Peripheral endoplasmic reticulum localization of the Gp78 ubiquitin ligase activity.
Gp78 (also known as AMFR and RNF45) is an E3 ubiquitin ligase that targets proteins for proteasomal degradation through endoplasmic reticulum (ER)-associated degradation (ERAD). In this study, we showed that gp78-mediated ubiquitylation is initiated in the peripheral ER. Substrate monoubiquitylation and gp78 CUE domain integrity restricted substrate to the peripheral ER, where CUE domain interactions and polyubiquitylation reduced gp78 mobility. Derlin-1 and derlin-2, which are involved in the retrotranslocation of ERAD substrates, localized to a central, juxtanuclear ER domain, where polyubiquitylated proteins accumulated upon proteasome inhibition. Transfer of polyubiquitylated substrate to the central ER was dependent on ubiquitin chain elongation and recruitment of the AAA ATPase p97 (also known as VCP). HT-1080 fibrosarcoma cells expressed elevated levels of endogenous gp78, which was associated with segregation of ubiquitylated substrate to the peripheral ER and its polyubiquitin-dependent redistribution to the central ER upon proteasome inhibition. Therefore, the peripheral ER is the site of gp78 ubiquitin ligase activity. Delivery of ubiquitylated substrate to the central ER was regulated by ubiquitin chain elongation and opposing actions of gp78 CUE domain interactions and p97 recruitment.
University of British Columbia, Life Sciences Institute, Vancouver, BC, Canada.
Pascal
P
St-Pierre
Thao
T
Dang
Bharat
B
Joshi
Ivan R
IR
Nabi
D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D021381:Protein Transport; D060285:Receptors, Autocrine Motility Factor / Q000378:metabolism*
C555000:AMFR protein, human; D060285:Receptors, Autocrine Motility Factor