Discussion
Thus far, the only known function of eEF2 is its role in mRNA translation by facilitating the GTP-dependent translocation of the ribosome. Unexpectedly, we found that, in addition to its presence in the cytoplasm, eEF2 also exists in the mitochondria (Fig. 1) and interacts with Drp1 to promote mitochondrial fission (Fig. 4). Although these novel findings appear to stray from the current knowledge about eEF2, they clearly show that this protein has an important regulatory role in mitochondrial dynamics. Noteworthily, novel functions of other elongation factors have reported recently. For instance, it was found that elongation factor-Tu could specifically bind to T6S integral membrane toxin, and this interaction is required for delivery of this toxin to target cells.(13) This finding provides another evidence that elongation factors may have the aptitude for interacting with other proteins, affecting certain cellular functions. It was shown in a previous study that silencing of eEF2 expression caused morphological alterations of mitochondria and other organelles,(14) but no further investigation on the role of eEF2 in mitochondria regulation has been reported so far. Here, we show that eEF2 is not only present in the cytoplasm, but also exists in the mitochondria and has a promotive role in mitochondrial fission. The positive regulation of mitochondrial fission by eEF2 was evidenced by our observation that knockdown of eEF2 increased the length of mitochondria (Fig. 2a, b and c) and forced expression of eEF2 decreased the length of this organelle (Fig. 2d). Expression of eEF2 also affected mitochondrial metabolism (Fig. 3) and cell proliferation (SFig. 5), the likely consequences of the altered mitochondrial fission mediated by eEF2 expression. Mechanistically, eEF2 appears to stimulate the activity of GTPase of Drp1 through its interaction with this major component of the fission machinery, as these two proteins are physically associated and co-localized (Fig. 4), and deletion of the GTP binding site of eEF2 substantially weakened its binding to Drp1 (Fig. 4) and impaired its ability to activate GTPase of Drp1 (Fig. 6) and to support mitochondrial fission (Fig. 5). Drp1 is a cytosolic protein, which upon activation translocates to the outer membrane of the mitochondria and plays a central role in inducing mitochondrial fission. Nevertheless, how precisely Drp1 is targeted and translocated from the cytosol to the mitochondria remains less clear. We show here that silencing eEF2 expression not only decreased the mitochondrial eEF2 but also Drp1 in the mitochondria (Fig. 4b); by contrast, forced expression of eEF2 increased the mitochondrial Drp1 (Fig. 4a). Therefore, it is likely that binding of eEF2 to Drp1 facilitates the translocation of Drp1 to the outer membrane of the mitochondria. It is known that phosphorylation of eEF2 by eEF2 kinase reduces its binding affinity to the ribosome, thereby inhibiting peptide elongation. Our experiments showed that the cells with depletion of eEF2 kinase had a significantly increase of mitochondrial fission (SFig. 4b), (15) suggesting that phosphorylation of eEF2 limits its interaction with Drp1. Additionally, it was reported that eEF2 can be phosphorylated by cyclin A-CDK2 at Se-595; thus, cyclin A-CDK2 might affect the eEF2-mediated mitochondrial fission as well.
As mitochondrial fusion and fission are important determinants of the functions of this organelle and defects in these processes are implicated in various human diseases such as cardiovascular, neurodegenerative, endocrine and neoplastic diseases,(16-18 ) our revelation of eEF2 as a new regulator of mitochondrial dynamics might provide a potential target for therapeutic intervention of those human disorders resulting from mitochondria dysfunction. Notably, small molecule inhibitors of eEF2 have just been developed recently,(19 ) and this may provide a promising opportunity to test their effects on the mitochondrial activity and the mitochondria-associated biological functions and their potential as therapeutic agents for the mitochondria-associated diseases.