The recently discovered hOXR1
gene represents a family of well-conserved eukaryotic genes whose function is proposed to include resistance to oxidative damage (42
). In this report, we demonstrate that the regulation of gene expression, protein localization, and function of Oxr1 is also conserved from yeast to humans.
Numerous proteins are localized to the mitochondria to counteract the deleterious effects of ROS, including glutathione peroxidase, thioredoxin, SOD, and multiple DNA repair enzymes (2
). Despite the seeming overabundance of these oxidative damage resistance functions, the yeast oxr1
Δ mutant remains sensitive to oxidative damage, indicating an important role for this gene in protecting cells from oxidative damage (42
). Consistent with this idea are the results of several microarray experiments addressing stress-induced gene expression in yeast. The scOXR1
gene has been shown to be induced under conditions of heat stress, stationary phase, and diauxic shift (6
). Interestingly, these same conditions have been reported to result in significant increases in ROS within the cell (12
). Our findings corroborate the microarray data and expand these observations by showing that scOXR1
is part of a stress response pathway turned on under conditions of ROS production and provide further evidence that scOxr1 protein serves to protect yeast cells from oxidative damage. We also demonstrate that the scOxr1 protein can be functionally replaced by its human orthologue containing the Oxr1 homology domain.
The mouse homologue of hOXR1
, was isolated by others in a screen for genes up regulated upon cell attachment to extracellular matrix. With the C7M antibody generated to domain II of C7, this protein was shown to localize to the nucleolus in several rodent cell lines (14
). We have used the C7C antibody produced from this study and shown it to recognize specifically mitochondrial protein in HeLa cells (Fig. ), as well as in Hep2 and COS cells. This is consistent with the Western blot data showing that the C7C antibody recognizes only one major protein in untreated HeLa cell extracts (Fig. B, untreated control lane). A second species is detectable after induction by oxidative or heat stress and also appears to be largely mitochondrial. No nucleolar staining is detectable, even after stress induction. Our results indicate that, in the cell lines tested, hOxr1 is associated with the mammalian mitochondria. That we observed no nucleolar staining with the C7C antibody suggests either that the nucleolar isoform of Oxr1 lacks the amino acid sequence recognized by C7C or that the antibody cannot access such sequences. Recent studies failed to detect nucleolar staining in human cells (Eva Engvall, personal communication) and are similar to our results with the C7C or C7M antibodies (Fig. and and data not shown). This suggests that the nucleolar staining in rodent cells is due to a species difference or is a species-related artifact. Mitochondrial localization of the Oxr1 homology domain is consistent with the finding that Oxr1 must be targeted to this cellular compartment for the antioxidant function of this domain in yeast.
As is the case with scOXR1
, the hOXR1
gene is induced by stress conditions in human cells. The first evidence of stress-induced expression of the hOXR1
gene came from immunofluorescence experiments with HeLa cells after hydrogen peroxide treatment (Fig. A). During a 1-h recovery from oxidative stress, hOxr1 protein visibly accumulated in the mitochondria. We also saw a more intense signal in the cytoplasm, which may be due to leakage of hOxr1 protein from the mitochondria, incomplete importation of all of the protein into the mitochondria, or the expression of a distinct cytoplasmic isoform of hOxr1. The latter possibility is consistent with the Western blot results showing the appearance of multiple Oxr1 bands following peroxide treatment (Fig. B). This Western blot finding also confirms the oxidative stress-induced accumulation of the 37.5-kDa hOxr1 protein. As in yeast, heat stress has been shown to lead to increased ROS and induction of antioxidant functions in mammalian cells (38
). We have shown that heat stress induces expression of hOxr1 protein in a manner very similar to that of oxidative stress (Fig. C). Although the induction of mitochondrial heat shock proteins by heat and oxidative stress is well known (16
), there is little evidence of mitochondrial proteins outside of this well-conserved protein family induced by both heat and oxidative stress. Also, the most well-characterized mitochondrial heat shock proteins are chaperonins (33
), and it is unclear what, if any, antioxidant activity they possess. hOxr1 may therefore represent one of a small set of proteins that are responsive to multiple stress conditions and provide protection against ROS in human mitochondria. In this respect, it is interesting that hOXR1
mRNA appears to be abundant in tissues with a relatively high respiration capacity (heart, skeletal muscle, brain; Fig. ), where it would be advantageous to counteract mitochondrial ROS production.
It has been hypothesized that ROS play a role in mediating cell death in mammalian cells, particularly through the mitochondrial apoptosis pathway (15
). Conditions that increase the amount of mitochondrial ROS production (for example, inhibition of the electron transport chain) lead to increased apoptosis. Conversely, depletion of mitochondrial antioxidant functions has also been shown to increase cell death by apoptosis (22
). Regulation of the mitochondrial redox state has been shown to be important for resistance to oxidative stress in S. cerevisiae
, as well as in mammals. Deletion of the mitochondrial thioredoxin reductase TRR2 in yeast causes increased sensitivity to hydrogen peroxide (34
), while homozygous mutation of mitochondrial thioredoxin (Trx-2) in mice results in elevated apoptosis and embryonic lethality (32
). We have found that targeting hOxr1 to the yeast mitochondria is necessary for complementing the hydrogen peroxide sensitivity of an oxr1
Δ mutant (Fig. A). The hOxr1 protein containing an N-terminal MTS is targeted to the yeast mitochondria (Fig. ) and exhibits wild-type resistance to peroxide, particularly at the highest doses tested. A strain expressing an identical copy of hOXR1
lacking the MTS is as sensitive to peroxide as is the oxr1
Δ mutant. These data suggest that the peroxide-induced lethality seen in yeast is mediated by a mitochondrial process, and mitochondrial localization of OXR1 function (either yeast or human) is required for wild-type resistance to peroxide damage. Furthermore, these results support the claim that the hOxr1 and scOxr1 proteins are functionally homologous. Our findings suggest that both hOxr1 and scOxr1 may be part of a mitochondrial stress response. Since hOxr1 is capable of providing yeast cells protection from oxidative damage when localized to the mitochondria, it is likely that it plays a similar role in oxidative stress resistance in human cells as well. It will be interesting to determine if hOxr1 is involved in the regulation of ROS production or detoxification and protection from oxidation-mediated apoptosis in human cells.