Impaired Interdomain Communication in Mitochondrial Hsp70 Results in the Loss of Inward-directed Translocation Force*

The essential mitochondrial Hsp70 (mtHsp70) is required for the import of mitochondrial preproteins into the matrix compartment. The translocation-specific activity of mtHsp70 is coordinated by its interaction with specific partner proteins, forming the import motor complex that provides the energy for unfolding and complete translocation of precursor polypeptide chains. A major biochemical characteristic of Hsp70-type chaperones is their nucleotide-regulated affinity to polypeptide substrates. To study the role of this allosteric regulation in the course of preprotein translocation, we have generated specific mtHsp70 mutations located within or close to the interface between the nucleotide-binding and the substrate-binding domains. Mitochondria isolated from the mtHsp70 mutants displayed severely reduced import efficiencies in vitro. Two of the mutants exhibited strong growth defects in vivo and were significantly impaired in the generation of an inward-directed, ATP-dependent import force on precursor proteins in transit. The biochemical properties of these two mutant proteins were consistent with defects in the transfer of conformational signals to the substrate-binding domain, resulting in a prolonged and enhanced interaction with imported substrate proteins. Furthermore, interference with the allosteric mechanism resulted in defects of translocation-specific partner protein interaction. We conclude that even a partial disruption of the interdomain communication in the mtHsp70 chaperone results in an almost complete breakdown of its translocation-driving properties.

crucial cellular functions under normal and stress conditions (1). Eukaryotic cells contain several Hsp70 paralogs that are generally involved in all stages of protein biogenesis, ranging from biosynthesis at the ribosomes, intracellular transport, and eventually proteolysis (2)(3)(4). Mitochondria of the model organism Saccharomyces cerevisiae contain three types of Hsp70 proteins, encoded by the genes SSC1, SSQ1, and SSC3 (ECM10) (5). Ssc3 is expressed at very low levels under normal growth conditions and its cellular function is so far unknown. Ssq1 has been shown to assist the assembly of iron/sulfur cluster cofactors in mitochondria. The most abundant and functionally important mitochondrial Hsp70 is Ssc1, generally referred to as mtHsp70. It performs most of the typical chaperone functions in terms of protein folding and quality control for polypeptides localized in the mitochondrial matrix compartment (5)(6)(7). In addition, Ssc1 is required for the import of all mitochondrial precursor proteins destined for the matrix (8). In yeast cells, deletion of SSC1 results in a lethal phenotype, a property that has been directly attributed to its crucial role in the import process. Mitochondrial preprotein translocation comprises a series of consecutive steps (9): after synthesis at cytosolic ribosomes, the N-terminal targeting signal of a precursor polypeptide is recognized at the mitochondrial surface by specific receptor proteins and inserted into the outer membrane translocation channel, the TOM 5 (translocase of the outer membrane) protein complex. Subsequent insertion of the presequence into the inner membrane translocase channel TIM23 (translocase of the inner membrane) is driven by the electric potential (⌬) across the inner membrane. Next, the polypeptide chain interacts with mtHsp70, which forms the core of the translocation motor complex (9,10), also termed PAM (presequence translocase-associated motor). By coupling translocation with ATP hydrolysis, the motor complex drives unfolding of the polypeptide chain and its transport into the matrix. Finally, the import process is completed by MPP-mediated (mitochondrial processing peptidase) proteolytic removal of the targeting sequence and further folding and assembly reactions. Similar to its bacterial ortholog DnaK, mtHsp70 activity is closely regulated by the interaction with specific co-chaperones. Substrate interaction and ATP hydrolysis during protein folding is assisted by Mdj1, a co-chaperone belonging to the so-called J-domain protein family (11). In addition, Pam18, another J-domain protein and a PAM component, stimulates the ATPase activity of mtHsp70 in the immediate vicinity of the translocation channel (12)(13)(14)(15). Release of ADP is stimulated by the nucleotide exchange factor Mge1, a homolog of the bacterial GrpE (16,17), strongly increasing the intrinsic ATPase activity of mtHsp70. Furthermore, the peripheral membrane protein Tim44, a component of the TIM23 complex, is required for the translocation-specific activity of mtHsp70. By its specific and stable interaction with Tim44, the soluble chaperone is tethered to the exit site of the preprotein translocation channel at the inner face of the mitochondrial inner membrane (18 -20). This interaction is essential for the motor activity of mtHsp70, indicated by the lethal phenotype of a TIM44 gene deletion in yeast.
The following aspects of the mtHsp70 reaction cycle have been shown to be essential for the mitochondrial protein import process (5,21): (i) interaction with the translocation channel via Tim44, (ii) regulation of the ATPase activity by the cofactors Pam18 and Mge1 and, most importantly, (iii) nucleotide-regulated interactions with the substrate protein, in this case the precursor polypeptide in transit. However, the details of the molecular mechanism enabling mtHsp70 to drive polypeptide movement and unfolding are still not entirely established (22,23). A basic model for the mtHsp70-assisted import reaction has been proposed, in which ATP-regulated binding of mtHsp70 to the incoming polypeptide chain serves as a passive molecular ratchet, rendering the bidirectional movement of the preprotein inside the translocation channel vectorial (24,25). However, detailed kinetic and mechanistic studies of the import process indicated a more active role of mtHsp70 during preprotein import. It was shown that the import motor conveys an inward-directed translocation force on the polypeptide in transit, not only contributing to its forward movement, but also to the active unfolding of C-terminal domains for passage through the translocation channel (26 -29).
Generally, the activity of Hsp70-type chaperones is based on their binding affinity to hydrophobic and unfolded segments in polypeptide substrates (30). Although substrate proteins are bound by the C-terminal substrate-binding domain (SBD) of Hsp70, the N-terminal nucleotide-binding domain (NBD) controls the conformation and substrate affinity of the SBD by an allosteric mechanism. In the ATP-bound state, the SBD is in an "open" conformation allowing rapid binding and release of substrate peptides, which results in an overall low substrate affinity. ATP hydrolysis in the NBD induces a "closed" conformation of the SBD, i.e. the peptide substrate is locked in the peptidebinding site with high affinity. The J-domain of DnaJ-like proteins has been shown to contribute significantly to this substrate docking process (31,32). Vice versa, peptide binding in the SBD enhances ATPase activity of the NBD. Although the allosteric interaction between NBD and SBD is crucial for Hsp70 function, details of its structural basis have emerged only recently. Previous studies had resulted in the separate determination of individual Hsp70 domain structures (33)(34)(35)(36). However, recent experiments resulted in the identification of structural elements in both Hsp70 domains that are crucial for mutual allosteric control (37)(38)(39)(40). The first crystal structure of a Hsp70 molecule comprising NBD and major parts of the SBD defined a domain interface region that contains several characteristic interaction sites between amino acid residues of the NBD and the SBD, respectively (41).
Based on the recent progress in the structural description of Hsp70-type chaperones, we were now able to directly address the importance of the allosteric mechanism in the course of the mitochondrial preprotein import reaction. As outlined above, the ATP-regulated substrate interaction is the key feature of mtHsp70 enabling it to drive translocation of a polypeptide chain into the mitochondrial matrix. We have introduced amino acid exchanges into the mtHsp70 NBD that are supposed to directly affect the allosteric regulation of substrate affinity. These mutations were analyzed with regard to their effects on the biochemical properties of mtHsp70 and its activity during mitochondrial preprotein import. Interference with the allosteric mechanism resulted in severe growth defects in vivo correlating with a strong reduction of preprotein import in organello. Hence, functional interdomain communication is strictly required for the essential role of the mtHsp70 chaperone machinery in driving mitochondrial preprotein import.
Isolation of Mitochondria and in Vitro Import-Yeast strains were grown in YPG (1% (w/v) yeast extract, 2% (w/v) peptone, and 3% (v/v) glycerol) to mid-log phase. Expression of the mutant Hsp70s analyzed in this study was induced by addition of 2% galactose 2-3 h prior to harvesting. Mitochondria were isolated as described (44), and stored in SEM buffer (250 mM sucrose, 1 mM EDTA, and 10 mM MOPS, pH 7.2) at Ϫ80°C. To induce the temperature-sensitive ssc1-3 phenotype, isolated mitochondria were subjected to heat shock at 37°C for 15 min immediately prior to all assays described in the following sections.
Radiolabeled precursor proteins were synthesized by in vitro transcription and translation in the presence of [ 35 S]methionine (TNT-coupled reticulocyte lysate system, Promega). Import assays were performed by incubation of precursor proteins with isolated mitochondria in import buffer at 25°C (250 mM sucrose, 80 mM KCl, 5 mM MgCl 2 , 2 mM ATP, 4 mM NADH, 10 mM KP i , pH 7.4, 10 mM MOPS-KOH, and 3% bovine serum albumin) at 25°C. Import reactions were stopped by addition of valinomycin and cooling on ice. Non-imported protein material was removed by an incubation with proteinase K (50 g/ml) for 10 min. All experiments using radiolabeled preproteins were analyzed by digital autoradiography and quantified with the help of the ImageQuant software (GE Healthcare).
For the analysis of the inward-directed import activity, recombinant precursor proteins (b 2 (167) ⌬ -DHFR) were imported in saturating amounts (29) in the presence of the dihydrofolate reductase (DHFR) ligand methotrexate (MTX) as described (45). After terminating the import reaction by addition of valinomycin, mitochondria were re-isolated and resuspended in import buffer in the presence of ATP. Samples were taken at the indicated time points and treated with proteinase K (50 g/ml) for 10 min. After re-isolation of mitochondria, proteins were separated by SDS-PAGE and detected by immunoblotting using an antibody against mouse DHFR (44).
Co-immunoprecipitation of mtHsp70 with Imported Proteins-Isolated mitochondria were resuspended in import buffer. Import of 35 S-labeled Su9(86)-DHFR for 5 min, subsequent lysis of mitochondria in 0.1% Triton X-100, 100 mM NaCl, 10 mM Tris/HCl, pH 7.5, 5 mM EDTA, and incubation with immobilized antibodies against mtHsp70 were performed as described (47). Bound proteins were eluted with SDS-PAGE sample buffer, separated by SDS-PAGE, and analyzed by digital autoradiography.
Nucleotide-and Substrate-binding Properties of mtHsp70-Binding of mtHsp70 to 5Ј-ATP-agarose 4B (Sigma) was performed in a buffer composed of 200 mM KCl, 30 mM Tris/HCl, pH 7.4, 5 mM MgCl 2 , 5% (v/v) glycerol, and 0.3% Triton X-100, as described (48). Bound proteins were eluted with the same buffer containing 3 mM ATP. Samples of the mitochondrial extracts and the eluates were subjected to trichloroacetic acid precipitation and analyzed by SDS-PAGE and immunoblotting using an antibody directed against the V5-tag.
Coupling of the Hsp70 model substrate RCMLA (reduced carboxymethylated ␣-lactalbumin; Sigma) to CNBr-activated Sepharose 4B (GE Healthcare) and binding of mtHsp70 to RCMLA-Sepharose was performed in 100 mM NaCl, 100 mM KCl, 50 mM sodium phosphate, pH 7.5, 5 mM MgCl 2 , 5% (v/v) glycerol, 0.1% (w/v) bovine serum albumin, and 0.1% Triton X-100, as described (48). Bound proteins were eluted with the same buffer containing 3 mM ATP or by SDS-PAGE sample buffer and incubation at 95°C. Samples of the mitochondrial extracts and the eluates were subjected to trichloroacetic acid precipitation and analyzed by SDS-PAGE and immunoblotting using an antibody directed against the V5-tag.
Tryptic Digest of Hsp70-Endogenous ATP was depleted from isolated mitochondria by addition of apyrase (10 units/ml) and oligomycin (20 M). Trypsin digestion was performed for 2, 5, 15, and 30 min at 20°C in the presence of the non-hydrolysable ATP analog AMP-PNP in import buffer, as described (19). After trichloroacetic acid precipitation, samples were analyzed by SDS-PAGE and Western blot. Detection of mtHsp70 and its degradation fragments was done by use of an antibody against Escherichia coli DnaK.
Interaction of mtHsp70 with Partner Proteins Mdj1, Mge1, and Tim44-Mitochondria were lysed in 0.3% Triton X-100, 30 mM Tris/HCl, pH 7.4, 100 mM KCl, 5% glycerol, and 1 mM phenylmethylsulfonyl fluoride. ATP was either depleted by incubation of intact mitochondria in the presence of apyrase (10 units/ ml) and oligomycin (20 M) prior to lysis, or maintained by addition of 2 mM ATP and 5 mM MgCl 2 in the lysis buffer. After a clarifying spin at 20,000 ϫ g, the extracts were incubated with Ni-NTA-agarose beads for 1 h with end-over-end shaking at 4°C. Samples were subsequently washed three times in 30 mM Tris/HCl, pH 7.4, 100 mM KCl, 5% glycerol, 35 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride. Bound proteins were eluted with 30 mM Tris/HCl, pH 7.4, 150 mM NaCl, and 350 mM imidazole. After trichloroacetic acid precipitation, proteins were separated by SDS-PAGE and detected by immunodecoration.
Chemical Cross-linking-Isolated mitochondria were resuspended in import buffer (250 mM sucrose, 10 mM MOPS-KOH, pH 7.2, 80 mM KCl, and 5 mM MgCl 2 ). Chemical cross-linking was performed on ice using disuccinimidyl suberate that was added from a 100-fold stock in dimethyl sulfoxide. Mitochondrial ATP was either depleted by incubation of mitochondria in the presence of apyrase (10 units/ml) and oligomycin (20 M) prior to cross-linking, or kept high by addition of 2 mM ATP, 4 mM NADH, 0.2 mg/ml creatine kinase, and 20 mM creatine phosphate during the cross-linking reaction. In the control sample, dimethyl sulfoxide without cross-linker was added. After 30 min of incubation on ice, excess cross-linker was quenched by addition of 25 mM Tris/HCl, pH 7.5, and subsequent incubation on ice for 15 min. For analysis of mtHsp70 cross-linking adducts, re-isolated mitochondria were solubilized with 1% SDS in 30 mM Tris/HCl, pH 7.4, 150 mM NaCl, and 5% glycerol for 15 min at 25°C. Samples were diluted 20-fold in 30 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5% glycerol, and 0.5% Triton X-100. After a clarifying spin at 20,000 ϫ g, the extracts were added to Ni-NTA-agarose beads. After 90 min incubation at 4°C with end-over-end shaking, beads were washed three times in 30 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5% glycerol, and 35 mM imidazole. Bound proteins were eluted with 30 mM Tris/HCl, pH 7.4, 150 mM NaCl, and 350 mM imidazole. After trichloroacetic acid precipitation, proteins were separated by SDS-PAGE and detected by Western blot.

Amino Acid Exchanges in the Nucleotide-binding Domain
Surface Interfere with mtHsp70 Function in Vivo-Chaperones from the Hsp70 family generally exhibit similar biochemical reactivities and a high overall amino acid sequence conservation. In particular, the amino acid residues contributing to the putative domain interface between the NBD and the SBD are virtually identical in bovine Hsc70, in the mitochondrial Hsp70 Ssc1 from yeast as well as in the Hsp70 ortholog DnaK from E. coli (supplemental Fig. S1). We modeled the structure of yeast Ssc1 on the basis of the bovine Hsc70 crystal structure comprising both domains of the molecule (41). Based on this model, we selected three regions of the mtHsp70 NBD located within or close to the proposed domain interface with the SBD. We hypothesized that the amino acid exchanges introduced into Ssc1 would specifically interfere with the allosteric signaling between NBD and SBD. The model structure, comprising both NBD and SBD of mtHsp70, its protein surface, and the mutated positions are indicated in Fig. 1A. According to the published crystal structure, the exchange of amino acids Asn 175 (Asn 151 in Hsc70) and Asp 176 (Asp 152 ) to alanine (mutant N175A/D176A) abolishes a salt bridge that is formed between Asp 176 of the NBD and Lys 544 (Lys 524 ) of the SBD (Fig. 1B). Altering residues Glu 240 (Glu 218 ) and Val 241 (Val 219 ), resulting in mutant E240A/V241A, is supposed to affect both a cluster of charge interactions (with Lys 546 and Arg 437 of the mtHsp70 SBD) as well as hydrophobic interactions at the interface between the two Hsp70 domains (e.g. Ala 542 within ␣-helix A of the SBD, see Fig. 1B). In addition, we chose to mutate residue Tyr 173 (Tyr 149 ) that is exposed on the molecular surface and located close to both the nucleotide-binding site and the putative domain interface (Fig. 1B), resulting in mutant Y173A.
Yeast cells are not viable in the absence of a functional mtHsp70. To test their in vivo growth phenotypes we expressed the NBD mutants in a strain background lacking the wild-type SSC1 gene. Mutant cells with a deletion of the full-length SSC1 open reading frame (ssc1⌬), expressing wild-type SSC1 from a plasmid containing the URA3 marker gene, were transformed with single copy plasmids encoding the NBD mutants under their endogenous promotor. The plasmid harboring the wildtype copy was selectively removed by growing the cells on 5-fluoroorotic acid, which selects against cells containing the URA3 marker gene. Ura3 converts 5-fluoroorotic acid into the toxic compound 5-fluorouracil. The growth phenotypes of the resulting yeast strains exclusively expressing the mutant mtHsp70s were assayed at different temperatures (Fig. 1C). Cells containing mutant N175A/D176A were able to grow at low temperature (25°C) but showed a pronounced growth defect already at physiological temperature (30°C), indicating a temperature-sensitive phenotype. Cells expressing mutant E240A/V241A were unable to grow at all temperatures tested, exhibiting the lethality of these mutations. The Y173A mutant cells, by contrast, displayed normal growth that was indistinguishable from cells expressing wild-type Ssc1. Hence, with the exception of mutant Y173A, the NBD mutations exhibited severe functional defects in vivo and could not substitute for the wild-type protein.
Import of Matrix-targeted Precursor Proteins Is Strongly Impaired in mtHsp70 NBD Mutants-MtHsp70 is strictly required for both membrane translocation and unfolding of preproteins destined for the mitochondrial matrix. We therefore analyzed the NBD mutants with regard to their efficiency of protein import into the mitochondrial matrix by in vitro import assays. However, due to the severe growth defects of mutants N175A/D176A and E240A/V241A we decided to coexpress the mutant proteins in the background of the conditional mutant ssc1-3 (43). The temperature-sensitive Ssc1-3 FIGURE 1. Phenotype of mtHsp70 mutants. A, surface representation of mtHsp70. The yeast mtHsp70 structure was modeled on the structure of bovine Hsc70 (Protein Data Bank code 1YUW) using the program SWISS-MODEL with the NBD in dark gray and the SBD in light gray. Indicated in blue are the surface-exposed NBD residues that have been mutated in this study and their molecular contacts within the SBD in green. B, magnification into the putative domain interface of mtHsp70. Indicated are the mutated NBD residues (framed) and their potential molecular contacts (italics) within the SBD (acidic residues, red; basic residues, blue; non-charged polar residues, green; hydrophobic residues, gray). C, in vivo complementation of ssc1⌬ strain by ATPase domain mutants. Plasmids encoding the wild-type SSC1 (WT), the NBD mutants (Y173A, ND175AA (N175A/D176A), and EV240AA (E240A/ V241A)), and an empty control plasmid (vector control) were inserted into ssc1⌬ cells by plasmid shuffling. Cells were grown on YPD plates containing 5-fluoroorotic acid at the indicated temperatures.
protein can be almost completely inactivated in isolated mitochondria by a short incubation at 37°C for 15 min that abolishes nucleotide interactions and blocks subsequent preprotein import. The NBD mutants and the control wild-type were expressed as fusion proteins containing a C-terminal V5 and a hexahistidine tag under control of an inducible promotor (GAL1/10). To avoid indirect negative effects due to an excess expression of the mutant proteins, synthesis of the mtHsp70 variants was induced only 2-3 h prior to isolation of mitochondria to protein levels similar to the endogenously expressed mtHsp70 (data not shown). For the import experiments we first employed the standard preprotein b 2 (167) ⌬ -DHFR, a fusion construct consisting of the targeting signal and the first 89 N-terminal residues of the mature cytochrome b 2 fused to the entire mouse DHFR molecule. Due to a 19-residue deletion in the intermembrane space sorting sequence (amino acids 47-65, indicated by a ⌬), b 2 (167) ⌬ -DHFR is translocated to the mitochondrial matrix and processed to an intermediate (i) form by MPP (49). In the matrix, an second processing step by the octapeptidyl peptidase (Oct1) takes place, resulting in the i*-form. For quantification, the bands for both intermediate forms (i and i*) were included in the quantification. The preprotein was synthesized in rabbit reticulocyte lysate in the presence of [ 35 S]methionine and incubated with mitochondria isolated from ssc1-3 cells co-expressing either plasmid-encoded wild-type or mutant mtHsp70s (see above). As shown in the upper panel of Fig. 2A, the fusion protein was imported into wild-type and NBD mutant mitochondria with similar efficiencies, excluding a dominant-negative phenotype caused by the co-expression of ssc1-3 and the NBD mutants. Mitochondria in the control samples ( Fig. 2A, lanes 5-8) were obtained from ssc1-3 cells transformed with the corresponding empty vector. After induction of the non-permissive phenotype by heat shock (see above) immediately prior to import, matrix import was drastically reduced in ssc1-3 mitochondria and in all three NBD mutants (Fig. 2,  A, lower panel, and B). Because all mitochondria employed in the import assays were derived from the ssc1-3 strain, the small background import activity observed in the control mitochondria containing the empty control plasmid was subtracted from each of the samples in all import quantifications. The E240A/ V241A mutant mitochondria reached about 35-40% of the wildtype import efficiency after 15 min of incubation, whereas the Y173A and the N175A/D176A mitochondria were at 20 -25 and 10 -15%, respectively, of the wild-type levels. As a control, we imported the construct b 2 (167)-DHFR, which carries the complete intermembrane space sorting signal and is transported to the intermembrane space. Due to its specific import pathway, b 2 (167)-DHFR utilizes both outer and inner membrane translocation channels but is imported independently of the mtHsp70 system (49). Import of this fusion construct was essentially normal for all mitochondria assayed, even under non-permissive conditions (supplemental Fig. S2A). Preprotein import into the matrix is strongly dependent on the electric potential (⌬) across the inner mitochondrial membrane. To exclude effects of the mtHsp70 mutants on the generation or stability of the potential, we measured ⌬ by use of the fluorescent dye diSC 3 (5) under non-permissive conditions. The fluorescence emission from mitochondriaassociated diSC 3 (5) is quenched by an established ⌬ across the inner membrane. Thus, ⌬ is reflected by the restoration of diSC 3 (5) fluorescence emission in response to addition of the uncoupler valinomycin. The NBD mutant mitochondria exhibited wild-type-like fluorescence quenching, indicating a normal generation and maintenance of ⌬ (supplemental Fig. S2B). Taken together, the high efficiency of intermembrane space transport and the intact membrane potential indicated that the observed impairment in matrix protein import is a specific consequence of defects in mtHsp70 function.
Additionally, we chose the precursor of the ATP synthase component F 1 ␤ as an example for a relatively large genuine mitochondrial preprotein. The radiolabeled preprotein was imported into ssc1-3 mitochondria co-expressing wild-type and mutant mtHsp70s under non-permissive conditions. Quantification of the import data revealed the same tendency as observed for the artificial b 2 (⌬)-DHFR fusion protein (Fig.  2C). However, the import defects were even more pronounced than those observed for the matrix-targeted b 2 -DHFR construct.
We also utilized a smaller preprotein comprising the presequence of the F 0 -subunit 9 of the F 1 F 0 -ATP-synthase from Neurospora crassa and the first 20 residues of the mature protein fused to DHFR (Su9(86)-DHFR). Due to its short unstructured N-terminal extension, it has been shown that productive interaction of the mtHsp70 motor with the preprotein in transit is particularly important for the generation of matrix-directed import activity (see below) and, hence, for full import efficiency (47). Generally, all three mutated mtHsp70s displayed severe defects in Su9(86)-DHFR import as well (Fig. 3A). However, a more detailed analysis of the translocation kinetics revealed interesting differences between the NBD mutants (Fig. 3B). Import into the E240A/V241A mitochondria displayed very low initial rates but was accelerated over longer incubation periods. However, the final yield of imported Su9(86)-DHFR remained lower than that observed for b 2 (167) ⌬ -DHFR, which contains a larger N-terminal extension. In contrast, import into the Y173A mitochondria was initiated with relatively high rates as compared with the other two mutants but reached saturation much faster. For this mutant, the final amount of imported polypeptides was very similar to the values obtained for b 2 (167) ⌬ -DHFR. In the case of N175A/D176A, Su9(86)-DHFR import was almost entirely abolished. In summary, the mutations introduced in the Ssc1 NBD convey severe general defects in the import of matrix-destined precursor proteins. However, the residual translocation activities revealed significant differences between the individual mutants, reflecting specific effects of the mutations on the molecular mechanism of mtHsp70.
NBD Mutants Are Deficient in the Formation of an Inward-directed Translocation Activity-The ability of the mutant mtHsp70s to generate an import driving force on preproteins in transit was analyzed by a specific assay that is based on the stable arrest of translocation intermediates inside the import channels (Fig. 3C). To maximally challenge the import machinery, saturating amounts of recombinant precursor proteins purified from E. coli cells were utilized (29). We employed mitochondria isolated from ssc1-3 cells expressing either wild-type or the NBD mutants as described above. After induction of the ssc1-3-phenotype by heat shock, the fusion protein b 2 (167) ⌬ -DHFR was accumulated inside the translocation channels by preincubation of the preprotein with the DHFR-ligand MTX. MTX stabilizes the native folding state of DHFR and, hence, prevents complete translocation of b 2 (167) ⌬ -DHFR into the mitochondrial matrix. In case of an active mtHsp70 translocation motor, the MTX-stabilized DHFR is maintained in close proximity to the surface of the outer mitochondrial membrane, thereby remaining inaccessible for externally added proteases (Fig. 3D). After dissipation of the inner membrane potential ⌬, proteinase K was added and the amounts of protease-resistant i-b 2 (167) ⌬ -DHFR was determined for each of the samples. In . Import of a short mitochondrial precursor protein, Su9(86)-DHFR. Import reactions into isolated mitochondria were performed as described in the legend to Fig. 2. A, long import kinetics. B, short import kinetics. Data represent average values of five independent experiments. C, assessment of the inward-directed translocation force exerted by wild-type and mutant mtHsp70s; diagram of the experimental procedure. The preprotein b 2 (167) ⌬ -DHFR, preincubated with the DHFR-ligand MTX, was incubated with isolated mitochondria (see legend to Fig. 2) at 25°C (Accumulation). Non-inserted preprotein was removed by reisolation. After dissipation of the membrane potential ⌬ incubation was continued in the presence of ATP, and samples were taken at the times indicated. Protease resistance of the translocation intermediates was assayed by treatment with proteinase K. D, generation of protease-resistant translocation intermediates by an active translocation motor as graphical depiction. Full translocation of the preprotein is prevented by the stably folded DHFR domain. The N-terminal part crosses both membranes (OM and IM) and contacts the import motor (PAM). The preprotein is not accessible to proteolysis by externally added proteinase K (PK) (TOM, translocase of the outer membrane; TIM23, translocase of the inner membrane (IM)). E, quantification of stably accumulated, processed i-b 2 (167) ⌬ -DHFR. The amount of i-b 2 (167) ⌬ -DHFR directly after the accumulation step was set to 100% for wild-type (WT) and each mutant. Error bars and standard deviations are derived from three independent experiments. OM, outer membrane. the case of mitochondria expressing wild-type mtHsp70, ϳ50 -60% of the initially accumulated preprotein intermediates were stably arrested in the translocation channel and remained largely protease-resistant even after 15 min of incubation, representing full translocation motor activity (Fig. 3E). In Y173A mitochondria, about 20% of the overall accumulated intermediates remained protease-resistant throughout the whole incubation period. With about 10% of stably arrested i-b 2 (167) ⌬ -DHFR the N175A/D176A mutant displayed significantly reduced pulling activity that rapidly decreased to levels indistinguishable from the ssc1-3 control mitochondria. In the case of E240A/V241A, only background amounts of protease-resistant intermediates could be detected even directly following the accumulation step. Taken together, the NBD mutations, in particular mutants N175A/D176A and E240A/V241A, caused a significant reduction in the generation of a translocation driving force, correlating with the strong import defects for preproteins destined for the mitochondrial matrix. In case of the mutant Y173A, a slightly improved ability to generate a translocation force is reflected by its high initial import rates.
NBD Mutants Exhibit an Altered Substrate Interaction Behavior during the Import Process-An important feature of the Hsp70 allosteric mechanism is the reduction in substrate affinity resulting from binding of ATP to the NBD (50). To assess the interaction properties of the mutant mtHsp70s with substrate polypeptides in the course of the import reaction, we performed co-immunoprecipitation assays (Fig. 4A), employing the radiolabeled fusion protein Su9(86)-DHFR as a substrate. Su9(86)-DHFR consists of the N-terminal 86 amino acid residues of subunit 9 of the N. crassa F 0 -ATPase and has two processing sites for MPP, resulting in the transient formation of a processing intermediate (i) in addition to the fully matured (m) form. Because about 50 amino acid residues are required to span both mitochondrial membranes, only the first processing site is accessible for MPP in an initial stage of the import reaction. At this stage of the import reaction, mainly the i-form of the precursor protein is formed. The second processing step is possible only after complete insertion of the DHFR moiety into the translocation channel, therefore representing a later stage of the import reaction.
After induction of the ssc1-3-phenotype, Su9(86)-DHFR was imported for 5 min at 25°C. Subsequently, the import reaction was terminated by addition of the uncoupler valinomycin. Samples were further kept at 25°C for distinct incubation periods to chase translocation intermediates into the matrix. Finally, mitochondria were lysed under native conditions in the absence of ATP and co-immunoprecipitations with antibodies directed against mtHsp70 were performed. After subtraction of background signals, represented by the material bound to preimmune control samples, the co-precipitated amounts of both i-and m-Su9(86)-DHFR were quantified. The respective total amounts of imported proteins for each of the samples were set to 100%. As expected, association of wild-type mtHsp70 with fully imported substrate proteins (m-form) was transient and decreased after a short incubation period (Fig. 4B). About 3% of the completely processed m-form could be co-precipitated after 1 min of post-import incubation. The interaction of m-Su9(86)-DHFR with the Y173A mutant was increased by a factor of two compared with wild-type (Fig. 4B). Binding of the N175A/D176A and E240A/V241A mutant proteins to imported and processed substrate proteins was enhanced by more than 3-fold after 1 min and still remained elevated after 15 min of post-import incubation (Fig. 4B). This behavior is consistent with an allosteric defect resulting in prolonged substrate association. Due to the extended conformation of polypeptide chains in transit and the presence of J-type cofactors at the inner face of the translocation pore complex, the overall affinity of mtHsp70 to incoming polypeptide chains is relatively high. Accordingly, we found a higher proportion of co-precipitated i-Su9(86)-DHFR in wild-type mitochondria compared with the m-form (Fig. 4C). In contrast to the differences observed for m-Su9(86)-DHFR binding, mutants Y173A and N175A/D176A interacted with the i-form with similar efficiencies as wild-type but exhibited a slightly destabilized interaction after longer incubations. Interestingly, mutant E240A/V241A displayed lower levels of co-precipitated i-Su9(86)-DHFR, indicating its  Fig. 2) under non-permissive conditions. After 5 min at 25°C, import was terminated and samples were further incubated at 25°C. Subsequently, mitochondria were lysed, and imported proteins bound to mtHsp70 were co-immunoprecipitated using anti-mtHsp70 antibodies. Precipitated proteins were assayed by SDS-PAGE and digital autoradiography. B and C, quantification of co-immunoprecipitated, fully processed m-Su9(86)-DHFR (B) and partially processed i-Su9(86)-DHFR (C). The amounts of i-and mSu9(86)-DHFR detected in the respective import control samples were set to 100% for each of the samples depicted. The amount of protein material that was nonspecifically binding to preimmune serum control samples was subtracted from the co-precipitation values. The data represent average values obtained from at least four independent experiments. inability to efficiently interact with the incoming precursor at the site of the translocation channel. Taken together, all three NBD mutants displayed an altered substrate interaction behavior that may be causative for the severe translocation defects. All mutants, particularly N175A/D176A and E240A/V241A, exhibited enhanced and stabilized binding to fully imported substrate proteins while, especially in the case of E240A/ V241A, the interaction with preproteins in transit was destabilized.
Biochemical Properties of N175A/D176A and E240A/V241A Are Consistent with Defects in the Allosteric Mechanism-To directly assess potential defects in the nucleotide-dependent regulation of substrate interaction, we analyzed the NBD mutants for their nucleotide interaction properties. We first analyzed ATP binding in wild-type and mutant mtHsp70s under steady state conditions by retention of the mtHsp70 constructs on ATPagarose columns. Mitochondrial extracts were prepared by lysis with Triton X-100 under native conditions, and subsequently incubated with immobilized ATP. Under nonpermissive conditions, the endogenous Ssc1-3 protein is not able to bind to ATP (19). Bound mtHsp70 variants were eluted by addition of excess free ATP and detected by Western blot and immunodecoration using anti-V5 antibodies. Wildtype mtHsp70 could be efficiently eluted from the agarose matrix (Fig.  5A, lane 6). Mutants Y173A and N175A/D176A exhibited ATPbinding efficiencies similar to the wild-type protein (Fig. 5A, lanes 8  and 9). The mutant E240A/V241A was able to interact with ATP-agarose as well, albeit with slightly reduced efficiency (Fig. 5A, lane 10). In accordance with the location of the respective amino acids at the domain interface, the NBD mutants did not exhibit significant effects on the steady-state interaction of the NBD with ATP.
The ability of the mutant proteins to release substrate proteins in response to ATP binding was assessed by use of RCMLA as a permanently unfolded Hsp70 model substrate. After lysis of mitochondria under native buffer conditions, the mitochondrial extracts were incubated with RCMLA coupled to Sepharose beads. Bound mtHsp70 was eluted from the Sepharose matrix by addition of excess free ATP. Samples were analyzed by Western blot and immunodecoration using antibodies against the V5 tag. As reported previously, a fraction of wildtype mtHsp70 interacted with the immobilized substrate was retained on the columns and eluted by the addition of ATP (Fig.  5B, lane 6). The Y173A mutant could be eluted from the model substrate with efficiency similar to the wild-type protein (Fig.  5B, lane 8). This mutant, hence, promotes ATP-induced signal transduction to the substrate-binding pocket, which in turn results in substrate release. For both, N175A/D176A and E240A/V241A, only very small amounts could be detected in the ATP eluates (Fig. 5B, lanes 9 and 10). Although with reduced efficiencies, the two mutants per se were able to inter-  2) were lysed under non-permissive conditions, and binding to ATP-agarose was assayed as described under "Experimental Procedures." Bound proteins were eluted by addition of 3 mM ATP. Load (10% of total) and eluate fractions were precipitated with trichloroacetic acid, and separated by SDS-PAGE. MtHsp70 was detected by Western blot and immunodecoration with anti-V5 antibodies. B, binding of wild-type and mutant mtHsp70 to RCMLA-Sepharose. Mitochondria (see legend to Fig. 2) were lysed under non-permissive conditions, and binding to RCMLA-Sepharose was performed as described under "Experimental Procedures." Bound proteins were first eluted with a buffer containing 3 mM ATP and subsequently with SDS-PAGE sample buffer (Laemmli). Samples of the mitochondrial extracts (load, 10% of total) and eluate fractions were precipitated with trichloroacetic acid, analyzed by SDS-PAGE and Western blot using anti-mtHsp70 antibodies. C, tryptic digest of wild-type and mutant mtHsp70s. The fragmentation behavior of wild-type and mutant mtHsp70 was analyzed in the presence of AMP-PNP as described under "Experimental Procedures." A control sample was taken prior to protease treatment (lanes 1-5). After trichloroacetic acid precipitation, proteins were separated by SDS-PAGE and the degradation products were analyzed by Western blot and immunodecoration with an antibody directed against E. coli DnaK. To visualize faint signals, two exposures of the chemiluminescence detection are shown. All results are representative for three independent experiments.
act with the immobilized model substrate (Fig. 5B, lanes 14 and  15). In both cases, the substrate interaction behavior correlated with an inefficient propagation of nucleotide-induced conformational changes from the NBD to the SBD.
Former studies on the conformational state of Hsp70s revealed their differential stabilities towards limited proteolytic digestion in dependence of the nucleotide binding state (41,47,51). In the presence of ATP or its non-hydrolysable analog AMP-PNP, wild-type mtHsp70 is partially digested to a fragment of 56 kDa in size, comprising segments of both NBD and SBD. The appearance of the 56-kDa fragment indicates the compact conformational state induced by ATP binding in which N-terminal parts of the substrate-binding domain are protected against proteolysis. Isolated mitochondria co-expressing the mutant mtHsp70s in the ssc1-3 background were first subjected to heat shock at 37°C. Subsequently, mitochondrial lysates were prepared under native conditions in the presence of AMP-PNP. The lysates were treated with limited amounts of trypsin for different time periods and the resulting fragmentation pattern was analyzed by Western blot using an antiserum against full-length E. coli DnaK. Under the chosen conditions, the endogenous Ssc1-3 protein was completely degraded as shown by the absence of a signal in the control lanes (Fig. 5C, lanes 22-25). As shown previously (47), wildtype mtHsp70 was largely resistant against proteolytic digestion and exhibited a proteolytic fragment of about 56 kDa, indicative for tight interdomain coupling (Fig. 5C, lanes 6 -9). The Tyr 173 mutant displayed a behavior very similar to wildtype mtHsp70, i.e. it showed the presence of the 56-kDa fragment (lanes 10 -13). In the N175A/D176A samples, the 56-kDa fragment appeared in very small amounts, whereas in the case of E240A/V241A it could be hardly detected at all even after long exposures (lanes 14 -17 and lanes 18 -21). The signal decrease of the full-length proteins in the case of these two mutants indicated lower overall stability. These differences in the proteolytic degradation pattern strongly indicate a defect in the formation of the ATP-induced compact state in the case of the two double mutants.
Impaired Interdomain Communication Affects the Interaction with Translocation-specific Partner Proteins-The translocation-specific activity of mtHsp70 is strongly dependent on its specific interaction with partner proteins forming the import motor complex. We therefore assayed the interaction behavior of the mutant mtHsp70s with partner proteins essential for protein import into the mitochondrial matrix. Protein complexes containing wild-type and mutant mtHsp70s were isolated by nickel-chelate affinity chromatography from mitochondrial extracts obtained from ssc1-3 cells co-expressing the mtHsp70 variants. Nucleotide conditions were manipulated by either depletion of endogenous ATP prior to lysis (Fig. 6A) or addition of Mg-ATP in the lysis buffer (Fig. 6B). Samples were analyzed by Western blot and immunodecoration against Mdj1, Tim44, and Mge1. Previous studies using the bacterial DnaK have implicated the region in which we introduced the NBD mutations in the interaction with the J-domain protein DnaJ (52). However, we did not observe significant differences between wild-type and NBD mutants concerning their interaction with the mitochondrial DnaJ ortholog Mdj1. The small amount of background binding present in the control lane was probably due to nonspecific interaction of Mdj1 with the column material. As shown previously, the Mdj1-mtHsp70 interaction was not sensitive to the presence of ATP in the lysis buffer (48). Hence, the introduction of single point mutations in the domain interface did not per se affect binding of mtHsp70 to DnaJ protein family members.
The complex between mtHsp70 and the nucleotide-exchange factor Mge1 is stable in the absence of ATP, whereas addition of ATP results in complex dissociation. Like the wildtype, the Y173A mutant formed a specific and nucleotide-sensitive complex with Mge1 (Fig. 6A, lane 7). In the case of N175A/D176A, formation of the Mge1 complex was reduced to a large extent although the residual Mge1 association remained sensitive to ATP (Fig. 6A, lane 8). For E240A/V241A, the interaction with Mge1 was virtually abolished (Fig. 6A, lane 9). The co-chaperone could only be detected in trace amounts in the eluate fraction. This deficiency in Mge1 binding has severe implications for the mtHsp70 ATPase cycle and, consequently, for the import reaction.
In the presence of peptide substrates, the interaction between mtHsp70 and the inner membrane translocase component Tim44 occurs in a nucleotide-dependent fashion. In the ATP-bound state, mtHsp70 dissociates from Tim44, whereas in the presence of ADP, or in the absence of nucleotides, it co-elutes with mtHsp70. Basically, all NDB mutants were able to interact with Tim44, displaying only slightly reduced binding compared with wild-type mtHsp70 (Fig. 6A, lanes 6 -9). Interestingly, mutant E240A/V241A showed a stabilized interaction with Tim44 even in the presence of ATP (Fig. 6B, lane 9). This observation may correlate with its inability to stably bind to Mge1. E240A/V241A seemed to be trapped in a particular stage of the enzymatic cycle, delaying or preventing nucleotide exchange and indirectly stabilizing the interaction with Tim44.
The transient interaction of mtHsp70 with the J-domain partner Pam18 was analyzed by chemical cross-linking using disuccinimidyl suberate (DSS) and intact mitochondria (Fig.  6C). Under in vivo conditions, Pam18 interacts with mtHsp70 (13) and stimulates its ATPase activity via its conserved J-domain. For purification of cross-linking adducts with the respective mtHsp70 mutant constructs, mitochondria were lysed under denaturing conditions and the resulting extracts were incubated with Ni-NTA-agarose. The eluates were analyzed for cross-linking products by Western blot and decoration with anti-Pam18 antiserum. Cross-linking with DSS yielded several Pam18-containing adducts between 100 and 120 kDa apparent molecular mass that could be co-purified with wild-type mtHsp70 (Fig. 6C, lanes 6 and 16). All of these adducts were generated only in the presence of ATP. The mtHsp70-Pam18 adducts were formed in the Y173A mutant with even slightly increased efficiency compared with wild-type (Fig. 6C, lanes 8  and 18). The mutants N175A/D176A and E240A/V241A exhibited a strongly decreased ability to form Pam18 adducts (Fig. 6C, lanes 9 -10 and 19 -20). Only after long exposure times, small amounts were detectable in the lanes containing the respective purified samples. Because Mdj1 binding was not affected in the mutants, we conclude that the mutations cause a specific Pam18-interaction defect instead of a general J-domain-interaction failure.

DISCUSSION
Mitochondrial Hsp70 (mtHsp70 or Ssc1 in yeast) is required for the import of mitochondrial preproteins into the matrix compartment (5,21). Its activity is based on the nucleotide-dependent interaction with preproteins that emerge on the matrix side of the inner membrane translocation channel. Recently available structural insights into Hsp70 interdomain communication (40,41) allowed us to address the consequences of the allosteric mechanism for the translocation-specific activity of mtHsp70 for the first time. We selected three regions within the NBD of mtHsp70 for site-directed mutagenesis. The mutant proteins were introduced into mitochondria of yeast cells to allow analysis of potential deleterious effects under physiological conditions. Two of the NBD mutants, N175A/D176A and particularly E240A/V241A, were unable to complement the growth defect of a ssc1 null mutation. All three mutants displayed severe in organello defects in regard of their ability to drive protein translocation into the mitochondrial matrix. Matrix import was most severely affected by the N175A/D176A mutant for all precursors tested, whereas E240A/ V241A differed in its residual import activity in dependence of the preprotein employed. Import into the Y173A mitochondria was characterized by fast saturation of the translocation machinery.
Interestingly, our analysis indicated that reduced protein translocation rates and defects in cell viability are not generally correlated. We suggest that the lethal phenotype is attributed to a specific activity of mtHsp70 in the import process. Apart from driving polypeptide movement through the translocation channel, mtHsp70 is also required for the unfolding of stably folded preprotein domains (26). Active unfolding implies the generation of an inward-directed translocation force on preproteins in transit (27,29). As was previously observed for the conditional mutant ssc1-2, in the absence of a translocation force import becomes limited by (low) spontaneous unfolding rates. In case of ssc1-2, this deficiency resulted in a lethal phenotype under non-permissive conditions (19,47). Indeed, the in vivo growth defects of the NBD mutants N175A/ D176A and E240A/V241A were closely reflected by their inability to exert an inward-directed translocation force.
The strong in organello import defect of the N175A/D176A mutant is likely correlated with its significantly enhanced and stabilized interaction with imported substrate proteins. This phenotype resembles the one observed for the temperature- and incubated with Ni-NTA-agarose beads. Bound proteins were eluted with 350 mM imidazole. Load (10% of total) and eluate fractions were precipitated with trichloroacetic acid, separated on SDS-PAGE, and analyzed by Western blot and immunodecoration with antibodies against the V5-tag, Mdj1, Tim44, and Mge1 as indicated. C, isolated wild-type and mutant mitochondria (see legend to Fig. 2) were incubated with the cross-linking agent disuccinimidyl suberate in the presence or absence of Mg-ATP. Mitochondria were solubilized in 1% SDS, and incubated with Ni-NTA-agarose beads in the presence of 0.5% Triton X-100. Bound proteins were eluted with 350 mM imidazole. Load (10% of total) and eluates were precipitated with trichloroacetic acid and separated on SDS-PAGE. Pam18-containing adducts were analyzed by immunoblotting with antibodies raised against Pam18. Monomeric Pam18 and Pam18-containing mtHsp70 adducts are indicated. All results are representative for at least five independent experiments. sensitive Ssc1-2 protein that is characterized by increased and prolonged interactions with imported proteins (27,47,53). Increased trapping of matrix proteins by mtHsp70 interferes with an efficient translocation reaction because the release of substrate proteins becomes limiting. Furthermore, this observation is consistent with a defect in the allosteric mechanism given that a breakdown of conformational signals caused by ATP binding in the NBD might retain the chaperone in its high affinity ADP state.
The biochemical properties exhibited by both N175A/ D176A and E240A/V241A correlated well with defective interdomain communication. Although ATP binding was not affected under steady-state conditions the two mutants did not support an ATP-induced substrate release and displayed a degradation behavior different from the wild-type protein. As can be deduced from the crystal structure of bovine Hsc70, mutations N175A/D176A and E240A/V241A affect close contact ionic and hydrophobic interactions that directly connect the NBD with helix A of the SBD (N175A/D176A and E240A/ V241A) and with an adjacent part of the SBD ␤-sheet subdomain (E240A/V241A). Both structural elements have been implicated in the allosteric mechanism because helix A forms the basis of the ␣-helical lid that covers the peptide-binding pocket formed by the ␤-sheet subdomain (54,55). Additionally, apart from directly disrupting molecular contacts between NBD and SBD, the mutations could affect interdomain communication by interfering with the insertion of the hydrophobic domain linker segment into a hydrophobic cleft between subdomains IA and IIA of the NBD. These NBD-linker interactions have recently been proposed to be involved in the allosteric mechanism (40,56). A partially equivalent mutation to N175A/D176A was analyzed in bovine Hsc70. Substitution of residue Asp 152 (corresponding to Asp 176 in Ssc1) by lysine resulted in a phenotype similar to N175A/D176A and E240A/ V241A, i.e. strongly reduced biological activity (in this case clathrin uncoating), and reduced nucleotide-dependent conformational changes as indicated by alterations in the proteolytic pattern of the chaperone (41).
The Y173A mutant, by contrast, displayed nucleotide-dependent substrate interactions under steady-state conditions and a proteolytic digestion pattern indistinguishable from the wild-type protein. Although not directly contributing to the domain interface, the residue Tyr 173 is closely located to a region that has been identified in the bacterial Hsp70 DnaK to form a switch-relay system, propagating nucleotide-induced signals to the surface of the NBD and altering peptide affinity in the SBD (39). The Y173A mutant showed wild-type activity in terms of ATP binding, in the ability to adopt the ATP-induced compact conformation, and an efficient J-domain interaction. Despite these apparent normal properties, import efficiency into the Y173A mitochondria was significantly reduced. Specifically, mutant Y173A displayed relatively normal initial import kinetics but reached saturation already after short incubation periods. Given that Y173A displays ATP-induced substrate release under steady-state conditions, this phenotype cannot be attributed to impaired signal transduction between NBD and SBD. Instead, it is most likely due to reduced enzymatic cycling at the import sites, i.e. a reduction in the nucleotide hydrolysis rate. This hypothesis is supported by the observed enhanced and prolonged interaction of the Y173A mutant with imported substrate proteins.
Functional interaction with specific partner proteins or cochaperones is a characteristic feature of Hsp70-type chaperones. The NBD mutants are all capable to bind to the essential inner membrane translocase component Tim44, which recruits otherwise soluble mtHsp70 to the translocation site and serves as fulcrum for the generation of an inward-directed translocation force (45,57). In contrast to wild-type mtHsp70, the interaction of E240A/V241A with Tim44 was only poorly affected by ATP, which correlates with the severe defects of this mutant in allosteric communication. In addition, the E240A/V241A mutant showed severe deficiencies in the interaction with the nucleotide-exchange factor Mge1, whereas mutants Y173A and N175A/D176A were not or less severely affected. Mge1 has been reported to be essential for the translocation reaction (58,59), most likely by its ability to activate ATP hydrolysis and hence recycling of mtHsp70 at the import site (60). In relation to the NBD mutations, the binding sites for Mge1 are located on the opposite face (48), indicating an indirect influence on cofactor interaction. Taken together, we suggest that the Mge1 and Tim44 interaction defects are rather mediated by an arrest in the mtHsp70 reaction cycle caused by interference with the allosteric mechanism.
Proteins of the DnaJ family have been shown to be intricately involved in the allosteric regulation of Hsp70 chaperones (11,31). J-proteins not only activate the ATPase activity of Hsp70type chaperones but contribute to the transduction of conformational signals from the NBD to the SBD, thereby enhancing substrate affinity (32). MtHsp70 has two prominent J-domain partner proteins: Mdj1, the genuine DnaJ homolog, is involved in protein folding reactions in the matrix (61,62), whereas the membrane-integrated Pam18 regulates mtHsp70 translocation activity at the import channel (12)(13)(14). In contrast to Mdj1, the Pam18-mtHsp70 interaction was sensitive to the presence of ATP (13,48), indicating a different interaction behavior of mtHsp70 with the individual J-domain partner proteins. The NBD mutations are located in a region that has been implicated in the interaction of the bacterial chaperone DnaK with DnaJ (52). However, mutations in the DnaK chaperone not only abolished DnaJ interactions, but also resulted in increased basal ATPase rates and in a reduced dissociation rate constant (k off ) for bound peptide substrate. We did not observe any effect of the mtHsp70 mutations on the steady-state interaction with Mdj1 in organello. Furthermore, according to the recently established crystal structure of bovine Hsc70 in the presence of the auxilin J-domain (63), the mutations we have introduced into the NBD do not directly interfere with J-domain contact sites (64). Interestingly, the crystal structure revealed a widespread displacement of NBD:SBD contacts in the presence of the J-domain that for example, directly affected the salt bridge formed by residues Asp 152 -Lys 544 (corresponding to Asp 176 in Ssc1). However, due to the interdependence of J-protein binding and the allosteric regulation of Hsp70 substrate affinity, it is conceivable that defects in interdomain communication affect both Hsp70 function directly and interaction with J-proteins indirectly. This is supported by the observation that mutant N175A/D176A as well as E240A/V241A did not bind to the translocase component Pam18, whereas Y173A displayed wildtype-like Pam18 interactions. We therefore conclude that the observed defects in Pam18 binding do not reflect a general inability of N175A/D176A and E240A/V241A to interact with J-domain co-chaperones but is rather explained by a retention of the chaperone in a non-productive state of the enzymatic reaction cycle state in which it is unable to interact with the translocase channel components of the inner membrane.
In summary, our data show that interference with the allosteric mechanism of mtHsp70 in its in vivo environment retained the chaperone in the high affinity state of its ATPase cycle. The resulting reduction of recycling rates at the import site severely compromised the ability of the import motor complex to generate an active translocation driving force on preproteins in transit. Indirectly, this defect would also lead to an impaired interaction with the nucleotide exchange factor Mge1 and the J-domain cofactor Pam18. In combination, these defects contribute to a significant decrease of the mtHsp70mediated protein import activity as well as to a severe growth reduction in vivo.