Defective mitochondrial gene expression can result from either mtDNA mutations or nuclear mutations that impact mtDNA expression or replication. The biological consequences of these defects are highly variable, as illustrated by the complex clinical presentation of mitochondrial disease symptoms in patients. For example, mtDNA mutations in two different tRNA genes can cause drastically different phenotypes and diseases, despite both mutations ultimately decreasing translation of all mtDNA-encoded proteins (7
). In fact, the same mtDNA mutation can generate dramatically diverse phenotypes in different patients or even different tissues within the same patient (7
). The implication of these observations is that subtle defects in mitochondrial gene expression can differentially impact respiration and in turn lead to a variety of deleterious downstream cellular consequences. To address this issue experimentally, we analyzed in detail two mtRNA polymerase ATD mutants previously shown to be defective in coupling mitochondrial transcription to translation. One of the strains, GS129, has relatively efficient but imbalanced translation (Fig. ), whereas the other strain, GS130, exhibits a global reduction in mitochondrial translation that is representative of the majority of ATD mutants (23
). Together, these two mutants represent strains with distinct perturbations in mitochondrial gene expression that differentially affect assembly of the OXPHOS system via disruption of the same process. While these two mutants have several phenotypes in common (e.g., decreased respiration and increased ROS production in stationary phase; Fig. , , and ), they have extremely different chronological life span defects and responses to overexpression of superoxide dismutase (SOD1
; Fig. , , , and ).
From this study we draw two primary conclusions. The first is that defective mitochondrial gene expression (specifically, defective coupling of transcription to translation) leads to increased ROS production and inhibition of respiration in stationary phase, ultimately limiting chronological life span. The second is that ROS themselves can lead to the complete inactivation of the respiratory chain and a dramatic loss of viability, but only if produced above a certain threshold level. Altogether, our results support the hypothesized “vicious cycle” of mitochondrial ROS production that, once initiated, is thought to lead to the progressive mitochondrial and cellular dysfunction seen in a number of human diseases and aging. The rationale for these conclusions is discussed in detail below.
In our initial characterizations of the ATD of mtRNA polymerase, we examined several point mutations and deletions in this domain. Most of these mutations cause a global reduction in mitochondrial translation due to an inability to properly couple transcription to translation during mitochondrial gene expression (22
). One mutation, rpo41-R129D
(strain GS129), stood out in these analyses in that it has the most pronounced glycerol growth phenotype (22
) (Fig. ). Experiments aimed at better understanding this unique ATD mutant were the starting point for this study. We found that GS129 has a severe chronological life span defect (Fig. ), imbalanced mitochondrial translation (Fig. ), severely decreased respiration in stationary-phase glucose (SD) cultures (Fig. ), increased ROS production (Fig. ), and enhanced sensitivity to oxidative stress (Fig. ). These results clearly establish that disruption of mitochondrial gene expression can limit life span and strongly suggest mitochondrial ROS are responsible for progressive cellular dysfunction (Fig. ).
FIG. 8. Model depicting how different defects in mitochondrial gene expression differentially affect yeast life span. Depicted is a scenario in which certain types of mitochondrial gene expression defects (e.g., loss of coupling between transcription and translation (more ...)
We ruled out the possibility that the stationary-phase respiration defect of GS129 was caused solely by an increased rate of petite mutant formation. Under the conditions in which our life span assays were carried out, the GS129 mutant has a slightly higher rate of petite formation than the wild type (~6% in GS129 versus ~1.6% in the wild type at day 1 stationary phase), consistent with increased levels of oxidative stress and potentially greater damage to mtDNA. However, the dramatic decrease in respiration and viability of GS129 in stationary phase (Fig. , and ) cannot be attributed to such a minimal relative increase in the percentage of petites.
We also point out that the growth rate of the wild-type and GS129 mutant strains are indistinguishable in the SD media used for this assay, and the maximum titers attained by both strains are nearly identical (data not shown), thus discounting the possibility that the differences in respiration observed are due to one strain reaching stationary phase before the other. Importantly, the GS129 mutant is capable of supporting a high level of respiration in stationary phase in glycerol (YPG) medium (Fig. ). Though direct comparisons of the wild type and GS129 are more difficult to make in this case (due to differences in growth rates of the two strains in YPG), it can be conservatively concluded that the GS129 mutant mtRNA polymerase can carry out significant gene expression that can support high levels of respiration under at least some conditions. GS129 must therefore be considered a conditional mutant with respect to respiration (i.e., oxygen consumption).
We sought to confirm our hypothesis that the life span defect in GS129 is a direct consequence of its increased ROS production. Therefore, we increased antioxidant defenses in this strain by overexpressing SOD1, SOD2, or the stress-response transcription factor gene MSN4 (Fig. and ). As predicted, decreasing ROS in GS129 stationary-phase cells by overexpression of SOD1, SOD2, or MSN4 each greatly extended the life span of this mutant (Fig. and ). Also, the glycerol growth phenotype of GS129 was greatly ameliorated by SOD overexpression (Fig. ), consistent with the interpretation that the heterogeneous colony size and presence of apparently abortive colonies in this mutant (Fig. and ) are due to stochastic ROS production and damage. However, perhaps unexpectedly, we found with either SOD or MSN4 overexpression that the inactivation of respiration normally exhibited by GS129 in stationary phase was also reversed (Fig. ). These observations lead us to the inevitable conclusion that ROS are actually causative in the cessation of respiration in GS129. That is, the respiration block in GS129 cannot be due to a complete lack of expression of an essential OXPHOS component, as reduction of ROS could hardly be imagined to complement this type of defect. Rather, we interpret these data as indicating that, under the nutrient-limited conditions of stationary phase, ROS generated as the result of a partially blocked and aberrant respiration in GS129 overwhelm antioxidant defenses and inactivate the remaining functional complexes, ultimately resulting in death (Fig. ). Under this scenario, overexpression of antioxidant defenses protects these complexes and prevents the complete inhibition of respiration.
The fact that overexpression of SOD1
both rescue the life span and respiration defects of GS129 is worthy of discussion. The almost complete rescue by SOD2
(a mitochondrial matrix enzyme) strongly suggests that superoxide production or accumulation in the mitochondrial matrix is the major contributor to the observed phenotypes. However, the partial rescue by SOD1
suggests that ROS outside the mitochondrial matrix are also important. Sod1p has been shown to localize to the mitochondrial intermembrane space as well as the cytoplasm, and the loss of Sod1p has been shown to compromise mitochondrial matrix proteins (21
). Also, it was recently suggested that mitochondrial superoxide may react with nitric oxide to produce peroxynitrite, a membrane-permeable molecule (19
). Therefore, it is possible that superoxide and/or peroxynitrite produced on the outer face of the inner membrane crosses into the matrix, where it can damage OXPHOS components and contribute to the vicious cycle. Also, it is probable that some of the deleterious effects of mitochondria-derived ROS in GS129 are occurring in the cytoplasm or the nucleus, and both Sod1p and Sod2p are capable of detoxifying these ROS before reaching their sites of action. Indeed, this is consistent with the diffuse localization of DHE fluorescence exhibited by GS129 (Fig. ) and the restoration of punctate staining by SOD2
overexpression (Fig. ).
Having characterized the GS129 mutant in detail, we next compared this revealing mutant to other mtRNA polymerase ATD mutants with similar yet distinct perturbations of mitochondrial gene expression. First, we found that three other ATD mutations (rpo41Δ2, rpo41Δ3, and rpo41-N152A/Y154A) also result in reduced chronological life span. The life span defects in these strains were virtually identical to one another (unpublished observations), and rpo41-N152A/Y154A (strain GS130) was characterized in greater detail as a representative of the group (Fig. ). While GS130 has a considerable life span defect (e.g., ~5- to 10-fold loss of viability at day 4), it has a substantially longer life span than GS129, which exhibits a >100-fold loss in viability at the same time point (Fig. ). GS130 is also sensitive to hydrogen peroxide (Fig. ), has increased ROS levels (Fig. ), and carries out substantially decreased respiration in stationary phase (Fig. ). Yet, like its life span defect, these phenotypes are less severe than those observed in GS129 (Fig. , , and ). However, the magnitude of the GS130 respiration defect is arguably on the same order as that observed in GS129 (compare Fig. and ).
Perhaps the most noteworthy difference between GS129 and GS130 is the inability of SOD1 or SOD2 overexpression to rescue the life span (Fig. ) and respiration (Fig. ) phenotypes of GS130. Our interpretation of these results is that the specific block in respiration in GS130 generates more ROS than the isogenic wild-type strain but never in quantities sufficient to overwhelm antioxidant defenses and initiate the vicious cycle (Fig. ). Therefore, ROS are not causative in either the inactivation of respiration in this mutant or its life span defect. Rather, it is likely that the decrease in the life span of GS130 is due solely to its inability to produce sufficient quantities of OXPHOS components and thus maintain normal amounts of respiration in stationary phase (Fig. ). This interpretation is consistent with our results showing that further decreases in respiration in GS130 (via SOD overexpression) shorten its life span (Fig. ).
Interestingly, GS130 actually carries out lower levels of mitochondrial translation than GS129 (23
) (Fig. ) and, unlike GS129, its mitochondrial RNA polymerase no longer interacts with the translation-coupling factor Nam1p (22
). Based on these differences, it is tempting to speculate that globally reduced translation (in GS130 and the other ATD mutants [23
]) represents a less severe defect with regard to ROS production and life span than efficient but imbalanced translation (Fig. , GS129). This interpretation is also consistent with the observation that a Cox1-specific imbalance in mitochondrial translation (in a nam1
null strain that is partially rescued by overexpression of SLS1
]) also leads to a stationary-phase respiration defect and dramatically decreased life span (see Fig. S1 in the supplemental material). This implies that perturbations in mitochondrial gene expression (due to mtDNA or nuclear mutations) that cause specific types of imbalanced production or assembly of OXPHOS components lead to extreme cellular dysfunction via a vicious cycle of oxidative stress (Fig. ). It is tempting to speculate further that the reduced mitochondrial translation of electron transport components of the OXPHOS system, in combination with increased translation of ATP synthase components (i.e., the translation defect observed in GS129 [Fig. ]), represents an unusually deleterious imbalance that greatly increases ROS production and aging.
In summary, this study has revealed novel insights into the mechanisms by which defective mitochondrial gene expression contributes to cellular dysfunction and aging. We provide strong evidence that improper assembly of the OXPHOS system (due to loss of coupling of transcription and translation during mitochondrial gene expression) leads to increased ROS production and substantial but conditional decline in respiration. However, the loss of cell viability (and the magnitude of the longevity defect, at least in yeast) resulting from such perturbations depends on the level of mitochondrial ROS production and whether a critical ROS threshold level is reached (Fig. ). Such a scenario may help explain the variable phenotypic expression (i.e., clinical presentation) of mtDNA mutations in human disease and aging, in that even subtle differences in mitochondrial gene expression can lead to grossly different outcomes depending on the precise nature of the defect. Finally, our results suggest a novel role for SOD in regulating respiration by maintaining proper ROS homeostasis. Increased SOD activity can either increase or decrease respiration depending on genetic background and environmental conditions (Fig. and ), implying that ROS may play a role in regulating respiration more complex than simply mediating damage to cellular components. Understanding the dynamic interplay between mitochondrial gene expression, respiration, and ROS in human disease and aging remains fertile ground for future investigation.