Mechanistic insights into ER-associated protein degradation

Abstract

Misfolded proteins of the endoplasmic reticulum (ER) are discarded by a conserved process, called ER-associated protein degradation (ERAD). ERAD substrates are retro-translocated into the cytosol, polyubiquitinated, extracted from the ER membrane, and ultimately degraded by the proteasome. Recent in vitro experiments with purified components have given insight into the mechanism of ERAD. ERAD substrates with misfolded luminal or intramembrane domains are moved across the ER membrane through a channel formed by the multispanning ubiquitin ligase Hrd1. Following polyubiquitination, substrates are extracted from the membrane by the Cdc48/p97 ATPase complex and transferred to the proteasome. We discuss the molecular mechanism of these processes and point out remaining open questions.

Figure 1. Overview of ERAD-L and –M

The scheme shows different steps in ERAD-L. Step 1: The N-glycan chain of a misfolded luminal glycoprotein is trimmed from eight to seven mannoses (M8 and M7, respectively) by glycosidases. Step 2: The generated terminal α1,6-linked mannose residue binds to Yos9, and the misfolded segment around the glycan attachment site binds to Hrd3. The substrate inserts into the Hrd1 channel with the help of Der1, which associates with Hrd1 through Usa1. Step 3: ERAD-M substrates are misfolded in their membrane-spanning segments (indicated by an “x”) and enter Hrd1 sideways. Step 4: Both ERAD-L and ERAD-M substrates are polyubiquitinated by Hrd1. Step 5: The Cdc48 ATPase is recruited to the ER membrane by binding of the Ufd1/Npl4 (UN) cofactor to the ubiquitin chain and by Cdc48 binding to Ubx2. Step 6: Cdc48 uses ATP hydrolysis to pull the polypeptide substrate out of the membrane, the complex of Cdc48 ATPase and substrate leaves the membrane, and a DUB trims the ubiquitin chain, allowing release of the substrate from Cdc48. Step 7: The substrate is degraded by the proteasome.

Figure 2. Structure of a Hrd1/Hrd3 complex

(A) Model of Hrd1 bound to the luminal domain of Hrd3, based on cryo-EM single-particle analysis [38]. The upper left panel shows a cartoon of the Hrd1/Hrd3 dimer, with the Hrd1 molecules in light blue and salmon, and the Hrd3 molecules in dark blue and red. The upper right panel shows a view from the cytosol. The lower left panel shows a space-filling model of the funnel of one Hrd1 molecule together with TM1 of the other. The lower right panel shows a view from the cytosol. (B) The left panel shows a cut through a space-filling model of Hrd1. Hrd1/Hrd3 allows an ERAD-M substrate to move into the cytosol (arrow). The right panel shows a cut through a space-filling model of the bacterial YidC protein, which allows TM segments to move from the cytosol into the membrane (arrow).

Figure 3. Substrate processing by the Cdc48 ATPase

The scheme shows different stages. Stage 1: The Cdc48 ATPase, containing an N domain and two ATPase domains (D1 and D2), forms a hexameric, double-ring structure that associates with one copy of the Ufd1/Npl4 (UN) cofactor. The central pore and the cis- and trans-sides are indicated. Stage 2: The ubiquitin (Ub) chain attached to a substrate (in green) binds to UN. The D1 ATPases are locked in the ATP-bound state with the N domains in the up-conformation, while the activity of the D2 ATPases is stimulated. Stage 3: The substrate is pulled through the central pore, causing polypeptide unfolding. Stage 4: The substrate is moved entirely to the trans-side of the ATPase ring. Ubiquitin is also unfolded and follows the substrate (blue line). Stage 5: ATP hydrolysis in D1 causes movement of the N domains into the down-conformation, allowing a DUB to trim the ubiquitin chain. The DUB Otu1 binds through its UBX-like domain to the N domain. Stage 6: Ubiquitin molecules emerging at the trans-side presumably refold, allowing the substrate to be recognized by shuttling factors and the proteasome.