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A Conserved Protein with AN1 Zinc Finger and Ubiquitin-Like Domains

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A Conserved Protein with AN1 Zinc Finger and Ubiquitin-Like Domains THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 47, pp. 33682–33696, November 22, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. A Conserved Protein with AN1 Zinc Finger and Ubiquitin-like Domains Modulates Cdc48 (p97) Function in the Ubiquitin-Proteasome Pathway*□S Received for publication, September 20, 2013, and in revised form, October 10, 2013 Published, JBC Papers in Press, October 11, 2013, DOI 10.1074/jbc.M113.521088 Bebiana Sá-Moura‡§1, Minoru Funakoshi‡2, Robert J. Tomko, Jr.‡3, R. Jürgen Dohmen¶, Zhiping Wuʈ, Junmin Pengʈ4, and Mark Hochstrasser‡5 From the ‡Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, the §Graduate Program in Areas of Basic and Applied Biology, Abel Salazar Biomedical Sciences Institute, University of Porto, Porto 4099-002, Portugal, the ¶Institute for Genetics, University of Cologne, Zülpicher Strasse 47, D-50674 Cologne, Germany, and the ʈDepartments of Structural Biology and Developmental Neurobiology, St. Jude Proteomics Facility, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 Background: New regulators of the ubiquitin-proteasome system (UPS) were sought in yeast. Results: Cuz1 (Cdc48-associated ubiquitin-like/zinc finger protein-1) interacts with the Cdc48/p97 ATPase and promotes endoplasmic reticulum-associated degradation. Conclusion: Cuz1 is a highly conserved Cdc48 cofactor that also binds proteasomes, especially in cells exposed to arsenite. Downloaded from Significance: This first characterization of the Cuz1 protein family links it to specific Cdc48/p97 complexes. Regulated protein degradation mediated by the ubiquitin- The ubiquitin-proteasome system (UPS)6 is an elaborate net- proteasomesystem(UPS)iscriticaltoeukaryoticproteinhomeo- work of enzymes and proteins that ensures the specific and stasis. Often vital to degradation of protein substrates is their timely degradation of proteins in eukaryotic cells (1). Substrate http://www.jbc.org/ disassembly, unfolding, or extraction from membranes. These proteins include both regulatory proteins that must be inacti- processes are catalyzed by the conserved AAA-ATPase Cdc48 vated for proper cell and organismal function and quality con- (also known as p97). Here we characterize the Cuz1 protein trol substrates such as misfolded or misassembled proteins (1). (Cdc48-associated UBL/zinc finger protein-1), encoded by a Attachment of ubiquitin polymers to substrates is generally previously uncharacterized arsenite-inducible gene in budding required for their efficient recognition by the 26 S proteasome, by guest on March 11, 2015 yeast. Cuz1, like its human ortholog ZFAND1, has both an AN1- a 2.6-MDa complex that ultimately degrades the substrate into type zinc finger (Zf_AN1) and a divergent ubiquitin-like domain short peptides and recycles ubiquitin (2). (UBL). We show that Cuz1 modulates Cdc48 function in the The 26 S proteasome is composed of two major subcom- UPS. The two proteins directly interact, and the Cuz1 UBL, but plexes: the 20 S proteasome core particle (CP) and the 19 S not Zf_AN1, is necessary for binding to the Cdc48 N-terminal regulatory particle (RP) (3, 4). The RP recognizes polyubiquity- domain. Cuz1 also associates, albeit more weakly, with the pro- lated substrates, and uses a heterohexameric ring of AAAϩ teasome, and the UBL is dispensable for this interaction. Cuz1- ATPases to unfold substrates and translocate them into the proteasome interaction is strongly enhanced by exposure of central chamber of the CP where the proteolytic sites are cells to the environmental toxin arsenite, and in a proteasome located. Additional subunits in the RP function either as intrin- mutant, loss of Cuz1 enhances arsenite sensitivity. Whereas loss sic receptors for polyubiquitin via their ubiquitin-binding of Cuz1 alone causes only minor UPS degradation defects, its Npl4-Ufd1 domains or as binding sites for more mobile extrinsic ubiquitin combination with mutations in the Cdc48 complex receptors or shuttle factors such as Rad23 (5). leads to much greater impairment. Cuz1 helps limit the accumu- Much of the machinery of the UPS has been identified, but lation of ubiquitin conjugates on both the proteasome and key parts of the system remain poorly understood. Prominent Cdc48, suggesting a possible role in the transfer of ubiquitylated among the open questions is the mechanism by which poly- substrates from Cdc48 to the proteasome or in their release ubiquitylated proteins are released by E3 ligases and transferred from these complexes. to ubiquitin receptors and the proteasome for degradation. The highly conserved, multifunctional Cdc48 protein, also called valosin-containing protein or p97 in mammals (6), is an AAA- * This work was supported, in whole or in part, by National Institutes of Health Grants GM083050 and GM046904 (to M. H.). ATPase that forms a homohexameric ring (7). It has been □S This article contains supplemental Tables S1–S3. shown to be required for UPS-mediated protein degradation of 1 Supported by a Portuguese Foundation for Science and Technology Fellow- several classes of substrates, including those degraded by the ship SFRH/BD/33538/2008. 2 Present address: Marine Products Kimuraya Co., Ltd., 3307 Watari, Sakaimi- endoplasmic reticulum-associated degradation (ERAD) and nato, Tottori 684-0072, Japan. 3 Supported in part by an American Cancer Society postdoctoral fellowship. 4 Supported by the American Lebanese Syrian Associated Charities. 6 The abbreviations used are: UPS, ubiquitin-proteasome system; CP, core parti- 5 To whom correspondence should be addressed: 266 Whitney Ave., New cle; RP, regulatory particle; ERAD, endoplasmic reticulum-associated degrada- Haven, CT 06520. Tel.: 203-432-5101; Fax: 203-432-5158; E-mail: mark. tion; UFD, ubiquitin-fusion degradation; UBL, ubiquitin-like domain; Cuz1, [email protected]. Cdc48-associated UBL/zinc-finger protein-1; co-IP, co-immunoprecipitation. 33682 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 47•NOVEMBER 22, 2013 A Novel Cdc48 Cofactor with AN1 Zinc Finger and UBL Domains ubiquitin-fusion degradation (UFD) branches of the UPS EXPERIMENTAL PROCEDURES (8–11). The general mechanistic function of Cdc48 appears to Yeast Strains, Plasmids, and Plasmid Constructions—Yeast be that of a “segregase,” a protein that uses the energy of ATP rich (YPD) and minimal (SD) media were prepared as described hydrolysis to unfold or disassemble protein complexes or to previously, and all yeast manipulations were carried out extract proteins from membranes (12, 13). according to standard procedures (28). Yeast chromosomal Cdc48 consists of a globular N-terminal domain; two type II gene deletions were made by PCR-mediated marker amplifica- AAA (ATPases associated with diverse cellular activities) tion and gene replacement in diploid cells; the resulting diploid ATPase domains called D1 and D2, which share 40% sequence heterozygotes were dissected to verify 2:2 marker segregation identity; and a disordered C-terminal tail (14, 15). Cdc48 inter- and to isolate haploid deletion strains. Complete lists of Saccha- acts with a plethora of cofactors, and these proteins mediate the romyces cerevisiae strains and plasmids used in this study are wide range of functions in which Cdc48 has been implicated. presented in supplemental Tables S1 and S2, respectively. Members of the largest family of Cdc48 cofactors are related by The YNL155W (CUZ1) and YOR052C genes were isolated by the UBX (ubiquitin regulatory X) domain; there are seven UBX PCR amplification from genomic yeast DNA, and inserted into proteins in Saccharomyces cerevisiae. Structural determination various plasmids. The absence of mutations was verified by DNA of the UBX domain, which binds specifically to the Cdc48 sequencing of the entire inserts. Plasmid pRS314-FLAG-Cuz1 was N-terminal domain (16), revealed a ␤-grasp fold similar to that derived from pRS314-Cuz1 using site-directed, ligase-independ- of ubiquitin (17). ent mutagenesis (SLIM) (29). DNA sequences encoding His6- The first characterized UBX domain-containing Cdc48 Cdc48 or His6-Cdc48(1–220) were PCR amplified from genomic cofactor was p47 (called Ubx1 or Shp1 in yeast), which is Downloaded from yeast DNA using an oligonucleotide that introduced a His6 tag and required for homotypic membrane fusion in the nuclear enve- then cloned into pET42b using NdeI and XhoI restriction sites, lope, Golgi, and ER (18, 19). Ubx2 is a transmembrane protein which removed the sequence for the GST tag from the plasmid. of the ER that helps recruit Cdc48 to ubiquitin-ligase com- DNA sequencing confirmed that the ORF contained no muta- plexes in the ER membrane (20), whereas Ubx5 appears to tions. Plasmid pGEX-KT was used to express full-length Cuz1 recruit Cdc48 complexes to chromatin sites of DNA repair (21). and different Cuz1 deletion variants as GST fusions in Esche- http://www.jbc.org/ Precise functions for the other UBX proteins have not yet been richia coli. CUZ1 sequences were obtained by amplifying the as clearly defined (see “Discussion”). desired DNA fragments from yeast genomic DNA and inserting In the ERAD and UFD pathways, a key Cdc48 cofactor is the them downstream of the GST coding sequence in pGEX-KT. Npl4-Ufd1 heterodimer (22, 23). One Cdc48 hexamer interacts To make pGEX-KT-Cuz1–4S, Cys to Ser codon mutations with one Npl4-Ufd1 heterodimer via a short binding site (BS1) were introduced into the CUZ1 sequence using two sequential in
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