Fig. 4.

Schematic summary of the spatial arrangement of p97-Ufd1-Npl4 in the ERAD pathway. The ER membrane (wavy gray line) hosts numerous transmembrane proteins that could be potential sites for the retrotranslocation of ERAD substrates (represented as blue rectangles). p97 D1 D2 domains are represented as purple hexagons, and N domains are represented as red cylinders. Ufd1-Npl4 is represented as a pink bilobe shape, which we suggest is interacting with a p97 N domain via its thinnest central region. We speculate that the binding of one Ufd1-Npl4 complex to p97 causes a rearrangement of other N domains to prevent further Ufd1-Npl4 binding and to promote interactions with other proteins (represented by different orientation of “red cylinder” N domains and red arrows). Membrane-bound proteins that have been shown to bind p97-Ufd1-Npl4 via p97 N domains are VIMP in mammals (13) and Ubx2 in yeast (14, 15). In yeast, p97-Ufd1-Npl4 can also interact with the E4 ligase, Ufd2 (in competition with Ufd3) via p97ΔN (blue arrows) (9, 43), and the deubiquitinating enzyme, Otu1 (simultaneously with Ufd3) via the p97 N domain (43). p97-Ufd1-Npl4 recognizes the ERAD substrate via 1-2 ubiquitin moieties (green circles) (9) at one region of Ufd1-Npl4 (UT3 domain). The green “fuzz” represents the ERAD substrate which may also interact with p97 itself. The green arrow represents recognition and movement of the ERAD substrate from the lumen to the cytosolic face of the ER. The red star represents the action of the E1-E3 enzymes on the ERAD substrate, covalently attaching ubiquitin moieties; the green star and green rings represent the possibility of further ubiquitin moieties being added [e.g., by E4 enzyme, Ufd2 (9)] and interacting with the Ufd1-Npl4-bound p97 N domain (although interactions with other N domains are not excluded). The action of the p97-Ufd1-Npl4 complex “pulling” the substrate into the cytosol (pink arrow) is essential for the delivery of the ERAD substrate to the 26S proteasome. Figure is not to drawn scale.