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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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Interaction of the RIC8A C-terminal domain with RIC1. Recombinant proteins and 32P-labeled tRNATyr were incubated in the indicated combinations and the products separated by native gel electrophoresis. (A) Coomassie stain. (B and C) parallel western blots probed with anti-RIC1 or anti-RIC8A antibody, respectively. (D) Autoradiogram.
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Figure 7: Interaction of the RIC8A C-terminal domain with RIC1. Recombinant proteins and 32P-labeled tRNATyr were incubated in the indicated combinations and the products separated by native gel electrophoresis. (A) Coomassie stain. (B and C) parallel western blots probed with anti-RIC1 or anti-RIC8A antibody, respectively. (D) Autoradiogram.

Mentions: To directly observe the interaction between these two receptors, recombinant RIC1 was incubated with either the expressed N- or C-terminal domain of RIC8A in the absence or presence of the 32P-labeled type I tRNATyr. The products were resolved by native gel electrophoresis, and the components of each complex were identified by western blotting and autoradiography. In presence of tRNATyr, RIC1 formed a binary complex tRY-1 (Figure 7, lane 1). Neither the N- nor the C-terminal domain reacted with the type I tRNA, as expected, since the former domain has binding specificity for type II tRNAs, while the latter does not have an RNA-binding site (Figure 7, lanes 2 and 3). Addition of the N-terminal domain to the binary complex did not affect the latter (Figure 7, lane 4), and this domain did not interact with RIC1 in the absence of tRNATyr (Figure 7, lane 6). In the absence of tRNATyr, the C-terminal domain failed to bind RIC1 (Figure 4, lane 7). However, in presence of the tRNA, a ternary complex containing it, as well as RIC1 and the C-terminal domain, was formed (Figure 7, lane 5). Thus, RIC8A is allosterically activated by interaction of its C-terminal domain with the tRNA–RIC1 complex.Figure 7.


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)

Interaction of the RIC8A C-terminal domain with RIC1. Recombinant proteins and 32P-labeled tRNATyr were incubated in the indicated combinations and the products separated by native gel electrophoresis. (A) Coomassie stain. (B and C) parallel western blots probed with anti-RIC1 or anti-RIC8A antibody, respectively. (D) Autoradiogram.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2553583&req=5

Figure 7: Interaction of the RIC8A C-terminal domain with RIC1. Recombinant proteins and 32P-labeled tRNATyr were incubated in the indicated combinations and the products separated by native gel electrophoresis. (A) Coomassie stain. (B and C) parallel western blots probed with anti-RIC1 or anti-RIC8A antibody, respectively. (D) Autoradiogram.
Mentions: To directly observe the interaction between these two receptors, recombinant RIC1 was incubated with either the expressed N- or C-terminal domain of RIC8A in the absence or presence of the 32P-labeled type I tRNATyr. The products were resolved by native gel electrophoresis, and the components of each complex were identified by western blotting and autoradiography. In presence of tRNATyr, RIC1 formed a binary complex tRY-1 (Figure 7, lane 1). Neither the N- nor the C-terminal domain reacted with the type I tRNA, as expected, since the former domain has binding specificity for type II tRNAs, while the latter does not have an RNA-binding site (Figure 7, lanes 2 and 3). Addition of the N-terminal domain to the binary complex did not affect the latter (Figure 7, lane 4), and this domain did not interact with RIC1 in the absence of tRNATyr (Figure 7, lane 6). In the absence of tRNATyr, the C-terminal domain failed to bind RIC1 (Figure 4, lane 7). However, in presence of the tRNA, a ternary complex containing it, as well as RIC1 and the C-terminal domain, was formed (Figure 7, lane 5). Thus, RIC8A is allosterically activated by interaction of its C-terminal domain with the tRNA–RIC1 complex.Figure 7.

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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