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INTRODUCTION |
The role of the presequence in targeting proteins to mitochondria
has been well established for a variety of species using chimeric
constructs containing passenger proteins and deletion studies where
removing the presequence abolished mitochondrial import (1, 2). This
process requires the presence of cytosolic factors (3-5) and
recognition event(s) at the surface of the mitochondria (6). Inside the
mitochondria precursors proteins are processed to a mature form by
specific peptidase(s), which requires information located within the
presequence for correct recognition of the cleavage site (1, 7).
Despite the fact that the sequence of a large number of presequences
are known, no consensus sequence for mitochondrial targeting and
subsequent processing are defined. This is because of large variations
in the length of presequences, which display no primary amino acid sequence homology (7). However mitochondrial targeting presequences share some common features such as enrichment of positively charged amino acids and a lack of negatively charged amino acids and display an
enrichment for alanine, leucine, and serine (7). Furthermore they are
normally predicted to have the ability to form an amphiphilic
-helix
(8).
Mitochondrial presequences can be roughly divided into two domains, an
N- and a C-terminal domain (8, 9). The N-terminal has the potential to
form the
-helix and is proposed to function in targeting of the
precursor (10). The C-terminal domain contains the information
necessary for proper cleavage of the presequence (10) but does not
exhibit any preference to a secondary structure (11). The functions of
both domains are independent of each other, but they may overlap
(9).
Positive charges and the position of the positive charge appear to be
important for mitochondrial targeting. Studies using site-directed
mutants of rat ornithine transcarbamylase precursor (pOTC),1 pig aspartate
aminotransferase, and rat aldehyde dehydrogenase indicate the critical
role of arginine residues located in the N-terminal part of the
presequence for import (12-14). Human pOTC appears to be different;
the mutation of some individual arginines (arginine 23) had a drastic
inhibitory effect on import, whereas changing arginine 26 to glycine
had no effect on import (15). In contrast, mutagenesis of arginine 23 to glycine of rat pOTC affected processing but not import (12). To our
knowledge this discrepancy has not been resolved. Experiments whereby
negatively charged residues are incorporated into the presequence also
affected import (16). This suggests that mitochondrial protein import depends on a net positive charge on the presequence that may be required at different stages of translocation (1), possibly through
interaction with the acid bristle of the translocation pore, as
suggested by Haucke and Lithgow (17).
As there is no known primary structure homology at the cleavage site
between different precursor proteins, it is intriguing how these sites
are recognized and cleaved by the general mitochondrial processing
peptidase (MPP). An arginine residue located two amino acids upstream
of the start of the mature protein has been proposed to act as the
processing signal (7). The mutagenesis studies outlined above indicated
that the arginine residues involved in processing were not required for
import. However certain arginine residues may overlap in function,
e.g. human pOTC, where changing an arginine residue resulted
in loss of import and processing (15).
Only a few studies have looked at the requirements for precursor
targeting to plant mitochondria. Studies with the F1
presequence from Nicotiana plumbaginifolia showed that the
first half of the 54 amino acid presequence could support mitochondrial
targeting although at a much lower efficiency (18). Likewise, the
superoxide dismutase from maize showed that only small deletions in the
C-terminal region of the presequence could be tolerated (19). Few
studies have addressed the requirement for processing of precursor
proteins in plants. The
2 arginine rule does hold for the soybean
alternative oxidase precursor (20). Surprisingly, processing of the
N. plumbaginifolia F1
was inhibited by
synthetic peptides, which had a predicted ability to form a helical
structure, suggesting a structural requirement for processing in this
case (21).
Therefore to define the requirements of a mitochondrial presequence for
targeting and processing in plants, we have carried out studies with
the soybean alternative oxidase precursor, an inner membrane protein
responsible for cyanide-insensitive respiration (22). We have carried
out site-directed mutagenesis with the authentic protein to avert any
potential problems with the nature of the passenger protein and linker
sequences (23). This is the first study to investigate the role of
specific residues required for plant mitochondrial import. Also it is
the first investigation of the processing requirements of plant MPP by
site-directed mutagenesis.
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MATERIALS AND METHODS |
Synthesis of the Soybean Alternative Oxidase Precursor and
Derivatives--
The alternative oxidase (AOX) from soybean contains a
presequence of 41 amino acids. Mutagenesis was performed using altered site-directed mutagenesis (Promega, Madison, WI) or quick-change site-directed mutagenesis (Stratagene) kits as per the manufacturer's instructions, and mutants were confirmed by sequencing.
35S-Labeled alternative oxidase precursor protein was
synthesized as described by Whelan et al. (24). The
mutations introduced were designated by the position relative to the
start site of the mature protein, followed by the three-letter
abbreviation for the amino acid introduced, i.e.
2Gly
means the amino acid glycine two residues upstream of the start of the
mature protein. A list of the mutations is shown in Fig.
1C.
Soybean and Rat Liver Mitochondrial Isolation and in Vitro
Imports--
Mitochondria were isolated from 7-day-old soybean
(Glycine max L. Merr, c.v. Stevens) cotyledons as described
by Day et al. (25). Import of the soybean alternative
oxidase mutants was performed as by Whelan et al. (20). Rat
liver mitochondria were isolated from approximately 30 g of
tissues (26). In vitro mitochondrial imports were performed
similar to the soybean mitochondrial imports, except that
dithiothreitol was omitted, as import was found to be lower in the
presence of dithiothreitol with rat liver mitochondria, and the 20-min
incubation of the reaction was performed at 30 °C. The results were
analyzed using a MacBAS 1000 according to manufacturer's instructions
(Fuji). Import studies with all mutant precursor proteins were also
accompanied by a valinomycin control (see Fig. 2) to ensure that all
PK-protected protein generated was dependent on a membrane potential
(data not shown).
Processing Studies and Western Blotting--
Isolated rat liver
mitochondria were resuspended in fractionation buffer (1 mM
methionine, 30 mM Tris-Cl, pH 8.0). The suspension was
cooled on ice for 5 min before sonication. Sonication was performed 3 times on a Microtip (Branson) setting of 4 for 10 s with a 2-min
cooling on ice inbetween. The fractions were separated by
centrifugation at 18,000 × g for 30 min at 4 °C.
The supernatant was collected and diluted 2-fold for use in processing
assays. Processing mix contained the following: 20 µg of rat matrix
fraction, 1 mM methionine, 0.5 mM
MgCl2, and 10 mM EDTA in a final volume of 10 µl. N-Ethylmaleimide was added to the rat matrix fraction to 5 mM, a concentration that inhibited the mitochondrial
intermediate peptidase but not the MPP (27, 28). The reaction was
incubated at room temperature for 15 min followed by the addition of
dithiothreitol to 10 mM to inactivate
N-ethylmaleimide. The mix was then incubated with 2 µl of
radiolabeled precursors. The samples were analyzed similar to the
import assay.
Processing of the soybean alternative oxidase and the mutant precursors
was performed using purified spinach MPP (29). Western analysis was
carried out with 40 µg of purified mitochondria using antibodies to
the alternative oxidase (30).
Densitometric Analysis--
Quantitation of imports was
performed as indicated by Dessi and Whelan (31).
Quantitations of imports were determined by densitometric analysis on
the raw image scans using the MacBas v2.0 software (Fuji). The
efficiency of import and processing was calculated based on density of
precursor and mature bands. The percentage of import was calculated as
follows (
PK and +PK denotes bands in the non-PK-treated and
PK-treated samples, respectively.
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(Eq. 1)
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The percentage import of the wild type precursor protein was set
to 100% and that of the mutant precursor proteins was calculated relative to the wild type. Import values that are less than 5% are not
indicated on the figures even though the band(s) is present on the figures.
Processing of the wild type and mutants were calculated as follows.
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(Eq. 2)
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The percentage of wild type processing was set to 100%, and the
percentage of processing of the mutants was calculated relative to the
wild type. All data taken fell within the linear range of the MacBAS
1000, and all assays were carried out at least three times.
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RESULTS |
The Presequence of the Alternative Oxidase--
The AOX
presequence was analyzed using MITOPROT (32) and Robson-Garnier
analysis (33) to determine the amphiphilicity and secondary structure,
respectively. The AOX presequence has the potential to form an
amphiphilic
-helix (32) (Fig.
1A) between residues 9 and 25 followed by no predictable structure after residue 26 (Fig.
1B), in agreement with the two-domain structure of
mitochondrial targeting presequences (8, 9). To demonstrate the
requirement for the presequence in mitochondrial targeting of the AOX,
the mature AOX form (AOX
P) was constructed. This was not imported
into isolated soybean and rat liver mitochondria (Fig.
2, A and B,
lanes 6 to 8) under conditions that clearly supported the import of the wild type AOX (Fig. 2, A and
B, lanes 1 to 3). (The identity of the
additional bands P' and M' will be discussed with the deletion mutants
below.) From this experiment it was concluded that the presequence was
responsible for targeting the AOX to the mitochondrion in a membrane
potential-dependent manner (Fig. 2, A and
B, lanes 4 and 5), and site-directed
mutagenesis was carried out to determine critical residues for import
and processing in plants and animals (Fig. 1C).

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Fig. 1.
Structural analysis of the soybean
alternative oxidase presequence. A, hydrophobic moment
plot analysis of the presequence calculated at a = 95°.
B, secondary structure prediction (see text). C,
mutant forms of the soybean alternative oxidase presequence.
Arrowhead represents the start site of the mature protein of
soybean alternative oxidase (45). The number along the
bottom refers to the position of the amino acid relative to the start
of the mature protein (see "Materials and Methods").
Dashes indicate amino acids removed. Preseq,
presequence.
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Fig. 2.
Import of the soybean alternative oxidase
precursor protein into soybean cotyledon mitochondria (Mit)
(A) and rat liver mitochondria (B) . Lanes 1 and 6, precursor protein. Lanes
2 and 7, precursor protein incubated with mitochondria.
Lanes 3 and 8, as lanes 2 and
7, with PK added. Lanes 4 and 9, as
lanes 2 and 7, with valinomycin (Val)
added. Lanes 5 and 10, as lanes 4 and
9, with PK added. P and M denotes
precursor and mature forms of AOX. Additional products observed are
labeled P' and M' (see text).
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Role of the
2 to + 2 Region in Targeting and Processing--
The
2 arginine was the main focus as it is common to other precursor
proteins (34). Import using isolated soybean mitochondria showed that
the greatest effect was seen with the
2Gly and
2Thr, where at least
30% inhibition was consistently observed (Fig. 3A, Table
I (note that the import efficiencies are
normalized as indicated under "Materials and Methods" because of
different precursor translation efficiencies). The mature form
generated upon import with these two mutants had a slower mobility
(higher Mr) compared with the wild type AOX,
indicating that processing upstream of the correct site had taken
place. When the processing site was deleted (
1+1 mutant), import
proceeded normally, except this time the mature form generated had a
greater mobility (lower Mr) compared with
the control. An overall affect of the various
2 mutations was the
accumulation of imported precursor forms, up to 40% in the case of the
2Gly,
2Thr,and 
1+1 mutants.

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Fig. 3.
Import of the mutant soybean alternative
oxidase precursor proteins with mutations in the 2 to the +2 region
into soybean cotyledon mitochondria (A) and into rat liver
mitochondria (B). Each panel represents a
different precursor protein, which is designated on top, containing
three lanes corresponding to the first three
lanes of import as indicated in Fig. 2. Import efficiency is
indicated and refers to imported precursor and mature bands in the last
lane in each panel as indicated under
"Materials and Methods." A circled minus sign or
plus sign above certain panels denotes mobility
of the imported mature form, which is lower or higher compared with the
wild type AOX, respectively. A circled double minus sign
denotes a much lower mobility.
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Table I
Import and processing efficiency of the precursor proteins used in
this study
The import efficiencies refer to the combined amount of the imported
mature and precursor product. The mutants are outlined in Fig.
1C, and the symbols used are as outlined in Figs. 3 and 4.
For Plant, import was with soybean cotyledon mitochondria and
processing was with purified spinach MPP. For Animal, import was with
rat liver mitochondria and processing was with rat liver mitochondrial
matrix fraction.
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Imports with the
2 mutant precursor proteins were also carried out
with isolated rat liver mitochondria (Fig. 3B, Table I). In
agreement with the soybean mitochondria experiments, no complete inhibition of import was observed, and import was inhibited by 20%
with most mutants. However, in the case of rat liver mitochondria, the
2Gly,
2Ala,
2Thr,
2Gln, and
2His mutants had slower mobility, and the 
1+1 mutant had a faster mobility (Fig. 3B).
Similar to soybean, for some mutants, significant amounts of imported precursor form was observed (Fig. 3B).
Processing studies of the
2 mutants were also conducted using
isolated spinach MPP (35) and a rat matrix fraction (36). The wild type
AOX was efficiently processed by the isolated spinach MPP but except
for the +2Gly and 
1+1 mutants, most of the
2 mutants inhibited
processing drastically (Fig.
4A, Table I). In contrast,
processing using the rat matrix fraction was not affected except for
the
2Phe mutant (Fig. 4B, Table I).

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Fig. 4.
Processing assays carried out with the
soybean alternative oxidase mutant precursor proteins with isolated
spinach MPP (A) and rat liver matrix fraction
(B). The efficiency of processing is expressed
compared with wild type alternative oxidase. The first lane
of each figure consist only of the wild type AOX precursor.
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Roles of Residues in the
10 to
2 Region--
The AOX
presequence contains three glycine residues at positions
4 to
6 and
a
10 arginine (a putative processing signal) (37). A
4Arg could not
complement the inhibition of import seen with the
2Gly (Fig.
5A), and even though the
efficiency of processing (in the import assay) was not increased with
the
2Gly,
4Arg mutant compared with the
2Gly mutant, the fidelity of processing was restored (Fig. 3A and 5A). The
10Gly mutant decreased import by 35%, similar to the inhibition seen
with the
2Gly (Fig. 5A). The double mutant
2,
10Gly
inhibited import by ~70%, and this could not be restored by the
presence of an arginine as shown in the
2,
10Gly,
4Arg mutant (Fig.
5A, Table I). Therefore the position of the
2 arginine
residue is important as well as the positive charge; if charge alone
was the only parameter, it would be expected that the
2,
10Gly,
4Arg would be imported with a similar efficiency to the
single mutants
2Gly or
10Gly.

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Fig. 5.
Import of the various soybean alternative
oxidase precursor proteins with mutation(s) at the 4 to 10 position
into soybean cotyledon mitochondria (A) and rat liver
mitochondria (B). Symbols are as for Fig. 3.
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The AOX presequence contains a
10 arginine, a putative processing
signal for two-step processing by MPP and mitochondrial intermediate
peptidase, respectively (37). A
7 serine residue is considered
important for two-step processing (38). Therefore we changed this
7
residue to a serine to see if the
10 arginine could act as a
processing signal. It was found the
7Ser,
2Gly mutant had the effect
that the majority of the product was processed upon import into soybean
mitochondria (Fig. 5A). However it had a slower mobility
than the wild type AOX, indicating processing upstream of the correct
site, possibly using the
10 arginine as a processing signal. In the
case of rat liver mitochondria, a similar effect was seen with the
2,
10Gly mutants in that import was inhibited; however, only by 40%
compared with 70% with soybean cotyledon mitochondria (Fig.
5B, Table I). Again, the presence of a
4Arg could not
restore processing of the
2Gly in the import assay (Fig.
3B), but the mature form generated with the
2Gly,
4Arg mutant was similar to that with wild type AOX (Fig. 3B and
5B). In the import experiments with rat liver mitochondria,
the
7Ser could restore the processing efficiency in the import assay
with the
2Gly (
7Ser,
2Gly mutant) and to a lesser degree with the
2Thr mutant (Fig. 3B and 5B).
In the rat liver import assays, precursors containing the
2Gly
mutation, except the
2Gly,
4Arg mutant, the mature form generated had a slower mobility compared with the wild type. This is in contrast
to the soybean import assays where only the
2Gly and
2Thr mutants
had this effect. However, in rat mitochondrial import, the presence of
the
10Gly mutation with the
2Gly affected the formation of the
mature product, whereby a slower mobility mature form was generated
(denoted by a double minus sign). This suggests that in the presence of
the
2Gly mutant, the wild type
10 arginine acted as a processing
signal. The presence of a
7Ser with the
2Gly (
7Ser,
2Gly mutant)
appears to have increased the efficiency at which the rat mitochondria
utilized the
10 arginine residue as a processing signal in the import
reaction. A serine in the
7 position would be predicted to change the
residues to that of the consensus required for
10 arginine processing
in animals (7). The
10Gly mutant alone had little effect on the
mobility of the product obtained (Fig. 5B).
The role of residues in the
2 to
10 region was also analyzed in
processing reactions (Fig. 4). In contrast to the import studies, the
4Arg mutant restored processing with spinach MPP, processing of the
2Gly mutant (16%) was increased by greater than 3-fold to
approximately 56% by the
4Arg mutant, and the processed product
generated had the same mobility as wild type AOX. The processing of the
2,
10Gly mutant was increased from 9 to 57% in the
2,
10Gly,
4Arg mutant. The ability of the
4Arg to restore
significant processing to both these mutants but have no effect on
import indicates that these arginine residues play a critical role in
import distinct from a role in processing. Similar to what was seen
with the import assays into soybean, the
7Ser mutant could restore
processing of the
2Gly mutant but not the
2Thr mutant.
In contrast to purified spinach MPP, processing by the rat matrix of
the
10Gly mutant was inhibited by 50%, and the
2,
10Gly mutant
was also inhibited by greater than 50%, generating a lower mobility
product (Fig. 4B). The other notable difference compared with spinach MPP was that the
4Arg did not restore the processing efficiency of the
2,
10Gly (but did change the product to that similar to wild type AOX), and the
7Ser did not restore the processed product of the
2Gly to the same mobility as wild type AOX. This may
be because of the fact that the
10 arginine can act as a processing
signal with rat matrix but not with spinach MPP.
Role of Positive Residues in the Presequence on Import and
Processing--
From import experiments using both soybean and rat
liver mitochondria, it was evident that changing any single positive
residue at the
20,
30, or
35 position had some (
25%)
inhibitory effect (Fig. 6) similar to the
level seen with changing the
2 or
10 arginines individually (Figs.
3 or 4). Changing two positive residues inhibited import into both
soybean and rat liver mitochondria, overall more so in the latter (Fig.
6, Table I). For rat liver mitochondrial import, complete inhibition
was seen with the
20,
30Gly mutant, and inhibition was almost
complete with the
30,
35Gly mutant. This is much greater than the
expected additive effect seen with the individual mutants. The double
mutants did inhibit import into soybean mitochondria, but it was
notable that import efficiency was twice as high compared with rat
liver mitochondrial import (Fig. 6, Table I). The triple mutant
completely abolished import with both sets of mitochondria.

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Fig. 6.
Import of soybean alternative oxidase
precursor proteins with mutation(s) at the 20, 30, and 35 regions
of the presequence into soybean cotyledon mitochondria (A)
and rat liver mitochondria (B). Symbols are as for
Fig. 3.
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Processing by purified spinach MPP was significantly inhibited by each
of the changes of the N-terminal positive residues. It would appear
that the double mutant, which included the
20 position
(i.e.
20,
30Gly, or
20,
35Gly), relieved this
inhibition of processing to some extent compared with the single mutant
(Fig. 4). The
30Gly mutant was not processed at all by spinach MPP, compared with 60% efficiency with the
20,
30Gly mutant. In contrast the
20,
30Gly mutant was processed at the least efficiency (along with the
10Gly) with rat liver matrix of all the mutants tested (Fig.
4, Table I). The triple mutant
20,
30,
35Gly was processed by rat
liver matrix and spinach MPP (Fig. 4, Table I), so the inhibition of
spinach MPP seen with the single and double mutants was relieved by the
triple mutant. This indicates that the inhibition of processing seen
with spinach MPP by changing positive residues in this region of the
presequence may have been a result of changes in the overall structure
of the presequence rather than any of the positive residues playing a
specific role in binding or catalytic process of MPP.
Is the PK Protected Precursor Form Truly Imported?--
One effect
of many of the single mutants in soybean was that the imported form was
largely precursor as opposed to mature form (Fig. 3A). It is
possible that the hydrophobic nature of the AOX protein may allow it to
be embedded into the outer membrane where it may display some PK
resistance (22). To investigate if the precursor form was truly inside
the mitochondria, we tested if the imported precursor form had the same
PK insensitivity as the mature imported AOX. When the PK concentration
was increased to 100 µg/ml, it was evident that the imported
precursor form of the
2Gly mutant displayed a similar degradation
profile as the imported mature form of wild type AOX (Fig.
7, A and B).
Furthermore we analyzed the state of the endogenous AOX protein in
soybean mitochondria under these conditions (Fig. 7C). The
endogenous AOX proteins are designated as AOX2 and AOX3, as determined
by direct N-terminal sequencing in a previous study (39). It was evident that under these conditions, the imported AOX (both precursor and mature forms from wild type and
2Gly mutant) displayed similar percentage degradation with increasing PK, as did the endogenous AOX in
the mitochondria. Therefore it can be concluded from these experiments
that the PK-protected precursor was truly imported into
mitochondria.

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Fig. 7.
PK titrations of imported product into soybean
cotyledon mitochondria (Mit) with wild type alternative
oxidase (A) and the 2Gly mutant (B). Lane
1, precursor protein alone. Lane 2, precursor protein
incubated with mitochondria. Lanes 3 to 7 represent the import assay treated with 4, 16, 25, 50, and 100 µg/ml
PK. C, endogenous AOX detected by Western blotting.
Lane 1, no mitochondria. Lane 2, mitochondria
with no added PK. Lanes 3 to 7, mitochondria
treated with PK with concentrations indicated above. The amount
detected with mitochondria treated with 4 µg/ml of PK is set to
100%, and other values are expressed relative to this amount.
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Structure Requirements Sufficient for the Import of AOX--
As
mentioned above, in vitro translation of the AOX precursor
(and mutants) was also accompanied with a lower molecular weight product (P') below the authentic precursor protein (Fig.
8A). Furthermore, import into
soybean mitochondria always resulted in a product with a higher
mobility than that of the authentic mature (M') form, which
is never present in rat liver mitochondria imports (Fig. 8A
versus 8B). However, this band is not
PK-protected and was generated in the absence of membrane potential
(Fig. 2A, lane 4). This indicated that it was not
necessary for the P' to cross the inner mitochondrial membrane in a

-dependent manner for the generation of M'.

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Fig. 8.
Import and processing of the AOX 9-36 and
AOX 1-17. A, import into isolated soybean cotyledon
mitochondria (Mit). Symbols are as in Fig. 2. B,
import into rat liver mitochondria. C, processing with
purified spinach MPP. D, processing with rat matrix
fraction.
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The P' protein is thought to be because of the internal methionine
initiating the start of translation (i.e. methionine at position 18) and not because of an early termination product. It is
possible that this form can be imported into the mitochondria and can
be processed to the authentic mature form. To investigate this we
created the
1-17 construct (Fig. 1C). As expected in the
in vitro translation, only one band was seen with the same mobility comparable with the P' seen with the wild type AOX
translation. When import into soybean and rat liver mitochondria were
performed, this construct was imported and processed to the correct
mature form with different efficiencies of 15 and 45%, respectively
(Fig. 8, A and B, lanes 7-9). This
indicated that the C-terminal 24 amino acids could support import into
rat liver mitochondria 3 times more efficiently than in soybean.
We then created a mutant in which the presequence encompasses only the
predicted
-helical region (AOX 9-36) (Fig. 1C), and imports were performed to test if this structural feature alone was
capable of directing the AOX precursor to the mitochondria. Translation
of this construct resulted in two bands with the lower mass band
because of an internal methionine (as discussed above). The import
figure shows the efficiency of import for each product; these values
cannot be added to give an overall efficiency, as they represent the
efficiency of import from each band (Fig. 8). The efficiency of import
presented in Table I is for the upper, authentic band. Import into
soybean and rat liver mitochondria showed 20 and 40% formation of
PK-protected mature forms, respectively (Fig. 8, A and
B, lanes 4 to 6). This indicated that
the helical structure supported import twice as much in rat liver as in
soybean. The mutagenesis studies outlined above showed the requirements of positively charged residues throughout the presequence for import
into soybean mitochondria.
The processing efficiency of the
1-17 mutant was largely
unaffected, presumably as the authentic processing site was still intact. As expected, the AOX 9-36 construct was not processed because
of deletion of the authentic processing site.
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DISCUSSION |
The transit sequence of the triose-phosphate translocator, which
has the predicted ability to form an amphiphilic
-helix, has been
shown to contain mitochondrial-targeting ability (40, 41). We have
carried out this study with the authentic "passenger" protein in an
attempt to avoid any differential import with passenger proteins (23,
40-42). We have also carried out experiments with rat liver
mitochondria to compare import into another higher eukaryote.
Import of the Site-directed Mutants--
Import of the various
site-directed mutants into soybean mitochondria indicated that positive
residues throughout the presequence were important for import. In fact,
changing any single positive residue resulted in a decrease in import
by 20 to 35%. With soybean mitochondria, changing the
2 arginine or
10 arginine had a slightly greater effect than changing the positive
residues upstream (Table I). Rat liver mitochondria displayed a similar
trend with the single mutants. Surprisingly for both soybean and rat
liver mitochondria, substitution of an arginine residue at the
4
position for a glycine could not restore the import ability of the
2Gly mutant, indicating that the position of this arginine residue
was important for import in this presequence. In the import experiments
with soybean mitochondria, the
2Gly mutant gave a mature product with
a slower mobility (apparent higher Mr) and a
significant amount of imported precursor form, but the
2Gly,
4Arg
mutant produced a mature form upon import that appeared similar to wild
type AOX. This double mutant (
2Gly,
4Arg) did not restore the import
or processing efficiency in the import experiments, just the size of
the mature product obtained. In contrast, with purified spinach MPP,
the
2Gly,
4Arg mutant was processed 4 times as efficiently as the
2Gly mutant alone and restored the processed product to the same size
as the wild type AOX.
The double
2 and
10 arginine mutations had an additive effect in
both soybean and rat liver. However, the effect was greater with
soybean mitochondria where import of
2,
10Gly mutant was inhibited
by 65%, compared with 40% seen with rat liver mitochondria. Mutating
two or more positive residues at the
20,
30, and
35 positions,
however, did have a greater inhibition on import than that predicted
from the individual mutants, and this effect was much greater with rat
liver mitochondria than that with soybean. The
20Gly,
30Gly, and
the
35Gly individual mutants gave 30, 10, and 10% inhibition of
import alone in rat liver, where any two inhibited import by 80% or
greater (Table I). In soybean, a similar effect was seen, but the
inhibition of import with the double mutants was not as drastic; all
double mutants imported into soybean mitochondria with twice the
efficiency as they did into rat liver mitochondria (Table I). The
triple mutant (
20,
30,
35Gly) of these residues completely
inhibited import into both sets of mitochondria.
The conclusion from the site-directed mutagenesis study on the effect
of import into soybean mitochondria with the AOX precursor are (i)
positive residues in the C-terminal region, at the
2 and
10
position relative to the mature protein, play an important role in
import; (ii) changing the position of the positive residue at the
2
position to
4 does not restore import.
Processing of the Site-directed Mutants--
Surprisingly, the
majority of mutants had a drastic effect on processing with purified
spinach MPP. All mutants of the
2 arginine inhibited processing by
80% or greater. Additionally the processed product in the
2Gly and
2Leu mutants had a slower mobility, strongly suggesting that
processing did not take place at the correct site. Even the
4Arg
mutant alone decreased processing by 40%, although this mutant had no
inhibitory effect on import. However the
4Arg mutant could restore
processing in the
2Gly mutant, the processing of the
2Gly,
4Arg
mutant being almost the same as the
4Arg mutant alone, which was
almost 4-fold higher than the
2Gly mutant. Additionally the mature
size product obtained had a similar mobility to wild type AOX. This
suggested that the
2 arginine residue in the wild type AOX plays two
different roles, a role in import, where it is essential in the
2
position, and a role in processing, where the position can be changed
to some degree. The other mutation that could restore processing was a
7Ser mutant; 80% of the imported product in the
7Ser,
2Gly mutant was mature form compared with only approximately 35% in the presence of the
2Gly mutant. In the processing assay, the
7Ser mutant restored processing of the
2Gly mutant from 16 to 65%.
We also carried out processing studies with rat liver matrix. In
contrast to spinach MPP, processing was efficient with the majority of
the mutants. We believe that the activity detected in the rat matrix
represented true processing as (i) no significant proteolysis was
evident; (ii) the size products generated on processing with the rat
matrix fraction extract corresponded closely to that generated upon
import with rat liver mitochondria (the same trend in mobility shift of
the mature form with various mutants was seen with the processing
experiments as in the import experiments); (iii) although the majority
of mutants had no great effect on processing the double
2,
10Gly
mutant inhibited processing by 50%.
2 and/or
10 arginine residues
have previously been characterized as processing signals with rat liver
MPP (7, 12, 15).
In conclusion the effect of the various site-directed mutants on
processing with purified spinach MPP were (i) that all
2 mutants had
a drastic effect on processing; (ii) that double mutants that could not
restore import could restore processing; (iii) that even positive
residues at the
20,
30, and
35 positions of the wild type AOX
seem to play some role in processing; and (iv) that the
10 arginine
residue does not act as a cryptic processing signal with spinach MPP,
even though an
4Arg mutant apparently can.
Does an Amphiphilic
-Helix Play a Crucial Role in Plant
Mitochondrial Targeting?--
The deletion mutant
1-17 does not
contain the positive residues at
35 and
30 positions. The second
construct was a 9 to 36 mutant encompassing the predicted amphiphilic
-helix region. Import into isolated soybean mitochondria indicated
that the
1-17 mutant and the 9 to 36 mutant were imported poorly.
This indicated that an amphiphilic
-helix alone was not sufficient
to support efficient import. In contrast both these deletion mutants
displayed significant import into rat liver mitochondria, with at least 45% efficiency of the wild type AOX. Although the
1-17 mutant had
the first portion of the potential amphiphilic
-helix deleted, it
was still predicted to contain some amphiphilic helical character. Therefore an
-helical amphiphilic element alone cannot support efficient import with soybean mitochondria, but it can with rat liver mitochondria.
Determinants of Import and Processing in Different
Species--
This study indicates that positives residues throughout
the presequence were required for import. This is further supported by
the fact that the amphiphilic
-helix alone could not support efficient import. Although a similar trend was seen with rat liver mitochondria, it was evident that the N-terminal residues play a more
prominent role in import than they did with soybean mitochondria. This
can be seen with the deletion constructs where the predicted amphiphilic
-helix region supported import into rat liver
mitochondria. Purified spinach MPP had a stringent requirement for
processing, with all
2 position mutations having a drastic effect.
The
10 arginine could not act as a cryptic processing signal,
although a
4Arg mutant and a
7Ser mutant could restore processing.
A direct comparison with rat MPP was not possible, as we used matrix extract; however it was apparent that the processing by this extract was not affected to the same degree by the mutations in AOX. It was
apparent from the import assays that the processing specificity differed between the two organisms, as the products obtained upon import into rat liver mitochondria differed in size to those obtained upon import into soybean mitochondria (Table I). The difference in
efficiency in processing the various mutants and the size products obtained in the import assays confirms that organism differences exist
in processing that cannot be attributed to the difference between a
purified MPP and a matrix extract.
With the plant processing studies there was a strong discrepancy
between the inhibition of cleavage obtained with the import assays and
that obtained with purified MPP. In the import assays with soybean
mitochondria, greater processing of the various mutant precursor
proteins was seen compared with the processing studies. Processing
could be restored without the restoration of import ability, indicating
that the affect on both processes may be different. However as more
efficient processing was seen in the import assays, it suggests that
(i) either additional isoforms of MPP or other processing peptidases
are present in plant mitochondria or (ii) that additional components
help MPP to process precursor proteins upon import into intact
mitochondria. In the first instance, we have reported a soluble
specific peptidase in both spinach and soybean that can process several
mitochondrial precursor proteins (43). It is possible that the
requirement for processing with the membrane-bound MPP is more strict,
as it is incorporated into a large multi-subunit complex, and that
another less complex, soluble peptidase does not have as strict
requirements. The results from processing with the rat liver matrix are
in agreement with this. The hypothesis that the membrane-bound MPP
represents the primitive situation and was moved to the matrix to
increase efficiency or range (44) is also in agreement with the results
obtained here. The second possibility is that additional factors such
as chaperones may bind to the presequence and present it in a proper conformation for processing to MPP. Again, processing with the rat
liver matrix, which may contain these factors, supports this hypothesis. However, as purified MPP processes the wild type AOX precursor efficiently in the absence of any added factors, it is
difficult to imagine that the single amino acid change affects binding
of a chaperone in the translation lysate to the extent that processing
is completely inhibited, but import, which has been shown to require
several chaperones, is much less affected (4).
The other important conclusion from this study is that an amphiphilic
-helix alone is not the only requirement for plant mitochondrial
import. Although a predicted helical region can support import with low
efficiency, it is not an absolute requirement, nor it is sufficient for
efficient import. Therefore, it cannot be presumed that precursor
protein from other organelles, such as chloroplasts, which have the
ability to form an amphiphilic helix, will necessarily have to be
specifically excluded from importing into plant mitochondria. The
question of how import specificity is maintained in plants has been
raised many times. It is important to note that specificity may be the
result of many steps. Here we have shown that the requirement for
import does not solely depends on an amphiphilic
-helix in plants.
Other steps such as binding to specific chaperone factors may also be involved in specificity, so that overall a number of discriminating steps maintain specificity (3). No single step alone may dictate 100%
specificity but may combine so that the overall specificity of import
is very high.