Protein interaction profiling of the p97 adaptor UBXD1 points to a role for the complex in modulating ERGIC-53 trafficking

Dale S Haines, J Eugene Lee, Stephen L Beauparlant, Dane B Kyle, Willem den Besten, Michael J Sweredoski, Robert L J Graham, Sonja Hess, Raymond J Deshaies

Research output: Contribution to journalArticle

22 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)M111.016444
JournalMolecular & cellular proteomics : MCP
Volume11
Issue number6
DOIs
Publication statusPublished - Jun 2012

Keywords

  • Adenosine Triphosphatases/antagonists & inhibitors
  • Benzoates/pharmacology
  • Carrier Proteins/chemistry
  • Cell Line, Tumor
  • Furans/pharmacology
  • Humans
  • Mannose-Binding Lectins/chemistry
  • Membrane Proteins/chemistry
  • Nuclear Proteins/antagonists & inhibitors
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping
  • Protein Transport
  • Pyrazoles/pharmacology
  • Quinazolines/pharmacology
  • Secretory Vesicles/metabolism
  • Ubiquitin-Activating Enzymes/antagonists & inhibitors
  • rab3 GTP-Binding Proteins/metabolism

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