Little is known about the orientation of LITAF in vesicles. The PPXY motifs of LITAF must be in the same compartment as theWWdomains of Itch so the two proteins can interact in vivo. Since Itch is suspected to be a cytosolic protein associated with internal membranes, we deduce that the Nterminus of LITAF, containing the PPXY motif, must also be found in the cytosol. If the hydrophobic stretch of amino acids found in the SLD of LITAF act as a transmembrane domain than the Cterminus of LITAF may be found on the luminal side of endosomes/lysosomes. Future studies will further explore the orientation of LITAF in vesicle membranes. Itch and Nedd4 are structurally similar proteins that are members of a conserved family of HECT ubiquitin ligases. Both contain an N-terminal C2 domain that may play a role in membrane targeting. They both contain 4 WW domains that mediate interactions with proline rich motifs along with a C-terminus HECT domain responsible for E3 ligase activity. Furthermore, both Itch and Nedd4 interact with LITAF, at least primarily through LITAF��s first PPXY motif. There are several possible explanations as to why LITAF can mediate the re-localization of Itch, but not Nedd4. First, given the high levels of structural similarities between Itch and Nedd4, it suggests that functional differences between Nedd4 and Itch are responsible for the different situations induced following an interaction with LITAF. Another possibility is that the targeting sequences of Nedd4 are ����stronger���� than the targeting sequences for Itch. This would imply that although LITAF and Nedd4 can interact, LITAF is not able to mediate the re-localization of Nedd4. Finally, in vivo, LITAF and Nedd4 may not be present in the same cellular compartments. If the two proteins cannot physically interact in vivo, then there is no possibility of LITAF mediating the relocalization of Nedd4. Our immunofluorescence data suggests that Nedd4 is found in the Golgi apparatus, but not in the trans-Golgi network. This may move Nedd4 out of the cycling pathway between the trans Golgi network/endosome/AZ 960 lysosome compartments precluding it from interacting with LITAF. Since the function of LITAF remains unknown, we can only speculate on the consequences that the re-localization of Itch has on Itch and LITAF function. Itch may be sorted from the trans- Golgi LY294002 PI3K inhibitor network to the lysosomes with the assistance of LITAF. LITAF and Itch may form a stable complex that translocates to the lysosome where Itch may or may not dissociate from LITAF within the lysosome for future degradation. It is also possible that LITAF retains Itch in the late endosomes/lysosomes. Itch has been found to localize within both endosomes and the trans-Golgi network. The presence of LITAF may limit movement of Itch and retain Itch within the late endosomes.