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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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Assembly competence of RIC8A mutants in vitro. (A and C) BN PAGE of RIC8A-knockdown complex (KD) reconstituted with indicated mutant proteins. WT, complexes from uninduced cells, Coomassie stain. (B) Western blot of gel run in parallel to (A), probed with anti-RIC8A antiserum (1:50). The amount of protein loaded on this gel was about one-third that in (A). RKD, knockdown RIC subcomplex; VKD, complex V from KD cells; RRC, reconstituted RIC. (D) SDS–PAGE profile of RKD and RRC (H1) (RIC reconstituted with H1 protein), compared to wild-type RIC (R). RIC subunits are numbered as in ref. 9.
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Figure 3: Assembly competence of RIC8A mutants in vitro. (A and C) BN PAGE of RIC8A-knockdown complex (KD) reconstituted with indicated mutant proteins. WT, complexes from uninduced cells, Coomassie stain. (B) Western blot of gel run in parallel to (A), probed with anti-RIC8A antiserum (1:50). The amount of protein loaded on this gel was about one-third that in (A). RKD, knockdown RIC subcomplex; VKD, complex V from KD cells; RRC, reconstituted RIC. (D) SDS–PAGE profile of RKD and RRC (H1) (RIC reconstituted with H1 protein), compared to wild-type RIC (R). RIC subunits are numbered as in ref. 9.

Mentions: In normal L. tropica, four mitochondrial complexes—RIC, and respiratory complexes III, IV and V can be resolved by BN electrophoresis [(7); Figure 3]. As described recently (15), complexes III and IV were absent in RIC8A-knockdown cells; complex V was truncated (species labeled VKD; Figure 3A); and an assembly intermediate of RIC (RKD), lacking various subunits in addition to RIC8A [(15); Figure 3D], was formed (Figure 3A). Addition of purified deletion fragments D1, D2 and D4 of RIC8A to the reconstitution reaction resulted in the specific retardation of the RIC subcomplex to form RRC containing the added fragment (as shown by western blotting; Figure 3B), indicating this fragment to be competent for assembly. In contrast, deletions D3, D5 and D6 were not assembled. Considering the map positions of D1–D6 (Figure 1A), these results place the essential assembly motif between residues 46 and 71, roughly coinciding with the position of helix α2 (54–64). Moreover, mutant H1 (lacking residues 40–46) was assembly competent, and was detected in second-dimension gels of the reconstituted complex (Figure 3D), but H2 (lacking residues 50–66) or point mutants P6 (K57A) and P7 (R61A), both within helix α2, was not (Figure 3C). Thus, helix α2 appears to be critical for assembly of RIC8A into the import complex.Figure 3.


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)

Assembly competence of RIC8A mutants in vitro. (A and C) BN PAGE of RIC8A-knockdown complex (KD) reconstituted with indicated mutant proteins. WT, complexes from uninduced cells, Coomassie stain. (B) Western blot of gel run in parallel to (A), probed with anti-RIC8A antiserum (1:50). The amount of protein loaded on this gel was about one-third that in (A). RKD, knockdown RIC subcomplex; VKD, complex V from KD cells; RRC, reconstituted RIC. (D) SDS–PAGE profile of RKD and RRC (H1) (RIC reconstituted with H1 protein), compared to wild-type RIC (R). RIC subunits are numbered as in ref. 9.
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Related In: Results  -  Collection

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Figure 3: Assembly competence of RIC8A mutants in vitro. (A and C) BN PAGE of RIC8A-knockdown complex (KD) reconstituted with indicated mutant proteins. WT, complexes from uninduced cells, Coomassie stain. (B) Western blot of gel run in parallel to (A), probed with anti-RIC8A antiserum (1:50). The amount of protein loaded on this gel was about one-third that in (A). RKD, knockdown RIC subcomplex; VKD, complex V from KD cells; RRC, reconstituted RIC. (D) SDS–PAGE profile of RKD and RRC (H1) (RIC reconstituted with H1 protein), compared to wild-type RIC (R). RIC subunits are numbered as in ref. 9.
Mentions: In normal L. tropica, four mitochondrial complexes—RIC, and respiratory complexes III, IV and V can be resolved by BN electrophoresis [(7); Figure 3]. As described recently (15), complexes III and IV were absent in RIC8A-knockdown cells; complex V was truncated (species labeled VKD; Figure 3A); and an assembly intermediate of RIC (RKD), lacking various subunits in addition to RIC8A [(15); Figure 3D], was formed (Figure 3A). Addition of purified deletion fragments D1, D2 and D4 of RIC8A to the reconstitution reaction resulted in the specific retardation of the RIC subcomplex to form RRC containing the added fragment (as shown by western blotting; Figure 3B), indicating this fragment to be competent for assembly. In contrast, deletions D3, D5 and D6 were not assembled. Considering the map positions of D1–D6 (Figure 1A), these results place the essential assembly motif between residues 46 and 71, roughly coinciding with the position of helix α2 (54–64). Moreover, mutant H1 (lacking residues 40–46) was assembly competent, and was detected in second-dimension gels of the reconstituted complex (Figure 3D), but H2 (lacking residues 50–66) or point mutants P6 (K57A) and P7 (R61A), both within helix α2, was not (Figure 3C). Thus, helix α2 appears to be critical for assembly of RIC8A into the import complex.Figure 3.

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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