Functional Division of Substrate Processing Cofactors of the Ubiquitin-Selective Cdc48 Chaperone  Sebastian Rumpf, Stefan Jentsch  Molecular Cell  Volume 21, Issue 2, Pages 261-269 (January 2006) DOI: 10.1016/j.molcel.2005.12.014 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Interactions between Cdc48 Cofactors (A) Pull-down experiments with GSTUfd2 and Ufd3GST. Equimolar amounts of GST, GSTUfd2, and Ufd3GST were prebound to glutathione beads and then incubated with yeast protein extract (400 mg each, NPL43myc background). After washes, beads were eluted with lysis buffer containing 1 M NaCl. Eluted proteins were analyzed by SDS-PAGE and Coomassie staining and identified by mass spectrometry (not shown) or Western blotting analysis (right panel). (B and C) Confirmation of Ufd2 and Ufd3 interactors. Anti-Ufd2 (B) or anti-Ufd3 (C) immunoprecipitates from wt cells expressing Npl43HA from the genomic locus were blotted with the indicated antibodies. Δufd2 and Δufd3 cells served as negative controls. (D and E) Mapping of cofactor binding sites on Cdc48. (D) GST-tagged full-length (FL) and truncated Cdc48 versions were incubated with the indicated Cdc48 cofactors and precipitated with glutathione beads. (E) Pull-downs with GST-cofactor fusions and Cdc48 ND1. Bound material was analyzed by Western blot. (F) Schematic representation of Cdc48 interaction domains. Molecular Cell 2006 21, 261-269DOI: (10.1016/j.molcel.2005.12.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Interaction Properties of Ufd3 (A) Ufd2 and Ufd3 compete for Cdc48. GSTCdc48 was incubated with constant amounts of Ufd2 and increasing amounts of Ufd3 (left panels) or vice versa (right panels). GSTCdc48 and bound proteins were precipitated with glutathione beads (bound) and analyzed by Coomassie blue staining. Inputs are 10% of the total. (B and C) Mutually exclusive Cdc48 binding. Immunoprecipitation with either Ufd2- specific antibodies (B) or Ufd3-specific antibodies (C) and detection by immunoblotting against the proteins as indicated. (D) The Cdc48 binding site of Ufd3. GST fusions of full-length (FL) Ufd3 or Ufd3 truncations were bound to glutathione beads and incubated with recombinant Cdc48. Bound material was eluted with sample buffer and detected by Coomassie staining. (E) Ubiquitin binding by Ufd3. GST fusions of full-length (FL) Ufd3 or truncations were bound to glutathione beads and incubated with ubiquitin chains assembled on Ubi-ProA. Bound material was eluted with sample buffer and detected with antibodies against Protein A. Molecular Cell 2006 21, 261-269DOI: (10.1016/j.molcel.2005.12.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Antagonistic Effects of Ufd2 and Ufd3 In Vivo (A) Effects of Ufd2 or Ufd3 overexpression on Ub-Pro-βgal degradation. Ub-Pro-βgal decay was measured by promoter shutoff and cycloheximide chase, and Ufd3, Ufd2, and Ufd2ΔU-box were expressed from the GAL promoter. Shp1 was used as a loading control. The asterisk denotes a deubiquitylated by-product, and the 90 kDa fragment is a typical proteolytic intermediate (Bachmair et al., 1986). The graph on the right shows quantifications of the blots. (B) Influence of Ufd3 truncation mutants on ubiquitin-Pro-βgal (Ub-P-βgal) degradation. The indicated truncation mutants were expressed from the GAL promoter, and Ub-P-βgal degradation was followed as above. (C) Free ubiquitin is depleted in Δufd3 mutants. Samples of the indicated mutant strains were taken at the indicated time points after addition of cycloheximide and analyzed by blotting against ubiquitin or Pgk1 as a loading control. (D) Genetic interactions between Δufd3, Δubp6, and Δufd2. Sevenfold serial dilutions of the indicated strains were spotted on SC-Trp or SC-Trp medium containing CHX and incubated at 34°C for 2 to 3 days. Molecular Cell 2006 21, 261-269DOI: (10.1016/j.molcel.2005.12.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Identification of Otu1, a Suppressor of Δufd3 (A) High-copy suppressors of Δufd3. The indicated proteins were expressed in Δufd3 cells from 2 μ plasmids under the control of their own promoter (DOA4), the CUP1 promoter (ubiquitin), or the ADH promoter (UBP6, OTU1, OTU2). Five-fold dilutions were spotted on SC-Trp medium or SC-Trp medium containing CHX and were incubated for 2 to 4 days at 37°C. (B) Otu1 increases ubiquitin levels in Δufd3 cells. Extracts from Δufd3 cells harboring the indicated genes on overexpression plasmids were blotted against ubiquitin or Pgk1 as a loading control. Bands were quantified with a CCD camera. Intensities from UFD3-expresssing samples were set to one, and the relative, normalized values of the other two samples are expressed as mean ± standard deviation (n = 3) in the graph. (C) Otu1 interactors by GST pull-down. Otu1 interactors were isolated and identified as described in Figure 1A. (D) Otu1 interactors by IP. Extracts from wt cells or cells expressing Otu13myc from the endogenous locus were precipitated with anti-myc antibodies and analyzed by Western blotting analysis. (E) Otu1 domain organization. (F) Mapping the Cdc48 binding site of Otu1. GSTOtu1 or truncation mutants were bound to glutathione beads and incubated with recombinant Cdc48, and bound proteins were analyzed by Coomassie blue staining. (G) Mapping the Otu1 binding site of Cdc48. Immobilized GST or the indicated GST fusion proteins were incubated with full-length or truncated Cdc48, and bound proteins were analyzed by Western blotting analysis with anti-Cdc48 antibodies. (H) Indirect interactions between Otu1 and Ufd3. Immobilized GSTOtu1 was incubated with recombinant Ufd3 in the absence (−) or presence (+) of Cdc48, and bound material was analyzed by Western blotting analysis with anti-Ufd3 antibodies. (I and J) DUB activity of Otu1. (I) Time course of Ub-AMC hydrolysis. (J) K48 or K63 Ub2–7 chains were incubated with the indicated proteins overnight at room temperature. Reactions were analyzed by Western blotting analysis with ubiquitin antibodies. Where indicated, ubiquitin aldehyde (Ubal) was included in the reaction. Molecular Cell 2006 21, 261-269DOI: (10.1016/j.molcel.2005.12.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Spt23 as a Substrate for Ufd3 and Otu1 (A) Mutations in Δufd3 and Δotu1 partially suppress Δufd2 sensitivity to oleic acid. The indicated strains harbored an integrative UBI4 plasmid to suppress possible side effects due to ubiquitin depletion. Five-fold serial dilutions of cells were spotted on YPD plates or YPD plates with 0.2% oleic acid and incubated at 33°C for 2 to 3 days. (B) Ufd3/Otu1 overexpression in Δufd2 cells. Δufd2 cells overexpressing the indicated proteins were spotted on SC-Trp-Ura medium or the same medium containing 0.2% oleic acid and grown for 2 to 3 days at 37°C. Note that Δufd2 cells are viable on SC plates containing oleic acid. (C) Influence of Ufd3 and Otu1 overexpression on Spt23 stability. mycSpt23 was expressed from the GAL promoter, and its decay was followed by promoter shutoff and CHX chase in cells overexpressing Ufd3 or Otu1. Spt23 decay was quantified using a CCD camera (see graph). (D) Spt23 interacts with Ufd3 and Otu1. mycSpt23 was expressed from the GAL promoter in wild-type or ufd1-2 cells and precipitated with myc-antibodies, and bound proteins were analyzed by immunoblotting. Molecular Cell 2006 21, 261-269DOI: (10.1016/j.molcel.2005.12.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Hypothetical Model Depicting the Potential Fates of Cdc48 Substrates and the Responsible Substrate-Recruiting and Substrate-Processing Cofactors Molecular Cell 2006 21, 261-269DOI: (10.1016/j.molcel.2005.12.014) Copyright © 2006 Elsevier Inc. Terms and Conditions