From the
The in vivo import of liver mitochondrial aldehyde
dehydrogenase was investigated in yeast by constructing fusion proteins
between its leader sequence and
It has been proposed that the leader sequences which allow
precursor proteins to be transported into mitochondria contain
amphilpathic
helices(1, 2, 3, 4, 5) . The
structure of three processed and three nonprocessed leaders sequences
have been determined by two-dimensional
NMR(6, 7, 8, 9, 10) . All are
composed of an N-terminal amphilphilic helix, supporting the early
prediction. The leader sequence for rat liver mitochondrial aldehyde
dehydrogenase (ALDH)
Investigators have often used yeast to
study
import(12, 13, 14, 15, 16) .
Leader sequences were fused to carrier proteins in order to ascertain
residues in the leader important for import. A common carrier protein
employed is
In frame fusions between ALDH and lacZ were isolated after transformation into E. coli strain MC1000 on plates containing X-gal. The precise fusion
joints were verified by DNA sequencing. All of the plasmids can be
shuttled between E. coli and yeast. The
To extract the
fusion protein from mitochondria, the mitochondria were diluted with an
equal volume of distilled water to give a hypotonic condition and were
lysed for 15-30 min by the addition of 0.1% Triton X-100 at
0-4 °C. They were then sonicated at 0 °C for 30 s two
times using a Fisher-Artek model 300 sonic dismembrator at a power
setting of 85% relative output. Membrane fragments and other debris
were removed by centrifugation at 48,000
Assaying for
We showed in the in vitro system that, if the
three-amino acid linker (RGP) of the native rat liver aldehyde
dehydrogenase leader sequence was missing, import occurred but not
processing(7) . The chimeric protein from this construct was
isolated from yeast mitochondria, but no sequence could be obtained,
because the N-terminal amino acid was found to be blocked. No attempt
was made to identify the blocking group, but Western blot analysis
showed that the leader was still present.
Fusion proteins composed of the leader sequence of a
mitochondrial precursor protein and a carrier protein have been used to
investigate import of proteins into
mitochondria(12, 13, 14, 15, 16) .
Investigators have often deleted portions of the leader sequence in an
attempt to determine the minimum number of amino acids necessary for a
leader sequence to function. If a construct containing a modified
leader was not imported, it was assumed that the section deleted was
vital.
We found
that, when the leader sequence from rat liver aldehyde dehydrogenase
was fused directly to
If proteolysis occurred prior to import it
would not be possible for the precursor protein to be translocated into
mitochondria. To test for this, the fusion proteins remaining in the
cytosol were isolated and sequenced. The chimeric proteins isolated
from yeast cytosol were missing some or all of the leader sequence.
Undoubtedly, the portion of the protein was removed by the action of a
protease located in the cytoplasm. We have previously shown that
deletion of residues at the N-terminal end of the leader sequence
prevented ALDH from being translocated into rat liver
mitochondria(6) . Thus the reason for the lack of import of the
fusion proteins employed in this study was the fact that the leader was
destroyed or removed by the action of proteases in the cell prior to
import.
It is impossible to state whether the proteolysis occurred
in one step by the action of an endopeptidase, or by the sequential
removal of single amino acids by the action of an N-terminal amino acid
peptidase. The argument against the latter is the fact that only one
peptide sequence was obtained for each protein. If the amino acids were
being removed sequentially, it could be expected that a very ragged
N-terminal sequence would have been found. We have no data to determine
whether or not the protease was specific. As shown in ,
when 0, 21-, or 71-amino acid residues from the mature portion of ALDH
were included, proteolysis occurred at different positions. Based on
the deduced amino acid sequence (24) the cleavage occurred
between an L-S, an S-A, and an A-R bond when 0, 21, and 71 residues of
ALDH were included in the chimera, respectively. If there were three
specific proteases involved, it could have been expected to have
obtained three different amino acid sequences when the construct
containing 71 amino acids was employed.
Even though proteolysis
occurred which prevented some of the protein from entering the
mitochondria, a portion of the newly synthesized precursor protein was
found in the mitochondria. Although only 7% of the protein was imported
into the mitochondria when the aldehyde dehydrogenase leader sequence
was fused directly to
The linker-deleted precursor was shown to
direct the import of ALDH into isolated rat liver mitochondria, but was
not removed by the action of the protease after import(7) . The
corresponding
Most investigators (37, 38, 39, 40) argue that import is a
post-translational event. There are some who have presented data which
can be interpreted as implying that mitochondrial import is a
co-translation event(15, 41) . Our finding that the
fusion proteins have been subjected to the action of a protease prior
to import is supportive of the notion that import is a
post-translational event. If the leader was entering mitochondria as it
was coming off the ribosome it would be protected from the action of
the cytosolic proteases. Similar import data were obtained when the
multicopy plasmid or the single-copy plasmid were employed. This
finding shows that proteolysis occurred independent of the rate of
synthesis of the precursor protein and was not simply a result of
protein being synthesized faster than it was being imported.
The
proteolysis observed in this study shows a potential difficulty in
investigating import of fusion proteins in yeast. Investigators cannot
really tell why import did not occur unless they were to sequence the
protein remaining in the cytosol. Our study was performed only with
Cellular fractionations and enzyme assays
were performed as described under ``Experimental
Procedures.'' Imports are given as percentage of the total
activity present in the combined fractions and calibrated by assaying
for the marker enzymes. The numbers represent the average of at least
two experiments. The experimental error was ±8% of the mean.
-galactosidase. Only 7% of the
protein was imported. If 21 or 71 amino acids from the mature portion
of aldehyde dehydrogenase were included in the construct, 40% was
imported. The protein remaining in cytosol was sequenced. When the
leader was fused directly to
-galactosidase, the first 7 residues
of the leader were missing. When 21 residues of mature aldehyde
dehydrogenase were included, the entire leader plus 6 residues of the
mature portion were missing; if 71 residues of mature aldehyde
dehydrogenase were included, the first residue found corresponds to the
66th residue of the mature portion. When the leader was fused directly
to
-galactosidase, no processing of the imported protein occurred,
and the N-terminal amino acid was blocked, presumably by acetylation.
If the 21-amino acid insert was included, processing occurred. A
modified leader sequence lacking the three-amino acid linker (RGP) was
imported but not processed, just as we found in vitro (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D.G.(1993) J. Biol. Chem. 268, 19906-19914). The less than 100%
import of pre-aldehyde dehydrogenase was due to the action of a
post-translational protease attack which prevented import by destroying
the leader peptide segment.
(
)was found to be composed
of three structural components(6) . These were two short
segments of
helices separated by a three-amino acid linker (RGP).
We investigated the necessity of each region by making deletions or by
constructing chimeric leader sequences with components of cytochrome
oxidase, a protein whose leader sequence also was determined by
two-dimensional NMR(8) . We concluded that the minimum
structural requirement of the leader peptide was just a two-turn
amphilpathic
helix at the N termini of the leader
sequence(11) . The role of the other portions of the leader was
proposed to stabilize the N-terminal helix. The experiments that led to
these conclusions were performed with isolated rat liver mitochondria
employing proteins synthesized in rabbit reticulocytes. Data obtained
from in vitro import studies are thought to represent what
occurs in vivo because the cytosolic factors necessary to
allow import to occur are supplied by the reticulocyte system. We felt
it would be advantageous to investigate the peptides in a true in
vivo system, rather than just the in vitro one employed,
to verify our observations.
-galactosidase because of the ease of assaying the
enzyme. Typically, mitochondria are separated from cytosol, and the
amount of
-galactosidase activity in each organelle is used as a
measure of import. The leader sequence from rat liver aldehyde
dehydrogenase and an altered sequence we employed in the in vitro studies were fused to
-galactosidase on a yeast expression
vector to test whether the observations made in the in vitro system were valid in vivo. It will be shown that the
fusion of the leader sequence directly to
-galactosidase produced
a protein that was poorly imported. In contrast, if some residues of
the mature portion of aldehyde dehydrogenase were included, then the
protein could be imported into mitochondria, but much of the fusion
protein remained in the cytoplasm.
Strain and Growth Condition
Saccharomyces
cerevisiae strain KX 147-2C (MAT leu1
LEU4
ura52(17) was kindly
provided by Prof. Gunter B. Kohlhaw of this department). Yeast cells
were grown at 30 °C in SD medium, which contained 0.67% yeast
nitrogen base without amino acids, 2% glucose, and supplemented with 2
mM leucine. Escherichia coli strains used were TG1
and MC1000(18) . E. coli was grown at 37 °C in LB
medium or 2
yeast tryptone medium. Ampicillin was added to a
final concentration of 100 µg/ml. The lithium acetate method (19) was used for yeast transformation. E. coli transformation was as described by Maniatis et
al.(20) .
DNA Manipulation
Standard DNA techniques were
performed as described elsewhere(20, 21) . Site-directed
mutagenesis was performed with the ``Muta-Gene'' mutagenesis
kit by Bio-Rad, which is based on the procedure of Kunkel(22) .
Oligonucleotides were synthesized on an Applied Biosystems model 380A
synthesizer by the Laboratory for Macromolecular Structure, Purdue
University. DNA sequencing was determined by the dideoxy
chain-termination method(23) , using single-stranded template or
alkali-denatured, double-stranded DNA and Sequenase (U. S. Biochemical
Corp.). Some DNA fragments were synthesized by the polymerase chain
reaction using Taq DNA polymerase performed for 30 cycles with
the following program: 1 min at 94 °C, 1 min at 52 °C, and 2
min at 72 °C.
Construction of Plasmid Vector
For mutagenesis and
sequencing, plasmid pYH305 and pYH305DE were constructed. For pYH305,
the 2.8-kb SacI-ScaI fragment from pGEM-3Z that
contained the 1.8-kb EcoRI fragment of pALDH of rat liver was
isolated and ligated to Bluescript SKII(+), which was cut with SacI and ScaI. For pYH305DE, an EcoRI site
in the middle of the pALDH coding region in pYH305 was removed by
mutagenesis. This just changed the codon for the same amino acid.
ALDH-lacZ Gene Fusion Constructions
The rat liver
ALDH cDNA has been cloned and the entire nucleotide sequence has been
determined (24). A set of plasmid pGEM-3Z containing the pALDH sequence
with various modified signal peptides has been constructed for in
vitro import study(11) . The plasmids were cut with EcoRI and PvuII. The 308 bp of EcoRI-PvuII fragments which include 19 amino acids of
the signal region and 71 amino acids of the ALDH coding region were
isolated by running digested DNA on 3% NuSieve GTG gel. They were then
ligated into the EcoRI and SmaI sites of plasmid
pSEYC102, a yeast centromere plasmid (10.5 kb), containing the yeast
URA3, CEN4-ARS1, pBR322 sequence and unique EcoRI, SmaI, and BamHI sites to the 5` side of the truncated lacZ gene. Plasmid pYB1 has a 1.7-kb BamHI fragment
including 645 bp of the leu2 promoter region, 637 bp of the leu2 coding region, and 400 bp of the bacterial sequence.
Using the 1.7-kb BamHI fragment as the template, a 0.64-kb EcoRI-SphI fragment of leu2 promoter was
synthesized by a polymerase chain reaction, and then this fragment was
inserted into the above ligated plasmids which had been digested with EcoRI and SphI. The resulting plasmids have a leu2 promoter-ALDH-lacZ fusion (Fig. 1). The
plas-mid without the DNA fragment of the leader is the same as the
native plasmid except the leader sequence has been removed by creating
a SphI site at the 18th amino acid position of leader region
by mutagenesis.
Figure 1:
Plasmid construction of leu2 promoter-ALDH-lacZ fusion. The 308-bp EcoRI-PvuII fragments containing 19 amino acids of
the signal peptides and 71 amino acids of mature ALDH coding region
were inserted between the EcoRI and SmaI sites of
plasmid pSEYC102 and proved to be in frame by sequencing. The 0.6-kb EcoRI-SphI fragment of the leu2 promoter was
synthesized by a polymerase chain reaction, using pYB1 as the template,
and inserted into the EcoRI and SphI
sites.
To construct a fusion protein with a shorter portion
of mature ALDH and to obtain high expression of the fusion protein,
other ALDH-lacZ gene fusions were constructed, and a multiple
copy plasmid was adopted. An oligonucleotide with sequence
CGGAATTCTCGAGGAGAAC, which corresponds to nucleotides from -653
to -634 and contains an EcoRI restriction site
(underlined), was used as the sense primer for the polymerase chain
reaction experiments. The antisense oligonucleotide for the
construction of the fusion proteins which contain 21 amino acid
residues of mALDH was AATCGGATCCTGGTTGCAGAAGACCTCGGG (corresponding to
amino acid residues 15-24 of mALDH) and for the construction of
the fusion proteins without any portion of mALDH was
GGGATCCAGCAGGCGGCTCAGGCGTGGCCC (corresponding to -8 to +2
amino acid residues), in which a BamHI restriction site
(underlined) was made, respectively. The plasmid DNAs shown in Fig. 1were used as templates. All the DNA fragments amplified by
polymerase chain reaction were then subcloned into vector pSEYC101
between the EcoRI and BamHI sites. These plasmids can
normally be maintained in multiple copies per yeast cell due to the
presence of the 2µ gene.
-galactosidase-positive E. coli transformants were
selected. The plasmid DNA isolated from them finally were transformed
to yeast XK147-2C and tested for
-galactosidase expression on a
yeast minimal medium containing X-gal. Yeast cells expressing
-galactosidase were verified by measuring enzymatic activity in
cell-free extracts.
Cellular Fractionation and Enzyme Assay
Yeast
cells were grown to 1.5 optical density units at 600 nm in SD medium
supplemented with leucine. Cells were fractionated into cytosolic and
mitochondrial fractions essentially according to the procedure of Daum et al.(25) . Spheroplasts were prepared from yeast
cells in the presence of Lyticase (Sigma). Finally, mitochondria were
resuspended in 20 mM HEPES, pH 7.0, 0.6 M mannitol
buffer. For enzyme assay, the reaction buffer contained 1% Triton
X-100. -Galactosidase activity was measured in Z buffer by the
method of Miller (26) at 30 °C. The recovery of mitochondria
relative to cytosol was monitored by assaying the mitochondrial marker
enzyme fumarase (27) and the cytosolic marker enzyme
glucose-6-phosphate dehydrogenase(28) .
Purification of
All subsequent steps were carried out at 0-4
°C in the presence of protease inhibitors (Pefabloc SC, 1 mg/ml; o-phenanthroline, 5 mM; leupeptin, 3 µg/ml;
pepstatin, 15 µg/ml; phenylmethane sulfonyl fluoride, 1
mM; EDTA, 1 mM). Nucleic acids were precipitated from
the post-mitochondrial supernatant fractions by adding slowly 2.5%
protamine sulfate with constant stirring to give a final concentration
of 0.1%. After stirring for 30 min at 4 °C, the solution was
clarified by centrifugation at 10,000 -Galactosidase-Fusion
Proteins
g for 15
min(29) . An equal volume of 100% (of saturation) ammonium
sulfate solution was added slowly with stirring. The precipitate was
collected by centrifugation at 20,000
g for 10 min.
The pellet was then suspended in Tris-HCl buffer (0.25 M NaCl,
10 mM Tris HCl, pH 7.6, 10 mM MgCl
, 1
mM EDTA, 10 mM 2-mercaptoethanol, 0.1% Triton X-100)
and dialyzed against 100 volumes of the same buffer for a minimum of 2
h while changing the buffer three times. The dialysate was passed
through a
-galactose analog affinity column(30) . The
column was washed with 10 bed volumes of the buffer, followed by 5 bed
volumes of the buffer without Triton X-100. The protein was eluted from
the column with 0.1 M sodium borate (pH 10) and promptly
precipitated with 100% saturated ammonium sulfate.
g for 30 min.
The supernatant was dialyzed against the same buffer used for affinity
chromatography and then passed through the substrate analog affinity
column.
Protein Sequencing
N-terminal protein sequencing
was performed by the Purdue University Biochemistry Department Protein
Sequencing Facility on samples electrophoretically transferred from 8%
SDS gels to a membrane as described previously(31) .
Miscellaneous Chemicals
All the restriction and
modifying enzymes were purchased from New England BioLabs,
Boehringer-Mannheim, Promega, or Life Technologies, Inc. A prestained
molecular weight standard for SDS-polyacrylamide gel electrophoresis
was from Bio-Rad. Alkaline-phosphatase conjugate anti-mouse IgG and
mouse anti--galactosidase monoclonal antibody were from Promega.
Rabbit anti-pALDH signal peptide antibody was previously prepared in
our laboratory. Protease inhibitor set was from Boehringer Mannheim. p-Aminophenyl
-D-thiogalactopyranoside-Sepharose
4B was from Sigma. Western blotting was as previously
described(32) . All other reagents were commercially available
analytical reagents.
The
plasmids for the gene fusion constructs which contained the leu2 promoter/N terminal native and modified precursor (Fig. 2)
ALDH were fused in frame to the 10th amino acid of lacZ (Fig. 1). -Galactosidase Expression in Yeast
-Galactosidase activity for all constructs
was found in both E. coli and yeast, and no activity was
expressed from the parent vector in these cells. The level of
-galactosidase expressed in crude yeast extracts from all of the
gene fusions was essentially identical. The reason is that these have
been constructed in the plasmid pSEYC102 which contains the yeast
centromere sequence CEN4 and the sequence ARS1 which
allows for the maintenance of essentially one copy of the plasmid per
cell.
Figure 2:
The
amino acid sequences of the signal peptides. Amino acids in italics are from the mature part of ALDH. Linker deletion is wild type
presequence without the (RGP) linker which connects the N- and
C-terminal helices.
The constructs containing the ALDH/-galactosidase fusion
was also cloned into the SEYC101 vector which are maintained in
multicopy in the cells. Activity assays showed that all constructs were
expressed to the same extent but at a level more than 10-fold higher
than with the single copy plasmid ().
-galactosidase activity in isolated mitochondrial and cytosolic
fractions allowed us to determine whether import occurred in
vivo. If the leader sequence of ALDH was not included in the
construct, 99% of the total
-galactosidase activity was found
associated with the cytosol fraction, showing that, as expected, the
enzyme could not be imported into mitochondria if it did not possess a
mitochondrial leader sequence.
Import of
The leader sequence of aldehyde dehydrogenase was fused
directly to the 10th residue of -Galactosidase Chimera Possessing a Leader
Sequence Requires the Presence of a Portion of the Mature
Protein
-galactosidase. It was unexpected
to find that for this construct only 7% of the
-galactosidase
activity was found in the mitochondrial pellet with the remainder in
the cytosolic fraction. The constructs containing 21 or 31 amino acids
of mature ALDH between the leader sequence and
-galactosidase were
imported to a much greater extent (40%). The distribution of
-galactosidase activity be-tween mitochondria and cytosol for the
ALDH chimera is presented in .
Amino Acid Sequence of the
Lack of import could have resulted from at
least two unrelated events. We showed that a disruption of the
N-terminal helix of the leader sequence affected its ability to
function(11) . Possibly, the three-dimensional structure of the
leader sequence was affected by the presence of -Galactosidase Chimera
Found in Cytosol
-galactosidase
being fused to it. Alternatively, if the leader sequence was modified in vivo prior to import, the precursor protein would not enter
mitochondria. To test for this possibility,
-galactosidase was
isolated from the cytosol and subjected to amino acid sequencing. When
the chimera with no intervening sequence between the leader and
-galactosidase was employed, the protein was found to be lacking
the N-terminal portion of the leader sequence. The first amino acid
identified corresponded to the 6th residue of the leader. The proteins
which were designed to contain intervening segments of mature aldehyde
dehydrogenase were found to be missing all of the leader sequence plus
part of the mature aldehyde dehydrogenase portion. When the construct
with 71 residues of mature ALDH was analyzed, the first amino acid
corresponded to the 66th residue of the mature portion of ALDH. When
the construct with 21 intervening residues was analyzed, the first
amino acid found corresponded to the 7th residue of the mature portion
of ALDH. These sequences are shown in and illustrated in Fig. 3.
Figure 3:
Cleavage site of the fusion protein
isolated from cytosol. The numbers 1-19 above the filled lines indicate the leader sequence. The numbers below
the single line indicate the intervening sequence of mature
ALDH in the fusion protein. The position of the first amino acid found
from sequencing the fusion protein is indicated by the numbers 66, 7, and 6,
respectively.
Amino Acid Sequences of Protein Isolated from
Mitochondria after Import
The -galactosidase from two
fusion proteins possessing the native leader sequence were isolated
from mitochondria and subjected to amino acid sequence analysis. The
first had the leader fused directly to
-galactosidase; the second
had the 21-amino acid insert. No sequence could be found from the
protein missing the insert, suggesting that the N-terminal residue was
blocked. A Western blot analysis was performed using an antibody
generated to recognize only the leader region to verify that the leader
sequence was still attached to
-galactosidase (Fig. 4).
Figure 4:
The Western blot analysis of the fusion
proteins in mitochondria. Samples were run on 8% SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose paper, and probed by
anti-signal peptide antibody (A) and anti--galactosidase
antibody (B). Mitochondrial fractions of pZ001 (no intervening
sequence for mature ALDH, lane 1), pZ211 (21 amino acids from
mature ALDH, lane 2), and pZ212 (linker deleted with 21 amino
acids from mature ALDH, lane 3) were loaded, respectively. The
band near 139 Kd is the fusion protein. The molecular weight
standards were prestained by Coomassie Blue (not
shown).
When the 21-amino acid portion of ALDH was included in the
construct, the leader sequence was removed by the mitochondrial
processing enzyme. The amino acid sequence corresponded to the first
residues of the mature portion of ALDH, showing that processing
occurred at the expected site located between RLL, the last 3 residues
of the leader, and SAA of the mature protein (I and Fig. 2). Western blot analysis using the antibody against the
leader sequence was negative, as expected, for a protein missing the
leader.
-Galactosidase is a commonly employed carrier protein
used to study import in yeast(12, 13, 30) .
There are reports in the literature showing that for some constructs
-galactosidase did not function well(12) . Some
investigators have suggested, although never proven, that the size
(1023 amino acids) may have been responsible for its not allowing a
leader sequence to function, for if the same peptide was fused to, say,
dihydrofolate reductase, in vitro import would occur. We
showed that the rate of import of precursors proteins could be a
function of the size of the mature portion of the protein (33). Thus
the large size of the
-galactosidase fusion protein may have
contributed to its not functioning well in some systems.
-galactosidase, little of the total enzyme
activity was found associated with yeast mitochondria. This was an
unexpected finding since the leader worked well in causing the import
of mature ALDH and orithine transcarbamylase into rat liver
mitochondria and into isolated yeast mitochondria.
(
)Douglas et al. (34), however, reported that
it was not possible to import F
-ATPase fused directly to
-galactosidase.
-galactosidase, no processing occurred after
import. This could have been a result of the destruction of the
processing site. When the leader was fused to
-galactosidase
through 21 amino acids of the mature ALDH, a sequence was obtained. The
amino acid sequence of the protein revealed that it was processed at
the expected site. This is the first proof that the actual site of
processing of the ALDH leader sequence was between the RLL and the SAA
residues. It has always been assumed that this was the site based upon
the amino acid sequence of the enzyme isolated from liver. The
potential uncertainty comes about from the fact that human liver
mitochondrial ALDH was reported to possess a ragged N-terminal sequence (35) and that rat and beef liver mitochondrial ALDH, isolated
from fresh tissue, had a blocked N-terminal (acetylated)
residue(36) .
-galactosidase fusion protein isolated from yeast
mitochondria after import was found to be blocked and not processed.
The blocking, presumed to be acetylation, could have occurred either
after import or prior to import. It is not known whether or not the
initiator methionine residue is removed in cytosol prior to import.
Detailed structure analysis of the blocked precursor proteins by mass
spectroscopic techniques could be used to address this question.
-galactosidase as the carrier protein. It is not possible to state
why the ALDH portion was so susceptible to cleavage or why the leader
was cleaved when fused directly to
-galactosidase. It is possible
that using a protein which was imported slowly could have made it more
susceptible to the action of nonspecific cytosolic proteases. A similar
study will have to be undertaken using other proteins to determine if
proteolysis is a problem common for all chimeric proteins expressed in
yeast or is unique to the system employed in this study.
Table: Distribution of fusion proteins between
mitochondria and cytosol
Table: 1768843040p4in
Not determinable.
Table: N-terminal sequencing of purified fusion
protein isolated from mitochondria
-D-galactoside; bp, base pair; kb, kilobase pair.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.