1. Volume 54, Issue 4, Pages 586-600 (May 2014)
K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking 
Wei-Chien Yuan, Yu-Ru Lee, Shu-Yu Lin, Li-Ying Chang, Yen Pei Tan, Chin-Chun Hung, Jean-Cheng Kuo, Cheng-Hsin Liu, Mei-Yao Lin, Ming Xu, Zhijian J. Chen, Ruey-Hwa Chen 
Molecular Cell 
Volume 54, Issue 4, Pages (May 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 54, 586-600DOI: (10.1016/j.molcel.2014.03.035)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
KLHL20 Is Localized to Golgi and Potentiates Anterograde Trafficking
(A) Confocal images of Cos-1 cells stained with DAPI and indicated antibodies (top). Bar, 20 μm. Colocalization of KLHL20 with each Golgi marker was quantified (bottom). Data are mean ± SD; n = 3, seven cells per group per experiment.
(B) Western blot analysis of Golgi-enriched fraction and whole-cell lysates (WCL) derived from the same number of 293T cells (TfR, endosome/PM marker; calnexin, ER marker; EEA1, early endosome marker).
(C) Cos-1 cells stably expressing indicated siRNAs were transfected with GFP-VSVG for VSVG transport assay and representative confocal images are shown. Bars, 20 μm. The knockdown efficiencies of KLHL20 siRNAs are shown in Figure S1D.
(D) Cells as in (C) were transfected with GFP-MPR for MPR transport assay. Confocal section with the maximum Golgi signal is shown. The boxed areas are enlarged to shown on the bottom. Bars, 10 μm.
(E and F) Cos-1 cells as in (C) were transfected with indicated KLHL20 constructs, GFP-VSVG (E) or GFP-MPR (F), and mCherry. Cells were subjected to VSVG or MPR transport assay, and the percentage of cell surface VSVG or peripheral MPR in mCherry-positive cells at 120 min (for VSVG) or 60 min (for MPR) after releasing from the temperature block was quantified (see Supplemental Experimental Procedures). Representative confocal images are shown in Figures S2C and S2D.
(G) VSVG transport assay in Cos-1 cells transfected with indicated siRNAs, GFP-VSVG, and mCherry. The expression levels of Cul3 are shown on the bottom. Data in (E)–(G) are mean ± SD, n = 3, 50 cells (E and G) or 30 cells (F) per group per experiment. See also Figures S1 and S2.
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4. Figure 2
Cul3-KLHL20 Complex Controls the Formation of Tubular-Carrier Precursors at TGN
(A and C) Time-lapse confocal images of Cos-1 cells stably expressing indicated siRNAs, transfected with GFP-VSVG (A) or GFP-MPR (C), and subjected to transport analysis as in Figure 1. The representative images revealing the sequence of events of tubule formation and scission are shown. The arrows indicate scission occurrence, and the arrowheads indicate the beginning of tubule formation. Images were applied by an inverted setup of software. Bars, 5 μm.
(B and D–F) Quantitation of the VSVG- or MPR-positive tubules emerged from TGN during 5 min period (left) and the cytoplasmic carriers observed at the starting time point (right) in each indicated group. Data are mean ± SD, n = 3, ten cells per group per experiment. See also Movie S1 and Movie S2.
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5. Figure 3 Cul3-KLHL20 Complex Targets Crn7 for a K33-Ubiquitination
(A) Coimmunoprecipitation analysis of the interaction between endogenous KLHL20 and endogenous Crn7 in 293T cells.
(B) GST pull-down analysis for the interaction of KLHL20 with Crn7. The input of GST fusion proteins and Crn7 protein are shown on the bottom and right, respectively.
(C and D) Analysis of Crn7 ubiquitination in 293T cells expressing indicated constructs and/or siRNAs. The ubiquitinated proteins were pull down under denaturing conditions by Ni-NTA agarose and analyzed by western blot.
(E) Western blot analysis of Crn7 levels in 293T cells stably expressing indicated siRNAs (top) or transfected with indicated constructs (bottom).
(F) Effects of indicated ubiquitin KR mutants or ubiquitin K-only mutants on KLHL20-mediated Crn7 ubiquitination. 293T cells were transfected with indicated constructs and Crn7 ubiquitination was analyzed as in (C).
(G) Tandem mass spectrum of a peptide derived from ubiquitinated Crn7 showing ubiquitin conjugation at the K33 residue of ubiquitin (left). Ratio of indicated ubiquitin linkages detected by MS analysis of ubiquitinated Crn7 purified from Cul3-KLHL20 overexpressing cells to that from control cells (right). The abundance of each ubiquitin linkage was calculated as described in Supplemental Experimental Procedures. The K6 linkage was not detected.
(H) Ubiquitinated Crn7 purified from 293T cells overexpressing Cul3 and KLHL20 was incubated with indicated DUBs and analyzed for Crn7 ubiquitination.
(I) Tandem mass spectra of peptides derived from ubiquitinated Crn7 showing ubiquitin conjugation at amino acids 472 (left) and 680 (right). Ions labeled with “0” indicate a neural loss of H2O. See also Figure S3.
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6. Figure 4
KLHL20-Mediated Crn7 K33-Ubiquitination Promotes F-Actin Assembly to Facilitate Post-Golgi Trafficking
(A and B) VSVG or MPR transport assay in Cos-1 cells stably expressing indicated siRNAs and transfected with indicated Crn7 constructs, GFP-VSVG (A) or GFP-MPR (B), and mCherry. Data are mean ± SD, n = 3, 50 cells (A) or 30 cells (B) per group per experiment. The knockdown efficiencies of Crn7 siRNAs are shown in Figure S3B.
(C and D) VSVG or MPR transport assay in indicated ubiquitin replacement cell lines treated with or without doxycycline. Data are mean ± SD, n = 3, 50 cells (C) or 30 cells (D) per group per experiment.
(E) Confoal analysis of TGN-associated F-actin in Cos-1 cells stably expressing indicated siRNAs and stained with DAPI, phalloidin, and anti-TGN46 antibody. Confocal section showing the maximum F-actin puncta in the TGN area was used for analysis. Representative confocal images are shown (left). Bar, 20 μm. The number and area of F-actin puncta were quantified and plotted (right).
(F and G) Analysis of TGN-associated F-actin puncta in Cos-1 cells stably expressing indicated siRNAs and transfected with indicated constructs and mCherry at a ratio of 10:1.
(H) Analysis of TGN-associated F-actin puncta in K33R ubiquitin replacement cells treated with or without doxycycline. Representative confocal images and quantitation data are shown. Bar, 20 μm.
(I) Analysis of TGN-associated F-actin puncta in indicated ubiquitin replacement cells treated with doxycycline.
Data in (E)–(I) are mean ± SD, n = 3, 11 cells (E, F, and H) or 15 cells (G and I) per group per experiment. See also Figures S4 and S5.
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7. Figure 5 K33-Ubiquitinated Crn7 Interacts with Eps15 at the Golgi
(A) Coimmunoprecipitation analysis of Crn7 ubiquitination and interaction with Eps15 in 293T cells transfected with indicated constructs.
(B) Analysis of Crn7 ubiquitination and interaction with Eps15 and AP1 in Golgi-enriched membranes prepared from 293T cells transfected with indicated constructs.
(C) Analysis of Crn7 ubiquitination and interaction with Eps15 in various ubiquitin replacement cells transfected with indicated constructs and treated with doxycycline.
(D) Analysis of endogenous Crn7 ubiquitination and interaction with endogenous Eps15 in 293T cells stably expressing indicated siRNAs and transfected with His-ubiquitin.
(E) Analysis of endogenous Crn7 ubiquitination and interaction with endogenous Eps15 in K33R ubiquitin replacement cells treated with or without doxycycline.
(F) Analysis of Crn7 ubiquitination and interaction with Eps15 in Golgi-enriched membranes prepared from 293T cells transfected with indicated constructs.
(G and H) In vitro pull-down assay with full-length Eps15 (G) or Eps15 tUIM alone (H) fused with GST and ubiquitinated Crn7. Ubiquitinated Crn7 was purified from 293T cells transfected with Flag-Crn7, Myc-Cul3, Myc-KLHL20, and His-ubiquitin using anti-Flag M2 beads followed by Ni-NTA agarose.
(I) In vitro pull-down assay with indicated GST fusion proteins and ubiquitinated Crn7 purified from indicated ubiquitin replacement cell lines transfected as in (G) and treated with doxycycline (left). The amounts of input proteins are shown on the right. See also Figure S6.
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8. Figure 6
Eps15 Is Critical for Crn7 TGN Recruitment, TGN-Pool F-Actin Assembly, and Post-Golgi Trafficking
(A) Confocal analysis of Crn7 distribution in Cos-1 cells stably expressing indicated siRNAs and/or constructs, and stained with DAPI and antibodies to Crn7 and TGN46 (top). Bar, 20 μm. The line scans of Crn7 and TGN46 fluorescence intensity (F. I.) across the TGN area (right) and the percentage of Crn7 fluorescence in the TGN area (middle) were analyzed by Image J software and plotted. KLHL20 expression levels are shown on the bottom. The specificity of Crn7 antibody for immunofluorescence is shown in Figure S7F.
(B and C) Quantification of Crn7 TGN localization in K33R ubiquitin replacement cells treated with or without doxycycline (B) or Cos-1 cells stably expressing indicated siRNAs (C). In (C), the Eps15 expression levels are shown on the bottom. Data in (A)–(C) are mean ± SD, n = 3, 30 cells per group per experiment.
(D) Quantification of Crn7 TGN localization in Cos-1 cells stably expressing indicated siRNAs, transfected with V5-Eps15, and costained with antibodies to Crn7, TGN46, and V5. Data are mean ± SD, n = 3, 40 cells per group per experiment.
(E) Quantification of TGN-associated F-actin puncta in Cos-1 cells stably expressing indicated siRNAs, transfected with indicated Eps15 constructs and mCherry, and stained with phalloidin and antibody to TGN46. Data are mean ± SD, n = 3, 15 cells per group per experiment. Representative confocal images for (B)–(E) are shown in Figures S7A, S7B, S7D, and S7E, respectively.
(F and G) Quantification of VSVG- or MPR-positive tubular-carrier precursors and cytoplasmic carriers during the trafficking assays performed in cells as in (D). Data are mean ± SD, n = 3, ten cells per group per experiment.
(H and I) VSVG or MPR transport assay of cells as in (D). Data are mean ± SD, n = 3, 50 cells (H) or 30 cells (I) per group per experiment. See also Figure S7.
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9. Figure 7
Enforced TGN Targeting of Crn7 Rescues Post-Golgi Transport in KLHL20-Depleted or K33 Ubiquitin Chain-Depleted Cells
(A) Confocal analysis of TGN-associated F-actin puncta in Cos-1 cells stably expressing KLHL20 siRNA, transfected with indicated constructs and treated with or without rapamycin. Bar, 20 μm.
(B) VSVG transport assay of Cos-1 cells as in (A), transfected with indicated constructs together with GFP-VSVG, and treated with or without rapamycin. Bar, 20 μm.
(C) Quantification of TGN-associated F-actin puncta in K33R ubiquitin replacement cells transfected with indicated constructs and treated with or without doxycycline and rapamycin.
(D) VSVG transport assay of K33R ubiquitin replacement cells transfected with indicated constructs together with GFP-VSVG, and treated with or without doxycycline and rapamycin.
(E) Model for KLHL20-mediated Crn7 ubiquitination in regulating TGN-associated F-actin assembly and post-Golgi trafficking. Data in (A)–(D) are mean ± SD, n = 3, ten cells (A and C) or 50 cells (B and D) per group per experiment.
Molecular Cell  , DOI: ( /j.molcel )
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