Abstract
ABSTRACTIntracellular trafficking of viruses and proteins commonly occurs via the early endosome in a process involving Rab5. The RNA Import Complex (RIC)-RNA complex is taken up by mammalian cells and targeted to mitochondria. Through RNA interference, it was shown that mito-targeting of the ribonucleoprotein (RNP) was dependent on caveolin 1 (Cav1), dynamin 2, Filamin A and NSF. Although a minor fraction of the RNP was transported to endosomes in a Rab5-dependent manner, mito-targeting was independent of Rab5 or other endosomal proteins, suggesting that endosomal uptake and mito-targeting occur independently. Sequential immunoprecipitation of the cytosolic vesicles showed the sorting of the RNP away from Cav1 in a process that was independent of the endosomal effector EEA1 but sensitive to nocodazole. However, the RNP was in two types of vesicle with or without Cav1, with membrane-bound, asymmetrically orientated RIC and entrapped RNA, but no endosomal components, suggesting vesicular sorting rather than escape of free RNP from endosomes. In vitro, RNP was directly transferred from the Type 2 vesicles to mitochondria. Live-cell imaging captured spherical Cav1− RNP vesicles emerging from the fission of large Cav+ particles. Thus, RNP appears to traffic by a different route than the classical Rab5-dependent pathway of viral transport.
Highlights
Many viruses, proteins and lipids are taken up through, or recycled from, the plasma membrane through endocytosis
Trafficking of RNA to mitochondria Cultured cells were incubated with the pcRNA1-RNA Import Complex (RIC) complex
In case of NSF-KD, uptake was normal but the RNA did not localize to mitochondria, indicating a block at an intermediate step. These results indicate that endocytosis of the RNP into cytosolic transport vesicles is dependent on caveolin 1 (Cav1), Dynamin 2 (Dyn2) and Filamin A (FilA), but independent of clathrin
Summary
Proteins and lipids are taken up through, or recycled from, the plasma membrane through endocytosis. A number of distinct uptake mechanisms have been described, including through clathrin-coated pits, caveole/lipid rafts and clathrin- or caveolin-independent pathways (Wieffer et al, 2009; Hansen and Nichols, 2009). Endocytic vesicles from the plasma membrane enter pre-existing sorting compartments from which the cargo emerges either free or in secondary vesicles that are targeted to the destination compartment. The transport of various cellular RNAs within and between the nuclear and cytosolic compartments involves ribonucleoprotein (RNP) complexes, motor proteins and cytoskeletal elements (Eliscovich et al, 2013). Genetic Engineering Laboratory, CSIR–Indian Institute of Chemical Biology, 4 Raja S. *Present address: Penn Institute for Regenerative Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA.
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