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J Biol Chem, Vol. 273, Issue 45, 29389-29393, November 6, 1998
From the Department of Biochemistry, Purdue University, West
Lafayette, Indiana 47907-1153
Mitochondrial leader sequences have been found to
be statistically enriched for positively charged residues, with only a
few known leader sequences possessing negatively charged residues. Mutational studies that have introduced negatively charged residues into various leader sequences have shown a general, but not absolute, trend toward reduced import. The leader sequence of rat liver aldehyde
dehydrogenase has been previously determined by NMR to form a
helix-linker-helix structure. A negative charge introduced into this
leader did not prevent import, provided that a net positive charge
remained in the N-helical segment. When the net charge of the
N-terminal helical segment was reduced to zero, import could be
recovered by removing the linker, which resulted in a longer, more
stable leader. This structural recovery of import was effective enough
to compensate for a net charge of zero within the first 10 residues,
even when a glutamate is the first charged side chain presented in the sequence.
The majority of mitochondrial proteins are encoded from nuclear
DNA and synthesized in the cytosol. These nascent proteins possess
nonidentical N-terminal amino acid sequences that contain sufficient
information to direct the protein from the cytosol to the mitochondria.
Although mitochondrial leaders do not share any primary sequence
identity, they are statistically rich in positively charged amino acid
residues and display the ability to form an amphiphilic secondary
structure (1, 2). Mitochondrial receptors, which recognize and interact
with these leader sequences, possess regions in their primary sequence
that are enriched with negatively charged residues (3). Although the
structures of the various import receptor components are unknown, it is
likely that certain receptor components bind the leader sequences
amphiphilically (4, 5). The enrichment of negative charges within
segments of the mitochondrial receptors implies an electrostatic
complementation to the positive charges of the leader sequence.
From a statistical basis, negatively charged amino acid residues are
infrequently found in leader sequences (1). However, a number of leader
sequences that have been investigated, such as isopropyl malate
synthase (6, 7), 10-kDa chaperonin (8), and rhodanese (9), possess a
minimum of 1 negatively charged residue. Mutational studies have
demonstrated both a tolerance and an intolerance to the introduction of
negative charges within leader sequences (10-13). The statistical fact
that naturally occurring leader sequences are biased to be rich in
positive charges, coupled with conflicting import results from the
mutagenic introduction of negatively charged residues, raises questions
as to how negative charges could be tolerated.
The leader sequence for the
pALDH1 has been studied
extensively both structurally and in two separate systematic mutational investigations (14-16). As shown in Fig. 1, the leader is comprised of
19 amino acids that can be induced into a helix-linker-helix amphiphilic structure in the presence of trifluroethanol or detergent micelles. Deletion of the linker results in a nonprocessible leader that is more stable and longer in helical content (17). Removal of the
linker results in a leader capable of importing aldehyde dehydrogenase
to greater levels than that of the native sequence (15). The enhanced
stability of the linker-deleted leader has been successfully used to
study structural aspects as a compensating factor for the loss of
positive charges through arginine to glutamine mutational substitutions.
The leader sequence of pALDH contains five arginines with two arginines
in each helix and one in the linker segment. It was found from a
mutational study that numerous arginine to glutamine substitutions
could be made with no apparent effect on import (15). Although each
helical segment contains an equal number of positive charges, the
N-terminal helical segment has been shown to provide the majority of
the necessary charge for efficiently targeting the leader to the
matrix. If both of the N-helical segment arginines were substituted
with glutamine residues, the ability to import was essentially
eliminated. This result implies that the net positive charge in the
N-helical segment is more important than the total positive charge
throughout the leader. In contrast, the linker-deleted structure could
import a passenger protein even if both of the N-terminal arginine
residues (Arg3 and Arg10) were mutated to
glutamines. The linker-deleted leader demonstrated that a longer, more
stable amphiphilic structure compensates for the absence of the
N-terminal positive charges. Based on 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.
Site-directed Mutagenesis--
The plasmid containing rat liver
mitochondrial pALDH, which was previously cloned into pGEM-3Z (Promega;
Ref. 18), was digested with BglII and HindIII to
remove the segment coding from amino acid residue 23 to the end the
mature protein. Mouse dihydrofolate reductase cloned into SP64 was
amplified by a polymerase chain reaction utilizing a 5' primer with a
BglII overhang and a 3' primer with a HindIII
overhang. The BglII- and HindIII-digested polymerase chain reaction fragment was ligated to the abovementioned vector, resulting in a plasmid that codes for the leader sequence of
pALDH, the first 22 amino acids of the mature segment of pALDH, followed by dihydrofolate reductase. Subsequent point mutations within
the leader sequence were performed by polymerase chain reaction with 5'
primers encoding a SphI overhang and the desired codon
change and a 3' primer with a BglII site. Oligonucleotides were obtained and purified as described previously (15). All mutational
constructs were verified by sequencing.
In Vitro Import--
Mitochondria were isolated from male Harlan
Sprague-Dawley Wistar rat livers as described previously (15).
Radiolabeled proteins were synthesized using the TNT Quick Coupled
transcription and translation system (Promega). Quantification of
import was performed using the band intensities from SDS-polyacrylamide
gel electrophoresis gels with a Bio-Rad phosphor imaging system. The level of import was defined as the ratio of the total counts of the
protease-protected bands:that of the total counts provided in the
assay. The import values obtained from the mutant leaders were
normalized to the values obtained from the native leader in the same
experiment. The averages shown in Table I reflect a minimum of three
separate import experiments.
Circular Dichroism--
Circular dichroism was performed on a
Jasco J-600 Spectropolarimeter. Peptides were obtained, purified, and
analyzed for mass and amino acid composition as described previously
(15). The experiments were performed at a peptide concentration of 20 µM at 25 °C in 50 mM phosphate buffer, pH
5.2 or pH 7.2. The percentage of TFE was based on volume. Three
baseline determinations were obtained and averaged at the given
percentage of TFE. Three measurements with the peptide under the same
percentage of TFE were obtained and averaged. A value of Neutral and Negatively Charged Amino Acid Mutations in the
Precursor Aldehyde Dehydrogenase Sequence--
The pALDH leader
sequence is structurally comprised of two amphiphilic helical segments
joined by a flexible, three-amino acid linker segment (Fig.
1). Each helical segment, referred to as
either the N-terminal or the C-terminal segment, contains two arginines, whereas the linker contains one arginine. It was our aim to
determine the effect on import by the introduction of negative charges,
particularly in the N-terminal helical segment, because this helix
segment is thought to be responsible for the positive charge required
for import (14, 15). We chose Ser7 as the residue to begin
the site-directed mutagenic introduction of a negative charge. A
mutagenic replacement of this Ser with Glu or Asp would retain the
amphiphilicity required for mitochondrial leader sequences, as
illustrated in Fig. 1 (4). As a control for replacing Ser7
with the charged residues, both Gln and Asn were substituted at the
same position. As shown in Fig. 2 and
summarized in Table I, the S7N and S7Q
mutants were capable of targeting the dihydrofolate reductase passenger
protein to the matrix to the same extent as the native leader. The S7E
mutant was imported to levels that averaged up to 84% of the native
leader, indicating a tolerance to a negative charge. The S7D mutant was
only capable of importing to about 30% of the native levels.
Previously, we showed that only one arginine was required in the
N-terminal domain of the pALDH leader sequence, because both the R3Q
and R10Q mutant leaders were imported as well as the native sequence
(15). The double mutant R3Q(R10Q) was impaired at import, demonstrating
a necessity for at least one positive charge in the first 10 amino
acids. If the positive charges of the arginines could interact with the
negative charge through ion pairing, this interaction could shield and
reduce the effect of the introduced negative charge. To test this
hypothesis, two mutants were made in which the flanking arginines were
substituted with glutamine, a neutral, helix-promoting residue. As
shown in Fig. 2, the R3Q(S7E) and R10Q(S7E) mutants were imported less
than the S7E mutant, indicating that both arginines were required to
achieve native import levels when a negatively charged amino acid was
present at position 7.
In the R3Q(S7E) and R10Q(S7E) mutants, the total charge of the leader
has been reduced from +5 to +3, whereas in the N-terminal helical
segment, the charge was reduced from +2 to 0. The N-helical segment of
the impaired R3Q(R10Q) leader also possessed a net charge of 0. If a
net charge within the N-terminal helical segment is more important than
the total charge of the leader, then we could potentially influence
this net charge through a S7E(R11E) mutant. Although, this additional
glutamic acid is not part of the N-terminal helical segment, its
presence could cancel the effect of the adjacent positive charge of
Arg10. As shown in Fig. 2, the S7E(R11E) mutant was
impaired at import. The import results with the mutants of S7E,
R3Q(S7E), and R10Q(S7E), as well as the R3Q(R10Q) previously studied,
converge to a model that supports a regional net charge effect in the
leader sequence of pALDH rather than the total number of positive
charges in the entire leader, as has been previously implied with the
ornithine transcarbamylase leader (19). However, S7E(R11E) does not
fully support the model of a regional N-terminal net charge because the
total charge of the leader may have been reduced too much.
To attempt to resolve whether S7E(R11E) was impairing import by
influencing either the total charge of the leader or the N-helical segment's net charge, we introduced an additional negative charge into
the C-helical segment of the S7E mutant. The two arginines in the C
helix of the native leader could be separately mutated to glutamines
without any effect on import, but the R14Q(R17Q) double mutant was
imported to 50% efficiency (15). We chose Arg14 to
preserve the processing site motif and thus made a S7E(R14E) leader. If
the total charge of the leader were more important than the net charge
of the N-helical segment, then we would expect import to be reduced
similar to that of S7E(R11E). If the net charge of the N-terminal
helical segment were providing the necessary positive charge, we would
expect import levels similar to that of R14Q(R17Q). As shown in Fig. 2,
neither of the expected results was observed. The S7E(R14E) mutant was
found to be more efficient at importing compared with the native
leader. In the S7E(R14E) mutant, the overall net charge of the native
leader sequence has been reduced, as in the S7E(R11E) mutant, to +2,
yet import was increased. What may have compensated for the C-terminal
net charge deficiency was a network of potential ion pairs between the
Glu and Arg residues that could form in both helical segments as well as between them. There are six potential ion pairs in the S7E(R14E) leader, and this ion pairing may have improved the stability of the
leader, especially through the linker segment. If the potential increase in structural stability were the factor in compensating for
net positive charge reduction in the C-helical segment, structural stability may also compensate for the loss of positive charge in the
N-terminal helical segment by removing the linker.
Removing the linker segment in the leader sequence of pALDH has been
shown to result in increased stability and helical length (17).
Additionally, it has been shown to compensate for the loss of positive
charges in the N-helical segment, as with the R3Q(R10Q) mutant (15).
Removing the linker from the R3Q(S7E) and R10Q(S7E) double mutants
restored import, as shown in Fig. 2. Although both mutants were
imported, the efficiencies were dissimilar. The
R10Q(S7E)-RGP mutant restored import to levels equivalent to
that of the linker-deleted leader, whereas the R3Q(S7E)-RGP mutant was imported to native levels. What may have been a contributing factor for the reduced import in the R3Q(S7E)-RGP leader
compared with that of R10Q(S7E)-RGP was that the first
N-terminal side chain bearing a charge was negative. However,
improving the apparent stability and increasing the helical length of
the leader can compensate for an N-terminal positive charge deficiency.
Circular Dichroism of the S7D, S7E, R3Q(S7E), and S7E(R14E)
Peptides--
The S7D and R3Q(S7E) leaders were impaired at import,
which may suggest that the ability of these leaders to be induced into a stable helix has been compromised by the substituted amino acid. Peptides corresponding to the S7D and R3Q(S7E) leaders were studied at
20 and 40% TFE to assess their ability to be induced into an
As summarized in Table II, the ability of
the S7D to be induced into a helical conformation at 20 and 40% TFE
was comparable to that of the native peptide at pH 5.2. At pH 7.2, the
S7D peptide displayed a greater fraction of peptide in a helical
conformation compared with that of the native leader at both 20 and
40% TFE. In contrast to the S7D peptide, the S7E peptide was
approximately 2-fold higher in helical population compared with that of
the native leader at both 20 and 40% TFE and was surprisingly
independent of the pH value.
The R3Q(S7E) peptide, in contrast to S7E, displayed a pH dependence on
helical conformation and was more readily induced into a helix at 20 and 40% TFE, pH 7.2. As with the S7D and S7E peptides at 20% TFE and
pH 7.2, the S7E and R3Q(S7E) peptides were indistinguishable at 20%
TFE, pH 5.2. Although there is a greater ability to form a helical
structure in R3Q(S7E) as compared with the S7E peptide, neither peptide
was as capable of being induced into a helix as well as the
linker-deleted leader. The circular dichroism results with peptides
S7D, S7E, and R3Q(S7E) show that their ability to form a helix has not
been compromised. Thus, the reason for the reduced import of S7D and
R3Q(S7E) is most likely to be charge related and not structurally related.
S7E(R14E) displayed greater levels of import, surpassing that of the
linker-deleted leader. The peptide S7E(R14E) displayed a pH dependence
on being induced into a helix. At 20% TFE and pH 5.2, the population
of peptide in a helical conformation was essentially identical to that
of S7E and R3Q(S7E). However, at 40% TFE and pH 5.2, the ability to
form a helix was approaching that of the linker-deleted peptide. At
40% TFE and pH 7.2, it was nearly identical to that of the
linker-deleted peptide. Analogous to the linker-deleted peptide, the
ability of S7E(R14E) to form such a stable structure is a likely
explanation for the enhanced import.
The mitochondrial import receptor complexes of translocator of the
outer membrane and translocator of the inner membrane from yeast have
been extensively studied, and the functional roles of each complex have
been partially uncovered (3). The translocator of the outer membrane
complex is thought to provide regions of net negative charge on both
sides of the outer membrane, which may serve to electrostatically guide
the leader to the intermembrane space. The translocator of the inner
membrane complex, which is also thought to contain acidic patches for
binding leaders, differs from the translocator of the outer membrane
complex with respect to the transfer mechanism of a leader across the
inner membrane. Translocation from the translocator of the inner
membrane complex to the matrix requires a The leader sequences of the mutants S7E and S7E(R14E) revealed a
remarkable tolerance to the presence of a negative charge. A previous
study demonstrated that the leader sequence of F1- We have shown that not all of the positive charges in the leader of
pALDH are required for import; R3Q, R10Q, R14Q, R17Q, and R14Q(R17Q)
are all functional leaders under in vitro conditions (15).
The double mutant R3Q(R10Q) was import impaired and led to the
suggestion of the necessity of at least one positive charge in the
N-helical segment. Both Arg3 and Arg10 are
required for the S7E mutant to function properly. A likely explanation
for the ability of the leader to tolerate the S7E substitution is that
the N-helical segment retains a sufficient, net positive charge. As
summarized in Table I, the S7E mutant is equivalent in N-helical
segment charge to the mutants of R3Q and R10Q. The mutants R3Q(S7E) and
R10Q(S7E) are equivalent to that of R3Q(R10Q) in that all three display
a net charge of 0 in the N-terminal helical segment.
Arguments for a net positive charge requirement in the N-terminal
helical segment were strengthened by the results obtained from the
S7E(R11E) and S7E(R14E) mutants. For both the S7E(R11E) and S7E(R14E)
mutants, the overall charge of the leader was reduced from +5 to +2.
The S7E(R14E) mutant displayed greater import and even surpassed the
import level of the linker-deleted structure. The introduction of the
two negative charges in the S7E(R14E) mutant may have potentially
improved the stability of the leader through a network of electrostatic
interactions within the leader, as illustrated in Fig.
3 and suggested by the circular dichroism experiments. In the S7E(R11E) mutant, the presence of a negative charge
was more likely influencing the net charge of the N-terminal helical
segment, even though it is not part of the structure determined from
the native leader determined by NMR (16).
Removing both positive charges from either the N-terminal helical
segment or the C-terminal helical segment of the aldehyde dehydrogenase
leader sequence impaired import. However, deleting the linker from the
R3Q(R10Q) or R14Q(R17Q) mutants restored import. This was also found
for the R3Q(S7E) and R10Q(S7E) mutants. The more stable helix, produced
by removing the linker, appeared to overcome charge deficits in that it
restored import when arginines were substituted by glutamines. Although
the linker-deleted leader could compensate for the reduction of
positive charges, the level of import depended upon whether a positive
or negative charge was the first charged residue in the sequence.
The S7D mutant of pALDH was not imported as well as the S7E mutant. A
structural investigation on a peptide corresponding to the S7D leader
showed no apparent loss in the ability of the peptide to be induced to
a helical structure as compared with the native peptide. There are two
plausible explanations for the observation that the S7D precursor
mutant is import deficient, yet structurally stable. Firstly, the
glutamic acid residue of S7E is capable of extending the charge toward
a hydrophilic environment, but the charge on the shorter aspartate
residue is unable to extend away from what may be a hydrophobic
environment. If the N-terminal helix of pALDH begins to interact
amphiphilically with the receptor proteins, the negative charge on the
aspartate residue may be placed in an unfavorable hydrophobic
environment. The glutamic acid and arginine residues are capable of
proper extension and potential ion paring, which may shield opposing
charge effects and add a favorable, albeit small, energetic
contribution to helix stability. An alternative, related explanation
for the reduced import of the S7D mutant could be that the charge was
not effectively shielded through potential electrostatic interactions
with the two arginines in the leader.
From the results of this study, we propose a model to explain how
negative charges may be tolerated in the leader sequence of pALDH.
Introducing a negative charge into the N-terminal helical segment
reduces the net charge of the helix in much the same manner that was
observed when the positive charges were substituted with glutamines in
absence of negative charges. Provided that a net positive charge is
maintained in the N-helical segment, negative charges are tolerated,
and import is maintained. When the presence of a negative charge
reduces the net charge of the N-terminal helical segment to 0, import
is impaired but can be restored by increasing the length and stability
of the leader. Although more structural information and import
properties are needed from other leader sequences that naturally
contain negative charges, the model is consistent with our previous
studies that have shown structure to be an important contributor to
offset any reduction in positive charge. If our model is correct,
mitochondrial leader sequences that naturally contain a number of
negative charges such as isopropyl malate synthase may have a greater
capacity to form a longer, more stable helix than those that are richer in positive charges.
*
This work was supported in part by National Institutes of
Health Grants GM53169 and AA10795. This is paper No. 15765 from the
Purdue University Agricultural 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 abbreviations used are:
pALDH, precursor
aldehyde dehydrogenase; pALDH-RGP, linker-deleted aldehyde
dehydrogenase leader; TFE, trifluroethanol.
A Regional Net Charge and Structural Compensation Model to
Explain How Negatively Charged Amino Acids Can Be Accepted within a
Mitochondrial Leader Sequence*
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
35,600 was
used to estimate the fraction of peptide in a helical conformation at
[
]222 (15).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (21K):
[in a new window]
Fig. 1.
Helical wheel projections of pALDH and
pALDH-RGP based on two-dimensional NMR structures.
A, the helix-linker-helix structure of the native pALDH
leader illustrates the number of charges to each helical segment and
the linker. The native leader carries a gross positive side chain
charge of +5. B, the pALDH-RGP leader sequence,
which has lost one positively charged residue and two helix-disrupting
amino acids. The resulting sequence forms a continuous helix that
surpasses the ability of the native sequence to be induced into a
secondary structure by TFE or detergent micelles.
View larger version (64K):
[in a new window]
Fig. 2.
SDS-polyacrylamide gel electrophoresis of the
mitochondrial import experiments of the native and mutant precursor
proteins. Labeled translated protein was incubated with rat liver
mitochondria for 30 min at 30 °C in a total volume of 100 µl. A
50-µl aliquot was removed and treated with proteinase K to digest
nonimported precursor protein. Phenylmethylsulfonyl fluoride was added
to terminate the proteinase K digestion before pelleting the
mitochondria and solubilization in SDS treatment buffer. For each
precursor, the left lane represents a known amount of
translated protein that was not exposed to mitochondria. The
middle lane is the protein obtained from the non-proteinase
K-treated mitochondrial pellet. The right lane is the
precursor protein obtained from the proteinase K-treated mitochondrial
pellet and represents imported protein.
Correlation between pALDH mutant import and residual positive
charge
-helix. Because the S7E leader was not impaired at import, we also
studied this leader to assess its ability to form a stable helical
structure and compare it with the S7D and R3Q(S7E) peptides. Two
different pH conditions were utilized. A pH of 5.2 was used for
comparison with the previous circular dichroism and NMR studies with
the native and linker-deleted peptides (15). A pH of 7.2 was utilized
to keep the glutamic residues ionized in the aqueous TFE medium and
thus potentially allow ion pairs to form within the peptides.
Circular dichroism of the S7D, S7E, R3Q(S7E), and S7E(R14E) peptides
]222, measured in aqueous TFE
prepared as (v/v) proportions with 50 mM phosphate buffer,
pH 5.2 and pH 7.2. The fraction of peptide in a helical conformation
with respect to the total is shown in parentheses.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(negative inside)
chemical potential across the inner membrane, used apparently for the
electrophoretic migration of the leader (20, 21). Utilizing the
helix-promoting glutamic acid residue while maintaining amphiphilicity
in all of the constructs, we demonstrated a tolerance to a
statistically uncommon acidic residue in a mitochondria leader sequence.
ATPase could
tolerate the mutational introduction of glutamic acids as single and
double point mutations (10). However, in another study, negative
charges introduced in the same leader interfered with import (11). The
conflicting results may be interpreted using arguments based on
amphiphilicity. In the case in which negative charges interfered with
the import of F1-ATPase, amphiphilicity was not maintained. This was
also observed in a mutant of malate dehydrogenase in which a Leu to Glu
mutation placed the negative charge on what appears to be the
hydrophobic portion of the helix (12). Conversely, the negative charges
that were tolerated in F1-ATPase were placed in a proper amphiphilic
position. An alternative explanation for the different results may
reside in a model of structural compensation for charge deficiencies to
be developed below.
View larger version (29K):
[in a new window]
Fig. 3.
Potential ion pairing within the S7E(R14)
mutant leader. The modeled charge relay between Ser7,
Arg10, Arg11, and Glu14 side chains
clustered between the two helices of the native leader is shown. Such a
clustering of the side chain charges within the S7E(R14E) mutant
potentiates a shielding of the effect of introducing two negative
charges in the native leader and models intramolecular electrostatic
interactions within the leader. The ionic pairing between these
residues leaves the N-helical segment with a net positive charge
essential for import.
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biochemistry,
Purdue University, West Lafayette, IN 47907-1153. Tel.: 765-494-1650;
Fax: 765-494-7897; E-mail: weiner{at}biochem.purdue.edu.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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