Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import

Abstract

Mitochondrial genomes generally encode a minimal set of tRNAs necessary for protein synthesis. However, a number of eukaryotes import tRNAs from the cytoplasm into their mitochondria. For instance, Saccharomyces cerevisiae imports cytoplasmic tRNA Gln into the mitochondrion without any added protein factors. Here, we examine the existence of a similar active tRNA import system in mammalian mitochondria. We have used subcellular RNA fractions from rat liver and human cells to perform RT-PCR with oligonucleotide primers specific for nucleus-encoded tRNA CUG Gln and tRNA UUG Gln species, and we show that these tRNAs are present in rat and human mitochondria in vivo. Import of in vitro transcribed tRNAs, but not of heterologous RNAs, into isolated mitochondria also demonstrates that this process is tRNA-specific and does not require the addition of cytosolic factors. Although this in vitro system requires ATP, it is resistant to inhibitors of the mitochondrial electrochemical gradient, a key component of protein import. tRNA Gln import into mammalian mitochondria proceeds by a mechanism distinct from protein import. We also show that both tRNA Gln species and a bacterial pre-tRNA Asp can be imported in vitro into mitochondria isolated from myoclonic epilepsy with ragged-red fiber cells if provided with sufficient ATP (2 mM). This work suggests that tRNA import is more widespread than previously thought and may be a universal trait of mitochondria. Mutations in mitochondrial tRNA genes have been associated with human disease; the tRNA import system described here could possibly be exploited for the manipulation of defective mitochondria.human ͉ MERFF D ifferent protein-synthesizing systems exist in the cytoplasm and organelles (mitochondria and chloroplasts) of eukaryotic cells. Reflecting the evolutionary origin of the organelle, the mitochondrial system is bacteria-like (1, 2). For instance, the tRNAs and aminoacyl-tRNA synthetases present in the mitochondria are closely related to their bacterial counterparts. The mitochondrial genome of higher eukaryotes encodes all of the tRNA species necessary for protein synthesis (3), whereas the aminoacyl-tRNA synthetases are nucleus-encoded and imported into the organelle. The mitochondrial protein-synthesizing system is important for the synthesis of a number of proteins for multienzyme complexes involved in oxidative phosphorylation (1). Since the original suggestion 40 years ago (4), tRNA import into mitochondria has been demonstrated in Tetrahymena, trypanosomatids, yeast, plants, and marsupials (reviewed in refs. 5 and 6). The detailed nature of the different tRNA import mechanisms is not yet known; however, ATP hydrolysis is always required. In the yeast system, the importance of some mitochondrial membrane proteins as well as of specific cytosolic proteins (enolase and lysyl-tRNA synthetase) has been established (reviewed in refs. 5 and 6). After the early observation of the presence of a cytoplasmic tRNA CUU Lys in Saccharomyces cerevisiae mitochondria (7), import o...

Biogenesis of glutaminyl-mt tRNA Gln in human mitochondria

“…This speculation is supported by the observation that mitochondrial extracts from mouse liver do not exhibit GlnRS activity (5). It was recently reported that cytoplasmic tRNA Gln is imported into human mitochondria (13), indicating an involvement of this tRNA in the biogenesis of Gln-tRNA Gln in human mitochondria. However, fate of the imported tRNA and the enzymatic pathway for generating mitochondrial Gln-tRNA Gln were unknown.…”

“…It was reported that cytoplasmic (cyto) tRNA Gln is imported into human mitochondria (13), indicating cyto tRNA Gln might be charged with Gln, and participate in mitochondrial translation, although we have revealed in this study that mt tRNA Gln is a major substrate in the indirect pathway in human mitochondria. To examine cyto tRNA Gln to be a substrate for mtGluRS and hGatCAB, we isolated cyto tRNA Gln from human placenta using the chaplet column chromatography (15), and carried out glutaminylation of cyto tRNA Gln by recombinant mtGluRS and hGatCAB.…”

“…It was reported that mammalian cyto tRNA Gln is imported into mitochondria (13). To investigate the fate of the imported cyto tRNA Gln , we examined cyto tRNA Gln to be converted to GlntRNA Gln in vitro using the recombinant mtGluRS and hGat-CAB.…”

Translating organellar glutamine codons : A case by case scenario?

“…14 Thus, if the mischarged E-tRNA Q , which is the obligatory substrate intermediate for the AdT, cannot be synthesized by the mitochondrion, there can't be a transamidation pathway responsible for Q-m tRNA Q formation in yeast. Following this report, earlier this year, it was shown that human mitochondria import, like yeast and protozoans, nuclear-encoded tRNA Q , 18 although, the human mitochondrial genome encodes its own tRNA Q species. Finally, the enzymatic pathway that generates human mitochondrial Q-m tRNA Q remains still unknown.…”

RNase P enzymes

“…The tRNAs GlnUAA and LeuUAA are enriched in mitoplast preps and tRNAGlnUAA is imported via a mechanism that is distinct from protein import. 26,27 Although the biological significance of importation of this tRNA is unknown, it demonstrates that RNA can be imported into the mitochondria. The import of larger structured RNAs such as the RNA components of MRP and RNase P into the mitochondria is controversial and remains to be fully resolved.…”