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Title: Endofin recruits clathrin to early endosomes via TOM1
Authors: Seet, L.-F 
Hong, W 
Keywords: adaptor protein
amino acid
aspartic acid
chimeric protein
clathrin heavy chain
cytoplasm protein
glutamic acid
polyclonal antibody
protein Tom1
Smad anchor for receptor activation
unclassified drug
binding affinity
binding site
carboxy terminal sequence
cell membrane transport
cell structure
controlled study
correlation function
gene deletion
gene fusion
gene overexpression
human cell
molecular biology
priority journal
protein analysis
protein binding
protein domain
protein function
protein motif
protein protein interaction
sequence homology
Binding Sites
Cell Line
Cell Line, Tumor
Clathrin Heavy Chains
Gene Expression
Intracellular Signaling Peptides and Proteins
Microscopy, Fluorescence
Protein Binding
Recombinant Fusion Proteins
Serine Endopeptidases
Issue Date: 2005
Citation: Seet, L.-F, Hong, W (2005). Endofin recruits clathrin to early endosomes via TOM1. Journal of Cell Science 118 (3) : 575-587. ScholarBank@NUS Repository.
Rights: Attribution 4.0 International
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 (362LEDEF366). Although residues 321-326 could function independently as a weak clathrin-binding motif, deletion of amino acids 300-321 or mutation of 362Leu and 364Asp to Ala residues reduced the binding of clathrin to TOM1. A fragment lacking amino acids 300-322 and containing 362Leu and 364Asp 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.
Source Title: Journal of Cell Science
ISSN: 00219533
DOI: 10.1242/jcs.01628
Rights: Attribution 4.0 International
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