View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1823 (2012) 117–124 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr Review The Cdc48 machine in endoplasmic reticulum associated protein degradation☆ Dieter H. Wolf ⁎, Alexandra Stolz Institut für Biochemie, Universität Stuttgart, Paffenwaldring 55, 70569 Stuttgart, Germany article info abstract Article history: The AAA-type ATPase Cdc48 (named p97/VCP in mammals) is a molecular machine in all eukaryotic cells that Received 8 July 2011 transforms ATP hydrolysis into mechanic power to unfold and pull proteins against physical forces, which Received in revised form 1 September 2011 make up a protein's structure and hold it in place. From the many cellular processes, Cdc48 is involved in, Accepted 2 September 2011 its function in endoplasmic reticulum associated protein degradation (ERAD) is understood best. This quality Available online 16 September 2011 control process for proteins of the secretory pathway scans protein folding and discovers misfolded proteins in the endoplasmic reticulum (ER), the organelle, destined for folding of these proteins and their further de- Keywords: AAA-type ATPase livery to their site of action. Misfolded lumenal and membrane proteins of the ER are detected by chaperones Cdc48 and lectins and retro-translocated out of the ER for degradation. Here the Cdc48 machinery, recruited to the p97/VCP ER membrane, takes over. After polyubiquitylation of the protein substrate, Cdc48 together with its dimeric Cdc48–Ufd1–Npl4 complex co-factor complex Ufd1–Npl4 pulls the misfolded protein out and away from the ER membrane and delivers ER-associated protein degradation (ERAD) it to down-stream components for degradation by a cytosolic proteinase machine, the proteasome. The – Ubiquitin proteasome-system (UPS) known details of the Cdc48–Ufd1–Npl4 motor complex triggered process are subject of this review article. This article is part of a Special Issue entitled: AAA ATPases: Structure and function. © 2011 Elsevier B.V. All rights reserved. 1. Introduction through the secretory pathway to its site of action or, when incorrect, is retained in the ER and retrograde transported across the ER membrane Proper function of the workhorses of a cell, the proteins, is essen- back into the cytosol, where it is rapidly degraded by the ubiquitin– tial for all organisms. Statistic folding errors during synthesis or fold- proteasome-system [8,12–17]. It is between retrograde transport to ing mistakes induced upon stresses like heat, oxidation or heavy the cytoplasm and degradation of the misfolded substrates by the pro- metal ions require a functional protein quality control machinery, teasome where an ATP consuming machine, the Cdc48–Ufd1–Npl4 which recognizes misfolded proteins and delivers them to an elimina- complex, acts as a motor required for substrate delivery to the protea- tion system [1,2]. Impairment of the quality control and elimination some. Cdc48 (yeast) was found as p97/VCP in mammals, TER94 in fly system leads to severe diseases in humans as are Alzheimer's-, and CDC-48 in nematodes. However, we will use the term Cdc48 Parkinson's-, Huntington's-, Creutzfeldt–Jakob- and many other dis- throughout this review article, irrespective of the organism used in eases [3,4]. About 30% of proteins of a eukaryotic cell are proteins of the cited articles. the secretory pathway, which function in the ER, the Golgi apparatus, the lysosome, the cell membrane or the exterior of the cell. The ER con- 2. Import of proteins into the ER, folding and quality control tains the protein folding factory for these proteins [5–7]. After import into the ER in an unfolded state, it is a specialty of proteins of the secre- Proteins of the secretory pathway are imported into the ER in an tory pathway to acquire certain modifications during the folding pro- either co-translational or post-translational fashion. The membrane cess, as is the formation of disulfide bonds and N-glycosylation. The translocation occurs via Sec61, a component of a large multiprotein status of protein folding is also efficiently scanned in this organelle on complex providing the channel for protein entry [7,18]. Global folding the level of exposed hydrophobic regions and glycan modification. An of the protein occurs right upon its entry into the ER lumen. At the array of chaperones, co-chaperones, oxido-reductases, glycan chain same time disulfide bonds are formed and sugar chains are added. modifying enzymes and lectins is constantly in a dynamic action to Major players of the folding process are chaperones and their co-factors, foldandscanaproteinforproperfolding[8–11]. After having passed protein disulfide isomerases, the oligosaccharyltransferase (OST) com- the quality control, a protein is either released for further transport plex, carbohydrate trimming enzymes and lectins [5–11,19–21]. Pro- teins unable to reach their final conformation are mainly degraded by a process named ERAD. ☆ This article is part of a Special Issue entitled: AAA ATPases: Structure and function. ⁎ Corresponding author at: Institut für Biochemie, Universität Stuttgart, Pfaffenwaldring The eukaryotic model organism yeast Saccharomyces cerevisiae has 55, D-70569 Stuttgart, Germany. Tel.: +49 711 685 64390; fax: +49 711 685 64392. been a pacemaker in studies on ERAD [12–17]. According to the loca- E-mail address: [email protected] (D.H. Wolf). tion of the misfolded protein domain relative to the ER, three types of 0167-4889/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2011.09.002 118 D.H. Wolf, A. Stolz / Biochimica et Biophysica Acta 1823 (2012) 117–124 ERAD substrates have been defined. (i) ERAD-L substrates carrying repertoire of ER-associated E3 ligases has significantly expanded their misfolded domain in the lumen of the ER, (ii) ERAD-M [34–36] making a high plasticity in the use of different recognition substrates having a misfolded domain in the ER membrane and pathways of misfolded ERAD substrates likely. Even the participation (iii) ERAD-C substrates exposing a misfolded domain into the cyto- of a cytosolic E3 in ERAD is possible [34,35,37]. plasm [22,23]. When a misfolded protein of the ER is discovered as such, either In yeast, two polytopic RING-finger type ER membrane embedded because it has been retrograde transported back to the ER surface or ubiquitin ligases, Hrd1/Der3 [24–27] and Doa10 [28,29] are required because in its membrane location it presents a misfolded domain to for polyubiquitylation of these substrates to render them to proteaso- the cytosolic environment, it is recognized by the respective E3 ubi- mal proteolysis. While ERAD-L and ERAD-M substrates are targets of quitin ligase [38] and polyubiquitylated. In yeast the major ubiquitin the ubiquitin ligase Hrd1/Der3, ERAD-C substrates are mainly targets conjugating enzymes working together with the two E3 ligases are of Doa10. The mammalian equivalents of the yeast ligase Hrd1/Der3 Ubc7 and Ubc6 [12–17]. A third ubiquitin conjugating enzyme, are HRD1 and gp78. TEB4 (MARCH-IV) is the equivalent of yeast Ubc1, has only been shown to work together with Hrd1/Der3. Ubc7 Doa10 [30,31]. However, the clear definition of two ERAD recognition is recruited to the ER membrane via the membrane protein Cue1 pathways was only possible for yeast up to now (Fig. 1). [34,39,40], while Ubc6 is tail-anchored in the ER membrane. The Substrates are not exclusively dependent on either E3 ligase sys- mammalian orthologues of Ubc6 and Ubc7 are Ube2j1 as well as tem, Hrd1/Der3 or Doa10. Both pathways exhibit some plasticity: A Ube2j2 and Ube2g1 as well as Ube2g2, respectively [34]. Doa10 substrate can for instance also in part be a target of the Prior to ubiquitylation, the different ERAD substrates have to be Hrd1/Der3 ligase [32,33]. As compared to yeast, the mammalian recognized and excluded from folding intermediates and properly Fig. 1. Cdc48 in ERAD of yeast. Misfolded proteins in the ER lumen (ERAD-L recognition pathway) and in the ER membrane, the misfolded domain facing the cytosol (ERAD-C recognition pathway), are discovered and polyubiquitylated at the outer surface of the ER by two polytopic membrane localized ubiquitin ligases, Hrd1/Der3 and Doa10, respectively. At this stage the ternary Cdc48–Ufd1–Npl4 motor complex takes over, makes contact with the misfolded protein and prepares it for delivery to the proteasome for degradation. Besides Ubx2, recruiting the Cdc48 complex to the ER membrane and by this bringing the complex into contact with Hrd1/Der3 and Doa10, the Der1 homologue Dfm1 is able to bind Cdcc48 via its SHP domain. For details, see text. D.H. Wolf, A. Stolz / Biochimica et Biophysica Acta 1823 (2012) 117–124 119 folded proteins. As compared to ERAD-C, the recognition pathway of of yeast Ubx2 in mammalian cells are UBXD2 and UBXD8 [84,85]. ERAD-L substrates is rather complicated. A standard substrate of this The mammalian ligases HRD1 and gp78, in contrast to the yeast pathway is CPY*, a mutated vacuolar (lysosomal) peptidase of yeast, equivalent Hrd1/Der3, possess Cdc48 binding motifs by themselves which has led to the discovery of the major principles of ERAD [41– being also able to recruit the ATPase machine to the ER membrane 43]. After post-translational import into the ER, the mutated protein is [86,87]. N-glycosylated at four sites. In general, folding in the ER is controlled by chaperones detecting hydrophobic patches on the surface of a pro- 3. The Cdc48 machine tein accompanied by a trimming process of the N-linked Glc3–Man9– GlcNAc2 carbohydrate [9,11,44–48]. Specifically, CPY* is contacted by The Cdc48 gene was first identified in a genetic screen in yeast the Hsp70 chaperone of the ER, Kar2, which is linked to its Hsp40 cofac- when searching for conditional mutations affecting the cell cycle tors Jem1 and Scj1 [49–51] (Fig.