Two ancient membrane pores mediate mitochondrial-nucleus membrane contact sites.

Coordination between nucleus and mitochondria is essential for cell survival, and thus numerous communication routes have been established between these two organelles over eukaryotic cell evolution. One route for organelle communication is via membrane contact sites, functional appositions formed by molecular tethers. We describe a novel nuclear-mitochondrial membrane contact site in the protozoan Toxoplasma gondii. We have identified specific contacts occurring at the nuclear pore and demonstrated an interaction between components of the nuclear pore and the mitochondrial protein translocon, highlighting them as molecular tethers. Genetic disruption of the nuclear pore or the TOM translocon components, TgNup503 or TgTom40, respectively, result in contact site reduction, supporting their potential involvement in this tether. TgNup503 depletion further leads to specific mitochondrial morphology and functional defects, supporting a role for nuclear-mitochondrial contacts in mediating their communication. The discovery of a contact formed through interaction between two ancient mitochondrial and nuclear complexes sets the ground for better understanding of mitochondrial-nuclear crosstalk in eukaryotes.

Nuclear Interactions: A Spotlight on Nuclear Mitochondrial Membrane Contact Sites.

Membrane contact sites (MCS) are critical for cellular functions of eukaryotes, as they enable communication and exchange between organelles. Research over the last decade unravelled the function and composition of MCS between a variety of organelles including mitochondria, ER, plasma membrane, lysosomes, lipid droplets, peroxisome and endosome, to name a few. In fact, MCS are found between any pair of organelles studied to date, with common functions including lipid exchange, calcium signalling and organelle positioning in the cell. Work in the past year has started addressing the composition and function of nuclear-mitochondrial MCS. Tether components mediating these contacts in yeast have been identified via comprehensive phenotypic screens, which also revealed a possible link between this contact and phosphatidylcholine metabolism. In human cells, and in the protozoan parasites causing malaria, proximity between these organelles is proposed to promote cell survival via a mitochondrial retrograde response. These pioneering studies should inspire the field to explore what cellular processes depend on the exchange between the nucleus and the mitochondrion, given that they play such central roles in cell biology.

Identification and Validation of Toxoplasma gondii Mitoribosomal Large Subunit Components.

Mitochondrial ribosomes are fundamental to mitochondrial function, and thus survival, of nearly all eukaryotes. Despite their common ancestry, mitoribosomes have evolved divergent features in different eukaryotic lineages. In apicomplexans, the mitochondrial rRNA is extremely fragmented raising questions about its evolution, protein composition and structure. Apicomplexan mitochondrial translation and the mitoribosomes are essential in all parasites and life stages studied, highlighting mitoribosomes as a promising target for drugs. Still, the apicomplexan mitoribosome is understudied, with one of the obstacles being that its composition is unknown. Here, to facilitate the study of apicomplexan mitoribosomes, we identified and validated components of the mitoribosomal large subunit in the model apicomplexan Toxoplasma gondii.

tRNA import across the mitochondrial inner membrane in T. brucei requires TIM subunits but is independent of protein import.

Mitochondrial tRNA import is widespread, but mechanistic insights of how tRNAs are translocated across mitochondrial membranes remain scarce. The parasitic protozoan T. brucei lacks mitochondrial tRNA genes. Consequently, it imports all organellar tRNAs from the cytosol. Here we investigated the connection between tRNA and protein translocation across the mitochondrial inner membrane. Trypanosomes have a single inner membrane protein translocase that consists of three heterooligomeric submodules, which all are required for import of matrix proteins. In vivo depletion of individual submodules shows that surprisingly only the integral membrane core module, including the protein import pore, but not the presequence-associated import motor are required for mitochondrial tRNA import. Thus we could uncouple import of matrix proteins from import of tRNAs even though both substrates are imported into the same mitochondrial subcompartment. This is reminiscent to the outer membrane where the main protein translocase but not on-going protein translocation is required for tRNA import. We also show that import of tRNAs across the outer and inner membranes are coupled to each other. Taken together, these data support the 'alternate import model', which states that tRNA and protein import while mechanistically independent use the same translocation pores but not at the same time.

The single CCA-adding enzyme of T. brucei has distinct functions in the cytosol and in mitochondria.

tRNAs universally carry a CCA nucleotide triplet at their 3'-ends. In eukaryotes, the CCA is added post-transcriptionally by the CCA-adding enzyme (CAE). The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes and therefore imports all of its tRNAs from the cytosol. This has generated interest in the tRNA modifications and their distribution in this organism, including how CCA is added to tRNAs. Here, using a BLAST search for genes encoding putative CAE proteins in T. brucei, we identified a single ORF, Tb927.9.8780, as a potential candidate. Knockdown of this putative protein, termed TbCAE, resulted in the accumulation of truncated tRNAs, abolished translation, and inhibited both total and mitochondrial CCA-adding activities, indicating that TbCAE is located both in the cytosol and mitochondrion. However, mitochondrially localized tRNAs were much less affected by the TbCAE ablation than the other tRNAs. Complementation assays revealed that the N-terminal 10 amino acids of TbCAE are dispensable for its activity and mitochondrial localization and that deletion of 10 further amino acids abolishes both. A growth arrest caused by the TbCAE knockdown was rescued by the expression of the cytosolic isoform of yeast CAE, even though it was not imported into mitochondria. This finding indicated that the yeast enzyme complements the essential function of TbCAE by adding CCA to the primary tRNA transcripts. Of note, ablation of the mitochondrial TbCAE activity, which likely has a repair function, only marginally affected growth.

Inducible orthogonal aminoacylation demonstrates that charging is required for mitochondrial tRNA import in Trypanosoma brucei.

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs have emerged as powerful means of site-specifically introducing non-standard amino acids into proteins in vivo. Using amino acids with crosslinking moieties this method allows the identification of transient protein-protein interactions. Here we have introduced a previously characterized evolved tyrosyl-tRNA synthetase/suppressor tRNATyr pair from E. coli into the parasitic protozoan Trypanosoma brucei. Upon addition of a suitable non-standard amino acid the suppressor tRNATyr was charged and allowed translation of a green fluorescent protein whose gene contained a nonsense mutation. - T. brucei is unusual in that its mitochondrion lacks tRNA genes indicating that all its organellar tRNAs are imported from the cytosol. Expression of the bacterial tyrosyl-tRNA synthetase in our system is tetracycline-inducible. We have therefore used it to demonstrate that cytosolic aminoacylation of the suppressor tRNATyr induces its import into the mitochondrion.

tRNA Biology in Trypanosomes.

Besides their medical importance, the parasitic protozoan Trypanosoma brucei and its relatives are experimentally highly accessible model systems for many cell biological processes. Trypanosomes are phylogenetically essentially unrelated to the popular model eukaryotes, such as yeast and animals, and thus show several unique features, many of which are connected to RNA. Here we review the tRNA biology of trypanosomes. Even though tRNAs were already discovered 60 years ago, owing to current technological advances in the field, research on tRNA biology has seen a Renaissance in recent years. First we discuss the extensive mitochondrial tRNA import process and the consequences it has for the parasite. Next we focus on trypanosomal aminoacyl-tRNA synthetases, some of which may be exploited as drug targets. Furthermore, we summarize what is known about trypanosomal tRNA modifications in both the cytosol and the mitochondrion. Finally, we provide an overview on the emerging field of tRNA-derived fragments and their possible function as translation regulators.