From the
We report studies of the import into mitochondria of MTF1, a
nucleus-encoded factor that markedly stimulates the specific
transcription of mitochondrial DNA. Unlike most of the mitochondrial
precursor proteins studied so far, import of MTF1 does not appear to
require a receptor on the outer membrane, membrane potential across the
inner membrane, or ATP hydrolysis. Its import is not affected by low
temperature. It lacks a cleavable presequence but translocates across
the inner membrane through its amino terminus; its sorting is
independent of hsp60. Our results indicate an unusual and distinct
import pathway for MTF1 into the yeast mitochondria.
Mitochondria have their own genome
(1) , which is
transcribed by a machinery that consists of a core polymerase
(2) and a mitochondrial transcription factor MTF1
(3) ,
both of which are encoded in the nucleus and are imported into the
mitochondria from the cytoplasm. MTF1 is a 43-kDa protein
required for specific mitochondrial transcription. Its predicted amino
acid sequence as specified by its gene and the amino-terminal amino
acid sequence of the mature protein purified from isolated mitochondria
indicates that following translocation, only the initiating methionine
is removed in transit to the mitochondria
(4) .
There are
several steps involved in the import of proteins into mitochondria and
their proper sorting within mitochondrial subcompartments. These
include binding of the precursor protein to a receptor on the outer
membrane; unfolding of the precursor protein into a translocation
competent conformation; translocation through contact sites between the
outer and the inner membranes, which is dependent on the
electrochemical potential across the inner membrane. During or after
translocation, the targeting sequence of the precursor is cleaved by
matrix-localized proteases to generate mature protein, which is then
refolded and sorted to its proper submitochondrial location and in many
cases is assembled into multimeric complexes
(5, 6) . In
some cases, the targeting sequence is included within the mature
protein sequence so that following import there is no reduction in the
size of the imported protein.
In this paper, we report that the
import of MTF1, a nucleus-encoded mitochondrial protein, takes place
through an unusual pathway, which does not require the receptor on the
outer mitochondrial membrane, the membrane potential across the inner
membrane, and ATP hydrolysis. Its sorting is independent of hsp60,
although it moves across the inner membrane.
While we have not
pinpointed the sequence(s) responsible for mitochondrial localization,
we have evidence that it is present at the amino-terminal half of the
protein. We are currently engaged in studying the more precise
definition of the targeting sequence.
There are other proteins that
enter the mitochondria independent of one or more features that
characterize the usual protein import mechanism or pathway. But none of
them is as fully independent of the features upon which mitochondrial
import depends, as is the case for MTF1. Import of cytochrome c into the inter membrane space is independent of a surface
receptor
(26, 27) . Import of subunit Va of cytochrome
c oxidase into the inner membrane also is independent of a
proteinaceous surface receptor
(11) . Transport of apocytochrome
c across the outer membrane is not dependent on the membrane
potential but requires functional cytochrome c heme lyase.
Import of subunit Va, on the other hand, is dependent on the membrane
potential for its import, although its requirement for ATP is very low
and it is completely imported at low temperature. Our results, taken
together, strongly indicate that the import pathway of MTF1 is quite
unusual compared with that of the majority of proteins studied so far
and is distinct even from the pathways used by either cytochrome c or subunit Va of cytochrome c oxidase, although it does
share some but not all of the import features with each of the latter
proteins.
Apocytochrome c, requiring no mitochondrial
surface receptor, probably inserts spontaneously into the mitochondrial
lipids of the outer membrane, dependent mainly on the anionic lipids
(27). It crosses the mitochondrial membrane reversibly and is fixed
inside the mitochondria by reaction with heme catalyzed by cytochrome
c heme lyase. It remains to be established whether similar
features characterize the transport of the transcription factor. If it
associates spontaneously with mitochondrial lipid, it does not appear
to distinguish between the lipids of the outer membrane and inner
mitochondrial membranes, since the protein is imported by mitoplasts
lacking intact outer membrane. This experiment would not, however,
distinguish transport through the outer membrane-inner membrane contact
sites, which are probably preserved in the mitoplasts. It remains to be
established whether MTF1 gene product crosses mitochondrial membranes
specifically or whether it can readily cross other cellular or
organellar membranes. Further experiments along these lines are in
process. If the mitochondrial transcription factor is able to cross
other membranes easily, it would be necessary to postulate some
fixation event to account for its mitochondrial targeting as is the
case for cytochrome c. Once imported, the transcription factor
may be fixed in the mitochondrial matrix by one of three possible
events: interaction with another macromolecule in the mitochondrial
matrix (DNA or protein), post-translational chemical modification
(e.g. acylation), or postimport folding catalyzed by an
intramitochondrial chaperone. Each of these possibilities is being
explored in further experiments.
The findings on MTF1 import are
thus quite surprising. Since much of the electrochemical gradient and
ATP are required for the preservation of a translocation-competent
conformation and since neither of these conditions is required for MTF1
import, one might postulate that MTF1 naturally assumes a
translocation-competent conformation. This could only be established by
physical analysis of the MTF1 synthetic product or by strategic
mutagenesis.
Another feature that deserves comment and may relate to
the conformation for translocation is the observation that proteinase K
but not trypsin digestion reveals a product that is slightly, but
reproducibly shortened. There is good reason to believe that this does
not represent cleavage of the amino-terminal pretargeting or targeting
sequence. First, the product isolated from intact mitochondria, when
compared with the sequence specified by the cloned gene, lacks only the
amino-terminal methionine. Second if removal of the presequence was the
basis for the small reduction in size, this should be revealed by
trypsin, as well as proteinase K proteolysis. Third, inhibition of the
matrix metalloprotease thought to be involved in processing of recently
imported mitochondrial precursors should prevent the observed reduction
in protein size. This was not the case. Since the newly imported
protein is in the matrix and not attached to the inner membrane, even
transiently, it seems that this reduction in size cannot be explained
as a transport intermediate that assumes a hairpin configuration with a
small portion of the carboxyl terminus accessible to proteinase K. The
basis for this apparent shortening must await further experimentation.
We thank Charlene Murphy for help in the preparation
of the manuscript.
Materials
Carbonyl cyanide
m-chlorophenylhydrazone (CCCP),(
)
valinomycin, and apyrase were purchased from Sigma.
Digitonin was purchased from ICN Pharmaceuticals Inc. The vector pGEM3
and rabbit reticulocyte lysate were purchased from Promega (Madison,
WI). [
S]Methionine (specific activity, 800
Ci/mmol) was purchased from Amersham Corp.
Isolation of Mitochondria
Mitochondria were
isolated from the yeast strain D-273-10B (ATCC 25657) according
to the method of Daum et al.(7) .
Isolation of MTF1 Gene by Polymerase Chain
Reaction
The whole coding sequence of the MTF1 gene was isolated
from yeast genomic DNA, prepared from the yeast strain D-273-10B
according to the method of Cryer et al.(8) , by the
polymerase chain reaction using two oligonucleotides chosen based upon
the published sequence
(3) .
In Vitro Coupled Transcription and Translation
The
coding sequence of the MTF1 gene was placed under the control of the
T promoter of the pGEM3 vector. The gene was transcribed
and translated in vitro as follows. Three µg of the
linearized plasmid DNA was transcribed in a 25-µl reaction mixture
containing 40 mM Tris-HCl, pH 7.5, 6 mM
MgCl
, 2 mM spermidine, 10 mM NaCl, 80
units of RNasin, 10 mM dithiothreitol, 500 µM
each of ATP, CTP, UTP, 80 µM GTP, and 500 µM
m
G(5`)ppp(5`)G using 40 units of T
RNA
polymerase at 37 °C for 30 min. The transcription reaction was
stopped by adding 11 units of DNase I followed by incubation at 37
°C for 10 min. One µl of this transcription reaction mixture
was used as a source of capped mRNA in a 25-µl translation mix that
contained 17.5 µl of rabbit reticulocyte lysate, 20 units of
RNasin, 0.5 µl of 1 mM amino acid mixture minus
methoinine, 20 µCi of [
S]methionine. The
reaction mixture was incubated at 30 °C for 2 h. The lysate
containing the labeled synthesized protein was then used for the in
vitro import assay. The same methodology was employed for the
coupled transcription/translation of control genes viz.
F
and ADP/ATP carrier (AAC).
In Vitro Import
Import of the in vitro synthesized protein into the isolated mitochondria was carried out
according to the method of Cheng et al.(9) . The import
buffer contained 0.6 M sorbitol, 3% bovine serum albumin, 10
mM MOPS, pH 7.2, 80 mM KCl, 2 mM ATP, and 1
mM NADH.
Pretreatment of Mitochondria with Trypsin
A
mitochondrial suspension (500 µl) containing 1 mg of mitochondrial
protein was incubated with 30 µg of trypsin at 25 °C for 15
min. 10-fold excess of soybean trypsin inhibitor and 0.2 mM
PMSF were then added and incubated at 25 °C for 3 min and at 4
°C for 10 min. The mitochondria were recovered by centrifugation in
a microcentrifuge; washed once with a buffer containing 0.6 M
sorbitol, 10 mM Tris, pH 7.5, 0.1 mg/ml soybean trypsin
inhibitor, plus 0.1 mM PMSF; suspended in a small volume of
the above mentioned buffer; and used for import after determination of
the protein concentration.
Treatment of Mitochondria and the Rabbit Reticulocyte
Lysate with Apyrase
150 µg of mitochondrial protein was
suspended in 200 µl of buffer containing 0.6 M sorbitol,
3% bovine serum albumin, 10 mM MOPS, pH 7.2, 80 mM
KCl, and 5 mM MgCl, incubated with 5 units of
apyrase at 25 °C for 15 min, and reisolated by centrifugation in a
microcentrifuge. Forty µl of rabbit reticulocyte lysate, containing
the labeled synthesized protein, was diluted 5-fold with a buffer that
contained 0.6 M sorbitol, 10 mM MOPS, pH 7.2, 80
mM KCl and incubated with 5 units of apyrase at 25 °C for
15 min.
Preparation of Mitoplasts
Mitochondria (1 mg of
protein) prepared as stated above, was resuspended in 300 µl of
buffer containing 0.1 M sorbitol, 10 mM Tris, pH 7.5,
and incubated on ice for 30 min. Mitoplasts were recovered by
centrifugation in a microcentrifuge and resuspended in a buffer
containing 0.6 M sorbitol, 10 mM Tris-HCl, pH 7.5,
and the protein concentration was measured.
Digitonin Treatment of Mitochondria
Mitochondria,
after import of the precursor protein, were reisolated and washed twice
with a buffer containing 0.6 M sorbitol, 10 mM
Tris-HCl, pH 7.5, resuspended in 100 µl of the same buffer
containing either 0.25 or 0.5% of digitonin, and placed on ice for 3
min. 500 µl of the above-mentioned buffer was added, followed by
centrifugation in a microcentrifuge at 4 °C. One set of pellets was
suspended in 100 µl of import buffer (3% bovine serum albumin, 0.6
M sorbitol, 10 mM MOPS, pH 7.2, 80 mM KCl)
and incubated with 40 µg of proteinase K on ice for 15 min followed
by the addition of 1 mM PMSF.
Estimation of Protein
The protein was estimated
according to the method of Lowry et al.(10) .
RESULTS
MTF1 Can Be Imported into the Isolated
Mitochondria
Many nucleus-encoded mitochondrial proteins are
synthesized with amino-terminal presequences for targeting to the
mitochondria. Usually the presequences are 20-80 amino acids long
and contain several basic and hydroxylated residues and no acidic
residues. However, it is still not clear which structural motif or
sequence determines the targeting function
(6) . MTF1, a
nucleus-encoded mitochondrial protein, contains an aspartic acid at
amino acid position 10 from the amino terminus, and the first 10 amino
acids contain only 1 basic residue (lysine) and 1 hydroxylated residue
(serine)
(3) , indicating either a very short or no cleavable
presequence. The amino-terminal amino acid sequence of the 43-kDa
protein is reported to be identical with that specified by the MTF1
gene, except for the removal of the initiating methionine
(4) .
We have investigated the import of the MTF1 gene product into isolated
mitochondria. The MTF1 gene in the pGEM3 vector was employed for
coupled trasnscription and translation (as described under
``Experimental Procedures''), and the resulting labeled
protein was used for the assay of import into the mitochondria isolated
from Saccharomyces cerevisiae. The results are illustrated in
Fig. 1
. Lane1 shows no product in the absence
of the added MTF1 mRNA. When the in vitro synthesized MTF1
mRNA was added, a 42.6-kDa protein was obtained (lane2). This protein was found to be associated with the
mitochondria after incubation at 25 °C (lane3),
but no reduction in size was observed. The protein was resistant to
added proteinase K (lane4). The mitochondria were
reisolated after import and lysed with Nonidet P-40 when the protein
was sensitive to proteinase K (lane5), indicating
translocation of labeled MTF1 inside the mitochondria. The imported
band could be immunoprecipitated by polyclonal antiMTF1 antiserum
(lane7), raised against recombinant MTF1
overexpressed in E. coli,(
)
but not by
preimmune serum (lane6) or by antiCEM1 antiserum
(lane8), an unrelated antibody raised against the
CEM1 gene
(12) .
Figure 1:
In vitro synthesis and
import of MTF1 into the isolated mitochondria. In vitro synthesized mRNA of MTF1 gene was used to synthesize the protein
in rabbit reticulocyte lysate in the presence of
[S]methionine (as described under
``Experimental Procedures''), and the labeled protein was
then used for the import into the mitochondria isolated from
D-273-10B cells. Lane1, 10 µl of the
translation mix without mRNA; lane2, 10 µl of
the reaction mix with mRNA; lane3, 20 µl of the
translation mix containing the labeled protein was incubated with 75
µg of mitochondrial protein in a 100 µl of import reaction
buffer at 25 °C for 30 min; lane4, same as in
lane3 and after import 40 µg of proteinase K was
added to the import reaction mix and incubated on ice for 30 min
followed by the addition of 1 mM PMSF; lane5, same as in lane3 and after import
the mitochondria were reisolated and disrupted with 1% Nonidet P-40 and
80 mM KCl followed by digestion with 40 µg of proteinase
K; lane6, same as in lane4 and
after the addition of PMSF the mitochondria were reisolated and
suspended in 50 µl of 3% SDS, boiled for 3 min, and diluted 14-fold
with a buffer (final concentration becomes 50 mM Tris, pH 7.5,
150 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA). 70 µl
of preimmune serum was then added, and the whole mixture was rotated
end-over-end overnight. 50 µl of protein A-Sepharose CL4B
suspension in the above-mentioned buffer was added and rotated for 12
h. Protein A-Sepharose beads were recovered by centrifugation in the
microcentrifuge, washed 3 times in the above-mentioned buffer, and then
boiled with Laemmli gel loading dye. Lane7, same as
in lane 6 but 70 µl of anti-MTF1 antiserum was used
instead of preimmune serum; lane8, same as in
lane6 and instead of preimmune serum, 70 µl of
antiCEM1 antiserum was added. All of the samples were then analyzed by
SDS-polyacrylamide gel electrophoresis followed by
fluorography.
Protease-digested Mitochondria Are Capable of Importing
MTF1
Functional studies with Neurosporacrassa and yeast have suggested the presence on the mitochondrial outer
membrane of specific receptors for the imported
proteins
(13, 14, 15, 16, 17) .
Binding of precursors to these receptors is one of the earliest steps
of protein import into the mitochondria. Does the import of MTF1
involve any proteinaceous receptor on the outer surface of the
mitochondria? Mitochondria were pretreated with trypsin and then used
for the assay of import of MTF1. F and ATP/ADP carrier
proteins were used as controls. The results depicted in
Fig. 2
indicate that when untreated mitochondria were used for the
import assay, the F
produced a mature form (smaller in
size than the precursor) (lane1), which is resistant
to exogenous proteinase K (lane2), indicating its
import into mitochondria. The ADP/ATP carrier, on the other hand, is
not reduced in size upon incubation with mitochondria (lane1) under import assay conditions, since it does not have
a cleavable presequence, but it is translocated to a site within the
mitochondria that renders it resistant to proteinase K (lane2). When trypsin- (30 µg) treated mitochondria were
used, both F
and ADP/ATP carrier failed to be imported
into the mitochondria as evidenced by the absence of protease protected
bands (lanes3 and 4). MTF1, on the other
hand, was able to be transported into both untreated (lanes1 and 2) and trypsin-pretreated mitochondria
(lanes3 and 4). MTF1 was found to be
imported also into the mitochondria pretreated with 200 µg of
trypsin (data not shown). This observation suggests that its import
probably does not require a receptor on the outer surface of the
mitochondria.
Figure 2:
Import of MTF1 is not sensitive to Trypsin
pretreatment. 40 µl of lysate containing the labeled protein
(either F or AAC, or MTF1) were incubated with 150
µg of mitochondrial protein either without pretreatment (lanes1 and 2) or pretreated with 30 µg of trypsin
(as described under ``Experimental Procedures'') (lanes3 and 4) in a 200 µl of import reaction
buffer at 25 °C for 30 min. After import, the reaction mix was
divided into two equal parts. 40 µg of proteinase K was added to
one part (lanes2 and 4), incubated on ice
for 30 min, followed by the addition of 1 mM PMSF.
Mitochondria were reisolated in each case and analyzed by SDS-PAGE
followed by fluorography.
Since the MTF1 is resistant to proteinase K after
import in trypsin-pretreated mitochondria, we examined the sensitivity
of in vitro synthesized labeled MTF1 to proteases by
incubating with different concentrations (5-40 µg) of
proteases viz. proteinase K and trypsin. Newly synthesized
MTF1 was found to be sensitive to even 5 µg of proteinase K,
although more than 5 µg of trypsin was needed to degrade it (data
not presented). This indicates that the results presented in
Fig. 1
and Fig. 2are not simply attributable to the ability
of MTF1 in the absence of detergent to assume a proteinase-resistant
conformation.
MTF1 Import Is Not Dependent on a Membrane
Potential
Translocation of precursor proteins into and across
the inner membrane depends on energization of the membrane provided by
the electrochemical potential. To determine if MTF1 requires a membrane
potential for its import, CCCP, an uncoupler of oxidative
phosphorylation, and valinomycin, an ionophore, were used during the
import. As a control for the effectiveness of these two agents, the
import of F subunit was studied alongside that of
MTF1. The results are depicted in Fig. 3. In the absence of
either agent, mitochondria produces mature F
(lane1), which is protease-protected (lane2). But in the presence of both CCCP and valinomycin,
mature form is not produced; rather, there is accumulation of the
precursor (lanes3 and 5), which is
sensitive to proteinase K (lanes4 and 6),
indicating that the import of F
is dependent on the
membrane potential. However, MTF1 is transported into a
protease-protected location within the mitochondria in the presence of
both CCCP (lanes3 and 4) and valinomycin
(lanes5 and 6), suggesting that MTF1 import
is independent of the membrane potential. The effect of inhibition of
the matrix-localized protease, responsible for the cleavage of the
presequences from the imported mitochondrial proteins, was examined
using 1,10-phenanthroline and EDTA. Under these conditions,
F
failed to be imported into the mitochondria, while
the import of MTF1 was not at all affected (lanes7 and 8).
Figure 3:
MTF1 can be imported in the absence of
membrane potential. 40 µl of lysate containing labeled protein
(either F or MTF1) was incubated with 150 µg of
mitochondrial protein in a 200 µl of import reaction buffer in the
absence of any agent (lanes1 and 2) or in
the presence of either 40 µM CCCP (lanes3 and 4), or 5 µM valinomycin (lanes5 and 6), or 250 µM
1,10-phenanthroline and 5 mM EDTA (lanes7 and 8) at 25 °C for 30 min. After import, the
reaction mixture was divided into two equal parts, and 40 µg of
proteinase K was added to one of them (lanes2,
4, 6, and 8) and incubated on ice for 30 min
followed by the addition of 1 mM PMSF. The mitochondria were
reisolated in each case and analyzed by SDS-PAGE followed by
fluorography.
Import of MTF1 Does Not Need ATP
It has been shown
that protein import into the mitochondria requires ATP along with an
energized inner membrane
(18) . Pfanner et al.(19) suggested that ATP is necessary both in the cytosol and in
the matrix (a) on the cytosolic side for the maintenance of
the translocation competent conformation of the precursor and
(b) for refolding and sorting of the precursors that involve
chaperone-like components. To investigate the requirement of ATP for
the import of MTF1, we depleted the mitochondria and the rabbit
reticulocyte lysate (containing the labeled precursor) of ATP by
treating them with apyrase. The effectiveness of functional ATP
depletion was assessed by examining the import of F in
parallel with that of MTF1. When untreated mitochondria were used in
the presence of exogenous ATP and NADH, F
was found to
be cleaved, and the mature form is accumulated inside the mitochondria
(Fig. 4, lanes2 and 3). This was
observed even in the absence of exogenous ATP and NADH (lanes4 and 5) since the mitochondria and the rabbit
reticulocyte lysate contain endogenous ATP. When this ATP was removed
by apyrase treatment, F
failed to be imported
(lanes6 and 7). MTF1, on the other hand,
was imported even in apyrase-treated mitochondria (lanes6 and 7), indicating that its import requires little if any
ATP.
Figure 4:
MTF1 can be imported in the absence of
ATP. 40 µl of lysate containing the labeled protein (either
F or MTF1) was incubated with 150 µg of
mitochondrial protein in a 200-µl import buffer in the presence
(lanes2 and 3) or absence (lanes4-7) of 2 mM ATP and 1 mM NADH
and incubated at 25 °C for 30 min. In the case of lanes6 and 7, both the mitochondria and the lysate,
containing the labeled protein, were pretreated with apyrase as
described under ``Experimental Procedures.'' After the
reaction, each reaction mix was divided into two equal parts, and one
of them was treated with 40 µg of proteinase K (lanes3, 5, and 7) incubated on ice for 30
min followed by the addition of 1 mM PMSF. The mitochondria
were reisolated and analyzed by SDS-PAGE followed by
fluorography.
Import of MTF1 Occurs Even at Low
Temperature
Temperatures lower than 20 °C reduce the
transfer of precursor proteins across the two mitochondrial membranes.
Translocation intermediates are trapped by performing import at lower
temperatures
(20, 21) . MTF1 import was examined at
different temperatures viz. 2, 10, and 25 °C. The extent
of import was found to be the same in each case (Fig. 5),
indicating that the import of MTF1 is not reduced by carrying out the
import at low temperature.
Figure 5:
Low temperature does not have any effect
on the import of MTF1. 40 µl of lysate containing the labeled MTF1
was incubated with 150 µg of mitochondrial protein in a 200-µl
import buffer at either 2 °C (lanes2 and
3), 10 °C (lanes4 and 5), or
25 °C (lanes6 and 7) for 30 min. Each
reaction mixture was then divided into two equal parts, and one of them
(lanes3, 5, and 7) was incubated
with 40 µg of proteinase K on ice for 30 min followed by the
addition of 1 mM PMSF. The mitochondria were reisolated and
analyzed by SDS-PAGE followed by
fluorography.
The Amino-terminal Two-thirds of MTF1 Contains the
Mitochondrial Targeting Sequence
Though many mitochondrial
proteins are synthesized with amino-terminal presequences that are
removed proteolytically within the mitochondrial matrix, some of these
proteins are synthesized without cleavable presequences, and they
include their targeting signals within their mature peptide
(6) .
Since MTF1 is translocated into the mitochondria without reduction in
size, we began to assess whether the targeting signal was present at
the amino or carboxyl-terminal half of the MTF1 gene product. The MTF1
gene was truncated from its 3`-end with unique restriction
endonucleases, and the resulting 5`-fragments were used for the coupled
transcription, translation, and import assay. Digestion of the MTF1
gene (1 kilobase) with BglII and BamHI produces 5`
889- and 671-base pair fragments, respectively. The corresponding
protein of the longer fragment (BglII) (obtained by coupled
transcription and translation using the fragment as a template), was
imported into the mitochondria as evidenced by its protection from
proteinase K digestion (Fig. 6, lanes2 and
3). In the case of the BamHI fragment, only a very
small proportion of the available protein was protected from protease
digestion (lane3), suggesting very limited import.
Both products were fully sensitive to proteinase K when the
mitochondria were reisolated after import and lysed with Nonidet P-40
before the addition of proteinase K, indicating that these peptides had
probably been transported to the interior of the mitochondria (lane4). It is notable, however, that the efficiency of import
with the truncated protein derived from the BglII fragment was
not as great, as is the case with the intact protein.
Figure 6:
Amino-terminal fragments of MTF1 can be
imported into isolated mitochondria. The MTF1 gene was digested with
either BglII or BamHI, and the resulting fragments
were used for in vitro transcription and translation as
described under ``Experimental Procedures.'' 60 µl of the
lysate containing the labeled protein was incubated with 225 µg of
mitochondrial protein in a 300-µl import buffer at 25 °C for 30
min. After import, the import reaction mixture was divided into three
parts (lanes2, 3, and 4). One of
them was treated with 40 µg of proteinase K and incubated on ice
for 30 min (lane3) followed by the addition of 1
mM PMSF. Mitochondria were reisolated from another part
(lane4), lysed with 1% Nonidet P-40 and 80
mM KCl, and then treated with 40 µg of proteinase K.
Mitochondria were reisolated from the other two, and all of the samples
were analyzed by SDS-PAGE followed by
fluorography.
Newly Imported MTF1 Is Internal to the Inner
Mitochondrial Membrane
Since MTF1 can be imported into
mitochondria devoid of ATP, in the absence of membrane potential and
even at low temperature, the question arises, in which of the four
mitochondrial compartments (outer membrane, inner membrane,
intermembrane space, and matrix) was MTF1 located after import under
those conditions? Mitochondria, after import of MTF1, were treated with
two different concentrations of digitonin followed by treatment with
proteinase K. AAC protein, which resides on the inner membrane facing
the inter membrane space, was used to validate the fractionation.
Fig. 7A shows that AAC, after import at 25 °C in the
presence of a membrane potential and ATP (lane1),
was protected from proteinase K (lane2) but was
degraded by proteinase K when the mitochondria were treated with either
0.25% digitonin (lane4) or 0.5% digitonin (lane6), showing that the AAC is accesssible to proteinase K
after digitonin treatment. Under the same conditions of import
(Fig. 7B), MTF1 was protected from proteolysis even
after treatment with 0.5% digitonin. In view of the unusual features of
MTF1 import, we checked that the protein was similarly resistant to
proteolysis even in the presence of valinomycin
(Fig. 7C) or CCCP (Fig. 7D) or in the
absence of ATP (Fig. 7E) and after treatment with 0.5%
digitonin (lane6). When the import was carried out
at low temp (5 °C) (Fig. 7F), in the presence of
CCCP (Fig. 7G) or valinomycin (Fig. 7H),
in the absence of ATP (Fig. 7I), or in the absence of
ATP but in the presence of CCCP (Fig. 7J), MTF1 was
still found to be resistant to proteinase K after treatment with both
0.25 and 0.5% digitonin. These data indicate that MTF1 was imported
across the inner membrane even when there was little or no
electrochemical gradient, no ATP, and at low temperature.
Figure 7:
Use of digitonin to subfractionate
mitochondria after import. 120 µl of lysate containing labeled
protein (either MTF1 or AAC) was incubated with 450 µg of
mitochondrial protein in a 600-µl import buffer at 25 °C
(A-E) or at 5 °C (F-J) for 30 min.
A, B, and F, in the presence of exogenous
ATP and NADH; C and H, in the presence of exogenous
ATP, NADH, and 5 µM valinomycin; D and
G, in the presence of exogenous ATP, NADH, and 40
µM CCCP; E and I, in the absence of
exogenous ATP and NADH (the mitochondria and the lysate were treated
with apyrase before import, as described under ``Experimental
Procedures,'' to remove the endogenous ATP); J, in the
absence of exogenous ATP and NADH but in the presence of 40
µM CCCP (the mitochondria and the lysate were treated with
apyrase before import to remove the endogenous ATP). After import, the
reaction mix was divided into six equal parts (lanes1-6). Two parts (lanes3 and
4) were treated with 0.25% digitonin, while two others
(lanes5 and 6) were treated with 0.5%
digitonin as described under ``Experimental Procedures.'' 40
µg of proteinase K was added to samples displayed in lanes2, 4, and 6 (after digitonin treatment)
and incubated on ice for 15 min followed by the addition of 1
mM PMSF. The mitochondria and the mitoplasts were reisolated
and analyzed by SDS-PAGE followed by
fluorography.
Newly Imported MTF1 Is Present in the Mitochondrial
Matrix
Next we have fractionated the mitochondria containing the
newly imported MTF1 to ascertain whether it is attached to the inside
of the inner membrane or present in the matrix. The results given in
Fig. 8
show that imported MTF1 is mostly present in the soluble
supernatant indicating its matrix localization. This corresponds to the
distribution of citrate synthase, an authentic matrix component (data
not shown).
Figure 8:
Localization of imported, proteinase K
resistant MTF1. Lane1, 10 µl of reticulocyte
lysate containing the labeled MTF1; lanes2-5,
700 µg of mitochondrial protein were incubated with 140 µl
reticulocyte lysate containing labeled MTF1 in a 700-µl import
reaction buffer containing ATP and NADH at 25 °C for 30 min. After
import, a 100-µl aliquot was removed (lane2).
240 µg of Proteinase K was added to the rest, which was incubated
on ice for 30 min followed by the addition of 1 mM PMSF. A
100-µl aliquot was removed (lane3). The
mitochondria from the remaining 500-µl reaction mix were reisolated
and mixed with 4.5 mg of untreated mitochondrial protein, which was
then subjected to submitochondrial fractionation according to the
method of Daum et al. (7) as follows. The mitochondria were
suspended in a buffer containing 0.1 M sorbitol, 10
mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA,
and 1 mM PMSF and incubated on ice for 20 min followed by
sonication for 15 s at 50 watts. The sonication was repeated 3 times
with 30 s of cooling between the sonications. The resulting extract was
centrifuged at 35,000 rpm for 1 h in Ti-50 rotor at 4 °C to
separate soluble supernatant from the membrane fraction. The proteins
from the soluble supernatant were precipitated by ice-cold
trichloroacetic acid. Equivalent amounts of proteins of membrane
fraction (lane4) and soluble supernatant (lane5) were used. The mitochondria were reisolated from the
samples displayed in lanes2 and 3, and all
of the samples were analyzed by SDS-PAGE followed by
fluorography.
MTF1 Can Be Imported into Mitoplasts
It has
recently been suggested that the outer and inner membranes of
mitochondria contain independent import
machineries
(22, 23) . So we examined whether mitoplasts,
which lack their mitochondrial outer membranes and hence their outer
membrane receptors, are competent to import MTF1. The results given in
Fig. 9A show that AAC can be imported successfully into
mitochondria (lane3) but not into the mitoplasts
(lane5). MTF1, on the other hand, is imported as
efficiently into the mitoplasts (lane5) as it is
into the mitochondria (lane3). The results
illustrated in Fig. 9B show that MTF1 can be imported
into the mitoplasts even at low temperature. The results of the
experiments with mitoplasts indicate that MTF1 can be transported
across the inner membrane of the mitochondria without the need for an
intact outer membrane.
Figure 9:
Import of MTF1 into the mitoplasts.
A, 40 µl of lysate containing labeled protein (either MTF1
or AAC) were incubated with 150 µg of either mitochondria
(lanes2 and 3) or mitoplasts (lanes4 and 5) in a 200-µl import buffer at 25
°C for 30 min. Each import reaction mixture was then divided into
two parts, and one of them (lanes3 and 5)
was treated with 40 µg of proteinase K on ice for 30 min followed
by the addition of 1 mM PMSF. The mitochondria or the
mitoplasts were reisolated and analyzed by SDS-polyacrylamide gel
electrophoresis followed by fluorography. B, 40 µl of
lysate containing the labeled MTF1 were incubated with 150 µg of
mitoplasts in 200 µl of import buffer at different temperatures.
The import reaction mixture was then divided into two equal parts, one
of which (lanes3, 5, and 7) was
treated with 40 µg of proteinase K on ice for 30 min followed by
the addition of 1 mM PMSF. The mitoplasts were reisolated and
analyzed by SDS-PAGE followed by fluorography. C, 80 µl of
lysate containing the labeled MTF1 were incubated with 300 µg of
either mitochondria (mt) (lanes2-5)
or mitoplasts (mp) (lanes6-13) in 400
µl of import buffer either at 25 or 5 °C for 30 min. The import
reaction mix was then divided into four equal parts and treated with
either proteinase K (lanes3, 4, 7,
8, 11, and 12) on ice for 30 min followed by
the addition of PMSF (1 mM final concentration) or trypsin
(lanes5, 9, and 13) at 4 °C
followed by the addition of trypsin inhibitor (10 the
concentration of trypsin). Mitochondria or mitoplasts were reisolated
and analyzed by SDS-PAGE followed by
fluorography.
A careful examination of most of the figures
so far presented indicates that proteinase K treatment modestly reduces
the length of MTF1 after import into either mitochondria or mitoplasts.
Similar reduction was observed when 5 µg of proteinase K instead of
40 µg was used. However, no reduction was observed when 40 µg
of trypsin was used instead of proteinase K (Fig. 9C).
Reduction was also not observed when truncated MTF1 (either
BglII or BamHI fragments) were used for import into
mitochondria (Fig. 6) or mitoplasts (data not shown). When
mitoplasts, prepared from steady-state mitochondria, were treated with
proteinase K followed by blotting and probing with antiMTF1 antibody,
we did not see any reduction in size of the endogenous MTF1 (data not
presented).
Sorting of MTF1 Is Independent of hsp60
There are
two types of sorting pathways for the proteins that are imported into
the mitochondria. The conservative sorting pathway involves
ATP-dependent interaction with the hsp60. The proteins that follow this
pathway have amino-terminal cleavable presequences and interact with
the MOM19 receptor preliminary to their entry into the mitochondria.
The nonconservative sorting pathway does not involve the interaction
with the hsp60. ADP/ATP carrier, which does not have a cleavable
presequence, follows this pathway for its import from outer to the
inner membrane via MOM72
(13, 14, 24) . The role
of hsp60 in the import of MTF1 was examined taking advantage of a
temperature-sensitive mutation in the hsp60 gene
(mna2-1)
(25) . In separate studies, we have shown that
this mutant is defective in the import of several proteins e.g. F subunit of ATP synthase, that are sorted by an
hsp60-dependent pathway.
(
)
MTF1 was imported at
37 °C into the mitochondria isolated from the mna2-1 pet
mutant grown at 37 °C (the nonpermissive temperature). The results
depicted in Fig. 10show that the import of MTF1 in contrast to
that of F
subunit (data not shown) is intact in
mna2-1 mitochondria at the restrictive temperature, indicating
an hsp60-independent pathway for MTF1 import into the mitochondria.
Figure 10:
Import of MTF1 into the mitochondria
isolated from the mna2-1 mutant grown at restrictive
temperature. Identical amounts of labeled MTF1 protein were used to
import (in a 100-µl import reaction buffer) into the mitochondria
(75 µg) isolated from the wild-type yeast 124 (lanes2-5) and the mutant mna2-1 (lanes6-9) grown at 37 °C. The import was carried out
at either 25 °C (lanes2, 3, 8,
and 9) or at 37 °C (lanes4-7) for
30 min. Two identical sets were used for import. One of them after
import was treated with 40 µg of proteinase K (lanes3, 5, 7, and 9) on ice for 30
min. The mitochondria were reisolated and analyzed by SDS-PAGE followed
by fluorography.
DISCUSSION
We have studied the requirements for the mitochondrial import
of the mitochondrial transcription factor. Our results indicate that
there are no specific requirements for this import. There is no
evidence for the requirement of an outer membrane receptor, for an
electrochemical gradient, for ATP, for hsp60. MTF1 is efficiently
imported into trypsin-treated mitochondria and into mitoplasts lacking
the bulk of the outer membrane. The independence of MTF1 import of each
of the factors normally involved in the import of prototypic proteins
destined for mitochondria suggests that this protein may exist in an
extended or loosely folded conformation both in its transport competent
form and perhaps also in its functional form.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.