Endofin, an endosomal FYVE domain protein

KIAA0305 is an uncharacterized member of the FYVE domain protein family. It is closely related to SARA, with about 50% identity in the carboxyl-termin al 800-amino acid region. Indirect immunofluorescence microscopy using poly clonal antibodies raised against KLAA0305 revealed that it is enriched in e arly endosomes. The Myc-tagged version is also faithfully targeted to the e arly endosome. We have tentatively called KLAA0305 endofin (for endosome-as sociated FYVE-domain protein). The association of endofin with endosomes is mediated by its FYVE domain because deletion mutants lacking the central F YVE finger motif are distributed in the cytoplasm. In addition, a single po int mutation in the FYVW finger motif at cysteine, residue 753 (C753S) is s ufficient to abolish its endosomal association. Its endosomal localization is also sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmanni n. Using in vitro liposome binding assays, we demonstrate that Myc-tagged e ndofin associates preferentially with phosphatidylinositol 3-phosphate, whe reas the C753S point mutant was unable to do so. We also show that endofin co-localizes with SARA but that they are not associated in a common complex because they failed to co-immunoprecipitate in co-expressing cells. Endofi n also does not associate with Smad2 nor behave like SARA in affecting tran sforming growth factor-beta signaling. At high levels of expression, both e ndofin and SARA can cause an endosome aggregation/fusion effect. In COS7 ce lls, which can support high levels of exogenous protein expression, both pr oteins can also cause other structural anomalies in the endocytic pathway, as represented by enlarged vesicular structures. These endosomal aggregates /fusions accumulated endocytosed epidermal growth factor. Taken together, t his report provides evidence to suggest that endofin and the highly related SARA are endosomal proteins with potential roles in regulating membrane tr affic.