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.