The type II iodothyronine deiodinase (D2) is a type I endoplasmic reticulum 
(ER)-resident thioredoxin fold-containing selenoprotein that activates thyroid 
hormone. D2 is inactivated by ER-associated ubiquitination and can be 
reactivated by two ubiquitin-specific peptidase-class D2-interacting 
deubiquitinases (DUBs). Here, we used D2-expressing cell models to define that 
D2 ubiquitination (UbD2) occurs via K48-linked ubiquitin chains and that 
exposure to its natural substrate, T4, accelerates UbD2 formation and 
retrotranslocation to the cytoplasm via interaction with the p97-ATPase complex. 
D2 retrotranslocation also includes deubiquitination by the p97-associated DUB 
Ataxin-3 (Atx3). Inhibiting Atx3 with eeyarestatin-I did not affect D2:p97 
binding but decreased UbD2 retrotranslocation and caused ER accumulation of 
high-molecular weight UbD2 bands possibly by interfering with the 
D2-ubiquitin-specific peptidases binding. Once in the cytosol, D2 is delivered 
to the proteasomes as evidenced by coprecipitation with 19S proteasome subunit 
S5a and increased colocalization with the 20S proteasome. We conclude that 
interaction between UbD2 and p97/Atx3 mediates retranslocation of UbD2 to the 
cytoplasm for terminal degradation in the proteasomes, a pathway that is 
accelerated by exposure to T4.