Abstract
TOM1 and its related proteins, TOM1-like1 (TOM1-L1) and TOM1-like2 (TOM1-L2), constitute a subfamily of the VHS domain protein family. We have recently shown that endofin, a FYVE domain protein associated with the early endosome, is able to recruit cytosolic TOM1 onto endosomal membranes. To reveal the biological consequence of endofin-mediated endosomal recruitment of TOM1, we have identified the clathrin heavy chain as a major interacting protein for TOM1. Optimal clathrin binding by TOM1 involves three sites: residues 300-321, 321-326 and a putative clathrin-binding box at residues 362-366 ((362)LEDEF(366)). Although residues 321-326 could function independently as a weak clathrin-binding motif, deletion of amino acids 300-321 or mutation of (362)Leu and (364)Asp to Ala residues reduced the binding of clathrin to TOM1. A fragment lacking amino acids 300-322 and containing (362)Leu and (364)Asp to Ala mutations lost the ability to interact with clathrin. Remarkably, overexpression of endofin led to a massive and specific recruitment of clathrin [but not dynamin, or the adaptor protein (AP) complexes, AP1, AP2 or AP3] onto endofin-positive endosomes. Although SARA is homologous to endofin, it did not interact with the C-terminal region of TOM1. Examination of chimeric proteins of endofin and SARA suggests that the C-terminal half of endofin is responsible for interaction with the C-terminal region of TOM1 and for recruitment of TOM1 and clathrin to endosomes. The correlation between the ability of endofin to interact with the C-terminal domain of TOM1 and clathrin recruitment suggests that endofin may recruit clathrin via TOM1. Indeed, a chimeric protein consisting of TOM1 fused to two FYVE domains derived from endofin has the ability to recruit clathrin onto endosomal structures. Moreover, we show that affinity-purified TOM1 antibody can abolish binding of clathrin to the C-terminal region of TOM1. Upon microinjection into cells, this antibody reduced the membrane association of clathrin. These results, taken together, suggest that TOM1 is an important molecule for membrane recruitment of clathrin, and that endofin is able to exploit this recruitment at the endosome.
Highlights
An emerging theme in cell biology is that many cytosolic proteins are dynamically recruited to the cytoplasmic side of specific membranes to mediate diverse intracellular processes
We found that TOM1 interacts with clathrin and that it bridges endofin to clathrin recruitment onto the endosome
Membrane recruitment of clathrin by TOM1 577 endosomal targeting of TOM1, we have used the GST pulldown affinity chromatography method to identify proteins that interact with the C-terminal region of TOM1
Summary
An emerging theme in cell biology is that many cytosolic proteins are dynamically recruited to the cytoplasmic side of specific membranes to mediate diverse intracellular processes. Endofin is a protein characterized by the presence of a phosphatidylinositol 3-phosphate (PI3P)-binding FYVE domain (Seet and Hong, 2001). It is recruited predominantly onto the early endosome and seems to regulate trafficking in the endocytic pathway, 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
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