Abstract
Misfolded proteins in the lumen of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol and polyubiquitinated before being degraded by the proteasome. The multi-spanning ubiquitin ligase Hrd1 forms the retrotranslocation channel and associates with three other membrane proteins (Hrd3, Usa1, Der1) of poorly defined function. The Hrd1 channel is gated by autoubiquitination, but how Hrd1 escapes degradation by the proteasome and returns to its inactive ground state is unknown. Here, we show that autoubiquitination of Hrd1 is counteracted by Ubp1, a deubiquitinating enzyme that requires its N-terminal transmembrane segment for activity towards Hrd1. The Hrd1 partner Hrd3 serves as a brake for autoubiquitination, while Usa1 attenuates Ubp1's deubiquitination activity through an inhibitory effect of its UBL domain. These results lead to a model in which the Hrd1 channel is regulated by cycles of autoubiquitination and deubiquitination, reactions that are modulated by the other components of the Hrd1 complex.
Highlights
Proteins translocated into the endoplasmic reticulum (ER) undergo quality control, such that only folded proteins are moved through the secretory pathway
The strongest increase of Hrd1 levels was seen with Ubp1, even though several other deubiquitinating enzyme (DUB) were expressed at a higher level than Ubp1 (Figure 1—figure supplement 1A)
Our results reveal a novel mechanism by which the central ER-associated protein degradation (ERAD) component Hrd1 is regulated (Figure 4E)
Summary
Proteins translocated into the endoplasmic reticulum (ER) undergo quality control, such that only folded proteins are moved through the secretory pathway. ERAD-L substrates contain misfolded domains in the ER lumen, ERAD-M substrates are misfolded within the membrane, ERAD-C substrates are membrane proteins with misfolded cytosolic domains, and ERAD-INM handles misfolded proteins in the inner nuclear membrane These pathways use different ubiquitin ligases: ERAD-L and -M use the Hrd ligase, ERAD-C the Doa ligase, and ERAD-INM the Asi ligase complex (Carvalho et al, 2006; Foresti et al, 2014; Huyer et al, 2004; Khmelinskii et al, 2014; Vashist and Ng, 2004). All pathways converge at the Cdc ATPase (p97 or VCP in mammals) (Bays et al, 2001; Jarosch et al, 2002; Rabinovich et al, 2002; Ye et al, 2001) This ATPase cooperates with a cofactor (Ufd1/Npl4) to extract polyubiquitinated substrates from the membrane (Stein et al, 2014)
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