Generation and Characterization of Novel Monoclonal Antibodies Recognizing UBXD1

Ramkumar, Poornima; Smith, Bennett A.; Akinbamidele, Anu C.; Kapcia, Joseph; Beauparlant, Stephen L.; Haines, Dale S.

doi:10.1089/hyb.2009.0035

Hybridoma

2009

DOI: 10.1089/hyb.2009.0035

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Generation and Characterization of Novel Monoclonal Antibodies Recognizing UBXD1

Poornima Ramkumar

Bennett A. Smith

Anu C. Akinbamidele

et al.

Abstract: UBXD1 is a recently identified adaptor for p97, a highly abundant and conserved member of the AAA family of ATPase that plays pivotal roles in a multitude of cellular processes involving the ubiquitin-proteasome pathway. Very little is known about the biochemical, cellular, and molecular functions of UBXD1. Here we report the generation of two mouse monoclonal antibodies, 5C3-1 and 2F8-24, that recognize UBXD1 using Western blotting, immunoprecipitation, and immunofluorescence.

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2012

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“…Constructs encoding amino-terminal FLAG tagged adaptors have been described previously (19). Antibodies used in experiments presented here are anti-FLAG mouse monoclonal antibody M2 (SIGMA), anti-UBXD1 mouse monoclonal antibody 5C3-1 (22), anti-ERGIC-53 H-245 rabbit polyclonal (Santa Cruz, Santa Cruz, CA), anti-p97 H-120 rabbit polyclonal (Santa Cruz), anti-Rab3GAP1 rabbit polyclonal (Novus, Littleton, CO), and anti-Rab3-GAP2 rabbit polyclonal (GeneTex, San Antonio, TX).…”

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Protein Interaction Profiling of the p97 Adaptor UBXD1 Points to a Role for the Complex in Modulating ERGIC-53 Trafficking

Haines

Lee

Beauparlant³

et al. 2012

Molecular & Cellular Proteomics

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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-

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confidence: 99%

Protein Interaction Profiling of the p97 Adaptor UBXD1 Points to a Role for the Complex in Modulating ERGIC-53 Trafficking

Haines

Lee

Beauparlant³

et al. 2012

Molecular & Cellular Proteomics

Self Cite

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Generation and Characterization of Novel Monoclonal Antibodies Recognizing UBXD1

Cited by 1 publication

References 11 publications

Protein Interaction Profiling of the p97 Adaptor UBXD1 Points to a Role for the Complex in Modulating ERGIC-53 Trafficking

Protein Interaction Profiling of the p97 Adaptor UBXD1 Points to a Role for the Complex in Modulating ERGIC-53 Trafficking

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