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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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Domain structure and organization in RIC8A. (A) Domains and subdomains: ss, mitochondrial targeting sequence; α1, α2, the two helices of the S15/NS1-like domain. Positions of various deletions (D) and point mutations (P) are shown. (B) Homology model of RIC8A N-terminal domain (right) templated on the hamster GluProRS repeat R1. Side chains of residues altered to alanine are indicated (mutant numbers in parenthesis). (C) Sequence alignment of the α1–α2 region of RIC8A with the appended S15/NS1 domain of eukaryotic aaRS. Different residue types are shaded differently: pink, basic; blue, acidic; green, hydrophobic. Residues involved in tRNA binding, or assembly, are indicated by + signs.
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Figure 1: Domain structure and organization in RIC8A. (A) Domains and subdomains: ss, mitochondrial targeting sequence; α1, α2, the two helices of the S15/NS1-like domain. Positions of various deletions (D) and point mutations (P) are shown. (B) Homology model of RIC8A N-terminal domain (right) templated on the hamster GluProRS repeat R1. Side chains of residues altered to alanine are indicated (mutant numbers in parenthesis). (C) Sequence alignment of the α1–α2 region of RIC8A with the appended S15/NS1 domain of eukaryotic aaRS. Different residue types are shaded differently: pink, basic; blue, acidic; green, hydrophobic. Residues involved in tRNA binding, or assembly, are indicated by + signs.

Mentions: The cloning of the RIC8A gene has been described (7). RIC8A fragments encompassing different structurally defined domains were PCR-amplified using appropriate sense and antisense primers (Table S1) from the parental clone of full-length gene, and inserted into the appropriate expression vector. The complete RIC8A gene includes a putative cleaved N-terminal mitochondrial targeting sequence [MTS; (8)]. Unidirectional deletions from the N terminus (mutants C, D1, D2 and D3; Figure 1A) lack the MTS; therefore, to express these proteins in Leishmania mitochondria, the PCR-amplified fragments were fused to the MTS (amino acid residues 1–30) of subunit RIC1 (7) in vector pUC(MTS-1) (Dhar,G. and Adhya,S. manuscript in preparation). The remaining clones (N, D4–D6, H1 and H2) contain the endogenous MTS of RIC8A. To construct internal deletions D4–D6, the corresponding amplified fragments were fused in-frame to the N-terminus of clone C (containing residues 82–202); this resulted in the creation of a 2-residue linker between the two fused domains. Internal deletions H1 and H2 were similarly constructed by fusing deletion fragments D6 and D5 with the N-terminus of fragments D2 and D3, respectively (Figure 1A). For bacterial expression, fragments were cloned downstream of the glutathione-S transferase (GST) gene of pGEX4T-1 (GE Life Sciences). For expression in Leishmania, the inserts were transferred in the sense orientation from pGEX4T-1 to expression vector pGET, between its HindIII and BamHI sites (7). Alanine-scanning point mutations P1–P7 were constructed using the wild-type RIC8A N-terminal domain (residues 1–82; derivative N in Figure 1A), cloned into the BamHI/EcoRI sites of pGEX4T-1, as template. Point mutagenesis was performed using PCR with mutagenic primers according to the method of Ito et al. (17). The mutant PCR-amplified fragment was cloned into pGEX4T-1. Details of mutagenesis protocols are provided in Supplementary Data. For expression in Leishmania, the relevant derivatives were excised from pGEX4T-1 or pUC19 backbone and inserted between the HindIII and BamHI sites of vector pGET, downstream of the inducible T7 RNA polymerase promoter.Figure 1.


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)

Domain structure and organization in RIC8A. (A) Domains and subdomains: ss, mitochondrial targeting sequence; α1, α2, the two helices of the S15/NS1-like domain. Positions of various deletions (D) and point mutations (P) are shown. (B) Homology model of RIC8A N-terminal domain (right) templated on the hamster GluProRS repeat R1. Side chains of residues altered to alanine are indicated (mutant numbers in parenthesis). (C) Sequence alignment of the α1–α2 region of RIC8A with the appended S15/NS1 domain of eukaryotic aaRS. Different residue types are shaded differently: pink, basic; blue, acidic; green, hydrophobic. Residues involved in tRNA binding, or assembly, are indicated by + signs.
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Figure 1: Domain structure and organization in RIC8A. (A) Domains and subdomains: ss, mitochondrial targeting sequence; α1, α2, the two helices of the S15/NS1-like domain. Positions of various deletions (D) and point mutations (P) are shown. (B) Homology model of RIC8A N-terminal domain (right) templated on the hamster GluProRS repeat R1. Side chains of residues altered to alanine are indicated (mutant numbers in parenthesis). (C) Sequence alignment of the α1–α2 region of RIC8A with the appended S15/NS1 domain of eukaryotic aaRS. Different residue types are shaded differently: pink, basic; blue, acidic; green, hydrophobic. Residues involved in tRNA binding, or assembly, are indicated by + signs.
Mentions: The cloning of the RIC8A gene has been described (7). RIC8A fragments encompassing different structurally defined domains were PCR-amplified using appropriate sense and antisense primers (Table S1) from the parental clone of full-length gene, and inserted into the appropriate expression vector. The complete RIC8A gene includes a putative cleaved N-terminal mitochondrial targeting sequence [MTS; (8)]. Unidirectional deletions from the N terminus (mutants C, D1, D2 and D3; Figure 1A) lack the MTS; therefore, to express these proteins in Leishmania mitochondria, the PCR-amplified fragments were fused to the MTS (amino acid residues 1–30) of subunit RIC1 (7) in vector pUC(MTS-1) (Dhar,G. and Adhya,S. manuscript in preparation). The remaining clones (N, D4–D6, H1 and H2) contain the endogenous MTS of RIC8A. To construct internal deletions D4–D6, the corresponding amplified fragments were fused in-frame to the N-terminus of clone C (containing residues 82–202); this resulted in the creation of a 2-residue linker between the two fused domains. Internal deletions H1 and H2 were similarly constructed by fusing deletion fragments D6 and D5 with the N-terminus of fragments D2 and D3, respectively (Figure 1A). For bacterial expression, fragments were cloned downstream of the glutathione-S transferase (GST) gene of pGEX4T-1 (GE Life Sciences). For expression in Leishmania, the inserts were transferred in the sense orientation from pGEX4T-1 to expression vector pGET, between its HindIII and BamHI sites (7). Alanine-scanning point mutations P1–P7 were constructed using the wild-type RIC8A N-terminal domain (residues 1–82; derivative N in Figure 1A), cloned into the BamHI/EcoRI sites of pGEX4T-1, as template. Point mutagenesis was performed using PCR with mutagenic primers according to the method of Ito et al. (17). The mutant PCR-amplified fragment was cloned into pGEX4T-1. Details of mutagenesis protocols are provided in Supplementary Data. For expression in Leishmania, the relevant derivatives were excised from pGEX4T-1 or pUC19 backbone and inserted between the HindIII and BamHI sites of vector pGET, downstream of the inducible T7 RNA polymerase promoter.Figure 1.

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