A Moonlighting Human Protein Is Involved in Mitochondrial Import of tRNA.

Maria Baleva,Nina Entelis,Benoît Masquida,Ali Gowher,Piotr Kamenski,Ivan Tarassov

doi:10.3390/ijms16059354

Abstract

In yeast Saccharomyces cerevisiae, ~3% of the lysine transfer RNA acceptor 1 (tRK1) pool is imported into mitochondria while the second isoacceptor, tRK2, fully remains in the cytosol. The mitochondrial function of tRK1 is suggested to boost mitochondrial translation under stress conditions. Strikingly, yeast tRK1 can also be imported into human mitochondria in vivo, and can thus be potentially used as a vector to address RNAs with therapeutic anti-replicative capacity into mitochondria of sick cells. Better understanding of the targeting mechanism in yeast and human is thus critical. Mitochondrial import of tRK1 in yeast proceeds first through a drastic conformational rearrangement of tRK1 induced by enolase 2, which carries this freight to the mitochondrial pre-lysyl-tRNA synthetase (preMSK). The latter may cross the mitochondrial membranes to reach the matrix where imported tRK1 could be used by the mitochondrial translation apparatus. This work focuses on the characterization of the complex that tRK1 forms with human enolases and their role on the interaction between tRK1 and human pre-lysyl-tRNA synthetase (preKARS2).

Highlights

Mitochondria are the centres of critical cellular processes, such as oxidative phosphorylation, apoptosis, fatty acids, amino acids and Fe–S cluster metabolisms
Mitochondria present genomes only encoding a fistful of proteins and RNAs dedicated to oxidative phosphorylation or mitochondrial translation
These results indicate that human enolases participate in the mitochondrial import pathway of tRK1 in human cells together with preKARS2, demonstrating the high similarity between the mechanisms occurring in yeast and human cells

Summary

Introduction

Mitochondria are the centres of critical cellular processes, such as oxidative phosphorylation, apoptosis, fatty acids, amino acids and Fe–S cluster metabolisms. The capacity of human cells to import tRK1 into mitochondria points to the possibility to use tRK1 as a vector to target foreign RNA into the mitochondrial matrix This strategy has been demonstrated experimentally by achieving replacement of the non-functional tRNALys in the case of the MERRF syndrome [9], or by the inhibition of the replication of mitochondrial genomic copies harbouring deletions or mutations [10,11,12]. From the two enolase isoforms in yeast, only Eno2p allows for tRK1 conformational rearrangement and targeting to preMSK, albeit their sequences are 97% identical This mechanism seems to be very close to a potential mechanism in human mitochondria since tRK1 import can be directed in vitro using human pre-lysyl-tRNA synthetase (preKARS2) and rabbit enolase [13]. These results indicate that human enolases participate in the mitochondrial import pathway of tRK1 in human cells together with preKARS2, demonstrating the high similarity between the mechanisms occurring in yeast and human cells

Results

Discussion

Expression and Purification of Recombinant Proteins

In Vitro Import Assay