AAA ATPase p97/Valosin-containing Protein Interacts with gp78, a Ubiquitin Ligase for Endoplasmic Reticulum-associated Degradation

Abstract

Endoplasmic reticulum-associated degradation (ERAD)is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosincontaining protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of polyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3␦, gp78 consistently enhances p97/VCP-CD3␦ binding and facilitates CD3␦ degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3␦, and abrogates VCP-CD3␦ interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3␦ degradation and leads to accumulation of polyubiquitinated CD3␦, suggesting a failure in delivering ubiquitinated CD3␦ for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.

The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site

“…Similarly, a truncation in which the G2BR is interrupted (595) stabilized gp78. A truncation that retained E2 binding (611) but lost the reported binding of gp78 to p97 (34) (Fig. 10, which is published as supporting information on the PNAS web site) was indistinguishable from WT gp78 in its own degradation (Fig.…”

Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

“…Similar binding was also observed for Rfm gp78-Myc (data not shown). Notably, p97/VCP, which cooperates with gp78 in ERAD (Zhong et al, 2004;Ballar et al, 2006), coimmunoprecipitated with CFTR⌬F508 when gp78-Myc was expressed in cells (Supplemental Figure 2). These results indicated that gp78 associates with CFTR⌬F508 and that it recruits p97 to CFTR⌬F508 complexes.…”

“…The association of gp78 with CFTR⌬F508 was mediated by its intrinsic CUE domain, which also has ubiquitin binding activity (Supplemental Figure 4; Zhong et al, 2004;Chen et al, 2006). To determine whether the ubiquitin binding region of the CUE domain overlapped the CFTR⌬F508 binding region, we generated a gp78 mutant in which the ubiquitin binding site in the CUE domain, MFP (amino acids 463-465), was mutated to AAR.…”

Unsupervised proteome analysis of human leukaemia cells identifies the Valosin-containing protein as a putative marker for glucocorticoid resistance

“…All proteins had been previously linked to malignancies and are involved in cellular processes like cell growth (RING finger protein and protein kinase C), regulation of the biological homeostasis including haematopoiesis and immunity (G-protein-coupled receptors), apoptosis (VCP) and processes related to cell division (Malate dehydrogenase). 9,11,[33][34][35] Since quantitative 2-DE was not performed, our results only indicate that these proteins are more frequently present in the average proteome of one responder group. This does not exclude that the respective protein will not be expressed in patients belonging to the other responder group, because proteins with a low expression level, for example, might accidentally escape software recognition.…”

“…40 Interaction of ubiquitin with the UT3 domain of the Ufd1 enhances VCPpolyubiquitin binding. 33 Furthermore, it regulates TNF-induced apoptosis and NFkB activation via IkBa degradation. 14 Thus, increased VCP expression in PPR could lead to a decreased apoptosis of leukaemic blasts.…”