A large body of evidence now indicates that mitochondrial translation is primarily, if not exclusively, a membrane-associated process (7
). In yeast, this is a complex process that involves not only membrane-associated ribosomes but also mRNA-specific translational activators and numerous other proteins involved in processing and stability of mRNA that are directly or indirectly associated with the inner membrane (4
). Our previous work (9
) in this area suggested that mtRNA polymerase is also intricately involved in the efficiency of translation not only directly, through synthesizing the requisite RNA species, but also via functions of the ATD in coordinating transcription with post-transcriptional events. In this report, we have elucidated a role for Sls1p in mitochondrial translation, and we provide additional new lines of evidence that support a model in which one function for the ATD of mtRNA polymerase is to nucleate a series of interactions involving Nam1p and Sls1p that are ultimately required to link mtRNA polymerase to the inner membrane to facilitate efficient mitochondrial protein synthesis. The data supporting these conclusions are discussed below.
A recent key observation that led to our initial proposal that the ATD of mtRNA polymerase is involved in coupling transcription to membrane-associated events is the ability of the membrane protein Sls1p to rescue the petite phenotype of nam1
Δ and ATD mutations when overexpressed (9
). Because Sls1p is required for proper assembly of the respiratory chain, but not for transcription or mtDNA maintenance per se
, it has been postulated to be involved in post-transcriptional steps of mitochondrial gene expression (17
). Our results (), which clearly demonstrate that our sls1
Δ strain has the same intron-processing defect we reported previously for nam1
Δ and ATD mutations (10
) and are globally deficient in labeling of mtDNA-encoded proteins in vivo
, establish a function for Sls1p in mitochondrial protein synthesis.
Next, we investigated whether the ability of Sls1p to rescue a nam1
Δ phenotype (9
) was linked to its role in mitochondrial translation identified here. Nam1p was shown by others (14
) to be involved in overall mitochondrial translation efficiency, accompanied by a severe defect in translation of Cox1p. We confirmed a role for Nam1p in overall mitochondrial translation () and went on to show that overexpression of Sls1p re-established a nearly wild-type mitochondrial labeling pattern in an nam1
Δ strain (), supporting a critical role for Sls1p in mitochondrial translational efficiency. Interestingly, overexpression of Sls1p in our nam1
Δ strain resulted in a Cox1p-specific translation defect () that is virtually identical to that originally reported by Asher et al.
), suggesting that the differences observed between these two strains may involve strain-dependent differences in endogenous expression of SLS1
The elucidation that Sls1p is involved in translation in cooperation with Nam1p () suggested to us that one function of the pathway of gene expression events involving Nam1p, Sls1p and the ATD that we have described previously (9
) is to facilitate delivery of transcripts to the translation machinery at the inner mitochondrial membrane. Two additional lines of evidence provided by this study support this conclusion. First, all mtRNA polymerase ATD mutations examined here resulted in translation-related defects, including a significant reduction in labeling of all mtDNA-encoded proteins in vivo
(), reduced steady-state levels of the mtDNA-encoded proteins Cox1p and Cox2p (), and, with the exception of the rpo41-E119A-C121A
mutation (discussed later), an increased sensitivity to the mitochondrial translation inhibitor erythromycin (). That the three ATD mutations that have translation defects in all three assays (rpo41
, and rpo41-N152A/Y154A
) are those that were shown previously to negatively impact Nam1p binding (10
) strongly suggests that the observed defects result from disruption of the proposed RNA-handeling pathway. Second, Sls1p is found associated with nucleoids in an ATD-dependent and energy-dependent manner (), strongly supporting the notion that efficient mitochondrial translation is occurring when Sls1p is found in a complex with those mtRNA polymerase molecules that are bound to mtDNA and presumably actively engaged in transcription of the mitochondrial genome.
Based on these data, we propose a revised model for mitochondrial gene expression involving these factors (). In this model, Nam1p is predicted to bind to the ATD of mtRNA polymerase to facilitate the interaction of a transcriptionally active, nucleoid-associated mtRNA polymerase with Sls1p at the inner mitochondrial membrane. Once this connection is established between mtRNA polymerase and Sls1p (which may involve additional factors), translation of the mRNA is accomplished in a transcription-coupled manner. The precise functions of Nam1p and Sls1p remain to be elucidated. However, based on the recent report by Fox and colleagues (18
), Nam1p may facilitate interactions between the nascent mRNA and COX
-specific translation activators. If this were the case, then it is tempting to speculate that, once a functionally coupled transcription/translation complex is fully established, Nam1p would dissociate in order to locate another template-bound mtRNA polymerase that has yet to be membrane-coupled. A transient nature to the Nam1p interactions is postulated based on the fact that Nam1p is not found as a nucleoid component (see Ref.25
; this study, data not shown) and is localized primarily to the matrix in mitochondrial fractionation studies (30
). At present we speculate that the most likely function for Sls1p in this regard is to serve as part of a membrane-anchoring point for mtRNA polymerase during active gene expression.
Model describing critical interactions required to coordinate transcription and translation at the inner mitochondrial membrane
Whereas the vast majority of data presented herein are consistent with this model, it is clear that mutations in the ATD can cause multiple defects in mitochondrial gene expression that are not as easily explained. First, although we were successful at eliminating mtDNA instability as an explanation for the observed overall translation defects seen in most of ATD mutant strains tested (), the rpo41-E119A-C121A
is a notable exception. This mutation resulted in a relatively large defect in steady-state accumulation of Cox1p and Cox2p (). In fact, its steady-state defect was comparable with that of the rpo41-N152A/Y154A
strain (), despite the fact that its in vivo
labeling capacity was substantially greater (), and it was not hypersensitive to erthyromycin (). This mutation also does not appear to disrupt Nam1p binding (10
). Altogether, these results suggest that the rpo41-E119A-C121A
defect is not due to disruption of the proposed translation-coordination function of the ATD but rather its mtDNA instability phenotype (). Second, overexpression of Sls1p is unable to rescue the in vivo
labeling defect in the rpo41
ATD mutant strains (data not shown) but does moderately increase the steady-state level of Cox1p and Cox2p in the ATD mutants (), suggesting that Sls1p has a role in assembly of the oxidative phosphorylation complexes as originally postulated by others (17
). These data indicate the ability to Sls1p to partially rescue the ATD mutant phenotypes (9
) is most likely through this second function and not it ability to reestablish normal membrane coupling of mtRNA polymerase through the ATD. One possibility is that overexpression of Sls1p may allow increased numbers of membrane complexes to form in the ATD mutant strains and, even though they are not “wild-type” complexes, facilitates assembly (and hence stability) of the proteins that do manage to get translated, thus partially rescuing the mutant phenotype. In the case of nam1
Δ strains, a similar scenario is envisioned where the ability of Sls1p to increase the number of membrane sites would, in principle, increase the probability that normally coupled complexes would form because the ATD is intact in these strains, thus providing an explanation for the nearly full rescue of the nam1
Δ phenotype by Sls1p overexpression (9
). This interpretation is entirely consistent with the proposal that Nam1p functions to facilitate formation of the membrane-coupled mtRNA polymerase complex involving Sls1p (), but itself is not an active member of the complex once it is formed. Finally, the rpo41-R129D
mutation results in only a moderate reduction in mitochondrial translation (data not shown), yet causes the most severe glycerol growth phenotype (10
), suggesting that this mutation in the ATD of mtRNA polymerase can compromise additional cellular functions.
In conclusion, we have elucidated important new aspects of how mitochondrial gene expression is accomplished in yeast. Our results indicate that the primary mechanism involves a complex series of interactions that ensure coordination of transcription and translation of mitochondrial transcripts at the inner mitochondrial membrane that is mediated through the ATD of mtRNA polymerase. It will be of great general importance to determine whether a similar mechanism is operating in human cells, where defects in mitochondrial gene expression can cause and exacerbate disease states and impact the aging process.