Monoubiquitylation of GGA3 by hVPS18 regulates its ubiquitin-binding ability.
GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins), constitute a family of monomeric adaptor proteins and are associated with protein trafficking from the trans-Golgi network to endosomes. Here, we show that GGA3 is monoubiquitylated by a RING-H2 type-ubiquitin ligase hVPS18 (human homologue of vacuolar protein sorting 18). By in vitro ubiquitylation assays, we have identified lysine 258 in the GAT domain as a major ubiquitylation site that resides adjacent to the ubiquitin-binding site. The ubiquitylation is abolished by a mutation in either the GAT domain or ubiquitin that disrupts the GAT-ubiquitin interaction, indicating that the ubiquitin binding is a prerequisite for the ubiquitylation. Furthermore, the GAT domain ubiquitylated by hVPS18 no longer binds to ubiquitin, indicating that ubiquitylation negatively regulates the ubiquitin-binding ability of the GAT domain. These results suggest that the ubiquitin binding and ubiquitylation of GGA3-GAT domain are mutually inseparable through a ubiquitin ligase activity of hVPS18.
Yogosawa
Satomi
S
Department of Neurochemistry, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan.
Kawasaki
Masato
M
Wakatsuki
Soichi
S
Kominami
Eiki
E
Shiba
Yoko
Y
Nakayama
Kazuhisa
K
Kohsaka
Shinichi
S
Akazawa
Chihiro
C
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; C407306:GGA adaptor proteins; D025801:Ubiquitin; C424437:VPS18 protein, human; D033921:Vesicular Transport Proteins; D044767:Ubiquitin-Protein Ligases; D020727:ADP-Ribosylation Factors; D008239:Lysine