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A novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network.

Guo Y, Zanetti G, Schekman R - Elife (2013)

Bottom Line: Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN.In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1.Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology , Howard Hughes Medical Institute, University of California-Berkeley , Berkeley , United States.

ABSTRACT
Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.DOI:http://dx.doi.org/10.7554/eLife.00160.001.

No MeSH data available.


Related in: MedlinePlus

Arfrp1 regulates TGN export of PTK7.(A) Sequence alignment of PTK7 from different species reveals aconserved tyrosine sorting motif in its predicted C-terminal cytosolicdomain. (B)–(G) COS7 cellswere transfected with control siRNA or siRNA against Arfrp1 andre-transfected after 48 hr with plasmids encoding PTK7-Myc-His. After anadditional 24 hr, cells were incubated at 20°C in the presence of 30μg/ml cyclohexmide for 4 hr then shifted to 32°C for 90 min.After incubation, cells were analyzed by immunofluorescence using antibodiesagainst His and TGN46. Size bar = 10 μm. (H) COS7cell lysates from cells transfected with control siRNA or siRNA againstArfrp1 were analyzed by immunoblotting with anti-Arfrp1 antibody and, as aloading control, anti-GM130 antibody. (I) The fraction of cellsshowing TGN-accumulated PTK7 was quantified after incubation at 32°C(mean ± SD; N = 3; over 150 cells were counted for each group).(J) Similar siRNA knockdown and temperature shiftexperiments were performed in COS7 cells transfected with HA-Frizzled 6. Theappearance of TGN-accumulated HA-Frizzled 6 was quantified in cells treatedwith control siRNA or siRNA against Arfrp1 after an incubation at 32°C(mean ± SD; N = 2; over 100 cells were counted for eachgroup).DOI:http://dx.doi.org/10.7554/eLife.00160.015
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fig8: Arfrp1 regulates TGN export of PTK7.(A) Sequence alignment of PTK7 from different species reveals aconserved tyrosine sorting motif in its predicted C-terminal cytosolicdomain. (B)–(G) COS7 cellswere transfected with control siRNA or siRNA against Arfrp1 andre-transfected after 48 hr with plasmids encoding PTK7-Myc-His. After anadditional 24 hr, cells were incubated at 20°C in the presence of 30μg/ml cyclohexmide for 4 hr then shifted to 32°C for 90 min.After incubation, cells were analyzed by immunofluorescence using antibodiesagainst His and TGN46. Size bar = 10 μm. (H) COS7cell lysates from cells transfected with control siRNA or siRNA againstArfrp1 were analyzed by immunoblotting with anti-Arfrp1 antibody and, as aloading control, anti-GM130 antibody. (I) The fraction of cellsshowing TGN-accumulated PTK7 was quantified after incubation at 32°C(mean ± SD; N = 3; over 150 cells were counted for each group).(J) Similar siRNA knockdown and temperature shiftexperiments were performed in COS7 cells transfected with HA-Frizzled 6. Theappearance of TGN-accumulated HA-Frizzled 6 was quantified in cells treatedwith control siRNA or siRNA against Arfrp1 after an incubation at 32°C(mean ± SD; N = 2; over 100 cells were counted for eachgroup).DOI:http://dx.doi.org/10.7554/eLife.00160.015

Mentions: In addition to Vangl2, Arfrp1 is known to regulate TGN-to-plasma membrane traffickingof VSVG and E-cadherin (Shin et al., 2005;Zahn et al., 2008; Nishimoto-Morita et al., 2009). Each of these cargo moleculescontains a basolateral sorting motif in the C-terminal cytosolic domain. Sequencealignment of protein tyrosine kinase 7 (PTK7), another plasma-membrane localizedregulator of planar cell polarity (Lu et al.,2004), revealed a conserved tyrosine sorting motif (YVDL) in its predictedcytosolic domain (Figure 8A). We used aC-terminal Myc-His-tagged PTK7 (PTK7-Myc-His) to examine the effect of Arfrp1depletion on traffic from the TGN. COS7 cells were transfected with control siRNA orsiRNA against Arfrp1 and re-transfected after 48 hr with plasmids encodingPTK7-Myc-His. These conditions achieved an siRNA-specific depletion of Arfrp1 (Figure 8H). At steady state, around 50% of cellsshowed both surface- and Golgi-localized PTK7 in control cells and this localizationpattern was not significantly changed in Arfrp1 knockdown cells. Given the highbackground of PTK7 delayed in the TGN in transfected COS7 cells, we adjusted theexperimental conditions using a 20°C incubation followed by cycloheximide tosynchronize a pool of newly-synthesized PTK7 in the TGN in control cells and Arfrp1knockdown cells. After incubation at 20°C, a majority of cells (80%) showedstrong accumulation of PTK7 at the TGN. After cells were returned to 32°C, asignificantly higher percentage accumulated PTK7 at the TGN when Arfrp1 was depletedthan in cells treated with control siRNA (Figure8B–G and Figure 8I, 12 ±8% vs 49 ± 6%). In contrast, the TGN localization of HA-Frizzled 6 was notenhanced by depletion of Arfrp1 (Figure 8J).These results suggest that Arfrp1 also regulates TGN export of PTK7.10.7554/eLife.00160.015Figure 8.Arfrp1 regulates TGN export of PTK7.


A novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network.

Guo Y, Zanetti G, Schekman R - Elife (2013)

Arfrp1 regulates TGN export of PTK7.(A) Sequence alignment of PTK7 from different species reveals aconserved tyrosine sorting motif in its predicted C-terminal cytosolicdomain. (B)–(G) COS7 cellswere transfected with control siRNA or siRNA against Arfrp1 andre-transfected after 48 hr with plasmids encoding PTK7-Myc-His. After anadditional 24 hr, cells were incubated at 20°C in the presence of 30μg/ml cyclohexmide for 4 hr then shifted to 32°C for 90 min.After incubation, cells were analyzed by immunofluorescence using antibodiesagainst His and TGN46. Size bar = 10 μm. (H) COS7cell lysates from cells transfected with control siRNA or siRNA againstArfrp1 were analyzed by immunoblotting with anti-Arfrp1 antibody and, as aloading control, anti-GM130 antibody. (I) The fraction of cellsshowing TGN-accumulated PTK7 was quantified after incubation at 32°C(mean ± SD; N = 3; over 150 cells were counted for each group).(J) Similar siRNA knockdown and temperature shiftexperiments were performed in COS7 cells transfected with HA-Frizzled 6. Theappearance of TGN-accumulated HA-Frizzled 6 was quantified in cells treatedwith control siRNA or siRNA against Arfrp1 after an incubation at 32°C(mean ± SD; N = 2; over 100 cells were counted for eachgroup).DOI:http://dx.doi.org/10.7554/eLife.00160.015
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig8: Arfrp1 regulates TGN export of PTK7.(A) Sequence alignment of PTK7 from different species reveals aconserved tyrosine sorting motif in its predicted C-terminal cytosolicdomain. (B)–(G) COS7 cellswere transfected with control siRNA or siRNA against Arfrp1 andre-transfected after 48 hr with plasmids encoding PTK7-Myc-His. After anadditional 24 hr, cells were incubated at 20°C in the presence of 30μg/ml cyclohexmide for 4 hr then shifted to 32°C for 90 min.After incubation, cells were analyzed by immunofluorescence using antibodiesagainst His and TGN46. Size bar = 10 μm. (H) COS7cell lysates from cells transfected with control siRNA or siRNA againstArfrp1 were analyzed by immunoblotting with anti-Arfrp1 antibody and, as aloading control, anti-GM130 antibody. (I) The fraction of cellsshowing TGN-accumulated PTK7 was quantified after incubation at 32°C(mean ± SD; N = 3; over 150 cells were counted for each group).(J) Similar siRNA knockdown and temperature shiftexperiments were performed in COS7 cells transfected with HA-Frizzled 6. Theappearance of TGN-accumulated HA-Frizzled 6 was quantified in cells treatedwith control siRNA or siRNA against Arfrp1 after an incubation at 32°C(mean ± SD; N = 2; over 100 cells were counted for eachgroup).DOI:http://dx.doi.org/10.7554/eLife.00160.015
Mentions: In addition to Vangl2, Arfrp1 is known to regulate TGN-to-plasma membrane traffickingof VSVG and E-cadherin (Shin et al., 2005;Zahn et al., 2008; Nishimoto-Morita et al., 2009). Each of these cargo moleculescontains a basolateral sorting motif in the C-terminal cytosolic domain. Sequencealignment of protein tyrosine kinase 7 (PTK7), another plasma-membrane localizedregulator of planar cell polarity (Lu et al.,2004), revealed a conserved tyrosine sorting motif (YVDL) in its predictedcytosolic domain (Figure 8A). We used aC-terminal Myc-His-tagged PTK7 (PTK7-Myc-His) to examine the effect of Arfrp1depletion on traffic from the TGN. COS7 cells were transfected with control siRNA orsiRNA against Arfrp1 and re-transfected after 48 hr with plasmids encodingPTK7-Myc-His. These conditions achieved an siRNA-specific depletion of Arfrp1 (Figure 8H). At steady state, around 50% of cellsshowed both surface- and Golgi-localized PTK7 in control cells and this localizationpattern was not significantly changed in Arfrp1 knockdown cells. Given the highbackground of PTK7 delayed in the TGN in transfected COS7 cells, we adjusted theexperimental conditions using a 20°C incubation followed by cycloheximide tosynchronize a pool of newly-synthesized PTK7 in the TGN in control cells and Arfrp1knockdown cells. After incubation at 20°C, a majority of cells (80%) showedstrong accumulation of PTK7 at the TGN. After cells were returned to 32°C, asignificantly higher percentage accumulated PTK7 at the TGN when Arfrp1 was depletedthan in cells treated with control siRNA (Figure8B–G and Figure 8I, 12 ±8% vs 49 ± 6%). In contrast, the TGN localization of HA-Frizzled 6 was notenhanced by depletion of Arfrp1 (Figure 8J).These results suggest that Arfrp1 also regulates TGN export of PTK7.10.7554/eLife.00160.015Figure 8.Arfrp1 regulates TGN export of PTK7.

Bottom Line: Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN.In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1.Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology , Howard Hughes Medical Institute, University of California-Berkeley , Berkeley , United States.

ABSTRACT
Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.DOI:http://dx.doi.org/10.7554/eLife.00160.001.

No MeSH data available.


Related in: MedlinePlus