An RNA-binding Respiratory Component Mediates Import of Type II tRNAs into Leishmania Mitochondria

Abstract

Transport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned. The C-terminal region of RIC8A is homologous to subunit 6b of ubiquinol cytochrome c reductase (respiratory complex III), while the N-terminal region has intrinsic affinity for type II, but not for type I, tRNAs. RIC8A is shared by the import complex and complex III, indicating its bi-functionality, but is assembled differently in the two complexes. Knockdown of RIC8A in Leishmania lowered the mitochondrial content of type II tRNAs but raised that of type I tRNAs, with downstream effects on mitochondrial translation and respiration, and cell death. In RIC8A knockdown cells, a subcomplex was formed that interacted with type I tRNA, but the negative regulation by type II tRNA was lost. Mitochondrial extracts from these cells were defective for type II, but not type I, import; import and regulation were restored by purified RIC8A. These results provide evidence for the relevance of allosteric regulation in vivo and indicate that acquisition of new tRNA-binding domains by ancient respiratory components have played a key role in the evolution of mitochondrial tRNA import.

The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria

“…Whether this putative receptor belongs to the TOM complex or not is an interesting question to answer in the future. Furthermore, an RNA import complex, including two bifunctional respiratory proteins, isolated from L. tropica inner mitochondrial membrane is implicated in tRNA import (12,13). However, because of the conflicting data reported so far (see above), identification of import factors in other trypanosomatids might help in understanding the complexity of mitochondrial tRNA import.…”

“…A 15-kDa putative import tRNA receptor has been reported in L. tropica, but this protein remains to be identified (10). More recently, it has been shown in L. tropica that a multisubunit RNA import complex (RIC) located on the inner mitochondrial membrane is implicated in tRNA import (11) and two subunits, RIC1 (a structural homologue to the ␣ subunit of F1 ATP synthase) and RIC8 (an homologue to subunit 6b of ubiquinol cytochrome c reductase) were identified (12,13). However, considering the contradictory data obtained so far, a deeper understanding of the import factors involved in different trypanosomatids will be important in the future.…”

Structural and Functional Association of Trypanosoma brucei MIX Protein with Cytochrome c Oxidase Complex

“…It suggests the possibility that we are dealing with a large complex within the inner mt membrane possessing more than one function or that some of the protein components have dual functions and may be found in different complexes. This is not an unusual circumstance in trypanosomatids, as some of the components of respiratory complexes III, IV, and V have been recently identified as being a part of a complex responsible for tRNA transfer into L. tropica mitochondria (5,20). Additionally, in the MIX-interacting complex, proteins such as cyclophilin, mt carrier protein, a chaperone, and a protein similar to the human Sec63 protein may be involved in the assembly of the complex or in mt import.…”

Proton-guided movements of tRNA within the Leishmania mitochondrial RNA import complex

“…The largest subunit, RIC1, binds type I tRNAs (21) and is essential for the import of this subset in vitro (18) as well as in vivo (21). The other tRNA subset (type II) is recognized by RIC8A (22). Binding of type II tRNAs to RIC8A is positively regulated by the RIC1–tRNA complex, while that of type I tRNAs is inhibited by RIC8A complexed with type II tRNA (18,22).…”