A bifunctional tRNA import receptor from Leishmania mitochondria

Abstract

In kinetoplastid protozoa, import of cytosolic tRNAs into mitochondria occurs through tRNAs interacting with membrane-bound proteins, the identities of which are unknown. The inner membrane RNA import complex of Leishmania tropica contains multiple proteins and is active for import in vitro. RIC1, the largest subunit of this complex, is structurally homologous to the conserved alpha subunit of F1 ATP synthase. The RIC1 gene complemented an atpA mutation in Escherichia coli. Antisense-mediated knockdown of RIC1/F1alpha in Leishmania resulted in depletion of several mitochondrial tRNAs belonging to distinct subsets (types I and II) that interact cooperatively or antagonistically within the import complex. The knockdown-induced defect in import of type I tRNAs was rectified in a reconstituted system by purified RIC1/F1alpha alone, but recovery of type II tRNA import additionally required a type I tRNA. RIC1/F1alpha formed stable complexes with type I, but not type II, tRNAs through the cooperation of its nucleotide binding and C-terminal domains. Thus, RIC1/F1alpha is a type I tRNA import receptor. As expected of a bifunctional protein, RIC1/F1alpha is shared by both the import complex and by respiratory complex V. Alternative use of ancient respiratory proteins may have been an important step in the evolution of tRNA import.

Fig. 1.

Structural and functional homology of RIC1 with ATP synthase subunit α. (A) RIC1 and AtpA conserved domain (COG0056) of F1 ATP synthase subunit α, showing N-terminal (N), ATP binding (AB), and C-terminal (C) domains. (B) Homology model of RIC1 secondary structure backbone compared to that of bovine F1 ATPase α subunit. (C) Complementation of E. coli AtpA mutation by Leishmania RIC1. Serial dilutions (from 10⁵ to 10 cells, left to right) of strain AN120 (AtpA), strain AN180 (the isogenic wild-type) or AN120 transfected with RIC1[39–574] or RIC1[109–574] were spotted on minimal media containing either glucose or succinate as carbon source. (D) Immunoblot of protein (100 μg) of total, mitochondrial (mito), or cytosolic (cyto) fractions of L. tropica promastigotes using anti-RIC1 serum as probe.

Fig. 2.

Antisense-mediated knockdown of RIC1/F1α. (A) Schematic diagram of the expression cassette of knockdown vector pGET(AS)RIC1/F1α. rDNA, ribosomal DNA spacer region; T7 pro, T7 RNA polymerase promoter; Tet O, tetracycline operator; RIC1/F1α AS, RIC1[109–574] in antisense orientation; TUB, β tubulin; ALD, aldehyde dehydrogenase; ACT SAS and ACT 3′ UTR, 5′ splice acceptor site and 3′ UTR, respectively, of actin gene; BLE, bleomycin resistance gene; Term, double T7 terminator. Arrows indicate transcription start sites. (B) RT-PCR assay of RIC1/F1α antisense RNA (Left), RIC1/F1α mRNA (Center), or RIC8A mRNA (Right) in pGET(AS)RIC1-transformed L. tropica 13–90 (10⁵ cell equivalent) grown for 24 h in absence (−) or presence (+) of tetracycline. (C) (Left) Total Coomassie-stained protein from 10⁷ cells and immunoblot with anti- RIC1/F1α (Upper) or anti-RIC8A (Lower). (Right) Mitochondrial protein blots probed with antibodies against RIC1/F1α, F1 β subunit, or complex II Fe-S protein. At the far right, mitochondrial fractions were treated with trypsin (20 μg/ml, 20 min, 4°C) in absence or presence of 0.5% Triton X-100. (D) Growth curves at 22°C. Open triangles, pGET-transformed L. tropica, −tet; filled triangles, pGET-transformed, +tet; squares, pGET(AS)RIC1-transformed, −tet; circles, pGET(AS)RIC1-transformed, +tet. (E) Total end-labeled mitochondrial tRNA in normal or RIC1-knockdown cells. (F) RT-PCR assay of tRNA^Tyr (tRY), tRNA^Trp (tRW), tRNA^Arg₁ (tRR), tRNA^Ile (tRI), tRNA^Val (tRV), and tRNA^Met-e (tRM-e) in cytosolic (C) or mitochondrial (M) RNA from 10³ RIC1/F1α (Left) or F1 ATPase β subunit (Right) knockdown cells. (G) [³⁵S]Methionine-labeled proteins from uninduced or tet-induced pGET(AS)RIC1-transformed cells cultured in absence (Left) or presence (Right) of cycloheximide. Chloramphenicol was additionally present in the cycloheximide-treated cultures as indicated, and the mitochondrial fractions were isolated (Right).

Fig. 3.

Requirement of RIC1/F1α for import of type I and II tRNAs. (A) Regulation of import by type I and II effector tRNAs. High specific activity import substrates (5 nM) were incubated in the absence or presence of low specific activity tRNA^Tyr or tRNA^Ile effector (0.5 nM) and assayed for import into RIC-reconstituted proteoliposomes. (B) Import reconstitution assay. Mitochondrial extract from uninduced (WT) or 24-h tet-induced (KD) pGET(AS)RIC1-transformed cells was incubated with or without recombinant RIC1/F1α or with BSA (BSA), in presence of liposomes, which were then assayed for import of tRNA^Tyr. (C) Import of the indicated tRNAs (5 nM) in absence or presence of low specific activity tRNA^Tyr effector (0.5 nM), into liposomes reconstituted with knockdown extracts in the absence or presence of recombinant RIC1/F1α.

Fig. 4.

Binding of tRNAs to RIC1/F1α. (A) EMSA of ³²P-labeled tRNAs (10 fmol) incubated with or without recombinant RIC1/F1α (50 fmol) in 5 μl. (B) Titration of tRNA^Tyr (10 fmol) with indicated amounts of RIC1/F1α (10-μl reactions). (C) (Left) Truncated versions of the RIC1/F1α gene expressed in E. coli stained with Coomassie blue. (Right) EMSA of ³²P-labeled tRNA^Tyr incubated with subfragments of RIC1/F1α alone or in combination (conditions as in A).

Fig. 5.

Relationship between the RNA import complex and respiratory complexes of Leishmania. (A) L. tropica mitochondrial protein (200 μg) run in 6% BN PAGE along with molecular weight markers (M). (B) The largest BN PAGE band was excised from BN gel, denatured and run on SDS/12% PAGE along with affinity purified RIC (AF). RIC band numbers are indicated at right. (C) Immunoblot of mitochondrial complexes probed with anti-RIC1/F1α or F1β antibody. (D) Second-dimension analysis of RIC and complex V, showing Coomassie-stained subunits (Left), anti-RIC1/F1α immunoblot (Center), and Northwestern blot probed with ³²P-labeled tRNA^Tyr (Right). (E) (Left) Leishmania and human mitochondrial complexes resolved by BN PAGE. (Right) Northwestern blot probed with ³²P-labeled tRNA^Tyr. (F) Dodecyl maltoside extracted mitochondrial complexes were incubated with ³²P- and BrdU-labeled tRNA^Tyr followed by UV-cross-linking, BN PAGE, transfer to nitrocellulose membrane, and autoradiography. Coomassie-stained complexes are shown at left.