©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Mitochondrial Protein Import Machinery
ROLE OF ATP IN DISSOCIATION OF THE Hsp70bulletMim44 COMPLEX (*)

(Received for publication, August 22, 1995; and in revised form, September 26, 1995)

Oliver von Ahsen Wolfgang Voos Hanspeter Henninger Nikolaus Pfanner (§)

From the Biochemisches Institut, Universität Freiburg, Hermann-Herder-Straße 7, D-79104 Freiburg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-Hsp70bulletMim44 complex contains ADP and that a nonhydrolyzable analog of ATP dissociates the mt-Hsp70bulletMim44 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-Hsp70bulletMim44 complex and that the reaction cycle includes an ATP-induced conformational change of mt-Hsp70.


INTRODUCTION

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 (^1)(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-Hsp70bulletMim44 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 ATPS, 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 ATPS was contaminated by ATP or that the ATPS 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 ATPS.

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-Hsp70bulletMim44 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.


MATERIALS AND METHODS

S. cerevisiae Strains and Isolation of Mitochondria

We used the strain PK81 (MATalpha ade2-101 lys2 ura3-52 leu2-3, 112 Deltatrp1 ssc1-2(LEU2), ``ssc1-2'') and the strain PK82 (MATalpha his4-713 lys2 ura3-52 Deltatrp1 leu2-3, 112; ``wild-type'') as described in Gambill et al.(1993). Published procedures were used for the growth of yeast cultures and isolation of mitochondria (Kang et al., 1990).

Affinity Purification of Anti-Mim44 Antibodies

The serum was diluted 5-fold with 10 mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl (TBS) and passed twice over a Mim44-Sepharose column (Blom et al., 1993; Rassow et al., 1994). The column was washed with 5 volumes of TBS, and bound antibodies were eluted by 1 volume of 100 mM glycine-HCl, pH 2.5. The eluted material was immediately neutralized by the addition of Tris-HCl, pH 8.0.

Covalent Coupling of Antibodies to Protein A-Sepharose

Affinity-purified antibodies from 1 ml of rabbit serum were incubated with 0.5 ml wet-volume protein A-Sepharose (Pharmacia Biotech Inc.) in 100 mM potassium phosphate, pH 7.5, for 1 h. After two washing steps with 0.1 M sodium borate, pH 9, antibodies and protein A-Sepharose were cross-linked with 5 mM dimethylpimelimidate in borate buffer for 30 min. The cross-linker was quenched by washing and incubation in 1 M Tris-HCl, pH 7.5, for 2 h. All steps were performed at room temperature. The preparation was stored in TBS at 4 °C.

Preparation of Mim44bulletHsp70 Complexes by Co-immunoprecipitation

Mitochondria were preincubated in 250 mM sucrose, 80 mM KCl, 5 mM MgCl(2), 20 mM MOPS-KOH, pH 7.2, together with 20 µM oligomycin and 5 units/ml apyrase for 15 min at 0 °C. After reisolation and washing in 250 mM sucrose, 10 mM MOPS, pH 7.2, 5 mM EDTA (SEM), the mitochondria were resuspended in lysis buffer (250 mM sucrose, 80 mM KCl, 5 mM EDTA, 20 mM MOPS-KOH, pH 7.2, and 0.1% (v/v) Triton X-100) at a protein concentration of 2 mg/ml and shaken for 15 min at 4 °C. After a clarifying spin of 10 min at 16,000 times g, the lysed mitochondria were added to anti-Mim44 antibodies prebound to protein A-Sepharose. The samples were incubated at 4 °C by end-over-end rotation for 1 h. After three washes in lysis buffer, dissociation studies were performed by a second incubation with different nucleotides or nonhydrolyzable analogs in 250 mM sucrose, 80 mM KCl, 5 mM MgCl(2), 20 mM MOPS-KOH, pH 7.2, 0.1% Triton X-100. Where indicated, KCl was replaced by NaCl, and MgCl(2) was replaced by EDTA.

Preparation of RCMLA-Sepharose

10 mg of reduced carboxymethylated alpha-lactalbumin (RCMLA, Sigma) were coupled to 0.25 g of CNBr-activated Sepharose 4B (Pharmacia) (extensively prewashed with 1 mM HCl) in 4 ml of 100 mM NaHCO(3), pH 8.3, 500 mM NaCl for 1 h at 25 °C. After washing in 100 mM Tris-HCl, pH 8.0, and blocking for 2 h in the same buffer, the preparation was washed in three alternating cycles with 0.1 M sodium acetate, pH 4.0, and 0.1 M Tris-HCl, pH 8.0, each washing buffer containing 500 mM NaCl. The preparation was stored as 1:3 suspension in blocking buffer.

RCMLA Binding Studies

50 µg of mitochondrial protein and 50 µg of RCMLA coupled to Sepharose were used per sample. The mitochondria were preincubated in 250 mM sucrose, 80 mM KCl, 5 mM MgCl(2), 20 mM MOPS-KOH, pH 7.2, together with 20 µM oligomycin and 5 units/ml apyrase for 15 min at 0 °C. After reisolation and washing, the mitochondria were lysed in 300 mM KCl, 50 mM sodium phosphate, pH 7.5, 5 mM MgCl(2), 0.3% (v/v) Triton X-100, and 1% (w/v) bovine serum Albumin. After a clarifying spin, RCMLA-Sepharose and mitochondrial lysate were shaken for 1 h at 37 °C. After three washing steps with 300 mM KCl, 50 mM sodium phosphate, pH 7.5, 5 mM MgCl(2), and 0.3% (v/v) Triton X-100, RCMLA-Sepharose-bound proteins were eluted by sample buffer and applied to SDS-PAGE.

Binding to ATP/ADP-Agarose

50 µg of mitochondrial protein and 0.5 mg of ADP- or ATP-agarose were used per sample. The mitochondria were preincubated in 250 mM sucrose, 80 mM KCl, 5 mM MgCl(2), 20 mM MOPS-KOH, pH 7.2, together with 20 µM oligomycin and 5 units/ml apyrase for 15 min at 0 °C. After reisolation and washing, the mitochondria were lysed in 300 mM KCl, 30 mM Tris-HCl, pH 7.5, 5 mM MgCl(2), 0.3% Triton X-100. A clarifying spin was performed, and the mitochondrial lysate was incubated for 30 min with ADP- or ATP-agarose by end-over-end rotation at 4 °C. The agarose pellets were washed three times in the same buffer, bound proteins were eluted by sample buffer and applied to SDS-PAGE.

Investigation of Hsp70 Conformation by Analysis of Tryptic Fragments

Mitochondria were pretreated with 20 µM oligomycin and 5 units/ml apyrase in order to remove endogenous ATP. They were lysed by shaking in 250 mM sucrose, 80 mM KCl, 5 mM MgCl(2), 20 mM MOPS-KOH, pH 7.2, 0.1% (v/v) Triton X-100 in the presence of different nucleotides for 15 min. A clarifying spin of 10 min at 16,000 times g was performed, and trypsin was added to a final concentration of 50 µg/ml. The samples were incubated for 20 min at room temperature in a mixer. Digestion was stopped by addition of 1 mM phenylmethylsulfonyl fluoride, and the samples were precipitated by trichloroacetic acid. mt-Hsp70 and its degradation products were detected by Western blotting.

Purification and Analysis of AMP-PNP

AMP-PNP (Sigma) was purified by anion exchange FPLC on a Pharmacia Mono Q column. The nucleotide was bound to the column in 50 mM MES-KOH, pH 6.0, and eluted with a linear gradient from 0 to 125 mM NaCl in 50 mM MES-KOH, pH 6.0, with a flow of 0.5 ml/min. The AMP-PNP-containing peak was collected, and the concentration was determined by measuring the absorbance at 260 nm.

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(4))(2)PO(4), 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.

[alpha-P]ATP Binding Studies

Mitochondria were preincubated with 250 µCi/ml [alpha-P]ATP for 30 min at 25 °C in presence of 8 µM antimycin A, 20 µM oligomycin, and 0.5 µM valinomycin. They were washed in 250 mM sucrose, 10 mM MOPS, pH 7.2, 5 mM EDTA (SEM), resuspended, and shaken for 5 min in lysis buffer (30 mM Tris-HCl, pH 7.4, 150 mM KCl, 5% glycerol, and 0.3% (v/v) Triton X-100) containing either 5 mM MgCl(2) or 5 mM EDTA. After a clarifying spin, the samples were subjected to immunoprecipitation. Precipitated proteins were eluted from the protein A-Sepharose by 2.5 M perchloric acid and neutralized with KOH. Nucleotides were separated by thin layer chromatography using polyethyleneimine plates 5579 from Merck. 0.5 M potassium phosphate, pH 4.2, was used as liquid phase.


RESULTS

Dissociation of the mt-Hsp70bulletMim44 Complex by a Nonhydrolyzable ATP Analog

We purified the mt-Hsp70bulletMim44 complex from detergent-lysed Saccharomyces cerevisiae mitochondria by affinity chromatography with anti-Mim44 antibodies (Fig. 1A, lane 1). The complex contained in addition the nucleotide exchange (release) factor Mge1 (mitochondrial GrpE) which is of critical importance for the regulation of Hsp70 by nucleotides (Fig. 1A, lane 1) (Kronidou et al., 1994; Schneider et al., 1994; Voos et al., 1994). The complex was dissociated by addition of Mg-ATP (Fig. 1A, lane 2, and Fig. 1B, column 8), but not by Mg-ADP or magnesium ions alone, even at millimolar concentration (Fig. 1B, columns 4 and 6). We conclude that the isolated complex was functional in specific dissociation.


Figure 1: Dissociation of mt-Hsp70bulletMim44 complex by AMP-PNP in a purified system. A, affinity-purified mt-Hsp70bulletMim44 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-Hsp70bulletMim44 complex. The Mim44bullet Hsp70 complex was isolated as described under ``Materials and Methods.'' After 3 washes, the complex was incubated in lysis buffer containing 5 mM MgCl(2) (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-Hsp70bulletMim44 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-Hsp70bulletMim44 complex by AMP-PNP requires the presence of potassium ions. A, purified AMP-PNP efficiently dissociates the complex. Immunoprecipitated mt-Hsp70bulletMim44 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(2), 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-Hsp70bulletMim44 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(2) 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-Hsp70bulletMim44 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-Hsp70bulletMim44 complex in the presence of potassium ions, but rather stimulated the dissociation. (^2)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-Hsp70bulletMim44 complex.

mt-Hsp70 Bound to Mim44 Does Not Carry ATP

To obtain direct evidence for the type of nucleotide bound to mt-Hsp70 in complex with Mim44, we incubated isolated mitochondria with [alpha-P]ATP, lysed the mitochondria, and isolated the complex between mt-Hsp70 and Mim44 by co-precipitation with antibodies directed against Mim44. Bound nucleotides were then analyzed by thin layer chromatography. Mt-Hsp70 associated with Mim44 had only ADP bound, no ATP (Fig. 4, lane 2). When the co-precipitation was performed in presence of the chelator EDTA, only very small amounts of nucleotides bound to the mt-Hsp70bulletMim44 complex were detected (Fig. 4, lane 1) that were in the background range obtained with preimmune antibodies (Fig. 4, lane 4). In parallel, we performed a precipitation with anti-mt-Hsp70 antibodies (more than 80% of mt-Hsp70 molecules are not bound to Mim44 (Rassow et al., 1994)) and found a presence of both ATP and ADP (Fig. 4, lane 3), indicating that Mim44-bound mt-Hsp70 is selectively in the ADP form. We conclude that mt-Hsp70 complexed to Mim44 does not contain ATP, but carries ADP or is nucleotide-free.


Figure 4: Mt-Hsp70 bound to Mim44 is in the ADP form. Mitochondria were preincubated with 250 µCi/ml [alpha-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(2) 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.



Binding of mt-Hsp70 to Mim44 Is Distinct from Its Binding to Substrate Proteins

We asked if Mim44 is bound to the substrate binding site of mt-Hsp70. We compared the influence of a mutation in the Hsp70 gene (ssc1-2) on binding of substrates and binding of Mim44. Cells from the temperature-sensitive S. cerevisiae strain ssc1-2 were grown at permissive conditions, and mitochondria were isolated. The mitochondria were incubated for 15 min at 37 °C to induce the mutant phenotype (Kang et al., 1990; Gambill et al., 1993). In parallel, isolated wild-type mitochondria were treated in the same manner. The mutant mt-Hsp70 was unable to bind to Mim44 (Fig. 5A, lane 2; Fig. 5B, column 1; Schneider et al.(1994)), but bound various substrate proteins at least as efficiently as wild-type mt-Hsp70: the permanently unfolded substrate reduced carboxymethylated alpha-lactalbumin (RCMLA) (Fig. 5A, lane 4; Fig. 5B, column 4), imported preproteins (Fig. 5B, column 2), and preproteins added to lysed mitochondria (Fig. 5B, column 3). We conclude that the ssc1-2 mutation, which leads to substitution of a conserved proline by a serine (residue 419) in the carboxyl-terminal domain of mt-Hsp70 (Gambill et al., 1993), blocks binding of mt-Hsp70 to Mim44, but not to various substrate proteins, supporting a view that Mim44 and substrates bind to different sites of mt-Hsp70.


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(1)-ATPase subunit beta 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.



Evidence for a Conformational Change of mt-Hsp70 by Binding of ATP in the Presence of K

Studies with the Hsp70 of the endoplasmic reticulum (BiP) and Escherichia coli DnaK (Kassenbrock and Kelly, 1989; Liberek et al., 1991; Buchberger et al., 1995) revealed nucleotide-dependent conformational changes of the Hsp70 monitored by the differential formation of proteolytic fragments. To assess the influence of nucleotides on mt-Hsp70, we lysed the mitochondria with nonionic detergent, performed a treatment with trypsin, and analyzed fragments by immunodecoration. Two major fragments were detected, a 56-kDa fragment and a 45-kDa fragment (Fig. 6A). All members of the Hsp70bulletDnaK family contain a highly conserved ATPase domain of about 400 amino acid residues (Bardwell and Craig, 1984) that form the protease-resistant amino-terminal domain of 44-45 kDa (Chappell et al., 1987). With an antibody directed against an amino-terminal peptide of mt-Hsp70, the assignment of the 45 kDa-fragment as amino-terminal ATPase domain was confirmed.^2 The carboxyl-terminal third of Hsp70bulletDnaK contains the putative substrate binding region (Wang et al., 1993). The 56-kDa fragment thus comprises parts of both the ATPase domain and the carboxyl-terminal domain. It was only formed in the presence of ATP (Fig. 6A, lane 2) or nonhydrolyzable analogs (Fig. 6A, lanes 3 and 4), but not with ADP (Fig. 6A, lane 5), suggesting a nucleotide-dependent conformational change of 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(2), 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(1) and f(2)) 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(2), 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).^2 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.


DISCUSSION

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-Hsp70bulletMim44 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-Hsp70bulletMim44 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 [alpha-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-Hsp70bulletMim44 complex.

The conclusion, however, is different from that by Schneider et al.(1994) who reported that the mt-Hsp70bulletMim44 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-Hsp70bulletMim44 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)). ATPS 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.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-761-203-5224; Fax: 49-761-203-5261.

(^1)
The abbreviations used are: Mim44, mitochondrial inner membrane protein of 44 kDa; AMP-PNP, 5`-adenylyl-beta,-imidodiphosphate; ATPS, adenosine 5`-O-(3-thiotriphosphate); DnaK, E. coli heat shock protein of 70 kDa; Mge1, mitochondrial GrpE (a co-chaperone of mt-Hsp70); mt-Hsp70, mitochondrial matrix heat shock protein of 70 kDa (in S. cerevisiae also termed Ssc1p); PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; FPLC, fast protein liquid chromatography; RCMLA, reduced carboxymethylated alpha-lactalbumin; MOPS, 4-morpholinopropanesulfonic acid.

(^2)
O. von Ahsen and N. Pfanner, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. E. A. Craig, K. Dietmeier, and M. Meijer for yeast strains and antisera, Drs. B. Bukau, T. Langer, M. Horst, G. Schatz, T. Rapoport, P. Dekker, J. Rassow, and U. Bömer for discussion, and Dr. J. Rassow for constructive comments on the manuscript.


REFERENCES

  1. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D. (1994) Molecular Biology of the Cell , 3rd Ed, p. 508, Garland Publishing Inc., New York
  2. Bardwell, J. C. A., and Craig, E. A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 848-852 [Abstract]
  3. Berthold, J., Bauer, M. F., Schneider, H. C., Klaus, C., Dietmeier, K., Neupert, W., and Brunner, M. (1995) Cell 81, 1085-1093 [Medline] [Order article via Infotrieve]
  4. Blom, J., Kübrich, M., Rassow, J., Voos, W., Dekker, P. J. T., Maarse, A. C., Meijer, M., and Pfanner, N. (1993) Mol. Cell. Biol. 13, 7364-7371 [Abstract]
  5. Buchberger, A., Theyssen, H., Schröder, H., McCarty, J. S., Virgallita, G., Milkereit, P., Reinstein, J., and Bukau, B. (1995) J. Biol. Chem. 270, 16903-16910 [Abstract/Free Full Text]
  6. Chappell, T. G., Konforti, B. B., Schmid, S. L., and Rothman, J. E. (1987) J. Biol. Chem. 262, 746-751 [Abstract/Free Full Text]
  7. Freeman, B. C., Myers, M. P., Schumacher, R., and Morimoto, R. I. (1995) EMBO J. 14, 2281-2292 [Abstract]
  8. Gambill, B. D., Voos, W., Kang, P. J., Miao, B., Langer, T., Craig, E. A., and Pfanner, N. (1993) J. Cell Biol. 123, 109-117 [Abstract]
  9. Glick, B. S. (1995) Cell 80, 11-14 [Medline] [Order article via Infotrieve]
  10. Glick, B., Wachter, C., Reid, G. A., and Schatz, G. (1993) Protein Sci. 2, 1901-1917 [Abstract/Free Full Text]
  11. Greene, L. E., Zinner, R., Naficy, S., and Eisenberg, E. (1995) J. Biol. Chem. 270, 2967-2973 [Abstract/Free Full Text]
  12. Kang, P. J., Ostermann, J., Shilling, J., Neupert, W., Craig, E. A., and Pfanner, N. (1990) Nature 348, 137-143 [CrossRef][Medline] [Order article via Infotrieve]
  13. Kassenbrock, C. K., and Kelly, R. B. (1989) EMBO J. 8, 1461-1467 [Abstract]
  14. Kronidou, N. G., Oppliger, W., Bolliger, L., Hannavy, K., Glick, B. S., Schatz, G., and Horst, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12818-12822 [Abstract/Free Full Text]
  15. Liberek, K., Skowyra, D., Zylicz, M., Johnson, C., and Georgopoulos, C. (1991) J. Biol. Chem. 266, 14491-14496 [Abstract/Free Full Text]
  16. Lithgow, T., Glick, B. S., and Schatz, G. (1995) Trends Biochem. Sci. 20, 98-101 [CrossRef][Medline] [Order article via Infotrieve]
  17. McCarty, J. S., Buchberger, A., Reinstein, J., and Bukau, B. (1995) J. Mol. Biol. 249, 126-137 [CrossRef][Medline] [Order article via Infotrieve]
  18. Nicholls, D. G. (1982) Bioenergetics , Academic Press, London
  19. O'Brien, M. C., and McKay, D. B. (1995) J. Biol. Chem. 270, 2247-2250 [Abstract/Free Full Text]
  20. Palleros, D. R., Reid, K. L., Shi, L., Welch, W. J., and Fink, A. L. (1993) Nature 365, 664-666 [CrossRef][Medline] [Order article via Infotrieve]
  21. Pfanner, N., and Meijer, M. (1995) Curr. Biol. 5, 132-135 [Medline] [Order article via Infotrieve]
  22. Pfanner, N., Tropschug, M., and Neupert, W. (1987) Cell 49, 815-823 [Medline] [Order article via Infotrieve]
  23. Pfanner, N., Craig, E. A., and Meijer, M. (1994) Trends Biochem. Sci. 19, 368-372 [CrossRef][Medline] [Order article via Infotrieve]
  24. Rassow, J., Maarse, A. C., Krainer, E., Kübrich, M., Müller, H., Meijer, M., Craig, E. A., and Pfanner, N. (1994) J. Cell Biol. 127, 1547-1556 [Abstract]
  25. Rassow, J., Voos, W., and Pfanner, N. (1995) Trends Cell Biol. 5, 207-212 [CrossRef]
  26. Scarpa, A. (1979) in Membrane Transport in Biology (Giebisch, G., Tosteson, D. C., and Ussing, H. H., eds) Vol. II, pp. 263-356, Springer Verlag, Berlin
  27. Schneider, H. C., Berthold, J., Bauer, M. F., Dietmeier, K., Guiard, B., Brunner, M., and Neupert, W. (1994) Nature 371, 768-774 [CrossRef][Medline] [Order article via Infotrieve]
  28. Stuart, R. A., Cyr, D. M., Craig, E. A., and Neupert, W. (1994) Trends Biochem. Sci. 19, 87-92 [CrossRef][Medline] [Order article via Infotrieve]
  29. Szabo, A., Langer, T., Schröder, H., Flanagan, J., Bukau, B., and Hartl, F.-U. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10345-10349 [Abstract/Free Full Text]
  30. Takenaka, I. M., Leung, S.-M., McAndrew, S. F., Brown, J. P., and Hightower, L. E. (1995) J. Biol. Chem. 270, 19839-19844 [Abstract/Free Full Text]
  31. Tyler, D. D. (1992) The Mitochondrion in Health and Disease , p. 182, VCH Publishers, Inc., New York
  32. Ungermann, C., Neupert, W., and Cyr, D. M. (1994) Science 266, 1250-1253 [Medline] [Order article via Infotrieve]
  33. Voos, W., Gambill, B. D., Guiard, B., Pfanner, N., and Craig, E. A. (1993) J. Cell Biol. 123, 119-126 [Abstract]
  34. Voos, W., Gambill, D. B., Laloraya, S., Ang, D., Craig, E. A., and Pfanner, N. (1994) Mol. Cell. Biol. 14, 6627-6634 [Abstract]
  35. Wang, T.-F., Chang, J.-h., and Wang, C. (1993) J. Biol. Chem. 268, 26049-26051 [Abstract/Free Full Text]
  36. Wilbanks, S. M., and McKay, D. B. (1995) J. Biol. Chem. 270, 2251-2257 [Abstract/Free Full Text]

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