Lys¹¹- and Lys⁴⁸-linked ubiquitin chains interact with p97 during endoplasmic-reticulum-associated degradation

The ATPase associated with various cellular activities p97 has a critical function in the cytoplasmic degradation of proteins misfolded in the ER (endoplasmic reticulum) through a mechanism known as ERAD (ER-associated degradation). During this process, p97 binds polyubiquitinated ERAD substrates and couples ATP hydrolysis to their dislocation from the ER as a prerequisite to destruction by the proteasome. The ubiquitin signals important for this process are not fully understood. In the present paper we report that p97 interacts with Lys¹¹- and Lys⁴⁸-linked ubiquitin polymers, but not those containing Lys⁶³ linkages. Disruption of p97 through siRNA-mediated depletion, dominant-negative overexpression or chemical inhibition results in the accumulation of Lys¹¹ and Lys⁴⁸ ubiquitin chains predominantly at the ER membrane, and is associated with ER stress induction. We show that a catalytically inactive deubiquitinating enzyme and p97 cofactor YOD1 enhances the accumulation of Lys¹¹- and Lys⁴⁸-linked polyubiquitin in the cytoplasm, at the ER membrane and bound to p97. In addition to general effects on p97-associated ubiquitin polymers, the ERAD substrate CD3δ is modified with both Lys¹¹ and Lys⁴⁸ ubiquitin chains prior to p97-dependent dislocation. Collectively, the results of the present study are consistent with a major role for p97 in the recognition of Lys¹¹ and Lys⁴⁸ polyubiquitinated proteins before their degradation by the proteasome.