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INTRODUCTION |
The majority of mitochondrial proteins are encoded by nuclear DNA
and are synthesized on cytosolic ribosomes as precursor proteins. As a
consequence of their cellular location during synthesis, mitochondrial
proteins must be specifically targeted to the organelle and be
imported. The N-terminal portion of these precursor proteins possesses
a leader sequence that contains sufficient information to be recognized
by the mitochondrial import apparatus, leading to import into
mitochondria. Mitochondrial presequences do not share any primary
sequence identity, but they do show a statistical bias of positively
charged amino acid residues, provided mostly through arginine residues;
very few presequences contain negatively charged amino acids (1). The
positive charges in the presequences have the potential to interact
with the negatively charged surface of mitochondrial membranes (2), the
mitochondrial import receptor (3), and the mitochondrial processing
peptidase (4, 5). Mitochondrial leader sequences also share an ability
to form an amphiphilic,
-helix (1, 6, 7). Numerous two-dimensional NMR studies of peptides corresponding to mitochondrial leaders show
that these leaders do form an amphiphilic helix and have provided more
structural detail in showing different structural motifs between leader
sequences (8-13). Thus, there is an apparent charge and structural
component needed to target precursor proteins to the mitochondria.
Mutations designed to study the role of positive charge, structure, or
hydrophobicity of leader sequences have been performed under in
vitro conditions (14-19). In this setting, numerous cytosolic components that assist the import process may be absent or in limited
quantity due to the conditions of the assay. Due to the membrane
binding property of leaders, in an in vivo setting the leader sequence may additionally need to discriminate among the various
organelles (6). Although there is an apparent preference of the leader
for the lipid composition found in mitochondria, potential nonspecific
interactions with membrane surfaces of other organelles may hinder the
process of locating mitochondria, especially in mutants designed to
study the import properties of the signal. Additionally, the total
number of positive charges of the leader may be more important in
vivo although in vitro experiments have shown that
numerous positive charges can be removed without apparent interference
with import. Thus, out of the diversity among all mitochondrial leader
sequences, positive charges, structural motif, and the hydrophobicity
of the leader may be balanced to minimize nonspecific membrane binding
that may not be detected in vitro (15).
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EXPERIMENTAL PROCEDURES |
Materials--
The pEGFP-N1 vector and
anti-GFP1 monoclonal antibody
were purchased from CLONTECH; Dulbecco's modified
Eagle's medium, calf serum, and trypsin-EDTA were obtained from Life
Technologies, Inc.; the antibiotics and HEPES were purchased from
Sigma; paraformaldehyde was from Aldrich; the SuperFect Transfection
Reagent was obtained from QIAGEN; the TNT coupled transcription and
translation system and the pGEM-7Zf(+) vector were obtained from
Promega; [35S] methionine and [
-35S]dATP
were purchased from Amersham Pharmacia Biotech; the SequiTherm EXCEL
DNA Sequencing kit was from Epicentre Technologies Company; the
alkaline phosphatase-conjugated goat anti-mouse IgG was from Bio-Rad;
nitrocellulose membranes were from Schleicher and Schuell; and the
Complete protease inhibitor mixture was from Roche Molecular Biochemicals. SuperScript II RNaseH
reverse transcriptase
was obtained from Life Technologies; random hexamers were obtained from
Promega; and the pET19b vector was from Novagen.
Cells--
HeLa cells were provided by Dr. Steven Broyles
(Department of Biochemistry, Purdue University). HeLa cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
calf serum, 100 units penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37 °C under an atmosphere of 5%
CO2 and 95% air.
Construction of Plasmids--
Either the native rat pALDH
presequence plus the first 23 amino acid residues from the mature
region (SP1) or the native ALDH presequence plus the mitochondrial
processing peptidase recognition site (SP2) were fused to the N
terminus of EGFP (a variant of native GFP) by the splicing by overlap
extension PCR method (20-22). PCR primers were designed to clone the
products into either pEGFP-N1 or pGEM-7Zf(+). PCR products were
digested by BglII/NotI and subcloned into the
pEGFP-N1 vector or digested by ApaI/XbaI and
subcloned into pGEM-7Zf(+). The native leader, R3Q, R10Q, R3Q(10Q),
pALDH-RGP, R3Q(10Q)-RGP, R14Q, R17Q, R14Q(R17Q), S7E, S7E(R11E) and
S7E(R14E) were made as SP1-EGFP constructs. The native leader, R3Q,
R10Q, and pALDH-RGP were also made as the SP2-EGFP construct.
Cloning of Rat Microsomal Aldehyde Dehydrogenase 3 cDNA by
Reverse Transcription-PCR--
The cDNA for rat microsomal ALDH3
was cloned by reverse transcription-PCR based on the published sequence
(23). Rat liver total RNA was isolated by Dr. Yie Lane Chen, formerly
of our laboratory. Total RNA (about 5 µg) was used for the synthesis
of the first strand of cDNA by following the protocol from Life
Technologies, Inc. Random hexamers were used for the reverse
transcription reaction, which was performed at 42 °C for 1 h.
The first strand of cDNA was then used as the template for PCR. Two
specific primers corresponding to the 5' (contains a NdeI
site) and 3' (contains a BamHI site) ends of the rat
microsomal ALDH3 cDNA sequence were used to amplify the full length
of the rat microsomal ALDH3.
The reverse transcription-PCR products were digested with
NdeI and BamHI and then cloned into the pET19b
vector. The amplified cDNA was confirmed by sequencing. The
constructs, SP1-EGFP, EGFP-ER, and SP1-EGFP-ER (Fig.
1), were constructed by the splicing by overlap extension PCR method (20-22) and cloned into the pEGFP-N1 vector. The SP1 R3Q, R3Q/R10Q, and linker-deleted (RGP from position 11 to 13 of the rat pALDH2 leader sequence was deleted) (
RGP)-EGFP-ER were also constructed. All constructs were confirmed by sequencing the
pALDH leader sequence, the N- and C-terminal EGFP fusion region, and
the the ER-targeting signal sequence.

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Fig. 1.
The N-terminal amino acid sequence of the
SP1- and SP2-EGFP constructs. A, the leader sequence of
the precursor ALDH is shown in boldface type,
while those of the mature portion of ALDH are shown in italics. The
processing site is indicated by the triangle above the
signal sequence. The hatched blocks below the leader show the two
helical segments of the leader between the flexible RGP linker. The
molecular weights of pALDH-EGFP, ALDH-EGFP, and EGFP are indicated by
the arrows and are rounded to whole numbers. B,
EGFP-ER and SP1-EGFP-ER constructs, used to test for co-translational
import, are illustrated to show the sequence of the 40 amino acids from
the rat microsomal ALDH3 ER signal.
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Direct Observation of EGFP Fluorescence in Cultured
Cells--
HeLa cells were plated on coverslips in six-well plates in
3 ml of growth medium (Dulbecco's modified Eagle's medium plus 10%
calf serum) the day before transfection. The cells were 40-80% confluent on the day of transfection. The cells were transfected with
2.5 µg of plasmid DNA at 37 °C for 2-3 h using the SuperFect Transfection Reagent from QIAGEN. Between 10 and 72 h after
transfection, the cells were washed twice with phosphate-buffered
saline, fixed on the coverslips with 4% paraformaldehyde in PBS at
room temperature for 30 min, and washed twice with phosphate-buffered
saline. Cells were observed with an Olympus BX60 fluorescence microscope.
Immunoblot Analysis--
Culture and transfection of HeLa cells
was performed as described above, except that 100-mm dishes and 10 µg
of plasmids were used for the transfection. The whole cell extracts
were separated by SDS-polyacrylamide gel electrophoresis and
electrotransferred onto a nitrocellulose membrane. Monoclonal antibody
against Aequoria victoria GFP was used as the primary
antibody; the alkaline phosphatase-conjugated goat anti-mouse IgG was
used as the second antibody.
Isolation of Mitochondria from HeLa Cells Expressing either the
Native or the Linker-deleted SP1-EGFP--
Culture and transfection of
HeLa cells were performed as described above, except that 150-mm dishes
and 20 µg of plasmid DNA were used for the transfection. 48 h
after transfection, the cells were washed twice with ice-cold
phosphate-buffered saline, harvested, and then resuspended in ice-cold
0.32 M sucrose and 5 mM HEPES, pH 7.4. The
subcellular fractionation was performed as described (24-27). The
cells were homogenized by using a Polytron homogenizer for 30 s at
10,500 rpm. The homogenate was centrifuged with an SS34 rotor at 3,000 rpm for 15 min to pellet the unbroken cells and the nucleus. The
supernatant was centrifuged with an SS34 rotor at 10,000 rpm for 20 min
to obtain the mitochondria-rich fraction.
Degradation of in Vitro Translated Proteins by the HeLa Cell
Cytosol Extracts--
HeLa cells were washed twice with ice-cold
phosphate-buffered saline, harvested, and then resuspended in ice-cold
0.32 M sucrose and 5 mM HEPES, pH 7.4. The
cells were homogenized by using a Polytron homogenizer for 30 s at
10,500 rpm. To remove the unbroken cells, the nucleus, and the
mitochondria, the homogenates were centrifuged with an SS34 rotor at
10,000 rpm for 20 min. The cytosolic supernatant was removed,
aliquoted, and saved at
70 °C. Radiolabeled SP1-EGFP,
SP1(R3Q)-EGFP, and EGFP were produced by the TNT coupled transcription
and translation with the pGEM7Zf(+) constructs in the presence of
[35S]methionine following the manufacturer's
instruction. The in vitro translated proteins were mixed
with the HeLa cell cytosol extracts in equal volume at room temperature
for fixed periods of time. EGFP was added in the both the SP1-EGFP and
SP1-(R3Q)-EGFP incubations to test for protease resistance over the
time points used. An aliquot was withdrawn and added to
SDS-polyacrylamide gel electrophoresis treatment buffer, which
contained protease inhibitors. The mixture was heated to 95 °C for 5 min. The samples were analyzed on a 12.5% SDS-polyacrylamide gel, and
labeled proteins were visualized by Bio-Rad phosphor imaging.
Subcellular Fractionation of HeLa Cells Expressing either EGFP-ER
or SP1-EGFP-ER--
Culture and transfection of HeLa cells were
performed as described above, except that 150-mm dishes and 20 µg of
plasmid DNA were used for the transfection. HeLa cells were transfected
by plasmids expressing either EGFP-ER or SP1-EGFP-ER. Between 48 and
72 h after transfection, the cells were washed twice with ice-cold
phosphate-buffered saline, harvested, and then resuspended in ice-cold
5 mM HEPES, pH 7.4, 0.32 M sucrose. The
subcellular fractionation was performed as described (24-27). The
cells were homogenized by using a Polytron homogenizer for 30 s at
10,500 rpm. The homogenate was centrifuged with an SS34 rotor at 3,000 rpm for 15 min; the pellet (P1) was the unbroken cells and the nucleus.
The supernatant was centrifuged with an SS34 rotor at 10,000 rpm for 20 min; the pellet (P2) was the mitochondria-rich fraction. The
supernatant was centrifuged with an TLA100.3 rotor at 100,000 rpm for
90 min; the pellet (P3) was the microsome-rich fraction, and the final
supernatant was the cytosol from HeLa cells. The different fractions
(homogenate, P1, P2, P3, and supernatant) were analyzed by a Western
blot. Succinate-cytochrome c dehydrogenase was used as the
mitochondria marker enzyme, and the enzyme assay was performed as
described (28). Lactate dehydrogenase was used as the cytosol marker
enzyme, and the enzyme assay was performed as described (29). NADPH
cytochrome P-450 reductase was used as the ER marker enzyme, and the
enzyme assay was performed as described (30, 31).
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RESULTS |
Detection of Fluorescence in HeLa Cells Transfected with EGFP and
the SP1 Constructs of pALDH-EGFP--
Cells that were transiently
expressing EGFP displayed an observable fluorescence that was
distributed throughout the whole cell, including the nucleus (Fig.
2), as shown by others (32). Specific
targeting of EGFP to any subcellular organelle was not observed. Cells,
which expressed the EGFP protein with an attached pALDH mitochondrial
leader sequence, displayed fluorescence localized to mitochondria.
Rhodamine 123, a mitochondrial specific stain was used to visually
confirm EGFP targeting to the organelle (data not shown). Portions of
some cells that expressed pALDH-EGFP appeared to have cytosolic
fluorescence. This was due to the fluorescence of mitochondria above
and below the focal plane of the cell. To further verify that the
pALDH-EGFP protein was localized to the mitochondria, cells that were
transiently expressing pALDH-EGFP were analyzed by a Western blot to
determine the molecular weight of the expressed protein. As shown in
Fig. 3, a band of 30 kDa was observed,
consistent with the imported and processed 32-kDa pALDH-EGFP chimera.
The fluorescence microscopy and Western blot results from the transient
expression of the EGFP and pALDH-EGFP demonstrated that the leader
sequence of pALDH could target EGFP into the mitochondria.

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Fig. 2.
Fluorescent microscopy of the HeLa cells
transiently expressing SP1 and SP2 forms of the pALDH-EGFP
chimeras. HeLa cells were cultured on coverslips and transfected
with 2.5 µg of plasmid DNA for the native and mutant forms of the
SP1-and SP2-EGFP constructs (first 16 photographs). The expressed EGFP, without a mitochondrial
signal, was distributed throughout the cell, including the nucleus. The
SP1-EGFP localized to mitochondria, which are seen either as small dots
or long cylinders. The absence of fluorescence in the cytosol and
nucleus is indicative of efficient import. The intracellular location
of the EGFP-ER and SP1-EGFP-ER proteins are shown in the
last four photos. The EGFP-ER was
localized to the ER that appeared as a lacy network of membranes. The
R3Q(R10Q)-EGFP-ER and ( RGP)-EGFP-ER constructs do not efficiently
target EGFP-ER to the mitochondria. The extent of ER localization is
more for R3Q(R10Q)- than ( RGP)-EGFP-ER as mitochondrial fluorescence
were not readily observed.
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Fig. 3.
Western blot of HeLa cells transiently
expressing SP1 and SP2 constructs. The whole cell extracts of HeLa
cells expressing the native and mutated SP1-EGFP (A) or
SP2-GFP chimeras (B) were separated by SDS-polyacrylamide
gel electrophoresis and identified by a Western blot. The
nontransfected HeLa cells were used as the negative control. The HeLa
cells expressing EGFP were used as the positive control. A,
the 30-kDa band corresponding to SP1-EGFP is the expected molecular
weight of the imported and processed protein. Absence of a detectable
precursor band (32 kDa) is indicative of efficient or near complete
import of the expressed protein. Lane 1, EGFP;
lane 2, HeLa cells (control); lane
3, SDS marker (34 kDa); lane 4,
SP1-EGFP; lane 5, SP1(R3Q)-EGFP; lane
6, SP1(R10Q)-EGFP; lane 7,
SP1(R3/10Q)-EGFP; lane 8, SP1( RGP)-EGFP;
lane 9, SP1( RGP, R3/10Q)-EGFP; lane
10, SP1(S7E)-EGFP; lane 11,
SP1(S7E/R11E)-EGFP; lane 12, SP1(S7E/R14E)-EGFP.
B, a 28-kDa band is the expected molecular weight of the
imported and processed SP2-EGFP protein. Lane 1,
HeLa cells (control); lane 2, SDS marker (34 kDa); lane 3, SP2-EGFP (30 kDa); lane
4, SP2-(R3Q)-EGFP; lane 5,
SP2(R10Q)-EGFP; lane 6, SP2( RGP)-EGFP;
lane 7, EGFP (28 kDa).
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It was shown that only one positive charge in the first helix of the
leader was required for import in vitro, since the single point mutations of R3Q and R10Q all imported to native levels, while
the R3Q(R10Q) mutant was severely impaired at import (5). As shown in
Fig. 2, under in vivo conditions, R3Q and R10Q were capable
of targeting EGFP to mitochondria, but cytosolic and nuclear fluorescence were observed, indicating that not all of the precursor proteins were imported. The R3Q and R10Q mutants were not imported at
similar levels, since the cytosolic fluorescence of EGFP appeared to be
greater with the R3Q mutant. The double mutant R3Q(R10Q) was the most
impaired of the three mutants, since fluorescence was mostly observed
in the cytosol.
The in vivo results with the Arg to Gln mutations in the
first helix were consistent with the in vitro results.
However, two unexpected properties emerged in subsequent in
vivo experiments. First, there was no apparent necessity for
positive charges in the C-helix of the native leader. In
vitro, the two arginines in the C-helix of the native leader could
be substituted with glutamines as single point mutations with no effect
on import, but substituting both positive charges with glutamine
resulted in a 2-fold reduction in import. However, in vivo,
substitution of both C-helix arginines with glutamine did not effect
import. The second unexpected result was that the linker-deleted leader was imported poorly in vivo, whereas it was found to import
better than the native leader in vitro. Removing the
helix-breaking residues of RGP in the sequence in the native leader
allows the leader to form a continuous helix. This property has been
shown to consistently recover in vitro import in mutations
of the native leader that reduced or eliminated import. In the in
vitro experiments, the R3Q(R10Q) mutation in the native leader
essentially eliminated import, but the R3Q(R10Q)-RGP leader restored
import to approximately 50% of native level. In the in vivo
experiments, the pALDH-RGP imported less than native as shown in Fig.
2, and the R3Q(R10Q)-RGP leader was essentially nonfunctional.
The import properties of the pALDH leader were shown to be surprisingly
tolerant to the mutagenic introduction of negatively charged amino
acids, provided that the charge of first helix of the leader remained
positive (33). As shown in Fig. 2, the import of the S7E was less than
native level, and the S7E(R11E) mutant did not appear to import,
consistent with the in vitro results. Although the first
helix of the leader remained positive in the S7E mutant, the loss of
import in the S7E(R11E) mutant was probably due to the negative charge
being placed adjacent to the first helix. In contrast to S7E(R11E), the
S7E(R14E) was imported in vivo. The S7E(R14E) mutant
surpassed the ability of the native leader to import in
vitro, but there appears to be only a slight reduction in import
from what was observed with the S7E mutant in vivo. The
S7E(R14E) mutant does not have the additional negative charge adjacent
to the first helix to influence the apparent critical positive charge
in the first helix. The results of these in vivo experiments
reveal that positive charges necessary for import are identical to
those necessary for locating the mitochondria and do not require all
five positive charges.
Subcellular Localization as Determined by Molecular Weight of the
EGFP Constructs--
After the precursor proteins are imported into
mitochondria, many of the leader sequences are proteolytically removed
by a matrix space peptidase. The pALDH leader sequence contains a
terminal -RLLS- sequence, which serves as the recognition site for the matrix-processing protein (5). As a direct measure of import in
transfected HeLa cells, we harvested cells expressing the various constructs of EGFP and analyzed them by a Western blot using monoclonal antibodies against EGFP. For the majority of the precursor proteins used in this in vivo study, a 30-kDa protein would be the
expected molecular mass of an imported and processed protein. The R17Q and R14Q(R17Q) mutants have lost the peptidase recognition motif, and
these leaders were not proteolytically removed by the peptidase in rat
liver mitochondria (5). Additionally, the pALDH-RGP and R3Q(R10Q)-RGP
leaders were also nonprocessable leaders, although they contain the
recognition site (5, 11). These nonprocessable proteins should retain
the leader after import and display a molecular mass of 32 kDa.
As shown in Fig. 3A (lane 4), the
native pALDH-EGFP leader resolved to the predicted, processed mass of
30 kDa. No observable precursor band of 32 kDa was observed, suggesting
that little, if any, of the precursor protein remained in the cytosol.
If a processable mutant leader was less capable of importing EGFP, we
would expect to observe a 32-kDa band and a 30-kDa band in the Western
blot, corresponding to the nonimported precursor and the imported and
processed protein, respectively. Mutant leader sequences that reduced
import displayed the expected 30-kDa band, indicative of import and
processing, but also displayed an unexpected 28-kDa band, Fig. 3. This
28-kDa band corresponds to the molecular mass of EGFP, which apparently
had lost the entire N-terminal fusion sequence due to nonspecific
proteolysis. For many of the mutant leader sequences, the intensity of
the 28-kDa band found in the Western blot paralleled the pattern of
cytosolic fluorescence shown in Fig. 2. Good import of the native
protein resulted in a single 30-kDa band, and less efficient import
resulted in both a 30- and 28-kDa band. Total loss of import resulted
in a single 28-kDa band.
An unexpected band was observed for the nonprocessed leaders,
pALDH-RGP, R17Q, and R14Q(R17Q). Data are shown only for pALDH-RGP in
Fig. 3A (lane 8). These proteins all
displayed a 30-kDa band, suggesting that they were processed. This was
not observed in our previous in vitro import studies using
rat liver mitochondria or processing studies with purified
mitochondrial processing peptidase (11, 34). A Western blot of isolated
mitochondria from HeLa cells expressing either the native or
linker-deleted EGFP constructs displayed a 30-kDa band for both
proteins (data not shown). Based on the current knowledge of the
processing motifs of matrix space processing enzymes, we cannot offer
an explanation for the removal of the leaders in some of the mutants
used in these experiments. However, independent of how the pALDH-RGP
leader could be processed, the experimental evidence does point to a
mitochondrial proteolysis as the source of the 30-kDa bands observed in
these Western blots.
N-terminal Cytosolic Degradation of pALDH-EGFP-expressed
Proteins--
The observation of cytosolic fluorescence in the
microscopy experiments with the import-impaired constructs indicates
that EGFP could properly fold and was resistant to proteolysis. To discern whether or not the mutations made in the leader were
susceptible to proteolysis, thereby corrupting the signal, the native
leader and the R3Q mutant were exposed to HeLa cell cytosol extracts as
described under "Experimental Procedures." As shown in Fig. 4, both pALDH-EGFP and the R3Q-EGFP
mutant were rapidly reduced to a slightly smaller molecular weight.
After 30 min, all of the leader sequence and the joining amino acids
were degraded, but the EGFPs were still present and resistant to
protease digestion (data not shown). The susceptibility to N-terminal
protease digestion of the native and the R3Q mutant were similar,
suggesting that the native leader could be just as likely to be rapidly
proteolyzed in vivo as it was in vitro.

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Fig. 4.
HeLa cell cytosolic proteolysis of pALDH and
R3Q in SP1 form. Shown are the time course of the proteolysis of
SP1-EGFP (A) and (R3Q)-EGFP (B). The individual
lanes of the SDS-polyacrylamide gel electrophoresis are presented as
densitometric trace with the peaks corresponding to the bands appearing
in the gel. Both SP1-EGFP and SP1-(R3Q)-EGFP show noticeable broadening
at 30, 60, and 90 s along with splitting of the SP1-EGFP and
SP1-(R3Q)-EGFP peaks indicative of proteolysis. EGFP remained present
and resistant to proteolysis even after 30 min (data not shown).
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SP2 Constructs of pALDH-EGFP and Mutant pALDH-EGFP--
The
susceptibility of both the native leader sequence and the R3Q mutant to
proteolysis with HeLa cell extracts were similar but did not clearly
indicate whether proteolysis occurred within the leader or within the
amino acids of the mature portion of pALDH. To address this issue, a
new chimeric of EGFP (SP2) was made in which six amino acids were used
to bridge the leader sequence to EGFP as illustrated in Fig. 1. As
shown in Fig. 2, the cellular fluorescence of the native leader, R3Q,
and the linker-deleted mutants in the SP2-EGFP constructs were
indistinguishable from the SP1 constructs.
In the Western blot using the SP2 constructs, the expected molecular
mass of the imported and processed leader is 28 kDa. We would expect to
see only a 28-kDa band and little, if any, 30-kDa precursor band if
they were imported well. For a protein that was not imported and was
retained in the cytosol, the molecular mass would be 30 kDa if the
leader were resistant to proteolysis. The Western blot from cells
transiently expressing the native leader, R3Q, and the linker-deleted
leader in SP2 form all displayed only a single 28-kDa band (Fig. 3).
This was the expected result for the native SP2-EGFP protein. Finding
no 30-kDa precursor band for the R3Q and linker-deleted SP2-EGFP
proteins, which were imported poorly, suggests that the leader sequence
was susceptible to cytosolic degradation. The import results with the
SP1- and SP2-EGFP constructs indicate that import needed to be rapid
enough to protect the leader from cytosolic proteolysis.
Co-translational Versus Post-translational Import--
The rapid
proteolysis of the leader, in addition to the protease-resistant
structure of EGFP, suggested that in vivo import needed to
be on the same time scale as protein synthesis. Using the same in
vivo system, we explored the hypothesis that pALDH-EGFP may have
been imported in a co-translational manner. A C-terminal ER signal was
fused to the C terminus of the SP1 pALDH-EGFP construct in order to
test if import was occurring before the protein was free from the
ribosome. The C-terminal ER targeting sequence was from rat microsomal
ALDH3, which is composed of the C-terminal 35 amino acids (25). These
C-terminal 35 amino acids contain both the transmembrane domain and the
ER targeting signal. If import were a post-translational event, the
EGFP passenger would be found in both mitochondria and ER. However, if
it were a co-translational event, then, despite having two attached
targeting signals, the EGFP passenger would only be found in the
mitochondria. The pALDH-EGFP-ER and EGFP-ER constructs are illustrated
in Fig. 1.
In the cells expressing EGFP-ER, the fluorescence was localized in what
appeared to be ER, which was observed as a lacy network of membranes
surrounding the nucleus. The ER localization of the expressed EGFP-ER
proteins was confirmed by subcellular fractionation. In cells
expressing the construct with both signals, SP1-EGFP-ER, the EGFP
fluorescence was found only in the mitochondria with no detectable ER
localization (Fig. 2). The cellular localization of the SP1-EGFP-ER
protein in mitochondria was consistent with a co-translational
mechanism, since no detectable ER or cytosolic localization was
observed with the dual signal protein.
To examine whether the C-terminal ER signal was functional in the
SP1-EGFP-ER construct, the ER targeting signal was fused to the less
efficient mitochondrial signals of SP1(R3Q), SP1(R3Q/R10Q), and
SP1(
RGP)-EGFP. Data are not shown for SP1(R3Q)-EGFP. When these
chimeras were expressed in HeLa cells, the EGFP fluorescence was
localized to both mitochondria and ER (Fig. 2). This verified that the
ER signal would function when both signals were presented on the same protein.
Subcellular Localization as Determined by Molecular Weight of the
Different ER Targeting Constructs--
The molecular weight difference
between the precursor and mature forms was used to indicate whether or
not the expressed chimeras were imported. The molecular mass of the
precursor SP1-EGFP-ER is 36 kDa, and that of the imported and processed
protein is 34 kDa. A Western blot of cells expressing SP1-EGFP-ER
showed only a 34-kDa band consistent with mitochondrial import (Fig.
5). Western blotting of cells expressing
the 32-kDa EGFP-ER protein showed a single band of 32 kDa, consistent
with ER targeting. This data is in agreement with the fluorescent
images that showed that the SP1-EGFP-ER construct was imported
exclusively into mitochondria and further supports a co-translation
import mechanism.

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Fig. 5.
Determination the molecular weight of the
SP1-EGFP, EGFP-ER, and SP1-EGFP-ER chimeras. Western blot of whole
cell extracts of HeLa cells expressing the SP1-EGFP, EGFP-ER,
SP1-EGFP-ER, and EGFP. The nontransfected HeLa cells were used as the
negative control, and the HeLa cells expressing EGFP was used as the
positive control. The 34-kDa molecular weight marker is not shown.
Lane 1, HeLa cells (control); lane
2, SP1-EGFP (30 kDa); lane 3, EGFP-ER
(32 kDa); lane 4, SP1-EGFP-ER (34 kDa);
lane 5, EGFP (28 kDa).
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Subcellular Fractionation of HeLa Cells Expressing either EGFP-ER
or SP1-EGFP-ER--
To confirm that SP1-EGFP-ER was not localized on
the ER membrane, the intracellular distributions of EGFP-ER or
SP1-EGFP-ER were analyzed by subcellular fractionation. The separation
of ER membranes from mitochondria was confirmed by assaying two typical marker enzymes, NADPH-cytochrome P450 reductase, which has been shown
to be an integral ER membrane protein (35), and a mitochondrial marker,
succinate-cytochrome c reductase (28). The purity of the
fractions was higher than 70%. The whole cell extracts and different
organelle fractions from HeLa cells expressing either EGFP-ER or
SP1-EGFP-ER were assayed by a Western blot (Fig.
6). From HeLa cells expressing the
EGFP-ER, a strong band was found in the ER-rich fraction (P3), and a
faint band was observed in the mitochondria-rich fraction (P2). This
was most likely due to some fragments of the ER membrane pelleting with
the mitochondrial fraction. Western blotting of HeLa cells expressing
SP1-EGFP-ER displayed strong bands associated with the P2 fraction and
no observable band in the P3 fraction. These results were consistent with the fluorescence microscopy observation that SP1-EGFP-ER was in
the mitochondria and not associated with the ER. All of these results
are consistent with co-translational mitochondrial import.

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Fig. 6.
The intracellular distribution of EGFP-ER and
SP1-EGFP-ER expressed in HeLa cells. Subcellular fractions (P1
(nuclei and unbroken cells), P2 (mitochondria-rich fraction), P3
(microsome-rich fraction), and supernatant (cytosol fraction)) were
isolated from cell homogenates as described under "Experimental
Procedures." The samples from each fraction were assayed by a Western
blot. The localization of the two proteins is consistent with the
microscopy experiments shown in Fig. 2. Lane 1,
HeLa cells (control); lane 2, prestained SDS
marker (34 kDa); lane 3, EGFP-ER, homogenate;
lane 4, EGFP-ER, P1; lane
5, EGFP-ER, P2; lane 6, EGFP-ER, P3;
lane 7, EGFP-ER, supernatant; lane
8, SP1-EGFP-ER, homogenate (34 kDa); lane
9, SP1-EGFP-ER, P1; lane 10,
SP1-EGFP-ER, P2; lane 11, SP1-EGFP-ER, P3;
lane 12, SP1-EGFP-ER, supernatant.
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DISCUSSION |
The basic notion of how pALDH was imported into mitochondria came
from mutagenic investigations and the use of model peptides for
synthetic membrane binding and two-dimensional NMR structural studies.
From these studies, the two helices of the pALDH leader were thought to
have different roles in the import process. The N-helix was proposed to
be responsible for targeting to the receptor, while the C-helix was
more responsible for binding and maintaining a stable N-terminal helix
(36, 37). Binding studies showed that the C-terminal helix was more
protected from amide exchange when bound to synthetic membranes, and
mutations involving the N-terminal charges did not reduce the ability
of the peptides to bind membranes as it did in the C-helix (5, 37).
Thus, it was modeled that the C-helix may bind the outer membrane and allow the N-helix to search for a receptor in two-dimensional space.
The positive charges in the first helix of the leader are essential for
both import and mitochondrial targeting in vivo, and there
is no apparent necessity for positive charges in the C-terminal helix
of the leader. Thus, the proposed membrane-binding model may not be
valid in vivo for pALDH. Surprisingly, and unexplainably, improving the structural properties of the leader through the linker-deleted leader reduced import efficiency and would not compensate for a reduction in positive charge as it consistently did
in vitro (5, 33).
What became apparent throughout this study was that mutant leaders that
resulted in reduced import efficiency were proteolyzed by cytosolic
enzymes. Thus, import needed to be sufficiently rapid to avoid
degradation of the signal. The remarkable resistance of EGFP to
cytosolic proteolysis suggests that it forms a stable tertiary
structure. Mitochondrial proteins must remain unfolded in the cytosol
in order to be imported. The EGFP passenger protein, however, will fold
regardless of whether or not it is successfully targeted into the
mitochondria. This is corroborated by a recent study, which has
demonstrated efficient autonomous folding of GFP as well as
chaperonin-mediated folding (38). This suggests that if pALDH-EGFP were
to be released into the cytosol for post-translational import, folding
would occur and would prevent import. Conversely, if protein synthesis
were occurring simultaneously with import, the leader would be
protected from proteolytic degradation, and the EGFP passenger protein
would not fold. The co-translational import of pALDH is further evident
from the import results obtained with a C-terminal ER signal attached
to the pALDH-EGFP protein that was not targeted to the ER. In the dual
signal construct of pALDH-EGFP-ER, the combination of rapid folding and
stability of EGFP, in addition to the alternative targeting signal that did not target the protein to the organelle ER, suggests that post-translational import of pALDH-EGFP is improbable.
Whether or not mitochondrial import can be co-translational is
controversial. Evidence supporting post-translational and
co-translational routes exist, but no direct in vivo
evidence proves which mechanism exists. This discrepancy is potentially
due to the fact that both routes of import are possible, but the import
studies have largely focused on one pathway. It is generally accepted
that mitochondrial import occurs post-translationally (39-50).
In vivo evidence exists for a post-translational mechanism,
since carbonyl cyanide m-chlorophenylhydrazone-treated cells
accumulated F1-
-ATPase in the cytosol and were then imported after
reestablishing potential (46). However, this same experiment with
cytochrome c1 demonstrated the opposite results
and also showed that the protein was proteolized if it accumulated in
the cytosol. It is clear that a post-translation route has been
established, and numerous cytosolic factors such as mitochondrial
stimulating factors and heat shock proteins assist the precursor
protein until import is achieved. However, numerous in vitro
and in vivo studies suggest that a co-translational import
mechanism might actually be required for some precursor proteins
(51-60). Ribosomal association with mitochondria has been observed in
yeast (51-53), and recently, it has been reported that ribosomes could
stably bind to purified rat liver mitochondria (61), which would be
consistent with a co-translational pathway. The mechanism by which a
leader is recognized early in protein synthesis and potentially routed
to either pathway remains to be discovered. It is clear that for a
leader to import in vivo by a post-translational mechanism, resistance of the leader sequence to protease degradation is required, and this requirement is not met by all leader sequences or the mature
portion of the protein. This study, in combination with studies from
other laboratories, provides strong evidence for a co-translational
mechanism coexisting with the established post-translational mechanism.