Prolonged ER stress down-regulates Ufd1, and depletion of Ufd1 by RNAi affects cell cycle progression. (A) Protein levels of endogenous Ufd1 were compared in HeLa cells treated with DMSO (−) or 0.5 μg/mL of TM (+) for 20 h by immunoblot analysis. GRP78 was used as a marker of UPR, and β-actin served as a loading control. (B) Immunoblot analysis of cell cycle markers in control and Ufd1-KD cells released from G1/S arrest after double-thymidine block. Control refers to cells infected with an empty shRNA plasmid. Levels of Ufd1 were quantified and are shown in arbitrary units (AU). (C) (Upper) Comparison of CDK1 and CDK2 kinase activity in control and Ufd1-KD cells released from G1/S arrest. CDK2 kinase reactions from control and Ufd1-KD cells were run in separate gels but were processed identically otherwise. Same for CDK1 kinase reactions. (Lower) Protein levels of Skp2 in control and Ufd1-KD cells. (D) Analysis of cell cycle markers in control and Ufd1-KD cells expressing FLAG-Ufd1. The melanoma cell line CHL1 expressing high levels of p27 was used solely as a positive control for p27 immunodetection.

Ufd1 interferes with the ubiquitination of Skp2 by recruiting the deubiquitinating enzyme USP13. (A) Mapping of the Ufd1 binding site to USP13 in vivo. USP13 was expressed with empty vector (lane 1) or with the indicated FLAG-Ufd1 C-terminal truncation mutants in HEK-293T cells (lanes 2–8). Ufd1-N215 denotes an Ufd1 truncation mutant containing the N-terminal amino acids 1–215, and so on. FLAG-Ufd1 immunoprecipitates were immunoblotted for USP13, p97, and FLAG. (B) Western blots of endogenous Skp2 in control (cont) and Ufd1-KD cells transfected with empty vector, FLAG-Ufd1, or mutant FLAG-Ufd1Δ261–280. (C) In vivo ubiquitination of Skp2 in control and Ufd1-KD cells overexpressing myc-Skp2 and HA-Ub with empty vector, FLAG-Ufd1, or FLAG-Ufd1Δ261–280. c-myc immunoprecipitates were immunoblotted for Ub. Levels of ubiquitination were quantified and are shown as AUs. (D) Mapping of the Ufd1 binding site to Skp2 in vivo. myc-Skp2 was expressed in HEK-293T cells with empty vector (lane 1) or the indicated FLAG-Ufd1 C-terminal truncation mutants (lanes 2–8). FLAG immunoprecipitates were immunoblotted for Skp2 and FLAG. (E) Coimmunoprecipitation of USP13 and Skp2 in vivo. USP13 was expressed with empty vector (lane 1) or together with myc-Skp2 (lanes 2 and 3). c-myc immunoprecipitates were immunoblotted for USP13 or Skp2. (F) Semiendogenous coimmunoprecipitation. HeLa cells were transfected with FLAG-Skp2, and FLAG immunoprecipitates were immunoblotted for endogenous USP13 and Ufd1. *Short exposure; **long exposure. (G) Evidence of ternary complex formation among USP13, Ufd1, and Skp2 by sequential immunoprecipitations. HeLa cells were transfected with FLAG-Ufd1, myc-Skp2, and USP13. FLAG immunoprecipitates were eluted with FLAG peptide. Eluates were then used for c-myc immunoprecipitation. Then 10% of FLAG-IP, 10% of eluate, and all of myc-IP were immunoblotted for USP13, Skp2, and Ufd1. (H) (Left) Immunoblots of endogenous Ufd1 in control and Ufd1-KD cells. (Middle) USP13 and myc-Skp2 were coexpressed in control cells (lanes 1 and 2) or Ufd1-KD cells (lane 3). Control cells were transfected with either half (*) or an equal amount (**) of the DNA used for transfection in Ufd1-KD cells. c-myc immunoprecipitates were immunoblotted for USP13. (Right) Control and Ufd1-KD cells were transfected with USP13 and myc-Skp2 together with empty vector (ev), FLAG-Ufd1, or the mutant FLAG-Ufd1Δ261–280. c-myc immunoprecipitates were immunoblotted for USP13 and Skp2.

ER stress regulates the Ufd1-Cdh1-Skp2-p27 axis to delay progression through G1. (A) HeLa cells were treated with DMSO or increasing concentrations of TM (0.05, 0.1, 0.25, and 0.5 μg/mL) for 20 h. Lysates were immunoblotted for Ufd1, Skp2, p27, and GRP78 (a marker of UPR). (B) Cells were treated with DMSO or with 0.5 μg/mL or 2.5 μg/mL of TM over a 24-h course. At the indicated time points, samples were collected for immunoblot and FACS analyses. Fig. S5C shows the corresponding quantification of G1 delay based on FACS analysis. (C) (Left) HeLa cells were treated with DMSO or with 2.5 μg/mL of TM alone or together with MG-132 (2 or 5 μM) for 12 h, and then collected for immunoblot analysis for the indicated proteins. (Right) HeLa cells transfected with control or Cdh1-specific shRNA for 24 h were treated with DMSO or with 2.5 μg/mL of TM alone or together with MG-132 (5, 10, or 15 μM) for 12 h. Total cell extracts were immunoblotted for the indicated proteins. (D) HeLa cells transfected with empty vector or FLAG-Ufd1 were treated with DMSO or TM (1 μg/mL for 8 h). (Upper) Western blot of Ufd1. (Bottom) Increase in G1 at 8 h after the addition of TM compared with DMSO treatment. Fig. S6C shows FACS histograms. (E) HeLa cells were transfected with either control or two different p27-specific shRNAs, as indicated, for 24 h. (Upper) Western blot analysis of p27 in control and p27-KD cells treated with TM (2.5 μg/mL for 8 h). (Bottom) Percent increase in G1 at 4 h and 8 h after TM addition relative to 0 h. The percentage of cells in G1 at 0 h is set as the baseline, with a 0% increase in G1. (F) Half-life analyses of GFP-CFTR and NHK proteins with CHX treatment in double thymidine-arrested (G1/S) or nocodazole-arrested (G2/M) HeLa cells. Total lysates were immunoblotted for GFP or α1-antitrypsin/NHK. Fig. S8A verifies cell cycle synchronization by FACS, and Fig. S8B quantifies protein half-lives. (G) Half-life of NHK protein in double thymidine-arrested G1/S HeLa cells or cells released from double-thymine arrest for 7 h (approximately corresponding to G2/M). Total lysates were immunoblotted for α1-antitrypsin/NHK. Fig S8C presents verification of cell cycle synchronization by FACS, and Fig. S8D shows quantification of NHK half-life. (H) In the proposed model, down-regulation of Ufd1 after prolonged ER stress reduces recruitment of USP13 to Skp2, thereby resulting in ubiquitin-dependent degradation of Skp2 by APC/C^Cdh1. Consequently, levels of p27 increase, contributing to G1 arrest that supports degradation of misfolded proteins.

Ufd1 interferes with the ubiquitination of Skp2 in vivo. (A) Quantification of Skp2 mRNA levels in control and Ufd1-KD cells by SYBR-green qRT-PCR. (B) Skp2 degradation assays in interphase Xenopus egg extracts supplemented with recombinant Cdh1, in the presence of the indicated amount of bacterially purified recombinant GST-Ufd1. Samples were obtained at the indicated times after ³⁵S-Skp2 was added to the extract. (C) (Upper) Half-life analysis of endogenous Skp2 protein with CHX treatment in empty vector- or FLAG-Ufd1–transfected HeLa cells released from nocodazole (noco) arrest. Total lysates were immunoblotted for Skp2 or FLAG. Quantification is shown below the immunoblots. (D) In vivo ubiquitination of Skp2. myc-Skp2 and HA-Ub were expressed in control and Ufd1-KD cells. Immunoprecipitates obtained with c-myc antibodies were immunoblotted for either ubiquitin (Ub) or c-myc to detect polyubiquitinated Skp2, denoted by (Ub)_n-Skp2. Control refers to cells infected with empty shRNA plasmid. (E) In vivo ubiquitination of Skp2. myc-Skp2 and HA-Ub were coexpressed with either empty vector or FLAG-Ufd1. Immunoprecipitates obtained with c-myc antibodies were immunoblotted for Ub. (Bottom) Expression of FLAG-Ufd1 in total cell extracts.

USP13 controls Skp2 levels via deubiquitination. (A) Immunoblots of endogenous Skp2 and exogenous USP13 in HeLa cells transfected with empty vector or untagged USP13. (B) Knockdown of USP13 in HeLa cells infected with lentivirus packaged with either empty vector (mock) or one of three individual USP13-specific shRNA vectors, as indicated. Immunoblots of endogenous USP13, Skp2, and p27 are shown. (C) In vitro deubiquitination of Skp2 by USP13 as described in Materials and Methods. Immunopurified FLAG-USP13 and immunoprecipitated myc-Skp2 were obtained from HeLa cells. Reactions were immunoblotted for Ub and Skp2.