Proteasomes of Autophagy-Deficient Cells Exhibit Alterations in Regulatory Proteins and a Marked Reduction in Activity.

Autophagy and the ubiquitin proteasome system are the two major processes for the clearance and recycling of proteins and organelles in eukaryotic cells. Evidence is accumulating that there is extensive crosstalk between the two pathways, but the underlying mechanisms are still unclear. We previously found that autophagy 9 (ATG9) and 16 (ATG16) proteins are crucial for full proteasomal activity in the unicellular amoeba Dictyostelium discoideum. In comparison to AX2 wild-type cells, ATG9-and ATG16- cells displayed a 60%, and ATG9-/16- cells a 90%, decrease in proteasomal activity. Mutant cells also showed a significant increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates. Here, we focus on possible reasons for these results. Reanalysis of published tandem mass tag-based quantitative proteomic results of AX2, ATG9-, ATG16-, and ATG9-/16- cells revealed no change in the abundance of proteasomal subunits. To identify possible differences in proteasome-associated proteins, we generated AX2 wild-type and ATG16- cells expressing the 20S proteasomal subunit PSMA4 as GFP-tagged fusion protein, and performed co-immunoprecipitation experiments followed by mass spectrometric analysis. The results revealed no significant differences in the abundance of proteasomes between the two strains. However, we found enrichment as well as depletion of proteasomal regulators and differences in the ubiquitination of associated proteins for ATG16-, as compared to AX2 cells. Recently, proteaphagy has been described as a means to replace non-functional proteasomes. We propose that autophagy-deficient D. discoideum mutants suffer from inefficient proteaphagy, which results in the accumulation of modified, less-active, and also of inactive, proteasomes. As a consequence, these cells exhibit a dramatic decrease in proteasomal activity and deranged protein homeostasis.

Domain Organization of the UBX Domain Containing Protein 9 and Analysis of Its Interactions With the Homohexameric AAA + ATPase p97 (Valosin-Containing Protein).

The abundant homohexameric AAA + ATPase p97 (also known as valosin-containing protein, VCP) is highly conserved from Dictyostelium discoideum to human and a pivotal factor of cellular protein homeostasis as it catalyzes the unfolding of proteins. Owing to its fundamental function in protein quality control pathways, it is regulated by more than 30 cofactors, including the UBXD protein family, whose members all carry an Ubiquitin Regulatory X (UBX) domain that enables binding to p97. One member of this latter protein family is the largely uncharacterized UBX domain containing protein 9 (UBXD9). Here, we analyzed protein-protein interactions of D. discoideum UBXD9 with p97 using a series of N- and C-terminal truncation constructs and probed the UBXD9 interactome in D. discoideum. Pull-down assays revealed that the UBX domain (amino acids 384-466) is necessary and sufficient for p97 interactions and that the N-terminal extension of the UBX domain, which folds into a ß0-α- 1-α0 lariat structure, is required for the dissociation of p97 hexamers. Functionally, this finding is reflected by strongly reduced ATPase activity of p97 upon addition of full length UBXD9 or UBXD9261-573. Results from Blue Native PAGE as well as structural model prediction suggest that hexamers of UBXD9 or UBXD9261-573 interact with p97 hexamers and disrupt the p97 subunit interactions via insertion of a helical lariat structure, presumably by destabilizing the p97 D1:D1' intermolecular interface. We thus propose that UBXD9 regulates p97 activity in vivo by shifting the quaternary structure equilibrium from hexamers to monomers. Using three independent approaches, we further identified novel interaction partners of UBXD9, including glutamine synthetase type III as well as several actin-binding proteins. These findings suggest a role of UBXD9 in the organization of the actin cytoskeleton, and are in line with the hypothesized oligomerization-dependent mechanism of p97 regulation.