Location of the Actual Signal in the Negatively Charged
Leader Sequence Involved in the Import into the Mitochondrial
Matrix Space*,
Abhijit
Mukhopadhyay
,
Thomas S.
Heard
,
Xiaohui
Wen
,
Philip K.
Hammen, and
Henry
Weiner§
From the Department of Biochemistry, Purdue University, West
Lafayette, Indiana 47907-2063
Received for publication, December 13, 2002, and in revised form, January 16, 2003
 |
ABSTRACT |
Proteins destined for the mitochondrial
matrix space have leader sequences that are typically present at the
most N-terminal end of the nuclear-encoded precursor protein. The
leaders are rich in positive charges and usually deficient of negative
charges. This observation led to the acid-chain hypothesis to explain
how the leader sequences interact with negatively charged receptor proteins. Here we show using both chimeric leaders and one from isopropyl malate synthase that possesses a negative
charge that the leader need not be at the very N terminus of the
precursor. Experiments were performed with modified non-functioning
leader sequences fused to either the native or a non-functioning leader of aldehyde dehydrogenase so that an internal leader sequence could
exist. The internal leader is sufficient for the import of the modified
precursor protein. It appears that this leader still needs to form an
amphipathic helix just like the normal N-terminal leaders do. This
internal leader could function even if the most N-terminal portion
contained negative charges in the first 7-11 residues. If the
first 11 residues were deleted from isopropyl malate synthase, the
resulting protein was imported more successfully than the native
protein. It appears that precursors that carry negatively charged
leaders use an internal signal sequence to compensate for the
non-functional segment at the most N-terminal portion of the protein.
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INTRODUCTION |
Most proteins found in the mitochondrial matrix space are encoded
by nuclear genes and are synthesized on cytosolic polysomes. The
preproteins typically carry N-terminal targeting sequences (leader
sequence) that direct the proteins to the translocase of the outer
mitochondrial membrane
(TOM),1 which consists of
receptors and a general import pore (1-5). A common feature of leader
sequences is the frequent occurrence of basic residues, particularly
arginine, a residue that is found three times more often in the
leaders than proteins (6). Because TOM proteins are negatively charged,
an acid-chain hypothesis was developed to suggest that positive charges
in the leader sequences are necessary for TOM to interact with
negatively charged receptor proteins (7).
The leader sequence for pALDH has been studied extensively in our
laboratory. As shown in Fig. 1, the leader is composed of 19 amino
acids that can be induced into a helix-linker-helix amphiphilic structure in the presence of trifluoroethanol or detergent micelles (8). Although each helical segment contains an equal number of positive
charges, the N-terminal helical segment has been shown to be necessary
to efficiently target the leader to the matrix space (9). If
both of the arginines of the N-helical segment are substituted with
glutamine, the ability to be imported is essentially eliminated,
showing that the net positive charge in the N-helical segment is more
important than the total positive charge throughout the leader (6).
Deletion of the linker results in a non-processable leader that is
longer in helical content (10) and is more capable of importing ALDH
than is the native sequence in a cell-free import assay (6). The
enhanced stability of the linker-deleted leader has been used
successfully to study structural aspects as a compensating factor for
the loss of positive charges resulting from arginine to glutamine
mutational substitutions. The linker-deleted structure can import a
"passenger" protein even if both of the N-terminal arginine
residues (Arg-3 and Arg-10) are mutated to glutamines (6). On the basis
of the positive charge versus structural compensation model
developed from our previous studies, glutamic and aspartic acid
residues were systematically introduced into the pALDH leader to
ascertain how negative charges may be tolerated. It was observed that
when serine was replaced with glutamic acid, the S7E mutant pALDH was
imported to an extent similar to pALDH; however, the R3Q,S7E double
mutant was poorly imported (11). This result implies that only a net
positive charge was required for the proper import of a precursor into the matrix. Although negatively charged amino acids typically are not
found in leader sequences, chaperonin 10 (12) and rhodanese (13) each
contain one negative charge. Different forms of yeast IPMS (coded by
Leu4 and Leu9) contain one and two negative
charges, respectively, in their leader sequences (14-16).
Mitochondrial IPMS provided us with a good model to study the mechanism
of import of a natural protein with negative charges within the
mitochondrial leader sequence.
In this study, we used several chimeric and mutant forms of pALDH and
IPMS containing neutral or negative charges in their leader sequences.
We will show that some precursors could be imported to the
mitochondrial matrix with a leader sequence that is not present at the
most N-terminal portion.
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MATERIALS AND METHODS |
Plasmid Constructions--
The native and mutant leader
sequences of ALDH in pGEM-3Z were amplified with a flanking
SphI site using native or mutant pALDH as a template. PCR
products and the vector containing pALDH or mutant pALDH were digested
with SphI, and the vector was subsequently treated with
alkaline phosphatase and ligated to the PCR products. The orientation
was confirmed by sequencing. LEU4 and LEU9 were amplified with Vent polymerase from genomic DNA isolated from Saccharomyces cerevisiae (ATCC 24657). These two genes were
ligated into the pGEM-3Z plasmid vector, respectively, between the
KpnI and HindIII sites for in vitro
transcription/translation and further mutational studies. The IPMS/ALDH
and Leu4/Leu9 chimeras were constructed by three separate polymerase
chain reactions that sequentially added additional coding fragments of
the LEU4 and LEU9 genes to DNA, encoding either the mature
portion of pALDH or the short form Leu4 or Leu9 protein (14). The final
PCR products were ligated to the pGEM-3Z plasmid between either the
KpnI and HindIII sites or the NcoI and
HindIII sites. All constructs were sequenced at the DNA
sequencing facility at either Purdue University or Iowa University.
In Vitro Mitochondrial Import--
Mitochondria were isolated
from S. cerevisiae (ATCC 24657) and stored at
70 °C
until used (17). Radiolabeled protein synthesis and in vitro
import were performed as described previously (18). Visualization and
analysis of import results from the SDS-PAGE were performed by using
the PhosphorImager System. Import data were analyzed with the
ImageQuant software from Amersham Biosciences.
Proteolysis Analysis of Leu4 and Leu9--
Radiolabeled protein
synthesis was performed as described in a reaction system with a total
volume of 12.5 µl. The protein synthesis was terminated by placement
of the reaction mixture on ice and the addition of ice-cold water to a
final volume of 50 µl. Before the addition of proteinase K, an 8-µl
aliquot was removed. Next, 6 µl of 20 mg/ml proteinase K was added to
the remaining 42 µl. At different time intervals, 10 µl of the
protease digestion sample was removed, and 3 µl of 100 mM
phenylmethyl-sulfonyl-fluoride was immediately added to terminate
proteolysis. An equal volume of 2× SDS treatment buffer was added. The
samples were heated to 100 °C for 5 min and then loaded on a 10%
SDS-PAGE.
NMR Spectrometry--
Two-dimensional NMR spectra were obtained
using a Varian Unity+ 600-MHz instrument. A peptide corresponding to
the N-terminal 21 residues of Leu4 was prepared at the Purdue
University Biochemistry Department and was purified by reverse-phase
high pressure liquid chromatography. The peptide was dissolved in equal
volumes of phosphate buffer (50 mM phosphate, 50 µM EDTA, 50 µM azide, pH 5.2) and
d3-TFE to a final concentration of ~1.8
mM. Chemical shifts were referenced to
2,2-dimethyl-2-silapentanesulfonic acid. Spectra were obtained at
temperatures ranging from 10 to 35 °C. Clean TOCSY spectra (19) used
30 and 70 ms mixing times, whereas mixing times
(
m) for NOESY spectra were 200 ms. Amide exchange experiments were performed using a Varian VXR 500-MHz instrument. The NMR sample described above was lyophilized and was
redissolved in a (1:1) mixture of 2H2O and
d3-TFE. The sample was immediately placed in the magnet, and five NOESY spectra (
m = 150 ms) were
acquired at 2-h intervals. Amide cross-peaks were observed in only the
first two NOESY data sets. The residues for which NH protons were
observed are identified in Fig. 3B. All two-dimensional
transformations were performed using Varian software from Varian
Associates, Inc.
 |
RESULTS |
Preproteins Can Be Imported to the Mitochondrial Matrix Space with
a Leader Sequence Not Present at the Very N Terminus--
A modified
pALDH leader with the R3Q,R10Q double mutation (B in Fig.
1) was shown to be barely capable of
allowing a preprotein to be imported to the mitochondria (6). This
leader was fused to the N terminus of pALDH to make chimeric protein C
(Fig. 1). The double leader strategy was used previously to study the
processing of the leader sequence by MPP (18). Here the purpose of
using it was to test whether or not a leader could be functional if it
were not present at the most N-terminal portion of a protein. We did
not expect that chimeric protein C would be imported into the
mitochondrial matrix, because we and others have shown that the
positive charges are needed at the N terminus of the presequence, presumably to interact with the TOM complex (6, 11, 20, 21). However,
chimeric protein C was found to be imported to the same extent as pALDH
(Fig. 2I, lane 4).
Two different mechanisms can be evoked to explain the import of
chimeric protein C. For the first mechanism it is suggested that when
an R3Q,R10Q-containing leader is fused with another pALDH leader, the
second helical segment of the first leader and the first helix of the
second leader form a long continuous helix (helix 2 in Fig.
1C) that might be responsible for import. For the second
mechanism it is suggested that although the native leader was not
present at the N terminus of C, it served as an internal leader
sequence.

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Fig. 1.
Leader sequence of rat liver pALDH and
schematic diagram of chimeric proteins. The sequences of native
pALDH and the linker deleted ( RGP) leaders are shown.
Below the leaders are the chimeric proteins used in this study with
numbered boxes to indicate helical domains. The percentage
import is relative to pALDH, defined as 100%. A, pALDH with
the helix-linker-helix motif. B, pALDH containing an
R3Q,R10Q double mutation. C, leader sequence of pALDH with
an R3Q,R10Q double mutation fused to pALDH.
D, leader sequence of pALDH with an
R3Q,S7E double mutation fused to pALDH. E, leader sequence
of pALDH with an R3Q,R10Q double mutation fused to another R3Q,R10Q
double mutant pALDH. G, the R3Q,R10Q-containing leader of
pALDH fused to pALDH with its C-terminal helix deleted. H,
an R3Q,R10Q-containing leader was fused with pALDH with a
proline-glycine linker between them. I, an
R3Q,R10Q-containing leader was fused with another R3Q,R10Q-containing
pALDH with a proline-glycine linker between them. J, an
R3Q,R10Q double mutant leader fused to pALDH (through a proline-glycine
linker) with its C-terminal helix deleted. K, an R3Q,R10Q double mutant
leader fused to another R3Q,R10Q double mutant leader (through a
proline-glycine linker) that had the C-terminal helix deleted.
L, an R3Q,R10Q,R14Q,R17Q-containing
leader fused to another R3Q,R10Q leader (through a proline-glycine
linker) that had the C-terminal helix deleted.
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Fig. 2.
In vitro import of chimeric pALDH
proteins. Import was performed as described under "Materials and
Methods." I, lanes 1 and 2 represent pALDH
(A), lanes 3 and 4 represent chimera
C, lanes 5 and 6 represent chimera
D, and lanes 7 and 8 represent chimera
E. Lanes 1, 3, 5, and
7 are for newly translated proteins, and lanes 2,
4, 6, and 8 are for those found after
import into mitochondria representing imported protein. II,
lanes 1 and 2 contain pALDH (A) and
lanes 3 and 4 contain chimera G. Lanes 1 and 3 are translated proteins and
lanes 2 and 4 contain protein bands found after
import. III, lanes 1, 3, and
5 are translated proteins of chimeras A,
H, and I, respectively, and lanes
2, 4, and 6 are the bands found after import
of A, H, and I, respectively.
IV, lanes 1 and 3 contain
A and J, respectively, and lanes 2 and
4 represent the imported bands of A and
J, respectively. V, lanes 1 and
3 represent translated proteins of A and
K, respectively, and lanes 2 and 4 represent protein found after import of A and K. VI, lanes 1 and 3 represent translated
proteins of A and L, respectively, and
lane 2 represents protein found after import of
A. No imported protein was observed with L
(lane 4).
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To determine which mechanism for the import of chimeric protein C was
valid, construct E (Fig. 1) was used. It contained two R3Q,R10Q leader sequences fused back to back, so that it possessed two
non-functioning leaders, and was shown to be imported as well as pALDH
(Fig. 2I, lane 8) as we reported previously (18).
Thus, for chimeric protein E the second long helix (helix 2 in Fig. 1E) and the third helix might have formed an efficient
leader. To determine the individual contribution of each helix and the specific region that acted as a leader, we made chimeric protein G
(Fig. 1). Chimeric G had one neutral helix at the N terminus followed
by a long helical segment. When chimeric protein G was added to
isolated mitochondria, it was imported to the matrix space and was
processed (Fig. 2II, lane 4). This result shows that the third helix in chimera E was not necessary; the helix, which
is a longer version of helix 2 in chimera B, acted as a leader in the
presence of a non-charged helical domain at the most N-terminal portion
of the preprotein.
From the data in Fig. 2II, we concluded that an internal
long helix was necessary for the import of G. Proline and glycine were
introduced to disrupt helix 2 in both constructs C and
E to produce H and I, respectively
(Fig. 1). Pure MPP cleaved at the two processing sites that were
present in each construct (data not shown), confirming that the long
helix was disrupted because MPP cannot cleave when the residues at the
processing site are in a helical environment (18, 22). Constructs
H and I were imported and processed in the
mitochondria (Fig. 2III, lanes 4 and
6). Although the long helix was disrupted, the two smaller helices in H and I (helix 2 and 3, Fig. 1) could
act as a leader because of their similarity to the helix-linker-helix
motif found in pALDH. Two more constructs were made to determine the
importance of the fourth helix as well as the internal
helix-linker-helix motif (helix 2 and 3). We removed the fourth helix
from H and I to make J and
K, respectively (Fig. 1). Both J and K
were imported into mitochondria (Fig. 2IV, lane 4 and 2V, lane 4, respectively). The second and
third small helices could form the helix-linker-helix motif and act as
a leader, because both precursors had an unfavorable helix at the most
N-terminal portion. Import again appeared to be a result of an internal
functioning leader.
In chimeric protein K, the second helix had two positive charges. To
study their importance, these residues were mutated to glutamines to
produce construct L (Fig. 1). Construct L
contained three small neutral helices and it was found, as expected,
not to be imported into mitochondria (Fig. 2VI, lane
4). No import of L was found, which implies that the
internal helix-linker-helix motif still needed some positive charges.
Our previous study showed that the S7E mutant of the ALDH leader was
imported along with the native pALDH. However, when the double mutation
R3Q,S7E was made, it imported just 16% compared with pALDH, which
shows that a net positive charge was necessary in the first helix of
pALDH for it to be imported well. Removal of the RGP linker to make the
leader a continuous helix (R3Q,S7E (
RGP)) allowed import of as much
as 50% compared with pALDH (11). Thus, the positive charge deficiency
could be overcome by introducing a long helix; in those studies, the
long helix was present at the very N terminus. To increase negative
charges at the very N terminus of a precursor protein we made a double
mutation, R3Q,S7E, at the N terminus of the first leader of a
two-leader containing ALDH to produce D (Fig. 1). When
chimeric protein D was incubated with mitochondria, it was imported
(Fig. 2I, lane 6), which indicates that not only
a neutral helix (chimera C) but also a negatively charged one (chimera
D) could be tolerated at the most N-terminal portion as long as an
internal leader was present.
All of the complex leaders used in this study were artificial, but
their presence allowed us to conclude that a neutral or even a
negatively charged helix could be tolerated at the most N-terminal
portion if a leader-like structure was present distal to the N
terminus. This finding is consistent with the proposal for the first
mechanism that an internal leader-like structure can be either a
long helix or a helix-linker-helix motif. To test the conclusions
obtained using the artificial leaders, a natural preprotein with a
negative charge leader sequence IPMS was employed.
Two-dimensional NMR of the IPMS4 Leader Residues 1-21--
The
N-terminal residues of the LEU4 and LEU9 gene are
illustrated in Fig. 3A. The
sequences before the second boldface methionine residue are considered
the mitochondrial leaders. The Leu4 short form starts at Met-31, and it
is assumed that for Leu9 the short form would start at Met-30. Both
leaders possess glutamates within the first 11 residues of the leader
sequence, with the Leu4 leader having two and glutamates Leu9 having
just one. The first 11 side-chain residues of Leu4 do not possess a net
positive charge. The leader was predicted to be a long helix by
secondary structure prediction methods (14). The helix would be
disrupted by the pair of proline residues. To investigate the
conformation of the N terminus of Leu4, a peptide corresponding to the
first 21 residues was studied by two-dimensional NMR. As with peptides
that were studied previously by NMR in our laboratory (6, 8, 10), a
tyrosine-alanine-amide sequence was added to the C-terminal portion of
the peptide.

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Fig. 3.
A, comparison of the N-terminal residues
of Leu4 and Leu9 long forms. The leader sequences of the
LEU4 and LEU9 gene long form products are
illustrated. The sequences before the second boldface
methionine are considered to be mitochondrial leaders. LEU4
and LEU9 gene short form products have the second
boldface methionine as their first amino acid residue,
respectively. Negatively charged glutamate residues are also presented
in bold. B, interresidue NOEs observed for IPMS4
(1-23) in 50% TFE. Two-dimensional NOESY spectra with 200 ms mixing
times were obtained using a Varian Unity+ 600-MHz instrument.
Unfilled bars indicate interactions that could not be
resolved. The heights of the bars in the row labeled
NHi-NHi+1 indicate
relative intensity. The row labeled NHExch
indicates amide resonances that were observed 1 h after the
peptide was dissolved in 50% TFE. C, in vitro
mitochondrial import of pALDH, Leu4, and Leu9. Lanes 1-3
contain pALDH, lanes 4-6 contain Leu4, and lanes
7-9 contain Leu9. The protein bands in lanes 1,
4, and 7 represent the
TNT-synthesized products; lanes 2, 5, and 8 represent the proteins after import; and lanes
3, 6, and 9 represent the supernatant after
the precipitation of mitochondria. The absence of protein bands in
lanes 3, 6, and 9 indicates that
unimported proteins were completely digested by the added
trypsin.
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High-resolution homonuclear two-dimensional NMR spectra were obtained
at 600 MHz in the presence of 50% TFE, which induced a secondary
structure that was not observed in aqueous buffer. Proton resonances
were assigned from TOCSY experiments (30 and 70 ms) at 20 °C, and
NOESY experiments were used to establish sequential resonance
assignments. Complete chemical shift assignments are available as
Supplemental Material. Secondary structure was assessed from
inter-residue interactions observed in the NOESY spectra illustrated
in Fig. 3B. These data were indicative of a
helical structure extending from Val-2 through Tyr-22, essentially the
entire length of the peptide. Compared with standard random coil values
(23), chemical shifts of H
were consistent with the general
impression formed from analysis of the NOESY data. The segment from K-3
to H-12 exhibited chemical shifts more closely associated with helical
character. Amide proton exchange experiments revealed that the region
from Ile-7 to Glu-11 was most resistant to exchange with 2H
in the solvent. These data provide an image of IPMS4 (1-21) that can
be induced to form a continuous helical conformation in TFE that is
most stable in the first 10-12 residues.
In Vitro Import of Leu4 and Leu9 into Yeast
Mitochondria--
To characterize the leaders of Leu4 and Leu9,
in vitro import assays were carried out under the conditions
described under "Materials and Methods." Rat pALDH was included as
a positive control. The number of potential radiolabeling sites in
pALDH and in Leu4 and Leu9 long forms was nearly the same (pALDH
contains 10 methionines, and the two Leu sequences contain 9 and 12 methionines, respectively).
Compared with pALDH (Fig 3C, lane 1), the
in vitro translation of Leu4 or Leu9 produced two protein
bands (Fig. 3C, lanes 4 and 7). The
lower protein band was postulated to be a product of translation
starting from the second methionine (Met-31 and Met-30, respectively).
The imported protein band of Leu9 (lane 8) had a molecular
weight almost identical to the long form precursor protein, a result
that is consistent with the leader being non-processable (14). pALDH
was imported and processed as expected (lane 2). To our
surprise, import was not observed with Leu4 (lane 5). This result was inconsistent with previously published data (14, 24). To
verify that import of Leu4 long form did not occur, radiolabeled
protein was added such that if 1% of the total counts provided in the
assay were taken up by mitochondria, it would be observable after a
12-18- h phosphorimage exposure. No import was observed under these conditions.
Cell-free Synthesis and Import of Leu4 and Leu9 Chimeric
Proteins--
To investigate the behavior of the Leu4 leader, a leader
exchange experiment was performed with the Leu4 purported leader (first
30 amino acids) fused to the Leu9 short form (Fig.
4A). The chimeric Leu4-Leu9
protein was found to be imported to the mitochondrial matrix space
(Fig. 4B, lane 5). On the basis of this result,
it was concluded that the first 30 amino acid residues of Leu4 could
target a passenger protein into mitochondria under in
vitro conditions. This observation is in contrast to what was found for the native protein where the same 30 residues were
ineffective in targeting its own short form protein into isolated
mitochondria under the same experimental system. To address this
discrepancy, two more chimeric protein constructs were made. Here,
either the Leu9 leader or the pALDH leader was fused to the Leu4 short
form as illustrated in Fig. 4A. The import results of these
two constructs were quite different in that the Leu9 leader could not
import the short form of Leu4, whereas the pALDH leader could (Fig.
4B, lanes 2 and 8). To assess the
import ability of Leu4 and Leu9 leaders, the N-terminal 40 residues of
Leu4 long form and a corresponding N-terminal 39 residues of Leu9 long
form were fused to the mature portion of ALDH, creating a
Leu4(1-40)ALDH chimera and a Leu9(1-39)ALDH chimera (Fig.
5). The reason we included the additional
10 residues was that the most N-terminal portion of a mature
mitochondrial protein appears to affect structure and import capability
(25, 26). Both of these constructs could be imported into mitochondria (Fig. 6A, lanes 2 and 11). These results showed that the N-terminal 40 and 39 residues of Leu4 and Leu9 can function as leader sequences.

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Fig. 4.
A, schematic illustrations of the native
and the IPMS chimeras. The long form Leu4 and Leu9 are represented by
two connected boxes. The first rectangle
represents the mitochondrial leader sequence, whereas the second
rectangle indicates the short form protein. The chimeric IPMS
proteins were constructed by exchanging the leaders of Leu4 and Leu9
and reconnecting the Leu4 leader before the Leu9 short form and the
Leu4 leader before the Leu9 short form. The pALDH-Leu4 was
constructed by placing the first 19 residues of pALDH, designated as
pALDH leader, before the Leu4 short form. Import of proteins is based
on a comparison with Leu9, which is defined as 100%. B,
in vitro import of IPMS chimeras. Chimeric proteins
synthesized in the TNT system are illustrated. Lanes
1-3 represent Leu9-Leu4 (L9-L4), lanes 4-6
represent Leu4-Leu9 (L4-L9) and lane 7-9
represent pALDH-Leu4. Lanes 1, 4 and 7 represent the TNT-synthesized products. The corresponding
proteins after import are shown in lanes 2, 5,
and 8, respectively. All of the chimeric proteins were
completely digested by trypsin, because no bands were found from the
corresponding supernatant after precipitation of the mitochondria in
lanes 3, 6, and 9, respectively.
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Fig. 5.
IPMS-ALDH chimeras. Both the first 40 residues of Leu4 long form and the first 39 residues of Leu9 long form
were truncated into several fragments. Each of these fragments was
placed before the mature portion of pALDH, forming five different
Leu4-ALDH chimeras and five different Leu9-ALDH chimeras. Import of
protein is based on Leu4 or Leu9, defined as 100%.
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Fig. 6.
Mitochondrial import of different
Leu-ALDH chimeras. The first 40 amino acid residues of Leu4 long
form or the first 39 amino acid residues of Leu9 long form were
truncated into several fragments and fused to mature ALDH separately
(Fig. 5). The expressed proteins are shown as follows. A,
lanes 1-3 are Leu4(1-40)ALDH, lanes 4-6 are
Leu4(1-21)ALDH, lanes 7-9 represent Leu4( 1-11)ALDH,
lanes 10-12 represent Leu9(1-39)ALDH, lanes
13-15 represent Leu9(1-20)ALDH, and lanes 16-18
represent Leu9( 1-11)ALDH. Lanes 1, 4,
7, 10, 13, and 16 represent
the TNT-synthesized products. Lanes 2,
5, 8, 11, 14, and
17 represent the protein bands after import. Lanes
3, 6, 9, 12, 15, and
18 represent the samples containing the supernatants after
mitochondrial precipitation. B, lanes 1-3
represent Leu4( 1-21)ALDH, lanes 4-6 represent
Leu9( 1-20)ALDH; lanes 7-9 represent Leu4(12-21)ALDH,
and lanes 10-12 represent Leu9(12-20)ALDH. Lanes
1, 4, 7, and 10 represent the
TNT-synthesized products, lanes 2, 5,
8, and 11 represent the protein bands after
mitochondrial import, and lanes 3, 6,
9, and 12 represent the samples containing
supernatant after mitochondrial precipitation.
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Characterization of the Leu4 and Leu9 Leaders--
Although the
first 11 residues of Leu4 do not possess a net positive charge, the
inducible long helical structure may compensate for the charge
deficiency similar to that shown with the ALDH constructs (11). Because
the primary sequences of the Leu9 and Leu4 leaders are nearly
identical, the first 20 residues of Leu9 were presumed to also be
capable of forming an
-helical structure. To localize the leader
fragment most responsible for the import signal, two other chimeras,
Leu4(1-21)ALDH and the corresponding Leu9(1-20)ALDH (Fig. 5), were
constructed and subjected to the import assay. To our surprise, these
two chimeras were barely imported into mitochondria (Fig.
6A, lanes 5 and 14).
To determine the locations of the mitochondrial targeting information
within the IPMS leaders, the 40 and 39 N-terminal residues of Leu4 and
Leu9 were manipulated (Fig. 5). When the first 11 residues were
deleted, the remaining leader segments of both Leu4 and Leu9 could
import ALDH as efficiently as, or even better than, the full-length
IMPS leader (Fig. 6A, lanes 8 and 17).
When the first 21 residues of Leu4 or the first 20 residues of Leu9
were removed, import again became poor (Fig. 6B, lanes
2 and 5). Neither the short fragment of residues 12-21
of the Leu4 leader nor residues 12-20 of the Leu9 leader could act as
a successful mitochondrial targeting signal on their own (Fig.
6B, lanes 8 and 11). On the basis of
the manipulation and subsequent import results, it is concluded that
the most N-terminal 11 residues of both Leu4 and Leu9 leaders are not
required to target mature ALDH into mitochondria, whereas the remaining
leader portion, residues 11-30, is necessary for passenger protein import.
 |
DISCUSSION |
During the past decade much has been learned about the leader
sequences of precursor proteins that are destined for the mitochondrial matrix space (3, 27). No reports exist, however, to explain how a
leader that does not possess a net positive charge at its N terminus is
imported. During our studies with the positive charge in the ALDH
leader, we often found that if the leader was made more helical it
would compensate for the removal of positive charges (6). No actual
structure for a precursor protein that possesses a cleavable leader
sequence has been determined. NMR has been used to show that a number
of peptides corresponding to leader sequences can indeed form
helices when bound to micelles or when in a hydrophobic environment (6,
8, 10, 28). Recently the structure of the aldehyde dehydrogenase leader
bound to TOM20 was shown to be helical, supporting the notion
that helicity is indispensable for its function (29).
A few precursor proteins do not possess positive charges in what could
be considered to be the classical N-terminal leader sequence. For
example, the non-processed precursors rhodanese (30) and chaperonin10
(31, 32) have no positive charges in the first eight residues and
actually have a negative charge in the leader. Leu4 leader has one
lysine residue but two negative charges in its first 11 residues. These
leader peptides were all found to be helical, consistent with our
notion that structure can somehow overcome positive charge deficiency.
Even if structure could overcome this situation, it is not
apparent how the TOM complex, which contains patches of negative
charges (7), can recognize the atypical leader.
We took advantage of the two-leader strategy that we used previously to
investigate the processing of leader peptides by MPP (18). Here a
modified non-functional leader was fused to the N termini of a native
or modified precursor protein. In this way, it was possible to
ascertain what part of the leader was actually needed by the import apparatus.
Previously, it was shown that the R3Q,R10Q double mutant pALDH was not
imported (6). Here we report that if this modified leader was fused to
the R3Q,R10Q pALDH, import was restored. This shows that
two non-functional leaders could actually be used to import a protein.
The reason this double leader was functional in import could be that
the internal leader-like structures shown in Fig. 1 actually were the
portion of the precursor that was involved in the translocation
process. No such structural motif existed in the original R3Q,R10Q
double mutant pALDH we previously investigated. From the various
chimeric precursors used, it is concluded that an internal helix that
is either a long continuous amphipathic helix or part of the
helix-linker-helix motif could act as the actual leader. No attempt was
made to determine how distal from the N terminus such a structure would function.
Using the strategy described above, it was possible to show that the
most N-terminal, non-functional segment could even possess a negative
charge such as found in the R3Q,S7E double mutant of pALDH. This
finding can then be used to explain how a natural precursor protein
could possess a net negative charge at the start of the N-terminal
sequence. It can be argued that the import apparatus simply ignores the
unfavorable region and interacts with the positively charged stable
helical domain that follows.
To test for the above mentioned model we used Leu4 and Leu9, natural
yeast proteins that possess negatively charged amino acids in the most
N-terminal segment of their leader sequence. NMR spectroscopy was used
to show that, as expected, the peptide corresponding to the leader of
Leu4 formed a stable
helix. Before the yeast genome was obtained,
only one form of the enzyme was known. The second gene product, Leu9
(which has 80% identity with Leu4), was identified later, but the
protein was not characterized (15). First, in vitro import
was used to show that Leu9, like Leu4, is a mitochondrial protein.
Unexpectedly, the Leu4 precursor was not imported under conditions that
allowed the Leu9 to be imported. A chimeric protein was made by fusing
the purported leader segment of Leu4 or Leu9 to mature ALDH. When the
first 21 residues of Leu4 (those found to be helical) were used, no import was found. But if the first 31 residues were used, import could
occur. In a similar manner, the first 30 residues of Leu9 could import
of mature ALDH, whereas the first 20 could not.
We were concerned with the fact that we could not obtain import with
Leu4 as others had reported (14, 24). The previously published
experimental procedure used total RNA for translation (14, 24) and
antibodies (33) to precipitate imported proteins. In contrast, the
current experiments all used pure cDNAs. Given the fact that the
Leu9 gene was not identified at the time the initial
experiments were performed and that total RNA was used, it is possible
that the Leu9 gene product was detected rather than the
product for Leu4. We conclude that under the in
vitro experimental procedures used in this study, Leu4 was not
imported to the mitochondrial matrix space.
The N-terminal portion of Leu4 and Leu9 provided a 21- or 20-residue
helix thought to provide the essential structure for a region
that does not possess a net positive charge. Our data revealed that the
N-terminal region containing the negative charges formed the most
stable part of the helix. However, the region that seemed most
responsible for import of Leu4 or Leu9 was not the most helical portion
of the leader, because the targeting signal seemed to appear after
residue 11. It is generally accepted that before import to the
mitochondria preproteins remain unfolded. A proteolysis assay was
performed with the TNT-synthesized Leu4. Proteinase K proteolysis
results indicated that the Leu4 was more resistant to digestion than
was the Leu4(
1-11) protein (data not shown). A possible explanation
for this phenomenon is that the most N-terminal 11 residues of Leu4
contribute to the overall structure of the protein. Without these
residues the native folding of these proteins might be changed, as
reflected by the instability of the protein and the proteinase K
sensitivity. Therefore, the proteolysis results showed that Leu4 was
folded and hence not imported to the mitochondria in an in
vitro experiment. Some proteins that fold rapidly have been found
to be imported to the mitochondria by a co-translocation pathway. The
pALDH leader fused to green fluorescent protein (a protein that folds
rapidly) was shown to be imported to the mitochondrial matrix space by
a co-translational pathway (34). Similarly, in vivo Leu4
could be translocated to the mitochondria through a co-translational pathway.
The acid-chain hypothesis was based on the fact that the leader
possessed positive charges at its N terminus, which could be important
for binding with the TOM complex. However, it has been shown that the
leader sequence of pALDH binds TOM20 through hydrophobic interactions
(29). TOM20 is the first receptor to be recognized by leader sequence
and does not appear to depend on the positive charges of the leader
sequence. It is possible that the other TOM protein may interact with
positive charges present in leader sequence. Here we show that if the
N-terminal segment is not positive, the translocator appears to
recognize not the N termini but the region of the leader sequences that are positively charged. It has been shown that inner and intermembrane space proteins use an internal signal after the most N-terminal matrix space targeting signal (35, 36). How the first 11 residues in
the case of Leu4 enter the translocation pore cannot be explained at
this time. If the model of import were co-translational and not
post-translational as we suggest, then it is possible that an unknown
cytosolic factor is necessary to insert the proteins into the TOM
apparatus. Although the data presented in this study cannot explain how
the precursor with negative charges interacts with the TOM proteins,
they do show that the information for import need not lie at the very N
terminus of the precursor protein.
 |
ACKNOWLEDGEMENT |
We thank Professor Guntar Kohlhaw, Purdue
University, for his help and advice when working with IPMS.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants AA10795 and GM 53269. This is journal paper 17006 from
the Purdue University Agriculture Experiment Station.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.
The on-line version of this article (available at
http://www.jbc.org) contains Table 1.
These authors contributed equally to this work.
§
To whom correspondence should be addressed: Dept. of Biochemistry,
Purdue University, 175 S. University Street, West Lafayette, IN
47907-2063. Tel.: 765-494-1650, Fax: 765-494-7897; E-mail: Hweiner@purdue.edu.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212743200
 |
ABBREVIATIONS |
The abbreviations used are:
TOM, translocase of the mitochondrial outer membrane;
pALDH, precursor
aldehyde dehydrogenase;
IPMS, isopropylmalate synthase;
MPP, mitochondrial processing peptidase;
TFE, trifluoroethanol;
long
form, preprotein containing the leader plus the mature part of IPMS;
short form, IPMS containing only the mature portion of IPMS;
IPMS4(1-21), a synthetic peptide corresponding to the N-terminal 21 residues of Leu4;
NOESY, Nuclear Overhauser effect spectroscopy;
TOCSY, total correlation spectroscopy.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.