Import of RNA into Leishmania mitochondria occurs through direct interaction with membrane-bound receptors.

Sridam Mahapatra,Samit Adhya

doi:10.1074/jbc.271.34.20432

Abstract

Cytoplasmic tRNAs are imported into the kinetoplast mitochondrion of Leishmania, but the mechanism of import is unknown, particularly whether RNA is transferred as a ribonucleoprotein complex through the protein import pathway or by a distinct receptor-mediated mechanism. Using isolated mitochondria, it was shown that a small, importable RNA, which is structurally homologous to tRNA, binds rapidly, specifically, and with high affinity to the mitochondrial surface in the absence of soluble protein factors to form an import intermediate. Two classes of binding site of apparent Kd 0.3 and 10 n, respectively, were distinguished. tRNA from Leishmania, but not yeast, competitively inhibited the binding. Northwestern blot analysis revealed the presence of a 15-kDa RNA binding protein on the mitochondrial surface. Whereas receptor binding was resistant to heparin and KCl, internalization was sensitive to both reagents. These results are consistent with the presence of a direct mechanism of receptor-mediated RNA import on Leishmania mitochondria.

Highlights

A unique characteristic of the mitochondrion of kinetoplastid protozoa such as Leishmania is the apparent lack of tRNA genes on mitochondrial DNA and the import of nuclear-encoded tRNAs from the cytoplasm [1,2,3,4,5,6,7,8]
For quantitative studies of the import mechanism, it is necessary to use a single species of RNA labeled to high specific activity; terminally labeled tRNA mixtures isolated from cellular RNA are unsuitable
We have investigated the presence of import receptors on the mitochondrial surface and examined the relationship between binding and internalization of RNA in order to distinguish between the co-import and direct import models

Summary

Introduction

A unique characteristic of the mitochondrion of kinetoplastid protozoa such as Leishmania is the apparent lack of tRNA genes on mitochondrial DNA and the import of nuclear-encoded tRNAs from the cytoplasm [1,2,3,4,5,6,7,8]. Import of a few tRNAs into the mitochondria of plants [9] and yeast [10] has been reported. Showing import of RNA in the absence of cytosolic proteins, in a sequence-specific, saturable, and ATP-dependent manner [11], suggested the second mechanism, but direct evidence was lacking. In addition to tRNAs and certain small ribosomal RNAs, synthetic transcripts from the 5Ј-untranslated region of the Leishmania ␤-tubulin gene are imported in vitro. For quantitative studies of the import mechanism, it is necessary to use a single species of RNA labeled to high specific activity; terminally labeled tRNA mixtures isolated from cellular RNA are unsuitable. Because high specific activity antisense transcripts can be synthesized in vitro, we have employed them as a sensitive probe for the import process

Methods

Results

Conclusion