eEF2 is present in the mitochondria
In study of the roles of eEF-2 kinase/eEF2 in stress response, we observed unexpectedly in several types of cells that, while eEF2 kinase was solely detected in the cytosol, eEF2 was found in both cytosol and mitochondria, as determined by Western blot of the mitochondria and cytosol preparations (Fig. 1a ). To validate this surprising finding, we performed the mitochondria immunofluorescence staining of eEF2, and showed that eEF2 co-localized with MitoTracker, a fluorescent dye that labels mitochondria within live cells (Fig. 1b). These results provide the clear evidence for the presence of eEF2 in the mitochondria.
Effects of eEF2 expression on the mitochondrial length.
To determine the functional significance of the presence of eEF2 in the mitochondria, we knocked down eEF2 using RNA interference or overexpressed eEF2 using its expression vector, and then examined whether there were any alterations in morphology of the mitochondria. We observed that as compared with the control cells, MCF-7 cells (Fig. 2a) and mouse embryonic fibroblasts (Fig.2b ) with knockdown of eEF2 had significantly increased mitochondrial lengths, suggesting an inhibition of mitochondrial fission. Similar effect of depletion of eEF2 on mitochondrial length was observed in LN-229 cells (SFig.1 ). Three eEF2-targeted siRNA were tested, and similar results were obtained (data not shown). Additionally, the electron microscopy (Fig. 2c ) and live cell imaging (SFig. 2 ) showed that the mitochondrial shapes were enlarged in the cells with depletion of eEF2 as compared with the control cells, also suggesting an alteration of mitochondrial dynamics. Effect of eEF2 knockdown on mitochondrial fission was also observed under hypoxic condition: while hypoxia promoted mitochondrial fission, knockdown of eEF2 inhibited the fission of this organelle (SFig. 3 ). By contrast, the mitochondrial length was shortened in the cells subjected to forced expression of eEF2 (Fig. 2d ). Additionally, the cells with depletion of eEF2 kinase, an enzyme that phosphorylates eEF2, not only showed loss of eEF2 phosphorylation (SFig. 4a)but also had shorter lengths of the mitochondria than the control cells (SFig. 4b ), suggesting that the non-phosphorylated form of eEF2 possesses a stronger capacity to support mitochondrial fission. Together, these experiments demonstrate a promotive role of eEF2 in mitochondrial fission.