The retrograde pathway is defined by the transport of proteins and lipids from the plasma membrane through endosomes to the Golgi complex, and is essential for a variety of cellular activities. Recycling endosomes are important sorting stations for some retrograde cargo. SMAP2, a GTPase-activating protein (GAP) for Arf1 with a putative clathrin-binding domain, has previously been shown to participate in the retrograde transport of the cholera toxin B-subunit (CTxB) from recycling endosomes. Here, we found that clathrin, a vesicle coat protein, and clathrin adaptor protein complex 1 (AP-1) were present at recycling endosomes and were needed for the retrograde transport of CTxB from recycling endosomes to the Golgi, but not from the plasma membrane to recycling endosomes. SMAP2 immunoprecipitated clathrin and AP-1 through a putative clathrin-binding domain and a CALM-binding domain, and SMAP2 mutants that did not interact with clathrin or AP-1 could not localize to recycling endosomes. Moreover, knockdown of Arf1 suppressed the retrograde transport of CTxB from recycling endosomes to the Golgi. These findings suggest that retrograde transport is mediated by clathrin-coated vesicles from recycling endosomes and that the role of the coat proteins is in the recruitment of Arf GAP to transport vesicles.

Endocytosis from the plasma membrane and the subsequent endosomal sorting are crucial for diverse cellular functions, such as nutrient uptake, signaling and cell adhesion. Retrograde transport is one of the endocytic pathways, and it delivers proteins and lipids from the plasma membrane to the Golgi via endosomes. The number of cargos known to use retrograde transport is rapidly increasing, thus expanding the functional roles of this pathway (Johannes and Popoff, 2008). For example, mannose 6-phosphate receptors (MPR46 and MPR300, also known as M6PR and IGF2R, respectively) bind newly synthesized lysosomal enzymes at the Golgi and shuttle them to the endosomes. At the endosomes, the enzymes disassociate from the receptors owing to the acidic pH of the luminal space and are then transported to lysosomes. However, the enzyme receptors are retrieved back to the Golgi by retrograde transport. The latter process allows for the next round of binding and delivery of lysosomal enzymes (Ghosh et al., 2003; Kornfeld and Mellman, 1989). Wntless, the sorting receptor for the morphogen Wnt, binds newly synthesized Wnt at the Golgi and transports it to the plasma membrane, where Wntless releases Wnt to the extracellular space. Wntless is then retrieved back to the Golgi through endosomes by retrograde transport, which allows for the next round of Golgi-to-plasma membrane delivery of Wnt (Eaton, 2008). Golgi proteins, such as TGN38 or TGN46 (both encoded by TGOLN2) (Ghosh et al., 1998; Stanley and Howell, 1993), GP73 (also known as GOLM1) (Puri et al., 2002) and furin (Molloy et al., 1994) also utilize retrograde transport to maintain their predominant Golgi localization. Additionally, some protein toxins, such as cholera toxin, Shiga toxin and ricin, exploit retrograde transport for the intoxication of host cells (Lencer and Tsai, 2003; Mallard et al., 1998; Sandvig et al., 1992; Sandvig and van Deurs, 2002). The toxins move from the plasma membrane through endosomes to the Golgi and ER then into the cytosol, where they exert their toxicity.

Recycling endosomes are emerging as an essential sorting station for some retrograde cargo proteins, such as MPR300 (Lin et al., 2004; McKenzie et al., 2012), the Shiga toxin B-subunit (STxB) (McKenzie et al., 2009,, 2012), and the cholera toxin B-subunit (CTxB) (Uchida et al., 2011); these proteins traffic sequentially from the plasma membrane to early endosomes, then to recycling endosomes and finally to the Golgi (Taguchi, 2013). Two recycling-endosome-localized proteins, evectin-2 (also known as PLEKHB2) and SMAP2, have recently been shown to be essential for the retrograde transport of CTxB at the recycling-endosome-to-Golgi step (Matsudaira et al., 2013; Uchida et al., 2011). Evectin-2 contains a pleckstrin homology (PH) domain which specifically binds phosphatidylserine (PtdSer) and this interaction is required for its function and localization to PtdSer-enriched recycling endosomes. SMAP2 localizes to the recycling endosomes by binding to evectin-2, and this recycling endosome localization is lost in evectin-2-knockdown cells. SMAP2 is a GTPase-activating protein (GAP) for Arf1, and contains a putative clathrin-interacting domain and an interaction domain for the clathrin assembly protein CALM (also known as PICALM), suggesting a possible role with clathrin.

Membrane transport between organelles is mediated by vesicles that are coated with specific proteins (Mellman, 1996; Rothman and Wieland, 1996). One of the best-characterized coat proteins is clathrin (Kirchhausen, 2000; Pearse and Robinson, 1990), which participates in a variety of membrane transport pathways (Brodsky, 2012). Clathrin is composed of three heavy chains and three light chains, which can polymerize to form a protein coat on membranes. The function of clathrin is coordinated by clathrin adaptor proteins (Pearse and Robinson, 1984; Traub, 2003). The clathrin adaptor proteins mediate the attachment of clathrin to membrane and recruit transmembrane cargo proteins into the coat. Two clathrin adaptor complexes, AP-1 and AP-2, have been extensively characterized. AP-2 localizes primarily to the plasma membrane and functions in endocytosis (Motley et al., 2003). AP-1 localizes to the trans-Golgi network (TGN) and, to a lesser extent, to endosomes (Ahle et al., 1988; Folsch et al., 2001; Haberg et al., 2008), and functions in membrane trafficking from the TGN (Gravotta et al., 2012). It is not known whether retrograde transport from recycling endosomes to the Golgi requires clathrin and clathrin adaptor proteins.

In the present study, we examined the requirement of clathrin in the retrograde transport of CTxB from recycling endosomes to the Golgi, given that SMAP2 has a putative clathrin-binding domain. We found that clathrin localizes to recycling endosomes in addition to the Golgi and is essential for the retrograde transport of CTxB from recycling endosomes to the Golgi. The recycling endosome localization of SMAP2 requires clathrin and AP-1, suggesting a role of coat proteins in the recruitment of Arf GAP to transport vesicles.

Clathrin and AP-1 are essential for the retrograde transport of CTxB from recycling endosomes

We examined whether CHC17 (also known as CLTC), the major isoform of clathrin heavy chain (for simplicity, hereafter named CHC), functions in the retrograde transport of CTxB from recycling endosomes to the Golgi. COS-1 cells were chosen to analyze the retrograde transport of CTxB. COS-1 cells are a cell line with distinctly separate organelles, which allows for the precise tracking of endosomal transport (Lee et al., 2015; Misaki et al., 2007; Uchida et al., 2011) (supplementary material Fig. S1). In COS-1 cells, CTxB reached the recycling endosomes after a 15–20-min chase and then moved to the Golgi after a 60-min chase (Uchida et al., 2011). The effect of knockdown of CHC on the retrograde transport of CTxB in COS-1 cells was examined. Among the three small interfering RNAs (siRNAs) designed against human CHC, siRNA #1 and #3 were effective at suppressing CHC expression (Fig. 1A). After a 60-min chase, Alexa-Fluor-594–CTxB colocalized with the Golgi marker GM130 (also known as GOLGA2), exhibiting the characteristic Golgi-ring appearance in control cells, as observed previously (Uchida et al., 2011). In contrast, CTxB did not reach the Golgi after a 60-min chase in cells depleted of CHC and, instead, accumulated in the recycling endosomes (Fig. 1A; supplementary material Fig. S2A). The Pearson's coefficients between CTxB and the Golgi protein GM130 after a 60-min chase were significantly lower in CHC-depleted cells than in control cells (Fig. 1B), whereas the Pearson's coefficients between CTxB and an recycling endosome protein Rab11 after a 60-min chase were significantly higher in CHC-depleted cells than in control cells (supplementary material Fig. S2B). CTxB reached the recycling endosomes after a 20-min chase in CHC-depleted cells, like in control cells (Fig. 1C; supplementary material Fig. S2C,D). These results suggest that clathrin is essential for the retrograde traffic of CTxB from recycling endosomes to the Golgi, but not from the plasma membrane to recycling endosomes.

Fig. 1.

Knockdown of CHC or AP-1γ impairs the retrograde transport of CTxB from recycling endosomes to the Golgi. (A) COS-1 cells were treated with control siRNA, CHC siRNA#1 or #3 for 48 h, or with AP-1γ siRNA#1 or #2 for 72 h. Cells were pulsed with Alexa-Fluor-594–CTxB for 3 min at 37°C then chased for 60 min. Cells were then fixed, permeabilized and stained for GM130. Nuclei were stained with DAPI (blue). Magnified images of boxed areas around the perinulcear region are shown in the right column. The lower panel shows the efficacy of knockdown with CHC or AP-1γ siRNAs. COS-1 cells were treated with the indicated siRNAs. Cell lysates were prepared and then immunoblotted with CHC or AP-1γ antibody. α-tubulin was used as a loading control. (B) Pearson's coefficients between CTxB and GM130 for the experiments shown in A (mean±s.d., n>20 cells from three independent experiments). ***P<0.001 (two-tailed Student's t-test). (C) COS-1 cells were treated with control siRNA or CHC siRNA#3 for 48 h, or AP-1γ siRNA#1 for 72 h. Cells were pulsed with Alexa-Fluor-594–CTxB for 3 min at 37°C and chased for 20 min. Scale bars: 10 μm.

Fig. 1.

Knockdown of CHC or AP-1γ impairs the retrograde transport of CTxB from recycling endosomes to the Golgi. (A) COS-1 cells were treated with control siRNA, CHC siRNA#1 or #3 for 48 h, or with AP-1γ siRNA#1 or #2 for 72 h. Cells were pulsed with Alexa-Fluor-594–CTxB for 3 min at 37°C then chased for 60 min. Cells were then fixed, permeabilized and stained for GM130. Nuclei were stained with DAPI (blue). Magnified images of boxed areas around the perinulcear region are shown in the right column. The lower panel shows the efficacy of knockdown with CHC or AP-1γ siRNAs. COS-1 cells were treated with the indicated siRNAs. Cell lysates were prepared and then immunoblotted with CHC or AP-1γ antibody. α-tubulin was used as a loading control. (B) Pearson's coefficients between CTxB and GM130 for the experiments shown in A (mean±s.d., n>20 cells from three independent experiments). ***P<0.001 (two-tailed Student's t-test). (C) COS-1 cells were treated with control siRNA or CHC siRNA#3 for 48 h, or AP-1γ siRNA#1 for 72 h. Cells were pulsed with Alexa-Fluor-594–CTxB for 3 min at 37°C and chased for 20 min. Scale bars: 10 μm.

We next examined the effect of downregulation of clathrin adaptor protein AP-1 based on the previous observation that AP-1 localized to recycling endosomes (Folsch et al., 2001). To suppress the function of AP-1, we knocked down the γ-subunit of AP-1 (AP-1γ) (Saint-Pol et al., 2004). Two siRNAs (siRNA#1 and #2) were designed against human AP-1γ, and both suppressed AP-1γ expression (Fig. 1A). In cells depleted of AP-1γ, CTxB did not reach the Golgi after a 60-min chase and instead accumulated in the recycling endosomes (Fig. 1A; supplementary material Fig. S2A). The Pearson's coefficients between CTxB and GM130 after a 60-min chase were significantly lower in AP-1γ-depleted cells than in control cells (Fig. 1B), whereas the Pearson's coefficients between CTxB and the recycling endosome protein Rab11 after a 60-min chase were significantly higher in AP-1γ-depleted cells than in control cells (supplementary material Fig. S2B). CTxB reached the recycling endosomes after a 20-min chase in AP-1γ-depleted cells, like in control cells (Fig. 1C; supplementary material Fig. S2C,D). These results suggest that AP-1γ is essential for the retrograde traffic of CTxB from recycling endosomes to the Golgi.

Clathrin and AP-1 localize to recycling endosomes

CHC and AP-1 might directly function in the retrograde transport of CTxB from recycling endosomes. We thus examined their localizations by immunostaining in COS-1 cells. Endogenous CHC colocalized well with the recycling endosome proteins, Rab11, transferrin (Tfn) and SMAP2 (Fig. 2A). Importantly, CHC showed the highest Pearson's coefficients with SMAP2 (>0.7). CHC partially colocalized with EGFP–Rab6A (a TGN protein) and Vps35 (an early endosome protein) (Fig. 2B), which is consistent with the presence of CHC at the Golgi and early endosomes (Brodsky, 2012). Endogenous AP-1γ also colocalized with the recycling endosome proteins Rab11, Tfn and SMAP2 (Fig. 3A). AP-1γ showed the highest Pearson's coefficients with SMAP2 (around 0.7), similar to CHC. AP-1γ partially colocalized with EGFP–Rab6A (Fig. 3B), which is consistent with the presence of AP-1 at the Golgi (Ahle et al., 1988; Folsch et al., 2001). AP-1γ showed little colocalization with Vps35.

Fig. 2.

Recycling endosome localization of endogenous CHC. (A) COS-1 cells were fixed with TCA, permeabilized, and then stained for CHC and the indicated proteins. For Tfn, cells were pulsed with Alexa-Fluor-647–Tfn for 30 min before being fixed and stained. Nuclei are indicated by fine white dotted lines. A fluorescence intensity profile along the line in the magnified image is shown in the right panel. The Pearson's coefficients were obtained using multiple images (n>12 cells). Data represent the mean±s.d. REs, recycling endosomes. (B) COS-1 cells were fixed with TCA, permeabilized and then stained for CHC. GFP–Rab6A (a TGN protein) and Vps35 [an early endosome (EE) protein] were used as Golgi and early endosome markers, respectively. Nuclei are indicated by fine white dotted lines. Scale bars: 5 μm.

Fig. 2.

Recycling endosome localization of endogenous CHC. (A) COS-1 cells were fixed with TCA, permeabilized, and then stained for CHC and the indicated proteins. For Tfn, cells were pulsed with Alexa-Fluor-647–Tfn for 30 min before being fixed and stained. Nuclei are indicated by fine white dotted lines. A fluorescence intensity profile along the line in the magnified image is shown in the right panel. The Pearson's coefficients were obtained using multiple images (n>12 cells). Data represent the mean±s.d. REs, recycling endosomes. (B) COS-1 cells were fixed with TCA, permeabilized and then stained for CHC. GFP–Rab6A (a TGN protein) and Vps35 [an early endosome (EE) protein] were used as Golgi and early endosome markers, respectively. Nuclei are indicated by fine white dotted lines. Scale bars: 5 μm.

Fig. 3.

Recycling endosome localization of endogenous AP-1γ. (A) COS-1 cells were fixed with TCA, permeabilized, and then stained for AP-1γ and the indicated proteins. For Tfn, cells were pulsed with Alexa-Fluor-647–Tfn for 30 min before being fixed and stained. Nuclei are indicated by fine white dotted lines. A fluorescence intensity profile along the line in the magnified image is shown in the right panel. The Pearson's coefficients were obtained using multiple images (n>12 cells). Data represent the mean±s.d. REs, recycling endosomes. (B) COS-1 cells were fixed with TCA, permeabilized and then stained for AP-1γ. GFP–Rab6A (a TGN protein) and Vps35 [an early endosome (EE) protein] were used as Golgi and early endosome markers, respectively. Nuclei are indicated by fine white dotted lines. Scale bars: 5 μm.

Fig. 3.

Recycling endosome localization of endogenous AP-1γ. (A) COS-1 cells were fixed with TCA, permeabilized, and then stained for AP-1γ and the indicated proteins. For Tfn, cells were pulsed with Alexa-Fluor-647–Tfn for 30 min before being fixed and stained. Nuclei are indicated by fine white dotted lines. A fluorescence intensity profile along the line in the magnified image is shown in the right panel. The Pearson's coefficients were obtained using multiple images (n>12 cells). Data represent the mean±s.d. REs, recycling endosomes. (B) COS-1 cells were fixed with TCA, permeabilized and then stained for AP-1γ. GFP–Rab6A (a TGN protein) and Vps35 [an early endosome (EE) protein] were used as Golgi and early endosome markers, respectively. Nuclei are indicated by fine white dotted lines. Scale bars: 5 μm.

We further verified the recycling endosome localization of CHC and AP-1 by exploiting the observation that nocodazole, a microtubule-depolymerizing agent, disperses recycling endosomes and then the Golgi to the cytoplasm in a time-dependent fashion (supplementary material Fig. S3A). Cells were pulsed for 30 min with Alexa-Fluor-647–Tfn to label recycling endosomes (Lee et al., 2015) and were then treated with 10 μM nocodazole for 30 min. Under this condition, Alexa-Fluor-647–Tfn was distributed into the cytoplasm where little GFP–Rab6A, used as a TGN marker, was observed, indicating the dispersal of recycling endosomes from the perinuclear region to the cytoplasm (Fig. 4A). GFP–Rab6A remained associated with the perinuclear area where little Alexa-Fluor-647–Tfn was observed. In the perinuclear area (Fig. 4A, box 2), CHC colocalized with GFP–Rab6A, which is consistent with the presence of CHC at the Golgi (Brodsky, 2012). In the cytoplasmic area (Fig. 4A, box 1), CHC colocalized with Alexa-Fluor-647–Tfn, as indicated by the arrows, showing that CHC was dispersed to the cytoplasm together with the recycling endosomes upon nocodazole treatment. These results support the hypothesis that clathrin localizes to recycling endosomes, in addition to the Golgi. We performed the same experiment with nocodazole for AP-1 and found that AP-1γ also localized to both recycling endosomes and the Golgi (Fig. 4B). Taken together, these results suggest that clathrin and AP-1 are directly involved in the retrograde transport of CTxB from recycling endosomes.

Fig. 4.

CHC and AP-1γ associate both with the Golgi and recycling endosomes. (A,B) COS-1 cells were transfected with GFP–Rab6A. Cells were pulsed with Alexa-Fluor-647–Tfn for 30 min and then treated for 30 min at 37°C with nocodazole. Cells were fixed with TCA, permeabilized and then stained for CHC (A) or AP-1γ (B). Nuclei are indicated by fine white dotted lines. Magnified images in the peripheral (box 1) or perinuclear (box 2) areas are shown in the lower row. Note that CHC and AP-1γ colocalized with Tfn in the peripheral areas, as indicated by arrows. Scale bars: 5 μm.

Fig. 4.

CHC and AP-1γ associate both with the Golgi and recycling endosomes. (A,B) COS-1 cells were transfected with GFP–Rab6A. Cells were pulsed with Alexa-Fluor-647–Tfn for 30 min and then treated for 30 min at 37°C with nocodazole. Cells were fixed with TCA, permeabilized and then stained for CHC (A) or AP-1γ (B). Nuclei are indicated by fine white dotted lines. Magnified images in the peripheral (box 1) or perinuclear (box 2) areas are shown in the lower row. Note that CHC and AP-1γ colocalized with Tfn in the peripheral areas, as indicated by arrows. Scale bars: 5 μm.

Clathrin and AP-1 are required for the recycling endosome localization of the Arf GAP SMAP2

SMAP2, an Arf GAP with a putative clathrin-binding domain (Natsume et al., 2006), localizes to recycling endosomes and is required for the retrograde transport of CTxB from recycling endosomes to the Golgi (Matsudaira et al., 2013). The present findings showing that SMAP2 strongly colocalized with CHC and AP-1γ (Fig. 2A; Fig. 3A), and that CHC and AP-1γ were essential for the retrograde transport of CTxB from recycling endosomes to the Golgi (Fig. 1A), led us to examine the relationship between SMAP2 and clathrin or AP-1.

In cells depleted of either CHC or AP-1γ, SMAP2 no longer localized to recycling endosomes (Fig. 5A). Under the same conditions, the abundance of SMAP2 did not change (Fig. 5B). These results suggest that clathrin and AP-1 are required for the recycling endosome localization of SMAP2. In cells depleted of SMAP2, CHC and AP-1γ still localized in the perinuclear area (Fig. 5C). The co-staining of AP-1γ and the recycling endosome protein Rab11 in cells depleted of SMAP2 showed that AP-1γ dissociated from Rab11-positive recycling endosomes (supplementary material Fig. S3B), but presumably still remained on the TGN.

Fig. 5.

CHC and AP-1γ regulate the perinuclear localization of SMAP2. (A) COS-1 cells were treated with control siRNA, CHC siRNA#3 or AP-1γ siRNA#1. Cells were then fixed, permeabilized and co-stained for SMAP2 and CHC, or SMAP2 and AP-1γ. Nuclei were stained with DAPI (blue). (B) COS-1 cells were treated with the indicated siRNAs for 72 h. Cell lysates were prepared and then immunoblotted for CHC, AP-1γ and SMAP2. α-tubulin was used as a loading control. (C) COS-1 cells were treated with SMAP2 siRNA for 72 h. Cells were then fixed, permeabilized and co-stained for SMAP2 and CHC, or SMAP2 and AP-1γ. Nuclei were stained with DAPI (blue). (D) SMAP2 (WT)–FLAG, CBm–FLAG, ΔCALM–FLAG or mock plasmid (pcDNA3-FLAG) was expressed in COS-1 cells for 24 h. The cell homogenates were solubilized by adding 1% NP-40. The lysates were then immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were separated by SDS-PAGE, and then blotted for CHC, AP-1γ and FLAG. GAPDH was used as a loading control. (E) COS-1 cells transfected with SMAP2 (WT)–FLAG, CBm–FLAG, or ΔCALM–FLAG were fixed with TCA, permeabilized and stained for CHC and FLAG. Nuclei were stained with DAPI (blue). Scale bars: 10 μm.

Fig. 5.

CHC and AP-1γ regulate the perinuclear localization of SMAP2. (A) COS-1 cells were treated with control siRNA, CHC siRNA#3 or AP-1γ siRNA#1. Cells were then fixed, permeabilized and co-stained for SMAP2 and CHC, or SMAP2 and AP-1γ. Nuclei were stained with DAPI (blue). (B) COS-1 cells were treated with the indicated siRNAs for 72 h. Cell lysates were prepared and then immunoblotted for CHC, AP-1γ and SMAP2. α-tubulin was used as a loading control. (C) COS-1 cells were treated with SMAP2 siRNA for 72 h. Cells were then fixed, permeabilized and co-stained for SMAP2 and CHC, or SMAP2 and AP-1γ. Nuclei were stained with DAPI (blue). (D) SMAP2 (WT)–FLAG, CBm–FLAG, ΔCALM–FLAG or mock plasmid (pcDNA3-FLAG) was expressed in COS-1 cells for 24 h. The cell homogenates were solubilized by adding 1% NP-40. The lysates were then immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were separated by SDS-PAGE, and then blotted for CHC, AP-1γ and FLAG. GAPDH was used as a loading control. (E) COS-1 cells transfected with SMAP2 (WT)–FLAG, CBm–FLAG, or ΔCALM–FLAG were fixed with TCA, permeabilized and stained for CHC and FLAG. Nuclei were stained with DAPI (blue). Scale bars: 10 μm.

The physical interaction of SMAP2 with CHC and AP-1γ was examined next. FLAG-tagged SMAP2 was expressed in COS-1 cells for 24 h. Cell lysates with detergent were then immunoprecipitated with anti-FLAG antibody. CHC was detected in the immunoprecipitates, confirming previous observations (Natsume et al., 2006) (Fig. 5D). AP-1γ was also detected in the immunoprecipitates.

SMAP2 has three distinct domains (Fig. 5D): an Arf GAP domain (amino acids 1–162), a putative clathrin-binding domain (amino acids 163–230) and a CALM-binding domain (amino acids 339–395). The putative clathrin-binding domain contains two clathrin-binding motifs [LLGLD (amino acids 187–191) and DLDLL (amino acids 210–214)]. We generated two SMAP2 mutants, each with a C-terminal FLAG tag. In the clathrin-binding domain mutant (CBm), all the amino acid residues in the LLGLD and DLDLL motifs were replaced by alanine residues. In the ΔCALM mutant, the CALM-binding domain was removed. As shown in Fig. 5D, both mutants did not immunoprecipitate CHC and AP-1. These results suggested that the putative clathrin-binding domain and the CALM-binding domain are required for the interaction of SMAP2 with CHC and AP-1.

We then examined the subcellular localization of the two SMAP2 mutants. As shown in Fig. 5E, both mutants no longer localized to recycling endosomes and were dispersed into the cytoplasm. Therefore, the interaction of SMAP2 with AP-1 and clathrin might be required for the localization of SMAP2 to recycling endosomes.

Evectin-2 is a recycling endosome protein with a PtdSer-specific PH domain and is essential for the retrograde transport from recycling endosomes (Uchida et al., 2011). In cells depleted of evectin-2, SMAP2 loses its recycling endosome localization and instead appears to be localized to the Golgi (Matsudaira et al., 2013). Given that CHC and AP-1γ are required for the localization of SMAP2 to recycling endosomes, we examined whether CHC and/or AP-1γ were also required for the localization of evectin-2 to recycling endosomes. As shown in Fig. 6, evectin-2 still localized at recycling endosomes in cells depleted of CHC or AP-1γ. Thus, the localization of SMAP2 at recycling endosomes might require an interaction with both evectin-2 and clathrin or AP-1.

Fig. 6.

CHC or AP-1γ knockdown does not affect the recycling endosome localization of evectin-2. COS-1 cells were treated with the indicated siRNAs. At 48 h after siRNA treatment, Myc-tagged evectin-2 was transfected and expressed for another 24 h. Cells were then fixed, permeabilized, and stained for Myc and syntaxin-5 (a Golgi protein). Nuclei were stained with DAPI (blue). Magnified images of boxed areas are shown in the right column. Scale bars: 10 μm.

Fig. 6.

CHC or AP-1γ knockdown does not affect the recycling endosome localization of evectin-2. COS-1 cells were treated with the indicated siRNAs. At 48 h after siRNA treatment, Myc-tagged evectin-2 was transfected and expressed for another 24 h. Cells were then fixed, permeabilized, and stained for Myc and syntaxin-5 (a Golgi protein). Nuclei were stained with DAPI (blue). Magnified images of boxed areas are shown in the right column. Scale bars: 10 μm.

Arf1 is essential for the retrograde transport of CTxB from recycling endosomes

SMAP2 has been suggested to function as an Arf1 GAP in vivo (Natsume et al., 2006). Therefore, we examined whether Arf1 was also involved in the retrograde transport of CTxB. Endogenous Arf1 localization in COS-1 cells showed a Golgi-ring appearance and colocalized with GFP–Rab6A (a TGN protein) (Fig. 7A), which is consistent with other reports (D'Souza-Schorey and Chavrier, 2006; Stearns et al., 1990). Arf1 did not colocalize with Tfn. We have previously shown that SMAP2 localizes predominantly to recycling endosomes and does not colocalize with the cis-Golgi marker GM130 (Matsudaira et al., 2013). Importantly, SMAP2 showed some colocalization with Arf1 (Fig. 7B), suggesting that SMAP2 partially localizes to the TGN.

Fig. 7.

Knockdown of Arf1 impairs the retrograde transport of CTxB from recycling endosomes to the Golgi. (A) COS-1 cells transfected with GFP–Rab6A were pulsed with Alexa-Fluor-647–Tfn for 30 min. Cells were then fixed with TCA, permeabilized and stained for Arf1. Nuclei are indicated by fine white dotted lines. Magnified images of the perinuclear areas are shown in the lower row. Scale bars: 5 μm. (B) COS-1 cells transfected with SMAP2–FLAG were fixed with TCA, permeabilized, and stained for Arf1 and FLAG. Nuclei are indicated by fine white dotted lines. Magnified images of the perinuclear areas are shown in the lower row. Scale bar: 5 μm. (C) COS-1 cells were treated with Arf1 siRNA#1 or #2 for 48 h. Cells were pulsed with Alexa-Fluor-594–CTxB for 3 min at 37°C then chased for 60 min. Cells were then fixed, permeabilized and stained for GM130. Nuclei were stained with DAPI (blue). Magnified images of boxed areas around the perinulcear region are shown in the right column. Scale bars: 10 μm. The lower panel shows the efficacy of knockdown with Arf1 siRNAs. COS-1 cells were treated with the indicated siRNAs. Cell lysates were prepared and then immunoblotted with Arf1 antibody. α-tubulin was used as a loading control. (D) Pearson's coefficients between CTxB and GM130 for the experiments shown in C (mean±s.d., n>20 cells from three independent experiments). ***P<0.001 (two-tailed Student's t-test).

Fig. 7.

Knockdown of Arf1 impairs the retrograde transport of CTxB from recycling endosomes to the Golgi. (A) COS-1 cells transfected with GFP–Rab6A were pulsed with Alexa-Fluor-647–Tfn for 30 min. Cells were then fixed with TCA, permeabilized and stained for Arf1. Nuclei are indicated by fine white dotted lines. Magnified images of the perinuclear areas are shown in the lower row. Scale bars: 5 μm. (B) COS-1 cells transfected with SMAP2–FLAG were fixed with TCA, permeabilized, and stained for Arf1 and FLAG. Nuclei are indicated by fine white dotted lines. Magnified images of the perinuclear areas are shown in the lower row. Scale bar: 5 μm. (C) COS-1 cells were treated with Arf1 siRNA#1 or #2 for 48 h. Cells were pulsed with Alexa-Fluor-594–CTxB for 3 min at 37°C then chased for 60 min. Cells were then fixed, permeabilized and stained for GM130. Nuclei were stained with DAPI (blue). Magnified images of boxed areas around the perinulcear region are shown in the right column. Scale bars: 10 μm. The lower panel shows the efficacy of knockdown with Arf1 siRNAs. COS-1 cells were treated with the indicated siRNAs. Cell lysates were prepared and then immunoblotted with Arf1 antibody. α-tubulin was used as a loading control. (D) Pearson's coefficients between CTxB and GM130 for the experiments shown in C (mean±s.d., n>20 cells from three independent experiments). ***P<0.001 (two-tailed Student's t-test).

Two siRNAs were designed against human Arf1 and both were effective at suppressing Arf1 expression in COS-1 cells (Fig. 7C). In the Arf1-depleted cells, CTxB did not reach the Golgi after a 60-min chase and instead accumulated in recycling endosomes (Fig. 7C). The Pearson's coefficients between CTxB and GM130 after a 60-min chase were significantly lower in Arf1-depleted cells than in control cells (Fig. 7D). These results suggest that Arf1 is required for the retrograde transport of CTxB from recycling endosomes to the Golgi.

In the present study, we demonstrated that clathrin and AP-1 are required for the retrograde transport from recycling endosomes to the Golgi. CTxB appeared to reach recycling endosomes in the clathrin- or AP-1-knockdown cells, similar to in control cells, suggesting that clathrin and AP-1 are not essential for the transport of CTxB from the plasma membrane through early endosomes to recycling endosomes. It has been shown that clathrin localizes to the TGN, early endosomes and the plasma membrane (Brodsky, 2012). We showed that CHC also localized to recycling endosomes. CHC colocalized with the recycling endosome proteins, Rab11, Tfn and SMAP2 in COS-1 cells (in which the Golgi, early endosomes and recycling endosomes are spatially distinct) (Lee et al., 2015; Misaki et al., 2007; Uchida et al., 2011). The recycling endosomes that were dispersed from the perinuclear region to the cytoplasm by nocodazole treatment remained positive for CHC. The localization of AP-1 to recycling endosomes (Folsch et al., 2001) was also confirmed in COS-1 cells. Thus, both clathrin and its adaptor protein AP-1 localize to recycling endosomes in COS-1 cells.

The roles of clathrin and AP-1 in endosomal membrane transport have been reported. For example, the transport of glucose transporter type 4 (GLUT4) from endosome-like vesicles to the plasma membrane is impaired by CHC knockdown (Li et al., 2007). CHC knockdown also suppresses the retrograde transport of STxB at early endosomes (Lauvrak et al., 2004; Popoff et al., 2007; Saint-Pol et al., 2004). In Caenorhabditis elegans, depletion of the clathrin-uncoating chaperone HSP-1 (a Hsc70 family protein) leads to the over-accumulation of CHC on early endosomes and causes missorting of Wntless into the degradation compartment (Shi et al., 2009). Depletion of the AP-1 subunit μ1A (an isoform encoded by AP1M1), alters the steady-state distribution of the mannose 6-phosphate receptors (MPR46 and MPR300) from the Golgi to endosomes (Folsch et al., 2001; Meyer et al., 2000) and impairs the retrograde traffic of furin at endosomes (Ishizaki et al., 2008). In yeast, AP-1 is essential for the retrieval of lysosomal enzymes from early endosomes to the Golgi (Foote and Nothwehr, 2006; Valdivia et al., 2002). In the present study, we found that knockdown of CHC or AP-1γ caused the accumulation of CTxB at recycling endosomes but not at early endosomes, indicating the requirement of both clathrin and AP-1 for the retrograde transport of CTxB from recycling endosomes to the Golgi. In contrast, the transport of STxB from early endosomes does not require AP-1 (Saint-Pol et al., 2004). Therefore, the endosomal transport system appears to use clathrin and AP-1 at different organelles, such as early endosomes and recycling endosomes, and their requirement for retrograde transport might depend on the cargo proteins.

In addition to clathrin, EpsinR (also known as CLINT1) and GPP130 (also known as GOLIM4) are essential for the retrograde transport of STxB from endosomes (Mukhopadhyay and Linstedt, 2012; Saint-Pol et al., 2004). EpsinR (Hirst et al., 2003; Kalthoff et al., 2002; Mills et al., 2003), a protein that interacts with AP-1 and clathrin, was also required for the retrograde transport of CTxB from recycling endosomes, whereas GPP130, a transmembrane protein that binds directly STxB, was not essential (supplementary material Fig. S2E–G) (Mukhopadhyay and Linstedt, 2012). Thus, the molecular machinery underlying the retrograde traffic of CTxB and STxB from endosomes might not be identical.

AP-1γ and CHC were present in the immunoprecipitates of SMAP2, an Arf GAP that is required for the retrograde transport of CTxB from recycling endosomes to the Golgi (Matsudaira et al., 2013). Knockdown of AP-1γ or CHC abolished SMAP2 localization to recycling endosomes, indicating that clathrin and AP-1 are required for the recycling endosome localization of SMAP2. These results suggest that, in addition to the well-known roles in the formation of transport vesicles, clathrin and AP-1 have an additional role in recruiting an Arf GAP to membranes. Evectin-2, which localizes to recycling endosomes and is crucial for retrograde transport (Uchida et al., 2011), also binds SMAP2 and is required for the recycling endosome localization of SMAP2 (Matsudaira et al., 2013). Thus, the localization of SMAP2 at recycling endosomes is determined by both evectin-2, and clathrin and AP-1. Of note, knockdown of CHC or AP-1γ redistributed SMAP-2 diffusively into the cytoplasm, whereas evectin-2 knockdown relocalized SMAP-2 to the Golgi-like compartments (Matsudaira et al., 2013). This suggests that in cells where evectin-2 is depleted, clathrin and AP-1 in the Golgi recruit SMAP2 to the Golgi.

We have recently shown that EHD1, a membrane fission protein (Grant and Caplan, 2008), is essential for the retrograde transport of CTxB from recycling endosomes to the Golgi (Lee et al., 2015). There are a few studies that suggest an interaction between EHD1 and clathrin. Recombinant EHD1 has been shown to bind CHC in extract from rat testes (Rotem-Yehudar et al., 2001). In Lampetra fluviatilis, perturbation of l-EHD, an orthologue of mammalian EHD1 and EHD3, inhibits synaptic vesicle endocytosis and causes accumulation of clathrin-coated pits with elongated necks, suggesting that the role of l-EHD is in promoting the budding of clathrin-coated vesicles (Ioannou and Marat, 2012; Jakobsson et al., 2011). Taking these findings into consideration, it is tempting to propose that clathrin and EHD1 function together to generate clathrin-coated vesicles at recycling endosomes. EHD1 is also required for the recycling transport of Tfn from recycling endosomes to the plasma membrane (Lee et al., 2015). We could not examine whether clathrin is required for the recycling transport of Tfn from recycling endosomes due to the severe defect in the internalization of Tfn from the plasma membrane to recycling endosomes in CHC-depleted cells (Motley et al., 2003). However, given that the recycling transport of GLUT4 needs clathrin (Li et al., 2007), clathrin and EHD1 might participate together in the recycling transport from recycling endosomes.

With regard to the Arf GAP function of SMAP-2 (Natsume et al., 2006), we found that the small GTPase Arf1 is required for the retrograde transport of CTxB from recycling endosomes in COS-1 cells. Arf1 is known to localize to the Golgi (Stearns et al., 1990) and we confirmed this exclusive localization to the Golgi in COS-1 cells. Given that SMAP2 partly localized to the TGN in addition to its primary localization in the recycling endosomes, SMAP2 might inactivate Arf1 on the TGN membranes. In fact, SMAP2-knockout mouse embryonic fibroblasts (MEFs) show increased levels of Arf1-GTP (Funaki et al., 2011). Interestingly, depletion of CHC, AP-1γ or evectin-2, which all lead to mislocalization of SMAP2 from recycling endosomes, also increased the Arf1-GTP levels (supplementary material Fig. S4A). Small G proteins, such as Arl1, participate in the tethering of transport vesicles to the acceptor membranes, which allows the subsequent fusion process (Behnia and Munro, 2005; Pfeffer, 2011). It is tempting to speculate that the ‘trans-inactivation’ of Arf1 at the TGN by SMAP2 on the retrograde transport vesicles is required for the completion of the tethering step. Presently, we cannot rule out the possibility that Arf1 at the Golgi delivers proteins to recycling endosomes that are required for the retrograde transport of CTxB from recycling endosomes. Other small G proteins that localize to recycling endosomes and are regulated by SMAP2 might also be involved.

Mammals have six Arf isoforms, Arf1–Arf6 (Arf2 has been lost in primates) (D'Souza-Schorey and Chavrier, 2006; Donaldson and Jackson, 2011; Tsuchiya et al., 1991). Among them, Arf1–Arf5 primarily localize and function at the Golgi (Donaldson and Jackson, 2011). In HeLa cells, knockdown of each Arf isoform does not affect the morphology of the Golgi and the localization of AP-1 to the Golgi. However, every combination of double knockdowns of Arf1, Arf3, Arf4 and Arf5 yields a distinct pattern of defects in anterograde trafficking through the Golgi, suggesting redundant roles for Arf proteins in membrane trafficking (Volpicelli-Daley et al., 2005). The double knockdown of Arf1 and Arf4 also affects the retrograde transport of Flag-tagged TGN38 from the plasma membrane to the Golgi (Nakai et al., 2013). In COS-1 cells, all Arf proteins except Arf2 are expressed (supplementary material Fig. S4B). In the present study, we found that single knockdown of Arf1 impaired the retrograde transport of CTxB from recycling endosomes to the Golgi, indicating a non-redundant role for Arf1 in membrane trafficking. The non-redundant role of Arf1 is also demonstrated by the observation that Arf1−/− mouse embryos die soon after implantation (Hayakawa et al., 2014).

The clathrin adaptor proteins recognize cytosolic sorting signals on transmembrane cargo proteins and recruit the proteins into the coat. AP-1 recognizes the tyrosine-based YxxØ motif (where x is any amino acid and Ø is an amino acid with a bulky hydrophobic side chain) and the dileucine motif [D/E]xxxL[L/I] of the cargo proteins (Ohno et al., 1995; Peden et al., 2001; Rapoport et al., 1998). Based on the requirement for AP-1 in the transport of CTxB from recycling endosomes, a cargo-receptor-like transmembrane protein might exist in recycling endosomes that binds AP-1 through the cytosolic region and CTxB through the luminal region, which allows AP-1- and clathrin-dependent retrograde transport of CTxB from recycling endosomes to the Golgi.

Plasmids

The Myc-tagged evectin-2 construct was as previously described (Uchida et al., 2011). N-terminal GFP-tagged Rab6A was a gift from Mitsunori Fukuda (Tohoku University, Japan). Mouse SMAP2 constructs [wild-type (WT) and clathrin-binding domain mutant (CBm)] were a gift from Masanobu Satake (Tohoku University, Japan). The mouse SMAP2 gene (WT and CBm) was amplified by PCR using the following primers: 5′-CCGGATATCATGACAGGCAAGTCGGTGAAG-3′ (sense primer, the EcoRV site is underlined) and 5′-CCGCTCGAGTTTCCACATCTGAGGACTGAG-3′ (antisense primer, the XhoI site is underlined), and subsequently cloned into pcDNA3 with FLAG-tag (Invitrogen) at EcoRV–XhoI sites. FLAG-tagged mouse SMAP2 ΔCALM was generated from FLAG-tagged mSMAP2 (WT) using 5′-GCTCGAGTCGACGACTACAAAGACG-3′ (sense primer) and 5′-ATAGCCTGCAGGCATGGCCATAGGG-3′ (antisense primer).

Antibodies

The following antibodies were used: mouse anti-CHC (Abcam); mouse anti-α-tubulin, mouse anti-AP-1γ, mouse anti-Myc (9E10) and rabbit anti-SMAP2 (Sigma); mouse anti-GM130 (BD Biosciences); rabbit anti-FLAG (Cell Signaling Technology); mouse anti-TfnR and rabbit anti-Rab11 (Zymed Laboratories); rabbit anti-syntaxin 5 (Synaptic Systems); sheep anti-TGN46 (Serotec); goat anti-VPS35 (Everest Biotech); mouse anti-GAPDH (Calbiochem); rabbit anti-GPP130 (COVANCE); rabbit anti-EpsinR (BETHYL); sheep anti-mouse-IgG conjugated to horseradish peroxidase (HRP) and donkey anti-rabbit-IgG conjugated to HRP (GE Healthcare), as well as Alexa-Fluor-conjugated secondary antibodies (Invitrogen).

Reagents

Alexa-Fluor-594–CTxB was purchased from Invitrogen. To prepare Alexa-Fluor-647–Tfn, human holo-Tfn (Sigma) was conjugated to Alexa Fluor 647 using Alexa Flour succinimidylester (Invitrogen) and then purified by PD-10 desalting columns (GE Healthcare). Nocodazole was purchased from Sigma and dissolved in dimethyl sulfoxide (Wako).

Cell culture

COS-1 cells (American Type Culture Collection) were cultured as previously described (Uchida et al., 2011).

Plasmid transfection

Cells were transiently transfected with plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

RNA interference

siRNA duplex oligomers (Nippon EGT) were as follows: CHC17 siRNA#1, 5′-AUCCAAUUCGAAGACCAAU-3′ (Motley et al., 2003); CHC17 siRNA#2, 5′-GGCUCAUACCAUGACUGAU-3′ (Anitei et al., 2010); CHC17 siRNA#3, 5′-GGGUGCCAGAUUAUCAAUU-3′ (Anitei et al., 2010); AP-1γ siRNA#1, 5′-ACCGAAUUAAGAAAGUGGU-3′ (Saint-Pol et al., 2004); Arf1 siRNA#1, 5′-ACCGUGGAGUACAAGAACA-3′ (Volpicelli-Daley et al., 2005); Arf1 siRNA#2, 5′-GATCAATTCTGCATGGTCA-3′; evectin-2 siRNA, 5′-CUGCAUGCUCCAGAUUGUU-3′; and SMAP2 siRNA 5′-GTTGTATATTAGGCAAACA-3′. s1142 (AP-1γ siRNA#2), s26169 (GPP130 siRNA#1), s26170 (GPP130 siRNA#2), s18645 (EpsinR siRNA#1), s18646 (EpsinR siRNA#2) and control siRNA (Silencer Negative Control no. 1 siRNA) were obtained from Ambion. GPP130 siRNA#3 and EpsinR siRNA#3 was obtained from Dharmacon (SMARTpool, g-005005-02, human genome). A total of 20 nM siRNA was introduced to COS-1 cells using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instruction. After 4 h, the medium was replaced by Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS) and cells were further incubated for 48 or 72 h for subsequent experiments.

Immunocytochemistry

Cells were washed with PBS briefly, fixed with 4% paraformaldehyde (PFA) in PBS at room temperature or 10% trichloroacetic acid (TCA) at 4°C for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. Blocking was performed with 3% BSA in PBS at room temperature for 30 min. Cells were then incubated with primary antibodies diluted in 3% BSA in PBS (for PFA-fixed cells) or Can Get Signal immunostain Solution A (TOYOBO) (for TCA-fixed cells) at 4°C for 12–16 h. After washing three times with PBS, cells were incubated with secondary antibodies at room temperature for 1 h, washed with PBS and then mounted in PermaFluor (Thermo).

CTxB uptake

Cells were washed with PBS briefly and pulsed with 1 μg/ml Alexa-Fluor-594–CTxB for 3 min. After washing twice with PBS, cells were chased for the indicated times in the medium without CTxB.

Tfn uptake

Cells were serum-starved for 30 min in DMEM and then incubated with 100 μg/ml Alexa-Fluor-647–Tfn for 30 min at 37°C. Cells were then washed twice with PBS prior to fixation.

Nocodazole treatment

Cells were serum-starved for 30 min in DMEM and then incubated with 100 μg/ml Alexa-Fluor-647–Tfn for 30 min at 37°C. Cells were then treated with 10 μM nocodazole in the presence of 100 μg/ml Alexa-Fluor-647–Tfn for 30 min or 60 min at 37°C prior to fixation.

Immunoprecipitation

COS-1 cells grown on 100-mm dishes were transfected with FLAG-tagged mSMAP2 (WT, CBm, or ΔCALM) or empty vector (pcDNA3-FLAG) using Lipofectamine 2000 (Invitrogen). At 24 h after transfection, cells were lysed in 1 ml of immunoprecipitation buffer (25 mM HEPES-KOH pH 7.33, 150 mM NaCl, 1 mM EDTA and 1% Triton X-100) with protease inhibitor (10 μg/ml of aprotinin, pepstatin and leupeptin). The cell lysates were centrifuged at 15,000 g for 20 min at 4°C, and the resultant supernatants were incubated for 3 h at 4°C with anti-FLAG M2 affinity gel beads (Sigma). The beads were washed four times with immunoprecipitation buffer.

Arf1 activation assay

COS-1 cells grown on 100-mm dishes were treated with control, CHC, AP-1γ or evectin-2 siRNA for 60 h using Lipofectamine RNAiMAX. To examine the Arf1-GTP levels, we used the Arf1 Activation Assay biochemistry kit (Cytoskeleton) (Takatsu et al., 2002). According to the manufacturer's instruction, cells were lysed and the cell lysates were centrifuged at 10,000 g for 2 min at 4°C. The resultant supernatants were incubated for 1 h at 4°C with GGA3-PBD beads. The beads were then washed, eluted and analyzed by SDS-PAGE and western blot analysis.

qRT-PCR

Total RNA was extracted from HeLa or COS-1 cells using Isogen II (Nippon EGT) and reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green PCR Master Mix (TaKaRa) and a Light-Cycler 480 (Roche Diagnostics). PCR products were separated on a 1% agarose gel, stained with ethidium bromide and visualized with UV light using an ImageQuant LAS4000 analyzer (GE Healthcare). The following primers were used: human Arf1, forward 5′-GTGACCACCATTCCCACCATAG-3′ and reverse 5′-TCATTGCTGTCCACCACGAAG-3′ (Islam et al., 2007); human Arf3, forward 5′-GAAACCTTCTCAAGAGCCTGATTG-3′ and reverse 5′-CCCACATCCCACACTGTAAAGC-3′ (Islam et al., 2007); human Arf4, forward 5′-GGGATGTTGGTGGTCAAGAT-3′ and reverse 5′-AGCAGCACTGCATCTCTCAA-3′ (Makpol et al., 2013); human Arf5, forward 5′-TGAAGTTGGGGGAGATTGTCAC-3′ and reverse 5′-CCTGAGTGTTCTGGAAGTAGTGCC-3′ (Islam et al., 2007); and human Arf6, forward 5′-GATTCGGGACAGGAACTGGTATG-3′ and reverse 5′-TGAAAGAGGTGATGGTGGCG-3′ (Islam et al., 2007).

Confocal microscopy

Confocal microscopy was performed using a laser scanning microscope (model LSM 510 META, Carl Zeiss Microimaging) with a 63×1.4 NA Plan-Apochromat oil immersion lens, or a TCS SP8 microscope (Leica) with a 63×1.2 NA Plan-Apochromat water immersion lens. With a LSM 510 META, excitation was performed with a 30 mW diode laser emitting at 405 nm, a 30 mW argon laser emitting at 488 nm, a 1.0 mW helium-neon laser emitting at 543 nm and a 15 mW helium-neon laser emitting at 633 nm. Emissions were collected using a 420–480 nm band-pass filter for DAPI, a 505–530 nm band-pass filter for Alexa Fluor 488 and GFP, a META detector from 561 to 615 nm for Alexa Fluor 594, and a META detector from 647 to 753 nm for Alexa Fluor 647. With the TCS SP8, excitation was performed with a 65 mW argon laser emitting at 488 nm. Emissions were collected at 500–600 nm using a Spectral detector.

Western blotting

Proteins were separated on a 12% polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 30 min using 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween-20 and 5% low-fat milk powder. The membranes were incubated with primary antibodies, followed by secondary antibodies conjugated to HRP. The proteins were visualized by chemiluminescence using an ImageQuant LAS4000 analyzer (GE Healthcare).

Fluorescent image analysis

Quantification of images was performed with ImageJ software (NIH). The fluorescence intensity and Pearson coefficients were determined with the RGB Profile Plot and JACoP plugins, respectively.

Statistical analysis

Statistical analysis was performed using two-tailed Student's t-test. P<0.05 was used as the criterion for statistical significance.

We thank Dr J. McKenzie (State of California Department of Pesticide Regulation, CA) and Dr Kenji Tanabe (Tokyo Women's Medical University, Tokyo) for comments on the manuscript.

Author contributions

T.M., T.T. and H.A. conceived of the experiments. T.M. and T.N. performed the experiments. T.M., T.N. and T.T. analyzed the data. T.T. and H.A. wrote the manuscript.

Funding

T.M. is supported by Research Fellowship for Young Scientists from Japan Society for the Promotion of Science DC1. This work was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (to H.A.); Grants-in-aid from the Japanese Ministry of Education, Culture, Sports, Science, and Technology [grant number 20370045 to H.A.]; and the Core Research for Evolutional Science and Technology (CREST) (to H.A.).

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Competing interests

The authors declare no competing or financial interests.

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