Endofin, an Endosomal FYVE Domain Protein

Abstract

KIAA0305 is an uncharacterized member of the FYVE domain protein family. It is closely related to SARA, with about 50% identity in the carboxyl-terminal 800-amino acid region. Indirect immunofluorescence microscopy using polyclonal antibodies raised against KIAA0305 revealed that it is enriched in early endosomes. The Myc-tagged version is also faithfully targeted to the early endosome. We have tentatively called KIAA0305 endofin (for endosome-associated FYVE-domain protein). The association of endofin with endosomes is mediated by its FYVE domain because deletion mutants lacking the central FYVE finger motif are distributed in the cytoplasm. In addition, a single point mutation in the FYVE finger motif at cysteine residue 753 (C753S) is sufficient to abolish its endosomal association. Its endosomal localization is also sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmannin. Using in vitro liposome binding assays, we demonstrate that Myc-tagged endofin associates preferentially with phosphatidylinositol 3-phosphate, whereas 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. Endofin also does not associate with Smad2 nor behave like SARA in affecting transforming growth factor-␤ signaling. At high levels of expression, both endofin and SARA can cause an endosome aggregation/fusion effect. In COS7 cells, which can support high levels of exogenous protein expression, both proteins 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, this report provides evidence to suggest that endofin and the highly related SARA are endosomal proteins with potential roles in regulating membrane traffic.

Endofin Recruits TOM1 to Endosomes

“…Immunoprecipitation followed by Western blotting were carried out as described previously (3). For co-immunoprecipitation of endogenous TOM1 and endofin, A431 cell lysate was incubated with 20 g of either anti-TOM1 or anti-endofin antibody as well as 20 l (bed volume) of protein A-Sepharose CL-4B (Amersham Biosciences) at 4°C overnight.…”

“…The truncated TOM1, TOM1-L1, or TOM-L2 constructs were also generated by PCR and cloned into the pGEX-4T vector (Amersham Biosciences) for expression in Escherichia coli. The cDNA encoding the endofin fragment from amino acids 815-1539 was generated by PCR and cloned into the pDMycneo vector as for the other endofin constructs described previously (3).…”

“…In many instances, therefore, the FYVE domain serves to localize the protein containing it to endosomes. In endofin, the FYVE domain appears to be necessary and sufficient to localize the protein to early endosomes (3). The FYVE domain is a modular motif that can function independent of its position on the molecular structure.…”

The Molecular Basis of the Differential Subcellular Localization of FYVE Domains

“…Mutational studies of many FYVE domain-containing proteins have indicated that FYVE domains play an important role in their binding to endosomes in the cell (21,31,32). Interestingly, however, among many FYVE domains identified so far, only FENS-1 (20), endofin (19), and SARA (22) FYVE domains have been reported to autonomously translocate to endosomal membranes when expressed ectopically. It is therefore unclear as to how these three isolated domains achieve unique endosomal targeting properties and how FYVE domains, in general, contribute to the membrane targeting of their host proteins in different ways.…”

“…As expected from the endosomal localization of PtdIns(3)P and its role in vesicle trafficking, a large number of FYVE domain-containing proteins, including EEA1, Hrs, and FENS-1, are involved in endocytic vesicle trafficking. Some FYVE domain-containing proteins also function in cytoskeletal regulation (faciogenital dysplasia 1) (23) and growth factor signaling (SARA (22) and endofin (19)). Sequence alignment of FYVE domains (see Fig.…”