(Received for publication, October 27, 1994; and in revised form, December 9, 1994)
From the
We have investigated the unusual import pathway of cytochrome c oxidase subunit Va (COXVa) into the yeast mitochondrial inner membrane by use of mutants that lack import receptors or are defective in matrix hsp70. (i) Mitochondria lacking the receptor MOM72 are not impaired in import of COXVa. Mitochondria lacking the main receptor MOM19 are moderately reduced in import of COXVa; this, however, is caused by a reduction of the inner membrane potential and not by a lack of specific receptor functions. (ii) Mitochondria defective in the unfoldase function of matrix hsp70 efficiently import COXVa, whereas mitochondria defective in the translocase function of the hsp70 are blocked in import of COXVa. A COXVa construct where the internal hydrophobic sorting signal is placed close to the presequence does not require either hsp70 function.
These results demonstrate that import of COXVa does not require MOM19 or MOM72, but they unexpectedly reveal a strong dependence on the translocase function of matrix hsp70. Two important implications about the characterization of mitochondrial protein import in general are obtained. First, the interpretation of import results with mutants lacking MOM19 have to consider effects on the membrane potential. Second, the distance between a matrix targeting sequence and a hydrophobic sorting sequence within a precursor appears to determine if the inner membrane sorting machinery can substitute for the translocase function of hsp70 or not.
The majority of nuclear-encoded mitochondrial preproteins are
synthesized with amino-terminal presequences that are characterized by
a prevalence of positively charged amino acid residues and the
potential to form an amphipathic -helix. The presequences direct
translocation of the polypeptide chains into or across the
mitochondrial inner membrane (``matrix targeting sequence'')
and are cleaved off by the processing peptidase in the matrix (Douglas et al., 1986; Hartl et al., 1989; Horwich, 1990;
Baker and Schatz, 1991; Pfanner et al., 1994). Nearly all
these cleavable preproteins analyzed so far require receptor proteins
on the outer membrane surface and the heat shock protein hsp70 in the
matrix (mt-hsp70) (
)for import into mitochondria.
Initial characterization of the biogenesis of subunit Va of cytochrome c oxidase (COXVa) suggested an exceptional behavior. Import of COXVa (i) was not inhibited by pretreatment of mitochondria with a protease known to degrade the surface receptors (Miller and Cumsky, 1991) and (ii) was not inhibited by a mutation in mt-hsp70 (Miller and Cumsky, 1993). COXVa appears to follow a novel targeting and sorting pathway into the mitochondrial inner membrane. However, alternative possibilities to explain the surprising results obtained with the import of COXVa are as follows. (i) The treatment of isolated mitochondria with protease not only removes the surface receptors MOM19 (MAS20) and MOM72 (MAS70), but may open cryptic import sites that allow non-physiological import of certain preproteins. Therefore, an import inhibition by removal of the surface receptors may be partially or completely reversed. To exclude this possibility, a selective removal of surface receptors is needed. (ii) The import of COXVa was not affected by a mutation in mt-hsp70 that inhibits import of other mitochondrial preproteins (Kang et al., 1990; Miller and Cumsky, 1993). A recent analysis assigned two distinct functions to mt-hsp70 in import of preproteins, facilitation of unfolding of preproteins during translocation (unfoldase function), and actual translocation across the inner membrane independently of the folding state of preproteins (translocase function) (Gambill et al., 1993; Voos et al., 1993). As the mutant mitochondria used were found to be solely defective in the unfoldase function (Gambill et al., 1993), it cannot be excluded that import of COXVa depends on the translocase function of mt-hsp70.
Here we tried to answer these questions about the import pathway of COXVa and to set its import into context with the general mitochondrial import pathway. To circumvent possible limitations of a biochemical analysis of the role of import receptors, we studied the biogenesis of COXVa with Saccharomyces cerevisiae mutants selectively lacking either MOM19 or MOM72 (Moczko et al., 1994). Moreover, we analyzed COXVa import in a temperature-sensitive S. cerevisiae strain defective in the translocase function of mt-hsp70 (Gambill et al., 1993). We show that import of COXVa (i) is indeed independent of the receptor functions of MOM19 or MOM72, but (ii) strictly requires the translocase function of mt-hsp70. A comparison of COXVa import with the general mitochondrial import pathway leads to important implications on the characterization of receptor mutants and the mechanisms of inner membrane protein sorting.
Import
of COXVa into mitochondria from a strain lacking MOM72 was analyzed.
The import occurred with comparable efficiency into MOM72 and
wild-type mitochondria (Fig. 1A, columns1 and 2). As controls for the specific effect of the
deletion of MOM72, we show that import of the non-cleavable preprotein
ADP/ATP carrier is inhibited (Fig. 1A, column4), while the import of cleavable preproteins is slightly
or not inhibited (Moczko et al., 1993, 1994). Cleavable
preproteins used were the
-subunit of the mitochondrial processing
peptidase (
-MPP) and a fusion protein between the presequence of
F
-ATPase subunit 9 and dihydrofolate reductase (Su9-DHFR) (Fig. 1A, columns6 and 8).
Figure 1:
Import of COXVa into
mutant mitochondria lacking MOM72 or MOM19. A, COXVa import
into MOM72 mitochondria occurs with wild-type efficiency.
Isolated, energized (2 mM ATP, 2 mM NADH)
mitochondria from wild-type (WT) or
MOM72 yeast (50
µg of protein) were incubated for 5 min at 25 °C with rabbit
reticulocyte lysates containing the radiolabeled precursor proteins of
COXVa, the ADP/ATP carrier (AAC),
-MPP, or Su9-DHFR.
Import was stopped with 1 µM valinomycin, and after
protease treatment (200 µg/ml proteinase K) of the mitochondria,
reisolation, and separation on SDS-polyacrylamide gels, fluorograms
were analyzed by densitometry. The import into wild-type mitochondria
was set to 100%, respectively. B, a moderate import reduction
of COXVa in
MOM19 mitochondria. The experiment was performed with
the precursors of COXVa,
-MPP, and Su9-DHFR as described above,
except that
MOM19 mitochondria were employed and a protease
treatment was omitted. A similar relation of import into wild-type and
MOM19 mitochondria was observed, when the mitochondria were
treated with proteinase K after the import reaction. p,
precursor; i, intermediate; m, mature form of a
protein.
The import of COXVa into MOM19 mitochondria, however, was
moderately reduced to about two-thirds of the import efficiency into
wild-type mitochondria (Fig. 1B, lanes1 and 2). The reduction was significant as determined from
the standard error of the means of several independent experiments (Table 2). The import of preproteins such as
-MPP and
Su9-DHFR, which are known to be imported via MOM19 (Moczko et
al., 1993), was more strongly inhibited by a deletion of MOM19 (Fig. 1B, lanes 3-6; Table 2).
Figure 2:
Assessment of the membrane potential in
mitochondria lacking MOM19. The membrane potential of
isolated
MOM19 and wild-type mitochondria was determined by
fluorescence photometry with the potential-sensitive dye
DiSC
(5) as described under ``Materials and
Methods.'' A pronounced fluorescence quenching (decrease) after
addition of the mitochondria (100 µg of protein) compared to the
levels obtained with 1 µM valinomycin/1 mM KCN
(in order to dissipate the membrane potential) indicates a high
.
Different
preproteins can have different requirements for the magnitude of the
membrane potential needed to drive import (Martin et al.,
1991). It can be argued that import of COXVa has the same dependence on
MOM19 as import of -MPP and Su9-DHFR and the weaker inhibition of
its import into
MOM19 mitochondria is just due to a lower
-requirement. To exclude this, we asked how the
-dependence of COXVa import was related to that of
-MPP
and Su9-DHFR. Wild-type mitochondria were partially uncoupled by
limiting concentrations of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). The higher the CCCP
concentration needed for a half-maximal inhibition of import, the lower
is the membrane potential needed for import (Martin et al.,
1991). COXVa import showed a CCCP inhibition curve similar to that of
-MPP, whereas Su9-DHFR could still be imported at lower membrane
potentials (Fig. 3). Therefore, the
-dependence of
COXVa is comparable to that of
-MPP and even higher than that of
Su9-DHFR. When the import efficiencies of the three preproteins into
MOM19 mitochondria were corrected for the reduction of
fluorescence quenching, i.e. the relative assessment of the
membrane potential, the import of COXVa was of wild-type efficiency (Table 2). As expected, the import of
-MPP and Su9-DHFR was
still strongly inhibited, in agreement with a dependence of their
import on the receptor function of MOM19 (Table 2).
Figure 3:
Dependence of COXVa import on the
mitochondrial membrane potential. Isolated wild-type mitochondria (50
µg of protein) were suspended in 1% BSA-containing import buffer in
the presence of 2 mM ATP, 2 mM NADH, and 20
µM oligomycin and partially uncoupled by adding increasing
concentrations of the protonophore CCCP as indicated (see
``Materials and Methods''). Then the S-labeled
precursor proteins COXVa,
-MPP, and Su9-DHFR were imported for 5
min at 25 °C, and the reactions were stopped with 1 µM valinomycin. After reisolation and SDS-PAGE separation of the
mitochondria, the processed proteins were quantified by densitometry of
the fluorograms. Samples receiving no CCCP represented 100% import
efficiency.
We
conclude that the reduction of COXVa import into MOM19
mitochondria is indirectly caused by a reduction of the membrane
potential and that the import of COXVa does not require the receptors
MOM72 and MOM19. In support of this, antibodies directed against MOM19
or MOM72 (Moczko et al., 1993) did not inhibit import of COXVa
into isolated mitochondria (not shown).
In agreement with a previous report (Miller and Cumsky, 1993),
import of COXVa into ssc1-2 mitochondria was not
inhibited (Fig. 4, lanes4 and 10).
However, import into ssc1-3 mitochondria was blocked (Fig. 4, lanes6 and 12). Proteolytic
processing of the presequence was also inhibited with ssc1-3 mitochondria (Fig. 4, lane6), indicating
that binding to mt-hsp70 is needed to drive COXVa deep enough into
mitochondria to expose the cleavage site to the matrix processing
peptidase. To exclude unspecific inhibitory effects of the ssc1-3 mitochondria on -dependent
translocation or on processing of preproteins, we analyzed the import
of a special test protein, a fusion between the 167 amino-terminal
amino acid residues of the precursor of cytochrome b
and entire dihydrofolate reductase (b
-DHFR).
This fusion protein has been shown to be imported independently of
functional mt-hsp70. The presequence of cytochrome b
is bipartite. A typical matrix targeting sequence in the first
half of the presequence is followed by a second sorting sequence which
includes a hydrophobic core and is required for directing transport of
cytochrome b
to the intermembrane space. A
hypothetical sorting component is assumed to bind to the sorting
sequence and to substitute for the translocase function of mt-hsp70
(Voos et al., 1993; Glick et al., 1993; Stuart et
al., 1994). Unfolding of b
-DHFR in the in
vitro system seems to need only low energy inputs, not requiring
the unfoldase function of mt-hsp70 (Voos et al., 1993). The
import of b
-DHFR into ssc1-2 and ssc1-3 mitochondrial preparations used in this study was
not affected (Fig. 4), demonstrating the import capabilities of
the mutant mitochondria as long as functional mt-hsp70 is not required.
We conclude therefore that import of COXVa requires the translocase
function of mt-hsp70.
Figure 4:
Import of COXVa into ssc1-3 mitochondria is strongly inhibited. Mitochondria from wild-type
yeast and from the temperature-sensitive mt-hsp70 mutants ssc1-2 and ssc1-3 (50 µg of protein)
were shifted to 37 °C for 10 min in order to stably induce their
phenotypes (Gambill et al., 1993). 1 µM valinomycin and 20 µM oligomycin dissipated the
membrane potential where indicated. Then the import of radiolabeled
COXVa precursor protein and of a fusion protein between the 167
amino-terminal residues of the cytochrome b precursor and entire dihydrofolate reductase (b
-DHFR) into the energized mitochondria was
performed for 3 min at 25 °C and stopped by addition of 1
µM valinomycin. One half of each sample was treated with
100 µg/ml proteinase K. The mitochondria were reisolated, subjected
to SDS-PAGE, and analyzed by fluorography and densitometry. The amount
of mature-sized protein in wild-type mitochondria was set to 100%. The
faint protease-sensitive band below mature COXVa (and
COXVa
in Fig. 5) is already present
in the reticulocyte lysate and is probably due to internal initiation
of translation (Hartl et al., 1987). p, precursor; m, mature protein;
, membrane
potential.
Figure 5:
COXVa with a deletion of the internal
sorting signal is inhibited in import into ssc1-3 mitochondria. The experiment was performed essentially as
described in the legend of Fig. 4with the exception that a
COXVa construct carrying a deletion of amino acid residues
101-118 was employed. p, precursor; m, mature
protein; , membrane potential.
COXVa contains a hydrophobic
segment in its carboxyl-terminal portion (residues 98-119) that
is required for directing the polypeptide into the mitochondrial inner
membrane (Glaser et al., 1990). A COXVa construct lacking most
of this inner membrane sorting sequence
(COXVa) was imported into the matrix
(Glaser et al., 1990). COXVa
showed the same dependence on the functions of mt-hsp70 as
wild-type COXVa, i.e. import of
COXVa
into ssc1-2 mitochondria was not significantly affected (Fig. 5, lanes4 and 10), while import into ssc1-3 mitochondria was inhibited (Fig. 5, lanes6 and 12).
Therefore, deletion of the sorting sequence does not influence the
dependence of COXVa import on the translocase function of mt-hsp70.
We then employed a construct where a major portion of the sequence
between the COXVa presequence (first 20 residues) and the hydrophobic
sorting signal was removed. In this construct
(COXVa) the sorting signal was placed close
to the presequence such that the matrix targeting signal and the
sorting signal were contained within the first 60 amino acid residues
of the preprotein. The close vicinity of the two distinct signals
resembles the situation of bipartite presequences of, for example,
cytochrome b
. The construct
COXVa
was imported into isolated wild-type
yeast mitochondria in a
-dependent manner (Fig. 6A, lanes 1 and 2). To test if
COXVa
was correctly inserted into the
mitochondrial inner membrane, we employed a specific assay developed
for authentic COXVa (Miller and Cumsky, 1991, 1993). After opening of
the intermembrane space by swelling of the mitochondria and addition of
proteinase K, only correctly localized COXVa gives rise to a fragment
that is about 3 kDa smaller than COXVa. The fragmentation indicates
removal of the carboxyl-terminal tail of COXVa behind the hydrophobic
sorting and membrane anchor sequence (Miller and Cumsky, 1991, 1993). Fig. 6B (lane2) shows that this
typical fragment was also formed from imported
COXVa
. Probably due to the modification of
COXVa close to the cleavage site of the matrix processing peptidase,
removal of the amino-terminal presequence from the construct was slow
and was thus not observed in the short import times used here (the
short import times are necessary to be in the kinetically linear range
for membrane translocation). At longer incubation times in the import
reaction, the expected proteolytic processing took place (not shown).
Figure 6:
Placement of the sorting signal close to
the presequence renders import of COXVa independent of functional
mt-hsp70. A, import of COXVa into
the ssc1-mutant mitochondria. A COXVa construct lacking amino
acid residues 26-89 was imported into wild-type, ssc1-2 and ssc1-3 mitochondria as described in the legend
of Fig. 4. All samples were treated with 100 µg/ml
proteinase K. Imported COXVa
(precursor
form) is indicated by an arrow.
, membrane
potential. B, characteristic fragmentation of correctly
imported COXVa
. The COXVa construct was
imported as mentioned above. After reisolating, the mitochondria in one
half of each sample were converted to mitoplasts by swelling in 25
mM saccharose, 1 mM EDTA, 10 mM MOPS, pH 7.2
(Blom et al., 1993), reisolated again, and incubated with 100
µg/ml proteinase K in BSA-containing import buffer. The
mitochondria were collected by centrifugation and separated by
SDS-PAGE. The proteolytic fragment formed by proteinase K is indicated
by an asterisk.
COXVa was also efficiently imported to a
protease-protected location in ssc1-2 and ssc1-3 mitochondria (Fig. 6A, lanes4 and 6). The import was inhibited by
dissipation of the membrane potential
(Fig. 6A, lanes3 and 5).
The insertion of COXVa
into the
mitochondrial inner membrane, assessed by formation of the proteolytic
fragment, was indistinguishable among wild-type, ssc1-2,
and ssc1-3 mitochondria (Fig. 6B, lanes2, 4, and 6). The import
behavior of COXVa
into ssc1-3 mitochondria is thus in strict contrast to that of authentic COXVa
and COXVa
.
S. cerevisiae mutants represent a powerful system to study intracellular protein sorting. Here we have characterized the unusual mitochondrial import pathway of cytochrome c oxidase subunit Va with yeast mutants defective in distinct import components. Our findings suggest a refined model of COXVa import and provide new insights into the properties of import receptor mutants and the mechanisms of membrane translocation driven by matrix hsp70.
Now three
mitochondrial preproteins are known that depend upon neither MOM19 nor
MOM72 for import. (i) Apocytochrome c seems to be translocated
through the lipids of the outer membrane and is trapped in the
intermembrane space by covalent attachment of heme mediated by
cytochrome c heme lyase (Nicholson et al., 1988;
Dumont et al., 1991; Jordi et al., 1992). (ii) The
precursor of MOM19 is imported into the receptor complex of the outer
membrane without a need for mature MOM19 or MOM72, but directly and
stably assembles with MOM38 (ISP42), the major component of the general
insertion pore in the receptor complex (Schneider et al.,
1991). (iii) COXVa seems to use yet another mechanism for translocation
across the outer membrane. To date, COXVa is the only
presequence-carrying preprotein that requires neither MOM19 nor MOM72.
The presequence of COXVa is positively charged, but is not predicted to
form an amphipathic -helix found with typical mitochondrial
presequences, which have positive charges clustered on one side of a
helix and hydrophobic residues clustered on the other side (Roise et al., 1986; von Heijne, 1986). Recent studies reinforced the
importance of an amphipathic
-helix for targeting of preproteins
via MOM19 (Brink et al., 1994). Other positively charged
sequences were shown to mediate a receptor-independent and low
efficient ``bypass'' import into mitochondria, which seems to
be mediated by the general insertion pore of the receptor complex
(Baker and Schatz, 1987; Pfaller et al., 1989). Typical
mitochondrial preproteins are also able to use this bypass for a small
fraction of their import in addition to the much more efficient
receptor-mediated import (Pfaller et al., 1989). The
efficiency of COXVa import is far above that observed for bypass
import. We speculate that the precursor of COXVa possesses a special
ability for direct insertion into and translocation through the general
insertion pore of the outer membrane and thus is imported without a
need for surface receptors.
We propose the following model for the
inner membrane translocation of preproteins containing a matrix
targeting sequence and a hydrophobic sorting sequence (Fig. 7A). The polypeptides are translocated as linear
chains. The membrane potential is required to drive
translocation of the amino-terminal presequence across the inner
membrane but is usually not sufficient to transport the presequence
deep enough into the matrix to allow proteolytic cleavage. mt-hsp70
binds to the preprotein segments emerging on the matrix side and
thereby promotes translocation of the complete presequence and of
mature portions of the preprotein. A component of the inner membrane
sorting machinery (MSM) selectively binds to the sorting sequence to
direct it into the inner membrane. Binding of the sorting component can
fully substitute for the translocase function of mt-hsp70 when the
sorting signal is close to the presequence (Fig. 7C).
That is, the initial
-driven translocation must bring the
sorting sequence close enough to the inner membrane sorting machinery
such that it can be recognized and bound. It is unknown what drives the
proposed translocase function of the sorting machinery, which is able
to function at very low ATP levels (Glick et al., 1993; Stuart et al., 1994). Possibly, the energy for driving import is
derived from binding of the sorting sequence to the sorting machinery
and its subsequent stable insertion into the lipid phase. In case the
sorting sequence is too far away from the presequence, the
-driven import of the linear polypeptide chain is not
sufficient to promote movement of the sorting signal toward the sorting
machinery (Fig. 7B), and therefore mt-hsp70 with its
broad reactivity for unfolded segments is essential to drive
translocation by binding to preprotein segments located closer to the
presequence.
Figure 7:
Hypothetical model about the role of
matrix hsp70 and the inner membrane sorting machinery in import of
preproteins with dual targeting information. A, the model
shows import of COXVa into the mitochondrial inner membrane driven by a
stepwise action of the membrane potential on the
presequence (positively charged box), binding of mt-hsp70 molecules to
unfolded preprotein segments, and binding of the mitochondrial inner
membrane sorting machinery to the hydrophobic sorting sequence (hatchedbox). B, when a mutated mt-hsp70 is
unable to bind to preproteins, MSM is not able to bind to the
preprotein as the distance to the sorting sequence is too large.
Translocation is blocked. C, when the sorting sequence is
moved close to the presequence, the
-driven translocation
transports the sorting sequence close enough to MSM such that binding
is possible also in the absence of functional mt-hsp70. Binding to MSM
can now substitute for the translocase function of mt-hsp70. GIP, general insertion pore in the outer membrane; IM, inner membrane; IMS, intermembrane space; MIM, mitochondrial inner membrane import machinery; MPP, mitochondrial processing peptidase; OM, outer
membrane. The exact topology of the mitochondrial sorting machinery is
unknown. The simplified model shown here assumes a location of MSM for
COXVa in the inner membrane. A substitution of MSM for the translocase
function of mt-hsp70 may also be possible when MSM is located on the
matrix side of the inner membrane.