Analysis of Npl4 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Npl4 in ERAD

Lass, Agnieszka; McConnell, Elizabeth J.; Fleck, Krista; Palamarchuk, Alla; Wójcik, Cezary

doi:10.1016/j.yexcr.2008.06.008

Cited by 14 publications

(11 citation statements)

References 49 publications

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“…Recalling that p47 is an inhibitor of the ATPase activity of p97/VCP, the latter is more likely to remain bound to p47 when ATP is limiting and thus inhibiting hydrolysis [56]. Conversely, binding of p97/VCP to UN promotes retrotranslocation of ER proteins and their ubiquitylation and chaperoning to the 26S proteasome in the ERAD pathway [41]–[43], [57]. This suggests that in an ATP-abundant environment, p97/VCP readily binds to UN over p47, to carry out removal of misfolded proteins or participate in its different roles in the nucleus such as mitotic progression or transcription factor activation.…”

Section: Discussionmentioning

confidence: 99%

ATP Binding to p97/VCP D1 Domain Regulates Selective Recruitment of Adaptors to Its Proximal N-Domain

Chia

Rao

et al. 2012

PLoS ONE

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p97/Valosin-containing protein (VCP) is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD). It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.

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Section: Discussionmentioning

confidence: 99%

ATP Binding to p97/VCP D1 Domain Regulates Selective Recruitment of Adaptors to Its Proximal N-Domain

Chia

Rao

et al. 2012

PLoS ONE

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“…Meanwhile another cytosolic protein complex have been found, containing an AAA-ATPase: the mammalian Cdc48p (also termed as p97 or VCP) (Goder et al, 2008;Shcherbik and Haines, 2007;Wilson et al, 2006) may be a key protein in the dislocation process. In complex with two other proteins, Npl4 and Ufd1 (Shcherbik and Haines, 2007;Lass et al, 2008;Cao et al, 2007) that bind ubiquitinated proteins, this complex (Cdc48p-Npl4-Ufd1) (Heubes and Stemmann, 2007;Nowis et al, 2006;Alzayady et al, 2005;Cao and Zheng, 2004) may be responsible for the breakdown of large protein complexes before proteolytic degradation of its elements. Experiments revealed that the process of translocation from ER by Cdc48p-Npl4-Ufd1 is ATP dependent; a comparable function has been attributed to chaperones of the Hsp70 family in importing newly synthesized proteins into the ER and mitochondria (Hood et al, 2003).…”

Section: Proteinsmentioning

confidence: 99%

The proteasomal system

Jung

Karademir

Grune

2009

Molecular Aspects of Medicine

364

311

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“…These retrotranslocation complexes also contain E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases that ubiquitylate the substrates as they emerge [2], [4]. The heterodimeric cofactor Ufd1-Npl4 may simultaneously interact with p97 and ubiquitylated substrates [31], [32] and assist p97 with substrate recruitment in ERAD [10] and other degradation pathways [33]. Then, peptide: N -glycanase associates with p97 and removes glycans from glycosylated ERAD substrates [34] before the substrate is finally transferred to the 26S proteasome and degraded.…”

Section: Introductionmentioning

confidence: 99%

The Tissue-Specific Rep8/UBXD6 Tethers p97 to the Endoplasmic Reticulum Membrane for Degradation of Misfolded Proteins

et al. 2011

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The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded secretory proteins via the ER-associated degradation pathway. This functional diversity of p97 depends on its association with various cofactors, and to further our understanding of p97 function it is important that these cofactors are identified and analyzed. Here, we isolate and characterize the human protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8 is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p97 to the ER membrane for efficient ER-associated degradation.

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Analysis of Npl4 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Npl4 in ERAD

Cited by 14 publications

References 49 publications

ATP Binding to p97/VCP D1 Domain Regulates Selective Recruitment of Adaptors to Its Proximal N-Domain

ATP Binding to p97/VCP D1 Domain Regulates Selective Recruitment of Adaptors to Its Proximal N-Domain

The proteasomal system

The Tissue-Specific Rep8/UBXD6 Tethers p97 to the Endoplasmic Reticulum Membrane for Degradation of Misfolded Proteins

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