5 S rRNA and tRNA Import into Human Mitochondria: COMPARISON OF IN VITROREQUIREMENTS

Nina S Entelis,Olga A Kolesnikova,Semih Dogan,Robert P Martin,Ivan A Tarassov

doi:10.1074/jbc.m103906200

Nina S Entelis, Olga A Kolesnikova + Show 3 more

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https://doi.org/10.1074/jbc.m103906200

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Abstract

In vivo, human mitochondria import 5 S rRNA and do not import tRNAs from the cytoplasm. We demonstrated previously that isolated human mitochondria are able to internalize a yeast tRNA(Lys) in the presence of yeast soluble factors. Here, we describe an assay for specific uptake of 5 S rRNA by isolated human mitochondria and compare its requirements with the artificial tRNA import. The efficiency of 5 S rRNA uptake by isolated mitochondria was comparable with that found in vivo. The import was shown to depend on ATP and the transmembrane electrochemical potential and was directed by soluble proteins. Blocking the pre-protein import channel inhibited internalization of both 5 S rRNA and tRNA, which suggests this apparatus be involved in RNA uptake by the mitochondria. We show that human mitochondria can also selectively internalize several in vitro synthesized versions of yeast tRNA(Lys) as well as a transcript of the human mitochondrial tRNA(Lys). Either yeast or human soluble proteins can direct this import, suggesting that human cells possess all factors needed for such an artificial translocation. On the other hand, the efficiency of import directed by yeast or human protein factors varies significantly, depending on the tRNA version. Similarly to the yeast system, tRNA(Lys) import into human mitochondria depended on aminoacylation and on the precursor of the mitochondrial lysyl-tRNA synthetase. 5 S rRNA import was also dependent upon soluble protein(s), which were distinct from the factors providing tRNA internalization.

Highlights

Introduction ofU34C mutation in tRK2 confers to this normally nonimported tRNA a capacity of import into yeast mitochondria [23, 60]
After incubation of 5Ј end-labeled human 5 S rRNA with mitochondria in the presence of human import directing proteins (HmIDPs) and ATP, we observed that a portion of the externally added RNA was protected from nucleases (Fig. 2)
Detection of labeled 5 S rRNA after treatment with nucleases cannot be explained by protection with proteins present in HmIDPs, because the same treatment in the absence of mitochondria leads to a complete hydrolysis of the RNA (Fig. 2)

Summary

Introduction

U34C mutation in tRK2 confers to this normally nonimported tRNA a capacity of import into yeast mitochondria [23, 60]. We observed that r2 was internalized by RNA. Maximum aminoacylationc tRK1 tRK2 r2 r8 tr trKHm Ͻ0.05. Ͻ0.05 a Efficiency of import was expressed as picomoles of imported RNA/mg of mitochondrial protein. B MSK binding efficiency was calculated as [B/(B ϩ N)]IDPs, where B is the radioactivity bound to the protein A-Sepharose beads and N is the radioactivity that was not bound (see “Experimental Procedures”). The control binding, [B/(B ϩ N)]BSA, was always Ͻ0.05. KRS was used to aminoacylate tRK1, tRK2, r2, and r8; tr was aminoacylated by purified recombinant MSK (mature form) and trKHm with partially purified mitochondrial LysRS

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