Selective Induction of Mitochondrial Chaperones in Response to Loss of the Mitochondrial Genome

Abstract

Molecular chaperones are known to play key roles in the synthesis, transport and folding of nuclearencoded mitochondrial proteins and of proteins encoded by mitochondrial DNA. Although the regulation of heat-shock genes has been the subject of considerable investigation, regulation of the genes encoding mitochondrial chaperones is not well defined. We have found that stress applied specifically to the mitochondria of mammalian cells is capable of eliciting an organelle-specific, molecular chaperone response. Using the loss of mitochondrial DNA as a means of producing a specific mitochondrial stress, we show by Western-blot analysis that mtDNA-less (e") rat hepatoma cells show an increase in the steady-state levels of chaperonin 60 (cpn 60) and chaperonin 10 (cpn 10). Nuclear transcription assays show that the upregulation of these chaperones is due to transcriptional activation. There was no effect on the inducible cytosolic Hsp 70, Hsp 72, nor on mtHsp 70 in @' ) cells, leading us to concluded that stress applied selectively to mitochondria elicits a specific molecular chaperone response. Heat stress was able to provide an additional induction of cpn 60 and cpn 10 above that obtained for the @ state alone, indicating that these genes have separate regulatory elements for the specific mitochondrial and general stress responses. Since the mitochondrial-specific chaperones are encoded by nuclear DNA, there must be a mechanism for molecular communication between the mitochondrion and nucleus and this system can address how stress is communicated between these organelles.Keywords: molecular chaperone ; mitochondria; stress response; heat shock ; gene activation. Molecular chaperones are found in all of the major sub-celMar compartments and have been shown to play essential functions in the folding and subcellular targeting of proteins. In addition, the ability of cells to survive stresses such as elevated temperatures, exposure to heavy metals and amino acid analogues is dependent on the induction of molecular chaperones (re- chaperones are essential for cell viability [lo, 15-17]. The upregulation of mitochondrial chaperones in response to metabolic insults such as heat shock, glucose deprivation, amino acid analogues and agents that impair energy metabolism [ 18 -191, suggest that they also play an important role in maintaining homeostasis in mitochondria when a general stress is applied to the cell. However, it is not known if mitochondrial chaperones are induced specifically in response to the application of a selective stress to mitochondria.Chaperone synthesis can be regulated in part by modulation of the message stability and the frequency of translation initiation, but is regulated mainly through specific transcriptional factors termed heat-shock factors (reviewed in [20]). The binding of heat-shock factors to DNA regulatory sequences, called heat-shock elements, promotes the expression of heat-shock genes, and is thought to be modulated by a number of processes such as oligomerisation, phosphorylation and i...

Isolation and characterisation of a cDNA encoding rat mitochondrial GrpE, a stress‐inducible nucleotide‐exchange factor of ubiquitous appearance in mammalian organs

“…As expected from previous metabolic labelling experiments [13,32], mt-Hsp70 mRNA levels increased in response to Azc treatment but not in response to heat shock whilst Hsp60 and Hspl0 mRNA levels increased in response to both treatments. These responses are in accord with nuclear run off experiments and Northern analysis we have performed in a parallel study [33] and indicates that the RT-PCR procedure, when performed carefully, will give a reliable indication of comparative transcript levels. The RT-PCR results obtained with the mt-GrpE specific primer set mirrored that obtained for mt-Hsp70 and thus indicated that the levels of mt-GrpE do not increase in response to heat shock but marginally in response to amino acid analogue treatment.…”

Metformin induced expression of Hsp60 in human THP-1 monocyte cells

“…The extraction of cellular protein, SDS-PAGE and Western blot analysis was carried out as essentially described in Martinus et al (1996). Briefly, total protein was extracted using TENT buffer (50 mM Tris, 250 mM NaCl, 5 mM EDTA, 1% Triton X-100 and freshly supplemented 0.4 mM phenylmethylsulfonyl fluoride).…”

“…An important cellular consequence of mitochondrial bioenergetic impairment is the up-regulation of the expression levels of mitochondrial-specific molecular stress protein Hsp60 (Martinus et al 1996). This protein is increasingly being implicated to play a key role as a powerful immunogen and immunomodulator in experimental models of arthritis, diabetes (type 1) and atheroscelerosis (Shamaei-Tousi et al 2007).…”

Unfolded protein responses in bacteria and mitochondria: A central role for the ClpXP machine

“…Thus, a distinct signal transduction pathway is required. To date the mitochondrial unfolded protein response has only been identified in animals, namely mammalian tissue culture cells and in the nematode worm, Caenorhabditis elegans (62,63,65). A disruption specifically to mitochondrial protein homeostasis activates neither the cell-wide heat shock response nor the ER-specific unfolded protein response (62,63,65).…”

“…However, mitochondrial-nuclear signaling does occur such as seen for the mitochondrial specific unfolded protein response (62,63). A disruption to protein homeostasis in the matrix compartment of mitochondria is sensed within the organelle and communicated to the nucleus resulting in the transcriptional upregulation of protein biogenesis and quality control factors such as processing enzymes, chaperones, and proteases (62)(63)(64)(65). This pathway is termed the mitochondrial unfolded protein response (UPR mt ) and like the envelope stress response in E. coli, it is a compartment specific stress response whereby the signal has to be transmitted across a membrane (two in the case of mitochondria) to activate transcription factors located in a different cellular compartment.…”