K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking

Please cite this article in press as: Yuan et al., K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.03.035

Molecular Cell

Article K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking Wei-Chien Yuan,1,2,5 Yu-Ru Lee,1,5 Shu-Yu Lin,1 Li-Ying Chang,1 Yen Pei Tan,1 Chin-Chun Hung,1 Jean-Cheng Kuo,3 Cheng-Hsin Liu,1 Mei-Yao Lin,1,6 Ming Xu,4 Zhijian J. Chen,4 and Ruey-Hwa Chen1,2,* 1Institute

of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 106, Taiwan 3Institute of Biochemistry and Molecular Biology, National Yang Ming University, Taipei 112, Taiwan 4Department of Molecular Biology, Howard Hughes Medical Institute, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA 5These authors contributed equally to this work and are co-first authors 6Present address: Synaptic Function Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorder and Stroke, National Institutes of Health, Bethesda, MD 20892, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.03.035 2Institute

SUMMARY

Ubiquitin chains are formed as structurally distinct polymers via different linkages, and several chain types including K33-linkage remain uncharacterized. Here, we describe a role for K33-polyubiquitination in protein trafficking. We show that the Cullin 3 (Cul3) substrate adaptor KLHL20 is localized to the transGolgi network (TGN) and is important for post-Golgi trafficking by promoting the biogenesis of TGNderived transport carriers. The Cul3-KLHL20 ubiquitin E3 ligase catalyzes a nondegradable, K33-linked polyubiquitination on coronin 7 (Crn7), which facilitates Crn7 targeting to TGN through a ubiquitindependent interaction with Eps15. Blockage of K33-chain formation, Crn7 ubiquitination, or disruption of Crn7-Eps15 interaction impairs TGN-pool F-actin assembly, a process essential for generating transport carriers. Enforced targeting of Crn7 to TGN bypasses the requirement of K33-ubiquitination for TGN-pool F-actin assembly and post-Golgi trafficking. Our study reveals a role of KLHL20-mediated K33-ubiquitination of Crn7 in post-Golgi transport and identifies a cellular recognition mechanism for this ubiquitin chain type. INTRODUCTION Protein ubiquitination is a posttranslational modification by tagging the 76 amino acid ubiquitin to the Lys residues in target proteins. As all Lys residues and the Met1 residue in ubiquitin can mediate the conjugation of another ubiquitin moiety, polyubiquitin chains with various lengths and linkages can be formed on substrates. Mass spectrometry (MS) analyses indicate that all ubiquitin linkages coexist in all seven cell types analyzed. These topologically distinct polymers can give rise to diverse cellular

functions, making ubiquitination as one of the most versatile posttranslational modifications in cells (Kulathu and Komander, 2012). For instance, K48-linked chains serve as proteolytic signal by targeting substrates for degradation in proteasomes (Hershko and Ciechanover, 1998). In contrast, K63-linked chains function as molecular platforms for protein-protein interactions important in various signaling pathways (Chen and Sun, 2009). Evidence has emerged that K11-linked chains also target substrates for proteasomal degradation (Wickliffe et al., 2011), whereas Met1-linked linear chains function nonproteolytically in NFkB and immune signalings (Rieser et al., 2013). However, the roles of the remaining ubiquitin chain types, including the K33-linked chains, are poorly understood. Furthermore, the diverse functions of ubiquitin linkages are mainly mediated by interactions with various ubiquitin binding proteins (Husnjak and Dikic, 2012), but the cellular recognition mechanism for K33-linked chains remains elusive. Ubiquitination is well known to function in protein trafficking. In both endocytic and secretory pathways, ubiquitin modification of membrane proteins serves as a sorting signal for their delivery to specific destinations through interaction with a number of ubiquitin-binding adaptor proteins (MacGurn et al., 2012), such as Eps15. Eps15 contains two ubiquitin-interacting motifs (UIMs) for binding ubiquitinated cargos. Besides UIMs, Eps15 possesses other interaction domains and acts as a scaffold for the recruitment and clustering of other clathrin adaptor proteins during the early stage of endocytosis (McMahon and Boucrot, 2011). Eps15 is also recruited to the trans-Golgi network (TGN) by interacting with AP1, which is critical for anterograde transport from TGN to endosomes and plasma membrane (PM) (Chi et al., 2008). However, the detailed roles of Eps15 in post-Golgi trafficking have not been characterized. TGN is a major sorting station of the secretory pathway, from which a plethora of cargos is delivered to different destinations via pleomorphic, post-Golgi tubular carriers. Carrier biogenesis involves the segregation of cargos into distinct microdomains, membrane deformation, elongation of the tubular-carrier precursors, and scission to release the carriers (Anitei and Hoflack, 2011; De Matteis and Luini, 2008). Accumulating evidence Molecular Cell 54, 1–15, May 22, 2014 ª2014 Elsevier Inc. 1

Please cite this article in press as: Yuan et al., K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.03.035

Molecular Cell K33-Polyubiquitination in Protein Trafficking

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 mm. 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). (legend continued on next page)

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Please cite this article in press as: Yuan et al., K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.03.035

Molecular Cell K33-Polyubiquitination in Protein Trafficking

indicates the contribution of actin cytoskeleton to the early stage of carrier biogenesis. The polymerization of F-actin, which is believed to direct toward the TGN membrane (Anitei et al., 2010), in conjunction with the myosin motors, exert mechanical force to assist membrane deformation (Anitei and Hoflack, 2012). A number of actin polymerization factors are recruited to TGN to promote the biogenesis of post-Golgi carriers (Anitei et al., 2010; Salvarezza et al., 2009). However, Hip1R is also recruited to TGN to prevent F-actin overassembly or misassembly at the surface of forming carriers (Carreno et al., 2004; Poupon et al., 2008). This finding implies the importance of temporal and/or spatial regulation of F-actin dynamics during carrier biogenesis, although the detail of such regulatory process has not been well understood. The multisubunit Cullin-RING ubiquitin ligases consist of the largest family of E3 ligases. In this E3 ligase complex, Cullin serves as a scaffold for linking the catalytic RING finger protein Roc1 and the substrate binding subunit. In Cullin 3 (Cul3)-based E3 ligase complex, the BTB domain-containing protein functions as the substrate binding subunit (Genschik et al., 2013; Pintard et al., 2004). Here, we report that the BTB protein KLHL20 participates in post-Golgi transport through a K33-linked polyubiquitination of an F-actin regulator coronin 7 (Crn7). Ubiquitinated Crn7 is recruited to TGN through an interaction with clathrin adaptor Eps15, thereby promoting F-actin assembly at TGN to contribute to post-Golgi trafficking. RESULTS KLHL20 Is Localized to TGN in an Arf-Family GTPase-Dependent Manner KLHL20 functions as a substrate adaptor of Cul3-based ubiquitin E3 ligase. Even though previous studies identified several cytoplasmic and nuclear substrates of KLHL20-based E3 ligase (Lee et al., 2010; Lin et al., 2011; Yuan et al., 2011), immunofluorescence staining revealed that endogenous KLHL20 was mainly distributed to the perinuclear region of Cos-1 cells (Figure 1A). This structure was partially costained with cis-Golgi marker GM130, trans-Golgi marker p230, and TGN marker TGN46, and the most precise colocalization was found with TGN46. The TGN localization of KLHL20 was also detected in several other cell types or by anti-Flag antibody for ectopic KLHL20 (see Figures S1A and S1B). The Golgi distribution of KLHL20 was further substantiated by a biochemical method using fractionated, Golgi-enriched membrane (Figure 1B). Notably, brefeldin A (BFA) treatment disrupted the perinuclear distribution of KLHL20, which occurred earlier than the disruption of Golgi complex (Figure S1C). This observation indicates that an Arf or

Arf-like (Arl) GTPase is required for the Golgi recruitment of KLHL20. Cul3-KLHL20 Complex Contributes to Anterograde Transport Since the Arf-family GTPases control many aspects of Golgimediated vascular trafficking (D’Souza-Schorey and Chavrier, 2006), we investigated the role of KLHL20 in trafficking. We first monitored the anterograde transport of ts045-VSVG-GFP (designated as GFP-VSVG hereafter), which is accumulated in the ER at 40 C and transported from ER via Golgi to PM upon shifting to a permissive temperature at 32 C (Hirschberg et al., 2000). At 15 min after temperature shift, the Golgi arrival of GFP-VSVG was observed in both control and KLHL20-depleted cells (Figure 1C). However, the transport of GFP-VSVG to PM was impaired in KLHL20-depleted cells, as detected by confocal or flow cytometry analysis of cell surface VSVG (Figures 1C, S1D, and S1E). Next, we monitored the transport of GFP-tagged mannose-6-phosphate receptor (GFP-MPR) from TGN to endosomes after releasing from a temperature block at 20 C. Similarly, KLHL20 depletion blocked TGN exit of GFP-MPR (Figure 1D). KLHL20 knockdown, however, did not alter the glycosylation of an anterograde cargo LAMP2 (Figure S1F), suggesting that KLHL20 affects cargo export rather than cargo modifications. Consistently, KLHL20 knockdown also impaired TGN exit of two other cargos, the GFP-tagged neural cell adhesion molecule (NCAM-GFP) and internalization-defective low-density lipoprotein receptor mutant (LDLR-A18-GFP) (Figures S1G and S1H). Since the Golgi complex also governs retrograde trafficking, we examined the influence of KLHL20 on the traffics of Cholera toxin B (CTxB) from PM to Golgi and Shiga toxin B (STxB) from PM via Golgi to ER. The endocytosed CTxB reached to Golgi at the same rate in control and KLHL20-depleted cells (Figure S2A). Similarly, neither the PM-to-Golgi nor the Golgi-to-ER transport of STxB was affected by KLHL20 knockdown (Figure S2B). Thus, KLHL20 contributes to anterograde trafficking from TGN to endosomes and PM without affecting retrograde trafficking. To dissect the mechanism by which KLHL20 regulates postGolgi trafficking, we evaluated whether this function is dependent on the formation of Cul3-KLHL20 complex. While reconstitution of wild-type KLHL20 into KLHL20-knockdown cells rescued anterograde transport of VSVG, MPR, NCAM, and LDLR, the Cul3 binding-defective mutant KLHL20m6 (Lee et al., 2010) failed to do so (Figures 1E, 1F, S1G, S1H, S2C, and S2D), even though this mutant was able to localize to Golgi (Figure S2E). Furthermore, Cul3 depletion impaired post-Golgi transport of VSVG (Figure 1G). These data collectively indicate a role of the Cul3-KLHL20 E3 ligase in anterograde trafficking.

(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 mm. 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 mm. (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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Please cite this article in press as: Yuan et al., K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.03.035

Molecular Cell K33-Polyubiquitination in Protein Trafficking

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 (legend continued on next page)

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Please cite this article in press as: Yuan et al., K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.03.035

Molecular Cell K33-Polyubiquitination in Protein Trafficking

Cul3-KLHL20 Complex Contributes to the Biogenesis of Post-Golgi Carrier Tubules To more precisely characterize the effect of KLHL20 on postGolgi trafficking, we determined its functional position in the anterograde transport process. Exit of cargos from TGN depends on the formation of tubular-carrier precursors which subsequently undergo a scission step to generate post-Golgi carriers (Anitei and Hoflack, 2011). Time-lapse image analysis was used to track the process of formation of post-Golgi carriers. In cells receiving control siRNA, GFP-VSVG- or GFP-MPR-containing tubular-carrier precursors were emanated from the TGN, followed by scission to release transport carriers (Figures 2A and 2C; Movie S1 and Movie S2). In addition, numerous GFP-VSVGor GFP-MPR-positive vesicular carriers were found in the cytoplasm (Figures 2A–2D). In contrast, the KLHL20 knockdown cells exhibited a significant decrease in the formation of tubular-carrier precursors, leading to the reduction of cytoplasmic carriers. Interestingly, short carrier precursors were occasionally seen in KLHL20-knockdown cells, but they failed to elongate and eventually retracted back to the TGN (Figures 2A and 2C; Movie S1 and Movie S2). Reconstitution of wild-type KLHL20, but not KLHL20m6, into KLHL20-depleted cells rescued the generation of VSVG- and MPR-containing tubular-carrier precursors and cytoplasmic carriers (Figures 2B and 2D). Furthermore, Cul3 knockdown phenocopied the effect of KLHL20 knockdown in the biogenesis of TGN-derived carrier tubules (Figures 2E and 2F). These findings point to a role of Cul3-KLHL20 E3 ligase in the formation and elongation of tubular-carrier precursors.

were not affected by KLHL20 overexpression or knockdown (Figures 3E and S3E). We thus analyzed the type of ubiquitin chain generated on Crn7 by the KLHL20-Cul3 complex. We first mutated each of the Lys residues on ubiquitin for testing their effects on Crn7 ubiquitination in cells overexpressing KLHL20 and Cul3. Strikingly, only the K33R ubiquitin could not support KLHL20-mediated Crn7 polyubiquitination (Figure 3F). We next used another set of ubiquitin mutants in which all but one Lys residues were replaced by Arg. Notably, while K48- and K63-ubiquitin could not significantly confer KLHL20-mediated Crn7 polyubiquitination, K33-ubiquitin promoted chain formation on Crn7 as potently as the wild-type ubiquitin (Figure 3F). To further demonstrate the generation of a K33-polyubiquitin chain on Crn7, we performed MS analysis of ubiquitinated Crn7 purified from control or KLHL20-Cul3-overexpressing cells. This analysis revealed that ubiquitin peptide with a di-Gly modification on the K33 residue was dramatically increased upon overexpression of Cul3 and KLHL20, whereas ubiquitin modification on other Lys residues was essentially unchanged or undetected (Figure 3G). Finally, we showed that a K29/K33-specific deubiquitinating enzyme (DUB) TRABID (Licchesi et al., 2012), but not its catalytic mutant, completely deconjugated ubiquitin chain on Crn7 purified from Cul3-KLHL20-overexpressing cells, whereas OTUB2, which acts preferentially on K63-linked chain and weakly on K48/K11-linked chains (Mevissen et al., 2013), did not display a significant effect (Figure 3H). Thus, our study provides multiple lines of evidence for the formation of a K33-linked polyubiquitin chain on Crn7 by the Cul3-KLHL20 E3 ligase.

Cul3-KLHL20 Complex Targets Crn7 for a K33-Linked Polyubiquitination To unravel the mechanism by which Cul3-KLHL20 complex regulates post-Golgi trafficking, we aimed to identify its substrate by performing a yeast two-hybrid screen using KLHL20 substratebinding domain, the kelch-repeat domain, as bait. Among the positive clones recovered, we were particularly interested in Crn7 (Figure S3A). Crn7 is crucial for post-Golgi transport of VSVG and MPR but not retrograde trafficking and promotes post-Golgi carrier formation (Rybakin et al., 2006). Crn7 knockdown also impaired TGN exit of LDLR and NCAM (Figures S3B and S3C). Thus, KLHL20 and Crn7 function similarly in postGolgi trafficking. Using coimmunoprecipitation analysis, we demonstrated the association of exogenous and endogenous KLHL20 with endogenous Crn7 (Figures 3A and S3D). In vitro pull-down analysis revealed a direct interaction of the two proteins (Figure 3B). Furthermore, coexpression of KLHL20 and Cul3 robustly stimulated Crn7 polyubiquitination, whereas replacement of KLHL20 with KLHL20m6 abolished this effect (Figure 3C). Conversely, depletion of KLHL20 diminished Crn7 ubiquitination (Figure 3D). These findings identify a role of Cul3-KLHL20 complex in Crn7 polyubiquitination. Despite the remarkable impact of KLHL20 on Crn7 polyubiquitination, the steady-state level and turnover rate of Crn7

KLHL20-Mediated Crn7 K33-Ubiquitination Contributes to the Post-Golgi Trafficking Next, we investigated whether promotion of Crn7 K33-ubiquitination is responsible for the function of KLHL20 in post-Golgi transport. The aforementioned MS analysis identified Crn7 K472 and K680 residues being modified by ubiquitin in Cul3KLHL20-overexpressing cells (Figure 3I). We therefore mutated each residue to investigate its influence on post-Golgi trafficking. While reconstitution of wild-type Crn7 or Crn7 K680R into Crn7depleted cells rescued VSVG transport, the Crn7 K472R mutant failed to do so (Figure 4A). Crn7 K472R also could not rescue MPR transport in Crn7-depleted cells (Figure 4B). To provide additional evidence for the importance of KLHL20-mediated Crn7 ubiquitination in post-Golgi trafficking, we sought to identify Crn7 mutant that cannot bind KLHL20. Coimmunoprecipitation analysis revealed a fragment encompassing residues 835–865 of Crn7 as the minimal KLHL20-binding region (Figure S4A). Importantly, the Crn7D835-865 mutant could not display a KLHL20-dependent ubiquitination (Figure S4B) and did not rescue the VSVG and MPR trafficking in Crn7-depleted cells (Figures S4C and S4D). These data collectively support a role of KLHL20-dependent Crn7 ubiquitination in post-Golgi trafficking. To demonstrate a requirement of K33-linked ubiquitination for post-Golgi trafficking, we utilized a previously developed

indicate scission occurrence, and the arrowheads indicate the beginning of tubule formation. Images were applied by an inverted setup of software. Bars, 5 mm. (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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Molecular Cell K33-Polyubiquitination in Protein Trafficking

(legend on next page)

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Molecular Cell K33-Polyubiquitination in Protein Trafficking

ubiquitin replacement system which allows simultaneous depletion of endogenous ubiquitin and expression of exogenous ubiquitin or its variants by doxycycline (Xu et al., 2009). Replacement of endogenous ubiquitin with a K33R mutant impaired postGolgi transport of VSVG, whereas replacement with the wildtype ubiquitin did not affect this trafficking (Figure 4C). Replacement with a K33R mutant also blocked TGN exit of MPR, LDLR, and NCAM (Figures 4D, S4E, and S4F). Notably, among the ubiquitin KR mutants, only K33R and K63R replacement impaired VSVG and MPR transport (Figures S4G and S4H). These results suggest an important and relatively specific role of K33-linked ubiquitination in post-Golgi trafficking, thus providing an additional layer of evidence for the relevance of KLHL20-mediated Crn7 K33-ubiquitination to this trafficking. KLHL20-Mediated Crn7 Ubiquitination Contributes to the Assembly of TGN-Associated F-Actin Next, we determined the mechanism by which KLHL20-mediated Crn7 ubiquitination potentiates post-Golgi trafficking. Formation of TGN tubular-carrier precursors is an actin-dependent process (Anitei and Hoflack, 2011, 2012). Coronin family proteins are F-actin regulators (Chan et al., 2011) and the Dictyostelium Crn7 binds F-actin to prevent its depolymerization (Shina et al., 2010). Since human Crn7 similarly bound F-actin in vitro (Figure S5A, top), we reasoned that KLHL20-mediated Crn7 ubiquitination may facilitate carrier biogenesis by regulating F-actin at TGN. In line with this hypothesis, KLHL20 depletion did not affect the anterograde transport of GFP-GPI (Figure S5B), which exits from TGN through an actin-independent mechanism (La´zaroDie´guez et al., 2007; Salvarezza et al., 2009). To directly access the influence of KLHL20 and Crn7 on the TGN-pool F-actin, we utilized two F-actin probes, F-tractin (Case and Waterman, 2011) and phalloidin. Consistent with previous reports (Almeida et al., 2011; Poupon et al., 2008), control cells exhibited F-actin puncta at TGN area, most of which were in the close vicinity of or overlapped with the TGN signal (Figures 4E and S5C). Remarkably, the number and size of TGN-associated F-actin puncta were reduced by depletion of KLHL20 or Crn7. Importantly, while reconstitution of wild-type KLHL20 and Crn7 into their knockdown cells completely rescued TGN-associated F-actin, KLHL20m6, Crn7 K472R, and Crn7D835-865 failed to do so (Figures 4F, 4G, and S5D). Since Crn7 K472R and Crn7D835-865 displayed in vitro F-actin stabilization activities

similar to the wild-type protein (Figure S5E), their inability to rescue post-Golgi trafficking is most likely due to a defect in ubiquitination. Furthermore, depletion of K33-linked ubiquitin chain, but not K63-linked chain, also downregulated TGN-associated F-actin puncta (Figures 4H and 4I). Thus, although K33and K63-linked ubiquitination events are both required for post-Golgi trafficking, they do not act at the same step in the trafficking process. Together, our study provides compelling evidence for the contribution of KLHL20-mediated Crn7 K33polyubiquitination to F-actin assembly at TGN. K33-Polyubiquitinated Crn7 Interacts with Eps15 at TGN One unanswered question is how Crn7 ubiquitination facilitates F-actin assembly at TGN. Notably, unmodified and polyubiquitinated Crn7 exhibited comparable F-actin binding and stabilization activities in vitro (Figure S5A, bottom; Figure S5F), suggesting that ubiquitination did not directly affect these functions. An alternative possibility is that ubiquitinated Crn7 regulates TGNassociated F-actin via interaction with a ubiquitin-binding protein. Eps15 and GGAs are known ubiquitin-binding proteins and are clathrin adaptors required for post-Golgi trafficking (Polo et al., 2002; Puertollano and Bonifacino, 2004). Immunoprecipitation of wild-type Crn7 from cells coexpressing ubiquitin and Cul3-KLHL20 complex revealed a coprecipitation of endogenous Eps15, but not GGA3 (Figures 5A and S6A). This coprecipitation was dependent on Crn7 ubiquitination, as it was markedly decreased in cells without overexpressing the KLHL20-Cul3 complex or by replacement of Crn7 with Crn7D835-865 or Crn7 K472R (Figures 5A and 5B). Interestingly, K472R mutation also blocked Crn7 interaction with the clathrin adaptor AP1, supporting a critical role of K472 ubiquitination for Crn7 binding to AP1 in vivo. We further showed that replacement of wild-type ubiquitin with a K33R mutant completely blocked basal and KLHL20-promoted interaction between Crn7 and Eps15, whereas replacement with a K48R mutant did not affect this interaction (Figure 5C). In line with these findings, depletion of KLHL20 or K33-linked ubiquitin chain not only decreased the ubiquitination of endogenous Crn7 but blocked the interaction between endogenous Crn7 and endogenous Eps15 (Figures 5D and 5E). Since the K33R ubiquitin was expressed at a level equivalent to that of endogenous ubiquitin in the ubiquitin replacement system (Figure S6B), our data support

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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Please cite this article in press as: Yuan et al., K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.03.035

Molecular Cell K33-Polyubiquitination in Protein Trafficking

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 GFPMPR (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 mm. The number and area of F-actin puncta were quantified and plotted (right). (legend continued on next page)

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Please cite this article in press as: Yuan et al., K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.03.035

Molecular Cell K33-Polyubiquitination in Protein Trafficking

the existence of Crn7 K33-ubiquitination and its crucial role in binding Eps15 in a physiological setting. The KLHL20-promoted interaction of Crn7 with Eps15 was also detected by using Golgienriched membranes (Figures 5B and 5F), thus demonstrating the location of this interaction. To determine whether this interaction is direct, in vitro pull-down assay was performed. Importantly, the ubiquitinated Crn7 purified from Cul3-KLHL20 overexpressing cells bound Eps15 but not Eps15DUIM, which harbors a deletion of its tandem-UIM (tUIM) (Figure 5G). Furthermore, the Eps15 tUIM segment, but not its mutant, was capable of binding polyubiquitinated Crn7 in vitro (Figure 5H). Thus, the Eps15 tUIM is sufficient and necessary for binding ubiquitinated Crn7. We further showed that Eps15 could pull down polyubiquitinated Crn7 purified from the ubiquitin replacement cells expressing wild-type ubiquitin, but not K33R ubiquitin, even though an equivalent amount of ubiquitinated Crn7 was introduced into the reaction (Figure 5I). This finding suggests a specificity of Eps15 for binding Crn7 modified by a K33-linked polyubiquitin chain in vivo, even though Eps15 tUIM did not preferentially bind K33-diubiquitin in vitro (Figure S6C). Together, our study supports that KLHL20-induced formation of a K33-linked ubiquitin chain on Crn7 facilitates its interaction with Eps15 at TGN. Interaction with Eps15 Facilitates the TGN Recruitment of Crn7 Crn7 exists in both cytosol and Golgi (Rybakin et al., 2006; Rybakin et al., 2008). We determined whether binding of ubiquitinated Crn7 to Eps15 contributes to the Golgi localization of Crn7. Importantly, depletion of KLHL20 attenuated TGN localization of Crn7, which was rescued by wild-type KLHL20 but not KLHL20m6 (Figure 6A). Depletion of K33-linked ubiquitin chain or Eps15 also decreased TGN localization of Crn7 (Figures 6B– 6D, S7A, and S7B). Reconstitution of the Eps15 knockdown cells with an Eps15 mutant that was defective in binding ubiquitinated Crn7 (Figure S7C) failed to rescue Crn7 TGN localization, whereas the wild-type Esp15 elicited a full rescue effect (Figures 6D and S7D). This mutant also could not rescue TGN-associated F-actin puncta, biogenesis of VSVG- and MPR-containing tubular carrier precursors and cytoplasmic carriers, and anterograde transport of VSVG and MPR (Figures 6E–6I and S7E), thus demonstrating a role of Eps15 tUIM in post-Golgi trafficking. Collectively, our findings support that ubiquitin-dependent interaction with Eps15 promotes the TGN recruitment of Crn7, which in turn regulates the TGN-pool F-actin to facilitate post-Golgi trafficking. Enforced Targeting of Crn7 to TGN Bypasses the Requirement of Crn7 K33-Ubiquitination for Post-Golgi Trafficking If the K33-ubiquitination functions as a signal for the recruitment of Crn7 to TGN, enforced TGN targeting of Crn7 would confer

post-Golgi trafficking without the need of this ubiquitination event. To test this possibility, we took advantage of a recently developed approach for acutely targeting protein to TGN by rapamycin-induced heterodimerization of mTOR FRB domain with FKBP12 (Szentpetery et al., 2010). When cells were cotransfected with TGN38-FRB-BFP and mRFP-FKBP12-Crn7, rapamycin induced Crn7 TGN localization via its association with the TGN protein TGN38 (Figure 7A). Importantly, this TGN targeting of Crn7 rescued TGN-pool F-actin and VSVG post-Golgi transport (Figures 7A and 7B). In control cells expressing TGN38-FRB-BFP and mRFP-FKBP12, rapamycin could not elicit these effects. Similarly, enforced TGN targeting of Crn7 in the K33R ubiquitin replacement cells reversed the defects in TGN F-actin assembly and VSVG post-Golgi transport (Figures 7C and 7D). Thus, the primary role of KLHL20-dependent K33ubiquitination is to bring Crn7 to TGN where it stabilizes F-actin to facilitate post-Golgi trafficking. DISCUSSION Our study identifies a function of KLHL20 in membrane trafficking (Figure 7E). We show that KLHL20 is localized to TGN and contributes to post-Golgi trafficking by promoting the biogenesis of TGN-derived tubular-carrier precursors. This function is attributed to the K33-ubiquitination of Crn7 mediated by Cul3-KLHL20 E3 ligase complex. This ubiquitination does not lead to Crn7 proteolysis but creates a binding surface for the tUIM of Eps15. Binding of the ubiquitinated Crn7 to Eps15 promotes Crn7 recruitment to TGN, where it exerts F-actin stabilization function to contribute to the assembly of TGN-pool F-actin, thereby facilitating the generation and elongation of TGNderived carrier tubules. Interestingly, among the two ubiquitination sites identified by MS, only K472 is critical for post-Golgi trafficking. One possibility is that ubiquitin chain conjugated on the other site is inaccessible to Eps15 tUIM. Regardless of the underlying mechanism for the distinct effect of ubiquitination sites, our study reveals a crucial role of Crn7 K33-ubiquitination in post-Golgi trafficking. Furthermore, since KLHL20 localization to Golgi is dependent on an Arf-family GTPase, we believe that this ubiquitination event is temporally controlled to coordinate with certain Arf-regulated processes, such as coat assembly, during the early stage of post-Golgi carrier biogenesis. Our finding that K33-ubiquitinated Crn7 does not undergo degradation is consistent with the fate of K33-ubiquitinated zchain of T cell antigen receptor (Huang et al., 2010) and the K29- and K33-ubiquitinated AMPK family kinases (Al-Hakim et al., 2008). Furthermore, a recent study on TRABID also supports a nonproteolytic fate of K33-ubiquitinated proteins (Licchesi et al., 2012). Although these studies indicate that the K33-linked chain is an inefficient proteolytic signal, the physiological role of this chain type remains elusive. To our knowledge,

(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 mm. (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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Molecular Cell K33-Polyubiquitination in Protein Trafficking

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. (legend continued on next page)

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Molecular Cell K33-Polyubiquitination in Protein Trafficking

the finding that K33-ubiquitinated Crn7 binds Eps15 tUIM to promote the assembly of TGN-associated F-actin represents the first description of a cellular recognition mechanism and the first demonstration of a positive signaling function of this atypical ubiquitin chain. Cul3-family ubiquitin ligases often target substrates for proteasomal degradation but can also catalyze monoubiquitination on certain substrates (Beck et al., 2013; Herna´ndez-Mun˜oz et al., 2005; Jin et al., 2012). Our identification of the K33-ubiquitination by Cul3-KLHL20 complex expands the versatility of this family of ligases. Similarly, Cul1-family ubiquitin ligases have been reported to form K63- and K11-linked chains on certain substrates (Wu et al., 2012; Zhang et al., 2013). Since the RING-type ubiquitin ligases rely on their E2s for determining the linkage specificity in chain formation, it is conceivable that the Cul3-KLHL20 complex can generate a K33-linked chain on Crn7 and a K48linked chain on several other substrates by cooperating with different E2s. The E2 enzyme specifically mediating K33-chain assembly has not been identified. Therefore, it remains elusive how KLHL20-based E3 ligase coordinates with different E2s to act on different substrates. The selectivity of Eps15 tUIM for binding different ubiquitin chains has not been reported. The distance between the two UIMs would fit the configuration of a K63-linked diubiquitin (Sims and Cohen, 2009), which is consistent with our finding. Of note, molecular modeling predicts that the K33-linked diubiquitin adopts an extended conformation (Fushman and Walker, 2010), similar to the K63-linked diubiquitin. This may explain our finding that Eps15 tUIM can also bind K33-linked diubiquitin, albeit with a lower affinity than K63-linked diubiquitin. Although this tUIM did not show a binding preference to K33-linked diubiquitin, pull-down experiment with ubiquitinated Crn7 purified from wild-type and K33R ubiquitin replacement cells suggests a preference of Eps15 for binding K33-ubiquitinated Crn7. One explanation for the seeming discrepancy is that the binding preference of Eps15 tUIM to diubiquitin and to a longer ubiquitin chain might be different. In keeping with this notion, the second UIM of Eps15 is double sided (Hirano et al., 2006), suggesting the ability of Eps15 tUIM to bind three ubiquitin moieties simultaneously. In addition, our pull-down analysis suggests a preference of Eps15 tUIM to bind Crn7 modified by a longer than shorter ubiquitin chain (by comparison of the input and pull down products). We show that ubiquitination does not affect the F-actin binding and stabilizing functions of Crn7, but enhances its TGN localization by binding to Eps15. In line with these findings, enforced targeting of Crn7 to TGN bypasses the requirement of K33ubiquitination for anterograde transport. Crn7 was reported to

interact with AP1 in vitro and in vivo (Rybakin et al., 2006). While this previous study suggests a role of AP1 in Crn7 TGN targeting, our study indicates that ubiquitin-dependent interaction of Crn7 with Eps15 is also required for this localization. Furthermore, the Crn7 ubiquitination site mutant is defective in binding AP1 in vivo, even though AP1 does not contain a ubiquitin-binding motif. This finding suggests a primary role of Eps15 in the TGN recruitment of Crn7. Once Crn7 is caught by Eps15, additional interaction with AP1 may ensure its retention on TGN. Such dual requirements for TGN targeting are observed with other proteins, such as arfaptins (Cruz-Garcia et al., 2013). In addition to promoting the TGN recruitment of Crn7, binding of ubiquitinated Crn7 to Eps15 may benefit the assembly of TGN-associated F-actin through an additional mechanism. As observed in endocytosis, Eps15 is concentrated at the edge, rather than the surface, of forming vesicles (McMahon and Boucrot, 2011; Tebar et al., 1996). At TGN, actin polymerization is thought to occur at the edge of forming tubular carriers, so that the pushing force generated by actin polymerization is directed toward the base of curved membrane to promote the elongation of carrier precursors (Anitei and Hoflack, 2011; Anitei et al., 2010). We reason that, through interacting with Eps15, ubiquitinated Crn7 is likely situated at the edge of forming carriers, where it stabilizes F-actin to promote the elongation of tubular-carrier precursors. Thus, our study reveals a function of Eps15 tUIM in vesicular trafficking, besides sorting ubiquitinated cargos. In addition, since Eps15 is a coat accessory protein, our findings suggest that Eps15 coordinates the coat assembly event with the downstream F-actin polymerization process during post-Golgi carrier biogenesis through its interaction with ubiquitinated Crn7. In summary, our study reveals a function of KLHL20-mediated, K33-linked ubiquitination of Crn7 in the assembly of TGN-associated F-actin to promote post-Golgi trafficking and identifies a cellular decoding mechanism for this ubiquitin chain type. Interestingly, a recent study indicates that the MAGE-L2-TRIM27 ubiquitin ligase targets WASH for K63-linked ubiquitination to potentiate endosomal F-actin nucleation and retromer-mediated transport (Hao et al., 2013). Nonproteolytic ubiquitinationinduced actin cytoskeleton remodeling might be a general mechanism for regulating various intracellular trafficking pathways. EXPERIMENTAL PROCEDURES Cell Culture, Transfection, and Stable Cell Lines HeLa, 293T, 293FT, A431, H1299, and Cos-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS). Stable cell lines were generated by lentivirus transduction. U2OSderived wild-type and K63R ubiquitin replacement cell lines were described previously (Xu et al., 2009), whereas other ubiquitin replacement

(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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Molecular Cell K33-Polyubiquitination in Protein Trafficking

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 mm. 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. (legend continued on next page)

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Molecular Cell K33-Polyubiquitination in Protein Trafficking

cell lines were generated by a similar strategy. These ubiquitin replacement cells were grown in DMEM containing 10% tetracycline-free FCS (GIBCO). Knockdown of endogenous ubiquitin and replacement with the expression of wild-type or mutant ubiquitin was performed with the addition of 1 mg/ml doxycycline for 48 hr. Transfection of 293T and 293FT was performed by the calcium phosphate method, whereas transfection of Cos-1 and ubiquitin replacement cells were performed by Lipofectamine 2000 reagent. Post-Golgi Trafficking Assays The post-Golgi transport of various GFP-tagged cargos was assayed in cells released from a temperature block. The detail methods are described in Supplemental Experimental Procedures. In Vivo Ubiquitination Assay For analyzing Crn7 ubiquitination, cells were transfected with various constructs together with His-ubiquitin and Flag-Crn7 and lysed under denaturing conditions by buffer A (6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4 [pH 8.0], and 10 mM imidazole). Lysates were incubated with Ni-NTA agarose for 2 hr at 4 C. The beads were washed once with buffer A, twice with buffer A/TI (1 vol buffer A: 3 vol buffer TI [25 mM Tris-HCl (pH 6.8), and 20 mM imidazole]), and three times with buffer TI, and then analyzed by western blot. In all experiments, equal expression of His-ubiquitin was verified. For simultaneously analyzing Crn7 ubiquitination and its interaction with Eps15, cells were lysed with RIPA buffer. The lysates were used for immunoprecipitation with anti-Flag antibody (for ectopic Crn7) or anti-Crn7 antibody (for endogenous Crn7), followed by western blot with antibodies to Eps15, His, or ubiquitin.

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SUPPLEMENTAL INFORMATION

Chan, K.T., Creed, S.J., and Bear, J.E. (2011). Unraveling the enigma: progress towards understanding the coronin family of actin regulators. Trends Cell Biol. 21, 481–488.

Supplemental Information includes seven figures, two movies, and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.molcel.2014.03.035.

Chen, Z.J., and Sun, L.J. (2009). Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286.

AUTHOR CONTRIBUTIONS

Chi, S., Cao, H., Chen, J., and McNiven, M.A. (2008). Eps15 mediates vesicle trafficking from the trans-Golgi network via an interaction with the clathrin adaptor AP-1. Mol. Biol. Cell 19, 3564–3575.

W.-C.Y. and Y.-R.L. contributed equally to this work. W.-C.Y. designed and performed cell biology experiments to demonstrate the functions of K33linked ubiquitination, and Y.-R.L. designed and performed biochemical experiments to elucidate the molecular mechanisms of K33-linked ubiquitination.

Cruz-Garcia, D., Ortega-Bellido, M., Scarpa, M., Villeneuve, J., Jovic, M., Porzner, M., Balla, T., Seufferlein, T., and Malhotra, V. (2013). Recruitment of arfaptins to the trans-Golgi network by PI(4)P and their involvement in cargo export. EMBO J. 32, 1717–1729.

ACKNOWLEDGMENTS

D’Souza-Schorey, C., and Chavrier, P. (2006). ARF proteins: roles in membrane traffic and beyond. Nat. Rev. Mol. Cell Biol. 7, 347–358.

We thank Jennifer Lippincott-Schwartz, Ludger Johannes, Satoshi Waguri, Guang-Chao Chen, Sandra Schmid, Angelika A. Noegel, Tamas Balla, Geri Kreitzer, Lih-Hwa Hwang, and Robin F. Irvine for constructs; the National RNAi Core Facility for shRNA constructs; the Academia Sinica Mass Spectrometry Facility for LC-MS/MS analysis; and Fang-Jen Lee and Ming-Daw Tsai for discussion. This work was supported by NSC Frontier Grant 1012321-B-001-007 and by the Academia Sinica Investigator Award. Received: October 4, 2013 Revised: January 28, 2014 Accepted: March 18, 2014 Published: April 24, 2014

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(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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Molecular Cell K33-Polyubiquitination in Protein Trafficking

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 mm. (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 mm. (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. (legend continued on next page)

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(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.

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