Endoplasmic Reticulum-associated Degradation of Pcal p, a Polytopic Protein, via Interaction with the Proteasome at the Membrane

Endoplasmic reticulum-associated degradation (ERAD) plays a critical role in the destruction of terminally misfolded proteins at the secretory pathway. The system also regulates expression levels of several proteins such as Pcalp, a cadmium exporter in yeast. To gain better insight into the mechanisms underlying ERAD of Pcalp and other polytopic proteins by the proteasome in the cytosol, our study determined the roles for the molecular factors of ERAD in dislodging Pcalp from the endoplasmic reticulum (ER). Inactivation of the 20S proteasome leads to accumulation of ubiquitinated Pcalp in the ER membrane, suggesting a role for the proteasome in extraction of Pcalp from the ER. Pcalp formed a complex with the proteasome at the membrane in a Doal0p E3 ligase-dependent manner. Cdc48p is required for recruiting the proteasome to Pcalp. Although the Ufd2p E4 ubiquitin chain extension enzyme is involved in efficient degradation of Pcalp, Ufd2p-deficient cells did not affect the formation of a complex between Pcalp and the proteasome. Two other polytopic membrane proteins undergoing ERAD, Steh*p and Hmg2p, also displayed the same outcomes observed for Pcalp. However, poly-ubiquitinated Cpyl*p, a luminal ERAD substrate, was detected in the cytosol independent of proteolytic activities of the proteasome. These results indicate that extraction and degradation of polytopic membrane proteins at the ER is a coupled event. This mechanism would relieve the cost of exposed hydrophobic domains in the cytosol during ERAD.