The Tissue-Specific Rep8/UBXD6 Tethers p97 to the Endoplasmic Reticulum Membrane for Degradation of Misfolded Proteins Louise Madsen1, Franziska Kriegenburg1, Andrea Vala1, Diana Best2, Søren Prag3, Kay Hofmann 4 , Michael Seeger5, Ian R. Adams2, Rasmus Hartmann-Petersen1* 1 Department of Biology, University of Copenhagen, Copenhagen, Denmark, 2 MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, Scotland, 3 Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal, 4 Bioinformatics Department, Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany, 5 Institut fu¨ r Biochemie, Charite´ – Universita¨tsmedizin Berlin, Berlin, Germany Abstract 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. Citation: Madsen L, Kriegenburg F, Vala A, Best D, Prag S, et al. (2011) The Tissue-Specific Rep8/UBXD6 Tethers p97 to the Endoplasmic Reticulum Membrane for Degradation of Misfolded Proteins. PLoS ONE 6(9): e25061. doi:10.1371/journal.pone.0025061 Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Received June 11, 2011; Accepted August 23, 2011; Published September 15, 2011 Copyright: 2011 Madsen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been supported financially by grants from the Lundbeck Foundation, the Novo Nordisk Foundation, the Medical Research Council and the Danish Council for Independent Research (Natural Sciences). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Kay Hofmann is employed by Miltenyi Biotec GmbH. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: rhpetersen@bio.ku.dk Introduction When correct folding of proteins or assembly of oligomeric proteins in the endoplasmic reticulum (ER) is disturbed, misfolded or unassembled proteins accumulate inside the ER lumen. If such proteins are allowed to linger, they may form insoluble aggregates and thus pose a serious threat to the cell. Normally, cells rapidly channel misfolded proteins back into the cytoplasm for degrada- tion by the ubiquitin-proteasome system [1–7]. The recognition of the misfolded proteins in the ER lumen and subsequent transport to the proteasome is termed ER quality control [3,5] and ER- associated degradation (ERAD) [1–5], respectively. Extraction of misfolded proteins from the ER relies on the ring-shaped homohexameric ATPase known as p97 or VCP (valosin- containing protein) in mammals and Cdc48 in yeast [8–12]. The p97 ATPase is a member of the AAA (ATPase associated with various activities) family of ATPases [13]. The monomer is a phylogenetically highly conserved and essential protein that carries two AAA modules, called D1 and D2. These link coordinated ATP-hydrolysis to conformational changes of the hexameric complex [14], in a fashion similar to other AAA-type ATPases [15]. The ATP-powered conformational changes allow p97 to drive the disassembly of protein complexes and segregate proteins from their binding partners [16,17]. This catalytic activity of p97, termed the ‘‘segregase’’ activity [10,18], is probably restricted to ubiquitylated proteins and is important for a number of cellular pathways, including membrane fusion [19], protein degradation [11,20], and transcription factor maturation [18,21]. Although p97 may bind ubiquitylated proteins directly [18], a series of p97 cofactors recruit and/or process substrates [22–24]. The functions of these cofactors are diverse, and each probably directs p97 activity to a particular cell function. For instance, the UBX domain protein, p47, directs p97 to functions in membrane fusion [19], while another UBX domain protein, Ubxd7, targets the HIF1a transcription factor for degradation [24]. With only one exception, Ubxd1 [25,26], all characterized UBX-domain proteins associate directly with p97 via their UBX domains [24,27,28]. During ERAD, p97 also relies heavily on cofactors. Initially, p97 associates with the cytosolic side of the ER membrane by interacting with transmembrane p97 cofactors such as VIMP and the UBX protein, Erasin [29,30]. These cofactors are tightly associated with numerous other proteins that probably form a channel through which the misfolded proteins traverse from the ER lumen to the cytosol. These retrotranslocation complexes also PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25061