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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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Schematic representation of the proposed role of the interaction motifs of RIC8A in assembly and import. Motifs 1–4 in RIC8A are lettered in blue. tRNA-I and tRNA-II are types I and II tRNAs, respectively. For details, see text.
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Figure 8: Schematic representation of the proposed role of the interaction motifs of RIC8A in assembly and import. Motifs 1–4 in RIC8A are lettered in blue. tRNA-I and tRNA-II are types I and II tRNAs, respectively. For details, see text.

Mentions: Thus, RIC8A/UCR6b has at least four functional motifs within the overall two-domain structure: one each for type II tRNA binding (helix α1 of the N-terminal domain), for stable interaction with RIC9 (possibly helix α2), for allosteric interaction with RIC1 (in the C-terminal domain), and for interaction with complex III subunits (designated as motifs 1–4, respectively, Figure 8). The assembly and function of RIC8A can then be visualized as a series of conformational changes resulting in stable or transient protein–protein or protein–RNA interactions. Free RIC8A is presumed to be an equilibrium mixture of two mutually exclusive conformations with active motif 2 or 4; thus it can assemble in either the import complex or complex III, respectively. In the RIC-assembled protein, the tRNA-binding site (motif 1) remains closed. Activation of this site is contingent upon the binding of a type I tRNA to receptor RIC1, with conformational changes in the latter, leading to its interaction with motif 3 of RIC8A, and transmission of the structural perturbation to, and activation of, motif 1, and binding of type II tRNA. Subsequent to this, as detailed in our recent study, the tRNA is transferred to RIC9 and thence through the membrane, in well-defined steps driven by an ATP-generated proton-motive force (15).Figure 8.


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)

Schematic representation of the proposed role of the interaction motifs of RIC8A in assembly and import. Motifs 1–4 in RIC8A are lettered in blue. tRNA-I and tRNA-II are types I and II tRNAs, respectively. For details, see text.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553583&req=5

Figure 8: Schematic representation of the proposed role of the interaction motifs of RIC8A in assembly and import. Motifs 1–4 in RIC8A are lettered in blue. tRNA-I and tRNA-II are types I and II tRNAs, respectively. For details, see text.
Mentions: Thus, RIC8A/UCR6b has at least four functional motifs within the overall two-domain structure: one each for type II tRNA binding (helix α1 of the N-terminal domain), for stable interaction with RIC9 (possibly helix α2), for allosteric interaction with RIC1 (in the C-terminal domain), and for interaction with complex III subunits (designated as motifs 1–4, respectively, Figure 8). The assembly and function of RIC8A can then be visualized as a series of conformational changes resulting in stable or transient protein–protein or protein–RNA interactions. Free RIC8A is presumed to be an equilibrium mixture of two mutually exclusive conformations with active motif 2 or 4; thus it can assemble in either the import complex or complex III, respectively. In the RIC-assembled protein, the tRNA-binding site (motif 1) remains closed. Activation of this site is contingent upon the binding of a type I tRNA to receptor RIC1, with conformational changes in the latter, leading to its interaction with motif 3 of RIC8A, and transmission of the structural perturbation to, and activation of, motif 1, and binding of type II tRNA. Subsequent to this, as detailed in our recent study, the tRNA is transferred to RIC9 and thence through the membrane, in well-defined steps driven by an ATP-generated proton-motive force (15).Figure 8.

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