Yeast Yeast 2004; 21: 127–139. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1071 Research Article Binding of Cdc48p to a ubiquitin-related UBX domain from novel yeast proteins involved in intracellular proteolysis and sporulation Anabelle Decottignies, Aude Evain and Michel Ghislain* Unit´ e de Biochimie Physiologique, Institut des Sciences de la Vie, Universit´ e Catholique de Louvain, Croix du Sud 2-20, B-1348 Louvain-la-Neuve, Belgium *Correspondence to: Michel Ghislain, Unit´ e de Biochimie Physiologique, Institut des Sciences de la Vie, Universit´ e Catholique de Louvain, Croix du Sud 2-20, B-1348 Louvain-la-Neuve, Belgium. E-mail: ghislain@fysa.ucl.ac.be Received: 22 January 2003 Accepted: 28 October 2003 Abstract The Cdc48/p97 AAA-ATPase functions in membrane fusion and ubiquitin-dependent protein degradation. Here, we show that, in yeast, Cdc48p interacts with three novel proteins, Cuil–3p, which contain a conserved ubiquitin-related (UBX) domain. Cui2p and Cui3p are closely related, interact with each other, and are localized at the perinuclear membrane. Cdc48p binds directly the UBX domain of Cui3p in vitro. Multiple deletions of the CUI1, CUI2 and CUI3 genes confer deficiency in sporulation and degradation of model ubiquitin–protein fusions. The Cuil–3 proteins were also found to interact with Ufd3p, a WD repeat protein known to associate with Cdc48p. Together, these results indicate that the Cuil–3 proteins form complexes that are components of the ubiquitin–proteasome system. Copyright  2003 John Wiley & Sons, Ltd. Keywords: Saccharomyces cerevisiae ; AAA-ATPase; Cdc48p; UBX domain; ubiquitin-dependent degradation Introduction Yeast Cdc48p and its mammalian homologue, p97, members of a family of A TPases a ssociated with different cellular a ctivities, or AAA-ATPases (Patel and Latterich, 1998), are required for ubiquitin- dependent protein degradation (Ghislain et al., 1996; Dai et al., 1998) and membrane fusion (Lat- terich et al., 1995; Acharya et al., 1995; Rabouille et al., 1995). The archaebacterial Thermoplasma acidophilum homologue, VAT, was the first of this group of proteins shown to be capable of folding and unfolding a model substrate by an ATP-dependent process (Golbik et al., 1999). The N-terminal domain of the archaebacterial AAA- protein is also sufficient to refold a permissive substrate independent of ATPase activity (Golbik et al., 1999). Recently, it was shown that Cdc48p can distinguish between the native and non-native conformation of model substrates in vitro, and that this chaperone-like function seems to be indepen- dent of ATPase activity (Thoms, 2002). The chaperone-like activity of Cdc48p/p97 is directed to different cellular pathways by interac- tion with specific protein adapters (Meyer et al., 2000). Cdc48p/p97 complexed with Ufd1 and Np14 proteins is required for endoplasmic retic- ulum (ER)-associated degradation (ERAD) of pro- tein (Ye et al., 2001; Bays et al., 2001; Rabinovich et al., 2002; Braun et al., 2002; Jarosch et al., 2002) and the ubiquitin-dependent cleavage and activation of yeast membrane-bound transcription factors, Spt23p and Mga2p (Hoppe et al., 2000; Hitchcock et al., 2001; Rape et al., 2001). In mam- mals, binding of p47 to p97 is required for post- mitotic fusion of Golgi membranes and the transi- tional ER (Kondo et al., 1997; Roy et al., 2000). It has recently been shown that the p97–Ufd1–Np14 and p97–p47 complexes contribute to distinct, sequential steps of nuclear envelope assembly (Het- zer et al., 2001). p47 contains a 80 amino acid domain that is structurally similar to ubiquitin, despite the lack of Copyright  2003 John Wiley & Sons, Ltd.