title: Destabilization of the VCP-Ufd1-Npl4 complex is associated with decreased levels of ERAD substrates.
abstract: p97/VCP associated with Ufd1-Npl4 is considered a key player in ER-associated degradation (ERAD). RNA interference (RNAi) of one component of the Ufd1-Npl4 heterodimer destabilizes the VCP-Ufd1-Npl4 complex inducing proteasome-dependent degradation of the other component and releasing free VCP. In contrast to RNAi of VCP, RNAi of Ufd1 or Npl4 depleting approximately 90% of the VCP-Ufd1-Npl4 complexes does not induce unfolded protein response, indicating that the Ufd1-Npl4 dimer is not involved in the regulation of ER function by VCP. RNAi of Ufd1 or Npl4 is associated with a 2-fold increase in the levels of polyubiquitinated proteins, which form dispersed aggregates often associated with calnexin-positive structures. However, contrary to the effects of proteasome inhibition, RNAi of Ufd1 or Npl4 does not induce an accumulation of alpha-TCR and delta-CD3, two ERAD substrates overexpressed in HeLa cells. Instead, a 60-70% decrease in their levels is observed. The decrease in alpha-TCR levels is associated with a 50% decrease of its half-life. Upregulation of the putative channel forming protein, derlin-1, may contribute to the increased degradation of ERAD substrates. To explain our findings, we propose a model, where association of emerging ERAD substrates with VCP-Ufd1-Npl4 is not required for their degradation but has a regulatory role.
authors.affiliation: Department of Anatomy and Cell Biology, Indiana University School of Medicine-Evansville, 8600 University Boulevard, Evansville, IN 47712, USA.
authors.forename: Dominika
authors.initials: D
authors.lastname: Nowis
authors.forename: Elizabeth
authors.initials: E
authors.lastname: McConnell
authors.forename: Cezary
authors.initials: C
authors.lastname: Wójcik
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport; D000251:Adenosine Triphosphatases; D017252:CD3 Complex / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D034622:RNA Interference; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000378:metabolism; D013379:Substrate Specificity; D014452:Ubiquitins / Q000378:metabolism*; D015854:Up-Regulation; D000074405:Valosin Containing Protein
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D017252:CD3 Complex; C472562:CD3delta antigen; D018797:Cell Cycle Proteins; C487709:DERL1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C514112:NPLOC4 protein, human; D009687:Nuclear Proteins; D011506:Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; C491497:UFD1 protein, human; D014452:Ubiquitins; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein