Cuz1/Ynl155w, a Zinc-dependent Ubiquitin-binding Protein, Protects Cells from Metalloid-induced Proteotoxicity The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Hanna, John, David Waterman, Marta Isasa, Suzanne Elsasser, Yuan Shi, Steven Gygi, and Daniel Finley. 2013. “Cuz1/Ynl155w, a Zinc- Dependent Ubiquitin-Binding Protein, Protects Cells from Metalloid- Induced Proteotoxicity.” Journal of Biological Chemistry 289 (3): 1876–85. https://doi.org/10.1074/jbc.m113.534032. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41483193 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 3, pp. 1876–1885, January 17, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Cuz1/Ynl155w, a Zinc-dependent Ubiquitin-binding Protein, Protects Cells from Metalloid-induced Proteotoxicity* Received for publication, November 9, 2013, and in revised form, November 27, 2013 Published, JBC Papers in Press, December 2, 2013, DOI 10.1074/jbc.M113.534032 John Hanna‡1, David Waterman§2, Marta Isasa§, Suzanne Elsasser§, Yuan Shi§, Steven Gygi§, and Daniel Finley§ From the ‡Department of Pathology, Brigham and Women’s Hospital, and §Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 Background: Protein misfolding, a universal threat to cells, is dealt with by the ubiquitin-proteasome system. Results: Ynl155w is a zinc-dependent ubiquitin-binding protein, interacts with proteasome and Cdc48, and is essential for surviving metalloids. Conclusion: Ynl155w may protect cells from metalloid-induced proteotoxicity by delivering ubiquitinated proteins to Cdc48 and proteasome. Significance: Ynl155w represents a novel stress response factor for misfolded proteins. Protein misfolding is a universal threat to cells. The ubiquitin- Cdc48 (p97 in higher organisms). Cdc48 has numerous cellular proteasome system mediates a cellular stress response capable functions, many of which require its central role as a force- Downloaded from of eliminating misfolded proteins. Here we identify Cuz1/ generating machine (4). For example, Cdc48 can extract pro- Ynl155w as a component of the ubiquitin system, capable of teins from membranes, chromatin, or larger protein assem- interacting with both the proteasome and Cdc48. Cuz1/ blies. Like the proteasome, Cdc48 interacts with numerous Ynl155w is regulated by the transcription factor Rpn4, and is co-factors that endow it with multiple activities including ubiq- required for cells to survive exposure to the trivalent metalloids uitin binding, deubiquitination, and ubiquitination (5). http://www.jbc.org/ arsenic and antimony. A related protein, Yor052c, shows similar We describe here a new component of the ubiquitin system, phenotypes, suggesting a multicomponent stress response path- Ynl155w, a zinc finger protein capable of directly interacting with way. Cuz1/Ynl155w functions as a zinc-dependent ubiquitin-bind- the proteasome and Cdc48. Ynl155w is co-regulated with other ing protein. Thus, Cuz1/Ynl155w is proposed to protect cells from elements of the ubiquitin-proteasome system, and its absence metalloid-induced proteotoxicity by delivering ubiquitinated sub- markedly compromises survival upon exposure to metalloid tox- by guest on October 4, 2019 strates to Cdc48 and the proteasome for destruction. icity. A second uncharacterized zinc finger protein, Yor052c, shows similar phenotypic properties, suggesting a multicompo- nent stress response pathway. Ynl155w functions as a zinc-depen- Protein misfolding is toxic to cells and has numerous causes dent ubiquitin-binding protein, suggesting the hypothesis that including heat, errors in translation, oxidation, genetic muta- Ynl155w protects cells from metalloid toxicity by delivering ubiq- tion, and aging. Heavy metals and metalloids (e.g. arsenic) are uitinated proteins to Cdc48 and the proteasome for destruction. ubiquitous environmental toxins that cause protein misfolding by directly binding proteins and altering their structures. The EXPERIMENTAL PROCEDURES recognition and elimination of these aberrant proteins is nec- Strains and Plasmids—Yeast strains and plasmids are listed essary for cell survival. in Table 1. Yeast were cultured at 30 °C unless otherwise indi- The ubiquitin-proteasome system is the major pathway for cated. YPD medium consisted of 1% yeast extract, 2% Bacto- selective protein degradation in eukaryotic cells (1, 2). Sub- peptone, and 2% dextrose. Synthetic medium consisted of 0.7% strates requiring destruction by this system are covalently mod- Difco Yeast Nitrogen Base supplemented with amino acids, ified by the small protein ubiquitin, which serves as a recogni- adenine, and 2% dextrose. Uracil was omitted from this tion motif for the proteasome (3). By harboring multiple medium for plasmid selection. enzymatic and non-enzymatic functionalities, the proteasome Recombinant Proteins—GST fusions proteins were expressed in is able to recognize ubiquitinated substrates, and unfold, deu- Escherichia coli and purified by glutathione-Sepharose affinity biquitinate, and proteolyze them into small peptides, while chromatography (6). Intact GST fusions were eluted with free largely releasing ubiquitin for reuse. glutathione; native proteins were generated by thrombin cleav- Prior to destruction by the proteasome, many substrates age. For the experiments shown in Fig. 4, purifications were require additional processing by the homohexameric ATPase carried out in the presence of 1 mM zinc sulfate. His12-Sumo- Cdc48 (a gift of T. Rapoport) was expressed in E. coli, purified by nickel-nitrilotriacetic acid affinity chromatography, and * This work was supported by National Institutes of Health Grant GM043601 (to D. F.). eluted with imidazole. 1 To whom correspondence should be addressed: 75 Francis St., Boston, Proteasome and Cdc48 Purification—The proteasome and MA 02115. Tel.: 617-525-8372; Fax: 617-432-1144; E-mail: jwhanna@ its subcomplexes were purified from yeast as described (6). partners.org. 2 Present address: Rosenstiel Basic Medical Sciences and Research Center and Endogenous, genomically encoded TAP-tagged Cdc48 was iso- Dept. of Biology, Brandeis University, Waltham, MA 02254. lated by IgG affinity chromatography (buffer 50 mM Tris-HCl, 1876 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 3•JANUARY 17, 2014 Ynl155w Protects Cells from Metalloid Toxicity TABLE 1 Strains and plasmids Downloaded from http://www.jbc.org/ by guest on October 4, 2019 pH 7.5, 1 mM EDTA), washed with buffer supplemented with challenged with Ynl155w species, washed with buffer contain- 0.5 M NaCl, and eluted by cleavage with TEV protease. Native ing 50 mM NaCl, and eluted with imidazole. gel analysis was carried out as described (7). Phenotypic Analysis of Yeast Mutants—Yeast spot assays Mass Spectrometry—Gel bands were excised, digested with were performed by serial dilution of overnight yeast cultures trypsin, and analyzed using a Q Exactive mass spectrometer. onto the indicated media, and culturing for the indicated peri- Spectra were processed using a Sequest-based in-house soft- ods of time. Sodium arsenite was dissolved in water; antimony ware pipeline. Database searching included all entries from the trioxide was dissolved in hydrochloric acid. yeast Uniprot database. Ubiquitin Binding Assays—His- and T7-epitope-tagged Ynl155w Binding Assays—Proteasome, proteasome subcom- ubiquitinated Sic1PY (ub-Sic1PY) was prepared as described (8). plexes, and TAP-tagged endogenous Cdc48 were immobilized Lys-48-linked free ubiquitin chains (ub2-ub7) were purchased on IgG resin, challenged with purified Ynl155w or GST- from Boston Biochem. Equivalent amounts of GST or GST- Ynl155w, as indicated, washed with the appropriate purifica- Ynl155w (50 ␮g) were immobilized on glutathione-Sepharose tion buffer (see above) supplemented with 50 mM NaCl, and resin in buffer (50 mM Tris-HCl, pH 7.5, 1 mM zinc sulfate), PY eluted by cleavage with TEV protease. GST fusion proteins challenged with equivalent amounts of ub-Sic1 (8 nM)or were immobilized on glutathione-Sepharose resin, challenged ubiquitin chains (5 ␮g), washed with buffer supplemented with with Ynl155w, washed in buffer supplemented with 50 mM 50 mM NaCl, and eluted with free glutathione. Metal chelation NaCl, and eluted with free glutathione (30 mM). His12-Sumo- was carried out for 30 min at 4 °C with 1,10-phenanthroline (5 Cdc48 was immobilized on nickel-nitrilotriacetic acid resin, mM) and EDTA (5 mM). JANUARY 17, 2014•VOLUME 289•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 1877 Ynl155w Protects Cells from Metalloid Toxicity Downloaded from http://www.jbc.org/ by guest on October 4, 2019 FIGURE 1. Interaction of Ynl155w with the proteasome. A, native gel electrophoresis of purified wild-type and spg5⌬ proteasomes (3 ␮g), followed by in-gel activity assay with the fluorogenic proteasome substrate, suc-LLVY-AMC. B, proteasomes from panel A were excised from the gel and analyzed by mass spectrometry, yielding three peptides from Ynl155w. C, interaction of purified Ynl155w (2.3 ␮g) with purified 26 S proteasome (5 ␮g). Upper panel, Coomassie- stained SDS-PAGE gel. Lower panel, anti-Ynl155w SDS-PAGE immunoblot. D, interaction