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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 Vangl2 and Frizzled 6 is protein kinase Ddependent.COS7 cells were co-transfected with GST-PKD2-KD and HA-Frizzled 6(A)–(D), GST-PKD3-KD and HA-Frizzled 6(E)–(H), GST-PKD2-KD and HA-Vangl2(I)–(L) or GST-PKD3-KD and HA-Vangl2(M)–(P). Day 1 after transfection, cellswere analyzed by immunofluorescence using anti-HA, anti-TGN46 and anti-GSTantibodies. Size bar = 10 μm.DOI:http://dx.doi.org/10.7554/eLife.00160.016
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fig9: TGN export of Vangl2 and Frizzled 6 is protein kinase Ddependent.COS7 cells were co-transfected with GST-PKD2-KD and HA-Frizzled 6(A)–(D), GST-PKD3-KD and HA-Frizzled 6(E)–(H), GST-PKD2-KD and HA-Vangl2(I)–(L) or GST-PKD3-KD and HA-Vangl2(M)–(P). Day 1 after transfection, cellswere analyzed by immunofluorescence using anti-HA, anti-TGN46 and anti-GSTantibodies. Size bar = 10 μm.DOI:http://dx.doi.org/10.7554/eLife.00160.016

Mentions: Protein kinase D (PKD) mediates membrane fission to generate TGN to cell surfacetransport carriers containing basolateral cargo molecules (Yeaman et al., 2004; Malhotraand Campelo, 2011). Expression of the kinase dead form of glutathioneS-transferase tagged PKD2 (GST-PKD2-KD) or PKD3 (GST-PKD3-KD) in COS7 cells resultedin the accumulation of HA-Vangl2 and HA-Frizzled 6 at the juxtanuclear area,colocalized with the TGN marker, TGN46 (Figure9). Thus, although Vangl2 and Frizzled 6 display distinct requirements forArfrp1 and AP-1, they both depend on PKD for traffic from the TGN. We suggest thatVangl2 (and PTK7) and Frizzled 6 are sorted by independent means into separatetransport vesicles but that they share a common mechanism for membrane fission toform these carriers.10.7554/eLife.00160.016Figure 9.TGN export of Vangl2 and Frizzled 6 is protein kinase Ddependent.


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 Vangl2 and Frizzled 6 is protein kinase Ddependent.COS7 cells were co-transfected with GST-PKD2-KD and HA-Frizzled 6(A)–(D), GST-PKD3-KD and HA-Frizzled 6(E)–(H), GST-PKD2-KD and HA-Vangl2(I)–(L) or GST-PKD3-KD and HA-Vangl2(M)–(P). Day 1 after transfection, cellswere analyzed by immunofluorescence using anti-HA, anti-TGN46 and anti-GSTantibodies. Size bar = 10 μm.DOI:http://dx.doi.org/10.7554/eLife.00160.016
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig9: TGN export of Vangl2 and Frizzled 6 is protein kinase Ddependent.COS7 cells were co-transfected with GST-PKD2-KD and HA-Frizzled 6(A)–(D), GST-PKD3-KD and HA-Frizzled 6(E)–(H), GST-PKD2-KD and HA-Vangl2(I)–(L) or GST-PKD3-KD and HA-Vangl2(M)–(P). Day 1 after transfection, cellswere analyzed by immunofluorescence using anti-HA, anti-TGN46 and anti-GSTantibodies. Size bar = 10 μm.DOI:http://dx.doi.org/10.7554/eLife.00160.016
Mentions: Protein kinase D (PKD) mediates membrane fission to generate TGN to cell surfacetransport carriers containing basolateral cargo molecules (Yeaman et al., 2004; Malhotraand Campelo, 2011). Expression of the kinase dead form of glutathioneS-transferase tagged PKD2 (GST-PKD2-KD) or PKD3 (GST-PKD3-KD) in COS7 cells resultedin the accumulation of HA-Vangl2 and HA-Frizzled 6 at the juxtanuclear area,colocalized with the TGN marker, TGN46 (Figure9). Thus, although Vangl2 and Frizzled 6 display distinct requirements forArfrp1 and AP-1, they both depend on PKD for traffic from the TGN. We suggest thatVangl2 (and PTK7) and Frizzled 6 are sorted by independent means into separatetransport vesicles but that they share a common mechanism for membrane fission toform these carriers.10.7554/eLife.00160.016Figure 9.TGN export of Vangl2 and Frizzled 6 is protein kinase Ddependent.

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