From the Department of Medicine, Division of
Endocrinology, University of Helsinki, Biomedicum Helsinki, 00029 HUS, Helsinki, Finland and ¶ CSC Scientific Computing Ltd., 02101, Espoo, Finland
Received for publication, January 7, 2003, and in revised form, January 24, 2003
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ABSTRACT |
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We have investigated the signal sequence for
mitochondrial transport of mutants (I12T, 78insC, IVS2-2a Protoporphyrinogen oxidase
(PPOX,1 EC 1.3.3.4) is the
penultimate enzyme in the heme biosynthesis (1). PPOX catalyzes the
six-electron oxidation of protoporphyrinogen IX to the planar, fully
conjugated macrocycle protoporphyrin IX in the inner membrane of the
mitochondrion and requires oxygen for its activity (2). Partial
deficiency of PPOX causes a disease, variegate porphyria (VP,
OMIMTM accession number 176200), that is inherited
as an autosomal dominant trait displaying incomplete penetrance (3).
The biochemical abnormalities found in VP patients include
overproduction and increased excretion of porphyrins and porphyrin
precursors. VP manifests clinically with photosensitivity and acute
attacks, which include various neuropsychiatric symptoms (4, 5).
PPOX is anchored to the inner membrane of mitochondria in eukaryotes
(6) with its active site facing the cytosolic side of the membrane (7).
The anchoring may involve amphipathic helical domains inserting PPOX
into the inner mitochondrial membrane. Alternatively post-translational
acylation may facilitate transient or permanent association of PPOX
with the membrane (8). The majority of proteins imported to
mitochondria contain a signal sequence of 20-60 amino acids in their
amino terminus that directs them into mitochondria (9). The
mitochondrial targeting signals of different polypeptides show no amino
acid sequence identity, but they have characteristic physicochemical
properties. They are enriched in positively charged, hydroxylated, and
hydrophobic residues; have no acidic residues; and usually are able to
form an amphiphilic secondary structure (10, 11). There is evidence that several amino-terminal mitochondrial targeting signals interact with the general import receptor Tom20 (translocase of outer membrane), which is a part of the TOM complex (12, 13) (for a review, see Refs. 14
and 15).
The amino terminus of PPOX contains a characteristic In this communication we have investigated the mitochondrial transport
of seven PPOX mutants by expressing the green fluorescent protein (GFP)
fusion proteins in COS-1 cells and studying their intracellular
localization. The mitochondrial targeting signal in the amino terminus
of PPOX was characterized, and a model for the interaction between the
targeting signal and the mitochondrial receptor Tom20 was proposed.
Seven mutations selected for this study were previously
identified from Finnish VP patients and expressed in Escherichia
coli and COS-1 cells (Table I) (21,
22).
c,
338G
C, R152C, 470A
C, and L401F) and the wild type
protoporphyrinogen oxidase (PPOX), which is the penultimate enzyme in
the heme biosynthesis. We constructed the corresponding green
fluorescent protein fusion proteins and studied their
intracellular localization in COS-1 cells. We showed that 28 amino
acids in the amino terminus of PPOX contain an independently functioning signal for mitochondrial targeting. The experiments with
amino-terminally truncated green fluorescent protein fusion proteins
revealed that amino acids 25-477 of PPOX contained an additional
mitochondrial targeting signal(s). We constructed a structural model
for the interaction between the amino-terminal end of PPOX and the
putative mitochondrial receptor protein Tom20. The model suggests that
leucine and isoleucine residues Leu-8, Ile-12, and Leu-15
forming an
-helical hydrophobic motif,
LXXXIXXL, were crucial for the recognition of
the targeting signal. The validity of the model was tested using
mutants L8Q, I12T, and L15Q disrupting the hydrophobic surface of the
LXXXIXXL helix. The results from
in vitro expression studies and molecular modeling were in
accordance supporting the hypothesis that the recognition of the
mitochondrial targeting signal is dependent on hydrophobic interactions
between the targeting signal and the mitochondrial receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
dinucleotide binding motif (Fig. 1A), which is often found
within flavine adenine dinucleotide-binding domains (16). The amino terminus of PPOX is also a putative mitochondrial targeting domain since it contains three basic residues and no acidic residues, and it
is capable of forming an
-helix (17). Of the more than 110 mutations
reported in the PPOX gene
worldwide,2 five are located
in this domain and potentially interfere with the mitochondrial
transport (18-22).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Characteristics of mutations
Normal and Mutant PPOX-GFP Constructs--
The normal and mutant
PPOX cDNAs were expressed as CT-GFP fusion proteins in COS-1
cells using the pcDNA3.1/CT-GFP vector (Invitrogen). The human
PPOX-pUC18 (kindly provided by Prof. S. Taketani, Kyoto Institute of
Technology, Kyoto, Japan) was digested with EcoRI and
XbaI, and the fragment including exons 1-7 was ligated as a
cassette to the pcDNA3.1/CT-GFP vector with T4 DNA ligase (New
England Biolabs, Beverly, MA). Exons 7-13 were amplified using
PPOX-pUC18 as a template and primers 1 and 2, which abolished the stop codon (Table II). The PCR fragment was
digested with XbaI and ligated
as a cassette into PPOX-(ex1-7)-pcDNA3.1/CT-GFP to obtain a
full-length PPOX-GFP. The constructs for the mutations I12T, R152C, and
470AC were made by digesting the mutated PPOX-pUC18 constructs (21,
22) with PpuMI and ligating each fragment with a mutation as
a cassette into the corresponding sites of the wild type PPOX-GFP. For
the 78insC mutation, the mutated PPOX-pUC18 was amplified with primers
3 and 4. The PCR fragment was digested with EcoRI and
XbaI and ligated as a cassette into the PPOX-GFP to replace
the wild type cDNA resulting in PPOX-(1-28)-GFP. The constructs
for the mutations IVS2-2a
c and 338G
C were made similarly using antisense primers 5 and 6, respectively. cDNA of each
construct was sequenced to confirm their authenticity.
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PPOX-GFP Constructs with Deletions or Missense Mutations in the
Amino Terminus--
For PPOX-(5-11)-GFP, exons 2-7 were
amplified using PPOX-pUC18 as a template and primers 7 and 8. The PCR
fragment was digested with EcoRI and Eco47III and
ligated as a cassette into PPOX-GFP. PPOX-(
5-24)-GFP was created
similarly using primers 8 and 9. For PPOX-(1-28)-L8Q-GFP, exons 2-7
were amplified using PPOX-pUC18 as a template with primers 4 and 10. The PCR fragment was digested with EcoRI and XbaI
and ligated as a cassette into PPOX-GFP to replace the wild type
cDNA. PPOX-(1-28)-L15Q-GFP was created similarly using primers 3 and 11, and PPOX-(1-28)-I12T-GFP was created with primers 3 and 4 together with PPOX-I12T-GFP as a template.
COS-1 Cell Culture and DNA Transfection-- COS-1 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal heat-inactivated bovine serum, penicillin (10,000 units/ml, Invitrogen), and streptomycin (10,000 units/ml). For transfection, the cells were seeded on a 3-cm six-well plate at 400,000 cells/well and grown overnight. The 50-70% confluent cells were transfected with 1.5 µg of the PPOX-GFP or mutated constructs by lipofection using FuGENE 6 transfection reagent (Roche Molecular Biochemicals). The pcDNA3.1/CT-GFP vector alone was used as a negative control in all experiments.
GFP Fluorescence, Mitochondrial Staining, and Confocal Laser
Scanning Microscopy--
For GFP fluorescence studies, COS-1 cells
were trypsinized and replated on glass coverslips 24 h after
transfection. For mitochondrial staining, 40 nM MitoTracker
Red CMXRos (Molecular Probes, Eugene, OR) was added to medium
for 45 min at 48 h post-transfection. The cells were fixed
immediately with 3% paraformaldehyde for 30 min at room temperature.
After fixation, the cells were washed three times in phosphate-buffered
saline and mounted in Mowiol 4-88 mounting medium (Calbiochem). The
cells were observed on a Leica TCS confocal laser scanning microscope
(Leica Microsystems GmBH, Heidelberg, Germany). Fluorescence of GFP was
excited using a 488 nm argon/krypton laser, and emitted fluorescence
was detected with a 500-530 nm band pass filter. For MitoTracker Red,
a 543 nm helium-neon laser was used for excitation, and fluorescence was detected with a 565-699 nm band pass filter. For three-dimensional imaging, a stack of 30 images with 0.20-µm distance was taken, and
the three-dimensional picture was constructed using Leica Confocal
Software 2.00.
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RESULTS |
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Model of the Interaction of PPOX with Tom20--
Analysis of the
amino-terminal sequence of PPOX using the Swiss-Model and MolMol
programs predicts that an 11-residue polypeptide, PPOX-(11-21),
forms an -helical structure where
hydrophobic residues are clustered together (Fig. 1) (Refs. 23 and 24;
www.expasy.org/swissmod/SWISS-MODEL.html, compared with
monoamine oxidase-B structure (Protein Data Bank entry 1GOS)
with 42.7% amino acid identity as a template).
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The model of PPOX-(6-23) bound to Tom20 was constructed in the Bodil
Modeling Environment
(www.abo.fi/fak/mnf/bkf/research/johnson/bodil.html) using the
coordinates of the NMR structure of rat Tom20 in a complex with an
11-residue recognition peptide from rat aldehyde dehydrogenase, pALDH-(12-22), as a template (Protein Data Bank entry 1OM2). pALDH-(12-22) contains a hydrophobic LXXLL motif in an
-helical conformation where leucines are in contact with a
hydrophobic groove on the surface of Tom20 (Fig.
2B) (25). Rat Tom20 shows a
98% amino acid identity with human Tom20, and the amino acids in the
vicinity of the hydrophobic groove are fully conserved (25).
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When the program Malign (26) in Bodil was used in the comparison of
PPOX-(1-23) and pALDH-(12-22), the sequence GPRLSRLLSYA in
pALDH-(12-22) aligns with the GGGISGLAASY part of the PPOX-(1-23) sequence at best. The most important determinant of this comparison was
alignment of the ISGLA motif in PPOX-(1-23) with the LSRLL motif in
pALDH-(12-22). ISGLA is the only part in PPOX-(1-23) that has a
hydrophobic surface in the -helical conformation similar to the
-helical LSRLL motif in pALDH-(12-22), which is needed for
the interaction with Tom20 (25). The model of the 18-residue PPOX-(6-23) was constructed using Sybyl 6.8 (Tripos Inc., St. Louis,
MO) (Fig. 2A).
The -helical model of PPOX-(6-23) was superimposed on the
coordinates of pALDH-(12-22) in a complex with Tom20 applying a constraint that the LXXLL motif in pALDH-(12-22) must be
aligned with the IXXLA motif in PPOX-(6-23). Sterical
clashes were removed manually by modifying the amino acid side chains
using Bodil Modeling Environment. The
-helical structure of
pALDH-(12-22) bound to Tom20 ends amino-terminally to Pro-13, whereas
in our model of PPOX-(6-23) (VVLGGGISGLAASYHLSR) the
-helix (underlined) continues an additional turn to facilitate the
contact of Leu-8 with Tom20 (Fig. 2C). Together the residues
8-15 (LXXXIXXL) of PPOX-(6-23) form a
continuous hydrophobic surface on one face of the
-helix (Fig.
2A). The validity of the model was tested using mutants Leu-8, Ile-12, and Leu-15 that disrupt the hydrophobic face of the
LXXXIXXL helix.
The Amino Terminus of PPOX Contains a Mitochondrial Targeting
Signal--
We investigated the transport of the wild type and seven
PPOX mutants to mitochondria by constructing GFP fusion proteins in
which GFP was located at the carboxyl-terminal end of PPOX (Fig.
3). PPOX-GFP and GFP construct alone were
used as controls in all experiments. COS-1 cells were transfected with
the constructs, and GFP fluorescence was analyzed with confocal laser
scanning microscopy to monitor subcellular localization of the
polypeptides. The cells expressing the wild type PPOX-GFP demonstrated
a typical filamentous mitochondrial pattern (27, 28), which was clearly distinguishable from the cytosolic pattern of GFP construct alone (Fig.
3, A and B). The localization was confirmed by
counterstaining with a mitochondrion-specific dye (Fig. 3C).
A pattern indistinguishable from PPOX-GFP was observed for each of the
seven mutants (Fig. 3D) indicating that transport to
mitochondria was not impaired in any of them. Since the truncated PPOX
fusion protein corresponding to the mutation 78insC in exon 2 (PPOX-(1-28)-GFP) contained only the first 28 residues of PPOX,
these amino acids must contain an independently functioning
mitochondrial targeting signal.
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The Amino Acids 25-477 of PPOX Contain an Alternative
Mitochondrial Targeting Signal(s)--
After localization of the
mitochondrial targeting signal in the amino terminus of PPOX, two
additional constructs were created where this region was removed either
totally (PPOX-(5-23)-GFP) or partially (PPOX-(
5-11)-GFP) (Fig.
4A). The experiments using fluorescence confocal microscopy demonstrated that these fusion proteins were associated with the mitochondria indicating that the
amino acids 25-477 of PPOX must contain an additional mitochondrial targeting signal(s).
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To make a hypothesis for the molecular recognition of the
amino-terminal mitochondrial targeting signal of PPOX, we constructed an -helical model of PPOX-(6-23) and a model of the interaction between PPOX-(6-23) and the mitochondrial import receptor Tom20. The
model places the LXXXIXXL (Leu-8, Ile-12, and
Leu-15) motif of PPOX-(6-23) in contact with the hydrophobic groove on
the Tom20 surface (Fig. 2B). We tested our structural model
by mutating the PPOX-(6-23) residues Ile-12 into threonine and
residues Leu-8 and Leu-15 into glutamine. These amino acid
substitutions disrupt the hydrophobic face of the
LXXXIXXL helix model (Fig. 4C), which should affect the capability of interaction with Tom20. Each mutation caused a disruption of PPOX transport into mitochondria (Fig. 4,
B and C) confirming that each of these residues
was essential for the PPOX targeting. In the amino terminus of PPOX,
the LXXXIXXL motif including residues Leu-8,
Ile-12, and Leu-15 appeared to be crucial for the putative interaction
between PPOX and Tom20.
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DISCUSSION |
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In this communication, we have investigated the signal sequence for the mitochondrial transport of the wild type PPOX and mutants by constructing the corresponding GFP fusion proteins. The intracellular localization of the seven mutants corresponding to clinically manifest VP showed no impairment of mitochondrial targeting. Because of the naturally occurring mutation 78insC, we were able to show that the first 28 amino acids in the amino terminus of PPOX contained sufficient information for transporting a reporter protein into mitochondria.
The predicted secondary structure of the PPOX amino terminus consists
of a motif (17) where the
-helix is a common structure
found in mitochondrial targeting signals. Our structural model of
interaction between the amino terminus of PPOX and Tom20 and the
experiments with amino-terminally mutated fusion proteins indicated
that the critical residues for the recognition of the PPOX targeting
signal include leucine and isoleucine residues that form a hydrophobic
motif, LXXXIXXL. Our findings support the
hypothesis that recognition of the mitochondrial targeting signal is
dependent on hydrophobic interactions with the mitochondrial receptor.
The NMR structure of rat Tom20 in a complex with the mitochondrial
presequence peptide revealed that an amphiphilic
-helical structure
of the presequences was important for binding to the receptor (25).
Subsequent mutagenesis studies showed that the hydrophobic residues
were essential for binding to Tom20, while the hydrophilic residues,
including two positively charged arginines in the presequence, were
dispensable (25). Consistently, in the amino-terminal recognition
sequence of PPOX, replacing a single hydrophobic leucine or isoleucine
with a hydrophilic residue of the same size could prevent the
mitochondrial transport.
The amino terminus of PPOX contains only three positively charged residues, which is less than in most presequences (29). It has been postulated that the positively charged residues could be involved in subsequent ionic interactions between the targeting signal and Tom22, which is also known to bind amino-terminal recognition sequences, especially their carboxyl-terminal parts, in a salt-sensitive manner (13, 30). In the case of PPOX, two positively charged residues, Arg-23 and Lys-29, which are located in the carboxyl-terminal part of the recognition sequence, could facilitate this interaction.
Surprisingly PPOX derivatives, where the amino-terminal
targeting signal was removed either totally or partially, were still located in the mitochondria. This implies that the residual part of
PPOX must contain an additional mitochondrial targeting signal(s) (29).
PPOX is a further example of mitochondrial proteins whose import is not
strictly dependent on the presence of an amino-terminal presequence
(29, 31-33). Secondary structure prediction of PPOX reveals several
internal leucine-rich -helical segments with a net positive charge.
Such segments can putatively form hairpin-like structures that mimic a
typical amphiphilic presequence and function as an internal
mitochondrial targeting sequence (34). Without knowledge of the
tertiary structure of PPOX it is, however, difficult to predict which
of these segments could be accessible to the receptor and serve as an
effective targeting signal.
Since the naturally occurring mutation I12T resides in the conserved
amino terminus of PPOX, the mutation could interfere with mitochondrial
transport and modify the phenotype of the disease. Our study shows that
although the I12T substitution is able to disrupt the mitochondrial
transport of the truncated PPOX, the corresponding full-length PPOX is
transported into mitochondria. In vitro expression studies
of the I12T substitution have shown a dramatic loss of the enzyme
activity both in prokaryotic and eukaryotic cells (21). A homozygous
patient with the I12T substitution has been identified with around 10%
residual PPOX activity measured from his lymphocytes (21). It would be
intriguing to hypothesize that in this patient the secondary
mitochondrial targeting signal(s) could serve as a backup system that
directs the polypeptide into mitochondria if the primary signal fails.
This transport may be, however, less specific and efficient (35) and
lead to non-optimal submitochondrial compartmentalization. This could
disrupt the final steps of the heme biosynthesis in the inner
mitochondrial membrane especially if the substrate channeling between
the last three enzymes of the pathway, namely coproporphyrinogen
oxidase, PPOX, and ferrochelatase, occurs through an enzyme complex as suggested by Ferreira et al. (7). Disruption of the enzyme complex would explain the low ferrochelatase activity (10% and 30-40% of normal) measured from the homozygous and heterozygous patients' erythrocytes, respectively.
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ACKNOWLEDGEMENTS |
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We thank Dr. Marc Baumann for excellent assistance in protein chemistry and Dr. Pekka Lehtovuori for assistance in molecular modeling.
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FOOTNOTES |
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* This study was supported by grants from the Finnish Cultural Foundation, the Magnus Ehrnrooth Foundation, the Instrumentarium Research Foundation, the Jalmari and Rauha Ahokas Foundation, the Research Funds and the Clinical Research Institute of the Helsinki University Central Hospital, the Biomedicum Helsinki Foundation, and the University of Helsinki.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Porphyria Research Center, Dept. of Medicine, University Central Hospital of Helsinki, Biomedicum Helsinki, P.O. Box 700, 00029 HUS, Helsinki, Finland. Tel.: 358-9-47171910; Fax: 358-9-47171921; E-mail: mikael.fraunberg@hus.fi.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M300151200
2 For mutations in the PPOX gene, see the Human Gene Mutation Database, www.uwcm.ac.uk/uwcm/mg/hgmd0.html.
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ABBREVIATIONS |
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The abbreviations used are: PPOX, protoporphyrinogen oxidase; GFP, green fluorescent protein; VP, variegate porphyria; Tom, translocase of outer membrane; pALDH, aldehyde dehydrogenase presequence peptide; CT, carboxyl terminus; IVS, intervening sequence.
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