G Proteins (Small)

The p97 D1 and D2 domains are depicted in dark and light pink, respectively, and the N domain in yellow

The p97 D1 and D2 domains are depicted in dark and light pink, respectively, and the N domain in yellow. position of p97 is indicated. *Positions of GST and of full-length UBXD9 and UBXD9 truncation constructs tagged with GST. Image_2.TIF (802K) GUID:?8E9DD6F0-6438-4ED0-A98F-A86A8C203359 Supplementary Figure 3: UBXD9 and UBXD9261C573 reduce the ATPase activity of p97. Data is presented as relative ATPase activity, mean values and SD of three experiments. For statistical analysis, the Dunnetts multiple comparison test, implemented in GraphPad Prism as analysis, was performed. *** 0.001. Image_3.TIF (83K) GUID:?7035FC09-122D-498F-AC14-DCF359F8DB8F Supplementary Figure 4: Analysis of IP experiments and proximity labeling proteomics. (A) GFP trap experiments with the soluble proteins from total cell lysates Erythrosin B of AX2 cells expressing GFP, UBXD9-GFP, or GFP-UBXD9. Top: SDS-PAGE and silver stain of proteins bound to the beads (Pellet). *Indicates the position of GFP-UBXD9 and # of GFP. +Indicates the position of the absent UBXD9-GFP silver band and of the visible UBXD9-GFP immunoblot band (see Western Blot). Bottom: Western blotting of proteins bound to the beads (Pellet). Endogenous UBXD9 of 95 kDa and GFP-tagged UBXD9 of 120 kDa were detected with the polyclonal “type”:”entrez-protein”,”attrs”:”text”:”UBX23520″,”term_id”:”2105906393″,”term_text”:”UBX23520″UBX23520 antibody. *Indicates the position of GFP-tagged UBXD9 and o of untagged UBXD9. (B) BioID experiments with soluble proteins from total cell lysates of AX2 and AX2/BirA-UBXD9 cells. Top: SDS-PAGE and silver stain of soluble proteins before (S1) and after (S2) incubation with streptavidin sepharose beads and proteins bound to the beads (Pellet). The position of BirA-UBXD9 is indicated. Bottom: Western blotting Erythrosin B of soluble proteins after (S2) incubation with streptavidin sepharose beads and of proteins bound to the beads (Pellet). Endogenous UBXD9 and BirA-tagged UBXD9 were detected with the polyclonal “type”:”entrez-protein”,”attrs”:”text”:”UBX23520″,”term_id”:”2105906393″,”term_text”:”UBX23520″UBX23520 antibody (top panel) and p97 with the polyclonal p97_8_6842 antibody (lower panel). Image_4.TIF (1.3M) GUID:?4888A8E6-BFBD-4B78-B2FA-1CAD08F36F6D Data_Sheet_1.PDF (87K) GUID:?62362BD9-3977-454B-840F-ED738EB78BD6 Data Availability StatementThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can Erythrosin B be found below:; Abstract The abundant homohexameric AAA + ATPase p97 (also known as valosin-containing protein, VCP) is highly conserved from Erythrosin B 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 UBXD9 with Erythrosin B p97 using a series of N- and C-terminal truncation constructs and probed the UBXD9 interactome in 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. p97. (A) p97 domain organization. N, N domain (yellow); D1, ATPase domain D1 (purple); D2, ATPase domain D2 (pink). Numbers indicate amino acid positions. (B) Schematic representation of the structure of the p97 homohexamer. Each monomer comprises a globular N domain (N) depicted in yellow and two ATPase domains, D1 (purple) and D2 (pink), forming two stacked rings. Surrounded by the rings, a central pore forms, which extends from the side (D1 domain) through the entire protein to the side (D2 domain). (C) Conformational states of the N domains are dependent on the nucleotide bound state of the D1 domains. Left: bound ADP induces down-conformation. Right: bound ATP induces up-conformation. For a long time, p97 was considered only as a segregase that extracts target proteins from complexes or membranes by ATP hydrolysis, Rabbit Polyclonal to Collagen II so that they can be degraded by the proteasome (Ye, 2006). However, now it is clear that the fundamental function of p97 is the unfolding of proteins in numerous protein.