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 Nterminal 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 subcellular trafficking of ERGIC-53, including promoting movement to the cell membrane. We propose that p97-

The N-terminal Region of the Ubiquitin Regulatory X (UBX) Domain-containing Protein 1 (UBXD1) Modulates Interdomain Communication within the Valosin-containing Protein p97

“…The H1/H2 binding region is ␣-helical and conserved among several species but unique for the UBXD1 cofactor. UBXD1 has recently been shown to also bind a population of ERGIC-53 at the plasma membrane with the very N-terminal amino acids (46). Intriguingly, this region overlaps with the H1/H2 binding site for p97, and therefore it would be interesting to clarify whether these differential interactions of UBXD1 represent alternative functions or whether they are functionally linked.…”

JNK-associated Leucine Zipper Protein Functions as a Docking Platform for Polo-like Kinase 1 and Regulation of the Associating Transcription Factor Forkhead Box Protein K1

“…To that end, SILAC and a quantitative solution-based LC-MS/MS interaction proteomics analysis (34,35) were performed in cells grown in media containing heavy or light isotope amino acids (Fig. 4A).…”

A proteolytic pathway that controls glucose uptake in fat and muscle

“…This results in methylation of p97, which negatively regulates its ATPase activity. This effect may regulate p97 ATPase activity at or near the ERES/ERGIC, and control the targeting of ubiquitylated cargos in endosomal and autophagy pathways [109-111]. Possibly, the ubiquitin-dependent sorting of GLUT4 may involve this mechanism [112].…”