An excellent monitoring system for surface ubiquitination-induced internalization in mammals.
BACKGROUND
At present, it is difficult to visualize the internalization of surface receptors induced by ubiquitination that is taken place at the plasma membrane in mammals. This problem makes it difficult to reveal molecular basis for ubiquitination-mediated internalization in mammals.


RESULTS
In order to overcome it, we have generated T-REx-c-MIR, a novel mammalian Tet-on B cell line using a constitutively active E3 ubiquitin ligase, c-MIR, and its artificial target molecule. By applying the surface biotinylation method to T-REx-c-MIR, we succeeded to monitor the fate of surface target molecules after initiation of ubiquitination process by doxycycline (Dox)-induced c-MIR expression. Target molecules that pre-existed at the plasma membrane before induction of c-MIR expression were oligo-ubiquitinated and degraded by Dox-induced c-MIR expression. Dox-induced c-MIR expression initiated rapid internalization of surface target molecules, and blockage of the internalization induced the accumulation of the surface target molecules that were newly ubiquitinated by c-MIR. Inhibition of the surface ubiquitination by down-regulating ubiquitin conjugating enzyme E2 impaired the internalization of target molecules. Finally, a complex of c-MIR and target molecule was detected at the plasma membrane.


CONCLUSIONS
These results demonstrate that in T-REx-c-MIR, surface target molecule is ubiquitinated at the plasma membrane and followed by being internalized from the plasma membrane. Thus, T-REx-c-MIR is a useful experimental tool to analyze how surface ubiquitination regulates internalization in mammals.
Goto
Eiji
E
Laboratory for Infectious Immunity, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan.
Mito-Yoshida
Mari
M
Uematsu
Mika
M
Aoki
Masami
M
Matsuki
Yohei
Y
Ohmura-Hoshino
Mari
M
Hotta
Hak
H
Miyagishi
Makoto
M
Ishido
Satoshi
S
D016827:CD8 Antigens / Q000378:metabolism; D002460:Cell Line; D004705:Endocytosis; D005434:Flow Cytometry; D006801:Humans; D025801:Ubiquitin / Q000378:metabolism*
D016827:CD8 Antigens; D025801:Ubiquitin