Endosome Independent - Vesicular Transport and Targeting of the Leishmania RNA Import Complex to Mammalian Mitochondria in Vivo and in Vitro

Abstract

The multi subunit RNA Import Complex (RIC) from the protozoal parasite Leishmania tropica is taken up by mammalian cells and trafficked to mitochondria, with functional RNA delivered to the matrix by an unknown mechanism. By live cell imaging of siRNA mediated knockdown cells we showed that intracellular vesicular transport of the ribonuclear protein complex is dependent on caveolin1, dynamin2 and filaminA but independent of components of the endosomal pathway such as EEA1, Rab5 or Rab7. Moreover, mitochondrial targeting was dependent on TOM20 and TOM22, two components of the outer membrane machinery. By immuno-selection we could distinguish two types of RNA loaded cytoplasmic vesicles, one containing caveolar components and the other lacking them. In vitro, transfer of RNP from the caveolin negative transport vesicles to mitochondria was dependent on ATP and on TOM22. RIC was transferred to TOM20 deficient mitochondrial membrane but the RNA was not imported into the matrix. These results imply that (1) endosomes are not involved in RIC trafficking and novel interactions between vesicle associated RIC and (2) TOM20 and TOM22 are required for transfer and positioning of RIC on the mitochondrial surface.