Interaction of eEF2 with Drp1
To explore the mechanism by which eEF2 promotes mitochondrial fission,
we first examined the effect of eEF2 expression on Drp1, a
dynamin-related GTPase that acts as a key driver of mitochondrial
fission. We found that the amount of mitochondrial Drp1 protein was
remarkably down-regulated, whereas the total or cytosol Drp1 protein was
unchanged, in the cells subjected to knockdown of eEF2 expression
(Fig. 4a ). By contrast, the mitochondrial Drp1 protein was
increased in the cells transfected with an eEF2 expression plasmid
(Fig. 4b ). These results suggest that eEF2 is crucial in
supporting Drp1 translocation to the mitochondria. Next, we tested the
possible association of eEF2 with Drp1 using the approaches of
co-immunoprecipitation and GST-associated precipitation.
Co-immunoprecipitation experiments demonstrated that eEF2 and Drp1 are
physically associated (Fig. 4c) , and GST-associated
precipitation using eEF2 or Drp1 GST fusions proteins also showed the
binding of eEF2 to Drp1 (Fig. 4d ). Moreover, we observed that
eEF2 and Drp1 co-localized at the mitochondria, as analyzed by
immunofluorescence staining and confocal microscopy (Fig. 4e ).
These results imply a physical association of eEF2 with Drp1 and suggest
that the effect of eEF2 on mitochondrial fission may be mediated through
its interaction with Drp1. As GTPase activity of Drp1 is essential for
mitochondrial fission and eEF2 has GTP binding site, we asked whether
eEF2 interacts with Drp1 through its GTP binding domain. To test this
possibility, we constructed an eEF2 mutant in which the GTP binding
motif (104-108; SPGHV) of eEF2 was deleted (Fig. 6a ) and
transfected this expression vector of the mutant into HEK293 cells. We
showed that deletion of the eEF2 GTP binding motif decreased the
association of eEF2 with Drp1 in vivo (Fig. 4c ) as well
as in vitro (Fig. 4d ). To further validate the role of
eEF2 in promoting mitochondrial fission, we tested the effect of the
deletion mutant of eEF2 on mitochondrial length. As shown inFig. 5a , this deletion mutant had no effect on mitochondria
fission as compared with wild-type eEF2. These results suggest that the
effect of eEF2 on mitochondrial fission is mediated through its
interaction with Drp1 at its GTP binding site. To confirm the
specificity of the effect of eEF2 on mitochondrial fission, we performed
rescue experiments using an RNAi-resistant eEF2 mutant in which 3
synonymous, single nucleotide changes were incorporated into the RNAi
target sequence of wild-type eEF2 cDNA. Fig. 5b shows that the
effect of eEF2-targeted siRNA on the mitochondrial length was partially
rescued in the cells transfected with this RNAi-resistant mutant of
eEF2.