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A mosaic of RNA binding and protein interaction motifs in a bifunctional mitochondrial tRNA import factor from Leishmania tropica.

Home P, Mukherjee S, Adhya S - Nucleic Acids Res. (2008)

Bottom Line: RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III).Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo.These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.

View Article: PubMed Central - PubMed

Affiliation: Genetic Engineering Laboratory, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India.

ABSTRACT
Proteins that participate in the import of cytosolic tRNAs into mitochondria have been identified in several eukaryotic species, but the details of their interactions with tRNA and other proteins are unknown. In the kinetoplastid protozoon Leishmania tropica, multiple proteins are organized into a functional import complex. RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III). We show that the N-terminal domain, unique to kinetoplastid protozoa, is structurally similar to the appended S15/NS1 RNA-binding domain of aminoacyl tRNA synthetases, with a helix-turn-helix motif. Structure-guided mutagenesis coupled with in vitro assays showed that helix alpha1 contacts tRNA whereas helix alpha2 targets the protein for assembly into the import complex. Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo. Moreover, a protein-interaction assay showed that the C-terminal domain makes allosteric contacts with import receptor RIC1 complexed with tRNA. These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.

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Effect of expressing RIC8A derivatives on tRNA import. (A) Nucleus-encoded mitochondrial tRNAs in normal (WT) or mutant RIC8A-expressing cells. Northern blot of mitochondrial RNA (5 × 106 cell-equivalents) probed with antisense oligonucleotides complementary to the indicated tRNAs: tR(X), tRNA specific for amino acid X (single-letter code). (B) Import of indicated 32P-labeled substrates, in the presence or absence of low-specific-activity effector, into liposomes reconstituted with extracts from RIC8A derivative-expressing cells (RNase protection assay).
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Figure 6: Effect of expressing RIC8A derivatives on tRNA import. (A) Nucleus-encoded mitochondrial tRNAs in normal (WT) or mutant RIC8A-expressing cells. Northern blot of mitochondrial RNA (5 × 106 cell-equivalents) probed with antisense oligonucleotides complementary to the indicated tRNAs: tR(X), tRNA specific for amino acid X (single-letter code). (B) Import of indicated 32P-labeled substrates, in the presence or absence of low-specific-activity effector, into liposomes reconstituted with extracts from RIC8A derivative-expressing cells (RNase protection assay).

Mentions: Northern blot analysis (Figure 6A) of nucleus-encoded mitochondrial tRNAs from normal or uninduced cells (WT) revealed differences in the steady-state levels of individual tRNAs (e.g. tRNAIle was significantly more abundant than tRNATyr); similar variations have also been observed in T. brucei mitochondria, and may reflect different import efficiencies (19). In H1-expressing cells the levels of some tRNAs, such as tRNAIle and tRNAVal, were significantly reduced or became undetectable, while those of other tRNAs, such as tRNATyr and tRNAArg, were elevated 2- to 2.5-fold (Figure 6A). These two groups of tRNAs were classified as type II and type I, respectively (12); thus, the import defect is typical of a nonfunctional type II receptor. This was verified by reconstituting liposomes with mitochondrial extracts from the overexpressing cells and assaying for in vitro import. Wild-type extracts induced import of the type I tRNATyr alone, but this import was inhibited by low concentrations of the type II effector tRNAIle; conversely, import of tRNAIle required the presence of type I effector (Figure 6B). In contrast, extracts from H1-expressing cells were incompetent in type II import even in the presence of type I tRNA, while there was no inhibition of type I tRNA import by type II effector (Figure 6B).Figure 6.


A mosaic of RNA binding and protein interaction motifs in a bifunctional mitochondrial tRNA import factor from Leishmania tropica.

Home P, Mukherjee S, Adhya S - Nucleic Acids Res. (2008)

Effect of expressing RIC8A derivatives on tRNA import. (A) Nucleus-encoded mitochondrial tRNAs in normal (WT) or mutant RIC8A-expressing cells. Northern blot of mitochondrial RNA (5 × 106 cell-equivalents) probed with antisense oligonucleotides complementary to the indicated tRNAs: tR(X), tRNA specific for amino acid X (single-letter code). (B) Import of indicated 32P-labeled substrates, in the presence or absence of low-specific-activity effector, into liposomes reconstituted with extracts from RIC8A derivative-expressing cells (RNase protection assay).
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Related In: Results  -  Collection

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Figure 6: Effect of expressing RIC8A derivatives on tRNA import. (A) Nucleus-encoded mitochondrial tRNAs in normal (WT) or mutant RIC8A-expressing cells. Northern blot of mitochondrial RNA (5 × 106 cell-equivalents) probed with antisense oligonucleotides complementary to the indicated tRNAs: tR(X), tRNA specific for amino acid X (single-letter code). (B) Import of indicated 32P-labeled substrates, in the presence or absence of low-specific-activity effector, into liposomes reconstituted with extracts from RIC8A derivative-expressing cells (RNase protection assay).
Mentions: Northern blot analysis (Figure 6A) of nucleus-encoded mitochondrial tRNAs from normal or uninduced cells (WT) revealed differences in the steady-state levels of individual tRNAs (e.g. tRNAIle was significantly more abundant than tRNATyr); similar variations have also been observed in T. brucei mitochondria, and may reflect different import efficiencies (19). In H1-expressing cells the levels of some tRNAs, such as tRNAIle and tRNAVal, were significantly reduced or became undetectable, while those of other tRNAs, such as tRNATyr and tRNAArg, were elevated 2- to 2.5-fold (Figure 6A). These two groups of tRNAs were classified as type II and type I, respectively (12); thus, the import defect is typical of a nonfunctional type II receptor. This was verified by reconstituting liposomes with mitochondrial extracts from the overexpressing cells and assaying for in vitro import. Wild-type extracts induced import of the type I tRNATyr alone, but this import was inhibited by low concentrations of the type II effector tRNAIle; conversely, import of tRNAIle required the presence of type I effector (Figure 6B). In contrast, extracts from H1-expressing cells were incompetent in type II import even in the presence of type I tRNA, while there was no inhibition of type I tRNA import by type II effector (Figure 6B).Figure 6.

Bottom Line: RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III).Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo.These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.

View Article: PubMed Central - PubMed

Affiliation: Genetic Engineering Laboratory, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India.

ABSTRACT
Proteins that participate in the import of cytosolic tRNAs into mitochondria have been identified in several eukaryotic species, but the details of their interactions with tRNA and other proteins are unknown. In the kinetoplastid protozoon Leishmania tropica, multiple proteins are organized into a functional import complex. RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III). We show that the N-terminal domain, unique to kinetoplastid protozoa, is structurally similar to the appended S15/NS1 RNA-binding domain of aminoacyl tRNA synthetases, with a helix-turn-helix motif. Structure-guided mutagenesis coupled with in vitro assays showed that helix alpha1 contacts tRNA whereas helix alpha2 targets the protein for assembly into the import complex. Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo. Moreover, a protein-interaction assay showed that the C-terminal domain makes allosteric contacts with import receptor RIC1 complexed with tRNA. These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.

Show MeSH
Related in: MedlinePlus