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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

TGN export of Frizzled-6 and Celsr1 is independent of the Arfrp1/AP-1machinery.(A)–(I). HeLa cells wereeither mock transfected (A–C) ortransfected with siRNA against Arfrp1 (D–F)or μ1-adaptin (G–I) andre-transfected after 48 hr with plasmids encoding GFP-Celsr1 andMyc-Frizzled 6. After an additional 24 hr, cells were analyzed byimmunofluorescence. Size bar = 10 μm.(J),(K). Cell lysates (250 μl) containing1 mg/ml proteins from COS7 cells co-transfected with HA-Vangl2 andGFP-Celsr1 (J) or HA-Vangl2 and GFP-Frizzled 6 (K)were incubated with glutathione beads bearing 1 μg of GST,GTPγS-loaded GST-Arfrp1 or GST-μ1. The entire sample of boundHA-Vangl2, GFP-Celsr1 or GFP-Frizzled 6 were detected by immunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.014
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fig7: TGN export of Frizzled-6 and Celsr1 is independent of the Arfrp1/AP-1machinery.(A)–(I). HeLa cells wereeither mock transfected (A–C) ortransfected with siRNA against Arfrp1 (D–F)or μ1-adaptin (G–I) andre-transfected after 48 hr with plasmids encoding GFP-Celsr1 andMyc-Frizzled 6. After an additional 24 hr, cells were analyzed byimmunofluorescence. Size bar = 10 μm.(J),(K). Cell lysates (250 μl) containing1 mg/ml proteins from COS7 cells co-transfected with HA-Vangl2 andGFP-Celsr1 (J) or HA-Vangl2 and GFP-Frizzled 6 (K)were incubated with glutathione beads bearing 1 μg of GST,GTPγS-loaded GST-Arfrp1 or GST-μ1. The entire sample of boundHA-Vangl2, GFP-Celsr1 or GFP-Frizzled 6 were detected by immunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.014

Mentions: Vangl2 and Frizzled-6 localize on opposing surfaces at cell–cell junctions inepithelial tissues. Because the TGN is a cargo sorting station, it is possible thatFrizzled-6 and Vangl2 may use different vesicle sorting machineries to exit the TGN.Unlike Vangl2, Frizzled 6 was inefficiently transported to the cell surface intransfected HeLa cells. However, when Frizzled-6 was co-expressed with Celsr1, anatypical cadherin, both proteins co-localized at cell junctions (Figure 7A–C) (Devenportand Fuchs, 2008). Unlike Vangl2, knockdown of Arfrp1 or μ1-adaptinhad no detectable effects on the localization of Frizzled-6 and Celsr1 (Figure 7D–I). Frizzled-6 and Celsr1 haveno known tyrosine- or dileucine-based sorting motifs in their cytosolic domains. Totest whether Arfrp1 or μ1-adaptin interact with Frizzled-6 or Celsr1, weperformed GST-pull down analysis as before. GST-Arfrp1 and GST-μ1 boundHA-Vangl2 but not GFP-Frizzled-6 or GFP-Celsr1 in cell lysates from COS7 cellsco-transfected with HA-Vangl2 and GFP-Celsr1 (Figure7J) or co-transfected with HA-Vangl2 and GFP-Frizzled 6 (Figure 7K). These results suggest that sorting ofFrizzled 6 and Celsr1 at the TGN is independent of the Arfrp1/AP-1 machinery.10.7554/eLife.00160.014Figure 7.TGN export of Frizzled-6 and Celsr1 is independent of the Arfrp1/AP-1machinery.


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)

TGN export of Frizzled-6 and Celsr1 is independent of the Arfrp1/AP-1machinery.(A)–(I). HeLa cells wereeither mock transfected (A–C) ortransfected with siRNA against Arfrp1 (D–F)or μ1-adaptin (G–I) andre-transfected after 48 hr with plasmids encoding GFP-Celsr1 andMyc-Frizzled 6. After an additional 24 hr, cells were analyzed byimmunofluorescence. Size bar = 10 μm.(J),(K). Cell lysates (250 μl) containing1 mg/ml proteins from COS7 cells co-transfected with HA-Vangl2 andGFP-Celsr1 (J) or HA-Vangl2 and GFP-Frizzled 6 (K)were incubated with glutathione beads bearing 1 μg of GST,GTPγS-loaded GST-Arfrp1 or GST-μ1. The entire sample of boundHA-Vangl2, GFP-Celsr1 or GFP-Frizzled 6 were detected by immunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.014
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fig7: TGN export of Frizzled-6 and Celsr1 is independent of the Arfrp1/AP-1machinery.(A)–(I). HeLa cells wereeither mock transfected (A–C) ortransfected with siRNA against Arfrp1 (D–F)or μ1-adaptin (G–I) andre-transfected after 48 hr with plasmids encoding GFP-Celsr1 andMyc-Frizzled 6. After an additional 24 hr, cells were analyzed byimmunofluorescence. Size bar = 10 μm.(J),(K). Cell lysates (250 μl) containing1 mg/ml proteins from COS7 cells co-transfected with HA-Vangl2 andGFP-Celsr1 (J) or HA-Vangl2 and GFP-Frizzled 6 (K)were incubated with glutathione beads bearing 1 μg of GST,GTPγS-loaded GST-Arfrp1 or GST-μ1. The entire sample of boundHA-Vangl2, GFP-Celsr1 or GFP-Frizzled 6 were detected by immunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.014
Mentions: Vangl2 and Frizzled-6 localize on opposing surfaces at cell–cell junctions inepithelial tissues. Because the TGN is a cargo sorting station, it is possible thatFrizzled-6 and Vangl2 may use different vesicle sorting machineries to exit the TGN.Unlike Vangl2, Frizzled 6 was inefficiently transported to the cell surface intransfected HeLa cells. However, when Frizzled-6 was co-expressed with Celsr1, anatypical cadherin, both proteins co-localized at cell junctions (Figure 7A–C) (Devenportand Fuchs, 2008). Unlike Vangl2, knockdown of Arfrp1 or μ1-adaptinhad no detectable effects on the localization of Frizzled-6 and Celsr1 (Figure 7D–I). Frizzled-6 and Celsr1 haveno known tyrosine- or dileucine-based sorting motifs in their cytosolic domains. Totest whether Arfrp1 or μ1-adaptin interact with Frizzled-6 or Celsr1, weperformed GST-pull down analysis as before. GST-Arfrp1 and GST-μ1 boundHA-Vangl2 but not GFP-Frizzled-6 or GFP-Celsr1 in cell lysates from COS7 cellsco-transfected with HA-Vangl2 and GFP-Celsr1 (Figure7J) or co-transfected with HA-Vangl2 and GFP-Frizzled 6 (Figure 7K). These results suggest that sorting ofFrizzled 6 and Celsr1 at the TGN is independent of the Arfrp1/AP-1 machinery.10.7554/eLife.00160.014Figure 7.TGN export of Frizzled-6 and Celsr1 is independent of the Arfrp1/AP-1machinery.

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