Endofin Recruits TOM1 to Endosomes

Abstract

Endofin is an endosomal protein implicated in regulating membrane trafficking. It is characterized by the presence of a phosphatidylinositol 3-phosphate-binding FYVE domain positioned in the middle of the molecule. To determine its potential effectors or binding partners, we used the carboxyl-terminal half of endofin as bait to screen a human brain cDNA library in a yeast two-hybrid system. Three clones that encode TOM1 were recovered. TOM1 is a protein closely related to the VHS (VPS-27, Hrs, and STAM) domain-containing GGA family. Although the function of the GGAs in mediating Golgi to lysosomal trafficking is well established, the subcellular localization and function of TOM1 remain unknown. Glutathione S-transferase pull-down assays as well as co-immunoprecipitation experiments confirmed that the carboxyl-terminal half of endofin binds specifically to the carboxyl-terminal region of TOM1. Neither SARA nor Hrs, two other FYVE domain proteins, interact with this region of TOM1. Moreover, endofin does not interact with the analogous region of two other members of the TOM1 protein family, namely, TOM1-like 1 (TOM1-L1) or TOM1-like 2 (TOM1-L2). The carboxyl-terminal region of TOM1 was used as immunogen to generate TOM1-specific antibody. This antibody can distinguish TOM1 from the other family members as well as coimmunoprecipitate endogenous endofin. It also revealed the primarily cytosolic distribution of TOM1 in a variety of cell types by immunofluorescence analyses. In addition, sucrose density gradient analysis showed that both TOM1 and endofin can be detected in cellular compartments marked by the early endosomal marker EEA1. A marked recruitment of TOM1 to endosomes was observed in cells overexpressing endofin or its carboxylterminal fragment, indicating TOM1 to be an effector for endofin and suggesting a possible role for TOM1 in endosomal trafficking.Endofin is a member of the FYVE domain family of proteins. The FYVE finger domain (1) is involved in interaction with the phospholipid phosphatidylinositol 3-phosphate, which is found in significant levels on endosomal membranes (2). 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. In the case of endofin, the FYVE domain sits in the middle of the molecule, thereby effectively dividing the molecule into two halves.Apart from a few exceptions like the proteins FGD1 (faciogenital dysplasia gene product 1) (4), Frabin (5), and DFCP1 (double FYVE-containing protein 1) (6), members of the mammalian FYVE domain family have, in general, two characteristics in common: the presence of the FYVE domain and the consequential location of the proteins on endosomal membranes. The presence of these proteins on endosomal structures imply that they have roles in trafficking and/or signaling involving e...

Endofin recruits clathrin to early endosomes via TOM1

“…In marked contrast to the GGAs, Hrs and STAMs, TOM1 and the related TOM1-L1 and TOM1-L2 are primarily distributed to the cytosol (Seet et al, 2004;Yamakami et al, 2003) (our unpublished observations), suggesting that either they function in the cytosol or their recruitment to specific membranes is highly regulated. Interestingly, TOM1 was recently reported to bind directly to ubiquitin, clathrin and tollip, a mediator of interleukin-1 signaling (Yamakami et al, 2003).…”

“…The construct containing glutathione S-transferase fused with the Cterminus of TOM1 (amino acids 300-492) [GST-TOM1(300-492)] has been described previously (Seet et al, 2004). All other GST fusion constructs containing increasingly truncated TOM1 C-terminus sequences or specific mutations were generated by PCR or by direct ligation of oligonucleotides onto the pGEX-4T-1 vector (Amersham Biosciences).…”

“…Anti-dynamin (Hudy 1) was also from Upstate Biotech and anti-AP2 and anti-AP3 were from Affinity Bioreagents and BD Transduction Laboratories, respectively. The rabbit anti-TOM1 antibody was raised as described previously (Seet et al, 2004). The AffiniPure rabbit anti-mouse IgG obtained from Jackson ImmunoResearch Laboratories was dialyzed extensively against PBS before being used for microinjecting cells.…”

“…It is recruited predominantly onto the early endosome and seems to regulate trafficking in the endocytic pathway, although the precise mechanism remains elusive. Major progress in defining the role and mechanism of the action of endofin in the endocytic pathway was made with the recent finding that endofin interacts with TOM1 (Seet et al, 2004). As this interaction leads to the recruitment of cytosolic TOM1 to the endosome, one of the roles of endofin is thought to involve the regulation of dynamic TOM1 recruitment onto the endosomes.…”

“…Our recent demonstration that TOM1 is recruited onto the endosome by its interaction with endofin (Seet et al, 2004) suggests that other TOM1-interacting proteins may be recruited to the endosomal membrane via endofin. In this report, we have addressed the biological consequence of endosomal recruitment of TOM1.…”