Limits...
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.

Show MeSH
Effects of expressing RIC8A mutants on mitochondrial complexes of L. tropica. (A–D), Complexes in cells expressing H1, H2, C-terminal domain or N-terminal domain, respectively. Left to right: western blot of total mitochondrial protein resolved by Tricine–SDS–PAGE, and probed with polyclonal anti-RIC8A antibody; 35S-methionine-labeled protein from wild-type or H1-expressing cells immunoprecipitated with nonimmune or anti-RIC8A serum; BN–PAGE profiles (Coomassie stain and western blot) of mitochondrial complexes; and subunit profiles (SDS–PAGE) of RIC and complex III. Subunit numbering as in ref. 9. Sizes of the endogenous (WT), H1 and H2 are 202, 195 and 185 amino acid residues, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Effects of expressing RIC8A mutants on mitochondrial complexes of L. tropica. (A–D), Complexes in cells expressing H1, H2, C-terminal domain or N-terminal domain, respectively. Left to right: western blot of total mitochondrial protein resolved by Tricine–SDS–PAGE, and probed with polyclonal anti-RIC8A antibody; 35S-methionine-labeled protein from wild-type or H1-expressing cells immunoprecipitated with nonimmune or anti-RIC8A serum; BN–PAGE profiles (Coomassie stain and western blot) of mitochondrial complexes; and subunit profiles (SDS–PAGE) of RIC and complex III. Subunit numbering as in ref. 9. Sizes of the endogenous (WT), H1 and H2 are 202, 195 and 185 amino acid residues, respectively.

Mentions: Inner membrane macromolecular complexes in sodium dodecyl maltoside-solubilized mitochondrial extracts (∼100 μg protein) were resolved by Blue Native (BN) electrophoresis, as described (7), and visualized by Coomassie blue staining, or probed with specific antibodies by western blotting. For second-dimension analysis, individual bands from the BN gel were excised, denatured in situ, and the subunits separated by Tricine SDS 16% PAGE (18); this system is particularly suitable for resolving low-molecular-weight proteins e.g. between the wild-type RIC8A (202 residues), H1 (195 residues) and H2 (185 residues) (Figure 5).


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)

Effects of expressing RIC8A mutants on mitochondrial complexes of L. tropica. (A–D), Complexes in cells expressing H1, H2, C-terminal domain or N-terminal domain, respectively. Left to right: western blot of total mitochondrial protein resolved by Tricine–SDS–PAGE, and probed with polyclonal anti-RIC8A antibody; 35S-methionine-labeled protein from wild-type or H1-expressing cells immunoprecipitated with nonimmune or anti-RIC8A serum; BN–PAGE profiles (Coomassie stain and western blot) of mitochondrial complexes; and subunit profiles (SDS–PAGE) of RIC and complex III. Subunit numbering as in ref. 9. Sizes of the endogenous (WT), H1 and H2 are 202, 195 and 185 amino acid residues, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Effects of expressing RIC8A mutants on mitochondrial complexes of L. tropica. (A–D), Complexes in cells expressing H1, H2, C-terminal domain or N-terminal domain, respectively. Left to right: western blot of total mitochondrial protein resolved by Tricine–SDS–PAGE, and probed with polyclonal anti-RIC8A antibody; 35S-methionine-labeled protein from wild-type or H1-expressing cells immunoprecipitated with nonimmune or anti-RIC8A serum; BN–PAGE profiles (Coomassie stain and western blot) of mitochondrial complexes; and subunit profiles (SDS–PAGE) of RIC and complex III. Subunit numbering as in ref. 9. Sizes of the endogenous (WT), H1 and H2 are 202, 195 and 185 amino acid residues, respectively.
Mentions: Inner membrane macromolecular complexes in sodium dodecyl maltoside-solubilized mitochondrial extracts (∼100 μg protein) were resolved by Blue Native (BN) electrophoresis, as described (7), and visualized by Coomassie blue staining, or probed with specific antibodies by western blotting. For second-dimension analysis, individual bands from the BN gel were excised, denatured in situ, and the subunits separated by Tricine SDS 16% PAGE (18); this system is particularly suitable for resolving low-molecular-weight proteins e.g. between the wild-type RIC8A (202 residues), H1 (195 residues) and H2 (185 residues) (Figure 5).

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