From the Institut für Physiologische Chemie der
Universität München, Goethestra
e 33, 80336 München, Germany
Proteins of the mitochondrial inner membrane
display a wide variety of orientations, many spanning the membrane more
than once. Some of these proteins are synthesized with
NH2-terminal cleavable targeting sequences
(presequences) whereas others are targeted to mitochondria via internal
signals. Here we report that two distinct mitochondrial targeting
signals can be present in precursors of inner membrane proteins, an
NH2-terminal one and a second, internal one. Using
cytochrome c1 as a model protein, we
demonstrate that these two mitochondrial targeting signals operate
independently of each other. The internal targeting signal, consisting
of a transmembrane segment and a stretch of positively charged amino
acid residues directly following it, initially directs the
translocation of the preprotein into the intermembrane space. It then
inserts into the inner membrane from the intermembrane space side in a

-dependent manner and thereby determines the orientation the protein attains in the inner membrane. Analysis of a
number of other presequence-containing protein of the inner membrane
suggest that they too contain such internal targeting signals.
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INTRODUCTION |
The inner membrane of mitochondria harbors a multitude of
proteins, which display a diverse range of orientations in the
membrane. A few of these proteins are encoded by the mitochondrial
genome, while the rest are encoded by the nucleus (1). The
nuclear-encoded proteins contain mitochondrial targeting signals which
ensure their targeting to mitochondria following their synthesis in the cytosol. Many of these proteins bear NH2-terminal
mitochondrial targeting signals (presequences) which, in addition to
their targeting function, facilitate the early steps in the

-dependent translocation across the inner membrane
(2, 3). Upon import into the matrix these signals are proteolytically
removed by the mitochondrial processing peptidase
(MPP)1 (4, 5). A wide range
of inner membrane proteins, however, do not have such cleavable
presequences, but are targeted to mitochondria by means of internal
signals. Very little is known about the nature of internal targeting
signals, although recently one such signal has been described for the
Bcs1p protein (6). A stretch of positively charged amino acid residues
located directly after the single transmembrane segment of the protein
serves to target Bcs1p to mitochondria, where it attains an
Nout-Cin orientation in the inner membrane.
This stretch has the potential to form an amphipathic helix, displaying
all positive charges on one side and a range of apolar ones on the
other. It is thought that the apolar face of the helical structure
interacts with the neighboring transmembrane segment to form a hairpin
loop structure which penetrates the translocase of the inner membrane
in a 
-dependent fashion (6).
Other proteins of the inner membrane of mitochondria also contain such
positively charged residues immediately following their transmembrane
segments; from the topological arrangement of the proteins these
residues are known to be localized on the matrix side of the inner
membrane. Interestingly some of the proteins containing such putative
internal mitochondrial targeting signals also bear
NH2-terminal cleavable presequences. This observation raises some important questions with regards to the role of internal mitochondrial targeting signals and intra-mitochondrial protein sorting. Can a preprotein contain more than one functional
mitochondrial targeting signal? If so, do these signals operate
independently of each other? What role do the internal signals play in
the attainment of the final orientation of these proteins in the inner
membrane?
To address these questions we analyzed the targeting and import
mechanisms of cytochrome c1. Cytochrome
c1, a subunit of the cytochrome
bc1 complex of the respiratory chain, is
anchored to the inner membrane in an Nout-Cin
orientation via a single transmembrane segment near its COOH terminus
(7). It is synthesized as a precursor protein, precytochrome
c1 which contains an NH2-terminal cleavable bipartite presequence (8, 9). The first part of this
presequence is a mitochondrial targeting signal which becomes proteolytically removed by MPP in the matrix to generate an
intermediate size cytochrome c1 (intermediate
size Cytc1). The second domain of the bipartite
sequence, a hydrophobic sorting sequence, directs the protein to the
inner membrane, whereby the NH2 terminus of the
intermediate size Cytc1 is maintained in the
matrix. Following the covalent addition of heme, intermediate size
Cytc1 undergoes a second processing event,
catalyzed by the Imp2p protease 2 (10), thus resulting in the release
of a free NH2 terminus in the intermembrane space. The
transmembrane segment at the COOH-terminal end of the protein serves to
anchor the protein to the inner membrane with the carboxyl terminus
exposed to the matrix. How this transmembrane segment becomes correctly
sorted during the import pathway of cytochrome
c1 was not clear until now.
We demonstrate here that this COOH-terminal transmembrane segment of
precytochrome c1, together with a stretch of
positively charged amino acids which directly follow it, constitute an
internal mitochondrial targeting signal. Consequently precytochrome
c1 bears two distinct targeting signals, an
NH2-terminal cleavable one and a second internal one. We
show here that these targeting signals operate independently from each
other and are both essential to achieve the efficient import and
attainment of the correct orientation of cytochrome
c1 in the inner membrane.
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EXPERIMENTAL PROCEDURES |
Isolation of Yeast Mitochondria--
Saccharomyces
cerevisiae wild-type strain (D273-10B) was grown in lactate medium
(11) at 30 °C. The Tim22(Gal10) strain (12) was grown at 30 °C in
minimal medium supplemented with 2% lactate, 0.1% glucose and either
in the presence (Tim22
) or absence (Tim22
) of 1% galactose for
five generations. The Tim23(fs) strain and its isogenic wild-type
strain (12) were grown at 24 °C in minimal medium with 2% lactate,
0.1% glucose, and 1% galactose. Cells were harvested at an
OD578 of ~1 and mitochondria were isolated, as described
previously (11). Isolated mitochondria were resuspended in 250 mM sucrose, 10 mM MOPS, pH 7.2, 1 mM EDTA (SEM buffer) at a protein concentration of 10 mg/ml.
Recombinant DNA Techniques and Plasmid Constructions--
The
recombinant DNA techniques applied were as described by Sambrook
et al. (13). Yeast precytochrome c1
was used in this analysis. All the cytochrome
c1-DHFR variants used were cloned by obtaining
the required cytochrome c1 regions through
designed polymerase chain reaction and ligating them before DHFR as
EcoRI/BamHI fragments. A DHFR derivative,
DHFRmut was used which bears a number of point mutations,
whereby Cys-7, Ser-42, and Asn-49 were replaced by Ser, Cys, and Cys,
respectively (14).
Precursor Proteins--
DNA encoding precursor proteins were
cloned in pGEM vectors and were transcribed with SP6 RNA polymerase.
All precursor proteins were then synthesized in rabbit reticulocyte
lysate (Promega Corp.) in the presence of [35S]methionine
(15).
Import into Mitochondria--
Import was performed essentially
as described (6). Import mixtures contained 2 mM NADH, 2 mM ATP, 0.25 mg/ml mitochondrial protein, and 1-2% (v/v)
reticulocyte lysate containing the radiolabeled precursor proteins and
was performed at 25 °C for the times indicated. Following import,
samples were treated with proteinase K (40 µg/ml) for 30 min at
0 °C either under nonswelling or hypotonic swelling conditions (see
below) (6). Samples were analyzed by SDS-PAGE and immunoblotting onto
nitrocellulose. The efficiency of swelling of the mitochondria was
assessed following immunodecoration of the blot with antisera against
endogenous cytochrome c peroxidase (soluble intermembrane
space protein) and Mge1p (matrix protein).
Depletion of Matrix ATP--
Isolated mitochondria were depleted
of their free matrix ATP, as described previously (16). Briefly,
mitochondria following dilution in import buffer were incubated at
25 °C for 3 min and then oligomycin (20 µM),
carboxyatractyloside (5 µM), and NADH (2 mM)
were successively added at 3-min intervals. When matrix ATP levels were
to be restored in depleted mitochondria an alternative depletion
protocol using oligomycin and apyrase was used as described before
(17). Matrix ATP was replenished by incubating the reisolated mitochondria in the presence of 4 mM ATP.
Miscellaneous--
Protein determination, SDS-PAGE, and Western
blotting were performed according to the published methods of
Bradford (18), Laemmli (19), Towbin et al. (20),
respectively. The detection of proteins after blotting onto
nitrocellulose was performed using the ECL detection system
(Amersham).
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RESULTS |
The COOH Terminus of Cytochrome c1 Contains an Internal
Mitochondrial Targeting Signal--
Analysis of the amino acid
sequence of cytochrome c1 indicated the presence
of a positively charged region immediately after the transmembrane
segment at the COOH-terminal end of the protein. This sequence
displayed the potential to form an amphipathic
-helical structure
with positively charged residues on one side and an abundance of apolar
amino acids on the opposite side (Fig.
1A). This arrangement of amino
acid residues resembles the internal mitochondrial targeting signal
recently described for the inner membrane protein, Bcs1p (6). To
address whether this region could also function as such an internal
import signal, a chimeric protein,
Cytc1-(TM-C)-DHFR, consisting of the final 63 residues of the COOH-terminal region of cytochrome
c1 fused to a mutated form of DHFR was
constructed (Fig. 1B). This region of cytochrome c1 (amino acid 247-309) encompasses the
transmembrane segment flanked by 26 residues at its NH2
terminus and directly followed by the positively charged segment (amino
acid residues 288-303), together with the final 6 residues of
cytochrome c1. The mutated form of DHFR bears a
number of point mutations which cause a destabilization in the folded
structure of the protein whereby in contrast to its wild-type
counterpart, the mutated DHFR remains sensitive to added proteases
(14).

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Fig. 1.
The COOH-terminal region of cytochrome
c1 contains an internal mitochondrial targeting
signal. A, -helical plot of amino acid residues 287-304
of cytochrome c1, + denotes positively charged
amino acids, apolar residues are circled. B, fusion protein Cytc1-(TM-C)-DHFR. The black area
denotes the transmembrane domain (amino acids 273-287), the
zigzag line denotes the internal targeting sequence (amino
acids 288-303), DHFR, mouse cytosolic dihydrofolate reductase (mutated
derivative, see "Experimental Procedures"). C, schematic
representation of the topology of
Cytc1-(TM-C)-DHFR in the mitochondrial inner
membrane after import. OM, outer mitochondrial membrane;
IM, inner mitochondrial membrane; IMS,
intermembrane space. D, radiolabeled
Cytc1-(TM-C)-DHFR (28 kDa) was imported into
isolated mitochondria (upper panel) or mitoplasts
(lower panel) for 10 min either in the presence (+ NADH) or
absence (+ valinomycin, val) of a membrane potential. After
import mitochondria and mitoplasts were reisolated, either mock-treated
or treated with proteinase K (PK, 40 µg/ml) under
nonswelling or swelling conditions, as indicated. All samples were
analyzed by SDS-PAGE and blotted onto nitrocellulose. Opening of the
intermembrane space was >95% efficient while the integrity of the inner membrane was not perturbed (data
not shown). 25 kDa, fragment generated of
Cytc1-(TM-C)-DHFR by proteinase K treatment of
mitoplasts; Std, 30% of the amount of radiolabeled
precursor added to each reaction.
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If imported and sorted by the same mechanism as authentic cytochrome
c1, Cytc1-(TM-C)-DHFR
(molecular mass 28 kDa) should be located in the inner membrane with an
Nout-Cin orientation (Fig. 1C).
Radiolabeled Cytc1-(TM-C)-DHFR was indeed
imported in a membrane potential (
)-dependent manner
into both isolated mitochondria and mitoplasts (Fig. 1D).
Hypotonic swelling of mitochondria following import combined with
protease treatment resulted in the degradation of the N-tail exposed to
the intermembrane space and protection of a larger fragment
corresponding to DHFR plus the transmembrane segment (approximately 25 kDa). A similar fragment was observed upon protease treatment after
import into mitoplasts. This result indicates that the COOH-terminal
DHFR had been imported across the inner membrane and had attained the
predicted Nout-Cin orientation.
It appeared necessary to exclude the possible existence of a cryptic
targeting signal in the passenger protein used above, the mutated form
of DHFR (DHFRmut). Furthermore, it was important to verify
that the positively charged amino acid segment (residues 288-303)
together with the neighboring transmembrane segment, were responsible
for the mitochondrial targeting observed. To this end, the following
set of chimeric proteins were constructed (Fig.
2A).

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Fig. 2.
Localization of the internal mitochondrial
targeting signal. A,
Cytc1-(TM-C)-DHFR-derived fusion proteins. The
black area denotes the transmembrane domain (amino acids
273-287), the zigzag line denotes the COOH-terminal
targeting sequence (amino acids 288-303), DHFR, see Fig.
1B; N-BCS1, amino acids 83-456 of the BCS1 protein.
B, the radiolabeled fusion proteins depicted in A
were imported into isolated mitochondria for 15 min at 25 °C either
in the presence (+ NADH) or absence (+ Val) of a
membrane potential. After import mitochondria were reisolated, either
mock-treated or proteinase K-treated under nonswelling or swelling
conditions, as indicated. All samples were analyzed by SDS-PAGE and
blotted onto nitrocellulose. Opening of the intermembrane space was
>95% efficient while the integrity of the inner membrane was not
perturbed (data not shown). Std, 30% of the amount of
radiolabeled precursor added to each reaction.
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The first construct, DHFRmut, was not imported to a
protease protected location, although it became associated with
mitochondria (Fig. 2B). Thus this unfolded mutated
derivative of DHFR does not contain a cryptic import signal. Second, a
Cytc1-(TM-C)-DHFR derivative in which the
transmembrane segment was deleted,
Cytc1-(
TM-C)-DHFR was also not imported into
isolated mitochondria (Fig. 2B). Third, placement of the
positively charged residues (residues 288-303) of cytochrome
c1 at the NH2 terminus of DHFR,
Cytc1-(288-309)-DHFR resulted in the efficient
import into mitochondria in a 
-dependent manner where
it was located in the matrix (Fig. 2B). Thus this stretch of
positively charged amino acids can act as an NH2-terminal mitochondrial targeting sequence. In a further derivative, the DHFR
moiety of Cytc1-(TM-C)-DHFR was exchanged for
another passenger protein, the matrix domain of Bcs1p (
N-BCS1). This
domain of Bcs1p does not contain any targeting information, as
previously demonstrated (6). The resulting fusion protein,
Cytc1-(TM-C)-(
N-BCS1), was efficiently
imported into mitochondria (Fig. 2B).
Taken together these observations rule out the possibility that a
cryptic import signal in DHFR becomes exposed when placed COOH-terminal
to the cytochrome c1 segment. Rather, the
positively charged segment of residues 288-303 of predicted
-helical structure together with the transmembrane segment functions
as an internal signal. We propose that this targeting signal forms a
loop structure in which the apolar face of the amphipathic
-helix
interacts with the hydrophobic transmembrane segment, similar to the
targeting signal of the Bcs1p (6).
The Complete Bipartite Presequence of Cytochrome c1
Together with the COOH-terminal Targeting Signal Leads to Attainment of
Correct Orientation--
Does this COOH-terminal internal
mitochondrial targeting signal function as a second independent
targeting signal in the cytochrome c1
preprotein? To address this question a series of fusion proteins were
constructed where the COOH-terminal segment followed by the DHFR moiety
(Cytc1-(TM-C)-DHFR) was fused to
NH2-terminal regions of precytochrome
c1 (Fig.
3A). When fused behind the
complete bipartite presequence,
pCytc1(1-64)-(TM-C)-DHFR, efficient import and
MPP processing of the cleavable matrix-targeting sequence was observed.
Hypotonic swelling in the presence of added protease resulted in the
degradation to a fragment (Fig. 3B, f) which could be
immunoprecipitated with DHFR specific antiserum (results not shown).
Thus the DHFR domain was located in the matrix, while the linker of
hydrophilic amino acid residues between the bipartite presequence and
the transmembrane segment remained in the intermembrane space,
accessible to the added protease (depicted in Fig. 3C). A
similar orientation was achieved after the import of
pCytc1(1-309)-DHFR, a chimeric protein of the
complete precytochrome c1 protein fused to DHFR
(Fig. 3, B and C). In contrast, when fused to
only the matrix-targeting domain of the cytochrome
c1 bipartite presequence pCytc1(1-35)-(TM-C)-DHFR, import into isolated
mitochondria in a 
-dependent fashion was observed
together with maturation by MPP (Fig. 3B). This chimeric
protein was, however, located completely in the matrix; the
NH2-terminal presequence had apparently overridden the
internal targeting and sorting signals as the
Nout-Cin orientation in the inner membrane had
not been achieved (depicted in Fig. 3C).

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Fig. 3.
The complete bipartite presequence of
cytochrome c1 together with the COOH-terminal
targeting signal leads to attainment of correct orientation in the
inner membrane. A,
Cytc1-(TM-C)-DHFR-derived fusion proteins
containing different regions of the NH2-terminal cleavable
presequence. The black area denotes the transmembrane domain
(amino acids 273-287), the zigzag line denotes the
NH2-terminal matrix-targeting signal (amino acids 1-28) or
the COOH-terminal targeting sequence (amino acids 288-303),
respectively; the shaded area denotes the hydrophobic
segment of the NH2-terminal bipartite presequence; DHFR,
see Fig. 1B; Imp2, inner membrane protease 2. B, the radiolabeled fusion proteins depicted in A
were imported into isolated mitochondria for 10 min as described in the
legend to Fig. 2B. p, precursor; i,
intermediate species; m, mature species; f,
COOH-terminal fragment generated by proteinase K treatment of
mitoplasts. Note, the lysate of
pCytc1(1-35)-(TM-C)-DHFR also contains a second
translation product, indicated by an asterisk (*), and
corresponds to Cytc1-(TM-C)-DHFR. C, schematic
representation of the topologies of the different fusion proteins
depicted in A in the mitochondrial inner membrane after
import. Abbreviations as in Fig. 1C.
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Thus, the COOH-terminal transmembrane segment can operate as an
internal targeting signal and become correctly sorted across the inner
membrane in the presence of the complete NH2-terminal bipartite presequence. In the absence of the hydrophobic sorting signal, i.e. in the presence of the NH2-terminal
matrix-targeting signal alone, the preprotein becomes targeted
completely to the matrix. Thus the COOH-terminal transmembrane segment
appears not to have the capacity to act as a reinsertion signal from
the matrix side of the inner membrane.
The Two Mitochondrial Targeting Signals of Cytochrome
c1 Act Independently of Each Other--
Can these two
targeting signals operate independently of each other and sequentially,
to achieve the correct orientation of cytochrome
c1 in the inner membrane?
In an attempt to dissect the operation of the two targeting signals, a
kinetic analysis of the import and sorting of radiolabeled pCytc1(1-64)-(TM-C)-DHFR into isolated
mitoplasts was performed at 12 °C. Rapid processing by MPP was
observed, indicating translocation of the NH2-terminal
region of pCytc1(1-64)-(TM-C)-DHFR across the
inner membrane (Fig. 4A). At
these early times the DHFR moiety remained protease accessible,
demonstrating it had not yet undergone translocation across the inner
membrane. Completion of import of this COOH-terminal domain was
observed at later times of incubation (depicted in Fig. 4B).
A similar kinetic dissection of NH2-terminal and
COOH-terminal targeting and import events was also achieved during the
import of pCytc1(1-309)-DHFR (results not
shown).

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Fig. 4.
Kinetic analysis of the import and sorting of
pCytc1(1-64)-(TM-C)-DHFR into mitoplasts at
12 °C. A, radiolabeled
pCytc1(1-64)-(TM-C)-DHFR was imported into mitoplasts at
12 °C for the times indicated. Samples were cooled on ice and were
either mock-treated to detect MPP processing, or treated with
proteinase K to remove non-inserted species or to generate the
COOH-terminal 25-kDa fragment of the inserted species, respectively.
Samples were analyzed by SDS-PAGE, fluorography, and laser
densitometry. B, schematic representation of the insertion
of the COOH terminus of pCytc1(1-64)-(TM-C)-DHFR into the
inner membrane. Abbreviations as in Fig. 1C.
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In conclusion, the internal mitochondrial targeting signal of
cytochrome c1 can act independently of the
NH2-terminal cleavable presequence. In addition, the
COOH-terminal transmembrane segment of cytochrome
c1 does not operate as a signal for export
following prior import into the matrix. Instead, it functions as an
insertion signal from the intermembrane space side of the inner
membrane, penetrating the inner membrane in a

-dependent fashion.
Translocation of the COOH Terminus into the Matrix Requires the
ATP-dependent Activity of mt-Hsp70--
Import of
preproteins across the inner membrane into the matrix is facilitated by
the ATP-dependent binding of mt-Hsp70. To address whether
the import and sorting of the COOH-terminal region of cytochrome
c1 requires mt-Hsp70, the import of the
DHFR-derived fusion proteins was analyzed in ATP-depleted mitochondria.
Matrix ATP depletion did not have an adverse effect on the import of Cytc1-(TM-C)-DHFR,
pCytc1(1-64)-(TM-C)-DHFR, and
pCytc1(1-309)-DHFR; the efficiency of import
was comparable to that in the control ATP-containing mitochondria (Fig.
5A). In contrast, hypotonic swelling of the mitochondria following import revealed that the translocation of the COOH-terminal DHFR into the matrix, facilitated by
the internal targeting signal, was severely inhibited in the absence of
the ATP-dependent activity of mt-Hsp70 (Fig. 5A,
f).

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Fig. 5.
Matrix ATP dependence of the insertion of the
COOH terminus. A, isolated mitochondria were incubated in
import buffer and were either depleted of matrix ATP ( ATP) or mock-treated (+ ATP), as described under
"Experimental Procedures." Samples were cooled on ice and
radiolabeled preproteins (depicted in Fig. 3A) were imported
for 5 min at 25 °C. After import, mitochondria were reisolated,
either mock-treated or proteinase K treated under nonswelling or
swelling conditions as indicated. Opening of the intermembrane space
was >95% efficient while the integrity of the inner membrane was not
perturbed (data not shown). Abbreviations are as described in the
legend to Fig. 3B. B, isolated mitochondria were depleted of
matrix ATP by an incubation with oligomycin and apyrase. Radiolabeled
preproteins were added and import was performed for 5 min at 25 °C.
Following a trypsin treatment (100 µg/ml) to remove non-imported
preprotein, mitochondria were reisolated, washed, resuspended in import
buffer, and divided into three aliquots. Further incubation was
performed for 15 min on ice (no chase) or at 25 °C after
replenishing of matrix ATP levels in the presence (chase
+ ) or absence (chase   ) of the membrane
potential. Mitochondria from all three samples were then reisolated and
subjected to a proteinase K treatment either under nonswelling or
swelling conditions. Samples were analyzed by SDS-PAGE, fluorography,
and laser densitometry. "Fragment in mitoplasts" represents the
amount of COOH-terminal 25-kDa fragment generated, expressed as % of total imported species.
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Consequently in the presence of the bipartite presequence or when
acting alone, the Cytc1-(TM-C)-DHFR-derived
fusion proteins can be imported into mitochondria in a
mt-Hsp70-independent manner, where their DHFR moiety accumulates in the
intermembrane space. Although translocation of the
NH2-terminal presequence leading to MPP processing can
occur independently of mt-Hsp70, passage of the COOH-terminal segments
across the inner membrane requires productive mt-Hsp70.
Once accumulated in the intermembrane space, such "ATP-depletion
intermediates" were further transported across the inner membrane
upon restoration of matrix ATP levels (Fig. 5B). The efficiency of this chase reaction was not affected by the prior dissipation of the membrane potential by valinomycin (Fig.
5B). Thus the internal targeting signal can penetrate the
inner membrane to beyond the 
-dependent step of
import without requiring mt-Hsp70. Upon doing so it becomes stabilized
and does not undergo retrograde translocation out of the inner
membrane. This insertion step may be sufficient to ensure the
translocation of the short COOH-terminal domain of the authentic
cytochrome c1 (22 amino acids); import and
sorting of cytochrome c1 has been reported to
occur in a matrix-ATP independent manner (21). On the other hand,
further translocation of the larger DHFR moiety at the COOH terminus
across the inner membrane requires the action of the ATP-driven
mt-Hsp70, but no longer the 
.
The Role of the Tim Translocases of the Inner Membrane in the
Translocation of the Internal Targeting Signal of Cytochrome
c1--
The mitochondrial inner membrane contains at least
two distinct preprotein translocases, which differ in their substrate
specificity. The Tim17/Tim23 translocase mediates the import of
NH2-terminal presequence-targeted preproteins and its
function is tightly coupled to that of the ATP-dependent
chaperone, mt-Hsp70 (22-25). On the other hand, insertion of proteins
of the carrier family into the inner membrane is facilitated by a
second translocase, of which the recently identified Tim22 is a
component (12). Does Tim22 translocase represent a general import site
for proteins with internal targeting signals?
In the yeast strain Tim22(Gal10), the Gal10 promoter was integrated
into the chromosome before the Tim22 gene, thus allowing regulated
expression of Tim22 by galactose (12). Mitochondria were isolated from
Tim22(Gal10) cells grown for five generations in the presence
(Tim22
) or absence of galactose (Tim22
). Tim22
mitochondria
display a strongly impaired ability to import the ADP/ATP carrier
protein (Fig. 6A and Ref. 12).
Import and correct sorting of Cytc1-(TM-C)-DHFR,
like the control NH2-terminal presequence-containing preprotein, pSu9(1-79)-DHFR, were unaffected in Tim22
mitochondria (Fig. 6A and Ref. 12). This result indicates that Tim22 is
not involved in the recognition and translocation of the internal targeting signal of cytochrome c1.

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Fig. 6.
The role of the Tim translocases of the inner
membrane in the translocation of the internal targeting signal of
cytochrome c1. A, isolated Tim22
and Tim22 mitochondria were preincubated for 5 min at 25 °C in
the presence of an ATP regenerating system. Samples were cooled on ice
and radiolabeled preproteins were imported at 25 °C. After import,
samples were cooled on ice and treated with proteinase K to remove
non-imported species. Samples were analyzed by SDS-PAGE, fluorography,
and laser densitometry. (Import into Tim22 mitochondria was set to
100%.) B, isolated Tim23(fs) and isogenic wild-type
mitochondria were preincubated for 30 min at 37 °C in the presence
of an ATP regenerating system to induce the ts phenotype of
the Tim23(fs) mitochondria. Samples were cooled on ice and radiolabeled
preproteins were imported at 25 °C. After import, samples were
cooled on ice and treated with proteinase K to remove non-imported
species. Samples were analyzed by SDS-PAGE, fluorography, and laser
densitometry. (Import into wild-type mitochondria was set to 100%.)
AAC, ADP/ATP carrier protein.
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To address the role of the Tim23/Tim17 translocase in this event,
Cytc1-(TM-C)-DHFR was imported into Tim23(fs)
mitochondria. These mitochondria harbor a variant of Tim23, bearing a
short COOH-terminal deletion due to a translation frameshift after
methionine 178 (12). Import of NH2-terminal
presequence-targeted preproteins into Tim23(fs) mitochondria is
drastically reduced, whereas the import of ADP/ATP carrier protein and
other members of the carrier family is as efficient as in wild-type
mitochondria (Fig. 6B and Ref. 12). Both import and sorting
of Cytc1-(TM-C)-DHFR, however, were unaffected
in mitochondria isolated from the Tim23(fs) mutant (Fig.
6B). Furthermore, treatment of mitoplasts prior to import to
remove the NH2-terminal hydrophilic domain of Tim23 which
is exposed to the intermembrane space, results in the inhibition of
both MPP processing and import of presequence-targeted preproteins; import of Cytc1-(TM-C)-DHFR in contrast was
unaffected by this treatment (results not shown). Taken together, these
results indicate that the import of the internal targeting signal of
cytochrome c1 does not display the same
requirements for Tim23 as presequence-targeted preproteins.
The COOH-terminal Targeting Signal Alone Is Not Sufficient to
Import Cytochrome c1 into Mitochondria--
It could be
speculated that the internal mitochondrial targeting signal present at
the COOH-terminal region is necessary and sufficient for sorting of
cytochrome c1 across both the outer and inner
membrane. Indeed cytochrome c1 from the
non-pathogenic trypanosomatids Crithidia fasciculata and
Bodo caudatus were reported to be synthesized without
NH2-terminal presequences (26). Is the
NH2-terminal presequence of cytochrome
c1 dispensable for import of yeast cytochrome
c1? We tested the ability of the internal mitochondrial targeting signal to import fusion proteins containing increasing lengths of the mature cytochrome c1
sequence at their NH2 terminus (Fig.
7A). The internal
mitochondrial targeting signal of these fusion proteins conferred
import of NH2-terminal regions of cytochrome
c1, with low, but distinct efficiency, its
capacity being reached at approximately 70 amino acid residues (Fig.
7B). Longer proteins become partially imported, whereby a
segment corresponding to the DHFR moiety is found in the matrix
(results not shown).

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Fig. 7.
The COOH-terminal targeting signal alone is
not sufficient to import cytochrome c1 into
mitochondria. A, fusion proteins derived from
Cytc1-(TM-C)-DHFR containing increasing lengths of the
mature cytochrome c1 sequence at their
NH2 terminus. The black area denotes the
transmembrane domain (amino acids 273-287), the zigzag line
denotes the COOH-terminal targeting sequence (amino acids 288-303).
Abbreviations as in Fig. 1B. B, kinetics of import of the
fusions proteins depicted in A. Radiolabeled fusion proteins were imported at 25 °C into isolated mitochondria for the times indicated. Samples were cooled on ice and treated with proteinase K to
remove non-imported species. Samples were analyzed by SDS-PAGE, fluorography, and laser densitometry.
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Thus the internal mitochondrial targeting signal can mediate the
initial import into the intermembrane space of only a limited COOH-terminal region of cytochrome c1. We
predict the additional NH2-terminal presequence is
essential to achieve complete import of the NH2-terminal
region of yeast cytochrome c1.
 |
DISCUSSION |
We describe here a novel mechanism of topogenesis of a precursor
protein destined for the inner membrane of mitochondria which contains
two distinct targeting signals, an NH2-terminal cleavable one and an internal one. These signals mediate the

-dependent insertion of different segments of the
preprotein across the inner membrane from the intermembrane space and
operate independently of each other. They play an essential role in the
intramitochondrial sorting, as only together they ensure the attainment
of the correct orientation of the protein in the mitochondrial inner
membrane.
The internal signal of cytochrome c1 is a
composite one; it consists of the transmembrane segment and the
positively charged stretch of amino acids directly following it. This
signal directs import of the COOH-terminal region of precytochrome
c1 initially into the intermembrane space (Fig.
8). In the presence of a 
, the
internal targeting sequence inserts across the inner membrane, presumably by adopting a hairpin loop structure. This is similar to the
internal import signal described for the Bcs1p (6). The internal
targeting signal operates from the intermembrane space side of the
inner membrane to sort the COOH-terminal region of cytochrome
c1 to the matrix and to anchor the transmembrane segment in the inner membrane. Our present evidence would speak against
the transmembrane segment operating as an export signal from the
matrix. Indeed if targeted to the matrix by an NH2-terminal matrix targeting sequence, the protein remains in the matrix and appears incompetent for subsequent sorting. This is in contrast to a
number of other inner membrane proteins whose transmembrane segments
have been demonstrated to facilitate export of hydrophilic segments
from the matrix to the intermembrane space (27-29).

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Fig. 8.
Model of the topogenesis of cytochrome
c1. See text for details. The black
area denotes the transmembrane domain (amino acids 273-287), the
zigzag line the COOH-terminal targeting sequence (amino
acids 288-303), the shaded area denotes the hydrophobic segment of the NH2-terminal bipartite presequence.
Abbreviations: IMS, intermembrane space; Imp2,
inner membrane protease 2;  , membrane potential; H,
heme group.
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The mechanisms of import and sorting of a number of
presequence-targeted inner membrane proteins appear to adhere to the
same principle. They contain potential internal mitochondrial targeting signals COOH-terminal to their second transmembrane segment, in addition to their NH2-terminal presequences. These proteins
include, for example, Yta10p (Afg3p), Yta12p (Rca1p) (30-33), and the
recently described Shy1p protein (34). Interestingly, Shy1p which spans the inner membrane twice appears to be divided into two functional domains. Both domains can be separately expressed to form a functional protein (34). This finding implies that both of these domains contain
their own independent mitochondrial targeting and submitochondrial sorting signals. We propose therefore that the positively charged stretch of amino acids located directly COOH-terminal to the second transmembrane segment of Shy1p forms an independent internal targeting signal, similar to that described here for cytochrome
c1.
Which protein translocase facilitates the passage of these internal
targeting signals across the inner membrane? Insertion of the internal
targeting signal of cytochrome c1 occurs in
mitochondria in which the Tim22 translocase was strongly reduced such
that members of carrier family were no longer imported; thus Tim22 does
not operate as a general translocase for those preproteins containing
internal targeting signals. Furthermore, the internal targeting signal
of cytochrome c1 does not display requirements for Tim23 which are similar to those of presequence-containing preproteins. The latter, in contrast, require the presence of the large
NH2-terminal hydrophilic domain of Tim23, suggesting it may
act as a presequence receptor. Our data do not exclude the possibility
that a membrane spanning domain of Tim23, which may be protected in the
trypsin pretreated mitoplasts, may suffice for the passage of the
internal targeting signal of cytochrome c1.
Alternatively, these data may suggest that Tim17 is involved in
mediating the recognition and translocation across the inner membrane
of these internal targeting signals in a manner independent of the
Tim23.
Finally, how can the observation of cytochrome
c1 containing a second independent targeting
signal be reconciled with earlier models of cytochrome
c1 sorting? Previously it had been suggested that the cytochrome c1 was first imported via
the matrix ("conservative sorting" model) (35). Together with the
NH2-terminal sorting signal, the transmembrane segment was
postulated to reinsert into the inner membrane from the matrix
resulting in the export of hydrophilic segment between them. We propose
the following alterations to the model of cytochrome
c1 sorting to take account of the observations made here (Fig. 8). Cytochrome c1 undergoes two
distinct sorting events to gain its final orientation in the inner
membrane. The COOH-terminal sorting event as described above occurs
from the intermembrane space and hence does not require prior import
into the matrix. The events in the sorting of the NH2
terminus are presently unresolved (Fig. 8). A number of lines of
evidence are in accordance with the NH2-terminal cleavable
sorting signal being imported into the matrix (35-38), however, it has
also been argued that the sorting signal becomes arrested in the TIM
machinery during the import step ("stop-transfer" model) (21, 39).
Whereas the model of conservative sorting may adequately describe the sorting of a number of nuclear-encoded proteins (27-29), both it, and
a stop-transfer type of model may be too simplistic to describe the
complex reactions in the sorting process of cytochrome
c1. We favor a model where the
NH2-terminal sorting signal does not become arrested during
import, but rather passes into the matrix by a default mechanism. Fig.
8 illustrates these alternative mechanisms which need to be addressed
in further experiments to study the multistep processes of cytochrome
c1 topogenesis.
We thank Sandra Weinzierl for excellent
technical assistance. We are grateful to Dr. Michael Brunner and
Christian Sirrenberg for making the Tim22(Gal10) and Tim23(fs) strains
available.