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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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Growth curves of uninduced (squares) or tetracycline-induced (triangles) promastigotes transformed with RIC8A derivatives. (A–D) Deletion mutants H1, H2, C-terminal and N-terminal domain, respectively.
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Figure 4: Growth curves of uninduced (squares) or tetracycline-induced (triangles) promastigotes transformed with RIC8A derivatives. (A–D) Deletion mutants H1, H2, C-terminal and N-terminal domain, respectively.

Mentions: Expression of H1 in L. tropica resulted in growth arrest after ∼72 h of tetracycline induction (Figure 4A) and reduction of O2 uptake (Table S2). In mitochondria from cells harvested at the earliest point of growth arrest (72 h), the normal profile of 3 OX PHOS complexes and RIC was replaced by one resembling the knockdown profile, i.e. absence of complexes III and IV, and replacement of RIC and complex V by two subcomplexes RH1 and VH1, respectively (Figure 5A). Under similar conditions of overexpression, the half-life of complex V is ∼12 h (Dhar,G. and Adhya,S. unpublished data), accounting for the near-complete disappearance of this complex by the time of growth arrest. Second-dimension analysis showed that the ectopic H1 replaced endogenous RIC8A in the RIC subcomplex (Figure 5A), and is thus competent for assembly.Figure 4.


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)

Growth curves of uninduced (squares) or tetracycline-induced (triangles) promastigotes transformed with RIC8A derivatives. (A–D) Deletion mutants H1, H2, C-terminal and N-terminal domain, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Growth curves of uninduced (squares) or tetracycline-induced (triangles) promastigotes transformed with RIC8A derivatives. (A–D) Deletion mutants H1, H2, C-terminal and N-terminal domain, respectively.
Mentions: Expression of H1 in L. tropica resulted in growth arrest after ∼72 h of tetracycline induction (Figure 4A) and reduction of O2 uptake (Table S2). In mitochondria from cells harvested at the earliest point of growth arrest (72 h), the normal profile of 3 OX PHOS complexes and RIC was replaced by one resembling the knockdown profile, i.e. absence of complexes III and IV, and replacement of RIC and complex V by two subcomplexes RH1 and VH1, respectively (Figure 5A). Under similar conditions of overexpression, the half-life of complex V is ∼12 h (Dhar,G. and Adhya,S. unpublished data), accounting for the near-complete disappearance of this complex by the time of growth arrest. Second-dimension analysis showed that the ectopic H1 replaced endogenous RIC8A in the RIC subcomplex (Figure 5A), and is thus competent for assembly.Figure 4.

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