A balance between protein synthesis and degradation is necessary to maintain cellular homeostasis. Failure to triage aberrant proteins may result in their accumulation and aggregation in the cytosol. The VCP-BAG6 complex facilitates a wide variety of ubiquitin-mediated quality control events at the ER; both prior to ER translocation as well as during ER associated degradation (ERAD). Yet, how ubiquitylated clients associated with BAG6 are recognized by VCP for proteasomal degradation is presently unknown. We have identified UBXN1 as the VCP adaptor in BAG6-dependent processes occurring prior to ER insertion but not during ERAD. Loss of VCP-UBXN1 results in the inappropriate stabilization of ubiquitylated BAG6 clients; their accumulation in insoluble aggregates and sensitizes cells to proteotoxic stress. Our results identify how VCP is specifically targeted to ubiquitylated substrates in the BAG6 triage pathway and suggest that degradation of ubiquitylated clients by the proteasome is reliant on UBXN1 association with ubiquitylated substrates and VCP catalytic activity.

The recognition and disposal of misfolded proteins is essential for the maintenance of cellular homeostasis. Perturbations in the pathways that promote degradation of aberrant proteins contribute to a variety of protein aggregation disorders broadly termed proteinopathies. The p97 AAA-ATPase in combination with adaptor proteins functions to identify ubiquitylated proteins and target them for degradation by the proteasome or autophagy. Mutations in p97 cause multi-system proteinopathies; however, the precise defects underlying these disorders are unclear. Here, we systematically investigate the role of p97 and its adaptors in the process of formation of aggresomes, membrane-less structures containing ubiquitylated proteins that arise upon proteasome inhibition. We demonstrate that p97 mediates aggresome formation and clearance and identify a novel role for the adaptor UBXN1 in the process of aggresome formation. UBXN1 is recruited to aggresomes and UBXN1 knockout cells are unable to form aggresomes. Loss of p97-UBXN1 results in increased Huntingtin polyQ inclusion bodies both in mammalian cells as well as in a C.elegans model of Huntington's Disease. Together our work identifies evolutionarily conserved roles for p97-UBXN1 in the disposal of protein aggregates.

Protein quality control is maintained by a number of integrated cellular pathways that monitor the folding and functionality of the cellular proteome. Defects in these pathways lead to the accumulation of misfolded or faulty proteins that may become insoluble and aggregate over time. Protein aggregates significantly contribute to the development of a number of human diseases such as Amyotrophic lateral sclerosis, Huntington’s and Alzheimer’s Disease. In vitro, imaging-based, cellular studies have defined key biomolecular components that recognize and clear aggregates; however, no unifying method is available to quantify cellular aggregates limiting our ability to reproducibly and accurately quantify these structures. Here we describe an ImageJ macro called AggreCount to identify and measure protein aggregates in cells. AggreCount is designed to be intuitive, easy to use and customizable for different types of aggregates observed in cells. Minimal experience in coding is required to utilize the script. Based on a user defined image, AggreCount will report a number of metrics: (i) total number of cellular aggregates, (ii) percent of cells with aggregates, (iii) aggregates per cell, (iv) area of aggregates and (v) localization of aggregates (cytosol, perinuclear or nuclear). A data table of aggregate information on a per cell basis as well as a summary table is provided for further data analysis. We demonstrate the versatility of AggreCount by analyzing a number of different cellular aggregates including aggresomes, stress granules and inclusion bodies caused by Huntingtin polyQ expansion.

The p97 ATPase extracts polyubiquitylated proteins from diverse cellular structures in preparation for destruction by the proteasome. p97 functions with Ufd1-Npl4 and a variety of UBA-UBX co-factors, but how p97 complexes assemble on ubiquitylated substrates is unclear. To address this, we investigated how p97 disassembles the CMG helicase after it is ubiquitylated during replication termination. We show that p97Ufd1-Npl4 recruitment to CMG requires the UBA-UBX protein Ubxn7, and conversely, stable Ubxn7 binding to CMG requires p97Ufd1-Npl4. This cooperative assembly involves interactions between Ubxn7, p97, Ufd1-Npl4, and ubiquitin. Another p97 co-factor, Faf1, partially compensates for the loss of Ubxn7. Surprisingly, p97Ufd1-Npl4-Ubxn7 and p97Ufd1-Npl4-Faf1 also assemble cooperatively on unanchored ubiquitin chains. We propose that cooperative and substrate-independent recognition of ubiquitin chains allows p97 to recognize an unlimited number of polyubiquitylated proteins while avoiding the formation of partial, inactive complexes.