TPR segments 5-7 and the C-terminal helical bundle are

reasonably well defined, whereas the N-terminal TPR domains 1-3 could only be

modeled approximately, as reflected in high average mobility factors (Table 2,

Supplement Figure S3A). There is, however, no evidence to support the unfolding of

11

considerable parts of the TPR segments. Previous circular dichroism data have shown

that the secondary structural content of both receptor conformations is almost identical

(Stanley et al., 2004).

A direct comparative analysis of the cargo-loaded and unloaded structures of the

Pex5p(C) receptor reveals major conformational changes upon cargo binding. Contrary

to previous hypotheses proposing that the TPR4 segment is a flexible hinge (Gatto et

al., 2000), these changes originate from three residue clusters in TPR segments 5 and 6,

rendering a rotation of about 20 degrees of the C-terminal TPR segments with respect to

N-terminal TPR segments (Figure 3D). As a result of this conformational change, the

7C-loop of the apo-structure is no longer capable of completing a ring-like structure of

Pex5p(C) as observed in the cargo complex, thus generating an open, snail-like

arrangement of the receptor (Figure 3C, Figure 4C). For instance, Gln586 and Ser600,

which interact with residues from the TPR1 segment in the cargo-loaded receptor

(Figure 5B), have moved by more than 8 Å in the apo-structure.

Since the overall arrangement of TPR segment–containing structures can be described

as a superhelical coil or solenoid (D'Andrea and Regan, 2003; Jinek et al., 2004), we

compared the underlying structural parameters of the cargo-free and cargo-loaded

structures of the Pex5p receptor. Our analysis revealed that the superhelical pitch is

about 30 Å in the apo-structure rather than 20 Å in the SCP2-Pex5p complex.

Furthermore, the N-terminal helices of TPR7 (556-568) and the C-terminal helical

bundle (601-613), which bear several residues that are involved in binding of the cargo

PTS1-motif, are moved by the equivalent of about two -helical pitches with respect to

the N-terminal TPR segments, leading to a displacement of part of the PTS1 motif

binding site by several Ångstroms (Figure 3D). The highly flexible arrangement of the

12

third and the fourth molecule found in the apo-Pex5p(C) crystals suggests that there

may be even more conformational freedom than that observed in the available apo-

crystal structures. These suggestions are consistent with our ITC data, indicating that

although the binding affinity is in the nanomolar range, there is only a small

contribution, or even a loss of entropy, during cargo binding (Table 1). Indeed, a recent

study has demonstrated coupled folding and ligand binding in at least one TPR array

(Cliff et al., 2005).

Our model allows for speculation on further possible structural changes. Although

TPR4 is not involved in the conformational changes evident from our comparative

analysis of the apo-and cargo-loaded Pex5p receptor structures, we cannot exclude the

possibility that there are steps during the target-import cycle that affect this receptor

segment as well. For instance, complete folding of the distorted TPR4 motif into the

canonical TPR domain structure would only require minor changes in the flexible loop

N-terminal to the TPR4 segment (Figure 5A). The resulting overall structure could

open up into a superhelical arrangement with a pitch in the order of 55 Å, reminiscent of

previous observations in another TPR segment–containing structure (Jinek et al., 2004).

In contrast to the observed changes in the receptor structures in the presence and in the

absence of cargo, the overall conformation of the cargo-loaded Pex5p(C) receptor

structure remains virtually identical regardless of whether it is bound only to the C-

terminal PTS motif (Gatto et al., 2000) or to a complete cargo target, as shown by the

Pex5p-SCP2 complex.

Residues from the 7C-loop are critical for in vivo PTS1 import

In contrast to several known peroxisome disease mutations wherein direct interactions

13

with the PTS1 motif are abolished, a patient with an inherited peroxisome biogenesis

disorder, infantile Refsum disease, was found to be impaired in the import of proteins

containing only the AKL- and KANL-type PTS1 motifs, such as SCP2 and catalase

(Shimozawa et al., 1999). In this patient, dysfunctional import into peroxisomes is

linked to mutation S600W in Pex5p. Comparative analysis of the cargo-loaded and

unloaded structures of the Pex5p(C) receptor reveals that Ser600, which is situated at

the base of the 7C-loop, plays a central role in connecting the C-terminal and N-

terminal TPR segments, to arrange the PTS1 binding site as well as the secondary

binding site at the C-terminal helical bundle in the Pex5p(C)/SCP2 complex (Figure

5B).

In order to validate the involvement of the 7C-loop in PTS1 target import we mutated

three residues (Gln586, Ser589, Ser600), which are involved in specific interactions of

this loop with other parts of the receptor in the cargo-bound bound conformation

(Figure 5B). As a control, we chose one single residue mutant from the TPR2 segment

(N382A), which previously was shown to be involved in PTS1 cargo import by the S.

cerevisiae Pex5p receptor (Klein et al., 2001). First, we measured the binding affinity of

the cargo SCP2 to the resulting Pex5p variants under in vitro conditions (Table 1). As

expected, no binding could be detected for the S600W mutant, while a more than ten-

fold reduced binding affinity was observed for the Q586R Pex5p variant, thus

demonstrating the critical involvement of this residue from the 7C-loop in SCP2 cargo

recognition as well. On the other hand, the cargo binding by the Pex5p S589Y was only

slightly reduced.

The same mutations were introduced in full-length Pex5p and the resulting variants

were expressed in a fibroblast cell line devoid of endogeneous PTS1 receptor.

14

Transfected cells were analyzed by fluorescence microscopy for their capacity to import

two established PTS1 peroxisome targets, catalase and SCP2 (Shimozawa et al., 1999).

While SCP2, due to its low molecular weight and absence of evidenced tendencies for

oligomerization, can be considered as a model target with only modest structural

requirements for import in its functional form, catalase forms a homo-tetrameric heme

containing assembly with a molecular mass of about 240 kDa, thereby demanding

considerable additional structural requirements for peroxisomal import (Purdue and

Lazarow, 1996). As a control, we tested Pex5p-dependent import of the PTS2-tagged

reporter protein, chloramphenicol acetyltransferase (CAcT) as well. To ensure that the

observed import defects are not because of a reduced amount of mutant due to lower

expression or increased turnover we have analyzed the expression of all Pex5p mutants

in a Pex5p-free cell line 24 hours after transfection with the corresponding plasmids by

immunoblotting (Supplement, Figure S4), demonstrating that all Pex5p mutants were

synthesized at their full-length. All mutant proteins are found at a steady-state level

comparable with wt Pex5p expressed from the same plasmid. The levels of expression

were significantly increased when compared with wt Pex5p under control of its

endogenous promotor.

The transfected wt Pex5p receptor correctly directed both PTS1 targets, catalase and

SCP2, as well as the PTS2-tagged CAcT into the peroxisomal matrix (Figure 6). As

expected, two of the 7C-loop mutants (Q586R, S589Y) and the PTS1 reference mutant

(N382A) mediated properly the import of PTS2 proteins but showed severe defects for

catalase targeting. Retarded import was observed for SCP2, most apparently when

expressing the Q586R mutant. During the first 24 hours after transfection the bulk of

GFP-SCP2 remained in the cytosol and only a few peroxisomes were detected by

fluorescence (Figure 6). The number of SCP2 containing peroxisomes further increased

15

with incubation time and after two to four days nearly all peroxisomes were labeled

(data not shown). Moreover, the Pex5p S600W mutant was not able to rescue either

catalase or SCP2 import during the whole time-course of the experiment (Figure 6).

By taking the our in vitro and in vivo data together, both lines of evidence demonstrate

the essential contribution of the Ser600 side chain in the peroxisomal import of PTS1

targets by the Pex5p receptor. Indeed, Ser600 is the only residue at the distal end of the

7C-loop that connects the C-terminal helical bundle with the TPR segments via a

specific hydrogen bond to a residue from the TPR1 motif, upon SCP2 cargo binding. In

structural terms, abolition of this interaction is expected to impair both the function of

the 7C-loop as closing element of the ring-like conformation of the cargo-bound

receptor as well as on the proper arrangement of the C-terminal helical bundle with

respect to the N-terminal TPR segments of the receptor. In contrast, the other two

mutants (Q586R, S589Y) are located at the N-terminus of the 7C-loop, which is

proximal to the preceding TPR segments. Gln586 is involved into a multi-hydrogen

bond network, involving residues from the first TPR segment and Ser600 (Figure 5B).

Our data show that abolition of specific interactions from these residues, in particular of

Gln586, leads to serious import defects as well. Comparison of the data for SCP2 and

catalase, however, indicates an amplification of the effect for the latter one, which is

expected to be more sensitive because of its oligomeric arrangement and requirement

for cofactor binding. Although a quantitative interpretation of this differential effect will

only be possible once a structure of receptor-catalase cargo complex becomes available,

it is intriguing to hypothesize on additional sorting effects for the import of different

PTS1 cargos, supporting or complementing previous observations (Kiel et al., 2004;

Knott et al., 2000; Otera et al., 2002).

16

Discussion

Pex5p receptor recognition of diverse PTS1 targets

We have analyzed our structural findings of the Pex5p import receptor to determine

whether, or to what extent, they infer general principles applicable for the translocation

of a variety of folded proteins into peroxisomes. For mSCP2, our data suggest a

bipartite recognition mechanism, via the C-terminal PTS1 motif and a secondary, less

conserved binding site that is distinct in terms of sequence and structure. Much attention

has previously focused on residues preceding the C-terminal tripeptide PTS1 motif,

suggesting that some of these may serve as determinants for altering binding affinity

(Lametschwandtner et al., 1998; Neuberger et al., 2003). However, the structure of the

Pex5p(C)/mSCP2 complex has not revealed any further specific cargo interactions with

the receptor beyond the C-terminal PTS1 motif. Although several hydrophobic

interactions can be observed these interactions may change in other PTS1 targets, in

which the orientation of the linker region between the PTS1 motif and the functional

domain structures may be different. As such, it is difficult to quantify possible

contributions of residues from the linker, connecting the core domain of SCP2 and the

PTS1 C-terminus to Pex5p receptor.

Recent data on the recognition of another peroxisome target, alanine:glyoxalate

aminotransferase (AGA), by the Pex5p receptor indeed support our findings on bipartite

cargo binding by a second topologically remote interaction site separated by about 40

residues in the AGA sequence (Huber et al., 2005). Since considerable variation has

been observed in the sequence within the PTS1 motif of different targets, it is plausible

17

to assume that there is a correlation between a weakened PTS1-Pex5p receptor

interaction and a need for secondary binding sites on the cargo surface.

A peculiar property of the Pex5p import system is its capability to allow translocation of

even large-folded proteins, either monomeric or oligomeric (Brocard et al., 2003;

Lazarow, 2003; Walton et al., 1995; Yang et al., 2001). Some PTS1 targets can be

imported into peroxisomes either by a PTS1-dependent or PTS1-independent

mechanism (Parkes et al., 2003). On the other hand, there has been recent biochemical

and structural evidence demonstrating that oligomerization of proteins established as

physiological peroxisomal targets may actually inhibit PTS1-driven translocation (Faber

et al., 2002; Modis et al., 1998). In this scenario, a possible blockade of secondary

binding sites, as, for instance, by oligomerization, appears to be more likely than the

PTS1 motif, as predicted by previous data (Parkes et al., 2003) and supported by the

Pex5p(C)/mSCP2 complex structure reported here.

The Pex5p import receptor cycle

The generally accepted model for PTS1-driven import of peroxisomal targets considers

at least four steps for one import cycle: (a) cargo recognition by the Pex5p receptor, (b)

cargo-loaded receptor docking and, possibly, integration into the peroxisomal

membrane, (c) cargo release into the peroxisomal lumen, and (d) recycling of the

unloaded receptor into the cytosol (Erdmann and Schliebs, 2005). The model proposes

two essential types of binding/unbinding events that are likely to be associated with

considerable conformational changes in the receptor: loading/unloading of the cargo and

docking/release of the receptor to/from the peroxisomal membrane. For canonical

PTS1-driven import of peroxisomal targets, loading/unloading of the cargo seems to be

18

confined to the C-terminal part of the receptor. Our comparative analysis of the near

atomic resolution structures of the Pex5p(C) receptor enables the unraveling of the

molecular parameters governing the conformational changes associated with cargo

loading. In contrast, data on the N-terminal part of Pex5p, which is thought to be largely

unfolded (Costa-Rodrigues et al., 2005), have indicated that this region is critically

involved in membrane docking/release of the receptor, either by interactions with other

docking factors of the translocon, such as Pex13p and Pex14p as well as Pex8p and

Pex17p (only demonstrated in yeast), or by direct interactions with the peroxisomal

membrane (Agne et al., 2003; Gouveia et al., 2003b; Schäfer et al., 2004). The model

implies that these interactions could have an effect on the affinity of the peroxisomal

target for the C-terminal part of the receptor, ultimately leading to cargo release. Pex8p

appears to play a key role in this process, either as a cargo release factor, as suggested

from in vitro data of the H. polymorpha Pex5p receptor (Wang et al., 2003) or as an

inducer of a subsequent translocon complex with additional components allowing cargo

release (Agne et al., 2003). Regardless the precise origin of the conformational changes

in the C-terminal part of the receptor, both the canonical nature of the loose

arrangement of the TPR domains and our direct comparative analysis of the cargo-

loaded and unloaded receptor structures suggest a ring-opening mechanism for

peroxisomal target release.

Common principles in protein translocation systems

A direct comparative analysis of the apo- and cargo-loaded conformations of the Pex5p

receptor has led to unraveling the molecular basis of loading the most widespread class

of peroxisomal targets—i.e. those containing a C-terminal PTS1 signal motif—onto the

receptor for import into peroxisomes. However, data from other peroxisome receptors

19

are too scarce to allow further investigations of common/divergent principles involved

in peroxisomal import. To date, the most widely investigated family of receptors have

been the karyopherins, which are involved in the signal-assembled import/export of a

wide range of targets into the nucleus (Chook and Blobel, 2001; Conti and Izaurralde,

2001). These receptors recognize nuclear localization signal (NLS) containing targets

either directly or via adaptor proteins, with cargo loading/unloading being regulated by

binding to RanGDP/GTP binding proteins. Unlike the case for nuclear transport, for

PTS-driven translocation by the Pex5p receptor, there is no evidence supporting

protein-mediated cargo translocation or involvement of further loading factors.

Nevertheless, the general principles for protein translocation inferred from our data are

surprisingly similar to those governing karyopherins. First, NLS peptides are bound in

an elongated coiled conformation, as is found here with PTS1. In addition, large parts of

karyopherin receptors comprise repeated -helical domain structures that are classified

by HEAT or ARM sequence motifs, which are analogous to those in the TPR segments

of the Pex5p receptor. In terms of overall arrangement of receptor recognition, our

findings on the cargo-Pex5p receptor complex are, for instance, reminiscent of the

structure of the cargo-loaded Kap()1 receptor, in which the circular structure of the

receptor binds an N-terminal fragment of the Kap() substrate adaptor into a central

tunnel-like cavity (Cingolani et al., 1999). Furthermore, recently determined structures

of the export receptor Cse1 in the apo-conformation (Cook et al., 2005), RanGTP-

bound form (Lee et al., 2005) as well as the ternary complex with RanGTP and

Kap60p (Matsuura and Stewart, 2004) have revealed unexpected structural insight into

the molecular parameters that support binding of the Kap60p adaptor, which, in turn,

inhibits binding of the NLS-containing targets. Comparison of these data with the

cargo-loaded/unloaded conformations of the Pex5p receptor reveal two further common

20

principles governing structural adaptations during the respective translocation cycles. In

both systems, overall conformational changes are triggered by local changes in

structural modules that may be propagated into modified arrangements with adjacent

modules. For instance, a comparison of the cargo-loaded structure with the unloaded

structure of karyopherin Cse1 have unraveled a conformational switch in the HEAT

repeat 8 (Conti and Izaurralde, 2001; Cook et al., 2005), leading to altered HEAT repeat

arrangements N- and C-terminal to the switch. Taken together, the comparative analysis

of the cargo-loaded and unloaded structures of the Pex5p receptor provides evidence for

a similar structural role of the TPR4 segment and adjacent TPR regions. Second, in both

systems—the karyopherins and the Pex5p receptor—conformational changes can be

described by alterations in the underlying geometrical parameters of each superhelical

domain arrangement. In both systems, binding of regulators, adaptors, and/or substrates

lead to changes in the overall superhelical pitch values that may be described as ring-

like, snail-type, and open arrangements (Conti and Izaurralde, 2001; Lee et al., 2005).

Thus, emerging evidence points to structural and functional similarities in the molecular

principles governing unrelated translocation receptors containing repeated helical

domain structures. Indeed, in the light of the recently detected common architectural

features of protein components of nuclear pore complexes and coated vesicles (Devos et

al., 2004; Devos et al., 2006) it may well be possible that there are common principles

in structural rearrangements in an increasing list of proteins with -solenoid

conformations, in which our findings on structural plasticity of the Pex5p import

receptor fit as well.

21

Experimental Procedures

Protein preparation

All versions of human Pex5p(C) (residues 315-639), human preSCP2 (residues 1-143),

and mSCP2 (residues 21-143) were expressed from a modified pET24d vector

(prepared by G. Stier, EMBL-Heidelberg) in E. coli BL21(DE3). Mutants N382A,

Q586R, S589Y and S600W of Pex5p(C) were created using the Quickchange XL-Site

Directed Mutagenesis Kit (Stratagene). The expressed proteins contained an N-terminal

His6-GST fusion, cleavable with tobacco etch virus (TEV) protease. Cultures were

grown in Tris-buffered LB medium supplemented with 1% (w/v) glucose, and induced

mid-log-phase with 0.5 mM IPTG for 6 hours at 21 °C. Following resuspension of the

cell pellets, cells were lysed by sonication in the presence of protease inhibitors. The

lysate was cleared by centrifugation, loaded onto a glutathione Sepharose 4B resin

(Pharmacia), and then eluted with 20 mM reduced glutathione. TEV protease was added

until fusion proteins were completely cleaved and the mixture was applied to Ni-NTA

agarose (QIAgen). The flow-through was subsequently subjected to gel filtration

through a Superdex 75 (16/60) column (Pharmacia).

Crystallization and X-ray data acquisition

Pex5p(C) and mSCP2 were mixed in a 2:3 molar ratio and dialyzed against 20 mM bis-

Tris-propane, 20 mM KCl, and 1 mM TCEP (pH 7.0). The protein mixture was then

concentrated to ~7 mg ml-1 total by ultrafiltration, and crystallization was carried out by

mixing 1 l protein with 1 l reservoir solution using the hanging drop vapor diffusion

method at 20 °C. Reservoir buffer conditions were optimized to 24% (w/v) PEG 3350,

22

175 mM NaCl and 100 mM bis-Tris (pH 6.5). Crystals grew within 4-6 weeks. Prior to

X-ray data collection, 10% (v/v) PEG 400 was added to the drops containing crystals

for 5-10 mins. Crystals of unliganded Pex5p(C) were obtained by mixing 1 l protein at

a concentration of 7 mg/ml with 1 l reservoir solution using the hanging drop vapor

diffusion method at 20 °C. Reservoir buffer conditions were optimized to 23% (w/v)

PEG 3350, 100 mM Tris-HCl (pH 8.75), and 0.22 mM octaethylene glycol monolauryl

ether. Crystals grew within 3-5 weeks.

X-ray data were collected at the synchrotron beamlines X13 at EMBL/DESY,

Hamburg, Germany, and at BL1 at BESSY, Berlin, Germany. Data were obtained from

single crystals at 100 K in a stream of gaseous nitrogen. Data were processed and scaled

using XDS (Kabsch, 1988), and then analyzed using SFCHECK (Vaguine AA et al.,

1999). For cross-validation of subsequent steps, 5% of the reflections from each data set

were randomly selected.

X-ray structure determination

Pex5p(C)/mSCP2 complex: The X-ray structure was solved by molecular replacement

using MOLREP (Vagin and Teplyakov, 1997). Initially, Pex5p(C) was located using the

PDB coordinates 1FCH (Gatto et al., 2000) as a model. Subsequently, SCP2 was found

using the PDB coordinates 1C44 (Choinowski et al., 2000) as a model. REFMAC-5

(Murshudov, 1997) was used to refine the structure, applying TLS parameterization

(Winn et al., 2001). Simulated annealing refinement was carried out in CNS (Bruenger

et al., 1998). Manual building and structure analysis were carried out in O (Jones et al.,

1991). Solvent molecules were constantly added both manually and by ARP/wARP

(Lamzin and Wilson, 1993). The structure quality was assessed using PROCHECK

23

(Laskowski et al., 1993). Residues 335-440 and 454-639 of Pex5p and residues 22-143

of SCP2 were included in the final model.

Unliganded Pex5p(C): In order to find a molecular replacement solution using the

program MOLREP (1994; Vagin and Teplyakov, 1997), the Pex5p coordinates of the

SCP2/Pex5p complex structure were split into two parts, spanning residues 335-440 and

454-637. All four copies of the C-terminal part, but only two copies of the N-terminal

part could be identified. Refinement of these molecule fragments was carried out using

REFMAC5 (Murshudov, 1997). The program O (Jones et al., 1991) was used for model

building and analysis. Analysis of the electron density after initial refinement and

rebuilding indicated weak electron density for the two missing N-terminal molecule

fragments, allowing the determination of the overall orientation of each domain based

on the densities of the known helices. The orientation of each N-terminal part relative to

the C-terminal part was essentially the same as in the two well defined molecules.

Refinement was continued by applying NCS restraints separately to the N- and C-

terminal halves of the four Pex5p monomers. Furthermore, the TLS refinement option

in REFMAC5 was used by defining each monomer as a single rigid body. Due to the

high amount of structural flexibility in the N-terminal parts of two of the four Pex5p

molecules in the crystal and the anisotropy of the X-ray diffraction data, the final R-

factors remained higher than those for the Pex5p/cargo complex. Programs of the CCP4

package (Collaborative Computational Project, Number 4, (1994) were also used for

structure manipulation, analysis, and validation.

NMR spectroscopy

24

Isotopically-labeled (90% 2H, 13C and/or 15N) SCP2 was prepared by growing bacteria

in minimal medium supplemented with [U-13C] glucose and/or 15NH4Cl in D2O.

Proteins/complexes were exchanged into 100 mM potassium phosphate (pH 6.5) by gel

filtration. Samples were used at concentrations of 0.2-1.0 mM. NMR spectra were

acquired at 22 °C (complex) or 37 °C (free SCP2) on Bruker spectrometers (DRX600

with cryogenic probe or DRX900 with triple resonance probe). The backbone chemical

shifts of preSCP2 and mSCP2 were based on BMRB entry 4438 (Garcia et al., 2000)

and extended using standard triple resonance experiments (Sattler M et al., 1999). The

assignments for SCP2 in the 50 kDa SCP2/Pex5p(C) complexes were obtained using

triple resonance and 15N-edited TROSY-NOESY experiments on samples comprising

2H,15N- or 2H,13C,15N-labeled SCP2 and unlabeled Pex5p(C). Chemical shift

perturbations ( = [(1H)2 + (1/5 15N)2]½ , in parts per million) were monitored in

two-dimensional 1H,15N-TROSY experiments. T1 15N relaxation was measured at 600

MHz with a spin-lock field strength of 2 kHz. Spin-label induced paramagnetic

relaxation enhancements were analyzed from intensity changes in 1H,15N-TROSY

experiments of SCP2 or SCP2/Pex5p(C) recorded in the presence of the fatty acid

derivative 5-doxyl stearic acid in the oxidized form and after reduction with ascorbic

acid (Battiste and Wagner, 2000).

Isothermal titration microcalorimetry

Proteins were co-dialyzed against 100 mM potassium phosphate (pH 7.4), 1 mM DTT.

SCP2 was pre-mixed with stearoyl-CoA (Sigma) in a small molar excess, and 2 M

stearoyl-CoA was added to the dialysis buffer to ensure uniform loading of SCP2.

Dialysates were degassed and the concentration measured by A280nm. ITC measurements

were conducted on a MicroCal VP-ITC using Pex5p(C) at 30-50 M as a sample and

25

SCP2 or PTS1 peptide (Sigma Genosys) at 350-750 M as the titration ligand.

Experiments were conducted at 35 °C using injection protocols found to saturate

Pex5p(C) with ligand. Ligand heats of dilution were subtracted and data fitted using

MicroCal Origin 5.0.

In vivo peroxisome import assays

Point mutations were introduced into the Pex5p sequence by using the Quickchange XL

– Site Directed Mutagenesis Kit (Stratagene). The pcDNA3 derived expression vector

pGD106 (Braverman et al., 1998) was used as a template to replace glutamine 586 by

arginine (Q586R; sense-primer: 5´-

GAGGCCCTGAACATGAGGAGGAAAAGCCGGGG-3´; antisense-primer: 5´-

CCCCGGCTTTTCCTCCTCATGTTCAGGGCCTC-3´), serine 589 by tyrosine

(S589Y; sense-primer: 5´-AACATGCAGAGGAAATACCGGGGCCCCCGGGG-3´;

antisense-primer: 5´-CCCCGGGGGCCCCGGTATTTCCTCTGCATGTT-3´), serine

600 by tryptophane (S600W; sense-primer: 5´-

GGAGGTGCCATGTGGGAGAACATCTGG-3´; antisense-primer: 5´-

CCAGATGTTCTCCCACATGGCACCTCC-3´) and asparagine 382 by alanin (N382A;

sense-primer: 5´-GTACCACCCAGGCAGAGGCTGAACAAGAACTATTAG-3´;

antisense-primer: 5´-CTAATAGTTCTTGTTCAGCCTCTGCCTGGGTGGTAC-3´).

mSCP2 was amplified by PCR using E.coli mSCP2 expression plasmid as a template

and the primers 5´-GATCTCGAGCCATGGGCTCTGCAAGTG-3´ and 5´-

TGAATTCAGAGCTTAGCGTTGCCTG-3´. The resulting fragment was digested with

restriction endonucleases XhoI/EcoRI and subcloned into the corresponding sites of

pEGFP-C1 plasmid (Clontech Laboratories, Inc.) thereby generating the expression

construct for the fusion protein EGFP-SCP2.

26

pPTS2-CAcT encoding an N-terminal PTS2 signal followed by the reporter protein

chloramphenicol acetyltransferase (CAcT) was described previously (Braverman et al.,

1998).

Human fibroblast cells were cultured at 37°C in Dulbecco modified Eagle’s medium

supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100.000 U/l penicillin,

and 100 mg/l streptomycin at 8% CO2. The human Pex5p-deficient skin fibroblast cell

line PBD005 (Dodt et al., 1995) was grown for one day on cover-slides in 60 mm

tissue-culture dishes before it was transfected with pPTS2-CAcT, pEGFP-SCP2 and one

of the Pex5p expression plasmids. Transfection was performed with 1-2 µg of each

plasmid DNA and 12 µl FuGENE 6 Transfection Reagent according to the

manufacturer’s instruction (Roche Diagnostics, Mannheim, Germany). At various time

points (24, 48, 72, 96 hours after transfection) cells were fixed on cover glasses with 3%

formaldehyde in phosphate-buffered saline (PBS), permeabilized with 1% Triton X-100

in PBS, and subjected to immunofluorescence microscopy and GFP life imaging.

Polyclonal rabbit antibodies against CAcT and sheep antibodies against human catalase

were purchased from Invitrogen (Germany) and The Binding Site (UK), respectively.

Secondary antibodies were conjugated with Alexa Fluor 594 or 488 (Invitrogen,

Germany).

All micrographs were recorded on a Zeiss Axioplan 2 microscope with a Zeiss Plan-

Apochromat 63x/1.4 oil objective and an Axiocam MR digital camera and were

processed with AxioVision 4.2 software (Zeiss, Jena, Germany).

Acknowledgments

27

We thank Ben Distel, André Klein, Ash Verma, Areti Malapetsas, Gunter Stier,

Christian Edlich, Christiane Sprenger, Elisabeth Becker, Bernd Simon and Elena Conti

for stimulating discussions and valuable support. This work was supported by the grants

HPRN-CT-2002-00252 (to M.W.) and LSHG-CT-2004-512018 (to R.E.) from the

European Commission, and grants Schl 584/1-1 and 1-2 (to W. Sch.) from the Deutsche

Forschungsgemeinschaft (DFG). We thank the DFG and the center for biomagnetic

resonance (BMRZ), Frankfurt, Germany, for access to the 900 MHz NMR instrument,

and BESSY, Berlin, Germany, for access to the synchrotron radiation beamline BL1.

Accession Numbers

Coordinates and structure factors have been deposited at the Protein Data

Bank with accession codes 2C0L and 2C0M .

28

References

Collaborative Computational Project, Number 4. (1994). The CCP4 suite: programs for

protein crystallography. Acta Crystallogr D Biol Crystallogr 50, 760-763.

Agne, B., Meindl, N. M., Niederhoff, K., Einwachter, H., Rehling, P., Sickmann, A.,

Meyer, H. E., Girzalsky, W., and Kunau, W. H. (2003). Pex8p: an

intraperoxisomal organizer of the peroxisomal import machinery. Mol Cell 11,

635-646.

Battiste, J. L., and Wagner, G. (2000). Utilization of site-directed spin labeling and

high-resolution heteronuclear nuclear magnetic resonance for global fold

determination of large proteins with limited nuclear overhauser effect data.

Biochemistry 39, 5355-5365.

Braverman, N., Dodt, G., Gould, S. J., and Valle, D. (1998). An isoform of Pex5p, the

human PTS1 receptor, is required for the import of PTS2 proteins into

peroxisomes. Hum Mol Genet 7, 1195-1205.

Brocard, C. B., Jedeszko, C., Song, H. C., Terlecky, S. R., and Walton, P. A. (2003).

Protein structure and import into the peroxisomal matrix. Traffic 4, 74-82.

Brown, N. P., Leroy, C., and Sander, C. (1998). MView: a web-compatible database

search or multiple alignment viewer. Bioinformatics 14, 380-381.

Bruenger, A., Adams, P., Clore, G., Delano, W., Gros, D., Grosse-Kunstleve, R., Jiang,

J.-S., Kuszewski, J., Nilges, M., Pannu, N., et al. (1998). Crystallography &

NMR System: a new software suite for macromolecular structure determination.

Acta Crystallogr D Biol Crystallogr D54, 905-921.

Choinowski, T., Hauser, H., and Piontek, K. (2000). Structure of sterol carrier protein 2

at 1.8 A resolution reveals a hydrophobic tunnel suitable for lipid binding.

Biochemistry 39, 1897-1902.

29

Chook, Y. M., and Blobel, G. (2001). Karyopherins and nuclear import. Curr Opin

Struct Biol 11, 703-715.

Cingolani, G., Petosa, C., Weis, K., and Muller, C. W. (1999). Structure of importin-

beta bound to the IBB domain of importin-alpha. Nature 399, 221-229.

Cliff, M. J., Williams, M. A., Brooke-Smith, J., Barford, D., and Ladbury, J. E. (2005).

Molecular recognition via coupled folding and binding in a TPR domain. J Mol

Biol 346, 717-732.

Conti, E., and Izaurralde, E. (2001). Nucleocytoplasmic transport enters the atomic age.

Curr Opin Cell Biol 13, 310-319.

Cook, A., Fernandez, E., Lindner, D., Ebert, J., Schlenstedt, G., and Conti, E. (2005).

The structure of the nuclear export receptor Cse1 in its cytosolic state reveals a

closed conformation incompatible with cargo binding. Mol Cell 18, 355-367.

Costa-Rodrigues, J., Carvalho, A. F., Fransen, M., Hambruch, E., Schliebs, W., Sa-

Miranda, C., and Azevedo, J. E. (2005). Pex5p, the peroxisomal cycling receptor,

is a monomeric non-globular protein. J Biol Chem.

D'Andrea, L. D., and Regan, L. (2003). TPR proteins: the versatile helix. Trends

Biochem Sci 28, 655-662.

Dammai, V., and Subramani, S. (2001). The human peroxisomal targeting signal

receptor, Pex5p, is translocated into the peroxisomal matrix and recycled to the

cytosol. Cell 105, 187-196.

Devos, D., Dokudovskaya, S., Alber, F., Williams, R., Chait, B. T., Sali, A., and Rout,

M. P. (2004). Components of coated vesicles and nuclear pore complexes share a

common molecular architecture. PLoS Biol 2, e380.

Devos, D., Dokudovskaya, S., Williams, R., Alber, F., Eswar, N., Chait, B. T., Rout, M.

P., and Sali, A. (2006). Simple fold composition and modular architecture of the

nuclear pore complex. Proc Natl Acad Sci U S A.

30

Dodt, G., Braverman, N., Wong, C., Moser, A., Moser, H. W., Watkins, P., Valle, D.,

and Gould, S. J. (1995). Mutations in the PTS1 receptor gene, PXR1, define

complementation group 2 of the peroxisome biogenesis disorders. Nat Genet 9,

115-125.

Eichler, J., and Irihimovitch, V. (2003). Move it on over: getting proteins across

biological membranes. Bioessays 25, 1154-1157.

Erdmann, R., and Schliebs, W. (2005). Peroxisomal matrix protein import: the transient

pore model. Nat Rev Mol Cell Biol 6, 738-742.

Faber, K. N., van Dijk, R., Keizer-Gunnink, I., Koek, A., van der Klei, I. J., and

Veenhuis, M. (2002). Import of assembled PTS1 proteins into peroxisomes of the

yeast Hansenula polymorpha: yes and no! Biochim Biophys Acta 1591, 157-162.

Frolov, A., Cho, T. H., Billheimer, J. T., and Schroeder, F. (1996). Sterol carrier

protein-2, a new fatty acyl coenzyme A-binding protein. J Biol Chem 271, 31878-

31884.

Garcia, F. L., Szyperski, T., Dyer, J. H., Choinowski, T., Seedorf, U., Hauser, H., and

Wuthrich, K. (2000). NMR structure of the sterol carrier protein-2: implications

for the biological role. J Mol Biol 295, 595-603.

Gatto, G. J., Jr., Geisbrecht, B. V., Gould, S. J., and Berg, J. M. (2000). Peroxisomal

targeting signal-1 recognition by the TPR domains of human PEX5. Nat Struct

Biol 7, 1091-1095.

Gould, S. J., and Collins, C. S. (2002). Opinion: peroxisomal-protein import: is it really

that complex? Nat Rev Mol Cell Biol 3, 382-389.

Gouveia, A. M., Guimaraes, C. P., Oliveira, M. E., Sa-Miranda, C., and Azevedo, J. E.

(2003a). Insertion of Pex5p into the peroxisomal membrane is cargo protein-

dependent. J Biol Chem 278, 4389-4392.

31

Gouveia, A. M., Guimaraes, C. P., Oliveira, M. E., Sa-Miranda, C., and Azevedo, J. E.

(2003b). Insertion of Pex5p into the peroxisomal membrane is cargo protein-

dependent. J Biol Chem 278, 4389-4392.

Holroyd, C., and Erdmann, R. (2001). Protein translocation machineries of

peroxisomes. FEBS Lett 501, 6-10.

Huber, P. A., Birdsey, G. M., Lumb, M. J., Prowse, D. T., Perkins, T. J., Knight, D. R.,

and Danpure, C. J. (2005). Peroxisomal import of human alanine: Glyoxylate

aminotransferase requires ancillary targeting information remote from its C-

terminus. J Biol Chem.

Jinek, M., Rehwinkel, J., Lazarus, B. D., Izaurralde, E., Hanover, J. A., and Conti, E.

(2004). The superhelical TPR-repeat domain of O-linked GlcNAc transferase

exhibits structural similarities to importin alpha. Nat Struct Mol Biol 11, 1001-

1007.

Jones, T., Zou, J.-Y., Cowan, S., and Kjeldgaard, M. (1991). Improved methods for

building protein models in electron density maps and the location of errors in

these models. Acta Crystallogr A A46, 110-119.

Kabsch, W. (1988). Evaluation of single crystal x-ray diffraction data from a position-

sensitive detector. J Appl Cryst 21, 916-924.

Kiel, J. A., Emmrich, K., Meyer, H. E., and Kunau, W. H. (2004). Ubiquitination of the

PTS1 receptor, Pex5p, suggests the presence of a quality control mechanism

during peroxisomal matrix protein import. J Biol Chem.

Klein, A. T., Barnett, P., Bottger, G., Konings, D., Tabak, H. F., and Distel, B. (2001).

Recognition of peroxisomal targeting signal type 1 by the import receptor Pex5p.

J Biol Chem 276, 15034-15041.

Knott, T. G., Birdsey, G. M., Sinclair, K. E., Gallagher, I. M., Purdue, P. E., and

Danpure, C. J. (2000). The peroxisomal targeting sequence type 1 receptor,

32

Pex5p, and the peroxisomal import efficiency of alanine:glyoxylate

aminotransferase. Biochem J 352 Pt 2, 409-418.

Krissinel, E., and Henrick, K. (2004). Secondary-structure matching (SSM), a new tool

for fast protein structure alignment in three dimensions. Acta Crystallogr D Biol

Crystallogr 60, 2256-2268.

Kunau, W. H., Agne, B., and Girzalsky, W. (2001). The diversity of organelle protein

import mechanisms. Trends Cell Biol 11, 358-361.

Lametschwandtner, G., Brocard, C., Fransen, M., Van Veldhoven, P., Berger, J., and

Hartig, A. (1998). The difference in recognition of terminal tripeptides as

peroxisomal targeting signal 1 between yeast and human is due to different

affinities of their receptor Pex5p to the cognate signal and to residues adjacent to

it. J Biol Chem 273, 33635-33643.

Lamzin, V., and Wilson, K. (1993). Automated refinement of protein models. Acta

Crystallogr D Biol Crystallogr D49, 129-147.

Laskowski, R., MacArthur, M., and Thormton, J. (1993). PROCHECK: a program to

check the stereochemical quality of protein structures. J Appl Cryst 26, 283-291.

Lazarow, P. B. (2003). Peroxisome biogenesis: advances and conundrums. Curr Opin

Cell Biol 15, 489-497.

Lee, S. J., Matsuura, Y., Liu, S. M., and Stewart, M. (2005). Structural basis for nuclear

import complex dissociation by RanGTP. Nature 435, 693-696.

Lensink, M. F., Haapalainen, A. M., Hiltunen, J. K., Glumoff, T., and Juffer, A. H.

(2002). Response of SCP-2L domain of human MFE-2 to ligand removal: binding

site closure and burial of peroxisomal targeting signal. J Mol Biol 323, 99-113.

Madrid, K. P., De Crescenzo, G., Wang, S., and Jardim, A. (2004). Modulation of the

Leishmania donovani Peroxin 5 Quaternary Structure by Peroxisomal Targeting

Signal 1 Ligands. Mol Cell Biol 24, 7331-7344.

33

Matsuura, Y., and Stewart, M. (2004). Structural basis for the assembly of a nuclear

export complex. Nature 432, 872-877.

Modis, Y., Filppula, S. A., Novikov, D. K., Norledge, B., Hiltunen, J. K., and Wierenga,

R. K. (1998). The crystal structure of dienoyl-CoA isomerase at 1.5 A resolution

reveals the importance of aspartate and glutamate sidechains for catalysis.

Structure 6, 957-970.

Murshudov, G. N., Vagin, A.A. & Dodson, E.J (1997). Refinement of macromolecular

structures by the maximum-likelihood method. Acta Crystallogr D53, 240-255.

Neuberger, G., Maurer-Stroh, S., Eisenhaber, B., Hartig, A., and Eisenhaber, F. (2003).

Motif refinement of the peroxisomal targeting signal 1 and evaluation of taxon-

specific differences. J Mol Biol 328, 567-579.

Otera, H., Setoguchi, K., Hamasaki, M., Kumashiro, T., Shimizu, N., and Fujiki, Y.

(2002). Peroxisomal targeting signal receptor Pex5p interacts with cargoes and

import machinery components in a spatiotemporally differentiated manner:

conserved Pex5p WXXXF/Y motifs are critical for matrix protein import. Mol

Cell Biol 22, 1639-1655.

Parkes, J. A., Langer, S., Hartig, A., and Baker, A. (2003). PTS1-independent targeting

of isocitrate lyase to peroxisomes requires the PTS1 receptor Pex5p. Mol Membr

Biol 20, 61-69.

Purdue, P. E., and Lazarow, P. B. (1996). Targeting of human catalase to peroxisomes

is dependent upon a novel COOH-terminal peroxisomal targeting sequence. J Cell

Biol 134, 849-862.

Sattler M, J, S., and C, G. (1999). Heteronuclear multidimensional NMR experiments

for the structure determination of proteins in solution employing pulsed field

gradients. Prog NMR Spectrosc 34, 93-158.

34

Schäfer, A., Kerssen, D., Veenhuis, M., Kunau, W. H., and Schliebs, W. (2004).

Functional similarity between the peroxisomal PTS2 receptor binding protein

Pex18p and the N-terminal half of the PTS1 receptor Pex5p. Mol Cell Biol 24,

8895-8906.

Schnell, D. J. (2000). Functions and origins of the chloroplast protein-import

machinery. Essays Biochem 36, 47-59.

Shimozawa, N., Zhang, Z., Suzuki, Y., Imamura, A., Tsukamoto, T., Osumi, T., Fujiki,

Y., Orii, T., Barth, P. G., Wanders, R. J., and Kondo, N. (1999). Functional

heterogeneity of C-terminal peroxisome targeting signal 1 in PEX5-defective

patients. Biochem Biophys Res Commun 262, 504-508.

Stanley, W. A., Sokolova, A., Brown, A., Clarke, D. T., Wilmanns, M., and Svergun, D.

I. (2004). Synergistic use of synchrotron radiation techniques for biological

samples in solution: a case study on protein-ligand recognition by the

peroxisomal import receptor Pex5p. J Synchrotron Radiat 11, 490-496.

Vagin, A., and Teplyakov, A. (1997). MOLREP: an automated program for molecular

replacement. J Appl Cryst 30, 1022-1025.

Vaguine AA, Richelle, J., and Wodak, S. (1999). SFCHECK: a unified set of

procedures for evaluating the quality of macromolecular structure-factor data and

their agreement with the atomic model. Acta Crystallogr D Biol Crystallogr D55,

191-205.

van der Klei, I., and Veenhuis, M. (2002). Peroxisomes: flexible and dynamic

organelles. Curr Opin Cell Biol 14, 500-505.

Walton, P. A., Hill, P. E., and Subramani, S. (1995). Import of stably folded proteins

into peroxisomes. Mol Biol Cell 6, 675-683.

35

Wang, D., Visser, N. V., Veenhuis, M., and van der Klei, I. J. (2003). Physical

interactions of the peroxisomal targeting signal 1 receptor pex5p, studied by

fluorescence correlation spectroscopy. J Biol Chem 278, 43340-43345.

Weller, S., Gould, S. J., and Valle, D. (2003). Peroxisome biogenesis disorders. Annu

Rev Genomics Hum Genet 4, 165-211.

Wickner, W., and Schekman, R. (2005). Protein translocation across biological

membranes. Science 310, 1452-1456.

Winn, M. D., Isupov, M. N., and Murshudov, G. N. (2001). Use of TLS parameters to

model anisotropic displacements in macromolecular refinement. Acta Crystallogr

D Biol Crystallogr 57, 122-133.

Yang, X., Purdue, P. E., and Lazarow, P. B. (2001). Eci1p uses a PTS1 to enter

peroxisomes: either its own or that of a partner, Dci1p. Eur J Cell Biol 80, 126-

138.

36

Tables

Table 1: Thermodynamic characterization of Pex5p interaction with PTS1-

containing ligands by ITC.

Pex5p receptor CargoH

(kJ/mol)

TS

(kJ/mol)

G

(kJ/mol)

Kd

(nM)

wild-type mSCP2 -42.4 -1.2 -41.2 109 ± 34

wild-type mSCP2(SCoA) -31.8 8.9 -40.8 124 ± 17

wild-type preSCP2 -35.9 6.2 -42.1 74 ± 9

wild-type PGNAKL -45.1 -8.7 -36.4 664 ± 37

N382A mSCP2 -27.3 10.8 -38.1 348 ± 54

Q586R mSCP2 -17.4 17.3 -34.5 1343 ± 321

S589Y mSCP2 -38.7 1.20 -39.9 173 ± 23

S600W mSCP2 no binding

Table 1 legend:

SCoA, stearoyl coenzyme A. The measured stoichiometries deviated less than 10%

from a 1:1 complex, except for the Pex5p (S600W) mutant. Because of the experimental

errors in protein concentration measurements the stoichiometry values were adjusted to

1.00.

37

Table 2: Crystallographic statistics

Pex5p(C):mSCP2 Pex5p(C)

X-ray data collection statistics

Space group P212121 P1

Unit cell dimensions [Å] 40.5, 68.6, 137.4 53.5, 85.6, 88.9, 71.2°, 90.0°, 73.4°

Resolution range [Å] 25.0-2.3 (2.4-2.3) 20.0-2.5 (2.6-2.5)

Rsym [%] 9.4 (49.8) 13.7 (53.8)

I/σI) 14.1 (3.8) 6.3 (1.7)

Completeness [%] 99.8 (100.0) 95.9 (85.9)

Data redundancy 6.0 (6.1) 2.2 (2.1)

Unique reflections 17,692 47,257

Refinement statistics

Resolution range [Å] 20.0-2.3 20.0-2.5

R-factor/R-free [%] 20.2/25.6 26.3/30.9

Protein atoms 3209 9483

Solvent atoms 99 147

Rmsd bond distances [Å] 0.006 0.013

Rmsd bond angles [°] 1.0 1.4

Average B factors [Å2]a

Pex5p(C) N/Cb 18/30 14, 23,76,76/15,19,41,42

SCP2 39 -

Solvent 24 15

Rmsd B factors of protein bonded atoms [Å2]

38

Main chain 2.2 0.5

Side chain 2.6 1.1

Ramachandran plot regions [%]

Most favored 89.1 88.3

Additional allowed 10.0 10.6

Generously allowed 0.6 1.0

Disallowed 0.3 0.1

Table 2 Legend:

a TLS refinement parameters have been applied.

b N, Pex5p(321-442); C, Pex5p(457-639).

39

Figure Legends:

Figure 1: Sequence/structure relationships in (A) human Pex5p(C) and (B) human

mSCP2. The positions of labeled secondary structural elements are depicted by

cylinders and arrows. Color coding, Pex5p(C), panel A: TPR1-TPR3, cyan; TPR4,

green; TPR5-TPR7, blue; 7C-loop, connecting TPR7 and the C-terminal helical bundle,

red; C-terminus, maroon. Color coding, mSCP2, panel B: Core domain, yellow; C-

terminus including PTS1 motif, orange. Residues of Pex5p and SCP2 involved in cargo

and receptor binding, respectively, have been identified using the program AREAMOL

of the CCP4 suite (Collaborative Computational Project, Number 4, (1994) and are

indicated in colors matching the bound sequence segments. Conserved residues have

been identified from multiple sequence alignments using BLAST/MVIEW (Brown et

al., 1998). In the ‘cons’ line, residues exhibiting 90% and 70 % homology to the

available sequences are indicated by upper case and lower case characters, respectively.

Residues that were identified by (Klein et al., 2001) and (Shimozawa et al., 1999) as

being involved in Pex5p receptor-cargo interactions are shown in red and blue colors.

TPR motif signature residues according to the criteria of (D'Andrea and Regan, 2003)

are underlined. Residue segments that function as hinge regions (496-500, 523-524,

533-537), triggering the conformational changes observed for the cargo-loaded and apo-

Pex5p(C) receptor are highlighted by orange bars that have been inserted into the

corresponding secondary structural elements.

Figure 2: Lipid binding to mSCP2 in the absence and presence of Pex5p(C), (A, B).

Binding of a spin labeled lipid molecule (5-doxylstearic acid, 5DSA) attenuates the

peak intensity in 1H,15N correlation spectra due to paramagnetic relaxation enhancement

40

(PRE). Spectra in the presence of oxidized (i.e. paramagnetic) and reduced lipid are

shown in green and black, respectively. Residues Thr105 and Gly106, which are located

in the centre of the lipid binding pocket, are entirely bleached. Gly139, which is

proximal to the PTS1 motif, experiences a large chemical shift perturbation in the

presence of Pex5p(C). It is less bleached in the Pex5p(C) complex, consistent with the

strongly reduced mobility of the C-terminal residues and the increased distance to the

lipid ligand. (C, D) Comparison of the lipid binding pocket of mSCP2, in the presence /

absence of Pex5p(C). The degree of attenuation of NMR signals due to PRE is colored

in green on a ribbon representation of mSCP2. Amide protons of residues with a more

than seven-fold reduction in peak intensities are depicted by green spheres.

Figure 3: Structures of the peroxisomal import receptor Pex5p(C) in the presence

(A,B,D) and in the absence (C,D) of the cargo mSCP2. Color coding, Pex5p(C):

TPR1-TPR3, cyan; TPR4, green; TPR5-TPR7, blue; 7C-loop, connecting TPR7 and the

C-terminal helical bundle, red; C-terminus, maroon. Color coding, mSCP2: Core

domain, yellow; C-terminus including PTS1 motif, orange. The orientation of the

receptor in (A) and (C) is identical. The ribbon of the Pex5p(C)/mSCP2 complex in (B)

has been rotated by 600 around a horizontal axis within the paper plane with respect to

the orientation in (A), to illustrate the mode of mSCP2 binding to the receptor. (D)

Superimposed Pex5p(C) receptor structures in the presence and in the absence of

mSCP2. The colors of the trace of the cargo-loaded conformation are as in panels A-C,

except that the conformational hinge regions are colored in orange. The trace of the apo-

Pex5p(C) structure is in gray, except for the 7C-loop, which is colored in faint red. The

coordinates of TPR segments 1-4 were used for structural superposition using the

program SSM (Krissinel and Henrick, 2004) (rmsd = 0.78 Å for 164 common residues).

41

The largest structural deviations of up to 8 Å are observed at the 7C-loop and adjacent

regions and are indicated by a red arrow.

Figure 4: Surface presentations of the peroxisomal import receptor Pex5p(C), in

the presence (A-B) and in the absence (C) of the cargo mSCP2. The right panel

structures are rotated by 45° with respect to those in the left panels by a horizontal axis

within the paper plane. The color codes are as in Figure 3. While the structure of the

Pex5p(C)/mSCP2 complex is shown in (A), only the structure of the cargo-loaded

conformation of the Pex5p(C) receptor is displayed in (B). The PTS1 and secondary

mSCP2 binding areas are mapped onto the Pex5p(C) surface in their respective colors

(orange, yellow). In the structure of the apo-Pex5p(C) receptor, the approximate

location of the PTS1 binding site, as determined from the Pex5p(C)/mSCP2 complex, is

indicated by an orange circle. Conformational changes of several residues at this site

lead to disappearance of the open tunnel, observed in the Pex5p(C)/mSCP2 complex

(B). In the apo-conformation, the 7C-loop region (red) is well separated from the

remaining TPR segments of the receptor.

Figure 5: Structural determinants of mSCP2 cargo loading onto Pex5p(C). (A)

Stereo view of the 2FO-FC electron density, using phases from the refined model and

contoured at 1 of the PTS1 motif from mSCP2 (gray) and some interacting residues

from Pex5p and ordered solvent molecules (dark green). (B) Pex5p(C)/mSCP2 complex

formation by two distinct interfaces; C-terminal PTS1 motif from mSCP2 (orange)–

central cavity of the circular TPR motif structure from Pex5p; secondary surface from

mSCP2–C-terminal helical bundle from Pex5p. Ser600 is in a central position between

the two surface patches, allowing the proper arrangement of the two cargo surface

patches of Pex5p to support binding of mSCP2. The C-terminus of the 7C-loop (red)

42

interacts by a few hydrogen bonds with the TPR1 segment. (C) TPR4 motif of

Pex5p(C), as observed in the cargo-loaded structure of the receptor. Specific

interactions between TPR3 and TPR4, generating a circular conformation of Pex5p(C),

are shown. Colors are as in Figures 3 and 4, except that some of the bonds of residues

from the C-terminal TPR motifs 5-7 and the 7C-loop are colored in gray to allow

illustrations of oxygen and nitrogen atoms. Hydrogen bonds are shown by dashed lines.

Figure 6: 7C- loop mutants lead to functional PTS1 import defects

Pex5p-deficient fibroblast cells from Zellweger patient PBD005 were co-transfected

with a PTS2-tagged CAcT expressing plasmid, pEGFP-SCP2 and plasmids expressing

either wt Pex5p or a range of different single residue mutants (N382A, Q586R, S589Y,

S600W). At 24 hours after transfection PTS2-CAcT (A, red color) and endogenous

catalase (B, red color) were labeled by immunofluorescence while EGFP-SCP2 was

detected by direct fluorescence (A and B, green color). In cells expressing wt Pex5p,

both marker proteins and EGFP-SCP2, co-localized in peroxisomes (A and B, yellow

color). All Pex5p mutants were capable to restore the PTS2 import defect of PEX5

deficient cells. In contrast, all mutants were impaired in catalase import and showed

more or less pronounced import defects for EGFP-SCP2. The strongest effect was

observed for the S600W mutant that led to complete mislocalization of both PTS1

proteins into the cytosol. The introduction of the 7C-loop mutation Q586R resulted in

an inefficient SCP2 import as indicated by the diffuse cytosolic staining and the very

few peroxisomes labeled in the representative cells. Strikingly, in cells expressing the

Pex5p mutants S589Y and N382A, both cytosolic and peroxisomal localizations of

SCP2 were found while the same cells were devoid of functional catalase import as

indicated by the lack of a congruent punctuate pattern.

α1Α α1Β α2Α α2Β

320 330 340 350 360 370 380 390 400 | | | | | | | H H | |Pex5 LTSATYDKGYQFEEENPLRDHPQPFEEGLRRLQEGDLPNAVLLFEAAVQQDPKHMEAWQYLGTTQAENEQELLAISALRRCLELKPDNQTcons ---------------np----------G------G-l--a-l--Eaa----P---eaW--LG------ene--ai-A------l-p-n--

α3Α α3Β α4Α α4B α5Α

410 420 430 440 450 460 470 480 490 |H H | | | | | | | |Pex5 ALMALAVSFTNESLQRQACETLRDWLRYTPAYAHLVTPAEEGAGGAGLGPSKRILGSLLSDSLFLEVKELFLAAVRLDPTSIDPDVQCGLcons al--La-s-tn------A---L--w----p-y----------------------------------v--l---A----p---D-D----L

α5Α α5Β α6Α α6Β α7Α α7Β

500 510 520 530 540 550 560 570 580 H H| | | H H H | H | H H | . H. H H |Pex5 GVLFNLSGEYDKAVDCFTAALSVRPNDYLLWNKLGATLANGNQSEEAVAAYRRALELQPGYIRSRYNLGISCINLGAHREAVEHFLEALNcons GvL--L--e---a---f--Al---P-d---Wn-lGa-lAN---s-eA--AY--AL---P---r--yN-g-s--n-g---ea------al-

αC1 αC2 αC3

590 600 610 620 630 | | . .H H | H H H H H |Pex5 MQRKSRGPRGEGGAMSENIWSTLRLALSMLGQSDAYGAADARDLSTLLTMFGLPQcons -------------------w--L--------------------------------

α1 α2 β1 β2 β3

10 20 30 40 50 60 70 80 90 | | H | H H H | | | | | |SCP2 MGFPEAASSFRTHQIEAVPTSSASDGFKANLVFKEIEKKLEEEGEQFVKKIGGIFAFKVKDGPGGKEATWVVDVKNGKGSVLPNSDKKADcons -----------------------------------------------vkk-------------------W--D-K-g-g---------aD

β4 α3 α4 β5 α5

100 110 120 130 140 | H H | | | LQ | SCP2 CTITMADSDFLALMTGKMNPQSAFFQGKLKITGNMGLAMKLQNLQLQPGNAKL Figure 1cons ------d-df----------------GK-K--Gn--l--KL------------ Stanley et al.

Figure 1

Figure 2

1 2 3 4 5 6 7 C

A B

C D

Figure 3 Stanley et al.

Figure 3

45 deg.

A: Pex5p (cargo) -mSCP2

B: Pex5p (cargo)

C: Pex5p (apo)

Figure 4 Stanley et al.

Figure 4

Figure 5

Figure 6

1

Recognition of a functional peroxisome type 1 target

by the dynamic import receptor Pex5p

Will A. Stanley1, Fabian V. Filipp2, Petri Kursula1, Nicole Schüller1, Ralf

Erdmann3, Wolfgang Schliebs3, Michael Sattler2, Matthias Wilmanns1*

1 EMBL-Hamburg Outstation, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany

2 Structural and Computational Biology Unit, EMBL-Heidelberg, Meyerhofstrasse 1,

69117 Heidelberg, Germany

3 Institute for Physiological Chemistry, Department of Systems Biology, Faculty of

Medicine, Ruhr University of Bochum, 44780 Bochum, Germany.

Supplementary Material

F) Supplemental Text and Figures

2

Supplementary Figure S1: NMR data characterizing SCP2-Pex5p(C) binding.

(A, B) Chemical shift difference (Δδ) vs. residue number between the free and

Pex5p(C) bound state of 0.5 mM 15N, 2H-labeled preSCP2 (A) and mSCP2 (B) were

monitored at a 1:1.2 cargo/receptor ratio. The PTS1 and secondary interactions sites

are depicted by red and blue bars, respectively. Residues with Δδ > 0.1 ppm are

colored red or blue in Figure 2D. Secondary structure elements of SCP2 are shown on

top. (C) Comparison of 15N T1ρ relaxation times for free mSCP2 and when bound to

Pex5p(C) measured at 22˚C at 600 MHz 15N frequency. The T1ρ values of flexible

terminal regions are significantly higher than the average values of residues in the

core of the domain. Due to the interaction with Pex5p(C), T1ρ values of residues in

the PTS1 tail are strongly reduced, indicating that they become highly ordered. The

large increase in the molecular weight of mSCP2 bound to Pex5p(C) (49.6 kDa)

versus free mSCP2 (13.4 kDa) results in slower molecular tumbling, which is

reflected in a general reduction of the average T1ρ values. (D) Strong chemical shift

perturbations of N-H NMR signals (Δδ >0.1 ppm) are colored on a surface

representation of mSCP2. The PTS1 interaction site is shown in red; the secondary

binding surface is shown in blue.

Supplementary Figure S2: (A, B) Relative peak intensities in 1H,15N TROSY

experiments recorded on free preSCP2 (A) and when bound to Pex5p(C) (B),

indicating that the pre-sequence remains highly flexible even when SCP2 is bound to

Pex5p(C). (C) {1H}-15N heteronuclear NOE data for free preSCP2 at 295 K as

described (Farrow et al. 2003). Chemical shift assignments for residues 1-25 were

obtained from triple resonance experiments on free preSCP2 and 15N-edited NOESY

3

experiments on free and Pex5p(C) bound preSCP2. Spectra were processed with

NMRPipe and analysed using NMRVIEW.

Supplementary Figure S3: Structural mobility changes in the Pex5p receptor

and mSCP2 upon receptor/cargo complex formation. (A), sequence/B-factor plot

of the structures of Pex5p(C) in the absence (gray, black) and in the presence of the

mSCP2 cargo (red). The residue B factors of the two Pex5p(C) molecules with well

defined N-terminal TPR segments 1-4 are shown by filled symbols. The residue B

factors of the two other Pex5p(C) molecules, in which the N-terminal TPR domains 1-

4 are mobile, are displayed with open symbols. (B), sequence/B-factor plot of the

structures of mSCP2 in the absence (black) and in the presence (red) of the Pex5p(C)

receptor. The residue B factors of the cargo-unloaded mSCP2 structure have been

taken from the coordinates of the PDB entry 1C44 (Choinowski et al., 2000). In (A)

and (B), the B factors of the Cα positions have been used for display. (C), ribbon

representations of the conformations of mSCP2 loaded onto the Pex5p(C) receptor

(left) and unloaded (right). The residue B factors are mapped on the two ribbons in

rainbow colors, ranging from blue (B = 15 Å2) to red (B = 60 Å2). While the N-

termini of mSCP2 are flexible in both conformations, the C-terminus bearing the

PTS1 motifs becomes the most rigid part of the structure upon loading onto the

Pex5p(C) receptor. The C-terminus of mSCP2 straightens into an extended

conformation pointing away from the remaining structure.

Supplementary Figure S4: Expression of Pex5p variants in human fibroblasts. Equal

amounts of whole cell lysates (10 ug protein) from Pex5p-free human fibroblasts

transfected transiently for 24 hours with plasmids expressing (1) wt Pex5p, (2)

4

S600W, (3) Q586R, (4) S589Y, (5) N382A, (6) empty pcDNA vector and from wild-

type cells (7) were subjected to Western-Blot analysis using antibodies directed

against human Pex5p and GFP. Monoclonal anti-GFP (JL-8) antibodies were obtained

from BD Biosciences, Pharmingen, Germany.

1 2 3 4 5 6 7 C

0

20

40

60

80

21 41 61 81 101 121 141

SCP2(Pex5p) SCP2(apo)

A

B C

Figure S3Stanley et al.

� ��

� � �

� ��

�������

1 2 3 4 5 6 7

Pex5p

GFP-SCP2

Figure S4 Stanley et al.