After 24 hours of incubation in 200 mM NaCl, EHD1 knock down and EH domain deletion overexpressing cells have lost viability, as quantified by Neutral Red staining [49], while wild-type seedlings are still viable, and EHD1 or the coiled-coil deletion over expressing cells possess even higher viability. Our results show that endocytosis is involved in plant salt stress coping mechanisms and indicate a role for EHD1 in salinity stress tolerance.DiscussionIn this work we examined the function of Arabidopsis EHD1, a AN 3199 biological activity protein we had previously isolated and found to be endocytosis related [25]. We had previously observed that EHD1 localizes to apparently endosomal vesicles in plant cells; here we show that EHD1 is localized to RabA and RabD positive vesicles. Mammalian EHD1 was reported to co-localize with mammalian Rab11 in the endocytic recycling compartment [50]. Additionally, mutant forms of EHD1 can affect Rab11 localization and cycling [50]. Though a per se endocytic recycling compartment has not been reported in plants, RabA1e has been reported to be highly BFA Licochalcone-A chemical information sensitive and mark a recycling endosome in plants [37] and indeed, this class of endosomes also contain plant EHD1, strengthening the indication that this class of endosome may have recycling functions in plant cells. Further, EHD1 was found to localize to endosomes which contain plant RabD2b. This could indicate an overlap between RabA and RabD on recycling endosomes in plant cells, or perhaps indicate that EHD1 has additional functions in cellular trafficking in plants, perhaps relating to ER to golgi and/or inter golgi trafficking pathwayswhich involve RabD [40]. Interestingly, mammalian EHD1 and EHD3, which are equally homologous to plant EHD1, were reported to be involved in retrograde transport from endosomes to golgi [35,36]. Possibly, plant EHD1 performs transport functions attributed to both mammalian EHD1 and EHD3. At any rate, there are many Rab proteins in plant cells and possible overlap in plant Rab functionalities has been discussed at length. It is also possible that organelles in which recycling and/or sorting processes take place in plant 1655472 cells can have additional functionalities. In addition to RabA/EHD1 containing endosomes, a golgi associated endosomal compartment to which EHD1 and RabD are localized in plants may mimic certain functions of the mammalian ERC. To further characterize the functionalities of a multi-domain containing protein, we created mutant protein forms possessing a deletion of the EH domain or the coiled-coil domain, both domains being responsible for protein-protein interactions [26,51,52]. We found that EHD1 lacking the coiled-coil domain continues to reside on endosomal structures and co-localize with RabA and RabD proteins, while EHD1 lacking the EH domain is almost completely excluded from these vesicles. The EH domain appears to be important for the localization of EHD1, as was demonstrated in mammalian cells [32,50]. Interestingly, an EH domain deletion mutant in mammalian EHD1 was found to lose its colocalization with Rab11, and further, to cause Rab11 to cluster in the peri-nulcear area, possibly as a result of impaired Rab11 recycling [50]. The same study found that a deletion mutant in the EH domain in mammalian EHD2 did not significantly affect the localization of the protein, and did not cause Rab11 to cluster in the peri-nulcear area, indicating that mammalian EHD2 does not affect recycling in the same manner as mammalian EHD1.After 24 hours of incubation in 200 mM NaCl, EHD1 knock down and EH domain deletion overexpressing cells have lost viability, as quantified by Neutral Red staining [49], while wild-type seedlings are still viable, and EHD1 or the coiled-coil deletion over expressing cells possess even higher viability. Our results show that endocytosis is involved in plant salt stress coping mechanisms and indicate a role for EHD1 in salinity stress tolerance.DiscussionIn this work we examined the function of Arabidopsis EHD1, a protein we had previously isolated and found to be endocytosis related [25]. We had previously observed that EHD1 localizes to apparently endosomal vesicles in plant cells; here we show that EHD1 is localized to RabA and RabD positive vesicles. Mammalian EHD1 was reported to co-localize with mammalian Rab11 in the endocytic recycling compartment [50]. Additionally, mutant forms of EHD1 can affect Rab11 localization and cycling [50]. Though a per se endocytic recycling compartment has not been reported in plants, RabA1e has been reported to be highly BFA sensitive and mark a recycling endosome in plants [37] and indeed, this class of endosomes also contain plant EHD1, strengthening the indication that this class of endosome may have recycling functions in plant cells. Further, EHD1 was found to localize to endosomes which contain plant RabD2b. This could indicate an overlap between RabA and RabD on recycling endosomes in plant cells, or perhaps indicate that EHD1 has additional functions in cellular trafficking in plants, perhaps relating to ER to golgi and/or inter golgi trafficking pathwayswhich involve RabD [40]. Interestingly, mammalian EHD1 and EHD3, which are equally homologous to plant EHD1, were reported to be involved in retrograde transport from endosomes to golgi [35,36]. Possibly, plant EHD1 performs transport functions attributed to both mammalian EHD1 and EHD3. At any rate, there are many Rab proteins in plant cells and possible overlap in plant Rab functionalities has been discussed at length. It is also possible that organelles in which recycling and/or sorting processes take place in plant 1655472 cells can have additional functionalities. In addition to RabA/EHD1 containing endosomes, a golgi associated endosomal compartment to which EHD1 and RabD are localized in plants may mimic certain functions of the mammalian ERC. To further characterize the functionalities of a multi-domain containing protein, we created mutant protein forms possessing a deletion of the EH domain or the coiled-coil domain, both domains being responsible for protein-protein interactions [26,51,52]. We found that EHD1 lacking the coiled-coil domain continues to reside on endosomal structures and co-localize with RabA and RabD proteins, while EHD1 lacking the EH domain is almost completely excluded from these vesicles. The EH domain appears to be important for the localization of EHD1, as was demonstrated in mammalian cells [32,50]. Interestingly, an EH domain deletion mutant in mammalian EHD1 was found to lose its colocalization with Rab11, and further, to cause Rab11 to cluster in the peri-nulcear area, possibly as a result of impaired Rab11 recycling [50]. The same study found that a deletion mutant in the EH domain in mammalian EHD2 did not significantly affect the localization of the protein, and did not cause Rab11 to cluster in the peri-nulcear area, indicating that mammalian EHD2 does not affect recycling in the same manner as mammalian EHD1.