Leishmania mitochondrial tRNA importers

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The International Journal of Biochemistry & Cell Biology 40 (2008) 2681–2685

Molecules in focus

Leishmania mitochondrial tRNA importers Samit Adhya ∗ Genetic Engineering Laboratory, Division of Molecular and Human Genetics, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700032, India Received 5 October 2007; received in revised form 15 October 2007; accepted 16 October 2007 Available online 20 November 2007

Abstract The RNA Import Complex (RIC) is a multi-subunit protein complex from the mitochondria of the kinetoplastid protozoon Leishmania tropica that induces transport of tRNA across natural and artificial membranes. Leishmania, Trypanosoma and related genera of the order Kinetoplastidae are early diverging, atypical eukaryotes with unique RNA metabolic pathways, including the import of nucleus-encoded tRNAs into the mitochondrion to complement the deletion of all organelle-encoded tRNA genes. Biochemical and genetic studies of RIC are contributing to greater understanding of the mechanism of import. Additionally, RIC was shown to act as an efficient delivery vehicle for tRNA and other small RNAs into mitochondria within intact mammalian cells, indicating its applicability to the management of diseases caused by mitochondrial mutations. © 2007 Elsevier Ltd. All rights reserved. Keywords: tRNA; Import; Mitochondria; Complex; Leishmania

1. Introduction Mitochondrial tRNA import occurs in a large number of species, including protozoa (Leishmania, Trypanosoma spp.), fungi (yeast), higher plants and marsupials (reviewed in Bhattacharyya & Adhya, 2004a; Mirande, 2007). Early in vitro and in vivo studies indicated differences in the transport mechanism in different systems. Hence, in yeast, soluble tRNA carriers are required, whereas plant and Leishmania mitochondria employ membrane-bound receptors. When a detergent extract of L. tropica inner mitochondrial membranes was chromatographed on a column of an immobilized RNA oligonucleotide containing a tRNA import signal, a large multi-protein aggregate (RIC) was retained, and could be eluted with high salt concentrations (Bhattacharyya ∗

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et al., 2003). RIC was functional for import of tRNAs into phospholipid vesicles in an ATP-dependent manner, and is currently the only mitochondrial import complex described in any system. This initial observation paved the way for the biochemical identification of the subunits by mass spectrometry, cloning of the candidate genes mined from the L. major genome sequence database, and confirmation by western blotting and gene knockdown experiments. 2. Molecular definition of RIC RIC can be obtained by several methods. In addition to the affinity procedure referred to above, RIC is resolved from other mitochondrial complexes by native gel electrophoresis (Goswami et al., 2006). Functional complexes can also be reconstituted from pure recombinant subunits (Mukherjee, Basu, Home, Dhar, & Adhya, 2007). RIC is composed of 11 major subunits, RIC1, 2, 3, 4A, 4B, 5-7, 8A, 8B and 9, of molecular mass 62–19 kDa

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Fig. 1. Working model of the Leishmania RNA Import Complex. The various subunits are numbered as in Mukherjee et al., 2007. The model is based on subunit contacts as determined by the assembly map and direct interaction assays (Mukherjee and Adhya, unpublished results). RIC1 and RIC8A are the two receptors that initially bind tRNA. Trimeric RIC6 and RIC9 form the translocation pore. RIC4A and RIC8B anchor the complex to the membrane. Membrane-embedded mitochondrion-encoded subunits 2 (dimeric), 4B (sub-stoichiometric) and 7 interact with RIC4A. The dispensable subunits RIC3 and RIC5 are assembled peripherally.

– in a defined stoichiometry adding up to a total mass of ∼580 kDa. Electron microscopy of purified preparations revealed globular particles of diameter ∼130 nm, some of which have fluid-filled cavities of ∼4.5 nm that could represent pores (Bhattacharyya et al., 2003). The detailed structure of the complex remains to be determined. The working model (Fig. 1) is based on our current knowledge of its subunits, their order of assembly and their known or surmised functions. Of the eight nucleus-encoded subunits, six (subunits 1, 4A, 6, 8A, 8B and 9) are essential for import (Table 1), as indicated by knockdown and in vitro reconstitution experiments (Mukherjee et al., 2007). RIC3 is a non-essential subunit encoded by one member of a multigene family encoding the M16 metalloproteinases, which includes the mitochondrial processing peptidase (MPP) and the core protein subunits 1 and 2 of complex III. Another dispensable component is RIC5, a trypanosomatid-specific protein. RIC also contains three mitochondrion-encoded subunits 2, 4B and 7. These proteins are highly hydrophobic, with predicted transmembrane ␣ helices; preliminary protein interaction assays suggest that they assemble around RIC4A in the membrane (S. Mukherjee and S. Adhya, unpublished data). They do not appear to be essential for tRNA import; however, a structural or regulatory role cannot be excluded. To date, about a dozen tRNA import factors have been identified in different organisms (Table 1). Strikingly, all except two of these proteins have established functions unrelated to tRNA import. In Leishmania, four of the

six essential RIC subunits are identical to specific components of complexes of oxidative phosphorylation (OX PHOS). In plants, preprotein import receptors TOM20 and TOM40 apparently function as membrane-bound tRNA receptors, while the Voltage-Dependent Anion Channel (VDAC) participates in tRNA translocation (Salinas et al., 2006). The outer and inner membrane preprotein import machineries (TOM and TIM complexes respectively) of yeast are also involved in tRNA translocation (Tarassov, Entelis, & Martin, 1995a). In this latter system, an aminoacyl tRNA synthetase and enolase, a glycolytic enzyme, act as soluble carriers to target the tRNA to the mitochondrial translocon (Tarassov, Entelis, & Martin, 1995b; Entelis et al., 2006). Mechanistically, the plant and protozoal systems are similar to each other, yet the factors are genetically unrelated. Thus, convergent evolution appears to have occurred in these systems, with conserved components of different ancient metabolic pathways being recruited to tRNA import. The coming together of multiple bifunctional (‘moonlighting’; Jeffery, 2003) in a single functional complex (RIC) is a unique example of evolutionary convergence. 3. Synthesis and assembly Six of the nine subunits of RIC are nucleus-encoded, and therefore synthesized on cytosolic ribosomes before being imported into mitochondria. The remaining three (RIC2, RIC4B and RIC7) are translated on mitochondrial ribosomes (Mukherjee et al., 2007). Assembly of the complex occurs in the organelle by ordered addition of nucleus-encoded subunits, as indicated by the isolation of assembly intermediates from cells knocked down for individual subunits (S. Mukherjee & S. Adhya, unpublished data). How the syntheses of the various subunits are coordinated remains a mystery. 4. Functional aspects The native complex, isolated by affinity chromatography, or reconstituted from recombinant subunits, is active for transport of tRNA across biological or artificial membranes. Additionally, various steps in the import process (Fig. 2) could be demonstrated using phospholipid vesicles reconstituted with the purified complex. 4.1. Specific tRNA binding and allosteric regulation Experiments with intact mitochondria or transiently transfected cells indicated the presence of at least two types of importable tRNA: type I tRNAs that

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Table 1 Identified tRNA import factors in different species Organism

Name

Identity

Function

References

Budding yeast

preMSK1p

Soluble tRNA carrier

Tarassov et al. (1995a)

Soluble tRNA carrier Membrane-bound GIP component (TOM complex) Inner membrane GIP component (TIM complex) Membrane-bound tRNA receptor (TOM complex) Membrane-bound tRNA receptor (TOM complex) tRNA binding protein required for translocation tRNA receptor ATPase

Entelis et al. (2006) Tarassov et al. (1995b)

RIC1/F1␣

Precursor mitochondrial isoform of lysyl tRNA synthetase Glycolytic enzyme Receptor for imported pre-protein on outer membrane Receptor for imported pre-protein on inner membrane Receptor for imported pre-protein on outer membrane Receptor for imported pre-protein on outer membrane Voltage-dependent anion channel (porin) F1 ATP synthase subunit ␣

RIC4A

Kinetoplastid-specific protein

RIC6/ISP RIC8A/UCR6b RIC8B RIC9/COXVI

Complex III iron sulfur protein Complex III subunit 6b Kinetoplastid-specific protein Complex IV subunit VI

eIF1a

Translation initiation factor 1a

Membrane assembly of complex Translocation pore tRNA receptor Complex assembly tRNA binding protein required for translocation tRNA carrier

enolase2p TOM20 (MOM19) TIM44 (MIM44/MPI1) Potato

TOM20 TOM40 VDAC

Leishmaniaa

Trypanosoma

a

Tarassov et al. (1995b) Salinas et al. (2006) Salinas et al. (2006) Salinas et al. (2006) Goswami et al. (2006); Goswami and Adhya (2006) Mukherjee et al. (2007) Mukherjee et al. (2007) Chatterjee et al. (2006) Mukherjee et al. (2007) Mukherjee et al. (2007) Bouzaidi-Tiali, Aeby, Charriere, Pusnik, & Schneider (2007)

Only the six essential subunits of RIC are shown.

Fig. 2. Current view of the tRNA import mechanism. RIC recognizes and binds to tRNA through receptor RIC1. tRNA binding triggers ATP hydrolysis by RIC1, and generation of a proton gradient (excess outside). The protonmotive force induces transfer of the tRNA to RIC9, and the subsequent translocation of thee tRNA through the pore. For simplicity, only the import of type I tRNA is shown. For details, see text.

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are imported by themselves, and type II tRNAs, the import of which is stimulated by type I tRNAs. Conversely, type II tRNAs inhibit the import of type I (Bhattacharyya, Chatterjee, & Adhya, 2002; Goswami, Chatterjee, Bhattacharyya, Basu, & Adhya, 2003). This reciprocal interaction was faithfully replicated in the liposome-RIC import system. RIC directly binds, and can be crosslinked to, type I tRNAs, as well as to a synthetic oligoribonucleotide containing a type I import signal, with nanomolar affinity. In contrast, loading of type II tRNAs is dependent on the presence of a type I tRNA. Antibody inhibition and gene knockdown experiments demonstrated the participation of subunits RIC1 and RIC8A as receptors for the binding and allosteric interactions of types I and II tRNAs, respectively (Bhattacharyya et al., 2003; Chatterjee et al., 2006; Goswami et al., 2006).

Recent experiments suggest that the import substrate, following the initial receptor-binding step, is transferred to RIC9 in the presence of ATP before being translocated through the pore (S. Basu & S. Adhya, unpublished data). The current model of the import mechanism (Fig. 2), based on the above findings, envisages the initial binding of tRNA through its import signal to its receptor, triggering RIC1-mediated ATP hydrolysis and generation of a proton gradient (ψ negative inside). The protonmotive force drives the transfer of the tRNA to RIC9, followed by the opening of the translocation pore to allow passage of the tRNA into the matrix.

4.2. tRNA dependent ATP hydrolysis

5. Biomedical applications

RIC, incorporated into liposomes, catalyses ATP hydrolysis in presence of type I tRNA (Bhattacharyya & Adhya, 2004b). Recombinant subunit RIC1 was shown to possess tRNA-dependent ATPase activity, and knockdown of RIC1 resulted in loss of this activity from mitochondrial membranes, indicating that RIC1 is the major, if not the sole, tRNA-dependent ATPase in Leishmania mitochondria (Goswami & Adhya, 2006).

A number of maternally inherited human disorders are caused by mitochondrial tRNA mutations (Taylor & Turnbull, 2005). The high efficiency of RIC as a tRNA transporter suggested its application as an inducer of the import of functional tRNAs into mutant human mitochondria to complement the genetic defect. In addition to Leishmania tRNAs, several human cytosolic tRNAs are imported by RIC into isolated human mitochondria (Mahata, Bhattacharyya, Mukherjee, & Adhya, 2005), presumably due to the presence of type I and/or type II import signals on these heterologous tRNAs. The imported cytosolic tRNA was functional in mitochondrial protein synthesis, repairing the translational defects caused by a pathogenic point mutation in the mitochondrial tRNALys gene. Importantly, RIC was efficiently taken up by mammalian cells through an endocytotic mechanism involving caveolin-1, a transport vesicle component, and targeted to mitochondria (Mahata, Mukherjee, Mishra, Bandyopadhyay, & Adhya, 2006). In cybrid lines containing tRNALys-mutant mitochondria derived from a patient with Myoclonic Epilepsy with Ragged Red Fibers (MERRF), RIC restored mitochondrial function to nearly wild-type levels (Mahata et al., 2006). This suggests the potential use of RIC to manage MERRF and other disorders such as maternally inherited diabetes that are caused by mitochondrial tRNA mutations. Recent studies suggest that RIC could be used to deliver small RNAs into the mitochondria of cells and tissues in vivo (S. Mukherjee, B. Mahato and S. Adhya, unpublished data). This may provide an important step towards mitochondrial gene therapy.

4.3. Generation of protonmotive force (pmf) In respiring mitochondria, electron transport between the respiratory complexes results in the generation of a trans-membrane gradient of protons across the inner membrane; the energy of this gradient is manifested as a membrane potential (ψ), and drives ATP synthesis by the F1Fo ATP synthase (complex V), as well as the transport of various metabolites. Under some conditions, complex V acts hydrolytically, generating ψ by hydrolyzing ATP. In vitro assays in several systems showed the sensitivity of tRNA import to uncouplers and inhibitors of oxidative phosphorylation that disrupt ψ, indicating the involvement of a transmembrane proton gradient in import (reviewed in Bhattacharyya & Adhya, 2004a). Purified RIC was shown to induce the uptake of the potential-sensitive dye rhodamine 123 into liposomes in the presence of ATP, indicating the generation of ψ (negative inside); this process was sensitive to oligomycin and to protonophores. Moreover, ATP in the import assay could be replaced by acidification of the external medium to pH ∼6, implying the impor-

tance of the proton gradient (Bhattacharyya & Adhya, 2004b). 4.4. Translocation of tRNA

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Acknowledgements Supported by DST grant# SR/SO/BB-28/2003, and CSIR Network Project NWP0038. I thank all GE lab members who have contributed to this research, and Sandip Kolay for the computer graphics. References Bhattacharyya, S. N., & Adhya, S. (2004a). The complexity of mitochondrial tRNA import. RNA Biology, 1, 84–88. Bhattacharyya, S. N., & Adhya, S. (2004b). tRNA-triggered ATP hydrolysis and generation of membrane potential by the leishmania mitochondrial tRNA import complex. Journal of Biological Chemistry, 279, 11259–11263. Bhattacharyya, S. N., Chatterjee, S., & Adhya, S. (2002). Mitochondrial RNA import in Leishmania tropica: aptamers homologous to multiple tRNA domains that interact cooperatively or antagonistically at the inner membrane. Molecular and Cellular Biology, 22, 4372–4382. Bhattacharyya, S. N., Chatterjee, S., Goswami, S., Tripathi, G., Dey, S. N., & Adhya, S. (2003). “Ping-pong” interactions between mitochondrial tRNA import receptors within a multiprotein complex. Molecular and Cellular Biology, 23, 5217–5224. Bouzaidi-Tiali, N., Aeby, E., Charriere, F., Pusnik, M., & Schneider, A. (2007). Elongation factor 1a mediates the specificity of mitochondrial tRNA import in T. brucei. EMBO Journal, 26, 4302–4312. Chatterjee, S., Home, P., Mukherjee, S., Mahata, B., Goswami, S., Dhar, G., & Adhya, S. (2006). An RNA-binding respiratory component mediates import of type II tRNAs into Leishmania mitochondria. Journal of Biological Chemistry, 281, 25270–25277. Entelis, N., Brandina, I., Kamenski, P., Krasheninnikov, I. A., Martin, R. P., & Tarassov, I. (2006). A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae. Genes and Development, 20, 1609–1620. Goswami, S., & Adhya, S. (2006). The alpha subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA. Journal of Biological Chemistry, 281, 18914–18917.

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