From the Endocrinologie, Métabolisme et Développement, CNRS-UPR 1524, 9 Rue J. Hetzel, 92190 Meudon, France
Received for publication, October 19, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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We have previously shown that the first 147 N-terminal residues of the rat liver carnitine palmitoyltransferase 1 (CPT1), encompassing its two transmembrane (TM) segments, specify both mitochondrial targeting and anchorage at the outer mitochondrial membrane (OMM). In the present study, we have identified the precise import sequence in this polytopic OMM protein. In vitro
import studies with fusion and deletion CPT1 proteins demonstrated that none of its TM segments behave as a signal anchor sequence. Analysis of
the regions flanking the TM segments revealed that residues 123-147,
located immediately downstream of TM2, function as a noncleavable,
matrix-targeting signal. They specify mitochondrial targeting, whereas
the hydrophobic TM segment(s) acts as a stop-transfer sequence that
stops and anchors the translocating CPT1 into the OMM. Heterologous
expression in Saccharomyces cerevisiae of several deleted
CPT1 proteins not only confirms the validity of the "stop-transfer" import model but also indicates that residues 1-82 of CPT1 contain a
putative microsomal targeting signal whose cellular significance awaits
further investigation. Finally, we identified a highly folded core
within the C-terminal domain of CPT1 that is hidden in the entire
protein by its cytosolic N-terminal residues. Functional analysis of
the deleted CPT1 proteins indicates that this folded C-terminal core,
which may belong to the catalytic domain of CPT1, requires TM2 for its
correct folding achievement and is in close proximity to residues
1-47.
In mammals, the mitochondrial carnitine palmitoyltransferase
(CPT,1 EC 2.3.1.21) 1 is the
key regulatory enzyme of long chain fatty acid oxidation (1). This
enzyme catalyzes the conversion of long chain acyl-CoA to
acylcarnitines, which permits, in cooperation with the
carnitine/acylcarnitine translocase and the CPT2, their transport from
the cytoplasm into the mitochondrial matrix to undergo Nuclear-encoded mitochondrial proteins are synthesized as precursors in
the cytosol and harbor signals that mediate primarily, via a specific
interaction with the outer mitochondrial receptors (Tom complex), their
mitochondrial targeting and specify their intramitochondrial sorting
(7). The targeting signals in matrix-destined preproteins are cleavable
N-terminal presequences, positively charged and that have the potential
to adopt amphipathic We have previously shown that the N-terminal domain (residues
1-147) of CPT1 contains all of the information for mitochondrial targeting, OMM insertion, and membrane orientation (20). In the present
study, we have identified the precise internal import signal of CPT1 by
using two complementary approaches. In vitro import assay of
fusion and/or deletion CPT1 proteins shows that its import into the OMM
does not occur by a "signal anchor" but by a "stop-transfer"
mechanism with the involvement of an internal matrix-targeting signal
located immediately downstream of TM2. Heterologous expression in
S. cerevisiae of several deleted CPT1 proteins not only
confirms the validity of this model but also allows new insights into
the folding of the C-terminal catalytic domain of CPT1. The functional
importance of certain residues within the N-terminal domain for
maintenance of a putative catalytic core is discussed.
Construction of Fusion and Deletion CPT1 Proteins
Escherichia coli DH5 pCPT1-(1-47)-OM-DHFR and pCPT1-(122-147)-OM-DHFR--
DNA
encoding the OM segment (amino acids 11-29) of S. cerevisiae Tom70 was amplified by PCR using the pOMD29 plasmid
(10) as template and the following primers: the 5'-primer 5'-CG GGA TCC
GCC ATT TTG GCT GCA-3' (introducing a BamHI restriction
site) and the 3'-primer 5'-AAA ATG AGC TCC TCG TGG TGG TTC TTT-3'
(including the SacI restriction site of DHFR in pOMD29). The
PCR product was digested by BamHI and SacI and
ligated into either pCPT1-(1-47)-DHFR or pCPT1-(122-147)-DHFR cut by
the same enzymes to generate pCPT1-(1-47)-OM-DHFR and
pCPT1-(122-147)-OM-DHFR.
pCPT1 pCPT1 pCPT1 pCPT1 pCPT1
cDNAs encoding the deleted CPT1 proteins were retrieved from pGEM4
as an EcoRI-SalI insert and subcloned into the
yeast expression vector pYeDP1/8-10 containing the full-length CPT1
(pYeDP-CPT1) (21) cut by the same enzymes to obtain pYe-CPT1 In Vitro Synthesis of Precursor Proteins and Import into
Mitochondria
Radiolabeled precursor proteins were synthesized by in
vitro transcription-translation using the TNT® SP6-coupled
reticulocyte lysate system (Promega) in the presence of
[35S]methionine (Amersham Pharmacia Biotech) according to
the manufacturer's protocols. Isolation of purified rat liver
mitochondria was performed as described previously (20). In
vitro import of radiolabeled proteins into mitochondria was
carried out for 30 min at 4 or 30 °C in import buffer as in Ref. 20.
Dissipation of the membrane potential ( Submitochondrial Localization of Imported Proteins
After import, samples were split into 5 eq aliquots (70 µg of
mitochondrial protein). Mitochondria were centrifuged at 12,000 × g for 5 min at 4 °C, washed in KCl buffer (250 mM sucrose, 10 mM Hepes, 80 mM KCl,
pH 7.6), and resuspended (0.5 mg of protein/ml). Mitochondria were then
diluted 10-fold either in KCl buffer (nonswollen mitochondria) or in a
swelling buffer (20 mM Hepes-KOH, pH 7.4) and subjected to
trypsin (100 µg/ml) when indicated. After a 20-min incubation on ice,
2.8 mg/ml soybean trypsin inhibitor (STI; Sigma) was added, and samples
were further kept on ice for 10 min. Mitochondria/mitoplasts were
reisolated by centrifugation and washed in EDTA buffer (250 mM sucrose, 10 mM Hepes, 1 mM EDTA,
pH 7.6). Mitoplasts were then solubilized by 0.5% (v/v) Triton X-100
(Sigma) in the absence or presence of trypsin (400 µg/ml). After
centrifugation, both the pellet and the supernatant were incubated 5 min at 65 °C to inhibit trypsin, and the solubilized proteins
present in the supernatant were trichloroacetic acid-precipitated.
Samples were submitted to SDS-PAGE, blotted onto nitrocellulose, and
analyzed by fluorography. The efficiency of swelling and solubilization
of the mitoplasts was assessed by immunostaining.
Yeast Culture and Subcellular Fractionation
The yeast expression vector pYeDP1-8/10 containing the various
deleted CPT1 proteins was used to transform S. cerevisiae
(haploid strain W303: MATa, his3, leu2, trp1, ura3, ade2-1,
can1-100) according to Ref. 22. Methods for yeast culture and
subcellular fractionation were as previously described (21, 23).
Miscellaneous Methods and Chemicals
Protein concentrations were determined by the method of Ref. 24
with bovine serum albumin as standard. Swelling of yeast mitochondria
and Western blotting were performed as described previously (21). The
antisera used were against the rat liver CPT1 (1/3000-1/10,000), the
yeast cytochrome b2 (1/1000), and the yeast
mtHSP70 (1/10,000). CPT activity was assayed at 30 °C as
palmitoyl-L-[methyl-3H]carnitine
formed from L-[methyl-3H]carnitine
(200 µM; 10 Ci/mol) and palmitoyl-CoA (80 µM) in the presence of 1% bovine serum albumin (w/v) as
described previously (21). Malonyl-CoA concentration was 150 µM. When indicated, mitochondria were solubilized by
0.5% Triton X-100 as described in Ref. 20 and centrifuged at
16,000 × g for 10 min at 4 °C to sediment the
insoluble membrane residues, and the supernatants were used for CPT assay.
Statistics
Results are expressed as means ± S.E. Statistical analysis
was performed using the Mann-Whitney U test.
The Mitochondrial Targeting Signal of CPT1 Resides within Residues
97-147--
To investigate the precise location of the mitochondrial
targeting signal of CPT1 within its N-terminal domain, we first asked whether each half of this domain could play an equivalent role in this
process. For this purpose, CPT1-(1-82) and CPT1-(97-147) were fused
to a non-OMM-related protein, the cytosolic mouse DHFR (Fig.
1A). CPT1-(1-147)-DHFR was
used as a positive control protein, since CPT1-(1-147) allowed import
of DHFR into the OMM in a temperature- and trypsin-sensitive surface
receptor-dependent manner, the latter being exposed on the
cytosolic face of mitochondria (20). Radiolabeled CPT1-(1-147)-DHFR,
CPT1-(1-82), and CPT1-(97-147) were synthesized in vitro
and used to perform import reactions to determine their specific import
requirements. In all cases, efficiency of import into the OMM was
assayed using the alkaline extraction method, which removes all
proteins that are not integrated into the membranes (10, 20). A
significant amount of alkaline-resistant CPT1-(1-82)-DHFR and
CPT1-(97-147)-DHFR was recovered at 30 °C when mitochondria were
added in the import reaction (Fig. 1B, compare lanes
2 and 5), suggesting that TM1 and TM2 of CPT1 allowed
membrane insertion of CPT1-(1-82)-DHFR and CPT1-(97-147)-DHFR,
respectively. Like CPT1-(1-147)-DHFR, the inserted CPT1-(1-82)-DHFR
and CPT1-(97-147)-DHFR were totally digested by exogenous added
trypsin (Fig. 1B, compare lanes 5 and
6), confirming their insertion into the OMM with DHFR exposed to the cytosol. However, the efficiency of OMM insertion of
CPT1-(1-82)-DHFR was decreased by 75% in comparison to what was
observed for CPT1-(1-147)-DHFR and CPT1-(97-147)-DHFR. Moreover, analysis of their import requirements showed that, like
CPT1-(1-147)-DHFR but unlike CPT1-(1-82)-DHFR, CPT1-(97-147)-DHFR
was imported into the OMM in a temperature- (Fig. 1B,
compare lanes 4 and 5) and trypsin-sensitive
receptor- (Fig. 1B, compare lanes 5 and
7) dependent manner. Thus, the second half of the
N-terminal domain of CPT1 conferred to DHFR the ability to interact
with the mitochondrial receptors and to be specifically inserted into
the OMM. Similarly, deletion of residues 83-148 in CPT1 abrogated
in vitro the ability of the protein to be imported into rat
liver mitochondria in a temperature- and trypsin-sensitive
receptor-dependent manner, whereas deletion of residues 1-82
did not (data not shown). These results suggest that the signal
sequence of CPT1 mediating mitochondrial targeting may reside within
residues 97-147.
The Transmembrane Segments of CPT1 Do Not Function as Signal Anchor
Sequences--
To determine whether one or both of the TM segments of
CPT1 function as a mitochondrial signal anchor sequence, we fused
CPT1-(48-75), CPT1-(97-122), and CPT1-(48-122) to DHFR (Fig.
2A). When import reactions
were performed in the absence of added mitochondria, a small amount of
CPT1-(48-75)-DHFR, CPT1-(97-122)-DHFR, and CPT1-(48-122)-DHFR was
recovered as alkaline-resistant forms (Fig. 2B, lane 2),
likely due to protein aggregation. Import in the presence of
mitochondria led to an increase in the level of these
alkaline-resistant forms (Fig. 2B, compare lanes
2 and 4) that were totally digested by trypsin (Fig.
2B, compare lanes 4 and 5). Thus, the
presence of TM1 and/or TM2 allowed insertion of DHFR into the OMM, the
latter facing the cytosol. However, in contrast to CPT1-(1-147)-DHFR and CPT1-(97-147)-DHFR (Fig. 1B), there was no significant
changes in the amount of inserted CPT1-(48-75)-DHFR,
CPT1-(97-122)-DHFR, and CPT1-(48-122)-DHFR when import was performed
with trypsin-pretreated organelles (Fig. 2C, compare
lanes 4 and 6). These results show that insertion
of TM1 and/or TM2 into the OMM does not require the trypsin-sensitive
surface receptors in contrast to CPT1. Thus, targeting of CPT1 to
mitochondria is not mediated by its TM segments, ruling out the
hypothesis that they may serve as signal anchor sequences. Therefore,
the mitochondrial targeting signal of CPT1 must reside in regions
flanking the TM segments.
The CPT1 Protein Is Targeted to Mitochondria by an Internal
Matrix-targeting Signal--
Hydrophobic cluster analysis is an
efficient method for predicting secondary protein structure and
segmentation (25). This method predicts two putative amphiphilic
Residues 122-147 Exert a Retention Force on the OMM
Surface--
We have previously reported that the N-terminal domain of
CPT1 participated in the determination of the
Ncyto-Ccyto membrane topology (20) (Fig.
5B). As shown in Fig.
2B, residues 48-122 alone, encompassing the two TM segments
and the connecting loop, seemed to be sufficient for attainment of the
Ncyto-Ccyto topology of CPT1. In the case of
S. cerevisiae Tom70, the Nin-Ccyto
orientation of the protein was reversed when its first 10 residues were
replaced by a strong matrix-targeting signal (28). Our aim was to test whether amphiphilicity of the regions flanking the TM segments of CPT1
could, in addition to the TM1-TM2 pairing, be an important determinant
for conferring protein topology. It has been shown that the unique TM
segment of Tom70 (residues 11-29, here termed as OM) allowed specific
Nin-Ccyto insertion of a reporter protein into
the OMM (20, 28). Our strategy was to determine whether residues 1-47
or 122-147 of CPT1 could reverse the membrane orientation of this OM
segment. For this purpose, CPT1-(1-47) and CPT1-(122-147) were fused
to the OM domain preceding the DHFR moiety (Fig. 5A). As
expected, the radiolabeled fusion proteins CPT1-(1-47)-OM-DHFR and
CPT1-(122-147)-OM-DHFR were efficiently membrane inserted following an
import reaction (Fig. 5B, compare lanes 2 and
3). Like CPT1-(1-147)-DHFR, the inserted
CPT1-(1-47)-OM-DHFR was totally digested by exogenously added trypsin
(Fig. 5B, lane 4). This implied that DHFR was
located on the cytosolic face of mitochondria and indicated that
residues 1-47 did not cause retention of the N terminus of the protein
on the cytosolic face of the OMM. These results were in agreement with
the observed Nin-Ccyto membrane topology of
CPT1-(1-82)-DHFR (Fig. 1B). Surprisingly, the
membrane-inserted CPT1-(122-147)-OM-DHFR was resistant to trypsin
treatment of intact mitochondria (Fig. 5B, lane 4), even in
the absence of a membrane potential (data not shown). Upon swelling of
mitochondria in the presence of trypsin, CPT1-(122-147)-OM-DHFR became
totally digested by the protease (Fig. 5B, lane 5), whereas
mtHsp70 remained protease-protected (data not shown). These results
suggested that DHFR was located in the intermembrane space compartment
and that CPT1-(122-147) was able to reverse the
Nin-Ccyto orientation of OM-DHFR. These experiments emphasize that CPT1-(122-147) but not CPT1-(1-47) may
participate together with residues 48-122 in the determination of the
membrane topology of CPT1.
Heterologous Expression of Various Deleted CPT1 Proteins in S. cerevisiae--
Yeast cells, a system devoid of endogenous CPT
activity, represent a suitable model to study the structure-function
relationships of the rat liver CPT1 (21, 29, 30). To validate
the importance of residues 123-147 of CPT1 for mitochondrial targeting
in an in vivo setting, CPT1
To examine the submitochondrial localization of the expressed deleted
CPT1 proteins, intact or swollen yeast mitochondria were submitted to
trypsin treatment (Fig. 7). The integrity
of the outer and inner mitochondrial membrane was checked by the inaccessibility of endogenous cytochrome b2
(intermembrane space protein) and mtHSP70 to trypsin proteolysis,
respectively. Upon trypsin treatment of intact mitochondria, CPT1 In the present study, we identify the import signal sequence that
specifies mitochondrial targeting of an OMM protein harboring two
The present results strongly support our working model of the import
pathway for CPT1 (Fig. 8). Initially, the
newly synthesized CPT1 is targeted to mitochondria by the means of its
internal import sequence (residues 123-147). Subsequently, the protein interacts with the OMM import machinery, as suggested by the inhibition of its import in trypsin-pretreated mitochondria. The determination of
the precise component(s) involved in this process awaits further analysis. Whereas the protein was specifically imported into the matrix
in the absence of any TM segment (CPT1
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ABSTRACT
INTRODUCTION
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DISCUSSION
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-oxidation.
CPT1 is tightly regulated by its physiological inhibitor, malonyl-CoA,
the first committed intermediate of fatty acid biosynthesis (1). The
liver mitochondrial CPT1 isoform is anchored into the outer
mitochondrial membrane (OMM) in an Ncyto-Ccyto
orientation via two
-helical hydrophobic transmembrane (TM) segments
(TM1, residues 48-75; TM2, residues 103-122). Its N terminus
(residues 1-47) and its large C-terminal domain (residues 123-773)
are facing the cytosol, whereas the loop connecting TM1 and TM2 is
exposed in the intermembrane space (2, 3). Apart from mitochondria,
microsomes and peroxisomes also contain membrane-bound malonyl-CoA-sensitive CPTs (4, 5), which share similar functional properties with the mitochondrial CPT1, have an identical molecular mass of about 88 kDa, and were immunoreactive with antibodies raised
against distinct linear epitopes of the mitochondrial CPT1 (6). Whether
these enzymes are identical or similar is still a matter of debate.
This raises the crucial question of how the mitochondrial CPT1 is
specifically imported into the OMM and whether multiple or hierarchical
targeting sequences could exist within a single polypeptide allowing
distinct subcellular locations.
-helices (8). By contrast, integral OMM
proteins are synthesized as noncleavable proteins (9) and therefore are
targeted to mitochondria by means of internal signals. How this is
accomplished is still not clear, although clues have begun to emerge
from studies of bitopic proteins, such as the Saccharomyces
cerevisiae Tom70 and Tom6 and the mammalian Bcl-2 protein. Their
targeting and insertion into the OMM have been shown to be mediated by
their unique hydrophobic TM segment that functions as a "signal
anchor sequence" selective for the OMM (10-12). An alternative to
the signal anchor sequence model is the combination of a
matrix-targeting signal with a hydrophobic stop-transfer sequence.
Primarily based on the import studies of artificial bitopic chimeric
proteins (13), this model has been shown to be valid for the
Neurospora crassa bitopic Tom22 protein (14). Very few
investigations have been performed on the nature of the targeting
and/or topogenic signals of integral polytopic OMM proteins over the
past 15 years. These proteins fall into two classes, namely those that
contain transmembrane
-sheets, such as porin and the yeast Tom40,
and those with
-helical hydrophobic TM segments. In the case of
porin and Tom40, limited information regarding structural determinants
of these
-barrel proteins is available (15-19), but the precise
nature of their targeting signals remains unclear. Bearing in mind the
structural difference between the two classes of polytopic OMM
proteins, it would appear unlikely that the targeting and/or topogenic
signals operate in a uniform manner. Thus, the rat liver CPT1 could be a useful model to study the mechanisms involved in mitochondrial targeting and membrane insertion of OMM proteins containing more than
one
-helical hydrophobic TM segment.
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ABSTRACT
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strain was used to propagate
various plasmids and their derivatives. The transcription plasmid pGEM4 (Promega) was used for cloning DNA fragments, making constructs, and
in vitro transcription/translation. All of the pGEM4
constructs were under the control of the SP6 promoter. All DNA
manipulations (restriction and ligation) were performed according to
the instructions provided by the manufacturers' protocols for the
respective enzymes. The same strategy was used to generate all
dihydrofolate reductase (DHFR) fusion constructs in which different
parts of the N-terminal end of CPT1 were fused to DHFR. cDNAs
coding for these regions were amplified by polymerase chain reaction
(PCR) using different primers and pGEM4-CPT1
3' (21) as template. All
5'-primers introduce an EcoRI restriction site (in front of
a start codon), whereas the 3'-primers contain a BamHI
restriction site, except for pCPT1-(33-47)-DHFR. DNA fragments
encoding CPT1-(1-32), CPT1-(1-47), and CPT1-(1-82) were amplified by
PCR using the same 5'-primer as previously described for
pCPT1-(1-147)-DHFR (20) and the respective 3'-primers as follows:
5'-GC GGA TCC GCA GAT CTG TTT GAG-3', 5'-CG GGA TCC CTT GAA CCG GAT
GAA-3', and 5'-CG GGA TCC GCC CAG GGA GGG-3'. The EcoRI-BamHI PCR products were subcloned into
pGEM4-DHFR (20) to generate pCPT1-(1-32)-DHFR, pCPT1-(1-47)-DHFR,
and pCPT1-(1-82)-DHFR. DNA fragments encoding CPT1-(97-122) and
CPT1-(97-147) were amplified using the same 5'-primer 5'-CG GAA TTC
ATG TCA AGC CAG ACG-3' and the following 3'-primers: 5'-GC GGA TTC CAT
GGT CAT GAT GAC-3' for CPT1-(97-122) and 5'-GC GGA TCC GGT GCT GCG GCT
CAT-3' for CPT1-(97-147). PCR products were then ligated into
pGEM4-DHFR to obtain pCPT1-(97-122)-DHFR and pCPT1-(97-147)-DHFR. The
latter 3'-primer was also used with the 5'-primer 5'-CG GAA TTC ATG CGC TAC TCG CTG-3' to amplify the DNA coding for CPT1-(122-147). The PCR
product inserted into pGEM4-DHFR creates pCPT1-(122-147)-DHFR. DNA
fragments encoding CPT1-(48-75) and CPT1-(48-122) were amplified using the same 5'-primer 5'-CG GAA TTC ATG AAT GGC ATC ATC ACT-3' and
the respective 3'-primers: 5'-GC GGA TCC GGC ATG CAT GGA TGA-3' and
5'-GC GGA TCC CTT CGT CTG GCT TGA-3'. DNA encoding CPT1-(76-102) was amplified using 5'-CG GAA TTC ATG AAA GTG GAC CCC TCC-3' as 5'-primer and 5'-GC GGA TCC CTT CGT CTG GCT TGA-3' as 3'-primer. The
PCR fragments were subcloned into pGEM4-DHFR to give
pCPT1-(48-75)-DHFR, pCPT1-(48-122)-DHFR, and pCPT1-(76-102)-DHFR,
respectively. The DNA fragment encoding CPT1-(33-47) was amplified
using pCPT1-(1-47)-DHFR as template, the 5'-primer 5'-CG GAA TTC ATG
CTG TCG GGG CTG CAC-3', and the 3'-primer 5'-TCC AAA CTT TTG GCA AGA
AAA-3' (including the unique SacI restriction site of DHFR
cDNA). The amplified EcoRI-SacI fragment was
then ligated into pCPT1-(1-47)-DHFR digested by EcoRI and
SacI to obtain pCPT1-(33-47)-DHFR. All of these DHFR fusion
constructs contain four extra amino acids (Gly, Ser, Gly, and Ile) in
the joining region.
82--
Deletion of the first 82 amino acids of CPT1,
CPT1
82, was achieved by amplifying the DNA coding for CPT1-(83-186)
by PCR using the 5'-primer 5'-CG GAA TTC ATG ATC GCA AAG ATC AGT-3'
(introducing an EcoRI restriction site) and the 3'-primer
5'-ATT CCA GGT ACC TGC TCA CAG-3' (containing the unique
KpnI restriction site of CPT1). The PCR product digested by
EcoRI and KpnI was ligated into pGEM4-CPT1
3'
cut by the same enzymes to generate pCPT1
82.
83-148--
DNA encoding CPT1-(1-82) was amplified by
PCR using the same 5'-primer previously used to generate
pCPT1-(1-147)-DHFR and the 3'-primer 5'-CGG GAT CCT GCC CAG GGA
GGG-3', introducing a BamHI restriction site. The
EcoRI-BamHI PCR fragment was subcloned into
pGEM4-CPT1
3' deleted from its large
EcoRI-BglII fragment which codes for the first
147 amino acids of CPT1. The resulting pCPT1
83-148 encodes a
protein in which amino acid 82 is fused to residue 149 and possesses
one extra amino acid (Arg) in the joining region.
31-148--
This construct was obtained by excising the
BglII-BglII fragment from pGEM4-CPT1
3' and
re-ligating the plasmid. This results in a CPT1 protein in which amino
acid 30 is fused to amino acid 149.
121--
DNA encoding CPT1-(122-147) was amplified by
PCR using pGEM4-CPT1
3' as template and the same 5'-primer as
previously used for pCPT1-(122-147)-DHFR and the 3'-primer 5'-AGC CAT
CCA GAT CTT GGT GCT GCG-3' containing a BglII restriction
site. This EcoRI-BglII PCR product was ligated
into pGEM4-CPT1
3' digested by EcoRI and BglII.
The resulting construct pCPT1
121 encodes a CPT1 protein deleted of
its 121 N-terminal amino acids.
123-148--
This plasmid encodes a CPT1 protein
deleted of residues 123-148. DNA encoding CPT1-(1-122) was amplified
by PCR using the same 5'-primer used for pCPT1-(1-147)-DHFR and the
3'-primer 5'-GCG GAT CCG CAT GGT CAT GAT GAC-3' introducing a
BamHI restriction site. This PCR product was cloned as an
EcoRI-BamHI fragment into pGEM4-CPT1
3'
digested with EcoRI and BglII to obtain
pCPT1
123-148. One amino acid (Arg) was introduced between residues
122 and 149.
82,
pYe-CPT1
83-148, pYe-CPT1
31-148, pYe-CPT1
121, and
pYe-CPT1
123-148. Each cDNA was placed under the control of the
inducible GAL10 promoter present in the vector. The fidelity of all
PCRs and the quality of DNA subcloning were confirmed by DNA sequencing.
) by 1 µM of
carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma),
mitochondrial pretreatment with trypsin (Sigma), postmitochondrial
treatment with trypsin, alkaline extraction with 0.1 M
Na2CO3, as well as analysis of the import
reactions by SDS-PAGE and fluorography were performed as described
previously (20).
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ABSTRACT
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Fig. 1.
CPT1-(97-147) contains a mitochondrial
targeting signal. A, CPT1-DHFR fusion proteins
CPT1-(1-147), CPT1-(1-82), and CPT1-(97-147) were fused to DHFR.
Zigzag lines denote amino acids 1-47, 76-102, and 123-147
of the N-terminal domain of rat liver CPT1. TM segments of CPT1 are
indicated by black squares. Numbers denote
residues of CPT1 after which the fusion to DHFR occurs. B,
import of CPT1 deletion proteins. Import of CPT1 fusion proteins was
carried out for 30 min at 4 (lane 4) or 30 °C
(lanes 2, 3, 5, 6, and 7) in the absence
( M; lanes 2 and 3) or presence of
freshly isolated rat liver mitochondria (80 µg) either directly
(lanes 4-6) or after a pretreatment with 50 µg/ml trypsin
for 20 min on ice to remove the mitochondrial surface receptors
(
R; lane 7). Following import, samples were
centrifuged and washed in KCl buffer. The samples were then split into
two equivalent aliquots and submitted to 0.1 M
Na2CO3 (pH 11.5; 0.2 mg of protein/ml) for 30 min on ice either directly (
Trypsin; lanes 2, 4, 5, and 7) or after a trypsin treatment (300 µg/ml)
(+Trypsin; lanes 3 and 6). After
centrifugation at 100,000 × g for 30 min at 4 °C,
integral membrane proteins were recovered in the pellet, and samples
were analyzed by SDS-PAGE and fluorography. 10%, percentage
of the amount of radiolabeled proteins added to each import reaction
(lane 1).
View larger version (25K):
[in a new window]
Fig. 2.
The N-terminal region of CPT1 does not
contain a signal anchor sequence. A, fusion protein
constructs used: CPT1-(48-75) corresponding to the TM1 segment of
CPT1, CPT1-(97-122) encompassing the TM2 segment, and CPT1-(48-122)
were fused to DHFR. Zigzag lines and black
squares are as in Fig. 1. Numbers denote residues of
CPT1 where the fusion to DHFR occurs. B, TM segments of CPT1
do not allow a specific import of DHFR into the OMM. Import of
radiolabeled proteins was performed as described in Fig. 1.
-strands within residues 1-32 (
1, residues 8-14;
2, residues
19-23) and three amphipathic
-helices (residues 33-47, 76-102,
and 123-147) flanking the TM segments (26). To test whether these
regions have the capacity to target a reporter protein to mitochondria,
we constructed a series of proteins in which CPT1-(1-47),
CPT1-(1-32), CPT1-(33-47), CPT1-(76-102), and CPT1-(122-147) were
fused to DHFR (Fig. 3A). We
used the Su9-DHFR protein, which consists of the presequence of
N. crassa F0-ATPase subunit 9 preceding DHFR
(27), as a positive control for protein import into the mitochondrial
matrix. In all cases, the efficiency of the import of these
radiolabeled fusion proteins into mitochondria was estimated by
determining their protection toward trypsin proteolysis. Su9-DHFR was
imported into the mitochondrial matrix, where it became processed to
its mature-form size, which was inaccessible to exogenously added
trypsin (Fig. 3B, compare lanes 4 and
8). Following import at 30 °C in the presence of
mitochondria, CPT1-(1-47)-DHFR, CPT1-(1-32)-DHFR, and
CPT1-(76-102)-DHFR were recovered in association with mitochondria but
were almost totally digested by exogenous trypsin (Fig. 3B,
compare lanes 4 and 8). This indicated that these
CPT1 residues were unable to drive DHFR into the mitochondria. By
contrast, CPT1-(33-47)-DHFR and CPT1-(122-147)-DHFR became largely
insensitive to added trypsin (Fig. 3B, compare lanes
4 and 8), demonstrating that they were at least
translocated across the OMM. Acquisition of the protease protection
occurred in a temperature- and receptor-dependent manner (Fig. 3B, compare lane 3-5) and needed the
presence of a membrane potential (
) (Fig. 3C, compare
lanes 3 and 4). To ascertain the location of
CPT1-(33-47)-DHFR and CPT1-(122-147)-DHFR to the matrix, mitochondria
were subfractionated by a swelling procedure in the presence of
trypsin. As expected from previous studies (21), the endogenous CPT1
protein was resistant to trypsin digestion in intact mitochondria (due
to a folded state of its large cytosolic C-terminal domain) (Fig.
3D, lane 3) and was partially degraded into an
83-kDa fragment when the OMM was disrupted upon swelling (Fig.
3D, f1 fragment). In contrast, the imported
CPT1-(33-47)-DHFR and CPT1-(122-147)-DHFR remained resistant to
trypsin proteolysis, as was the endogenous matrix-soluble HSP70
protein (mtHSP70). When the inner membrane of mitoplasts was further
solubilized by Triton X-100, the imported CPT1-(33-47)-DHFR and
CPT1-(122-147)-DHFR were totally degraded by trypsin, whereas a
proteolytic fragment was generated for mtHSP70 (Fig. 3D, lane
8), confirming their localization into the matrix. These results
indicate that residues 33-47 and 122-147 behave as matrix-targeting
signals. Surprisingly, when residues 1-32 were added to
CPT1-(33-47)-DHFR, import of DHFR into the mitochondrial matrix was
totally inhibited (Fig. 3B). Thus, the matrix-targeting
function of residues 33-47 was abrogated by the first 32 amino acids
of CPT1. This explained why residues 33-47 did not support import of
CPT1-(1-82)-DHFR (Fig. 1B) or CPT1
83-147. To study
further the role of residues 122-147 in mitochondrial targeting,
import of CPT1
121 was analyzed (Fig. 3A). In contrast to
CPT1
31-148 (data not shown) or CPT1
150 (20), CPT1
121 was
efficiently imported into mitochondria in a process that was dependent
upon the temperature, the presence of trypsin-sensitive surface
receptors, and a membrane potential (Fig. 3, B and
C). Swelling experiments confirmed that the imported CPT1
121 was located into the matrix compartment (Fig.
3D). These results demonstrate that residues 122-147
function as a matrix-targeting signal, driving the import of a reporter
protein into the matrix in the absence of any of the TM segments of
CPT1. As soon as one TM segment was present, such as in
CPT1-(97-147)-DHFR, protein translocation across the mitochondrial
membranes was arrested, leading to the insertion of the corresponding
protein into the OMM (see Fig. 1B). To confirm the essential
role of residues 122-147 in mitochondrial targeting, we compared the
receptor dependence of the import of CPT1 and CPT1
123-148 (Fig.
4). In contrast to the full-length CPT1,
deletion of residues 123-148 within CPT1 abrogated the ability of the
protein to be imported in a trypsin-sensitive receptor-dependent manner (Fig. 4B, compare
lanes 3 and 4). Thus, residues 123-147 specify
in vitro the mitochondrial targeting of CPT1, whereas its
hydrophobic TM segment(s) likely acts as a stop-transfer sequence that
stops and anchors the translocating protein into the OMM.
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Fig. 3.
The N-terminal domain of CPT1 contains two
matrix-targeting signals. A, fusion and deletion
protein constructs: Su9-DHFR corresponds to the matrix-targeting signal
of N. crassa F0-ATPase subunit 9 fused to DHFR
(27). CPT1-(1-47), CPT1-(1-32), CPT1-(33-47), CPT1-(76-102), and
CPT1-(122-147) were fused to DHFR, whereas residues 1-121 were
deleted within CPT1 (CPT1 121). Zigzag lines were used as
in Fig. 1A. Numbers denote residues of CPT1 after which the
fusion to DHFR occurs. B, import of CPT1-(33-47)-DHFR,
CPT1-(122-147)-DHFR, and CPT1
121 is temperature- and mitochondrial
receptor-dependent. Import of radiolabeled proteins was
carried out as described in Fig. 1. Following import, mitochondria were
washed, centrifuged, and split into 2 eq aliquots, and trypsin (200 µg/ml) treatment was performed when indicated. After inactivation of
the protease with STI (4 mg/ml), mitochondria were reisolated,
submitted to SDS-PAGE, and analyzed by fluorography. p,
precursor; m, mature form of Su9-DHFR. 10%,
percentage of input lysate of each radiolabeled protein (lane
1). C,
dependence of import of
CPT1-(33-47)-DHFR, CPT1-(122-147)-DHFR, and CPT1
121. Import of the
radiolabeled proteins was performed in the presence (
CCCP;
lanes 2 and 3) or in the absence
(+CCCP; lane 4) of a membrane potential (
).
Following import at 30 °C, untreated mitochondria were split into 2 eq aliquots. The first one was washed and directly submitted to
SDS-PAGE (lane 2). The second one as well as CCCP-pretreated
mitochondria were subjected to trypsin treatment (+Trypsin;
lanes 3 and 4) as described in B. Samples were then analyzed by SDS-PAGE and fluorography.
10%, percentage of input lysate of each radiolabeled
protein (lane 1). D, the imported
CPT1-(33-47)-DHFR, CPT1-(122-147)-DHFR, and CPT1
121 are located in
the matrix. After import of the radiolabeled proteins, their
submitochondrial localization was determined as described under
"Experimental Procedures." Mitochondria (lanes 2 and
3) and mitoplasts (lanes 4, 5, and 7)
were incubated in the absence (
Trypsin (1); lane
2) or presence of 100 µg/ml trypsin (+Trypsin (1);
lanes 3-5 and 7). All samples were reisolated by
centrifugation and washed in EDTA buffer supplemented with STI, except
for lane 7. The first 3 aliquots (lanes 2-4;
T, total) were directly analyzed by SDS-PAGE, and the last
two were solubilized by 0.5% Triton X-100 (+TX-100;
lanes 5 and 7) at 4 °C for 10 min. Trypsin
(400 µg/ml) was added when indicated (+Trypsin (2);
lane 7), and samples were kept on ice for another 10 min.
Samples were centrifuged to recover the pellet (P;
lanes 5 and 7) and the supernatant (S;
lanes 6 and 8) that was trichloroacetic
acid-precipitated. Samples were submitted to SDS-PAGE, blotted onto
nitrocellulose, and analyzed by fluorography. Immunostaining with the
endogenous CPT1 and the mtHSP70 was then performed. 10%,
percentage of input lysate of each radiolabeled protein (lane
1). f* denotes fragment of HSP70 generated by trypsin
treatment of solubilized mitoplasts. f1 and
f2 are CPT1-processed species.
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Fig. 4.
CPT1-(123-148) mediates interaction with the
mitochondrial trypsin-sensitive receptors. A, residues
123-148 were deleted within CPT1 (CPT1 123-148). Zigzag
lines and black squares were used as in Fig.
1A. B, import of CPT1
123-148 is
receptor-independent. Import of radiolabeled proteins was carried out
for 30 min at 30 °C in the absence (
M) or presence
(+M) of mitochondria either directly (lanes 2 and
3) or after a pretreatment with trypsin
(Pre-Trypsin; lane 4). Following import, all the
samples were submitted to alkaline extraction and analyzed by SDS-PAGE
and fluorography. 10%, percentage of input lysate of each
radiolabeled protein (lane 1).
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Fig. 5.
CPT1-(122-147) behaves as a membrane
retention signal. A, fusion protein constructs used:
CPT1-(1-47)-OM-DHFR and CPT1-(122-147)-OM-DHFR. CPT1-(1-47) and
CPT1-(122-147) were fused to the signal anchor domain (OM)
of S. cerevisiae Tom70 preceding DHFR. Zigzag
lines were outlined as described in Fig. 3A. Black
square denotes OM (residues 11-29 of Tom70p). Numbers
denote residues of CPT1 after which the fusion to OM-DHFR occurs.
B, CPT1-(1-47)-OM-DHFR and CPT1-(122-147)-OM-DHFR have
opposite OMM orientation. Following import of radiolabeled proteins at
30 °C, mitochondria were split into 4 eq aliquots (80 µg) and
washed in KCl buffer. The 1st aliquot was directly analyzed by SDS-PAGE
(lane 2), whereas the 2nd aliquot was submitted to alkaline
extraction (+Na2CO3; lane
3) as described in Fig. 1B. The 3rd aliquot was treated
with trypsin (+Trypsin; lane 4) as described in
Fig. 3D, and the last aliquot was submitted to swelling in
the presence of trypsin (+Swelling; lane 5). All
the samples were then analyzed by SDS-PAGE and fluorography.
10%, percentage of input lysate of radiolabeled proteins
(lane 1).
82, CPT1
83-148,
CPT1
121, CPT1
123-148, CPT1
31-148, and the full-length CPT1
were expressed in S. cerevisiae. Immunodetection of the
yeast-expressed proteins in crude homogenates showed that proteins of
the predicted sizes were expressed, except for CPT1
31-148 (Fig.
6A, lane 7). Thus, in
agreement with our in vitro import experiments, deletion of
residues 31-148 led to a protein that was unable to be targeted
correctly to mitochondria and hence might be rapidly degraded within
the cells. Analysis of the subcellular distribution of the
yeast-expressed full-length CPT1 shows that, by contrast to our
previous reports (20, 21), the amount of CPT1 recovered in the
microsomal fraction represents about 27% of the total expressed
protein (Fig. 6, B and C). One possible
explanation for this discrepancy was that immunoblotting was performed
more stringently in the present experiment, allowing increased
sensitivity in the detection of the expressed protein. Recovery of CPT1
in microsomes was not due to a mitochondrial contamination of the
microsomal fraction, as shown by the subcellular distribution of the
mtHSP70 that was representative of all the constructs (Fig.
6B). Moreover, microsomal recovered CPT1 exhibited CPT
activity (2.49 ± 0.27 nmol/min/mg of protein) that was almost totally (93%) inhibited by 150 µM malonyl-CoA (0.17 ± 0.02 nmol/min/mg of protein), ruling out that CPT1 was recovered as
aggregated protein. When analyzing the subcellular distribution of the
various deleted CPT1, we kept in mind that CPT1 harbors two TM
segments, and hence partial deletions within the N-terminal domain
might affect targeting and membrane anchorage differentially. Deletion of residues 83-148 led to a complete reverse subcellular distribution when compared with the full-length CPT1 since about 93% of
CPT1
83-148 was recovered in the microsomal fraction (Fig. 6,
B and C). Upon deletion of 123-148, both
mitochondrial and microsomal fractions contained equivalent amounts of
the expressed protein, underlying the absence of privileged
mitochondrial targeting (Fig. 6, B and C).
Conversely, deletion of residues 1-82 or 1-121 did not alter mitochondrial targeting, whereas their recovery in the microsomal fraction was almost totally abolished (Fig. 6, B and
C). These results confirmed our in vitro
experiments (Fig. 1 and Fig. 3) and supported the conclusion that
residues 123-148 were essential for mitochondrial targeting of
CPT1. The finding that deletion of residues 1-82 abrogated microsomal
location of CPT1 whereas CPT1
83-148 was most exclusively recovered
in this fraction was puzzling, since both proteins contain a TM segment
that could allow their nonspecific anchorage at the microsomal
membranes. The present observation suggests the presence of a
putative microsomal targeting signal within residues 1-82 of CPT1.
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Fig. 6.
Heterologous expression of deleted CPT1
proteins in S. cerevisiae. A,
immunodetection of rat liver CPT1 and deleted CPT1 proteins expressed
in S. cerevisiae. The SDS-PAGE gel was run using either
homogenate (20 µg of protein) from control yeast cells transformed
with the empty vector (lane 1) or from the different yeast
strains expressing the full-length CPT1 (lane 2), CPT1 82
(lane 3), CPT1
121 (lane 4), CPT1
123-148
(lane 5), CPT1
83-148 (lane 6), or
CPT1
31-148 (lane 7). Proteins were transferred onto
nitrocellulose, and the blot was probed with the rat liver CPT1
antibody. B, subcellular localization of the deleted CPT1
proteins expressed in S. cerevisiae. Yeast cells expressing
the full-length CPT1, CPT1
82, CPT1
83-148, CPT1
121, or
CPT1
123-148 were fractionated into homogenate (H),
mitochondria (Mt), cytosol (C), and microsomes
(Mc). Samples (20 µg of protein) were analyzed by SDS-PAGE
and immunoblotting with the rat liver CPT1 and yeast mtHSP70
antibodies. Results are representative of three to four different
experiments, and the subcellular distribution of mtHSP70 is
representative for all constructs. C, bands from Western
blots from three to four different experiments, as in B,
were quantified by scanning densitometry. Since mitochondria,
microsomes, and cytosol accounted, on average, for 9, 13, and 78% of
the total homogenate protein, the signal detected in the mitochondrial
and microsomal fractions has to be corrected accordingly to analyze the
ratio of CPT constructs targeted to mitochondria versus
microsomes. The recovery of the expressed protein in the mitochondrial
(gray bars) and microsomal (open bars) fractions
was expressed as percentage of total expressed protein.
82,
CPT1
83-148, and CPT1
123-148 were digested by the protease, and
a proteolytic fragment of about 60-kDa was generated (f2
fragment) that remained membrane-anchored and detected by our CPT1
antibody raised against residues 317-430 (Fig. 7). These results
indicated that (i) these deleted CPT1 proteins were anchored into the
OMM with their C-terminal domain exposed to the cytosol, (ii) residues
123-148 are not essential for achievement of the correct membrane
topology of CPT1, and (iii) a highly folded core exists within the
cytosolic C-terminal domain of CPT1. However, the generation of the
f2 fragment was less efficient in the case of CPT1
83-148,
suggesting a partial unfolding of the C-terminal domain of this deleted
protein. By contrast, CPT1
121 remained trypsin-protected even upon
swelling and became totally digested by the protease when the inner
membrane of mitoplasts was solubilized by Triton X-100 (Fig. 7).
This showed that CPT1
121 was efficiently imported into the
mitochondrial matrix but did not harbor a folded core. As shown in Fig.
3D for the native rat liver CPT1 protein, no
trypsin-resistant 60-kDa fragment was generated in intact yeast
mitochondria expressing CPT1 (Fig. 7). Consequently, the trypsin
cleavage site at the cytosolic C terminus, previously observed for
CPT1
82, was inaccessible in the entire protein. It became unmasked
only after the cleavage by trypsin of the loop connecting TM1 and
TM2 (f1 fragment), allowing the protease to generate the 60-kDa
fragment (f2 fragment) (Fig. 3D and Fig. 7). These
results indicate the existence of a highly folded core within the
cytosolic C-terminal domain of CPT1 that is hidden by the presence of
residues 1-82. Moreover, among the deleted CPT1 proteins, only
CPT1
82 was functionally active but showed a decreased malonyl-CoA
sensitivity (Table I). The absence of CPT
activity in intact yeast mitochondria expressing CPT1
121 could be
due to its matrix location (Fig. 7). To determine whether CPT1
121
was still functionally active despite its unfolding, CPT activity was
measured in solubilized yeast mitochondria expressing CPT1
121 or
CPT1 as positive control. Whereas solubilization of mitochondria by 5%
Triton X-100 inactivated the yeast-expressed CPT1 (20), we found that
0.5% Triton X-100, which is the concentration used for determining the
submitochondrial localization and the trypsin resistance of CPT1
121
(Fig. 7), allowed CPT1 to be solubilized in an active and
malonyl-CoA-sensitive form, when compared with intact mitochondria
(Table I). By contrast to CPT1, the solubilized CPT1
121 was totally
inactive (Table I), suggesting that unfolding of the protein led to its
inactivation. In conclusion, these results confirm our in
vitro import experiments and demonstrate that residues 123-148
function in vivo as a matrix-targeting signal specifying the
mitochondrial targeting of CPT1, whereas its TM segment(s) acts as an
anchoring signal allowing CPT1 insertion into the OMM.
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Fig. 7.
Submitochondrial localization of the
expressed deleted CPT1 proteins. Mitochondria (50 µg of protein)
isolated from yeast cells expressing CPT1, CPT1 82, CPT1
83-148,
CPT1
121, or CPT1
123-148 were incubated for 30 min at 4 °C
under either iso- (
Swelling) or hypo-osmotic
(+Swelling) conditions in the absence (
Trypsin)
or presence (+Trypsin) of trypsin (10 µg/ml). After
addition of STI, samples were sedimented, washed, electrophoresed on
SDS-PAGE, and analyzed by Western blot using anti-CPT1 antibody. When
Triton X-100 (TX-100; 0.5% v/v) was present during the
swelling procedure, the sample was directly centrifuged at the end of
the incubation. Both pellet (P) and supernatant
(S) fractions were incubated 5 min at 65 °C to inhibit
trypsin, and solubilized proteins present in the supernatant were
trichloroacetic acid-precipitated before being analyzed by SDS-PAGE and
immunoblotted. Marker proteins were cytochrome
b2 (Cyt.b2) for the intermembrane
space and mtHSP70 for the matrix. f1 and
f2 denote the respective 83- and 60-kDa fragments of
CPT1 generated by trypsin. Results are representative of three to four
different experiments.
CPT1 activity and malonyl-CoA inhibition of the deleted CPT1 proteins
expressed in S. cerevisiae
82, CPT1
83-148, CPT1
123-148, or
CPT1
121. CPT activity was assayed with 80 µM
palmitoyl-CoA and 200 µM carnitine in the absence or
presence of 150 µM malonyl-CoA, using either intact
mitochondria or mitochondria solubilized in 0.5% Triton X-100, as
described under "Experimental Procedures." Results are means ± S.E. of 3-4 separate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical hydrophobic TM segments, the rat liver CPT1. The concordance of the two approaches used clearly shows that neither TM1
nor TM2 of CPT1 constitutes a signal anchor sequence selective for the
OMM, in contrast to the bitopic Tom70 and Bcl2 proteins. This
emphasizes that unknown features other than simple hydrophobicity alone
may explain why a TM segment functions as an OMM signal anchor
sequence. The present data show that the mitochondrial targeting of
CPT1 is mediated by residues 123-147. These residues exhibit several
characteristic features of mitochondrial matrix-targeting signals. We
also identify a second noncleavable matrix-targeting signal within
residues 33-47. However, the presence of residues 1-32 totally
abrogates the matrix targeting function of residues 33-47, both
in vitro and in vivo. Epitope
Val14-Lys29 of CPT1 has been shown to be
sterically masked within the native protein, unless the extreme N
terminus of the protein is proteolytically cleaved (3, 6). Residues
1-32 might form a tight loop structure in which the hydrophobic sides
of the two highly conserved amphipathic
-sheets are facing, Gly and
Pro residues present between these two
-sheets acting as structural
breakers (26). We propose that the amphipathic
-helical residues
33-47 are in contact with this tight
-sheet loop. Such a physical
interaction should lead to an embedding of epitope
Val14-Lys29 and should mask the matrix
targeting function of residues 33-47.
121), the presence of TM2
proximal to residues 123-147 (CPT1
82) led to an OMM insertion of
the protein. This clearly shows that TM2 at least acts as a stop-transfer sequence that arrests protein translocation during import
across the OMM. At this stage, our working model includes two possible
variations. In the "step by step model" integration of the TM
segments would occur sequentially (Fig. 8a), whereas pairing
of TM1 and TM2 may be a prerequisite before membrane insertion in the
"single concerted step model" (Fig. 8b). The hairpin
structure will exhibit a higher hydrophobic moment that would favor
bilayer integration. Such a concerted partitioning of the TM segments has been described for the
-barrel Tom40 (19) and for the inner mitochondrial membrane carrier proteins (31). As reported for Tom40
(19), denaturation of the radiolabeled CPT1 precursor with urea
partially decreased the efficiency of its
import2 that favors the
single concerted step model. Although further experimental evidences
are required to discriminate between these two possibilities, the
present work clearly demonstrates that the TM segments of CPT1 act as
stop-transfer sequences that arrest protein translocation during import
across the OMM.
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Fig. 8.
Model for the import pathway of CPT1.
See text for details. Abbreviations used are: OMM, outer
mitochondrial membrane; R, import receptors. The black
area denotes the TM segments of CPT1; the internal import sequence
is represented by a helix, and * states for trypsin cleavage
site.
The distribution of charges on either side of a membrane anchor is responsible for the orientation of proteins of both the bacterial inner membrane and the endoplasmic reticulum membrane (32, 33). However, residues 1-47, 76-102, and 123-147 of CPT1 all bear an identical net positive charge of +3. Therefore, another putative topogenic determinant could have been the amphiphilicity of the regions flanking the TM segments that may exert on the mitochondrial surface a "retention signal" functionally similar to those created and analyzed by Shore and coworkers (13, 28). The fact that residues 1-82 of CPT1 can adopt either a Nin-Ccyto or Ncyto-Cin topology in the CPT1-(1-82)-DHFR and full-length CPT1 suggests that residues 1-47 do not contain any topogenic information. Indeed, CPT1-(123-147), but not CPT1-(1-47), was able to exert in vitro such a retention signal since its fusion to the unique TM segment of the yeast Tom70 led to the inversion of its membrane topology. However, deletion of residues 123-147 within the N-terminal domain of CPT1 did not alter its membrane topology. Although we cannot exclude that CPT1-(123-147) may participate in the process of membrane insertion, our results indicate that the presence of both TM1 and TM2 is sufficient for achievement of the correct Ncyto-Ccyto topology of CPT1.
Besides the identification of the import signal sequence specifying the import of CPT1 into the OMM, the present study shows that a minor proportion of the yeast-expressed CPT1 was recovered as a functional enzyme in microsomes. Deletion of the first 82 N-terminal residues abolished completely this microsomal targeting, the resulting protein being recovered only into the mitochondrial fraction, whereas deletion of residues 83-148 had the opposite effect. Similar results were also obtained by Zammit and co-workers3 by using another yeast expression system (Pichia pastoris). Subcellular distribution of isoenzymes is usually achieved by the expression of two (or more) closely nuclear-related genes. However, the product of a single gene can be targeted to different locations due to the use of alternative transcription-translation initiation sites, alternative splicing, or multiple targeting signal sequences (7). Although the identity of the microsomal CPT1 remains obscure, our results suggest that residues 1-82 of the mitochondrial CPT1 may contain a putative microsomal targeting signal. Further work is required to determine whether the microsomal CPT1 corresponds to a misrouting of the mitochondrial isoenzyme, and if the mitochondrial and microsomal CPT1s are encoded by a single gene. If it is not the case, the reminiscence of such a signal within the mitochondrial enzyme might result from the evolution of an ancestral CPT1 gene.
Finally, the present study allows new insights into the folding of
the C-terminal catalytic domain of CPT1. Following mitochondrial targeting and OMM insertion, CPT1 must fold correctly to attain its
native functional conformation that is characterized by a highly folded
state resistant to trypsin proteolysis (Fig. 8c). Our
current findings indicate the existence of a highly folded core in its
cytosolic C-terminal domain, as emphasized by the generation of a
trypsin-resistant 60-kDa fragment upon trypsin treatment of intact
mitochondria expressing CPT182. Possible trypsin sites occur
C-terminal to Arg-595 or -598 and Lys-631 or -634. Trypsin was able to
digest the full-length CPT1 at these sites only when the loop
connecting TM1 and TM2 was previously cleaved by the protease. This
suggests that the highly folded core within the cytosolic C-terminal
domain of CPT1 is hidden in the native protein by its cytosolic first
N-terminal residues. Several lines of evidence support the idea that
this folded domain may belong to the catalytic core of CPT1. First,
CPT1 remained active and malonyl-CoA-sensitive when solubilized by a
low Triton X-100 concentration that maintained its C-terminal domain
folded. Second, deletion of residues 1-82 did neither alter the
folded core nor the catalytic activity but decreased the malonyl-CoA sensitivity. This is in agreement with previous studies suggesting that
the catalytic domain of CPT1 resides within its cytosolic C-terminal
domain, whereas its extreme N terminus is important for malonyl-CoA
sensitivity (3, 20, 34). Third, deletion of residues 1-121 or 83-148
that encompassed TM2 altered both folding of the C-terminal domain and
CPT activity. The fact that residues 171-186 contain the
(LI)PX(LVP)P(IVTA)PX(LIVM)X(DENQAS)(ST)(LIVM)X2(LY) motif, which corresponds to the carnitine/choline acyltransferase family signature 1 (2), may explain why CPT1
123-148 was inactive despite the presence of the folded core. Indeed, deletion of residues 123-148 led to a shift of residues 171-186 to the OMM that likely alters the catalytic activity of the enzyme independently of the trypsin-resistant folded core.
The present study confirms our previous observation that the N-terminal
domain of CPT1 is essential to maintain an optimal conformation for
catalytic function (20). We show here that TM2 is essential to achieve
the correct folding of this putative catalytic core and that residues
1-47 may be in close proximity to this domain, preventing trypsin from
having access to residues at position 595, 598, 631, or 634. The
extreme protease resistance that characterizes the native CPT1 is
likely due to intramolecular interactions between either the cytosolic
N- and C-terminal domains of the enzyme or between TM1 and TM2.
Additionally, it could result from an oligomerization of the enzyme, as
reported for porin (16). It is now clear that -helical TM segments
of membrane proteins can participate in highly specific interactions
that drive their folding and/or oligomerization and contribute to an
increasingly diverse set of functional roles (35, 36). Whether the TM
segment(s) of CPT1 fulfill such functional interactions with either
each or with other OMM proteins needs to be determined.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Prof. W. Neupert (Munich, Germany) for the gift of the pGEM4Su9-DHFR plasmid and the antibodies against the yeast cytochrome b2 and HSP70; Prof. G. Shore (Montreal, Canada) for the pOMD29 plasmid; and Dr. P. Urban (Gif sur Yvette, France) for the yeast expression vector pYeDP1/8-10. We also thank L. Bernard and N. Marchand for taking care of the animals.
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FOOTNOTES |
---|
* 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.
Recipient of a doctoral fellowship from the Ministère de
l'Education Nationale, de la Recherche et de la Technologie, and of
the Fondation pour la Recherche Médicale.
§ To whom correspondence should be addressed. Tel.: 33 1 45 07 51 68; Fax: 33 1 45 07 50 39; E-mail: pripbuus@infobiogen.fr.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M009555200
2 C. Prip-Buus, I. Cohen, and J. Girard, unpublished results.
3 N. T. Price and V. A. Zammit, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: CPT, carnitine palmitoyltransferase; OMM, outer mitochondrial membrane; TM, transmembrane; DHFR, dihydrofolate reductase; PCR, polymerase chain reaction; CCCP, carbonyl cyanide m-chlorophenylhydrazone; PAGE, polyacrylamide gel electrophoresis; STI, soybean trypsin inhibitor.
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REFERENCES |
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