The experiments described here implicate the mitochondrially-localized peptide transporter, HAF-1, in signaling the stress of unfolded/misfolded proteins in the mitochondrial matrix to the nucleus and establish a correlation between mitochondrial unfolded protein stress and HAF-1-dependent efflux of peptides derived from proteolysis of matrix proteins. Importantly, HAF-1's contribution to peptide efflux from stressed mitochondria is readily integrated with the requirement for ClpP-mediated proteolysis in the UPRmt. Together these findings suggest a model whereby the efflux of peptides derived from stress-induced proteolysis is important to signaling the UPRmt.
The combination of impaired activation of mitochondrial chaperone genes and selectively enhanced sensitivity to mitochondrial stress observed in haf-1
deleted worms is precisely the phenotype expected of loss of a gene implicated in signaling the UPRmt
. It is a rare phenotype, as there are many more genes whose loss-of-function causes mitochondrial stress and increased sensitivity to further stress; but these are associated with enhanced signaling in the UPRmt
(Yoneda et al., 2004
; Benedetti et al., 2006
; Haynes et al., 2007
HAF-1's involvement in peptide export from the mitochondrial matrix is consistent both with its homology to the yeast mitochondrial peptide transporter, Mdl1p, and with the defect in peptide export observed in vitro from mitochondria isolated from haf-1
mutant worms. The assay used to measure peptide export has several features that bear on the plausibility of a link between UPRmt
signaling and peptide export: Peptide export is dependent on ATP, which is consistent with a role for ClpXP in peptide generation and HAF-1 (an ATPase) in their export. The minor differences in average peptide size we observed, compared with those reported for bacterial ClpAP in vitro (Choi and Licht, 2005
) may reflect different enzymes (ClpXP in C. elegans
mitochondria), different substrates (endogenous mitochondrial proteins in our experiments, versus GFP-SsrA in the in vitro experiments) or a bias introduced by the HPLC purification procedure.
Furthermore, peptide efflux is highly temperature dependent and therefore consistent with a role for protein unfolding in the generation of substrates for proteolysis and peptide export. This temperature dependence has also been observed in yeast, where there is an additional suggestion that labile, unfolded proteins are the preferred source of peptides for Mdl1-dependent export (Augustin et al., 2005
). Thus, the rate of peptide export is suited to report on the protein-folding environment in the mitochondrial matrix and to serve a proximal role in signaling the UPRmt
The significance of peptide export may be different in yeast and C. elegans
. In the former, many of the peptides exported originate from proteins that have domains in the inter-membrane space (Augustin et al., 2005
). And, the inter-membrane space protease, Yme1p, makes an important contribution to the generation of peptides which then diffuse through the semi-porous mitochondrial outer membrane to the cytosol (Young et al., 2001
). By contrast, in C. elegans
, matrix proteins appear to be the major contributors to the flux of peptides from mitochondria under conditions of thermal stress (). It is interesting to note that a loss-of-function mutation of ymel-1
), the only identifiable C. elegans
homolog of the yeast inter-membrane space protease Yme1p, had no effect on UPRmt
signaling and exhibited no synthetic interactions with the haf-1(ok705)
mutation (data not shown). Thus, the discovery of HAF-1's role in peptide export and the UPRmt
provides a plausible clarification for the previously-noted role for ClpP in the UPRmt
, though other proteases that were missed in our screen may make important contributions to the process.
Several caveats apply: The phenotype of ClpP (clpp-1) deficiency is much more severe than that of haf-1 deficiency. In the context of the UPRmt, ClpP loss-of-function blocks the redistribution of the transcription factor DVE-1 in the nuclei of cells experiencing mitochondrial stress, whereas DVE-1 redistribution is not discernibly affected by haf-1 deletion. Together these suggest that dve-1 contributes to the UPRmt independently of haf-1 and that the latter may signal in the nucleus by alternative means. A more thorough survey of transcription factor encoding genes by RNAi feeding identified an additional gene required for UPRmt signaling. The encoded bZIP-containing protein, ZC376.7, is concentrated in the nuclei of stressed worms in a manner that is dependent on clpp-1 and haf-1. Further analysis of the mechanisms regulating ZC376.7 may provide clues to missing components of the signal transduction pathway downstream of HAF-1.
How might peptide efflux be sensed to produce downstream signaling and influence ZC376.7 localization? In the cytosol peptides are rather short lived, but in mammalian cells some mitochondrial-derived peptides survive long enough to be transported into the ER and are presented on the cell surface in MHC-I complexes (Herget and Tampe, 2007
). Thus, a cytosolic/nuclear peptide receptor with some specificity for mitochondrial-derived peptides could signal downstream of the ClpP and HAF-1 steps suggested by this study (). Alternatively, signaling might proceed through a sensor, not of the peptides themselves but of the rate at which they flux through HAF-1. HAF-1 and its yeast homolog Mdl1p appear to have broad substrate specificity, but this last hypothetical mechanism side-steps the specificity problem by its indifference to the identity of the peptides transported. It is important to keep in mind that HAF-1 may also transport other substrates, in addition to peptides. For example, it has been suggested that the related mammalian ABC-me transports heme across the mitochondrial inner membrane (Shirihai et al., 2000
). In one fanciful scenario, HAF-1 could transport a ligand (e.g. heme) that is released from a matrix carrier protein by ClpP digestion under stress conditions and it is such a ligand that activates signaling in the UPRmt
. Either way, the aforementioned hypotheses predict the existence of downstream components of the UPRmt
that respond to a peptide ligand, peptide efflux or a non peptide ligand transported by HAF-1.