The Disaggregation Activity of the Mitochondrial ClpB Homolog Hsp78 Maintains Hsp70 Function during Heat Stress

doi:10.1016/j.jmb.2006.01.008

J. Mol. Biol. (2006) 357, 793–807

The Disaggregation Activity of the Mitochondrial ClpB Homolog Hsp78 Maintains Hsp70 Function during Heat Stress Birgit von Janowsky1,2, Tamara Major1,2, Karin Knapp1 and Wolfgang Voos1* 1

Institut fu¨r Biochemie und Molekularbiologie, HermannHerder-Str. 7, Universita¨t Freiburg, 79104 Freiburg Germany 2

Fakulta¨t fu¨r Biologie Universita¨t Freiburg, 79104 Freiburg, Germany

Molecular chaperones are important components of mitochondrial protein biogenesis and are required to maintain the organellar function under normal and stress conditions. We addressed the functional role of the Hsp100/ClpB homolog Hsp78 during aggregation reactions and its functional cooperation with the main mitochondrial Hsp70, Ssc1, in mitochondria of the yeast Saccharomyces cerevisiae. By establishing an aggregation/disaggregation assay in intact mitochondria we demonstrated that Hsp78 is indispensable for the resolubilization of protein aggregates generated by heat stress under in vivo conditions. The ATP-dependent disaggregation activity of Hsp78 was capable of reversing the preprotein import defect of a destabilized mutant form of Ssc1. This role in disaggregation of Ssc1 is unique for Hsp78, since the recently identified, Hsp70-specific chaperone Zim17 had no effect on the resolubilization reaction. We observed only a minor effect of the second mitochondrial Hsp100 family member Mcx1 on protein disaggregation. A “holding” activity of the mitochondrial Hsp70 system was a prerequisite for a successful resolubilization of aggregated proteins. We conclude that the protective role of Hsp78 in thermotolerance is mainly based on maintaining the molecular chaperone Ssc1 in a soluble and functional state. q 2006 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: mitochondria; preprotein import; protein aggregation; Hsp70; Hsp78

Introduction Mitochondria play a crucial role in the cellular metabolism by providing ATP and by performing many, in part essential, biosynthetic reactions.1 Recent research revealed that mitochondria also participate in important regulative processes like apoptosis.2 Hence, maintenance of mitochondrial activity by preserving protein content and function is a key aspect of cellular survival. Being of endosymbiontic origin, mitochondria contain an extensive set of metabolic and biosynthetic Present address: K. Knapp, Schwarz Pharma AG, 40789 Monheim, Germany. Abbreviations used: Hsp, heat shock protein; mtHsp70, mitochondrial Hsp70; DHFR, dihydrofolate reductase; Clp, caseinolytic protease. E-mail address of the corresponding author: [email protected]

enzymes. However, their genome encodes only a very limited number of endogenous proteins. Most mitochondrial proteins are synthesized in the cytosol and transported through the mitochondrial membranes to their final destination. An elaborate proteinaceous machinery, consisting of receptors, membrane channels and many other cofactors, ensures the effective transport of precursor proteins through the mitochondrial membranes.3–5 Molecular chaperones perform crucial functions at key steps of this protein biogenesis pathway. In addition, they are important components of a protein quality control system that is responsible for the maintenance of mitochondrial protein function under normal and stress conditions. In this protective role, molecular chaperones cooperate with oligomeric, ATP-dependent proteases that are responsible for the removal of irreversibly damaged polypeptides.6–8 A dysfunctional or insufficient protein quality control system

0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.

794 has been implicated in major pathological processes in human cells that are caused by the accumulation of protein aggregates.9,10 A major component of the chaperone system is the mitochondrial Hsp70 (mtHsp70), an essential protein, encoded by the gene SSC1 in yeast. Ssc1 is required for the import of all preproteins destined for the mitochondrial matrix. Here, its main role is to provide the ATP-generated driving force for the polypeptide translocation reaction.3,11 It is also involved in protein folding reactions, resulting in assembly and full activity of the respective substrate proteins.12 The folding function of Hsp70s is closely correlated with their role in the stabilization of misfolded or denatured proteins under stress conditions.13 In this case, an interaction with Hsp70 can prevent aggregation and may mediate refolding to the active conformation.14,15 Members of another ubiquitous protein family, the Hsp100 or Clp chaperones, are prominent components of protein quality control mechanisms.16 Some members of the Hsp100 protein family, in particular the bacterial chaperones ClpA, ClpX and their homologs, associate physically with the protease ClpP and become part of the active protease complex. Their function during proteolysis is focused on the recognition and unfolding of the respective protease substrate proteins.17–21 A specific subclass of Hsp100 chaperones generally deals with the deleterious effects of stress conditions but does not interact directly with proteases. The chaperone ClpB is able to prevent the toxic effects connected with the accumulation of protein aggregates. In Saccharomyces cerevisiae, homologs of ClpB have been identified in the cytosol (Hsp104) and in mitochondria (Hsp78). ClpB and its homologs belong to the AAA (ATPases associated with a variety of cellular activities) protein family.22 Their protective function against thermal stress is based on their capability to resolubilize aggregated proteins and facilitate their refolding to the active conformation. The first indications of this remarkable activity were obtained with Hsp104, which is the main component in the protection of cytosolic proteins against thermal inactivation in eukaryotic cells.23,24 Biochemical in vitro experiments demonstrated that the disaggregation reaction catalyzed by ClpB is performed in a close cooperation with the Hsp70 chaperone system.25–28 The functional role of the mitochondrial ClpB family member Hsp78 in the cellular environment still remains to be established. A deletion mutant of Hsp78 in yeast has no obvious growth phenotype at normal conditions and under heat stress.29 However, combination of a conditional lethal mtHsp70 mutation, ssc1-3, with an hsp78D deletion resulted in a severe growth phenotype even at permissive conditions.30,31 Based on the observation that the growth defect of the ssc1-3 mutant was partially rescued by the overexpression of Hsp78, a functional cooperation of Hsp78 with mtHsp70 is apparent, although the mechanistic basis for this effect has not been clarified so far. It has been

Hsp78-mediated Protein Disaggregation

proposed that Hsp78 could directly replace mtHsp70 and take over its functions in protein import.31 On the other hand, Hsp78 has been shown to be involved in the protection of respiratory competence and genome integrity under conditions of severe heat stress.32 The finding that Hsp78 was necessary to restore mitochondrial DNA replication after thermal inactivation of the DNA polymerase33 has corroborated this observation. Experiments in vitro, using purified Hsp78, indeed demonstrated its ability to reactivate heat-denatured model proteins like luciferase in cooperation with mtHsp70.34 Although these experiments indicate a role of Hsp78 in the maintenance of mitochondrial protein synthesis, the biochemical basis of its role during preprotein import and its functional cooperation with the major mitochondrial chaperone mtHsp70 (Ssc1) is not resolved to date. We addressed the protective role of Hsp78 during heat stress conditions by studying aggregation and disaggregation reactions in intact mitochondria. Different types of reporter substrates were imported into isolated mitochondria and their distribution between soluble and pellet fraction was assayed. The effects of mitochondrial Hsp100 mutant proteins on the solubility of destabilized proteins were tested. While the function of the mtHsp70 system had some effect on the prevention of aggregation, we observed a resolubilization of aggregated proteins that was strictly dependent on Hsp78. Mitochondria with a deletion of the mitochondrial ClpX homolog, Mcx1, showed no effect on the aggregation but were partially defective in the disaggregation reaction. The recently identified mtHsp70-specific chaperone Zim17 was not required for the resolubilization reaction of an aggregated destabilized Ssc1 mutant. High matrix ATP levels were required for both prevention of aggregation and disaggregation. Thus, the mitochondrial chaperones mtHsp70 and Hsp78 closely cooperate in maintaining aggregation-prone proteins in a soluble state.

Results The destabilized mtHsp70 mutant Ssc1-3 is reactivated by Hsp78 The mitochondrial Hsp70, Ssc1, is required for the import of precursor proteins across the mitochondrial membranes into the matrix compartment. Mitochondria from the temperature-sensitive mutant strain ssc1-3 are virtually unable to drive the translocation of preproteins under nonpermissive conditions due to a single point mutation (G56S) in the ATPase domain of Ssc1.35 The reporter protein Su9-DHFR, consisting of the 70 amino-terminal residues of the Fo-ATPase subunit 9 from Neurospora crassa fused to the mouse dihydrofolate reductase (DHFR), showed a typical twostep processing during import into wild-type and mutant mitochondria at the permissive temperature (Figure 1(a)). With increasing time of a heat

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pretreatment at 37 8C before the import reaction, processing of Su9-DHFR to the mature form was inhibited in ssc1-3 mitochondria and the intermediate form, which is located in the intermemstarted to accumulate. brane space, 35 Overexpression of Hsp78 in ssc1-3 cells via an inducible promotor improved the import activity of ssc1-3 mitochondria substantially. However, the import defect of the ssc1-3 mutation could not be completely overcome by Hsp78, indicated by the successive loss of import efficiency of the ssc1-3/ Hsp78 mitochondria at longer pre-incubation times at non-permissive conditions. The protective effect of Hsp78 on the import activity of Ssc1-3 was only possible in the presence of high matrix ATP levels (Figure 1(b)). If the heat pretreatment was performed after depletion of matrix ATP, the translocation activity of Ssc1-3 was not recovered by the overproduction of Hsp78, although the import reaction was performed in the presence of ATP. This indicates that the reactivation of Ssc1-3 by Hsp78 is an active, ATP-dependent process. The preprotein translocation function of mtHsp70 is dependent on a direct interaction with the translocated preproteins during the import reaction. We tested the interaction of Ssc1-3 with Su9-DHFR

Figure 1. Hsp78 partially recovers mtHsp70 import function in ssc1-3 mitochondria after induction of the temperature-sensitive phenotype. (a) Import and processing of Su9-DHFR in ssc1-3 mitochondria with normal and elevated Hsp78 levels. Wild-type (lanes 1, 4, 7, 10) and ssc1-3 (lanes 3, 6, 9, 12) mitochondria as well as ssc1-3 mitochondria overexpressing Hsp78 (lanes 2, 5, 8, 11) were incubated as indicated before import of radiolabeled Su9DHFR was initiated. Samples were analyzed by SDS-PAGE and autoradiography. Precursor (p), intermediate form (i) and mature form (m) of Su9-DHFR are indicated. (b) ATP requirement for the protective function of Hsp78. Su9-DHFR was imported into mitochondria isolated from the indicated strains after preincubation at 25 8C (lanes 1–3) or 37 8C (lanes 4–9) in the presence (lanes 1–6) or absence (lanes 7–9) of ATP. (c) Quantification of imported preproteins bound to mtHsp70 by co-immunoprecipitation. Imported Su9DHFR was precipitated with antibodies against mtHsp70 from ssc1-3 mitochondria containing normal (lanes 1 and 3) or elevated (lanes 2 and 4) levels of Hsp78 after the indicated pre-incubation conditions.

during import by co-immunoprecipitation using anti-Hsp70 antibodies (Figure 1(c)). Under nonpermissive conditions the amount of preproteins bound to Ssc1-3 was strongly increased in the mitochondria overexpressing Hsp78. The increased import efficiency of the mitochondria containing elevated levels of Hsp78 thus correlated directly with the improved preprotein interaction of Ssc1-3, indicating that Hsp78 had a repair effect on the activity of the mtHsp70 mutant. Hsp78 is required for resolubilization of aggregated mtHsp70 Since Hsp78 was involved in the maintenance of mtHsp70 import function, we asked if we could observe direct effects of Hsp78 on the aggregation behavior of Ssc1-3 in intact mitochondria under heat stress. We used an in organello aggregation/ disaggregation assay (Figure 2(a)) that relies on the analysis of individual substrate proteins after their import into isolated mitochondria. The use of intact organelles allowed us to monitor the effect of chaperones on specific reporter proteins under conditions identical to the in vivo situation. We analyzed wild-type mitochondria and mitochondria

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preprotein

Figure 2. Disaggregation of Ssc13 is dependent on Hsp78. (a) Scheme of the in organello mitochondria 15 min 25˚C 10 min 42˚C 60 min 25˚C aggregation/disaggregation assay. PK treatment + recovery – recovery After import of precursor proC teins, mitochondria were resuspended in a buffer containing lysis and high speed spin 15 min at 100,000 x g an ATP regenerating system and subjected to a heat shock to pellet supernatant induce protein aggregation. The mitochondria were further incubated at 25 8C for 60 min to allow disaggregation to occur. Samples were taken at the indicated (b) 4 1 2 3 5 6 positions and processed immediately by lysis and centrifugation supernatant imported Ssc1-3 to separate aggregated and solpellet uble matrix proteins. (b) Autoradiogram of an in organello C – + C – + recovery aggregation/disaggregation experhsp78∆ WT iment with wild-type (lanes 1–3) and hsp78D (lanes 4–6) mitochondria. Shown are supernatants (upper panel) and pellets (lower panel) of the control sample before heat treatment (C, lanes 1 (c) 80 and 4), aggregation (Krecovery, lanes 2 and 5) and disaggregation WT 60 (Crecovery, lanes 3 and 6) samples. (c) Kinetics of the dis40 aggregation reaction in isolated wild-type (black diamonds) and hsp78∆ 20 hsp78D (gray squares) mitochondria. After import and heat shock, 0 mitochondria of both strains were 0 20 40 60 80 100 incubated at 25 8C and aliquots recovery time (min) were taken after 15, 30, 60 and 90 min. The amount of aggregated Ssc1-3 directly after heat shock was set 100% and the percentage of disaggregated material was determined by SDS-PAGE and autoradiography.

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lacking Hsp78 (hsp78D) for their ability to prevent aggregation of imported, radiolabeled Ssc1-3 following import and heat shock. In addition, we tested their ability to resolubilize aggregated forms of Ssc1-3 that may have formed during the heat treatment. After import Ssc1-3 molecules aggregated significantly stronger in hsp78D mitochondria after heat shock treatment compared to wild-type (Figure 2(b)). Whereas the aggregates were efficiently resolubilized within 1 h of incubation at 25 8C in wild-type mitochondria, no disaggregation could be observed in the absence of Hsp78. We performed a time-course experiment of the Ssc1-3 resolubilization reaction (Figure 2(c)). Within the first 90 min after heat shock, we observed a continuous increase in the amount of soluble Ssc13 in wild-type mitochondria. In contrast, in hsp78D mitochondria only background levels of soluble Ssc1-3 could be detected throughout the experiment, demonstrating that the resolubilization defect has not been caused by a delay of the reaction in hsp78D mitochondria. We also observed an approximately 30% higher disaggregation efficiency of Ssc1-3 in mitochondria overexpressing Hsp78 compared to wild-type mitochondria (data

not shown), which is in good correlation with the increased preprotein translocation efficiency observed in ssc1-3/Hsp78 mitochondria (Figure 1). In summary, while only a weak protective effect of Hsp78 in preventing aggregation of Ssc1-3 was observed, the experiments indicated that the mitochondrial ClpB homolog is essential for the resolubilization of already aggregated Ssc1-3. The disaggregation of Ssc1-3 by Hsp78 thus contributes to the amount of functional Ssc1-3 proteins in the matrix compartment after heat stress, improving the ability of ssc1-3 mitochondria to import preproteins. We asked if the disaggregation function of Hsp78 is also effective on the wild-type form of Ssc1. We performed an analysis of aggregation and disaggregation of endogenous mitochondrial proteins in wild-type and hsp78D mitochondria. Coomassie blue staining of aggregated proteins separated by SDS-PAGE revealed considerable aggregation of many mitochondrial proteins after heat shock at 42 8C. However, no significant difference was observed in the pattern of aggregated proteins between wild-type and hsp78D mitochondria (data not shown). When we directly analyzed

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Role of the chaperones Mcx1 and Zim17 in the resolubilization of Ssc1-3 Additionally to Hsp78, yeast mitochondria possess a second member of the Hsp100 class of proteins, the ClpX homolog Mcx1.36 In bacteria, ClpX associates with the ClpP protease to form a functional chaperone–protease complex responsible for the ATP-dependent degradation of substrate polypeptides.16 Since an equivalent of the ClpP protease is lacking in yeast mitochondria, the function of Mcx1 is not clear. We therefore investigated a potential role of Mcx1 in protein aggregation and disaggregation. We compared wild-type mitochondria (Figure 4(a), lanes 1–3) and mitochondria lacking Hsp78 (Figure 4(a), lanes 4–6), Mcx1 (Figure 4(a), lanes 7–9) or both (Figure 4(a), lanes 10–12) for their ability to protect imported radiolabeled Ssc1-3 during heat stress. In contrast to Hsp78, Mcx1 did not protect imported

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the behavior of endogenous Ssc1 under heat shock conditions in intact mitochondria, we found a substantial amount of Ssc1 associated with the highspeed centrifugation pellet. During the recovery period almost all of the aggregated Ssc1 proteins were resolubilized to the supernatant (Figure 3(a), lanes 1 and 2). Although the amount of aggregated Ssc1 in the pellet was not increased in hsp78D mutant mitochondria, the disaggregation of wild-type Ssc1 was strictly dependent on the presence of Hsp78 (Figure 3(a), lanes 3 and 4). The presence of Ssc1 in the aggregation pellet can have two reasons: (i) it coaggregates with thermolabile proteins due to its chaperone activity; or (ii) it is itself denatured by heat stress and aggregates. Most likely, both scenarios occur under in vivo conditions. However, Ssc1 showed a pronounced tendency to form aggregates when we analyzed the purified protein (Figure 3(b)). Under the buffer conditions used, already about 30–40% of Ssc1 were found in the pellet at 4 8C. Heat treatment strongly increased the amount of aggregated Ssc1. At least in the absence of mitochondrial cofactors, the nucleotide state of Ssc1 had no major influence on its aggregation behavior. It is therefore conceivable that Ssc1 itself becomes a substrate of Hsp78 under heat shock conditions. We observed the resolubilization effect of Hsp78 also with other members of the mitochondrial mtHsp70 system. When we monitored the resolubilization of endogenous Mge1 and Mdj1 from protein aggregates we found that both proteins were removed from the pellet fraction in an Hsp78-dependent manner (Figure 3(c)). Although only minor effects of a lack of Hsp78 could be observed concerning the aggregation behavior of the overall protein population, the disaggregation ability of Hsp78 affected the complete mitochondrial Hsp70 system. Interestingly, in contrast to Ssc1, merely negligible amounts of Hsp78 were found associated with the aggregate pellet under the heat shock conditions tested (Figure 3(c)), indicating that the activity of Hsp78 may only require a transient interaction with the protein aggregates.

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Figure 3. Disaggregation of endogenous mitochondrial chaperones by Hsp78. (a) Ssc1 is efficiently removed from the pellet after heat shock in an Hsp78-dependent manner. The in organello aggregation/disaggregation assay was performed with the indicated mitochondria and the amount of endogenous Ssc1 in the pellet fraction after heat shock (K recovery, lanes 1 and 3) and recovery phase (C recovery, lanes 2 and 4) was determined by SDS-PAGE and Western blot. (b) MtHsp70 is prone to aggregation after heat shock in vitro. Purified Ssc1 was subjected to a heat treatment of 42 8C for 10 min (HS) in the presence (C) or absence (K) of Mg-ATP. Aggregation was analyzed by highspeed centrifugation as described. Proteins were separated by SDS-PAGE and stained with Coomassie blue. Lane 1 represents 50% of the total protein material. (c) Disaggregation of endogenous proteins in wild-type and hsp78D mitochondria. After performing the aggregation/disaggregation assay as described in the legend to Figure 2(a), supernatants and pellets were analyzed for the indicated proteins by SDS-PAGE and Western blot. Shown are loading controls of the supernatants directly after heat shock (lanes 1 and 2) as well as disaggregation samples after 0 min (lanes 3 and 7), 30 min (lanes 4 and 8), 60 min (lanes 5 and 9) and 180 min (lanes 6 and 10) of recovery.

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Ssc1-3 from heat-induced aggregation since the degree of aggregation was equal to the wild-type situation (Figure 4(b)). However, resolubilization during the recovery phase was partially impaired in mcx1D mitochondria (Figure 4(c)), suggesting a minor involvement of Mcx1 in the recovery after protein aggregation. The double deletion of both mitochondrial Hsp100 chaperones, Hsp78 and Mcx1, had the same effect as the deletion of Hsp78 alone. The experiments indicate that Hsp78 is essential to dissolve protein aggregates formed during heat stress, whereas the second Hsp100 chaperone, Mcx1, may have a supportive role, acting downstream of Hsp78 by facilitating the refolding of the disaggregated proteins. Recently it has been shown that the protein Zim17 is required to keep mtHsp70 in a soluble state.37,38 To investigate its potential role in the disaggregation of Ssc1-3, we performed the in

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Figure 4. Role of Mcx1 and Zim17 in protection of Ssc1-3. (a) Aggregation and disaggregation of Ssc1-3 in Hsp100 mutants. Shown are autoradiograms of supernatants (upper panel) and pellets (lower panel) of an in organello aggregation/disaggregation assay after import of radiolabeled Ssc1-3 into the indicated mitochondria. Samples are labeled as described in the legend to Figure 2. (b) Quantification of the Ssc1-3 aggregation assay as shown in (a). The sum of Ssc1-3 in supernatant and pellet of the control sample was set 100% and the percentage of signal intensity decrease in the supernatant fraction of the aggregation sample was determined. (c) Quantification of Ssc1-3 resolubilization in the experiment shown in (a). The amount of aggregated Ssc1-3 after heat shock was set 100% and the percentage of resolubilization after the recovery phase was calculated for the supernatants. (d) Influence of Zim17 on aggregation of imported radiolabeled Ssc1-3. After the aggregation/disaggregation assay with wild-type and zim17D mitochondria the percentage of aggregated Ssc1-3 was determined as described for (b). (e) Resolubilization of aggregated Ssc1-3 in dependence on Zim17. Quantification of the disaggregation reaction in wild-type and zim17D mitochondria after an in organello aggregation/disaggregation experiment as described for (c).

organello aggregation/disaggregation assay with mitochondria lacking Zim17. Compared to wildtype mitochondria, aggregation of Ssc1-3 was slightly increased in the zim17D strain (Figure 4(d)), confirming its role in supporting the solubility of Ssc1 in mitochondria. However, no defect in the resolubilization of Ssc1-3 could be observed in the absence of Zim17 (Figure 4(e)), indicating that Zim17 is not involved in the disaggregation reaction. Imported artificial reporter proteins are disaggregated by Hsp78 To investigate if the protective function of Hsp78 during heat stress is a general effect on soluble, aggregation-prone proteins in the mitochondrial matrix, we tested if an artificial reporter protein was also recognized by Hsp78 as a substrate. We used

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Figure 5. Destabilized artificial reporter proteins are disaggregated by Hsp78 and targeted for degradation. (a) Schematic representation of the b2(107)D-DHFR constructs. Shown are presequence (grey), matrix processing site (MPP), deletion of the intermembrane space targeting signal (indicated by a buckled line), the segment derived from cytochrome b2 (striped) and the mouse DHFR (checkered). The destabilized variant b2(107)D-DHFRds carries the three point mutations C7S, S42C and N49C in the DHFR domain (*) which prevent stable folding of the construct. (b) Aggregation/disaggregation of imported b2-DHFR fusion proteins. Recombinant b2(107)D-DHFR (left panel) or b2(107)D-DHFRds (right panel) were imported into the indicated mitochondria and the aggregation/disaggregation assay was performed as described. Samples were analyzed by SDS-PAGE and Western blot using antibodies specific for DHFR. (c) Hsp78-dependent resolubilization of aggregated b2(107)D-DHFRds. Aggregate pellets corresponding to lanes 8, 9, 11, and 12 of Figure 5(b) are shown. Samples were analyzed as described above. Indicated are the imported and processed protein (I, lane 1) and the aggregate pellets before (K, lane 2) and after (C, lane 3) recovery for 60 min at 25 8C in wild-type (WT) and hsp78D mitochondria. (d) Kinetics of the disaggregation reaction in isolated wild-type (black diamonds) and pim1D (gray squares) mitochondria. After import of b2(107)D-DHFRds and heat shock mitochondria were incubated at 25 8C for 120 min and aliquots were taken after 60, 90 and 120 min. After SDS-PAGE and Western blot the amount of aggregated protein in the supernatant directly after heat shock was set 100% and the percentage of disaggregated material was determined. (e) Resolubilization rates are similar in wild-type and pim1D mitochondria. The reporter substrates b2(107)D-DHFRds and Ssc1-3 were imported into the indicated mitochondria. An aggregation/ disaggregation assay was performed in organello as described in Material and Methods. Shown are the aggregate pellets before (C, lane 1) and after (K recovery, lane 2) an incubation for 10 min at 42 8C. Resolubilization is indicated by the reduction of the aggregated protein material after 60 min recovery at 25 8C (C recovery, lane 3). The reporter protein b2(107)D-DHFRds was detected by Western blot and Ssc1-3 by autoradiography.

fusion proteins between the amino-terminal part of cytochrome b2 and DHFR from mouse (Figure 5(a)). In the construct b2(107)D-DHFR the part derived from cytochrome b2 comprises the first 107 aminoterminal amino acid residues, providing a mitochondrial targeting sequence and the processing site for the matrix processing peptidase (MPP). Targeting to the intermembrane space is prevented by a deletion of 19 amino acid residues, resulting in localization of this protein to the mitochondrial matrix. The amino-terminal cytochrome b2 part is fused to DHFR by a short linker. After import into isolated mitochondria the DHFR domain of b2(107)D-DHFR folds to its native conformation and the protein is soluble in the matrix. A variant of this protein, b2(107)D-DHFRds, contains three point mutations in the DHFR domain (Figure 5(a)) and cannot acquire a stable folding state,39 making

it prone to aggregation under conditions of heat stress. The DHFR fusion proteins were expressed and purified from Escherichia coli cells. When imported in saturating amounts into isolated wild-type mitochondria, b2(107)D-DHFR remained mostly soluble in the matrix upon heat shock, whereas it aggregated to approximately 30% in hsp78D mitochondria after heat treatment (Figure 5(b), left panel). No solubilization was observed in the absence of Hsp78, confirming the requirement of Hsp78 for disaggregation reactions. In contrast to the wild-type DHFR fusion protein, the destabilized variant b2(107)D-DHFRds aggregated strongly in both strains due to the large amount of imported destabilized protein material. In wild-type mitochondria efficient disaggregation occurred with nearly 50% of the imported protein being recovered

800 in the soluble fraction after 1 h of incubation at 25 8C (Figure 5(b), lane 9). Again, mitochondria lacking the chaperone Hsp78 were not able to resolubilize the aggregated b2(107)D-DHFRds (Figure 5(b), lane 12). The dependence of the disaggregation reaction on Hsp78 was confirmed by the analysis of the aggregate pellets (Figure 5(c)). While a substantial amount of b2(107)D-DHFRds disappeared from the pellet fraction in wild-type mitochondria, the amount of aggregated material remained the same in hsp78D. Thus, Hsp78 is generally required for disaggregation of unstable endogenous and artificial proteins in the mitochondrial matrix. Substrates of the protease Pim1 are targeted for degradation after solubilization by Hsp78 The import experiments indicated that the destabilized mutant Ssc1-3 regained enzymatic activity after resolubilization by Hsp78. Recently we showed that Hsp78 was also involved in the degradation of substrate proteins by the mitochondrial protease Pim1.39 Pim1, a homolog of the bacterial Lon, is the major protease of the mitochondrial matrix and has been shown to recognize b2-DHFR constructs as degradation substrates.39,40 We therefore asked if some of the resolubilized polypeptides could be directly targeted for degradation. We performed a timecourse experiment of the in organello aggregation/ disaggregation assay after import of b2(107)DDHFRds into isolated wild-type and pim1D mitochondria. Since pim1D mitochondria are respiratory deficient, we used a comparable rhoK wild-type strain as control. Due to the limited ability to synthesize endogenous ATP, we observed a generally lower degree of protein disaggregation in rhoK mitochondria compared to wild-type mitochondria. However, the amount of proteins in the supernatant during the recovery period was significantly increased in the protease-deficient pim1D mitochondria (Figure 5(d)), indicating that in wild-type mitochondria a part of the resolubilized protein material was targeted for degradation. To exclude the possibility that the elevated amount of soluble reporter proteins in pim1D mitochondria was due to an increased resolubilization efficiency, we directly analyzed the protein amounts in the aggregate pellets (Figure 5(e)). Using b2(107)D-DHFRds as a substrate, the amount of protein in the pellet after the recovery period was higher in pim1D mitochondria than in wild-type mitochondria, indicating a slightly lower resolubilization activity. A similar observation was made using the imported mutant protein Ssc1-3 as a substrate for disaggregation. Here, the resolubilization efficiencies were essentially the same (52% disaggregation in wild-type; 58% in pim1D) although we observed a stronger aggregation of Ssc1-3 in pim1D mitochondria. We conclude that at least in the case of disaggregated proteins that are not able to acquire a native

Hsp78-mediated Protein Disaggregation

folding state, the resolubilized polypeptide material is degraded by the matrix protease Pim1. The role of the mitochondrial Hsp70 Ssc1 in prevention of aggregation and disaggregation It has been shown by in vitro experiments that chaperones of the Hsp70 and Hsp100 families cooperate closely in aggregate solubilization.25,41 To determine the contribution of the mitochondrial Hsp70 Ssc1 in protection against heat damage we performed the in organello aggregation/disaggregation assay with mitochondria from two Hsp70 temperature-sensitive mutant strains, ssc1-2 and ssc1-3.35 The protein Ssc1-3 is not functional for import or folding under non-permissive conditions due to its mutation in the ATPase domain. In contrast, the mutant Ssc1-2 carries a point mutation in the peptide-binding domain, resulting in an increased binding affinity for substrate proteins and a folding defect of newly imported preproteins. We imported the fusion protein b2(107)D-DHFRds under permissive conditions into isolated mitochondria as a reporter for protein aggregation. After completion of the import reaction, the mitochondria were treated with a heat shock of 42 8C for 10 min that both induced aggregation of the imported reporter protein and generated the temperature-sensitive phenotype of the mutant mtHsp70 proteins. In ssc1-3, only 60% of the imported b2(107)D-DHFRds was soluble after import, indicating a partial defect of Ssc1-3 even under permissive conditions.42 As observed before, b2(107)D-DHFRds aggregated heavily in all strains after heat shock (Figure 6(a)). However, due to its high binding affinity to unfolded preproteins, Ssc1-2 exerted a protective effect as approximately 15% of the imported reporter protein remained soluble compared to less than 5% in the other strains (Figure 6(a), lanes 2, 5 and 8). During the recovery period, both wild-type and ssc1-2 mitochondria were able to solubilize considerable amounts of the aggregated b2(107)D-DHFRds, whereas no disaggregation was observed in ssc1-3 mitochondria (Figure 6(a), lanes 3, 6 and 9). We conclude that at least a residual activity of mtHsp70 is required for a successful resolubilization of aggregated proteins. In contrast to a completely deactivated mtHsp70, represented by the ssc1-3 mutation, the Ssc1-2 mutant retains a high binding activity to imported preproteins although Ssc1-2 cannot support a folding reaction.43 The high disaggregation activity of the ssc1-2 mitochondria indicated that binding of mtHsp70 to aggregation-prone proteins is required for their subsequent solubilization in cooperation with Hsp78. ATP is required both for prevention of aggregation and disaggregation Because chaperones of the Hsp70 and Hsp100families both require ATP hydrolysis for their full activity, we were interested in the role of ATP

801

Hsp78-mediated Protein Disaggregation

1 2 3

(a)

4 5 6

7

8 9

soluble b2(107)∆-DHFRds (% of imported)

b2-(107)∆-DHFRds 100 80 60 40 20 0

C – + WT

C – + ssc1-2

C – + ssc1-3

recovery

(b) import

aggregation

disaggregation

ARS

ARS + + ATP

preprotein

apy/oli + – ATP

mitochondria

ARS – + ATP apy/oli – – ATP apy/oli – recovery

C

+ recovery

(c) 1 2 3

4 5 6

7 8 9 10 11 12

1 2 3

4 5 6

7 8 9

10 11 12

Ssc1-3

soluble Ssc1-3 (% of imported)

100 80 60 40 20 0

C

–+ ++

C

–+ – –

C

–+ + –

C

–+ –+

WT

C

–+ ++

C

–+ ––

C

–+ +–

C

– + recovery – + ATP

hsp78∆

Figure 6. Influence of mtHsp70 and ATP on the solubility of destabilized proteins. (a) Dependence of aggregation and disaggregation on Ssc1. Recombinant b2(107)D-DHFRds was imported into the indicated mitochondria and the in organello aggregation/disaggregation assay was performed as described. Shown are Western blot signals for b2(107)DDHFRds and quantification of supernatants in control (C), aggregation (K recovery) and disaggregation (C recovery) samples. Due to the reduced solubility of the reporter protein after import into the ssc1-3 mutant a longer exposition after immunodecoration is shown for this strain. (b) Scheme of the in organello aggregation/disaggregation assay to determine ATP dependence of the aggregation and disaggregation reaction, respectively. Radiolabeled Ssc1-3 precursor was imported and the assay incubations were performed either in the presence of an ATP regenerating system (ARS) or after depletion of matrix ATP by addition of 10 units/ml apyrase and 0.1 mM oligomycin (apy/oli). (c) Autoradiogram and quantification of the supernatants of the experiment described in (b) using wild-type (left panel) and hsp78D (right panel) mitochondria.

hydrolysis in both prevention of aggregation and aggregate resolubilization. We imported radiolabeled Ssc1-3 as an aggregation substrate into isolated wild-type and hsp78D mitochondria and examined aggregation and disaggregation in the presence of high matrix ATP levels or in the absence

of ATP (Figure 6(b)). When ATP was present during heat shock and recovery, approximately 55% of the imported Ssc1-3 remained soluble in wild-type mitochondria, whereas only 40% of the reporter protein escaped aggregation in hsp78D mitochondria (Figure 6(c), lanes 1–3). As observed before,

802 about half of the aggregated material was resolubilized within 1 h at 25 8C in wild-type mitochondria but no disaggregation was monitored in the absence of Hsp78. If the mitochondria were depleted of ATP after import but before the heat treatment, Ssc1-3 aggregated almost completely in both strains, indicating a strong dependence of the prevention of aggregation on the ATP level in the matrix independently of the presence of Hsp78 (Figure 6(c), lanes 4–6). When mitochondria were depleted of ATP just prior to the recovery phase, no disaggregation was observed in both strains, showing the requirement of the solubilization reaction for ATP. In fact, the amount of soluble protein even decreased further during the incubation at 25 8C (Figure 6(c), lanes 7–9). Interestingly, when ATP was re-supplied directly prior to the recovery period no resolubilization occurred if the heat shock had been performed in the absence of ATP (Figure 6(c), lanes 10–12). Thus, a high matrix ATP level was essential for both the prevention of aggregation and disaggregation. In addition, the nucleotide state of the mitochondria during the heat stress treatment strongly influenced the efficiency of subsequent resolubilization reactions.

Discussion Maintenance of mitochondrial protein function is dependent on the cooperative action of molecular chaperones in the matrix compartment. While mtHsp70, in yeast encoded by SSC1, is essential for the membrane translocation of preproteins and their subsequent folding steps,3,44 the cellular role of the Hsp100/Clp chaperone family member Hsp78 has not been completely established to date. Our experiments, utilizing a novel in organello aggregation/disaggregation assay in intact mitochondria, demonstrate that Hsp78 is required for the resolubilization of aggregated polypeptides after thermal stress under in vivo conditions. This disaggregation activity not only increases the solubility of the affected polypeptides but also facilitates their refolding to the active conformation, as was shown with the temperature-labile mtHsp70 mutant protein Ssc1-3. We previously observed that mutant forms of Ssc1 show a high tendency to form aggregates in the absence of Hsp78.30 We were now able to directly show that Hsp78 is required for the resolubilization of aggregated Ssc1-3 during the recovery period after induction of heat stress. We conclude that the previously observed repression of the temperature-sensitive lethal phenotype of ssc1-3 cells by overexpression of Hsp7831 is based on the reactivation of the import function of the aggregation-prone Ssc1-3 protein. Although more stable than Ssc1-3, also the wild-type form of Ssc1 showed a tendency to aggregate under heat stress conditions, both in vitro as a purified protein and in organello within intact mitochondria. In fact, a proteomic analysis of protein aggregation in yeast mitochondria showed Ssc1 as a major component of

Hsp78-mediated Protein Disaggregation

the aggregate pellets (T.M. & W.V., unpublished results). Taken together, a prominent aspect of Hsp78 function in mitochondria is the restoration of the highly specific and essential chaperone activity of mtHsp70 that is required for multiple pathways of mitochondrial protein biogenesis.4,11 A reactivation effect after protein aggregation in vitro and in vivo is typical for members of the ClpB protein family like Hsp78.23,24,45 In bacteria and in the eukaryotic cytosol, it has been shown that aggregated model proteins can be solubilized and reactivated in a concerted, ATP-dependent reaction requiring ClpB and the Hsp70 chaperone system. However, the molecular mechanism of this disaggregation activity is still a matter of debate. Recent experiments indicate that the disaggregation mechanism is intrinsically coupled with the molecular architecture of the large oligomeric, ring-shaped ClpB protein complex. 46 The disaggregation reaction is most likely based on the threading of the aggregated polypeptide chain through a small central pore of the ring-shaped ClpB protein complex.28,47 Although there are some differences in the primary structure between ClpB and Hsp78, most notably the lack of an amino-terminal domain in Hsp78, the overall molecular architecture of the Hsp78 complex in vitro is quite similar to ClpB.48 It is therefore conceivable that Hsp78 employs a similar mechanism of disaggregation. Although the mutant protein Ssc1-3 represents a major substrate, the disaggregation effect is a general phenomenon of Hsp78 activity. Hsp78 was also able to successfully resolubilize an aggregated model protein containing a destabilized DHFR reporter domain. Since similar reporter proteins have been widely used as substrates for proteolysis reactions in mitochondria, we could address the question of a potential coordination of the disaggregation activity with protein degradation reactions in the framework of a protein quality control system. The protective effect of ClpB during thermotolerance in bacteria has been linked to its ability to catalyze the re-acquisition of enzymatic activity after aggregation.28 A similar effect takes place in mitochondria in the case of the aggregation-prone mutant Ssc1-3. However, in contrast to the bacterial ClpB, Hsp78 has additionally been described to be involved in proteolytic reactions in the mitochondrial matrix. The degradation of foreign or damaged proteins by the matrix protease Pim1 was strongly reduced in mutant mitochondria lacking Hsp78.39,49 Previously it has been shown that Hsp70 chaperones closely cooperate with the protease Pim1 or its bacterial homolog Lon on a functional level.40,50,51 Here, they perform a stabilizing role on denatured polypeptides that favors eventual degradation over aggregation. Although Hsp78 most likely has no “holding” activity on destabilized polypeptides, we could show a direct functional cooperation with the Pim1 protease also in the case of polypeptide disaggregation. We observed a significant increase in the amount of resolubilized protein material in mitochondria

Hsp78-mediated Protein Disaggregation

lacking the protease Pim1, indicating that a major amount of the polypeptides that have been disaggregated by Hsp78 are targeted for degradation. However, the mechanistic basis of the cooperative action of Hsp78 and the protease Pim1 remains to be clarified. Despite a significantly higher overall stability than Ssc1-3 (data not shown), we could demonstrate that wild-type Ssc1 aggregated under heat stress conditions and was also resolubilized in a reaction dependent on Hsp78 during recovery from heat stress. Under the in vivo conditions used it is likely that the localization of Ssc1 to the aggregate pellet is caused by a combination of two different events. First, Ssc1 as such might be destabilized under the conditions used and in part subject to aggregation. Second, due to its chaperone function, i.e. binding to misfolded polypeptides, Ssc1 sediments together with the aggregation-prone proteins in the centrifugation assay. Interestingly, recent experiments demonstrated that the nucleotide-free state of mtHsp70 has a significant tendency to form aggregates in vivo.37 This observation resembles the behavior of the Ssc1-3 mutant protein that is not able to bind nucleotides under non-permissive conditions due to the point mutation in its ATPase domain.52 In fact, yeast mitochondria contain an Hsp70specific chaperone, called Zim17 or Hep1, that was shown to prevent the aggregation of Hsp70 family members in the matrix compartment.37,38 The biochemical mechanism of this protective function is not known so far. However, our data excluded an Hsp70-specific disaggregation activity for Zim17. Hsp78 might then be needed to retain Ssc1 including its co-chaperones in the soluble and active state. Besides Hsp78, mitochondria contain a second member of the mitochondrial Hsp100 family, Mcx1, a homolog of the bacterial ClpP partner-chaperone ClpX. 36 Due to the absence of ClpP in mitochondria, it was speculated that Mcx1 might solely perform a chaperonelike function. Our experiments showed no influence of Mcx1 on protein aggregation and only a minor effect on the disaggregation of unstable proteins after exposure to elevated temperatures. In general, maintenance of protein function under stress conditions comprises a combination of two separated chaperone activities, (i) prevention of aggregation by keeping damaged proteins in a soluble state and (ii) resolubilization of aggregated polypeptides. The classical holding activity of Hsp70-type chaperones stabilizes denatured or aggregation-prone substrate proteins and prevents their inactivation by aggregation.13,14,53 Usually, a molar excess of chaperone molecules is required for this function. In contrast, the resolubilization of aggregated proteins usually requires only substoichiometric amounts of chaperones. Optimal reactivation of substrates in vitro could even be achieved in the presence of 40 times less ClpB hexamers than Hsp70 molecules.25 However, a discussion of the relative amounts of chaperones versus substrates in

803 the case of the disaggregation reaction has to be regarded with caution since the concentration of the protein aggregates is generally unknown. Due to the essential nature of the main mitochondrial Hsp70, Ssc1, and its involvement in multiple cellular processes, a direct analysis of its protective function during aggregation in intact mitochondria is difficult. However, by the analysis of the mtHsp70 temperature-sensitive mutant mitochondria we were able to show a different influence of the mutants ssc1-2 and ssc1-3 on the aggregation behavior of imported reporter proteins. We observed a slightly lower aggregation tendency in ssc1-2 mitochondria that is in good correlation with the observed higher binding efficiency of Ssc1-2 to newly imported proteins.43 Interestingly, ssc1-2 mitochondria showed also a strongly improved disaggregation efficiency during the recovery period. In this mutant an increased “holdase” activity seems to be resulting in two effects. An enhanced binding of Ssc1-2 resulted at least in a partial prevention of aggregation. In addition, it is conceivable that even in the case that an interaction with Ssc1-2 is not sufficient to prevent aggregation, an increased amount of Hsp70 chaperones associated with the substrate proteins would allow a more efficient resolubilization reaction. In contrast, the mutant Ssc1-335,52 with its defective ATPase domain is neither able to prevent aggregation nor to support the disaggregation reaction. The mutation in Ssc1-3 inhibits nucleotide binding under non-permissive conditions, resulting in absent substrate binding and the complete deactivation of chaperone activity. The observation that a non-functional Ssc1 mutant is defective in prevention of aggregation and resolubilization of aggregated proteins confirms the necessity of the Hsp70 system for both processes. In contrast to mtHsp70, the ClpB homolog Hsp78 does not associate in large amounts with aggregated proteins in mitochondria. Its role is more specific, focused on the resolubilization of aggregated polypeptides and thereby repairing the damage resulting from heat stress. The chaperone activities of Hsp78 as well as mtHsp70 are dependent on the presence of ATP although the mechanisms of nucleotide involvement are completely different.14,16 Our results showed that a high ATP level in the mitochondrial matrix is absolutely required for protection against aggregation during heat stress and resolubilization of aggregated proteins in the recovery phase. Interestingly, resupplying ATP during the recovery phase was not able to restore the disaggregation activity when mitochondria had been depleted of ATP during the heat stress treatment. As already indicated by the analysis of the mtHsp70 mutant mitochondria, the ATP-requirement during aggregation seems to be mainly correlated with the holdase function of mtHsp70 (Ssc1). If mtHsp70 was not able to associate with the aggregated proteins in ATPdepleted mitochondria, similar to the ssc1-3 phenotype, a later resolubilization seemed not possible even in the presence of functional Hsp78.

804 In summary, the chaperones mtHsp70 and Hsp78 form an intricate functional network in mitochondria that is responsible for maintaining protein solubility in the mitochondrial matrix after heat stress (Figure 7). In general, the system within mitochondria closely resembles the situation in bacterial cells where it has been shown that the two chaperones ClpB and Hsp70 (DnaK) cooperate to prevent aggregation and to reactivate aggregated proteins.54 The biochemical mechanism of the cooperation of the two types of chaperones is not completely clear. In principle, the reactivation of aggregated proteins requires two mechanistically different activities. First, the aggregated protein has to be solubilized and second, the soluble but denatured polypeptide needs to fold back to its native conformation. Initially it was assumed that Hsp70 is primarily needed to assist the refolding step, but recent in vitro evidence indicates an earlier involvement of the Hsp70 system already during the solubilization step.28,41 Our experiments correlate well with the model of an early requirement for the interaction of disaggregation substrates with Hsp70, potentially already during the heat stress conditions. We could demonstrate that Hsp78 has a significant disaggregation activity under in vivo conditions. One of the main substrates is the mitochondrial chaperone mtHsp70. By supporting its essential functions in protein import and folding, Hsp78 contributes prominently to the maintenance of mitochondrial protein function under heat stress

Hsp78-mediated Protein Disaggregation

conditions. Hsp78 and the mtHsp70 system form a cooperative ATP-dependent chaperone network responsible for the protection of mitochondrial proteins against aggregation. On the other hand, the involvement of Hsp78 in protein degradation by the mitochondrial protease Pim1 indicates the targeting of resolubilized damaged proteins for proteolytic removal. Hence, Hsp78 represents a functional link between the two major activities of the mitochondrial protein quality control system.

Materials and Methods Isolation of yeast mitochondria The S. cerevisiae strains used are listed in Table 1. Deletion mutants of the respective chaperone proteins were generated using standard methods of yeast genetic manipulation. Mitochondria were isolated as described before55 after growth in YP medium (1% (w/v) yeast extract, 2% (w/v) Bacto-peptone) containing either 3% (v/v) glycerol (WVY38, WVY39, WVX40, WVY41, YPH499, BG1700) or 2% (w/v) glucose (PK81, PK82, PK83, KR01, KR02) at 24 8C (PK81, PK82, PK83, YPH499, BG1700) or 30 8C (all other strains), respectively. The open reading frame of Hsp78 was cloned into the plasmid pYES2 (Invitrogen) and transformed into the yeast strains PK82 (WT) and PK83 (ssc1-3) as indicated. Cells were grown on synthetic minimal medium containing glycerol as a carbon source. Overexpression of Hsp78 was induced by the addition of 2% (w/v) galactose to the growth medium.

Figure 7. Schematic drawing indicating the functional roles of the mitochondrial chaperones Hsp78 and mtHsp70 (Ssc1) in maintaining protein stability. Depending on environmental conditions (e.g. heat stress), folded polypeptides can denature to a misfolded state and eventually aggregate. Misfolded proteins interact with mtHsp70 and are stabilized against aggregation or even refolded. In other cases, the misfolded proteins might become degraded. Hsp78 is able to resolubilize aggregated polypeptides in concert with mtHsp70. Depending on the conformational state the polypeptides are either refolded or targeted for degradation with the help of Hsp78.

805

Hsp78-mediated Protein Disaggregation

Table 1. Yeast strains Strain

Genotype

WVY38 WVY39 WVY40 WVY41 PK81 PK82 PK83 YPH499 BG1700 KRY01 KRY02

his3-D200, leu2-D11, ura3-52, trp1-D63 his3-D200, leu2-D11, ura3-52, trp1-D63, mcx1DTkanMX his3-D200, leu2-D11, ura3-52, trp1-D63, hsp78DTURA3 his3-D200, leu2-D11, ura3-52, trp1-D63, hsp78DTURA3, mcx1DTkanMX MATa, ade2-101, lys2, ura3-52, leu2-3, 112, Dtrp1, ssc1-2(LEU2) MATa, his4-713, lys2, ura3-52, Dtrp1, leu2-3, 112 MATa, ade2-101, lys2, ura3-52, leu2-3, 112, Dtrp1, ssc1-3(LEU2) MATa, ade2-101, his3-D200, leu2-D1, ura3-52, trp1-D63, lys2-801 MATa, ade2-101, his3-D200, leu2-D1, ura3-52, trp1-D63, lys2-801 zim17DTADE2 MATa, ade2-101, his3-D200, leu2-D1, ura3-52, trp1-D63, lys2-801, rhoK MATa, ade2-101, his3-D200, leu2-D1, ura3-52, trp1-D63, lys2-801, pim1DTHIS3, rhoK

Import of preproteins into isolated mitochondria Recombinant cytochrome b2 -DHFR preproteins were expressed and purified as described.39 35S-labeled Ssc1-3 was synthesized in rabbit reticulocyte lysate (GE Biosciences) by in vitro transcription/translation after PCR on ssc1-3 genomic DNA using a forward primer that contained the SP6 promotor sequence. Su9-DHFR was transcribed in vitro from a pGEM transcription plasmid. Import of recombinant and radiolabeled precursor proteins was performed according to published procedures.39,55,56 To test the influence of the conditional mutants of Ssc1 on the import efficiency, isolated mitochondria were incubated at 37 8C directly before the start of the import reaction. To determine the amount of imported preproteins bound to mtHsp70, a co-immunoprecipitation reaction was performed according to published procedures.35 In organello aggregation/disaggregation assay with imported substrate proteins After import of substrate preproteins, mitochondria were treated with 100 mg/ml proteinase K (PK) for 10 min at 0 8C to digest non-imported preproteins, washed once with SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM Mops-KOH (pH 7.2)) supplied with 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and resolubilized in 300 ml of prewarmed resuspension buffer (250 mM sucrose, 80 mM KCl, 5 mM MgCl2, 3% BSA, 10 mM Mops–KOH (pH 7.2)) containing an ATP regenerating system (3 mM ATP, 4 mM NADH, 20 mM creatine phosphate, 200 mg/ml creatine kinase). A control sample corresponding to 15 mg of mitochondria was taken after resuspension. The remaining sample was incubated at 25 8C for 5 min followed by 10 min at 42 8C to induce aggregation and an aliquot corresponding to 15 mg of mitochondria was taken. The original sample was then incubated at 25 8C for 60 min to allow disaggregation and another aliquot corresponding to 15 mg of mitochondria was withdrawn. Mitochondria from control, aggregation and disaggregation samples were re-isolated, washed with SEM buffer supplied with 0.5 mM PMSF and lysed in 200 ml ice-cold lysis buffer (0.3% Triton X-100, 30 mM Tris–HCl (pH 7.4), 80 mM KCl, 5% glycerol, 0.5 mM PMSF, 5 mM MgCl2, 3 mM ATP, protease inhibitors) by shaking for 5 min at 4 8C. Soluble and aggregated matrix proteins were separated by a high speed spin at 100,000g for 15 min. The supernatant was precipitated with 10% trichloroacetic

Source Ro¨ttgers et al.39 Ro¨ttgers et al.39 Ro¨ttgers et al.39 This work Gambill et al.35 Gambill et al.35 Gambill et al.35 Sanjua´n et al.38 Sanjua´n et al.38 Ro¨ttgers et al.39 Ro¨ttgers et al.39

acid. Precipitated supernatants and pellets were analyzed by SDS-PAGE.

Quantification Radiolabeled reporter proteins were detected after SDS-PAGE by autoradiography and quantified using Image Quant 5.2. For immunodetection of cytochrome b2-DHFR constructs an affinity-purified antiserum against DHFR was applied. Signals were detected by using Lumi-LightPLUS Western Blotting Substrate and quantified with the Image Master 1D software (GE Biosciences). The sum of substrate amounts in supernatant and pellet before heat shock was set 100%.

In vivo disaggregation assay of endogenous mitochondrial proteins 80 mg of mitochondria were suspended in 250 ml of resuspension buffer (250 mM sucrose, 80 mM KCl, 5 mM MgCl 2, 10 mM Mops–KOH (pH 7.2)) and subjected to heat shock at 42 8C for 10 min followed by an incubation at 25 8C for 0 min, 30 min, 60 min or 180 min in resuspension buffer containing the ATP regenerating system described above. Mitochondria were then re-isolated and lysed in 45 ml of lysis buffer (0.5% Triton X-100, 30 mM Tris–HCl (pH 7.4), 200 mM KCl, 5 mM EDTA, 0.5 mM PMSF, protease inhibitors). After a high speed spin at 100,000g for 15 min supernatants were taken off and pellets were reextracted by shaking them in 50 ml of lysis buffer for 5 min, followed by another spin at 100,000g for 15 min. One third of the supernatants and the complete pellets were analyzed by SDS-PAGE and Western blot. Endogenous proteins were detected by immunodecoration with specific antibodies. To test the in vitro aggregation of Ssc1, Ssc1 containing a C-terminal hexahistidine tag was purified from yeast mitochondria as described.57 1.5 mg of purified Ssc1 (f. c. 0.2 mM) were suspended in 100 ml of incubation buffer (250 mM sucrose, 10 mM Mops–KOH (pH 7.2), 80 mM KCl, 0.02 mg/ml BSA) supplied either with 3 mM ATP and 5 mM MgCl2 or with 5 mM EDTA. Aliqouts of 50 ml of the samples were withdrawn as a control, the remaining samples were then subjected to a heat shock at 42 8C for 10 min. Aggregated and soluble protein was then separated and processed as described above. Protein aggregation was analyzed by SDS-PAGE and Coomassie staining as described above.

806

Acknowledgements We thank Dr C. Meisinger for providing the zim17D yeast strain and Dr E. A. Craig for the ssc1 conditional mutant strains. Dr B. Guiard provided the plasmid for the recombinant expression of the preprotein b2(107)D-DHFR. The purified Ssc1 was generously provided by Dr Y. Li. N. Zufall contributed to the work with excellent technical expertise. We are grateful to Dr N. Pfanner for critically reading the manuscript. The work was performed with funding by the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm 1132, grant VO 657/4-2 to W.V.).

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Edited by M. Yaniv (Received 14 October 2005; received in revised form 22 December 2005; accepted 4 January 2006) Available online 19 January 2006