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.