To establish a comprehensive picture of the molecular mechanisms governing protein homeostasis, two different experimental aspects have to be taken into account. First, due to the specific conformational properties of each individual cellular protein, the behavior of endogenous proteins has to be studied in its natural environment. Second, the proteins’ dependence on components of cellular protein quality control, in particular under stress conditions, needs to be established. Although the enzymatic components of mitochondrial protein quality control are relatively well-studied (reviewed in Voos, 2009
), less evidence about the vulnerability of specific mitochondrial proteins exists, in particular under in vivo conditions. In our study, we aimed at a comprehensive characterization of aggregation processes in mitochondria. To maintain a defined environment as close as possible to the natural state, the experiments were performed using intact and energized isolated mitochondria from the model organism S. cerevisiae
. Performing aggregation assays in organello provides numerous advantages: defined conditions, realistic protein concentrations, and full activity of the protein quality control system. Furthermore, because most mitochondrial proteins are nuclear-encoded and must be imported into the organelle, adaptive mechanisms by changes in cellular protein expression can be ruled out.
Aggregated polypeptides were separated by a high-velocity centrifugation of detergent-lysed mitochondria, a technique that has already been used previously to characterize protein aggregation in bacteria and in yeast mitochondria (Mogk et al.
; von Janowsky et al.
). Our first goal was to identify mitochondrial proteins that aggregate during various stress conditions. On a general level, our results showed that the majority of mitochondrial proteins remain soluble during heat stress. However, we discovered a set of eight mitochondrial proteins that were abundant in the aggregate pellet as candidates for temperature-induced aggregation. We have found three components of the TCA cycle (Aco1, Kgd1, and Kgd2), two enzymes that are involved in branched-chain amino acid synthesis (Ilv2 and Leu4), mitochondrial glycerol-3-phosphate dehydrogenase (Gut2), an enzyme of glycerol metabolism, and the major mtHsp70 chaperone (Ssc1). The proteins Aco1 and Ilv2 also showed a temperature-dependent aggregation behavior in intact cells, essentially ruling out the possibility that the observed aggregation of Aco1 and Ilv2 is due to the use of isolated mitochondria. The reduced degree of aggregation in intact cells indicates that the overall metabolic state of the cells has at least some influence on the functional activity of the mitochondrial protein quality control system, most probably via the supply of nutrients required for sufficient ATP production. Other mitochondrial proteins that form large enzyme complexes, like pyruvate dehydrogenase, were sedimented under all temperature conditions, indicating that their presence in the pellet is not due to true aggregation. Interestingly, the inactivation of a limited number of mitochondrial proteins while the majority remains unaffected suggests that aggregation is a very specific rather than a random process. Like in a previous study addressing protein aggregation in bacteria (Mogk et al.
), we observed an enrichment of high molecular weight proteins (>50 kDa) in the aggregate pellets. It may be speculated that large proteins with higher structural complexity are more vulnerable to partial unfolding that may result in aggregation. Interestingly, bacterial homologues of Aco1 and Kgd1 were also shown to aggregate at temperature-stress conditions like incubation at 45°C (Mogk et al.
). The bacterial homologue of Ilv2 was not identified as an aggregation-prone protein. However, its specific stability under heat stress was not determined, so a certain sensitivity to aggregation cannot be ruled out. Overall, respective mitochondrial and bacterial proteins appear to behave similarly during heat stress conditions. Taken together, our results indicate that important metabolic pathways in mitochondria can become compromised due to the aggregation of respective key components.
As a first approach, our analysis was aimed at the identification of abundant aggregation candidates. It is expected that the described negative effects on certain metabolic processes represent only the tip of the iceberg because the behavior of most proteins of low abundance, in particular regulative components, could not be characterized so far. However, the decrease of metabolic activity can have a profound impact on the cellular level. It has been described that a decrease of Aco1 activity is a major hallmark of aging in animal mitochondria (Yan et al.
; Yarian et al.
). Our experiments with yeast mitochondria indicate that aggregation processes contribute to the decline of aconitase function and activity. Similarly, the activity of KGDHC was reduced after heat shock, at least partially, due to protein aggregation. Interestingly, it was reported that KGDHC activity was also reduced in brain mitochondria from patients suffering from Alzheimer’s disease (Huang et al.
), indicating a possible role of KGDHC components’ aggregation in the pathogenesis of this disorder. Taken together, our observations emphasize that aggregation of mitochondrial enzymes plays a role in the development of mitochondrial dysfunction in neurodegenerative diseases and during aging.
As another important aspect of mitochondrial stress, we also examined the effects of ROS because these radicals lead to chemical modifications and inactivation of polypeptides. This is of particular importance because oxygen radicals and mitochondrial dysfunction have been described as a major causative agent in the development of several human diseases as well as aging processes (Lin and Beal, 2006
). In general, no significant overall effect of oxidative stress on protein solubility was detected (data not shown). However, we found that both Ilv2 and Aco1 aggregated after treatment with oxidative stressors like menadione or hydrogen peroxide, correlating with previous results that aconitase is readily inactivated by oxygen radicals both in yeast and in mammalian mitochondria (Bota and Davies, 2002
; Bender et al.
). Because Aco1 contains an oxidant-sensitive Fe/S cluster as a prosthetic group (Nulton-Persson and Szweda, 2001
), it is conceivable that its structural integrity is altered or compromised during oxidative stress, resulting in misfolding and aggregation. Interestingly, ROS-induced aggregation of these proteins was strongly increased if Sod2, the major radical scavenger of the mitochondrial matrix, was deleted. Our results correlated well with a study that identified both Aco1 and Ilv2 as mitochondrial proteins that show high levels of carbonylation, a typical marker of oxidative protein modification, if Sod2 is absent (O’Brien et al.
). Our observations showed that Sod2 is able to detoxify externally added ROS and therefore protects mitochondrial proteins from resulting destabilization and aggregation. Detoxification of superoxide radicals by Sod2 can therefore be regarded as a major defense mechanism in securing protein homeostasis under oxidative stress conditions.
The fact that during stress conditions important metabolic pathways in mitochondria are affected by aggregation of their enzymatic components raises the question which components contribute to the maintenance of mitochondrial homeostasis and functionality under adverse conditions. We have therefore examined the roles of the major components of the mitochondrial protein quality control system in the matrix compartment. The fact that a depletion of ATP levels increased aggregation suggests a protective effect of the ATP-dependent chaperone and protease components. Hsp70-type chaperones generally exhibit a high affinity to unfolded polypeptide segments exposing hydrophobic amino acid residues, thereby contributing to the stability of damaged substrate proteins (Bukau et al.
). Our experiments demonstrated that the soluble matrix chaperone mtHsp70, or Ssc1 in yeast (Voos and Röttgers, 2002
), is able to inhibit, at least partially, the aggregation of the reporter substrate Ilv2. This confirms observations where artificial reporter proteins like luciferase were protected by mtHsp70 from denaturation and refolded after heat treatment in vitro (Kubo et al.
; Liu et al.
). Due to the particular mechanism of protein import, mitochondrial proteins enter the matrix in an unfolded conformation (Schwartz et al.
). Any delay in the subsequent folding steps would render newly imported preproteins susceptible to denaturation and/or aggregation. Indeed, newly imported Ilv2 polypeptides showed a slightly increased tendency to form aggregates at mild temperature stress, an effect that was significantly enhanced in mtHsp70-defective mutant mitochondria. Because mtHsp70 is the first chaperone a newly imported polypeptide chain encounters (Horst et al.
; Strub et al.
), a defective chaperone function would directly impede the folding reaction, escalating the problem of aggregation. Also in bacteria, the Escherichia coli
Hsp70 homologue DnaK is prominently involved in prevention of protein aggregation (Mogk et al.
; Tomoyasu et al.
). In particular, at 42°C in the absence of DnaK, aggregation rates were significantly increased (Mogk et al.
). However, in this study, the “holding” effect of the mitochondrial Hsp70 system was most pronounced at physiological conditions. At higher temperatures, this protective effect was either overcome by the larger amount of aggregated polypeptides or by a certain heat sensitivity of the chaperone itself. The Hsp60 chaperone system has also been shown to be prominently involved in the folding of mitochondrial proteins after import has been completed. Interestingly, Hsp60 seemed to have an even higher influence on the aggregation propensity of mitochondrial proteins at all temperatures as the mtHsp70 system, indicating that Hsp60 is a prominent component of the mitochondrial protein quality control system. As was already shown in screening experiments for mitochondrial substrates (Dubaquié et al.
), the mitochondrial enzyme Aco1 was strongly dependent on the activity of Hsp60. Interestingly, due to the large size of Aco1, with a molecular mass of ~82 kDa, a nonconventional mechanism of the Hsp60 complex is needed for the assistence of Aco1 folding (Chaudhuri et al.
Another chaperone component that has been described in mitochondria and has been implicated in aggregation reactions is the AAA+ chaperone Hsp78, a homologue of the bacterial ClpB protein (Krzewska et al.
; von Janowsky et al.
). Generally, it is believed that ClpB and its homologues restore protein function by assisting the resolubilization and refolding of formerly aggregated polypeptides (Goloubinoff et al.
; Krzewska et al.
). Based on this hypothesis, no direct effect of Hsp78 on protein aggregation would be expected. Indeed, only a very small difference could be detected regarding the aggregation of Ilv2 in WT and hsp78
Δ mitochondria. This correlates with other studies in bacteria, where the homologue ClpB also could not prevent aggregation as such but rather executes the resolubilization of aggregates together with the Hsp70 system (Mogk et al.
). This protective effect of the disaggregtion activity of Hsp78 on mitochondrial functions in vivo has been well established. Hsp78 confers thermotolerance to mitochondria by restoring heat-sensitive processes like mitochondrial translation or DNA replication (Schmitt et al.
; Germaniuk et al.
), and it assists the chaperone activity of mtHsp70 (von Janowsky et al.
). Our results suggest that also amino acid synthesis and the TCA cycle are at least partially sensitive to heat, which raises the question of whether these pathways might be recovered by Hsp78-dependent disaggregation.
Proteolysis is suggested to be a major process that prevents the accumulation of damaged polypeptides before they can form aggregates (Luo and Le, 2010
). In our hands, the absence of the ATP-dependent protease Pim1, localized in the matrix of yeast mitochondria, increased aggregation of both model proteins, whereas overexpression of Pim1 drastically reduced the aggregation of both Ilv2 and Aco1. These observations suggest that proteolysis and aggregation are competing processes. In this model, Pim1 would recognize misfolded proteins that cannot be stabilized by the Hsp70 system anymore, and subsequently degrade them before they can form aggregates. This conclusion is supported by the finding that, in case of Ilv2, despite a general dependence of aggregation on the presence of Pim1, no significant difference in the aggregation rates of newly imported and steady-state polypeptides could be detected. The protective effect of Pim1 is most probably based on its ability to recognize unstructured segments of its substrates (von Janowsky et al.
), which is the basis for the initiation of its proteolytic activity on the substrate polypeptide. Moreover, a functional cooperation of Pim1 with Hsp70 has been reported, indicating that binding of unfolded proteins is followed by their degradation if refolding fails (Wagner et al.
). In addition, Pim1 has been discussed as the primary weapon against oxidatively modified proteins (Bota et al.
; Bayot et al.
; Bender et al.
). ROS-dependent damages in proteins are mostly covalent modifications like carbonylation, where refolding to the functional state is impossible. The results regarding the effect of Pim1 on aggregation are in good agreement with results from studies aimed at the identification of the targets of proteolytic degradation in mitochondria. Aco1 has formerly been reported to be a target of the Pim1/Lon protease both in yeast and human mitochondria, and also Ilv2 was found to be a substrate (Bota and Davies, 2002
; Major et al.
In summary, we thus propose a specific scenario for the maintenance of mitochondrial protein homoeostasis (). As a primary event, the direct interaction of damaged proteins with mtHsp70 retains them in the soluble state, whereas Pim1 acts like a quality control valve during stress conditions, degrading proteins that the Hsp70 system is not able to protect from aggregation. Hsp78 then acts as a clean-up chaperone, restoring the function of mitochondrial proteins that have aggregated because they have escaped the efforts of stabilizing chaperones. These components of mitochondrial protein quality control therefore form a cooperative functional network that represents a crucial aspect in securing the functional integrity of the organelle.
FIGURE 8: Overview of the protein quality control system of the mitochondrial matrix. During stress conditions, proteins become unfolded and subsequently aggregate because of irregular intermolecular interactions of exposed hydrophobic patches. mtHsp70 promotes (more ...)