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 α subunit of F1 ATP synthase. The RIC1 gene complemented an atpA mutation in Escherichia coli . Antisense-mediated knockdown of RIC1/F1α 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/F1α alone, but recovery of type II tRNA import additionally required a type I tRNA. RIC1/F1α formed stable complexes with type I, but not type II, tRNAs through the cooperation of its nucleotide binding and C-terminal domains. Thus, RIC1/F1α is a type I tRNA import receptor. As expected of a bifunctional protein, RIC1/F1α 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.

The mechanism of active transport of transfer RNA (tRNA) across membranes is largely unknown. Factors mediating the import of tRNA into the kinetoplast mitochondrion of the protozoon Leishmania tropica are organized into a multiprotein RNA import complex (RIC) at the inner membrane. Here, we present the complete characterization of the identities and functions of the subunits of this complex. The complex contains three mitochondrion-and eight nuclear-encoded subunits; six of the latter are necessary and sufficient for import. Antisense-mediated knockdown of essential subunits resulted in the depletion of mitochondrial tRNAs and inhibition of organellar translation. Functional complexes were reconstituted with recombinant subunits expressed in Escherichia coli. Several essential RIC subunits are identical to specific subunits of respiratory complexes. These findings provide new information on the evolution of tRNA import and the foundation for detailed structural and mechanistic studies.

Transport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned. The C-terminal region of RIC8A is homologous to subunit 6b of ubiquinol cytochrome c reductase (respiratory complex III), while the N-terminal region has intrinsic affinity for type II, but not for type I, tRNAs. RIC8A is shared by the import complex and complex III, indicating its bi-functionality, but is assembled differently in the two complexes. Knockdown of RIC8A in Leishmania lowered the mitochondrial content of type II tRNAs but raised that of type I tRNAs, with downstream effects on mitochondrial translation and respiration, and cell death. In RIC8A knockdown cells, a subcomplex was formed that interacted with type I tRNA, but the negative regulation by type II tRNA was lost. Mitochondrial extracts from these cells were defective for type II, but not type I, import; import and regulation were restored by purified RIC8A. These results provide evidence for the relevance of allosteric regulation in vivo and indicate that acquisition of new tRNA-binding domains by ancient respiratory components have played a key role in the evolution of mitochondrial tRNA import.

During myogenesis, satellite stem cells (SCs) are induced to proliferate and differentiate to myogenic precursors. The role of energy sensors such as the AMP-activated protein kinase (AMPK) and the mammalian Target of Rapamycin (mTOR) in SC activation is unclear. We previously observed that upregulation of ATP through RNA-mediated mitochondrial restoration (MR) accelerates SC activation following skeletal muscle injury. We show here that during regeneration, the AMPK-CRTC2-CREB and Raptor-mTORC-4EBP1 pathways were rapidly activated. The phosho-CRTC2-CREB complex was essential for myogenesis and activated transcription of the critical cell cycle regulator cyclin D1 (Ccnd1). Knockdown (KD) of either mTORC or its subunit Raptor delayed SC activation without influencing the differentiation program. KD of 4EBP1 had no effect on SC activation but enhanced myofiber size. mTORC1 positively regulated Ccnd1 translation but destabilized Ccnd1 mRNA. These antithetical effects of mTORC1 were mediated by two microRNAs (miRs) targeted to the 3= untranslated region (UTR) of Ccnd1 mRNA: miR-1 was downregulated in mTORC-KD muscle, and depletion of miR-1 resulted in increased levels of mRNA without any effect on Ccnd1 protein. In contrast, miR-26a was upregulated upon mTORC depletion, while anti-miR-26a oligonucleotide specifically stimulated Ccnd1 protein expression. Thus, mTORC may act as a timer of satellite cell proliferation during myogenesis. R egeneration of skeletal muscle following injury involves formation of new myofibers as well as patch repair of old injured fibers by fusion of satellite cell-derived myoblasts (1). The program of differentiation of satellite cells involves sequential expression of myogenic regulatory factors (MRFs) and their target genes (2-5). Mitochondrial biogenesis occurs at a specific time prior to the formation of myofibers (4, 6), suggesting an energy-requiring step, but the specific role of ATP in differentiation remains unknown. Eukaryotic cells contain a number of enzymes that sense the energy status, particularly, the pools of AMP and ADP (AMPactivated protein kinase [AMPK]) or of ATP (mammalian Target of Rapamycin [mTOR] kinases) (7,8). It is possible that mitochondrial activity, through modulation of the adenylate pool, impacts these energy sensors, thereby activating or inhibiting downstream pathways.Although energy sensors such as AMPK and mTORC are known to regulate cellular energy homeostasis and cell growth (9), there are indications that they could also modulate specific developmental processes. It was shown early that the mTORC inhibitor rapamycin inhibits growth of myofibers in regenerating adult muscle (10); such inhibition was overcome by expression of a rapamycin-resistant mTORC gene (11). Genetic ablation of mTORC (12), or of the mTORC1 subunit Raptor (13), or of the mTORC1 target S6K (14) results in severe muscle atrophy. These studies indicate that myofiber growth and maturation are regulated by mTORC1 acting through S6K in vivo but provide no information on the specific eff...

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