The ubiquitin-interacting motifs target the endocytic adaptor protein epsin for ubiquitination.
The covalent attachment of ubiquitin to proteins is an evolutionarily conserved signal for rapid protein degradation. However, additional cellular functions for ubiquitination are now emerging, including regulation of protein trafficking and endocytosis. For example, recent genetic studies suggested a role for ubiquitination in regulating epsin, a modular endocytic adaptor protein that functions in the assembly of clathrin-coated vesicles; however, biochemical evidence for this notion has been lacking. Epsin consists of an epsin NH(2)-terminal homology (ENTH) domain that promotes the interaction with phospholipids, several AP2 binding sites, two clathrin binding sequences, and several Eps15 homology (EH) domain binding motifs. Interestingly, epsin also possesses several recently described ubiquitin-interacting motifs (UIMs) that have been postulated to bind ubiquitin. Here, we demonstrate that epsin is predominantly monoubiquitinated and resistant to proteasomal degradation. The UIMs are necessary for epsin ubiquitination but are not the site of ubiquitination. Finally, we demonstrate that the isolated UIMs from both epsin and an unrelated monoubiquitinated protein, Eps15, are sufficient to promote ubiquitination of a chimeric glutathione-S-transferase (GST)-UIM fusion protein. Thus, our data suggest that UIMs may serve as a general signal for ubiquitination.
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Carla E
CE
Oldham
Robert P
RP
Mohney
Stephanie L H
SL
Miller
Richard N
RN
Hanes
John P
JP
O'Bryan
D033942:Adaptor Proteins, Vesicular Transport; D020816:Amino Acid Motifs; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila; D004705:Endocytosis; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D033921:Vesicular Transport Proteins; D014981:Xenopus
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D009479:Neuropeptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; C114154:epsin