Báo cáo khoa học: Lpx1p is a peroxisomal lipase required for normal peroxisome morphology potx

Lpx1p is a peroxisomal lipase required for normal
peroxisome morphology
Sven Thoms
1,
*, Mykhaylo O. Debelyy
1
, Katja Nau
1,
†, Helmut E. Meyer
2
and Ralf Erdmann
1
1 Institut fu
¨
r Physiologische Chemie, Abteilung fu
¨
r Systembiochemie, Ruhr-Universita
¨
t Bochum, Germany
2 Medizinisches Proteomcenter, Ruhr-Universita
¨
t Bochum, Germany
Peroxisomes are ubiquitous eukaryotic organelles that
are involved in lipid and antioxidant metabolism.
They are versatile and dynamic organelles engaged in
the b-oxidation of long and very long chain fatty
acids, in a-oxidation, bile acid and ether lipid synthe-
sis, and in amino acid and purine metabolism [1].
Peroxisomes are a source of reactive oxygen species,
and are involved in the synthesis of signalling mole-
cules in plants. Remarkably, peroxisomes are the only

site of fatty acid b-oxidation in plants and fungi.
Human peroxisomal disorders can be categorized
as either single-enzyme disorders or peroxisomal
biogenetic defects [2]. Single-enzyme disorders, for
example Refsum disease caused by a defect of
phytanoyl CoA hydroxylase, or X-linked adrenoleu-
kodystrophy caused by a defect in a peroxisomal
ATP-transporter. Biogenetic defects are mostly caused
by mutations in the peroxisomal biogenesis genes,
the PEX genes, that code for peroxins [3]. Peroxi-
somal disorders are associated with morphological
Keywords
lipase; peroxin; peroxisome; proteomics;
PTS1
Correspondence
R. Erdmann, Abteilung fu
¨
r
Systembiochemie, Ruhr-Universita
¨
t
Bochum, Universita
¨
tsstr. 150,
44780 Bochum, Germany
Fax: +49 234 32 14266
Tel: +49 234 322 4943
E-mail:
Present address
*Universita

¨
tsmedizin Go
¨
ttingen, Abteilung
fu
¨
rPa
¨
diatrie und Neuropa
¨
diatrie, Georg-
August-Universita
¨
t, Germany
†Forschungszentrum Karlsruhe, Institut fu
¨
r
Toxikologie und Genetik, Germany
(Received 20 September 2007, revised 22
November 2007, accepted 30 November
2007)
doi:10.1111/j.1742-4658.2007.06217.x
Lpx1p (systematic name: Yor084wp) is a peroxisomal protein from Saccha-
romyces cerevisiae with a peroxisomal targeting signal type 1 (PTS1) and a
lipase motif. Using mass spectrometry, we have identified Lpx1p as present
in peroxisomes, and show that Lpx1p import is dependent on the PTS1
receptor Pex5p. We provide evidence that Lpx1p is piggyback-transported
into peroxisomes. We have expressed the Lpx1p protein in Escherichia coli,
and show that the enzyme exerts acyl hydrolase and phospholipase A activ-
ity in vitro. However, the protein is not required for wild-type-like steady-

state function of peroxisomes, which might be indicative of a metabolic
rather than a biogenetic role. Interestingly, peroxisomes in deletion mutants
of LPX1 have an aberrant morphology characterized by intraperoxisomal
vesicles or invaginations.
Abbreviations
BPC, 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-undecanoyl)-sn-glycero-3-phosphocholine (bis-BODIPY-FL C
11
-PC);
DGR, 1,2-O-dilauryl-rac-glycero-3-glutaric acid (6-methyl resorufin) ester; DPG, 1,2-dioleoyl-3-(pyren-1-yl)decanoyl-rac-glycerol;
PNB, p-nitrobutyrate.
504 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS
peroxisomal defects such as inclusions or invagin-
ations [4,5].
Peroxisomal import of most matrix proteins depends
on the PTS1 (peroxisomal targeting signal type 1)
receptor Pex5p, which recognizes the PTS1 localized at
the very C-terminus [6,7]. The three-amino-acid signal
SKL (serine–lysine–leucine) was the first PTS1 to be
discovered, and is in many cases sufficient for directing
a protein to peroxisomes. Most PTS1 are tripeptides of
the consensus [SAC][KRH][LM] located at the extreme
C-terminus.
A second matrix protein peroxisomal targeting sig-
nal (PTS2) is present in considerably fewer peroxi-
somal proteins. PTS2 is usually located within the first
20 amino acids of the protein, and has been defined
as [RK][LVIQ]XX[LVIHQ][LSGAK]X[HQ][LAF] [8].
PTS2-bearing proteins are recognized by the cytosolic
receptor Pex7p.
Systems biology approaches led to the identification

of Lpx1p as an oleic acid-inducible peroxisomal matrix
protein of unknown function [9,10]. The gene sequence
of LPX1 predicts a lipase motif of the GxSxG type
that is typical for a⁄ b hydrolases [11,12]. Using mass
spectrometry, we identify Lpx1p as present in peroxi-
somes, and analyse its peroxisomal targeting. We show
that it acts as a phospholipase A, and, by electron
microscopy and morphometry, we provide the first evi-
dence for an interesting peroxisomal phenotype of the
Dlpx1 deletion mutant.
Results
Identification of Lpx1p in peroxisomes by mass
spectrometry
We identified Lpx1p (lipase 1 of peroxisomes;
EC 3.1.1.x) in a follow-up study to an exhaustive pro-
teomic characterization of peroxisomal proteins [13].
This involved purification of peroxisomes from oleic-
acid induced Saccharomyces cerevisiae, and subsequent
membrane extraction using low- and high-salt buffers.
Low-salt-extractable proteins were solubilized in SDS
buffer, and separated by RP-HPLC [14]. Proteins in
individual HPLC fractions were further separated by
SDS–PAGE, and protein bands were cut out and anal-
ysed by mass spectrometry. Lpx1p (systematic name:
Yor084wp) was extractable by low salt and identified
together with the peroxisomal aspartate aminotransfer-
ase Aat2p in HPLC fraction 7 at a molecular mass of
approximately 45 kDa (Fig. 1A) [15].
The predicted molecular mass of Lpx1p is 44 kDa.
It carries a peroxisomal targeting signal type 1, gluta-

mine–lysine–leucine (QKL) (Fig. 1B,D). The amino
acid sequence comprises the lipase motif GHSMG of
the general GxSxG type [11,16] with the central serine
being part of the catalytic triad. This lipase motif is
indicative of a⁄ b hydrolase family members [12].
Hydrophobicity predictions [17] indicate a pronounced
hydrophobic region in the central domain, consisting
of amino acids 154–177 with the core region 164LLI-
LIEPVVI173 (Fig. 1C).
By homology searches with other prokaryotic and
eukaryotic hydrolases (not shown) using profile hidden
Markov models [18], we identified a conserved histi-
dine that is probably part of the catalytic triad of the
active site (Fig. 1B). The third member of the catalytic
triad could not be identified by sequence-based
searches.
PTS1-dependent targeting of Lpx1p
to peroxisomes
The majority of the Lpx1p in a cell homogenate was
pelleted at 25 000 g, consistent with an organellar
localization of the protein (Fig. 2A). In this experi-
ment, more of the peroxisomal soluble thiolase Fox3p
(EC 2.3.1.x) than of Lpx1p appears to be present in
the supernatant. This is probably due to partial peroxi-
some rupture during preparation, and might indicate
that Lpx1p, in contrast to Fox3p, is loosely associated
with the peroxisomal membrane.
The peroxisomal localization of Lpx1p had been
demonstrated indirectly by immuno-colabelling of a
heterozygous C-terminally Protein A-tagged version

of Lpx1p in a diploid strain [10]. Peroxisomal locali-
zation under these conditions would depend on the
presence of copies of Lpx1p that are not blocked by
a C-terminal tag, and by the interaction of Lpx1p
with itself (piggyback import). We wished to analyse
whether Lpx1p directly localized to peroxisomes, and
cloned LPX1 for expression from a yeast shuttle
plasmid using an N-terminal GFP tag. This fusion
protein was localized to peroxisomes in a Dlpx1 dele-
tion strain (Fig. 2B), indicating that Lpx1p by itself
targets to peroxisomes. Peroxisomal localization of
Lpx1p was abolished when Lpx1p was expressed with
a C-terminal tag (Fig. 2C), indicating that the
C-terminus has to be free for Pex5p-dependent
import. Peroxisomal localization was abolished in the
absence of Pex5p (Fig. 2C), and was not affected by
the absence of Pex7p (Fig. 2C), indicating that its
targeting to peroxisomes is dependent on the PTS1
pathway.
We confirmed the peroxisomal localization of Lpx1p
by subcellular fractionation. On a sucrose density gra-
dient, GFP–Lpx1p co-migrated with Fox3p (alternative
S. Thoms et al. Peroxisomal lipase Lpx1p
FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS 505
name: Pot1p), with Pex11p, and with the catalase (EC
1.11.1.6) activity peak in the same density fraction at
about 1.225 gÆcm
)3
(fraction 10) (Fig. 2D). The activ-
ity of the mitochondrial marker fumarase (EC 4.2.1.2)

together with the mitochondrial Mir1p showed a
clearly separate peak at a density of 1.192 gÆcm
)3
in
fraction 14 (Fig. 2D).
Lpx1p was identified from low-salt-extractable mem-
branes (Fig. 1A), and the amount of Lpx1p that is not
membrane-associated or found in the non-peroxisomal
low-density fractions (Fig. 2D; fractions 19–29) is low
compared to Fox3p.
Although the QKL C-terminus of Lpx1p does not
match the PTS1 consensus [SAC][KRH][LM], a QKL
terminus is able to target a test substrate to peroxi-
somes [19]. Lpx1p is one of four S. cerevisiae proteins
that end in QKL (Fig. 1D), and is probably the only
one that is localized to peroxisomes.
Self-interaction of Lpx1p
C-terminally tagged Lpx1p localizes only to peroxi-
somes when endogenous copies of the protein are pres-
ent [10]. This suggests piggyback import of Lpx1p,
which, in turn, would rely on self-interaction of Lpx1p.
We tested this hypothesis by two-hybrid analysis of
LPX1. Neither the fusion of Lpx1p with the GAL4
binding domain nor its fusion with the activation
domain were auto-activating (Fig. 3A). The strains
expressing both fusions exhibit a strong two-hybrid
interaction signal, exceeding that of the control PEX11
with PEX19 (Fig. 3A). Because complex formation
Fig. 1. Identification of Lpx1p from Saccharomyces cerevisiae
peroxisomes by proteomics. (A) Isolation of putative peroxisomal

proteins by preparative chromatographic separation. Low salt-
extractable peroxisomal proteins were solubilized by SDS and
separated by reverse phase HPLC. Polypeptides of selected frac-
tions were separated by SDS–PAGE and visualized by Coomassie
blue staining. Only the first 13 lanes of the HPLC profile are shown
[15]. The band marked by an asterisk contains the peroxisomal
proteins Lpx1p (predicted molecular mass 44 kDa) and Aat2p (pre-
dicted molecular mass 44 kDa) in HPLC fraction 7 at a molecular
mass of approximately 45 kDa. (B) Alignment of the LPX1 gene
with a Mycoplasma genitale (Mg) gene encoding a putative ester-
ase ⁄ lipase (AAC71532) and with the putative triacylglycerol lipase
AAB96044 from Mycoplasma pneumoniae (Mp). Identical amino
acids are indicated by an asterisk and similar amino acids are indi-
cated by a colon and full point, depending on degree of similarity.
The conserved GxSxG lipase motif is shaded in grey. The lipase
motif contains the putative active-site serine. The arrowhead
indicates the probable active-site histidine, as determined from
alignments using eukaryotic esterase lipase family members (not
shown). The third member of the catalytic triad could not be identi-
fied by sequence-based analysis. (C) Hydropathy plot of Lpx1p.
A Kyte–Doolittle plot was calculated with window size of 11.
Values > 1.8 may be regarded as highly hydrophobic regions.
(D) Termini of all four QKL proteins from S. cerevisiae. Only Lpx1p
is predicted to target to peroxisomes. Positions relative to the
(putative) PTS1 are indicated. Grey boxes, lysine in position -1 and
valine in position -5 are probably required to target Lpx1p to
peroxisomes.
Peroxisomal lipase Lpx1p S. Thoms et al.
506 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS
might play a significant role in peroxisomal (piggyback)

protein import [20], we determined the size of the Lpx1p
complex by gel filtration of cell lysates of oleate-induced
cells on a Superdex 200 column. We found that the
majority of Lpx1p is not present in high-molecular-
mass complexes, but elutes at molecular masses cor-
responding to monomers, dimers and trimers (Fig 3B).
The two-hybrid interaction probably reflects the com-
plex formation. However, our identification of low-
molecular-mass complexes of Lpx1 does not exclude
the possibility that higher-molecular-mass complexes
are transiently formed during topogenesis of the protein.
Lpx1p is not required for peroxisome biogenesis
Having shown that Lpx1p is targeted to peroxisomes
by the soluble PTS1 receptor, we wished to determine
whether Lpx1p is required for the biogenesis of peroxi-
somes. We first tested the Dlpx1 knockout for growth
on oleate. However, Lpx1p is dispensable for growth
on oleate as the only carbon source (Fig. 4A). To
determine the influence of Lpx1p on peroxisome bio-
genesis in more detail, post-nuclear supernatants were
prepared from wild-type and Dlpx1 strains. The post-
nuclear supernatants were analysed by Optiprep gradient
analysis and subsequent tests of gradient fractions for
peroxisomal catalase and mitochondrial cytochrome c
oxidase (EC 1.9.3.l; Fig. 4B). None of these marker pro-
teins indicated a significant change in the abundance or
density of peroxisomes or mitochondria, suggesting
that peroxisomal and mitochondrial biogenesis remain
functional after deletion of the LPX1 gene.
Lipase activity of Lpx1p

Characteristic GxSxG motifs and similarities with
a ⁄ b hydrolases in the predicted protein sequence sug-
gest that Lpx1p is an esterase, possibly a lipase
[11,12,16]. To directly investigate Lpx1p, we expressed
the full-length protein as a fusion protein with a C-ter-
Fig. 2. Localization and PTS1-dependent targeting of Lpx1p to peroxisomes. (A) Immunological detection of GFP–Lpx1p in a sedimentation
experiment. A cell-free homogenate (T) was separated into supernatant (S) and an organelle-containing pellet fraction (P) by centrifugation at
25 000 g (30 min). Amounts corresponding to equal T content of each fraction were analysed by SDS–PAGE and western blotting with
antibodies against GFP and the peroxisomal marker protein oxoacyl CoA thiolase, Fox3p (alternative name: Pot1p). (B) Lpx1p is localized to
peroxisomes. Coexpression of PTS2-dsRed and GFP–Lpx1p in yeast cells. Cells were grown on ethanol to induce the expression of PTS2-
dsRed. (C) Import of Lpx1p into peroxisomes is dependent on Pex5p and independent of Pex7p. Lpx1p was expressed as either a C-terminal
fusion (top images) or N-terminal fusion (bottom images) with GFP. In the Dpex5 deletion mutant, Lpx1p cannot be imported into peroxi-
somes, irrespective of the position of the tag (right). Deletion of PEX7 does not influence Lpx1p targeting if the PTS1 is not blocked by GFP
(top left). GFP fusion proteins that are not targeted to peroxisomes mislocalize to the cytosol. Bar = 2 lm. (D) Sucrose density gradient anal-
ysis of GFP–LPX1-transformed yeast. A cell-free organelle sediment from oleate induced cells was analysed on a density gradient with
sucrose concentrations form 32 to 54% w ⁄ v. Individual fractions were analysed for catalase activity (peroxisomal marker) and fumarase
activity (mitochondrial marker). In addition, the presence of GFP–Lpx1p, Fox3p, Pex11p (peroxisomal membrane protein) and Mir1p (mito-
chondrial phosphate carrier) was tested by western blotting and immunodetection.
S. Thoms et al. Peroxisomal lipase Lpx1p
FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS 507
minal hexahistidine tag in Escherichia coli and purified
the protein using immobilized metal-ion affinity chro-
matography (Fig. 5A). The protein was further puri-
fied by gel filtration on a Superdex 200 column
(Fig. 5B). Gel filtration indicated the propensity of
Lpx1p to oligomerize in vitro, albeit to a much lower
extent than in yeast whole-cell lysates (compare
Figs 3B and 5B).
Purified protein was used for the generation of poly-
clonal antibodies in rabbit. Antisera recognized a pro-

tein of about 43 kDa, indicating that the antiserum is
specific for Lpx1p. We used these antibodies to con-
firm that the endogenous yeast Lpx1p is induced by
oleic acid (Fig. 5A).
To analyse the enzyme activity of Lpx1p, we assayed
the E. coli-expressed protein for esterase activity, using
p-nitrophenyl butyrate (PNB) as the test substrate. PNB
can be hydrolysed by esterases, yielding free p-nitro-
phenol, which can be determined photometrically at
410 nm. Lpx1p hydrolysed the test substrate with a
K
M
of 6.3 lm and V
max
of 0.17 lmolÆs
)1
(Table 1).
Lpx1p is strongly induced by oleic acid, regulated by
stress-associated transcription factors [21], and aligns
with human epoxide hydrolases (EC 1.14.99.x; not
shown). We found that Lpx1 hydrolysed the epoxide
hydrolase substrate [22] 4-nitrophenyl-trans-2,3-epoxy-
3-phenylpropyl carbonate (NEPC) (data not shown),
but we consider that this activity is non-specific,
because it could not be blocked by the specific epoxide
hydrolase inhibitor N,N’-dicyclohexylurea (DCU) (data
not shown).
To test for lipase activity, we used 1,2-dioleoyl-3-
(pyren-1-yl)decanoyl-rac-glycerol (DPG) as a substrate.
DPG contains the eximer-forming pyrene decanoic

acid as one of the acyl residues. Upon cleavage, the
free pyrene decanoic acid shows reduced eximer
fluorescence. Lpx1p exerts lipase activity towards
DPG of 5.6 pmolÆh
)1
Ælg
)1
(Table 1). For comparison,
we measured the lipase activity of commercial yeast
Candida rugosa lipase towards DPG and found an
Fig. 3. Lpx1p interacts with itself. (A) Two-hybrid assay. Full-length
Lpx1p was fused to the GAL4 binding or activation domain and co-
expressed in a yeast strain with Escherichia coli b-galactosidase
under the control of a GAL4-inducible promotor. b-galactosidase
activity was measured in lysates of doubly transformed strains. No
signal was obtained when LPX1 was combined with empty
vectors. Positive control: interaction of Pex19p with Pex11p.
(B) Size-exclusion chromatography of a wild-type cell lysate of
oleate-induced cells. The lysate was fractionated by gel filtration
on a Superdex 200 column and tested by immunoblotting with
anti-Lpx1p antiserum. The molecular masses indicated were
interpolated from a calibration curve and correspond well with
monomeric, dimeric and trimeric forms of Lpx1p. The relative
distribution of the three forms was quantified using NIH Image
(National Institutes of Health, Bethesda, MD, USA). The elution vol-
ume is indicated in millilitre.
Fig. 4. Absence of pex phenotype in a Dlpx1 deletion. (A) Growth
on plates with oleate as the only carbon source. Wild-type, Dlpx1 or
Dpex1 control stains were spotted in equal cell numbers in series of
10-fold dilutions on oleate or ethanol plates. Absence of growth and

oleic acid consumption (halo formation) indicates a peroxisomal
defect. Control: growth assay on ethanol. (B) Optiprep density gradi-
ent centrifugation analysis of postnuclear supernatants prepared
from oleate-induced wild-type and Dlpx1 strains. All fractions were
analysed using catalase (peroxisome) and cytochrome c oxidase
(mitochondria) enzyme assays. The peroxisomal and mitochondrial
densities were not measurably altered by LPX1 deletion.
Peroxisomal lipase Lpx1p S. Thoms et al.
508 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS
activity of 2.0 pmolÆh
)1
Ælg
)1
under the same assay con-
ditions (Table 1).
We sought to confirm lipase activity by testing
Lpx1p in a clinical assay for pancreatic lipase. The
assay uses the substrate 1,2-O-dilauryl-rac-glycero-
3-glutaric acid (6-methyl resorufin) ester (DGR) in a
desoxycholate-containing buffer. Lpx1p did not hydro-
lyse this substrate under the assay conditions (Table 1).
Next we tested for phospholipase C activity in a
coupled enzyme assay with phosphatidylcholine as the
substrate. In this assay, phospholipase C converts
phosphatidylcholine to phosphocholine and diacylglyc-
erol. Alkaline phosphatase hydrolyses phosphocholine
to form choline, which is then oxidized by choline
oxidase to betaine and hydrogen peroxide. The latter,
in the presence of horseradish peroxidase, reacts with
10-acetyl-3,7-dihydrophenoxazine to form fluorescent

resorufin. This assay, as well as a similar assay for
phospholipase D, gave negative results for Lpx1p
(Table 1).
Finally, we tested phospholipase A (EC 3.1.1.4)
activity using the substrate 1,2-bis-(4,4-difluoro-5,7-
dimethyl-4-bora-3a,4a-diaza-sindacene-3-undecanoyl)-
sn-glycero-3-phosphocholine (bis-BODIPY-FL C
11
-PC,
BPC). BPC is a glycerophosphocholine with BODIPY
dye-labeled sn-1 and sn-2 C
11
acyl chains. Cleavage
reduces dye quenching and leads to a fluorecence
increase at 530 nm upon excitation at 488 nm. Lpx1p
exerts phospholipase A activity of 7.9 pmolÆh
)1
Ælg
)1
.
As a control enzyme, we used commercial porcine
pancreas lipase, which hydrolysed 195 pmolÆh
)1
Ælg
)1
.
In summary, Lpx1p shows acyl esterase, lipase and
phospholipase A activity towards PNB, DPG and
BPC, respectively.
Altered peroxisome morphology in deletion

mutants of LPX1
Lastly, we analysed electron microscopic (EM) images
of knockouts of LPX1. To our surprise, a large
number of Dlpx1 peroxisomes showed an abnormal
morphology. The peroxisomes appear vesiculated
Fig. 5. Protein expression, antibody genera-
tion and oleate induction of Lpx1p. Expres-
sion of Lpx1p in Escherichia coli. (A) Lpx1p
was expressed as a fusion protein with a
C-terminal hexahistidine tag and purified by
His-trap chromatography. The purified Lpx1p
(lane 1) was used to generate polyclonal
antibodies in rabbit that recognize the puri-
fied recombinant protein (lane 4). Endoge-
nous Lpx1p in whole yeast lysates is
recognized only after induction of peroxi-
somes and Lpx1p by oleate (lane 2 versus
lane 3). Molecular masses are shown in
kDa. (B) Second purification step: gel filtra-
tion on Superdex 200 column. The elution
profile indicates that most of the protein
behaves as a monomer, but a small propor-
tion forms dimers and trimers.
Table 1. Esterase, lipase, and phospholipase activity of Lpx1p.
Esterase activity was measured using PNB (p-nitrobutyrate) as a
substrate. K
M
and V
max
values were calculated using Michaelis–

Menten approximations. Lipase activity was determined using DPG
as a substrate. Activity was measured from two independent pro-
tein preparations in triplicate. Candida rugosa lipase (CRL) was used
as a positive control for lipase measurement. (Pancreas) lipase
activity assays used DGR in a coupled enzyme assay as a sub-
strate. Phospholipase C and D (PLC and PLD) activities were mea-
sured in coupled enzyme assays using phosphatidylcholine (PC).
Phospholipase A measurements used BPC (bis-BODIPY-FL C
11
-PC)
as a test substrate. Porcine pancreas lipase (PPL) was used as a
control.
Enzyme Substrate Activity
Activity parameters
(pmolÆh
)1
Ælg
)1
)
Lpx1p PNB Acyl esterase K
M
6.3 lM;
V
max
0.17 lmolÆs
)1
Lpx1p DPG (Triacylglycerol)
lipase
5.6 ± 1.5
CRL DPG (Triacylglycerol)

lipase
2.0 ± 0.1
Lpx1p DGR (Pancreas) lipase Below detection limit
Lpx1p PC PLC Below detection limit
Lpx1p PC PLD Below detection limit
Lpx1p BPC PLA 7.9
PPL BPC PLA 195
S. Thoms et al. Peroxisomal lipase Lpx1p
FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS 509
(Fig. 6B), and either contain intraperoxisomal vesicles
or their membrane is grossly invaginated. On average,
one vesiculated peroxisome is visible in every fifth
mutant cell (Fig. 6E). When the average number of
altered peroxisomes is counted, we find that every
third peroxisome shows this vesiculation phenotype
(Fig. 6D). This is a high percentage, considering the
fact that the peroxisomes were viewed in thin micro-
tome sections. In three dimensions, every single peroxi-
some might contain a vesicular membrane or
indentation that escapes notice in two-thirds of the
‘two-dimensional’ sections.
The average number of peroxisomes per cell is
insignificantly increased in Dlpx1 (2.95 versus 2.76,
Fig. 6C). Wild-type cells did not contain any vesicu-
lated peroxisome (Fig. 6A,D,E). The drastic phenotype
of Dlpx1 is reminiscent of the peroxisomal morphology
found in peroxisomal disorders.
Discussion
Lpx1p is a peroxisomal protein with an unusual
PTS1

LPX1 is one of the most strongly induced genes fol-
lowing a shift from glucose to oleate, as determined by
serial analysis of gene expression (SAGE) experiments
[9]. The oleate-induced increase in mRNA abundance
is abolished in the Dpip2 Doaf1 double deletion strain,
indicating that its induction is dependent on the tran-
scription factor pair Pip2p and Oaf1p [9]. The Lpx1p
protein itself is induced by oleic acid as determined
using a Protein A tag [10] or by use of an antibody
raised against Lpx1p (see Results).
Lpx1p does not conform to the general PTS1 con-
sensus. The other three QKL proteins in S. cerevisiae
are probably not peroxisomal (Fig. 1D): Efb1p
(systematic name: Yal003wp) is the elongation factor
EF-1b [23], Rpt4p (Yor259cp) is a mostly nuclear
19S proteasome cap AAA protein [24], and Tea1p
(Yor337wp) is a nuclear Ty1 enhancer activator [25].
However, QKL is sufficient to sponsor Pex5p binding
[19]. Why are these QKL proteins not imported into
peroxisomes? This is probably due to the upstream
sequences. Lpx1p has a lysine at position -1 (relative
to the PTS1 tripeptide) and a hydrophobic amino acid
at position -5 (Fig. 1D). These features promote Pex5p
binding and are not found in the other three QKL
proteins (Fig. 1D) [19]. Our views were confirmed
by applying a PTS1 prediction algorithm (http://
mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp)
[26], which predicted peroxisomal localization for
Lpx1p only of the four proteins listed in Fig. 1D.
Fig. 6. Peroxisome morphology phenotype of the Dlpx1 deletion.

Absence of LPX1 leads to drastic peroxisomal vesiculation or invagi-
nation. Electron microscopic images of cells from (A) wild-type and
(B) Dlpx1. All cells were grown on medium with 0.1% oleic acid. Per-
oxisomes are marked by arrowheads. Bar = 2 lm. (C) Comparison of
per cell peroxisome numbers in wild-type and Dlpx1 strains. (D) Aver-
age number of vesicles per peroxisome (wild-type, n = 94; Dlpx1,
n = 142). In Dlpx1, about every third peroxisome contains a vesicle.
(E) Percentage of cells with vesicle-containing peroxisomes. Roughly
one in five Dlpx1 cells carries peroxisomes with a vesicle or invagi-
nations (wild-type, n = 34; Dlpx1, n = 48). px, peroxisome(s).
Peroxisomal lipase Lpx1p S. Thoms et al.
510 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS
Lipase activity and cellular function of Lpx1p
Lpx1p could be involved in various processes: (a)
detoxification and stress response, (b) lipid mobiliza-
tion, or (c) peroxisome biogenesis. As Lpx1p expres-
sion may be regulated by Yrm1p and Yrr1p [21], a
transcription factor pair that mediates pleiotropic drug
resistance effects, we speculate that Lpx1p is required
for a multidrug resistance response that did not show
a phenotype in our experiments. We could, however,
exclude epoxide hydrolase activity for Lpx1p, because
hydrolysis of the epoxide hydrolase test substrate
was not affected by a specific epoxide hydrolase
inhibitor.
We investigated the dimerization of Lpx1p in the
context of piggyback protein import into peroxisomes.
Self-interaction (dimerization) is frequently found in
regulation of the enzymatic activity of other lipases
such as C. rugosa lipase or human lipoprotein lipase

[27,28]. The putative active-site serine of Lpx1p is
located next to the region of highest hydropathy, sug-
gesting that Lpx1p is a membrane-active lipase that
contributes to metabolism or the membrane shaping of
peroxisomes.
Peroxisomes are sites of lipid metabolism. It is thus
not surprising to find a lipase associated with peroxi-
somes. Our experiments show that Lpx1p has triacyl-
glycerol lipase activity; however, activities towards the
artificial test substrates DPG and DGR were low. Our
evidence for phospholipase A activity of the enzyme,
together with the EM phenotype, suggest that Lpx1
has a more specialized role in modifying membrane
phospholipids.
Recently, a mammalian group VIB calcium-indepen-
dent phospholipase A2 (iPLA
2
c) was identified that
possesses a PTS1 SKL and a mitochondrial targeting
signal [29,30]. The enzyme is localized in peroxisomes
and mitochondria, and is involved, among others, in
arachinoic acid and cardiolipin metabolism [31,32].
Knockout mice of iPLA
2
c show mitochondrial ⁄ cardio-
logical phenotypes [33]. It will be exciting to determine
whether human iPLA
2
c and yeast Lpx1p are function-
ally related.

We have provided evidence that peroxisomes are still
functional in the absence of LPX1. This suggests a
non-essential metabolic role for Lpx1p in peroxisome
function. The morphological defect found in electron
microscopic images of a deletion of Lpx1p
(peroxisomes containing inclusions or invaginations) is
symptomatic of a yeast peroxisomal mutant, and is
reminiscent of the phenotypes found in human peroxi-
somal disorders [4,5]. Out data suggest that Lpx1p is
required to determine the shape of peroxisomes.
Experimental procedures
Strains and expression cloning
The S. cerevisiae strains BY4742, BY4742 Dyor084w,
BY4742 Dpex5, BY4742 Dpex7 and BY4742 Dpex1 were
obtained from EUROSCARF (Frankfurt, Germany). S. ce-
revisiae strain BJ1991 (Mata leu2 trp1 ura3-251 prb1-1122
pep4-3) has been described previously [34].
Genomic S. cerevisiae DNA was used as a PCR template
for PCR. For construction of pUG35-LPX1 (LPX1–GFP),
PCR-amplified YOR084w (primers 5¢-GCTCTAGAATG
GAACAGAACAGGTTCAAG-3¢ and 5¢-CGGAATTCCA
GTTTTTGTTTAGTCGTTTTAAC-3¢) was subcloned into
EcoRV-digested pBluescript SK
+
(Stratagene, La Jolla,
CA, USA), and then introduced into the XbaI and EcoRI
sites of pUG35 (HJ Hegemann, Du
¨
sseldorf, Germany). For
construction of pUG36-LPX1 (GFP–LPX1), PCR-amplified

YOR084w (primers 5¢-GAGGATCCATGGAACAGAACA
GGTTCAAG-3¢ and 5¢-CGGAATTCTTACAGTTTTTGT
TTAGTCGTTTTAAC-3¢) was subcloned into EcoRV-
digested pBluescript SK
+
, and then cloned into the BamHI
and EcoRI sites of pUG36 (HJ Hegemann).
pET21d-LPX1 was constructed by introducing PCR-
amplified YOR084w (primers 5¢-GAATCCATGGAACAG
AACAGGTTCAA-3¢ and 5¢-CGGTACCGCGGCCGCCA
GTTTTTGTTTAGTCGTTTT-3¢) into the NcoI and NotI
sites of pET21d (EMD Chemicals, Darmstadt, Germany).
For construction of pPC86-LPX1 and pPC97-LPX1,
YOR084w was amplified using primers 5¢-CCCGGGAAT
GGAACAGAACAGGTTCAAG-3¢ and 5¢-AGATCTTTA
CAGTTTTTGTTTAGTCGTTTT-3¢, and introduced into
pGEM-T (Promega, Mannheim, Germany). The ORF was
excised using XmaI and BglII, and introduced into pPC86
and pPC97 [35]. All constructs were confirmed by DNA seq-
uencing. pPTS2-DsRed has been described previously [36].
Image acquisition
Samples were fixed with 0.5% w ⁄ v agarose on microscopic
slides. Fluorescence microscopic images were recorded on
an Axioplan2 microscope (Zeiss, Ko
¨
ln, Germany) equipped
with an aPlan-FLUAR 100 x ⁄ 1.45 oil objective and an
AxioCam MRm camera (Zeiss) at room temperature. If
necessary, contrast was linearly adjusted using the image
acquisition software Axiovision 4.2 (Zeiss).

Protein purification and antibody generation
Lpx1p was expressed from pET21d-LPX1 in BL21(DE3)
E. coli. Cells were harvested by centrifugation (SLA3000,
4000 g, 15 mins), and resuspended in buffer P (1.7 mm
potassium dihydrogen phosphate, 5.2 mm disodium hydro-
gen phosphate, pH 7.5, 150 mm sodium chloride) containing
S. Thoms et al. Peroxisomal lipase Lpx1p
FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS 511
a protease inhibitor mix (8 lm antipain-dihydrochloride,
0.3 lm aprotinin, 1 lm bestatin, 10 l m chymostatin, 5 lm
leupeptin, 1.5 lm pepstatin, 1 mm benzamidin, and 1 mm
phenylmethane sulfonylfluoride) and 50 l g Æ mL
)1
lysozyme,
22.5 lgÆmL
)1
DNAse I and 40 mm imidazole. Cells were
sonicated 20 times for 20 s each using a 250D Branson
digital sonifier (Danbury, CT, USA) with an amplitude
setting of 25%. After removal of cell debris (SS34,
27 000 g, 45 min) the supernatant was clarified by 0.22 lm
filtration (Sarstedt, Nu
¨
mbrecht, Germany) and loaded
on Ni-Sepharose columns (GE Healthcare, Munich,
Germany) equilibrated with buffer W (buffer P containing
300 mm sodium chloride, 1 mm dithiothreitol, 40 mm
imidazole). The column was washed in buffer W until no
further protein was eluted. Recombinant Lpx1p was eluted
by a continuous 40–500 mm imidazole gradient based on

buffer W. Peak fractions (identified by SDS–PAGE) were
pooled and concentrated using VivaSpin concentrators
(30 kDa cutoff, Sartorius, Go
¨
ttingen, Germany). Lpx1p
was further purified by gel-filtration chromatography.
Protein was stored at 0 °C. For the production of poly-
clonal antibodies, gel bands corresponding to 150 lg
protein were excised and used for rabbit immunization
(Eurogentec, Seraing, Belgium).
Size-exclusion chromatography
For analysis of endogenous Lpx1p by gel filtration, 5 mL of
a glass bead lysate of oleate-induced BY4742 wild-type cells
in buffer A (buffer P, pH 7.3, 300 mm sodium chloride) with
a protease inhibitor mix were injected into a HiLoad 16 ⁄ 60
Superdex 200 prepgrade column (GE Healthcare) and eluted
using buffer A at a flow rate of 1 mL
)1
Æmin and a fraction
size of 2 mL. Fractions were analysed by SDS–PAGE and
Western blotting. A 500 lL aliquot of the concentrated
Ni-Sepharose eluate of Lpx1p from E. coli expression was
purified in the same buffer under the same conditions. For
estimation of Lpx1p complex sizes, molecular masses were
interpolated from a calibration curve generated using
ovalbumin (45 kDa), carboanhydrase (29 kDa), trypsin
inhibitor (20.1 kDa), lactalbumin (14.2 kDa) and aprotinin
(6.5 kDa) as molecular mass standards.
Enzyme assays
Esterase activity was determined using 0.5 mm p-nitrophe-

nyl butyrate (Sigma-Aldrich, Seelze, Germany) in NaCl ⁄ P
i
(pH 7.4) in a total volume of 200 lLat37°C. The amount
of free p-nitrophenol was determined at 410 nm in 96-well
plates. Michaelis–Menten kinetics were analysed using
GraphPad Prism4 (Graph Pad Software, San Diego, CA,
USA).
Lipase activity was determined using 0.5 mm DPG (Mar-
ker Gene Technologies, Eugene, OR, USA) in 0.1 m gly-
cine, 19 mm sodium deoxicholate, pH 9.5, in a total volume
of 200 lLat37°C. Hydrolysis of DPG was followed in
96-well plates at 460 nm with 360 nm excitation using
a Sirius HT fluorescence plate reader (MWG Biotech,
Ebersberg, Germany). Lipase activity towards DPG was
measured in assay setups containing 2–10 lg Lpx1p (from
two independent protein preparations), with C. rugosa
triacylglycerol lipase (Lipase AT30 Amano, 1440 UÆmg
)1
,
Sigma-Aldrich) as a control.
Phospholipase A activity was measured using bis-
BODIPY FL C
11
-PC (Molecular Probes ⁄ Invitrogen,
Eugene, OR, USA) as the substrate. The assay setup con-
tained 70 lg Lpx1p in 50 lL assay buffer (50 mm Tris,
100 mm sodium chloride, 1 mm calcium carbonate, pH 8.9)
together with 50 lL substrate-loaded liposomes. Liposomes
were prepared by injecting 90 lL of an ethanolic mixture of
3.3 mm dioleyl phosphatidylcholine (Avanti Polar Lipids,

Alabaster, AL, USA), 3.3 mm dioleyl phosphatidylglycerol
(Avanti Polar Lipids) and 0.33 mm bis-BODIPY FL C
11
-
PC into 5 ml assay buffer. Substrate turnover was mea-
sured at 528 nm emission after 488 nm excitation. Activity
was calculated from the initial velocity. Porcine pancreas
phospholipase A
2
(Fluka ⁄ Sigma-Aldrich, Buchs, Swizer-
land) was used as a control.
Density gradient centrifugation
Gradient centrifugation was carried out essentially as
described previously [37]. Briefly, oleate-induced yeast cells
were converted to spheroblasts using 25 UÆg
)1
Zymoly-
ase 100T (MP Biomedicals, Illkirch, France). Spheroblasts
were gently ruptured by Potter–Elvehjem homogenization,
and centrifuged at low speed (3 · 10 min at 600 g)to
generate postnuclear supernatants. These supernatants, con-
taining 5 mg protein, were loaded on a 32–54% sucrose
gradient (Fig. 2D) or an Optiprep gradient (Fig. 4B) and
centrifuged for 3 h at 19 000 g (Sorvall SV288, 19 000 rpm,
4 °C). The gradient was fractionated into about 29 frac-
tions of 1.2 mL. Fractions were analysed using enzyme
assays for oxoacyl CoA thiolase, catalase, fumarase and
cytochrome c oxidase [37].
Other methods
Mass spectrometry and high-pressure lipid chromatography

have been described previously [14,15,38,39]. Subcellular
fractionation, yeast two-hybrid assays and electron micros-
copy were carried out as described previously [37].
Acknowledgements
We thank Elisabeth Becker, Monika Bu
¨
rger and Uta
Ricken for technical assistance. We thank Sabine Wel-
ler and Hartmut Niemann for reading the manuscript.
We extend our thanks to three anonymous reviewers
Peroxisomal lipase Lpx1p S. Thoms et al.
512 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS
who helped to improve the manuscript. This work
was supported by the Deutsche Forschungsgemeins-
chaft (Er178 ⁄ 2-4) and by the Fonds der Chemischen
Industrie.
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