Cells respond to a wide variety of stresses through the transcriptional activation of genes that harbour stress elements within their promoters. While many of these elements are shared by genes encoding proteins representative of all subcellular compartments, cells can also respond to stresses that are speci®c to individual organelles, such as the endoplasmic reticulum unfolded protein response. Here we report on the discovery and characterization of a mitochondrial stress response in mammalian cells. We ®nd that the accumulation of unfolded protein within the mitochondrial matrix results in the transcriptional upregulation of nuclear genes encoding mitochondrial stress proteins such as chaperonin 60, chaperonin 10, mtDnaJ and ClpP, but not those encoding stress proteins of the endoplasmic reticulum. Analysis of the chaperonin 60/ 10 bidirectional promoter identi®ed a CHOP element as the mitochondrial stress response element. Dominant-negative mutant forms of CHOP and overexpression of CHOP revealed that this transcription factor, in association with C/EBPb, regulates expression of mitochondrial stress genes in response to the accumulation of unfolded proteins.
The role of cytosolic factors in protein targeting to mitochondria is poorly understood. Here, we show that in mammals, the cytosolic chaperones Hsp90 and Hsp70 dock onto a specialized TPR domain in the import receptor Tom70 at the outer mitochondrial membrane. This interaction serves to deliver a set of preproteins to the receptor for subsequent membrane translocation dependent on the Hsp90 ATPase. Disruption of the chaperone/Tom70 recognition inhibits the import of these preproteins into mitochondria. In yeast, Hsp70 rather than Hsp90 is used in import, and Hsp70 docking is required for the formation of a productive preprotein/Tom70 complex. We outline a novel mechanism in which chaperones are recruited for a specific targeting event by a membrane-bound receptor.
Mitochondria cannot be made de novo but replicate by a mechanism of recruitment of new proteins, which are added to preexisting subcompartments. Although mitochondria have their own DNA, more than 98% of the total protein complement of the organelle is encoded by the nuclear genome. Mitochondrial biogenesis requires a coordination of expression of two genomes and therefore cross talk between the nucleus and mitochondria. In mammals, regulation of mitochondrial biogenesis and proliferation is influenced by external factors, such as nutrients, hormones, temperature, exercise, hypoxia, and aging. This complexity points to the existence of a coordinated and tightly regulated network connecting different pathways. Communications are also required for eliciting mitochondrial responses to specific stress pathways. This review covers the mechanisms of mitochondrial biogenesis and the way cells respond to external signals to maintain mitochondrial function and cellular homeostasis.
In an accompanying paper, we show that the mitochondrial Unfolded Protein Response or mtUPR is initiated by the activation of transcription of chop through an AP-1 element in the chop promoter. Further, we show that the c/ebpβ gene is similarly activated and CHOP and C/EBPβ subsequently hetero-dimerise to activate transcription of mtUPR responsive genes. Here, we report the discovery of six additional mtUPR responsive genes. We found that these genes encoding mitochondrial proteases YME1L1 and MPPβ, import component Tim17A and enzymes NDUFB2, endonuclease G and thioredoxin 2, all contain a CHOP element in their promoters. In contrast, genes encoding mitochondrial proteins Afg3L2, Paraplegin, Lon and SAM 50, which do not have a CHOP element, were not up-regulated. Conversely, genes with CHOP elements encoding cytosolic proteins were not induced by the accumulation of unfolded proteins in mitochondria. These results indicate that mtUPR responsive genes appear to share a requirement for a CHOP element, but that this is not sufficient for the regulation of the mtUPR. A more detailed analysis of promoters of mtUPR responsive genes revealed at least two additional highly conserved, putative regulatory sites either side of the CHOP element, one a motif of 12 bp which lies 14 bp upstream of the CHOP site and another 9 bp element, 2 bp downstream of the CHOP site. Both of these additional elements are conserved in the promoters of 9 of the ten mtUPR responsive genes we have identified so far, the exception being the Cpn60/10 bidirectional promoter. Mutation of each of these elements substantially reduced the mtUPR responsiveness of the promoters suggesting that these elements coordinately regulate mtUPR.
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...
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