Analysis of the mitochondrial proteome under heat stress

Because we have established that heat stress caused functional defects in mitochondria, we determined the effects of heat stress on the structural integrity of the mitochondrial proteome by identifying polypeptides that are prone to aggregate at elevated temperatures. As described above, we treated isolated mitochondria for 20 min at physiological heat stress temperatures of 42 and 25 °C as control. Afterward, mitochondria were lysed by detergent treatment and centrifuged at 125,000 × g to separate aggregated proteins, which go to the pellet fraction, from soluble proteins, which remain in the supernatant (15, 19). To detect quantitative changes in the mitochondrial proteome, we used 2D DIGE, which allows the simultaneous separation of three different fluorescently labeled samples on one gel to exclude intergel variations. The gels were scanned with dye-specific wavelengths, and gel matching was performed to detect and quantify the protein spots in the respective samples. Spot detection and determination of spot intensities were performed using the Delta2D software (DECODON). For the quantitative analysis, the data of two independent experiments, each consisting of triplicates for stressed and unstressed samples were used. On average, a total of 300–400 spots/gel have been detected. Corresponding protein spots were manually excised from Coomassie-stained master gels containing proteins of isolated HeLa mitochondria. Excised spots were digested by trypsin and analyzed by MS. To obtain a consistent and comparable protein spot pattern, we analyzed the polypeptides present in the supernatant fractions of the different samples. A reduced or absent spot intensity in the supernatant of heat-treated mitochondria compared with the control sample would indicate potential aggregation-prone polypeptides that lost their solubility. The short time of incubation at elevated temperature essentially excluded the chance that the reduction of protein amount was caused by degradation processes.

An overlay of the 2D gels obtained after heat-shock treatment allowed a direct comparison of mitochondrial polypeptide solubility. Proteins present in the soluble proteome of the heat-stressed sample are displayed in green, and those of the control sample were stained in red, resulting in a yellow color overlay picture for protein spots that did not show a change in abundance after the heat shock (Fig. 2A). Therefore red spots would represent proteins present in reduced amounts in the heat stress samples indicating a potential temperature-dependent aggregation. Overall, the abundance of soluble proteins did not change significantly after the heat-shock treatment, indicating a high general temperature stability of the mitochondrial proteome, However, there were 11 protein spots that were consistently reduced in the soluble mitochondrial proteome after heat stress in all six experiments, of which four proteins could be identified by MS (see also Fig. S2). Two of the proteins, Pdia3 (protein disulfide-isomerase A3) and Hyou1 (hypoxia up-regulated protein 1), were proteins, which were assigned by the database to be localized in the lumen of the endoplasmatic reticulum (ER). This was likely because the used standard method for isolation of mitochondria by differential centrifugation is not able to completely remove all contaminating ER proteins from the mitochondria fraction. However, two of the identified proteins were mitochondrial proteins. The mitochondrial Lon protease homolog (LonP1) showed a partial reduction after heat shock. The most prominent protein spot, which completely disappeared from the supernatant after heat treatment, was the mitochondrial elongation factor Tu (Tufm). This result was consistent for all six gels (Fig. 2A, detail panels on the right).

Open in a separate window

Figure 2.

Proteomic analysis of the mitochondrial proteome under heat stress. A, 2D DIGE spot pattern of heat-treated mitochondria. Gel shows an overlay of total soluble protein samples from control (25 °C; red) and heat-treated (20 min at 42 °C; green) mitochondria. The gel shown is a representative example of three experiments. Protein spots of the mitochondrial elongation factor Tu (Tufm) are shown enlarged for all six gels analyzed (right panels). M_r, relative molecular mass. B, quantification of selected protein spots. Shown are relative differences of spot intensities between control and heat-stressed mitochondria. Average spot intensities in control samples were set to 100%. Spot detection and quantification were done using the Delta2D software (DECODON). Error bars indicate the S.E. The significance was calculated via one-way analysis of variance using the Dunnett's multiple comparisons test. A p value of ≤0.001 was considered significant and is indicated by asterisks.

We also compared the results for the 17 identified mitochondrial proteins (Table 1 and Fig. S2) and quantified the abundance in the soluble proteome after heat shock compared with the control. The spot intensities for each gel were normalized by the total sum of spot intensity at 25 °C for each gel (Fig. 2B). There was no significant decrease of abundance in the soluble proteome after heat shock for the majority of proteins, whereas a significant decrease in abundance was observed for the protease LonP1 (~40%) and Tufm (~70%) after heat shock. The protein amounts of Sdha (flavoprotein subunit of the succinate dehydrogenase), Hsp60 (60-kDa heat shock protein), and HspA9 (70-kDa heat shock protein) showed only a small decrease of ~5–15%, whereas the other proteins exhibited no significant decrease in the soluble fraction after heat stress compared with the control.

Overall, these results of the proteomic analysis showed that the mitochondrial proteome is relatively resistant against heat stress because the vast majority of mitochondrial proteins are not prone to aggregate. Strikingly, one of the mitochondrial proteins, the translation-related protein Tufm, exhibited a very thermolabile behavior by aggregating completely under heat stress.

Aggregation behavior of mitochondrial elongation factors during stress

Having determined that Tufm is the most prominent protein disappearing from the soluble fraction under heat-stress conditions, we wanted to confirm whether this behavior was due to protein aggregation and what relevance a potential aggregation has on the functionality of affected mitochondria. To get more information about a temperature-dependent aggregation of Tufm compared with other mitochondrial proteins, we incubated isolated mitochondria at a control temperature (25 °C), as well as under mild and strong heat-shock conditions (37 °C, 42 °C, and 45 °C). Although a temperature of 37 °C can be considered as physiological in HeLa cells, our results (Fig. 1C) have indicated that isolated mitochondria already showed some temperature sensitivity under this condition.

Isolated HeLa mitochondria were lysed after heat treatment, and aggregated proteins (pellet) were separated from the soluble proteins (supernatant) by high-speed centrifugation and analyzed by SDS–PAGE and Western blotting. In addition to Tufm, the aggregation behavior of the elongation factor Ts (Tsfm), which is associated with Tufm during translation inducing GDP to GTP exchange (20), as well as the mitochondrial 39S ribosomal protein 38 (Mrpl38) was investigated. A significant amount of Tufm and Tsfm, as well as Mrpl38, was recovered in the pellet already at 37 °C, and their aggregation tendency increased further with higher temperatures (Fig. 3A). Notably, at 42 °C Tufm and Mrpl38 were not detected anymore in the supernatant fraction, indicating a complete aggregation. The mitochondrial matrix proteins Aco2 (aconitate hydratase) and Hsp60 also started to aggregate at 37 °C, but not as strong as Tufm. Whereas Aco2 was completely aggregated after incubation at 45 °C, part of Hsp60 stayed soluble. The translocase of the inner mitochondrial membrane Tim23 served as a control, which showed no temperature-dependent aggregation.

Open in a separate window

Figure 3.

Aggregation behavior of mitochondrial elongation factors during stress. A, aggregation under heat stress. Isolated mitochondria were incubated for 20 min at the indicated temperatures, and aggregates were separated by centrifugation at 20,000 × g. Total lysates (T), supernatants (Sup), and pellets (Pel) were analyzed by SDS–PAGE and Western blotting. B, aggregation under oxidative stress. Isolated mitochondria were treated with menadione as indicated for 20 min and analyzed as described above. C, ATP dependence of Tufm aggregation. Isolated mitochondria were heat-stressed for 20 min at 42 °C in presence and absence of ATP (final concentration, 5 mm) and analyzed as described above. The asterisk by lane 4 indicates that less sample was loaded because of a loss of sample material during the procedure. D, GTP dependence of Tufm aggregation. Isolated mitochondria were heat-stressed in presence and absence of GTP (final concentration, 2.5 mm) and analyzed as describe above.

To investigate whether other stress conditions such as the formation of reactive oxygen species also induced an aggregation of mitochondrial proteins, mitochondria were treated with menadione. Isolated mitochondria were incubated with different menadione concentrations, and aggregated and soluble proteins were separated and analyzed as described above. Under oxidative stress conditions, we observed no aggregation for the mitochondrial matrix proteins Aco2 and Sod2 (superoxide dismutase), as well as for the outer membrane control protein Vdac (voltage-dependent anion-selective channel protein) (Fig. 3B). Whereas just a small subset of Hsp60 aggregated after treatment with high menadione concentrations, a significant amount of the elongation factor Tufm aggregated in a concentration-dependent manner, starting at 0.5 mm menadione. However, compared with the temperature behavior of Tufm, there was still a substantial amount of soluble protein detected in the supernatant, indicating that Tufm may be more sensitive to heat than to oxidative stress.

Molecular chaperones are the most prominent cellular enzymes involved in aggregation prevention and refolding of heat-denatured proteins (21). Because most molecular chaperones require ATP for their activity, we wanted to determine whether there is an ATP dependence of Tufm aggregation. Isolated HeLa mitochondria were heat-stressed at 42 °C in the presence and absence of ATP. Aggregates and soluble proteins were isolated and analyzed as described before. It was shown that although there was an ATP effect after heat shock visible for the shock protein Hsp60, which was more stable in the presence of ATP, there was no change of the Tufm as well as Tsfm, Mrpl38, and Aco2 aggregation (Fig. 3C), indicating that at least under the chosen conditions there is no aggregation-protective effect for the analyzed proteins mediated by mitochondrial chaperones.

Tufm is a GTPase, which hydrolyzes GTP as an energy source for conformational changes in the process of transferring the aminoacyl-tRNA to the A site of the ribosome (20). Because the presence or absence of guanine nucleotides might influence protein stability of Tufm, we also determined the influence of GTP on its temperature-dependent aggregation. Isolated mitochondria were heat-stressed in the absence and presence of GTP, and aggregates were isolated and analyzed as described above. As shown in Fig. 3D, there was no GTP dependence of the aggregation of Tufm at 42 °C, as well as for Tsfm. The ribosomal protein Mrpl38 was already present in the pellet fraction at 25 °C in the same amount as after heat shock, and there was no visible change in the presence of GTP.

Time dependence of aggregation under heat stress

The kinetics of the mitochondrial protein aggregation was studied by incubation of isolated mitochondria for several time points from 0 to 60 min at heat stress and control conditions. Aggregated proteins were analyzed by SDS–PAGE and Western blotting. The percentage of aggregation was calculated by the amount of aggregated protein compared with the total protein amount (Fig. 4). The mitochondrial elongation factors Tufm and Tsfm both exhibited remarkable fast aggregation kinetics. These proteins started to aggregate already after 2 min of incubation at 42 °C and practically reached a maximal level already after 5 min. The protein amounts in the pellet did not increase significantly over time anymore, indicating a very thermolabile behavior. The ribosomal protein Mrpl38 also showed fast aggregation kinetics with ~50% aggregation after 5 min, which increased over time up to 70%. However, 10% of Mrpl38 was already detected in the pellet fraction without any heat stress, indicating a temperature-independent tendency to sediment under the used centrifugation conditions, most likely because of its association with ribosomal particles. It should be noted that Tufm, Tsfm, and Mrpl38 showed also some aggregation at control conditions (25 °C) after longer incubation times, indicating an intrinsic low stability. In contrast to the former proteins, Aco2 and Hsp60 showed slower aggregation kinetics after heat shock. Whereas just 15–20% of these proteins were found aggregated after 5 min, the amount of aggregated protein increased over time up to 90% (Aco2) and 60% (Hsp60) after 60 min of heat shock. Tim23 served as a control for a heat-stable protein that only showed a minor aggregation propensity (less than 20%) even after 1 h of heat shock. These observations indicate that in particular the translation factors Tufm and Tsfm are extremely thermosensitive and aggregate very quickly under heat-shock conditions.

Open in a separate window

Figure 4.

Time dependence of aggregation under heat stress. After incubation of isolated mitochondria at 42 °C for the indicated times, aggregation rates of the indicated mitochondrial proteins were analyzed by SDS–PAGE and Western blotting. The percentage of aggregation was calculated by the ratio of aggregated protein (pellet) compared with the total protein (supernatant + pellet). Shown are mean values of three experiments; error bars indicate S.E.

Properties of Tufm aggregates

To analyze the nature of the Tufm aggregates, we analyzed their sedimentation behavior by differential centrifugation. Isolated mitochondria were subjected to a heat treatment as described before, and after lysis of the mitochondria, aggregated proteins were fractionated at different centrifugal forces to get information about size and density of the formed aggregates. Whereas Tufm and Tsfm were soluble at control conditions and just small amounts were detected in the pellet fraction after 125,000 × g, after heat shock aggregates were already detected after a low-spin step (5 min at 1600 × g), suggesting that they represent rather big aggregates (Fig. 5A, left panel). After 20 min of centrifugation at 20,000 × g, the Tufm aggregates were completely sedimented, which caused us to keep this as standard condition for isolating aggregates in the following experiments. Because it could be assumed that elongation factors might also sediment because of a possible attachment to the ribosome in the process of translation, we compared the sedimentation behavior of Tufm with the ribosomal protein Mrpl38. In contrast to Tufm, at control conditions (25 °C), Mrpl38 was mainly detected in the pellet fraction after 40 min centrifugation at 125,000 × g. This indicated that the presence of Mrpl38 in the pellet is due to the sedimentation of whole ribosomal subunits after high-speed sedimentation. However, after heat stress Mrpl38 also partially aggregated, as indicated by sedimentation already at lower centrifugation speeds. Still, a significant amount of Mrpl38 could be found in the putative ribosome pellet, whereas none of the elongation factor components were present in this fraction, at least under heat-stress conditions. Hence, the sedimentation behavior of Tufm and Tsfm represented a genuine aggregation phenomenon independent of the behavior of ribosomal proteins. This result was confirmed by analyzing the heat-stress aggregates by sucrose gradients, which also showed an independent aggregation behavior of Tufm and Mrpl38 (Fig. S3); in addition, there was no influence of high salt concentrations on the heat-induced Tufm aggregation detected, indicating no ionic binding between the protein and the ribosome present (Fig. S4).

Open in a separate window

Figure 5.

Nature of Tufm aggregates after heat stress. A, analysis of aggregate size after heat stress. Isolated mitochondria were incubated for 20 min under stress (42 °C) and control (25 °C) conditions. Protein aggregates were fractionated by differential centrifugation (supernatants of each centrifugation step were transferred to the next centrifugation step). B, protease resistance of Tufm aggregates. Isolated mitochondria were incubated for 20 min at the indicated temperatures. Mitochondria were lysed with 0.5% Triton X-100, and the extracts were treated with the indicated concentrations of proteinase K. The resulting proteolytic fragments of Tufm and a matrix control protein (Trap1) were detected by SDS–PAGE and Western blotting. C, degradation of mitochondrial proteins during heat stress. Intact energized mitochondria were incubated for up to 6 h at 30 °C (control) and 42 °C (heat stress). Protein was precipitated with TCA and analyzed by SDS–PAGE and Western blotting using the indicated antibodies. D, recovery of aggregation after heat shock. Isolated mitochondria were heat-stressed at 42 °C for 20 min and afterward incubated for up to 2 h at 30 °C. During the recovery incubation, mitochondria were energized and supplied with ATP. Supernatants (Sup) and pellets (Pel) were separated by centrifugation and analyzed by SDS–PAGE and Western blotting.

Although the less thermosensitive proteins Aco2 and Hsp60 were still partial soluble after heat stress, the stress-induced aggregates were also found already in the low-speed pellets (5 min of centrifugation at 20,000 × g). The control protein Tim23 was not aggregating after heat stress (Fig. 5A, right panel). That the pelleted material of Tufm represented genuine aggregates was confirmed by their resistance against the proteolytic digestion of the proteinase K. Heat-treated mitochondria were lysed with the nondenaturing detergent Triton X-100 and then incubated with different proteinase K concentrations. Whereas Tufm under control conditions (25 °C) was completely digested to a smaller fragment already at very low protease concentrations, it remained completely resistant against digestion after heat shock (Fig. 5B). In comparison, the mitochondrial matrix protein Trap1 was only partially digested by proteinase K under both control and stress conditions, indicating an intrinsic protease resistance. An investigation of the mitochondrial protein levels over 6 h under heat-stress incubation (42 °C) and control (30 °C) conditions showed that there was no change in the total protein amounts (Fig. 5C). This verified that the decrease in the soluble amounts of the proteins Tufm and Tsfm was indeed caused by a heat-induced aggregation and not due to proteolytic degradation.

Because the elongation factor Tufm exhibited a strong aggregation propensity after heat stress, we wanted to investigate whether the solubility of the protein could be recovered over time after the heat stress has ceased. For this purpose, heat-stressed mitochondria were subjected to a recovery incubation at 30 °C for 15, 60, and 120 min. During recovery, mitochondria were supplied with an ATP-regenerating system and mitochondrial substrate molecules to keep them fully energized. Aggregates and soluble proteins were separated as described before by centrifugation and analyzed by SDS–PAGE and Western blotting. In the tested time range, no resolubilization of aggregated Tufm nor of the other tested, less aggregation-prone proteins Aco2 and Hsp60 was observed (Fig. 5D), indicating that the aggregated polypeptides could not be quickly disaggregated.

Flow cytometry

Membrane potential was assayed by an incubation of cultures cells for 10 min at 37 °C with 0.5 μm TMRE. As control, a sample was treated with valinomycin (final concentration, 0.1 μm) for 30 min at 37 °C. The generation of ROS was determined using the mitochondrial superoxide indicator MitoSOX^TM (Thermo Fisher Scientific). The cells were incubated with the indicator dye for 10 min at 37 °C. As positive control, the cells were treated with 100 μm menadione for 15 min at 37 °C. The cells were harvested by trypsinization and washed twice in PBS containing 0.2% (w/v) BSA. Fluorescence of 20,000 cells for each sample was analyzed using the flow cytometer CyFlow space CY-S3001 (Partec).

Mitochondrial aggregation assay

Mitochondria were isolated from cultures cells essentially as described (59). For each sample 30 μg of fresh mitochondria were resuspended in 100 μl of resuspension buffer (500 mm sucrose, 160 mm potassium acetate, 40 mm HEPES/KOH, pH 7.6, 10 mm MgAc, 5 mm glutamate, 5 mm malate, 1 mm DTT). For analysis of the nucleotide dependence, the resuspension buffer was supplemented with 5 mm ATP, creatine phosphate (20 mm), and creatine kinase (4 μg/ml). Analysis of the GTP dependence was performed in detergent-lysed mitochondria (lysis buffer: 0.5% (v/v) Triton X-100, 200 mm KCl, 30 mm Tris/HCl, pH 7.4, 0.5 mm phenylmethylsulfonyl fluoride (PMSF), 1× protease inhibitors) supplemented with 10 mm MgAc and 2.5 mm GTP. Mitochondria were either preincubated for 20 min at indicated temperatures (25 °C-45 °C) or in the presence of menadione (0–2 mm) at 25 °C. After incubation, mitochondria were reisolated by centrifugation (12,000 × g, 10 min, 4 °C), and pellets were lysed in lysis buffer containing 5 mm EDTA, by vigorous shaking for 10 min at 4 °C. Soluble proteins were separated from aggregated proteins by centrifugation at 20,000 × g for 20 min at 4 °C. The supernatants were precipitated with TCA, and pellets were re-extracted in 100 μl of lysis buffer by shaking as described above. After a second centrifugation step at 20,000 × g for 20 min at 4 °C, supernatants were discarded, and the pellet was resuspended in 1× Laemmli SDS–PAGE buffer (2% (w/v) SDS, 10% (v/v) glycerol, 60 mm Tris/HCl, pH 6.8, 5% (w/v) β-mercaptoethanol, 0.02% (w/v) bromphenol blue). Samples were analyzed by SDS–PAGE and Western blotting.

To distinguish between aggregate sizes, different centrifugation steps were applied. After the first low-spin step at 1,600 × g for 5 min, the supernatant was collected and centrifuged at 20,000 × g for 5 min, following a 20-min centrifugation at the same spin. After the last spin at 125,000 × g for 40 min, the supernatant was precipitated with TCA. All pellets were resuspended in 1× Laemmli buffer and analyzed as described above.

For recovery from aggregation, 30 μg of mitochondria were resuspended in 100 μl of incubation buffer (250 mm sucrose, 80 mm potassium acetate, 20 mm HEPES/KOH, pH 7.6, 5 mm magnesium acetate, 5 mm glutamate, 5 mm malate, 5 mm KP_i, pH 7.4, 2 mm ATP, 1 mm DTT) to keep them fully energized. After heat stress for 20 min at 42 °C, the samples were incubated for up to 2 h at 30 °C. Samples were lysed, centrifuged, and analyzed as described above.

Proteinase K treatment of mitochondria

For determination of the integrity of mitochondria, mitochondria were treated after heat stress with 1.25 μg/ml proteinase K for 20 min. Treatments were stopped by 15 min of incubation with 1.5 mm PMSF and following precipitation by TCA. To assess proteinase resistance of polypeptides, mitochondria were lysed with lysis buffer containing 5 mm EDTA and incubated with proteinase K. Protease treatment was stopped by adding PMSF, and samples were precipitated with TCA.

Membrane potential measurement

30 μg of mitochondria were resuspended in potential buffer (600 mm sorbitol, 20 mm KPi, pH 7.2, 10 mm MgCl₂, 10 mm glutamate, 5 mm malate, 0.1% (w/v) BSA) and incubated with 1 μm TMRE for 10 min at 30 °C. Negative control samples were treated with valinomycin for 10 min at 30 °C prior TMRE incubation. All samples were washed with potential buffer and TMRE fluorescence (excitation, 540 nm; emission, 585 nm) was measured in a microplate reader (Infinite M200 PRO, Tecan).

Determination of ATP content in isolated mitochondria after heat shock

The mitochondrial ATP content was measured by a luciferase based ATP assay (ATP determination kit; Invitrogen). Isolated energized mitochondria were either analyzed directly or after 20 min of incubation at 25 °C or 42 °C. The ATP assay was performed according to the manufacturer's instructions, and luminescence was measured in a microplate reader.

Enzymatic assays

Activities of MDH and KGDHC were measured in isolated mitochondria (100 μg/sample for MDH and 200 μg/sample for KGDHC activity assay) after incubation for 20 min at 25 °C (control) or 42 °C (heat shock) in resuspension buffer. After reisolation of the mitochondria at 12,000 × g for 10 min at 4 °C, the mitochondrial pellets were lysed in 50 μl of homogenization buffer (0.5% Triton X-100, 50 mm Tris/HCl, pH 7.4) and shaken at maximum speed for 10 min at room temperature. The respective reaction mixtures (MDH activity assay: 50 mm Tris/HCl, pH 7.4, 0.5 mm oxalacetic acid, and 75 μm NADH; KGDHC activity assay: 50 mm Tris/HCl, pH 7.4, 50 mm KCl, 5 mm α-ketoglutarate, 2 mm MgCl₂, 0.3 mm thiamine pyrophosphate, 0.3 mm CoA, and 2 mm NAD⁺) were prewarmed at 37 °C, the lysed mitochondria were added, and enzyme activities were measured at 340 nm every 15 s for 10 min in a photometer (BioPhotometer plus, Eppendorf) by either decrease of NADH absorption (MDH) or increase of NADH absorption (KGDHC).

Blue Native SDS–PAGE

40 μg of isolated mitochondria were resuspended in resuspension buffer, incubated for 20 min at 25 °C (unstressed) or 42 °C (heat stress), and afterward solubilized in digitonin lysis buffer (0.4% (w/v) digitonin, 50 mm NaCl, 20 mm Tris/HCl, pH 7.4, 2 mm EDTA, pH 8.0, 1 mm PMSF, 10% (v/v) glycerol) by pipetting (20×) and shaking for 10 min at 4 °C at maximum speed. 10× loading dye (5% (w/v) Coomassie Blue G-250, 500 mm ϵ-amino n-caproic acid, 100 mm Bis-Tris/HCl, pH 7.0) was added, and samples were applied to a 5–16% polyacrylamide gradient gel. The samples were analyzed by Western blotting using antibodies against subunits of the respiratory chain complexes as indicated.

2D gel electrophoresis

Isolated mitochondria (150 μg total protein) were dissolved in resuspension buffer and incubated for 20 min at 25 °C (control) or 42 °C (heat stress). After centrifugation at 12,000 × g for 10 min at 4 °C, mitochondria were lysed in lysis buffer (0.5% Triton X-100, 200 mm KCl, 30 mm Tris/HCl, pH 7.4, 5 mm EDTA, 0.5 mm PMSF, 1× protease inhibitors) by vigorous shaking for 10 min at 4 °C. The aggregated proteins were separated ultracentrifugation at 125,000 × g for 40 min. Supernatants were precipitated with TCA and resuspended in IEF lysis buffer (7 m urea, 2 m thiourea, 4% (w/v) CHAPS, 10 mm DTT, 1× protease inhibitors, 40 mm Tris/HCl, pH 8.8) and lysed by 10 min of shaking at maximum speed at 4 °C. Control, heat-stressed, and standard samples were labeled with CyDye DIGE Fluor minimal dyes (GE Healthcare). The pooled samples were diluted in rehydration buffer (7 m urea, 2 m thiourea, 2% (w/v) CHAPS, 20 mm DTT, 0.8% (v/v) Pharmalyte^TM 3–11 NL, small amount of bromphenol blue) and applied to an 18-cm nonlinear, pH 3–11 Immobiline^TM dry strip (GE Healthcare) in an IPGphor isoelectric focusing system (GE Healthcare). In parallel, one sample containing 400 μg of total mitochondria was precipitated with TCA, resuspended in rehydration buffer, and applied to a strip as described above. Protein spots were detected by Coomassie Blue G-250 staining and manually excised for analysis by MS.

Immobiline strips were rehydrated in-gel rehydration for 12 h at 30 V. Afterward protein focusing was conducted stepwise for respectively 1 h at 200, 500, and 1000 V followed by a slow increase (2 h 30 min) to 8000 V for 3 h and finally decrease to 500 V for 2 h 30 min. Focused strips were incubated in equilibration buffer (6 m urea, 1.5 mm Tris/HCl, pH 8.8, 50% (v/v) glycerol, 1% (w/v) SDS) containing 1% (w/v) DTT for equilibration followed by an incubation in equilibration buffer containing 2.5% (w/v) iodacetamide for reduction. Both incubations were performed for 15 min in the dark. Proteins were separated by SDS–PAGE on a 11% polyacrylamide gel for 1 h at 0.5 W/gel followed by 1.5 W/gel overnight at 20 °C in an Ettan^TM DALTsix system (GE Healthcare). Proteins were detected by fluorescence scanning using the Fluoro image analyzer FLA-5100 (Fujifilm), and the obtained protein spots were analyzed by the Delta2D software (DECODON). For each gel spot, the intensities were normalized by the total sum of spot intensity at 42 °C divided by the total sum of spot intensity at 25 °C, and the average of six different gels and the S.E. were calculated. The significance was calculated using GraphPad Prism 6 software via one-way analysis of variance of all samples to a control of 100% (Dunnett's multiple-comparison test). p values of ≤0.001 are considered significant and indicated by asterisks. Protein spots of interest were manually excised and analyzed by MS (see the supporting “Experimental procedures”).

Preprotein import into mitochondria

In vitro import was performed as described (59), using the radiolabeled substrate proteins Su9(70)-DHFR, Trap1, and Mdh2, which were synthesized by in vitro transcription and translation in rabbit reticulocyte lysate. For each import reaction 50 μg of fresh isolated energized mitochondria were used that were either heat-shocked before the import reaction to assess the import efficiency or after the import reaction to test whether fresh imported proteins were prone to aggregate. The heat shock was performed in resuspension buffer for 20 min at respective temperatures. Control samples without membrane potential (Δψ) mitochondria were pretreated with antimycin, valinomycin, and oligomycin. The import reaction was performed for 40 min at 30 °C and stopped by adding 0.5 μm valinomycin. Where indicated, the samples were treated with 50 μg/ml proteinase K for 30 min at 4 °C to digest nonimported proteins. Samples were analyzed by SDS–PAGE and autoradiography.

We are grateful to M. Fuhrmann and B. Gehrig for expert technical assistance. We thank W. Jaworek, K. Pollecker, and Dr. G. Cenini for discussion and critical comments on the manuscript.