Just as the loss of protein-coding genes to the nucleus instigated a need for protein import, loss of tRNA-coding genes is coupled with tRNA import into mitochondria. Translation of the few proteins encoded in the mitochondrial genome requires, depending on the genetic code and the wobble rules, at least 20–22 different tRNAs. Experimental analysis in a number of different species confirmed that this has been achieved with an import pathway for tRNAs (Schneider & Marechal-Drouard 2000
; Entelis et al. 2001b
; Tarassov et al. 2007
; Salinas et al. 2008
). While it would be possible in principle, there is no evidence in any system for a functional transfer of mitochondrial tRNA genes to the nucleus; instead, imported tRNAs derive from cytosolic tRNAs that are also essential for cytosolic translation. The import of cytosolic tRNAs therefore must have preceded the loss of mitochondrial tRNA genes.
(a) Occurrence of mitochondrial tRNA import
Complete mitochondrial genome sequences are available for more than a 1000 different species of eukaryotes (O'Brien et al. 2009
). Bioinformatic analysis of these sequences can predict the number of mitochondrial tRNA genes and match these to the codons that are used by the corresponding mitochondrial translation systems. The conclusion of such an analysis is that most eukaryotes lack some of the essential mitochondrial tRNA genes in their mitochondrial genomes (). Experimental analysis in a number of these systems has shown that this lack is compensated for by import of the corresponding cytosolic tRNAs. Thus, imported tRNAs always represent a small fraction of the cytosolic tRNA pool. Exclusive mitochondrial localization of a nucleus-encoded tRNA has so far not been found, though a tantalizing possibility exists in the recently described Chlamydomonas reinhardtii
UUU (Vinogradova et al. 2009
Figure 4. Occurrence of mitochondrial tRNA import. Unrooted phylogenetic tree of the six eukaryotic supergroups (indicated in capitals). Branching order reflects the phylogenetic relationship of taxons but branch length is not to scale. Bioinformatic analysis of (more ...)
The phylogenetic distribution of mitochondrial tRNA, as predicted by bioinformatics, shows that contrary to popular belief the process is very widespread. At least some tRNAs are imported in the vast majority of species in probably all six eukaryotic supergroups (mitochondrial genome sequences for Rhizaria are still missing) (). It is organisms with a complete set of mitochondrial tRNA genes that are exceptional rather than the ones lacking them! Essentially, all these exceptions are opisthokont species and yet even within the opisthokonts, we find taxons such as the Cnidaria and the Chaetognatha (arrow worms) that have lost all but one or two mitochondrial tRNA genes. In addition, within the Fungi and the Bilateralia, there are many examples of individual species that have lost at least some mitochondrial tRNA genes whereas their close relatives have kept the whole set ().
It is likely that the original endosymbiont had a complete set of tRNA genes, and loss of mitochondrial tRNA genes is expected to be irreversible. Thus, having a complete set of mitochondrial tRNAs genes represents the ancestral situation. It follows that the loss of mitochondrial tRNA genes and import of the corresponding cytosolic tRNAs are derived traits. Based on this, we conclude that mitochondrial tRNA import has a polyphyletic evolutionary origin: it was invented many times in different branches of the eukaryotic evolutionary tree.
(b) Limits of bioinformatic analysis
One of the caveats on bioinformatic analyses aimed at the occurrence of mitochondrial tRNA import is that modifications of the wobble nucleotide, which cannot be predicted, can change the decoding capacity of a tRNA, making it difficult to match it to a specific codon set. Some mitochondrial tRNAs have unconventional structures (Wolstenholme et al. 1987
) or undergo RNA editing (Bullerwell & Gray 2005
) and are therefore hard to recognize. Moreover, the existence of a mitochondrial tRNA gene does not preclude mitochondrial import of a cytosolic tRNA that is able to read the same codons. In fact, a scenario like this may even have been an obligatory evolutionary intermediate that subsequently allowed the loss of the corresponding mitochondrial tRNA genes.
The limitations to predict scenarios for tRNA import can best be illustrated in the yeast S. cerevisiae
. The yeast mitochondrial genome encodes an apparently complete set of mitochondrial tRNAs. Nevertheless, yeast mitochondria import a small fraction of one of two cytosolic tRNALys
isoacceptors (Tarassov et al. 1995b
). Import of this tRNA is redundant under standard growth conditions but becomes essential when cells are grown at elevated temperature (Kamenski et al. 2007
). Import of the tRNALys
represents the best studied case of mitochondrial tRNA import in any species (discussed below). A recent study suggests that besides the tRNALys
also a small fraction of the cytosolic tRNAGln
is imported into yeast mitochondria (Rinehart et al. 2005
). The function of the imported tRNAGln
is presently unknown. However, surprisingly, it seems to be imported by a different pathway than the tRNALys
Moreover, a complex situation is also found in humans. Just as in yeast, the human mitochondrial genome encodes a complete set of mitochondrial tRNAs. Recent evidence, however, suggests that a fraction of the cytosolic tRNAGln
is imported into mitochondria (Rubio et al. 2008
). In addition to tRNA import, import of the RNA subunits of RNase P (Puranam & Attardi 2001
) and RNase MRP (Chang & Clayton 1989
) as well as the 5S rRNA (Magalhaes et al. 1998
) has been suggested. In the case of RNase P, these claims are highly controversial as mammalian mitochondrial RNase P has recently been shown to lack an RNA subunit (Holzmann et al. 2008
). Import of 5S rRNA is also surprising as no 5S rRNA has been found in mitochondrial ribosomes of mammals (Sharma et al. 2003
). However, import of 5S rRNA has been analysed in some detail using an in vitro
system and it has been proposed that the RNA might be co-imported with an as-yet-unknown protein (Entelis et al. 2001a
While it is clear that the described cases are of great interest and need to be further investigated, they are exceptions and, at least in the case of tRNAs, bioinformatics remains a valid tool to analyse the distribution of mitochondrial import on a global scale.
(c) Non-random loss of mitochondrial tRNA genes
The extent of tRNA gene loss from the mitochondrial genome is variable (). The most extreme situations are found in some poriferan species, Cnidaria, Chaetognatha and the green algae Pseudendoclonium akinetum
, which have retained only one or two mitochondrial tRNA genes, and in the trypanosomatids and apicomplexans that lack mitochondrial tRNA genes altogether (O'Brien et al. 2009
Even though the loss of specific mitochondrial tRNA genes does not show a defined phylogenetic pattern, it follows a distinct order when analysed across all taxa. This can best be explained by the fact that lost mitochondrial tRNA genes of bacterial genetic origin are compensated for by import of a fraction of cytosolic tRNAs that are of eukaryotic origin. The evolutionary origin of tRNAs can formally be demonstrated only for few species that show domain specific features. However, circumstantial evidence such as the fact that nucleus-encoded tRNAs are transcribed by RNA polymerase III suggests that all imported tRNAs are of eukaryotic descent.
Compensation can only be successful if the imported tRNA can be functionally integrated into the bacterial-type translation system of mitochondria. For the initiator tRNAMet
, this is difficult. Translation initiation is very different in eukaryotes and in systems of bacterial origin, the latter requiring a specific initiator tRNAsMet
that carries a formylated methionine (Mayer et al. 2001
). This means that even if a cytosolic eukaryotic initiator tRNAMet
would be imported into mitochondria, it would not be functional as it could not be formylated. This explains why the single tRNA gene that has been retained in mitochondria of many Cnidaria, Chaetognatha and P. akinetum
is the tRNAMet
Many mitochondria show variations from the classic genetic code, the most frequent one being a reassignment of the stop codon UGA to tryptophane (Knight et al. 2001
). Decoding of the reassigned codon requires an anticodon change in the mitochondrial tRNATrp
which for that reason cannot simply be replaced by its cytosolic counterpart that lacks this change. Thus, most mitochondrial genomes have kept the gene for the tRNATrp
Can the concept that the overall frequency of mitochondrial tRNA gene loss is determined by the efficiency of functional integration of an imported tRNA into the mitochondrial translation system be extended to all tRNAs? It has been suggested that overall the loss of mitochondrial tRNA genes follows a specific order which could be explained by the differential capabilities of mitochondrial aminoacyl-tRNA synthetases to recognize imported eukaryotic-type tRNAs (Schneider 2001a
). In such a scenario, the loss of a mitochondrial tRNA gene would essentially be driven by how good its imported counterpart can be aminoacylated in the mitochondrion. The model is supported by the observation that the frequency of the loss of a specific mitochondrial tRNA gene is positively correlated with the similarities of the corresponding bacterial-type and eukaryotic-type aminoacyl-tRNA synthetases as well as the similarities of their corresponding identity elements on the tRNA (Schneider 2001a
Thus, the difficulty to functionally integrate specific imported tRNAs into the mitochondrial translation system represents a barrier for mitochondrial tRNA gene loss. Translation initiation and mitochondrial codon reassignments appear to be especially strong handicaps for imported tRNAs. Surprisingly, however, two taxonomic groups, trypanosomatids (Schneider 2001b
) and apicomplexans (Feagin 2000
; Crausaz-Esseiva et al. 2004b
), have lost all mitochondrial tRNA genes, indicating that these barriers are not absolute (see below).
(d) Mitochondrial targeting of tRNAs
The mechanisms of mitochondrial tRNA import in the various systems have recently been expertly reviewed (Salinas et al. 2008
). Here, we provide a condensed discussion of this subject with the emphasis on aspects we consider important in the evolutionary context. To discuss how tRNAs are imported into mitochondria, it is helpful to subdivide the process into three temporally and spatially ordered steps: (i) targeting of the tRNA to the mitochondrion, (ii) membrane translocation, and (iii) integration of the imported tRNA into the mitochondrial translation system.
The number of imported tRNAs ranges from one only to the whole set and is species-specific. Interestingly, in some taxons such as plants, the import specificity can differ even in closely related species. However, in all species, at least a few tRNAs still exist that are exclusively cytosolic indicating the need of the cell to select a subset of tRNAs for mitochondrial import. This selection depends on targeting signals on the tRNAs and is mediated by proteins. There are only three systems where the signals that are both necessary and sufficient for mitochondrial tRNA import have been characterized in detail.
(i) The first case concerns import of a fraction of one of two tRNALys
isoacceptors into yeast mitochondria (Martin et al. 1979
). Mitochondrial targeting of the tRNALys
requires specific binding to Eno2p, an isoform of the glycolytic enzyme enolase (Entelis et al. 2006
). Enolase delivers the tRNA to the surface of mitochondria. There, the tRNA is released and can now bind to the precursor of mitochondrial lysyl-tRNA synthetase to which it has a higher affinity than to enolase. The precursor of mitochondrial lysyl-tRNA synthetase is translated in the vicinity of mitochondria and acts as a carrier for import (see below). The specific binding of the two proteins to imported tRNALys
is primarily determined by acceptor stem and anticodon nucleotides that differ between the two tRNALys
isoacceptors (Entelis et al. 1998
(ii) The second example is the trypanosomatid T. brucei
in which all mitochondrial tRNAs are derived from cytosolic ones (Hancock & Hajduk 1990
; Tan et al. 2002b
). The initiator tRNAMet
and the tRNASec
, however, are not imported (Bouzaidi-Tiali et al. 2007
). An in vivo
analysis has shown that the single T-stem nucleotide pair at position 51 : 63 is both necessary and sufficient to determine the localization of trypanosomal tRNAs (Crausaz-Esseiva et al. 2004a
). Thus, a U : A nucleotide pair at this position, found in the initiator tRNAMet
, specifies a cytosolic localization, whereas any other base pair indicates a mitochondrial localization. Interestingly, the U51 : A63 nucleotide pair in the cytosolic initiator tRNAMet
has previously been characterized as an anti-determinant for elongation factor 1a (eEF1a) binding (Drabkin et al. 1998
). In line with this, it was shown that mitochondrial targeting of trypanosomal tRNAs requires interaction with eEF1a. This also explains the cytosolic localization of the tRNASec
which lacks the U51 : A63 cytosolic localization signal but nevertheless does not bind to eEF1a as it has its own specialized elongation factor. Thus, in T. brucei
, eEF1a besides its housekeeping function in translation elongation has a second function in selecting a subpopulation of cytosolic tRNAs for mitochondrial import. Moreover, in vivo
analysis has shown that eEF1a-mediated targeting of tRNAs to the mitochondria is an obligatory step for membrane translocation of tRNA to occur.
(iii) The third example is Tetrahymena
which contains three very similar tRNAGln
isoacceptors. Two of them with the anticodons UUA and CUA are cytosol-specific and recognize the stop codon UCA which has been reassigned to glutamine in the nucleus of Tetrahymena
. The third tRNAGln
with the anticodon UUG recognizes the standard glutamine codons and is in part imported into mitochondria (Rusconi & Cech 1996a
). In an in vivo
analysis, it was shown that the anticodon UUG of the imported tRNAGln
is both necessary and sufficient to induce import of any of the three tRNAGln
molecules (Rusconi & Cech 1996b
). However, no protein interacting with the import signal has been identified yet.
These three examples illustrate that the targeting signals on the tRNA and the targeting factors are not conserved between the different systems. This is no surprise because it reflects the very different specificities of mitochondrial tRNA import in the three systems. Moreover, finding different targeting signals and mechanisms is in line with the presumed polyphyletic origin of mitochondrial tRNA import. However, despite these differences, there are also some striking similarities between yeast and trypanosomatids. Targeting of the tRNAs to the mitochondria is in both cases essential for subsequent membrane translocation of the tRNA. Moreover, targeting is mediated by cytosolic housekeeping proteins that perform a second function. Further, research is needed to show whether this common principle of mitochondrial tRNA targeting can be extended to even more organisms.
There are a number of other in vivo
studies that have attempted to identify the cis
-elements on the tRNAs that induce their import into mitochondria (Dietrich et al. 1996
; Lima & Simpson 1996
; Delage et al. 2003
). However, these studies are generally less complete and thus their interpretation is difficult. Moreover, targeting has also been analysed by in vitro
import assays (Mahapatra et al. 1998
; Rubio et al. 2000
; Bhattacharyya et al. 2002
). These assays were in most cases done in the absence of cytosol and therefore in this respect may not reflect the in vivo
situation. Overall, the studies mentioned above identified all major tRNA domains as being important for mitochondrial tRNA targeting in one or the other system and therefore further illustrate the non-conserved nature of the tRNA targeting signals.
(e) Extent of mitochondrial tRNA localization
Studies in trypanosomatids (Tan et al. 2002b
) and Chlamydomonas
(Vinogradova et al. 2009
), which import all or nearly all of their mitochondrial tRNAs, revealed large variations between the extent of mitochondrial localization of individual tRNAs. In both organisms, these variations were not correlated with the cytosolic concentration of the tRNAs. This raises two questions: why is the extent of mitochondrial localization of different tRNAs so variable and how is it regulated?
(i) The steady-state levels of Chlamydomonas
cytosolic tRNAs correlate with the codon usage of nuclear genes as has been shown in other organisms as well. However, in Chlamydomonas
, the same is true in mitochondria and the frequency of specific codons in mitochondrial genes appears to correlate with the levels of imported tRNAs that read them (Vinogradova et al. 2009
). As the nuclear and the mitochondrial codon usage are different, this requires differential mitochondrial localization of tRNAs. Interestingly, in T. brucei
, a similar study failed to reveal such a correlation (Tan et al. 2002b
). However, as in this study only a subset of tRNAs was analysed, the question may need to be reinvestigated. Thus, at least in Chlamydomonas
, the extent of mitochondrial localization may serve to adapt tRNA abundance to the mitochondrial codon usage.
(ii) What is responsible for the differential localization of imported tRNAs is not known in any system. Different mitochondrial steady-state levels of tRNAs could in principle be achieved by different import efficiencies or by regulating tRNA stability after import. A study in Leishmania
suggests that it might be the former. Cytosolic leishmanial tRNAGlu
have a thiomodified uridine at the wobble position whereas their imported counterparts are lacking this modification (Kaneko et al. 2003
). Thus, it was proposed that the thiomodified uridine acts as an antideterminant that prevents mitochondrial tRNA import. However, in vivo
evidence for this attractive proposal is yet to emerge.
(f) Membrane translocation of tRNAs
tRNAs destined to be imported into mitochondria must be translocated across the outer and the inner mitochondrial membranes. From in vitro import experiments, we know that this process requires ATP and in most but not all cases the membrane potential. Moreover, protease treatment of mitochondria prevents tRNA import indicating that it is mediated by proteins. Information on the nature of these protein factors is available in three systems: S. cerevisiae, Leishmania and plants.
(i) Saccharomyces cerevisiae
After targeting to the mitochondrial surface, the tRNALys
is released from enolase and binds to the precursor of mitochondrial lysyl-tRNA synthetase (pre-MSK) (Tarassov et al. 1995b
). Subsequently, the folded tRNA is co-imported together with pre-MSK across the protein import channel (Entelis et al. 1998
). This may seem surprising as it is difficult to see how the interaction between pre-MSK and the imported tRNALys
can be maintained during mitochondrial protein import which requires unfolding of the transported protein. However, the evidence for the co-import model is very convincing. In vivo
and in vitro
import of the tRNALys
strictly depends on the precursor of pre-MSK. The involvement of Tom20 and Tim44, two components of the protein import machinery, has directly been shown in vivo
and in vitro
(Tarassov et al. 1995a
). Thus, in S. cerevisiae
, pre-MSK has two functions, it aminoacylates the mitochondria-encoded tRNALys
and it is responsible for import of the cytosolic tRNALys
. Recent experiments have shown that the two functions can be separated and are associated with distinct regions of the pre-MSK molecule (Kamenski et al. 2007
). The imported tRNALys
cannot be aminoacylated inside mitochondria. Yet, as it can only bind to pre-MSK when aminoacylated, it is imported as a functional tRNA that can take part in protein synthesis, even though recycling is not possible. Interestingly, the other tRNA that is imported into mitochondria of S. cerevisiae
, the tRNAGln
, is not co-imported with protein but by an as-yet-unknown mechanism (Rinehart et al. 2005
The tRNA import pathway in Leishmania tropica
has been elucidated in great detail by the group of S. Adhya. It can only be summarized here, for more information refer to Bhattacharyya & Adhya (2004)
, Mirande (2007)
and Adhya (2008)
. The inner membrane tRNA import machinery of L. tropica
appears to consist of an unconventional protein complex of approximately 580 kDa, termed tRNA import complex (RIC), that was initially characterized by affinity chromatography using an RNA oligonucleotide consisting of an in vitro
-defined tRNA-import signal (Goswami et al. 2006
). Mass spectrometry analysis revealed that RIC consists of 11 major subunits, eight of which are nucleus-encoded and three that are mitochondria-encoded (Mukherjee et al. 2007
). Six of the former are essential for tRNA import and four are identical to subunits of different respiratory complexes (iron sulphur protein and subunit 6b of complex III, cytochrome oxidase subunit 6 of complex IV and F1α subunit of complex V, the ATP synthase complex). Ablation of either of the six essential subunits by an unusual conditional antisense knockdown strategy reduced the level of mitochondrial tRNAs to zero within 24 h. Moreover, the functional RIC complex could be reconstituted into liposomes with recombinant subunits expressed in Escherichia coli
. Omission of any of the six essential factors abolished ATP-dependent import of tRNAs into liposomes. How tRNAs are transported across the outer membrane has not been addressed.
Characterization of the tRNA import machinery of the Leishmania
mitochondrial inner membrane is a truly amazing feat. However, a closer analysis of the published data raises a number of questions. Conditional ablation of specific leishmanial mRNAs has been tried by many groups using various strategies without success. Yet, in the L. tropica
strain used by the Adhya group, the antisense strategy seems to work extremely efficiently. Moreover, reconstitution of the RIC complex—consisting of six different proteins—into liposomes was done starting from denatured proteins that were eluted from sodium dodecyl sulphate (SDS) gels and refolded (Mukherjee et al. 2007
). While this might not be impossible, we are not aware of any precedent where such an approach has worked. Finally, L. tropica
and T. brucei
are closely related. We therefore would expect to find the same tRNA import machinery in both species. However, while the tRNA import machinery has not been identified in T. brucei
, at least three of the trypanosomal RIC orthologues are not expressed in the bloodstream stage of the parasite (Panigrahi et al. 2009
), even though it does import tRNAs. Thus, there is much left to be sorted out regarding the tRNA import machinery of trypanosomatids.
Recently, it was shown that antibodies against the voltage-dependent anion channel (VDAC), the metabolite transporter of the outer mitochondrial membrane, inhibited import of tRNAs into isolated plant mitochondria (Salinas et al. 2006
). Consistent with these results, recombinantly expressed VDAC was able to bind tRNAs. In vitro
import of tRNAs was also inhibited using antisera against Tom20 and Tom40, two conserved components of the mitochondrial outer membrane protein translocation machinery. However, the fact that in vitro
import does not require a mitochondrial precursor protein together with tRNA import competition studies showed that, unlike in yeast, tRNAs and proteins are not co-imported. Based on these results, it was suggested that VDAC may be a major component of the tRNA import channel whereas Tom20 and Tom40 may function as import receptors (Salinas et al. 2006
In summary, it appears clear that, in agreement with the postulated polyphyletic origin of mitochondrial tRNA import, the membrane translocation mechanism of tRNAs is not conserved between the different organisms. Moreover, there is no evidence for a dedicated tRNA import machinery in any system. Instead, its components, just as the ones required for tRNA targeting, appear to be housekeeping components performing a second function.
(g) Functional integration of imported tRNAs
Imported tRNAs are always of the eukaryotic type, whereas the mitochondrial translation system is of bacterial descent. Many tRNAs might in principle be functionally interchangeable between the cytosol and the mitochondria. However, for the initiator tRNAMet and for tRNAs that read codons that differ from the standard genetic code, this is not the case. Trypanosomatids and apicomplexans did not retain any mitochondrial tRNA genes. Their mitochondrial translation system therefore depends exclusively on imported eukaryotic-type tRNAs and as a consequence requires unique evolutionary adaptations. Some of these adaptations have been characterized in T. brucei and are discussed below.
Even though there is no bacterial-type initiator tRNAMet
in trypanosome mitochondria, translation initiation requires a formylated tRNAMet
. The tRNA that becomes formylated is a fraction of the imported eukaryotic-type elongator tRNAMet
. Formylation is catalysed by an unusual tRNAMet
-formyl-transferase that selectively formylates elongator-type tRNAMet
and therefore has a substrate specificity diametrically opposed to conventional formyl-transferases (Tan et al. 2002a
). Thus, in T. brucei
, the elongator tRNAMet
has three distinct functions that depend on its localization. In the cytosol, it functions as a conventional eukaryotic elongator tRNAMet
, whereas after import in the bacterial-type translation system of mitochondria it is used as both initiator and elongator tRNAMet
, depending on whether it is formylated or not (Martin 2002
). The formylated methionine on the imported elongator tRNAMet
, but no specific feature of the tRNA itself, is the main determinant that is recognized by an apparently conventional bacterial-type initiation factor 2 (Charrière et al. 2005
). Thus, the unusual formyl-transferase seems to be the only required adaptation allowing the use of elongator tRNAMet
in translation initiation.
In mitochondria of trypanosomatids, the stop codon UGA has been reassigned to tryptophane. The organellar tRNATrp therefore has to decode UGA in addition to the normal tryptophane codon UGG. It is not obvious how this can be achieved by an imported cytosolic tRNATrp that does not recognize the UGA stop codon.
Trypanosomatids solve this problem by a mitochondria-specific RNA editing event that converts the CCA anticodon of the imported tRNATrp
to UCA (Alfonzo et al. 1999
; Charrière et al. 2006
). This allows the tRNA to decode both UGG and UGA codons. However, the CCA anticodon is an identity determinant for the eukaryotic tryptophanyl-tRNA synthetase. Thus, unlike most other imported tRNAs of trypanosomes, the edited tRNATrp
in mitochondria cannot be charged by an aminoacyl-tRNA synthetase that is dually targeted to the cytosol and the mitochondrion. Instead, trypanosomatids evolved a highly diverged eukaryotic-type tryptophanyl-tRNA synthetase that is specific for mitochondria and that, unlike its cytosolic counterpart, can aminoacylate both edited and unedited tRNATrp
(Charrière et al. 2006
). For trypanosomatids loosing the mitochondrial tRNATrp
gene was therefore very costly as it required the evolution of a specific enzyme that edits the tRNATrp
as well as of a novel type of eukaryotic tryptophanyl-tRNA synthetase.
Many more adaptations of the mitochondrial translation system to imported tRNAs are likely to exist. Studies of how imported tRNAs are functionally integrated into the mitochondrial translation system have so far been restricted to trypanosomatids. It would be interesting to extend them to apicomplexans which are faced with the same problems. Apicomplexans belong to a different eukaryotic supergroup than trypansomatids and therefore may have found different solutions. We believe that investigating the consequences tRNA import imposes on mitochondrial translation is of great interest. Most trypanosomatids and apicomplexans are clinically important pathogens. Thus, the parasite-specific adapations of the mitochondrial translation system may offer novel drug targets. Moreover, exploring the limits of adaptation of a bacterial-type translation systems to eukaryotic components will help to reveal the fundamental requirements of translation.