The observation that perturbation of the folding environment in mitochondria up-regulates the expression of nuclear genes encoding mitochondrial chaperones led to the discovery of the mitochondrial unfolded protein response (UPRmt) (Zhao et al.,2002; Kuzmin et al.,2004; Yoneda et al.,2004). In mammalian cells, the expression of a truncated folding-deficient form of ornithine transcarbamylase (ΔOTC) specifically up-regulated the mitochondrial chaperones Hsp60/10, Hsp40, and the protease ClpP. This signaling pathway likely involves the sensing of mitochondrial stress in the mitochondrial matrix, transduction of the stress signal to the nucleus, and the specific expression of the genes encoding for mitochondrial chaperones. It has been demonstrated that the transcription factors CHOP and C/EBPβ regulate the expression of the mitochondrial chaperones in the nucleus (Zhao et al.,2002). However, CHOP is conserved only in vertebrates, which led to the suggestion that other signaling pathways are involved in the UPRmt (Yoneda et al.,2004).
To identify the signaling components of the UPRmt, an RNAi screen of C. elegans chromosome I was performed to identify genes required for activation of the hsp-6 (Hsp70) and hsp-60 transcriptional GFP fusions. The genes, whose knock-down resulted in up-regulation of chaperone reporters were either encoding for proteins directly involved in the mitochondrial biogenesis, processing, and folding, or were components of multimeric complexes (Yoneda et al.,2004). The modifiers that contribute to the folding environment would be expected to activate molecular chaperones. By comparison, knock-down of components of multi-subunit protein complexes would be expected to cause an imbalance of subunits that could result in misfolding and aggregation of the partner proteins and thus increasing the number of chaperone client proteins. In subsequent genome-wide screens, eight components of the UPRmt signaling pathway were identified: (1) ubl-5, a ubiquitin-like protein, (2) dve-1, a homeobox containing transcription factor, (3) clpp-1, encoding the proteolytic component of the mitochondrial Clp protease, (4 and 5) the AAA+ partner Clp-ATPases, ClpX1 (K07A3.3) and ClpX2 (D2030.2), (6) haf-1, encoding an ABC transporter, (7) the bZip transcription factor ZC376.7, and (8) F54C8.5, a GTPase homologous to Rheb (Benedetti et al.,2006; Haynes et al.,2007,2010; see Fig. 3). Induction of mitochondrial stress promotes the accumulation of UBL-5 in nuclei, indicating that UBL-5 acts at a nuclear-localized step of the UPRmt (Benedetti et al,2006). DVE-1 displays a nuclear redistribution upon mt stress induction and directly binds to the promoter of the chaperone genes, hsp-6 and hsp-60. In addition, DVE-1 interacts with UBL-5, indicating that both act together in the transcriptional activation of the mitochondrial chaperone genes. UBL-5 appears to activate its own expression, leading to a feed forward regulation of the signal transduction. The function of ClpXP can be placed upstream of DVE-1 and UBL-5 since knock-down of clpp-1 (ClpP), K07A3.3 (ClpX1), and D2030.2 (ClpX2) attenuates the induction of ubl-5, hsp-6, and hsp-60 and also prevents the redistribution of DVE-1 and its binding to the promoter regions of hsp-6 and hsp-60 (Haynes et al.,2007,2010). It was proposed that the peptides generated by the proteolytic activity of ClpXP are then transported into the cytosol by the ABC transporter HAF-1 to transmit the UPRmt signal to the nucleus. Since the degradation of mitochondrial proteins results in an efflux of peptides in yeast by a homologous transporter (Mdl1p), it was speculated that a potential peptide efflux by HAF-1 could play a role in the UPRmt (Young et al.,2001; Haynes et al.,2010). Mitochondria isolated from C. elegans, indeed, exhibited an ATP-dependent release of peptides, which was abolished upon knock-down of either clpp-1 or haf-1. Down-regulation of haf-1 also attenuated ubl-5 expression upon mt stress, supporting the role of haf-1 in the signaling of UPRmt (Haynes et al.,2010). Notably, haf-1 had no influence on the nuclear redistribution of DVE-1, emphasizing that there are other yet unidentified regulators of the UPRmt. Additional screens have also revealed, ZC376.7, a bZip transcription factor as a regulator of the UPRmt. Upon mt stress, ZC376.7 relocated from the cytosol to the nucleus, which was attenuated by knock-down of clpP-1 and haf-1, thus placing ZC376.7 downstream of the proteolytic signaling cascade (Haynes et al.,2010). Little is known about the activity of the eighth and last component of the UPRmt, Rheb, but it seems to attenuate the UPRmt and might gain a role to shut off the UPRmt with cessation of the stress conditions. Knock-down by RNAi of F54C8.5 (RHEB) promotes the nuclear redistribution of DVE-1, the induction of ubl-5, and the complex formation of DVE-1 and UBL-5 (Haynes et al.,2007).
While there has been much recent progress, numerous questions remain: (1) on the initial sensing of mt stress: Does proteotoxic stress result in general misfolding of proteins, which are recognized by ClpXP or is there a specific substrate?, (2) characterization of the signal transduction events from the mitochondria to the nucleus: are there receptors for mitochondrial-specific peptides/components released by ClpXP-HAF-1 in the cytosol/nucleus? How are these peptides protected from cytosolic peptidases?, (3) identification of the specific targets of ZC376.7: Do the number and specificity differ between ZC376.7 and UBL-5/DVE-1?, and (4) the role of Rheb. A model for the signal transduction pathway of the UPRmt is summarized in Figure 3.