title: Protein interaction profiling of the p97 adaptor UBXD1 points to a role for the complex in modulating ERGIC-53 trafficking.
abstract: UBXD1 is a member of the poorly understood subfamily of p97 adaptors that do not harbor a ubiquitin association domain or bind ubiquitin-modified proteins. Of clinical importance, p97 mutants found in familial neurodegenerative conditions Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia and Amyotrophic Lateral Sclerosis are defective at interacting with UBXD1, indicating that functions regulated by a p97-UBXD1 complex are altered in these diseases. We have performed liquid chromatography-mass spectrometric analysis of UBXD1-interacting proteins to identify pathways in which UBXD1 functions. UBXD1 displays prominent association with ERGIC-53, a hexameric type I integral membrane protein that functions in protein trafficking. The UBXD1-ERGIC-53 interaction requires the N-terminal 10 residues of UBXD1 and the C-terminal cytoplasmic 12 amino acid tail of ERGIC-53. Use of p97 and E1 enzyme inhibitors indicate that complex formation between UBXD1 and ERGIC-53 requires the ATPase activity of p97, but not ubiquitin modification. We also performed SILAC-based quantitative proteomic profiling to identify ERGIC-53 interacting proteins. This analysis identified known (e.g. COPI subunits) and novel (Rab3GAP1/2 complex involved in the fusion of vesicles at the cell membrane) interactions that are also mediated through the C terminus of the protein. Immunoprecipitation and Western blotting analysis confirmed the proteomic interaction data and it also revealed that an UBXD1-Rab3GAP association requires the ERGIC-53 binding domain of UBXD1. Localization studies indicate that UBXD1 modules the sub-cellular trafficking of ERGIC-53, including promoting movement to the cell membrane. We propose that p97-UBXD1 modulates the trafficking of ERGIC-53-containing vesicles by controlling the interaction of transport factors with the cytoplasmic tail of ERGIC-53.
authors.affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 North Broad Street, Philadelphia, 19104 Pennsylvania, USA.
authors.forename: Dale S
authors.initials: DS
authors.lastname: Haines
authors.forename: J Eugene
authors.initials: JE
authors.lastname: Lee
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Beauparlant
authors.forename: Dane B
authors.initials: DB
authors.lastname: Kyle
authors.forename: Willem
authors.initials: W
authors.lastname: den Besten
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sweredoski
authors.forename: Robert L J
authors.initials: RL
authors.lastname: Graham
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071183:Autophagy-Related Proteins; D001565:Benzoates / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D005663:Furans / Q000494:pharmacology; D006801:Humans; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D021381:Protein Transport; D011720:Pyrazoles / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D022142:Secretory Vesicles / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism
chemical_list: C523934:4(4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl)-benzoic acid ethyl ester; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000071183:Autophagy-Related Proteins; D001565:Benzoates; D002352:Carrier Proteins; D005663:Furans; C084004:LMAN1 protein, human; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; C558357:N2,N4-dibenzylquinazoline-2,4-diamine; D009687:Nuclear Proteins; D011720:Pyrazoles; D011799:Quinazolines; C533572:UBXN6 protein, human; D000251:Adenosine Triphosphatases; C404942:p97 ATPase; D020692:rab3 GTP-Binding Proteins; D044764:Ubiquitin-Activating Enzymes