(Received for publication, August 22, 1995; and in revised form, September 26, 1995)
From the
Interaction of preproteins with the heat shock protein Hsp70 in
the mitochondrial matrix is required for driving protein transport
across the mitochondrial inner membrane. Binding of mt-Hsp70 to the
protein Mim44 of the inner membrane import site seems to be an
essential part of an ATP-dependent reaction cycle. However, the
available results on the role played by ATP are controversial. Here we
demonstrate that the mt-Hsp70Mim44 complex contains ADP and that
a nonhydrolyzable analog of ATP dissociates the mt-Hsp70
Mim44
complex in the presence of potassium ions. The previously reported
requirement of ATP hydrolysis for complex dissociation was due to the
use of a nonphysiological concentration of sodium ions. In the presence
of potassium ions, mt-Hsp70 undergoes a conformational change that is
not observed with a mutant Hsp70 defective in binding to Mim44. The
mutant Hsp70 is able to bind substrate proteins, differentiating
binding to Mim44 from binding to substrate proteins. We conclude that
binding of ATP, not hydrolysis, is required to dissociate the
mt-Hsp70
Mim44 complex and that the reaction cycle includes an
ATP-induced conformational change of mt-Hsp70.
Most mitochondrial proteins are synthesized on cytosolic
polysomes and imported into the organelle (summarized in Pfanner et
al.(1994), Stuart et al.(1994), and Lithgow et
al.(1995)). The preproteins are recognized by receptors on the
mitochondrial surface and translocated across the mitochondrial outer
membrane through a general insertion pore. Translocation across the
mitochondrial inner membrane is mediated by the integral membrane
proteins Mim17 (Sms1) and Mim23 (Mas6) and the peripheral membrane
protein Mim44 ()(Isp45) in cooperation with matrix Hsp70.
Recently it was shown that a fraction of mt-Hsp70 reversibly binds to
Mim44 in a 1:1 complex (Kronidou et al., 1994; Rassow et
al., 1994; Schneider et al., 1994). Binding to Mim44
seems to be an essential part of a mt-Hsp70 reaction cycle that
promotes unfolding and translocation of precursor polypeptides (Glick,
1995; Pfanner and Meijer, 1995; Rassow et al., 1995).
The
interaction of mt-Hsp70 with Mim44 is regulated by ATP; however, the
role of ATP in this process has been controversial. On the one hand,
Schneider et al.(1994) reported that the mt-Hsp70Mim44
complex was stable in the presence of a nonhydrolyzable analog of ATP,
AMP-PNP, and that the analog competed the dissociation by hydrolyzable
ATP. They concluded that hydrolysis of ATP was needed to dissociate the
complex. On the other hand, Kronidou et al.(1994) and Rassow et al.(1994) reported a dissociation of the complex by
addition of the analog ATP
S, leading to the conclusion that
binding of ATP, but not hydrolysis, triggered the release of mt-Hsp70
from Mim44. It was not excluded, however, that the ATP
S was
contaminated by ATP or that the ATP
S which contains
phosphoanhydride bonds like ATP was slowly hydrolyzed (Palleros et
al., 1993) (while AMP-PNP contains phosphoimide bonds between the
two terminal phosphate groups). The situation became even more
confusing as under certain conditions the addition of ADP or even
magnesium ions without nucleotides promoted a release of mt-Hsp70 from
Mim44 (Rassow et al., 1994; Schneider et al., 1994).
The controversial results may be caused by different experimental
conditions and technical limitations of the systems used, such as the
presence of pre-existing nucleotides in the mitochondrial extracts,
possible contaminations, or slow hydrolysis of ATP
S.
Since
mt-Hsp70 and Mim44 are thought to form the core of the mitochondrial
import motor, a characterization of the energetics of their interaction
and clarification of the controversial issues will be of critical
importance on the way toward a molecular understanding of protein
translocation into mitochondria. For this report we optimized the
system to characterize the release of mt-Hsp70 from Mim44. We found
that only in the presence of sodium ions, ATP hydrolysis is necessary,
whereas, in the presence of potassium ions, binding of AMP-PNP promotes
efficient dissociation of the mt-Hsp70Mim44 complex. Binding, not
hydrolysis, of ATP was shown to induce a conformational change in
mt-Hsp70 that seems to promote its release from Mim44. Moreover, by use
of a mt-Hsp70 mutant we demonstrate that the binding of mt-Hsp70 to
Mim44 is distinct from its binding to substrate proteins.
The purity of
nucleotides was analyzed by ion-pair reversed-phase HPLC on a Waters
Nova Pak C-18 column with a 150-mm length, 3.9-mm diameter, and a
particle size of 4 µm. The runs were performed with a linear
gradient from 0 to 40% (v/v) methanol and 100 to 60% (v/v) 6 mM tetrabutylammonium hydroxide, 20 mM
(NH)
PO
, pH 6.0, with a flow rate of
1 ml/min for 20 min at 40 °C. The nucleotides were detected by
measuring the absorbance at 262 nM.
Figure 1:
Dissociation of mt-Hsp70Mim44
complex by AMP-PNP in a purified system. A, affinity-purified
mt-Hsp70
Mim44 complex, including Mge1. The complex was purified
using antibodies directed against Mim44 as described under
``Materials and Methods.'' Sample 1 contained 5
mM EDTA, and sample 2 contained 5 mM Mg-ATP. B, specific dissociation of the mt-Hsp70
Mim44 complex.
The Mim44
Hsp70 complex was isolated as described under
``Materials and Methods.'' After 3 washes, the complex was
incubated in lysis buffer containing 5 mM MgCl
(columns 3-10) or 5 mM EDTA (columns
1 and 2) and 5 mM nucleotides as indicated for
30 min. Mim44-bound and eluted mt-Hsp70 (trichloroacetic
acid-precipitated) was analyzed by SDS-PAGE and Western blotting with
antiserum specific for mt-Hsp70. The total amount of mt-Hsp70 in pellet
and supernatant was set to 100%,
respectively.
Surprisingly, Mg-AMP-PNP led to
an efficient release of mt-Hsp70 from Mim44 (Fig. 1B, column 10). We asked if the effect of AMP-PNP could be
explained by contamination with ATP and analyzed the purity of the
commercially available AMP-PNP by HPLC. We found a contamination with
ATP of 0.5% (Fig. 2, profile b). Moreover, we
removed the small amount of contaminating ATP by anion exchange FPLC
and demonstrated the purity by HPLC (Fig. 2, profile
a). The ATP-free AMP-PNP efficiently dissociated the
mt-Hsp70
Mim44 complex (Fig. 3A), ruling out the
possibility that the dissociation was caused by contamination with ATP.
Figure 2: Chromatographic analysis and purification of AMP-PNP. Shown are the profiles of ion pair reversed phase HPLC of AMP-PNP purified by anion exchange FPLC (a), commercially available AMP-PNP (contamination with ATP of 0.52% as calculated by determination of peak area and relative absorbance at 262 nm) (b), and a mixture of ADP, AMP-PNP, and ATP (c). The nucleotides were identified by standard runs with only one component.
Figure 3:
Dissociation of the mt-Hsp70Mim44
complex by AMP-PNP requires the presence of potassium ions. A,
purified AMP-PNP efficiently dissociates the complex.
Immunoprecipitated mt-Hsp70
Mim44 complex was washed three times
and incubated for 3 min by end-over-end rotation in 250 mM sucrose, 80 mM KCl, 20 mM MOPS-KOH, pH 7.2, 0.1%
(v/v) Triton X-100, 5 mM MgCl
, and nucleotide as
indicated (FPLC-purified AMP-PNP). Mim44-bound Hsp70 was analyzed by
SDS-PAGE and Western blotting. B, comparison of the influence
of potassium and sodium ions. The mt-Hsp70
Mim44 complex was
isolated. After two washes in 250 mM sucrose, 80 mM KCl, 20 mM MOPS-KOH, pH 7.2, 0.1% (v/v) Triton X-100, and
5 mM EDTA, the complex was washed once in 250 mM sucrose, 80 mM KCl or 80 mM NaCl (as indicated),
20 mM MOPS-KOH, pH 7.2, 0.1% Triton X-100, and 5 mM MgCl
and then incubated for 30 min in the same buffer
in the presence of AMP-PNP as indicated. Mim44-bound mt-Hsp70 was
analyzed by SDS-PAGE and Western blotting.
How can it be explained that Schneider et al.(1994) did not
observe a release of mt-Hsp70 from Mim44 by AMP-PNP? A comparison of
the experimental conditions revealed that different monovalent ions had
been applied in the buffers. Schneider et al.(1994) used
sodium ions at a concentration of 100 mM which is
nonphysiological for mitochondria, whereas our studies were done in the
presence of 80 mM potassium ions, a concentration considered
to be typical for the mitochondrial matrix (Scarpa, 1979; Nicholls,
1982). We thus directly compared the influence of potassium and sodium
ions on the effect of AMP-PNP on the mt-Hsp70Mim44 complex and
indeed found that AMP-PNP was inefficient in dissociating the complex
in the presence of sodium ions (Fig. 3B). The presence
of substrate, such as a mitochondrial presequence peptide, did not
prevent the AMP-PNP-induced dissociation of the mt-Hsp70
Mim44
complex in the presence of potassium ions, but rather stimulated the
dissociation. (
)We conclude that in the presence of
potassium ions ATP hydrolysis is not required for release of mt-Hsp70,
but that binding of ATP leads to dissociation of the
mt-Hsp70
Mim44 complex.
Figure 4:
Mt-Hsp70 bound to Mim44 is in the ADP
form. Mitochondria were preincubated with 250 µCi/ml
[-
P]ATP for 30 min at 25 °C while the
endogenous ATP synthesis was inhibited. The mitochondria were washed in
SEM and lysed in 30 mM Tris-HCl, pH 7.4, 150 mM KCl,
5% glycerol, and 0.3% (v/v) Triton X-100, containing either 5 mM EDTA or 5 mM MgCl
as indicated. After a
clarifying spin, the samples were subjected to immunoprecipitation with
anti-Mim44, anti-Hsp70, or preimmune serum as indicated. Bound
nucleotides were analyzed by thin layer chromatography and storage
phosphorimaging technology.
Figure 5:
A mutant Hsp70 (Ssc1-2p) binds substrate
proteins, but does not bind to Mim44. A, Ssc1-2p does not bind
to Mim44, but binds to RCMLA-Sepharose. Mitochondria from wild-type (WT) or the mutant strain ssc1-2 were incubated for
15 min at 37 °C, ATP was depleted, and the mitochondria were lysed
as described under ``Materials and Methods.'' After a
clarifying spin, co-immunoprecipitations with antibodies against Mim44
were performed (samples 1 and 2) or binding to
RCMLA-Sepharose was performed for 1 h at 37 °C (samples 3 and 4). The total amount of mt-Hsp70 (20% of the material
used for binding to RCMLA-Sepharose) in wild-type and ssc1-2 mitochondria is shown in samples 5 and 6. B.
Comparison of binding of mt-Hsp70 from wild-type and ssc1-2 mutant to Mim44 and different substrates proteins. Samples 1 and 4 were performed as described for A, and the
ratio between ssc1-2 and wild-type of bound mt-Hsp70 is shown. Sample 2, the S-labeled fusion protein Su9-DHFR
containing a mitochondrial presequence and the passenger protein
dihydrofolate reductase (Pfanner et al., 1987) was imported
into isolated mitochondria for 5 min as described (Kang et
al., 1990; Gambill et al., 1993). The mitochondria were
reisolated, washed, and lysed in 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 0.1% (v/v) Triton X-100.
After a clarifying spin, an immunoprecipitation with antibodies
directed against mt-Hsp70 (Gambill et al., 1993; Voos et
al., 1994) was performed. Sample 3, mitochondria were
lysed in 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5
mM EDTA, and 0.1% (v/v) Triton X-100. The
S-labeled precursor of F
-ATPase subunit
was denatured in 8 M urea, 10 mM Tris-HCl, pH 7.5,
for 30 min at room temperature and diluted into the mitochondrial
lysate. This was then subjected to immunoprecipitation with anti-Hsp70
antibodies. Analysis of samples 2 and 3 was done by
SDS-PAGE and storage phosphorimaging analysis of autoradiograms. Shown
is the ratio between ssc1-2 and wild-type of preprotein bound
to mt-Hsp70.
Figure 6:
A conformational change of mt-Hsp70,
assessed by tryptic fragmentation, is favored by AMP-PNP in the
presence of potassium ions. A, tryptic fragments of mt-Hsp70.
Mitochondria were depleted of ATP and lysed in 250 mM sucrose,
80 mM KCl, 20 mM MOPS, pH 7.2, 5 mM
MgCl, and 0.1% (v/v) Triton X-100. After a clarifying spin,
the indicated nucleotides (5 mM) were added, and the samples
were incubated for 15 min at 4 °C. Then trypsin was added to a
final concentration of 50 µg/ml as indicated and described under
``Materials and Methods.'' Analysis was by SDS-PAGE and
Western blotting with antibodies directed against mt-Hsp70. Two major
fragments (termed f
and f
)
were observed. B, stabilization of the 56-kDa fragment by
AMP-PNP in the presence of potassium ions. The experiment was performed
as described above except that, in samples 4-6, KCl was
replaced by 80 mM NaCl.
We asked if the differential effect of potassium and sodium ions on the function of mt-Hsp70 is also reflected in a conformational change of the chaperone. In the presence of potassium ions, AMP-PNP favored the formation of the 56-kDa fragment (Fig. 6B, lane 2), whereas in the presence of sodium ions significantly smaller amounts of the 56-kDa fragment were observed (Fig. 6B, lane 5).
The mutant Hsp70 Ssc1-2p was rapidly digested by trypsin to the 45-kDa fragment (Fig. 7A, lanes 2-4). In contrast to wild-type Hsp70, the full-size chaperone was less stable toward proteolytic attack since it was largely degraded to the 45-kDa form (Fig. 7A), while a significant fraction of wild-type Hsp70 was not degraded by trypsin under the conditions used (Fig. 6, A and B). The characteristic 56-kDa fragment was not observed with Ssc1-2p (Fig. 7A). The 45-kDa fragment, however, was of higher stability in Ssc1-2p than in wild-type Hsp70 (Fig. 7A and Fig. 6B and data not shown). Interestingly, Freeman et al.(1995) observed a high stability of the ATPase domain in a Hsc70 mutated in a carboxyl-terminal motive which was shown to be necessary for the interaction between the ATPase domain and substrate binding domain.
Figure 7:
The mutant mt-Hsp70 Ssc1-2p is unable to
form the 56-kDa fragment, but can bind nucleotides. A, tryptic
fragmentation. The experiment was performed as described in the legend
of Fig. 6A except that mitochondria from the ssc1-2 mutant were used. B, Ssc1-2p binds to ADP- and
ATP-agarose as efficiently as wild-type mt-Hsp70. Mitochondria were
incubated for 15 min at 37 °C. The mitochondria were then depleted
of ATP and lysed in 300 mM KCl, 20 mM Tris-HCl, pH
7.5, 5 mM MgCl, 0.3% (v/v) Triton X-100. After a
clarifying spin, the lysate was incubated for 30 min at 4 °C with
0.5 mg of ATP- or ADP-agarose by end-over-end rotation as described
under ``Materials and Methods.'' 50% of the total amount of
mitochondrial lysate added to ATP- or ADP-agarose is shown in lanes
5 and 6 as standard. Analysis was done by
immunodecoration with antibodies directed against
mt-Hsp70.
We conclude that binding of ATP induces a conformational state in
Hsp70 (assessed by formation of the 56-kDa fragment by trypsin) that is
stabilized by AMP-PNP in the presence of potassium ions. The mutant
Ssc1-2p which is unable to bind to Mim44 does not adopt this
conformational state. Ssc1-2p does not seem to be impaired in binding
of nucleotides since it was bound to ATP-agarose and ADP-agarose as
efficiently as wild-type mt-Hsp70 (Fig. 7B, compare lanes 3 and 4 to lanes 1 and 2)
(another mutant mt-Hsp70 (ssc1-3) with an amino acid
alteration in the ATPase domain neither binds to ATP-agarose nor
ADP-agarose). These results are consistent with a view that
the amino acid alteration in the carboxyl-terminal third of Ssc1-2p
impairs an ``interdomain communication'' (Buchberger et
al., 1995; Freeman et al., 1995) that mediates the
nucleotide-dependent conformational change of mt-Hsp70.
We have characterized the role of ATP in the interaction
between matrix Hsp70 and the mitochondrial inner membrane protein
Mim44. We affinity-purified the mt-Hsp70Mim44 complex such that
it was free from unbound nucleotides, but still contained the
nucleotide exchange factor Mge1. Mg-ATP or the nonhydrolyzable analog
Mg-AMP-PNP efficiently promoted the dissociation of the
mt-Hsp70
Mim44 complex at (sub)micromolar concentrations, whereas
neither Mg-ADP nor magnesium ions alone were able to promote a
dissociation (even at millimolar concentrations of ADP). Of critical
importance is the purity of the analog. We therefore excluded that the
dissociating effect of the chemically stable analog AMP-PNP was caused
by contaminations with ATP. This indicates that binding of ATP is
sufficient to release mt-Hsp70 from Mim44. The conclusion was directly
confirmed by loading mitochondria with
[
-
P]ATP. Mt-Hsp70 bound to Mim44 was
selectively found in the ADP form. Our results provide strong evidence
that binding of ATP, not hydrolysis, causes the dissociation of the
mt-Hsp70
Mim44 complex.
The conclusion, however, is different
from that by Schneider et al.(1994) who reported that the
mt-Hsp70Mim44 complex was stable in the presence of AMP-PNP.
Schneider et al.(1994) used sodium ions at 100 mM concentrations, whereas the mitochondrial Na
concentration is about 1-4 mM; they did not
include potassium ions which are about 40-120 mM in
mitochondria (Scarpa, 1979; Nicholls 1982; Tyler, 1992). We reasoned
that the differential use of monovalent cations may provide the
explanation for the controversial nucleotide dependence. Indeed we
found that the dissociating effect of AMP-PNP on the
mt-Hsp70
Mim44 complex required the presence of potassium ions and
did not occur in the presence of sodium ions. Comparable effects of the
ions have also been observed with the homologs of mt-Hsp70 in bacteria
and the eukaryotic cytosol (the cytosolic K
concentration is
140 mM, and the cytosolic
Na
concentration is
5-15 mM (Alberts et al., 1994)). ATP
S was able to promote
the release of substrate from DnaK in the presence of potassium ions,
but not sodium ions (Palleros et al., 1993). The ATPase
activity of Hsc70 was minimal in the absence of potassium ions and
maximal at 100 mM potassium (O'Brien and McKay, 1995).
Wilbanks and McKay(1995) reported that two potassium ions were present
in the nucleotide binding cleft of the crystallized ATPase domain of
bovine brain Hsc70. Furthermore, Szabo et al.(1994) showed the
release of luciferase from DnaK by AMP-PNP, McCarty et al. (1995) demonstrated in a double-label experiment that substrate
release precedes ATP hydrolysis by DnaK, and Greene et
al.(1995) reported that it is ATP binding rather than hydrolysis
which causes dissociation of peptide from bovine brain Hsp70.
The comparable nucleotide dependence for release of substrate and Mim44 from mt-Hsp70 raised the possibility that Mim44 was just interacting with the substrate binding site of mt-Hsp70. This question is of direct relevance for the models on the role of mt-Hsp70 in driving polypeptides across the mitochondrial membranes. In one model, the polypeptides are sliding back and forth in the import channels, and binding by mt-Hsp70 traps them in the matrix (Stuart et al., 1994; Ungermann et al., 1994). This ``Brownian ratchet'' model is compatible with a situation that Mim44 is bound to the substrate binding site of mt-Hsp70. Prior to binding a preprotein, mt-Hsp70 must then be released from Mim44 (Berthold et al., 1995). In another model, mt-Hsp70 bound to Mim44 simultaneously binds the precursor polypeptide and, by a conformational change, exerts a pulling force on the polypeptide (``pulling by Hsp70'') (Pfanner et al., 1994; Glick, 1995; Pfanner and Meijer, 1995). The latter model explains the role of mt-Hsp70 in promoting unfolding of preprotein domains during translocation across the mitochondrial membranes (Glick et al., 1993; Voos et al., 1993; Glick, 1995; Pfanner and Meijer, 1995). The model is only possible, however, when Mim44 and substrate proteins are binding to different sites of mt-Hsp70 as a pulling force on a preprotein can only be generated when mt-Hsp70 is able to simultaneously bind to a membrane anchor (i.e. Mim44) and the preprotein. Indeed, we were able to demonstrate that the interaction of mt-Hsp with Mim44 is strikingly different from the interaction of mt-Hsp70 with substrate. A mutant mt-Hsp70 (Ssc1-2p) which is unable to bind to Mim44 efficiently binds substrate proteins. These results are thus compatible with a pulling model of mt-Hsp70 function. The suggestion that mt-Hsp70 may have at least two different binding sites may also be supported by recent work of Takenaka et al.(1995). They showed evidence that bovine Hsc70 bound to two chemically quite different types of peptides.
Support for the pulling model is also provided by the observation of nucleotide-induced conformational changes of mt-Hsp70, assessed by the differential formation of tryptic fragments. In particular, a 56-kDa fragment which comprises parts of both the amino-terminal ATPase domain and the carboxyl-terminal domain of mt-Hsp70 is observed only in the ATP-form, not in the ADP-form. AMP-PNP in the presence of potassium ions strongly favors this conformation. The mutant Ssc1-2p does not undergo this conformational change. Since Ssc1-2p is not blocked in binding to either nucleotides or to substrate proteins, we suggest that the single amino acid alteration in the carboxyl-terminal domain (Gambill et al., 1993) blocks an ``interdomain communication'' (Buchberger et al., 1995; Freeman et al., 1995) which regulates conformational changes in the mt-Hsp70 molecule. This may be an explanation for the inability of Ssc1-2p to interact with Mim44.
Our results suggest the following conclusions. (i) The interaction of mt-Hsp70 with substrate proteins is significantly different from that with Mim44. (ii) Part of the mt-Hsp70 reaction cycle can now be described, i.e. the release from Mim44. Binding, not hydrolysis, of ATP to Mim44-associated mt-Hsp70 induces a conformational change in the chaperone that promotes its dissociation from Mim44. We suggest that hydrolysis of ATP by the soluble mt-Hsp70 recycles the chaperone for new rounds of binding to Mim44 at the import site of the mitochondrial inner membrane.