Characterization of erasin (UBXD2): a new ER protein that promotes ER-associated protein degradation

Abstract

Ubiquitin regulator-X (UBX) is a discrete protein domain that binds p97/valosin-containing protein (VCP), a molecular chaperone involved in diverse cell processes, including endoplasmic-reticulum-associated protein degradation (ERAD). Here we characterize a human UBX-containing protein, UBXD2, that is highly conserved in mammals, which we have renamed erasin. Biochemical fractionation, immunofluorescence and electron microscopy, and protease protection experiments suggest that erasin is an integral membrane protein of the endoplasmic reticulum and nuclear envelope with both its N- and C-termini facing the cytoplasm or nucleoplasm. Localization of GFP-tagged deletion derivatives of erasin in HeLa cells revealed that a single 21-amino-acid sequence located near the C-terminus is necessary and sufficient for localization of erasin to the endoplasmic reticulum. Immunoprecipitation and GST-pulldown experiments confirmed that erasin binds p97/VCP via its UBX domain. Additional immunoprecipitation assays indicated that erasin exists in a complex with other p97/VCP-associated factors involved in ERAD. Overexpression of erasin enhanced the degradation of the ERAD substrate CD3δ, whereas siRNA-mediated reduction of erasin expression almost completely blocked ERAD. Erasin protein levels were increased by endoplasmic reticulum stress. Immunohistochemical staining of brain tissue from patients with Alzheimer's disease and control subjects revealed that erasin accumulates preferentially in neurons undergoing neurofibrillary degeneration in Alzheimer's disease. These results suggest that erasin may be involved in ERAD and in Alzheimer's disease.

Selenoprotein K Binds Multiprotein Complexes and Is Involved in the Regulation of Endoplasmic Reticulum Homeostasis

“…2, A and E). Our observation that SelK is specific for Derlin-1 adds SelK to the list of candidates that mediate the association of p97 with the ER membrane proteins, including GP78, HRD1 (16,20), erasin (21), and SelS (14 -16). Furthermore, the different SelK and SelS specificity for Derlins could determine the nature of the substrate that is translocated through the specific channel.…”

“…Extraction of many misfolded, ubiquitinated proteins to the cytosol requires the presence of a p97 (also known as VCP) complex, a hexameric, cytosolic ATPase containing two cofactors (Ufd1 and Npl4) (19). This complex may be associated with the site of ubiquitination on the ER membrane by interaction with either ER membrane integral ubiquitin ligases, for instance, GP78 and HRD1 (16,20), or with other integral proteins, such as Derlin-1 (16); erasin, also known as UBXD2 (21); and selenoprotein S (SelS) (22), also known as VIMP (14 -16).…”

Protein Folding and Quality Control in the ER

“…p97 binds to several ERAD components, including Derlin-1, VIMP (SelS), UBXD2 (Erasin), and UBXD8, and recruits several ubiquitin-chain modifiers including E3 ligases (gp78, HRD1, etc. ), chain elongation factors (Ufd2, E4 ubiquitin ligase), and deubiquitinases (YOD1, Ataxin-3, VCIP135) (Liang et al 2006;Mueller et al 2008;Schuberth and Buchberger 2008;Ernst et al 2009). Ubiquitinated substrates are transferred to the proteasome by shuttle proteins, known as HR23A/B or Ubiquilin-1 (Rad23 and Dsk2 in yeast), which contain ubiquitinassociated (UBA) and ubiquitin-like (UBL) domains that bind to polyubiquitin chains and the proteasome subunits (Rpn10/13, Rpt5), respectively (Deveraux et al 1994;Lam et al 2002;Raasi and Wolf 2007;Husnjak et al 2008;Finley 2009;Lim et al 2009).…”

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

“…Opti-Prep Gradient Fractionation-Opti-Prep gradient fractionation was performed as previously described (46). SH-SY5Y cells were homogenized in 1 ml of fractionation buffer (10 mM HEPES/KOH, pH 7.4, 1 mM EDTA) containing 250 mM sucrose.…”