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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

μ1-adaptin directly interacts with Vangl2 C-terminal cytosolicdomain in an YYXXF-motif dependent manner.(A) Cell lysates from COS7 cells transiently transfected withplasmids encoding HA-Vangl2 wild-type or the indicated Vangl2 mutantconstructs were incubated with glutathione beads bearing similar amounts ofGST or GST-μ1. The entire sample of bound HA-Vangl2 was evaluated byimmunoblotting with anti-HA antibody. (B) Yeast two-hybridanalyses recapitulated the results of the GST-pull down assay. Serialdilutions of the yeast colonies co-expressing the indicated constructs weredotted on the correspondent selective media. Pictures were taken after 3days of growth. (C) Purified MBP-Vangl2 C-terminus wild type,or the indicated mutant constructs were incubated with glutathione beadsbearing GST-μ1. The entire sample of bound MBP-Vangl2 was evaluated byimmunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.010
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fig4: μ1-adaptin directly interacts with Vangl2 C-terminal cytosolicdomain in an YYXXF-motif dependent manner.(A) Cell lysates from COS7 cells transiently transfected withplasmids encoding HA-Vangl2 wild-type or the indicated Vangl2 mutantconstructs were incubated with glutathione beads bearing similar amounts ofGST or GST-μ1. The entire sample of bound HA-Vangl2 was evaluated byimmunoblotting with anti-HA antibody. (B) Yeast two-hybridanalyses recapitulated the results of the GST-pull down assay. Serialdilutions of the yeast colonies co-expressing the indicated constructs weredotted on the correspondent selective media. Pictures were taken after 3days of growth. (C) Purified MBP-Vangl2 C-terminus wild type,or the indicated mutant constructs were incubated with glutathione beadsbearing GST-μ1. The entire sample of bound MBP-Vangl2 was evaluated byimmunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.010

Mentions: The tyrosine-based sorting motif is known to interact with the μ subunit of theAP complexes (Bonifacino and Lippincott-Schwartz,2003). To test whether μ1-adaptin interacts with Vangl2 via the YYXXFmotif, we performed GST pull-down assays using purified GST-μ1 and lysates fromCOS7 cells transiently transfected with HA-Vangl2 wild-type or mutant constructs.HA-Vangl2 wild type specifically bound GST-μ1 (Figure 4A). The interaction between Vangl2 and GST-μ1 was severelyreduced when crucial residues of the YYXXF motif were mutated, whereas alaninesubstitutions of the adjacent dileucine amino acids had no effect (Figure 4A). Yeast two-hybrid analysis confirmedthat μ1-adaptin interacted with Vangl2 and mutation of the basolateral sortingmotif, including the restrictive F283A substitution, inhibited this interaction(Figure 4B). The less restrictive singletyrosine mutant, Vangl2 (Y280A), interacted weakly with μ1-adaptin whereasmutating both tyrosine residues blocked interaction (Figure 4B). To test whether the Vangl2 cytosolic domain directly interactswith μ1-adaptin, we purified GST-μ1 and MBP-tagged Vangl2 C-terminaldomain proteins. The MBP-Vangl2 C-terminal domain bound GST-μ1 whereas mutationof the YYXXF motif blocked this interaction (Figure4C), consistent with a direct and signal-dependent interaction. Theinteraction pattern correlated well with the Vangl2 mutant localization analysis intransfected cells (Figure 3B–V).10.7554/eLife.00160.010Figure 4.μ1-adaptin directly interacts with Vangl2 C-terminal cytosolicdomain in an YYXXF-motif dependent manner.


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)

μ1-adaptin directly interacts with Vangl2 C-terminal cytosolicdomain in an YYXXF-motif dependent manner.(A) Cell lysates from COS7 cells transiently transfected withplasmids encoding HA-Vangl2 wild-type or the indicated Vangl2 mutantconstructs were incubated with glutathione beads bearing similar amounts ofGST or GST-μ1. The entire sample of bound HA-Vangl2 was evaluated byimmunoblotting with anti-HA antibody. (B) Yeast two-hybridanalyses recapitulated the results of the GST-pull down assay. Serialdilutions of the yeast colonies co-expressing the indicated constructs weredotted on the correspondent selective media. Pictures were taken after 3days of growth. (C) Purified MBP-Vangl2 C-terminus wild type,or the indicated mutant constructs were incubated with glutathione beadsbearing GST-μ1. The entire sample of bound MBP-Vangl2 was evaluated byimmunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.010
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fig4: μ1-adaptin directly interacts with Vangl2 C-terminal cytosolicdomain in an YYXXF-motif dependent manner.(A) Cell lysates from COS7 cells transiently transfected withplasmids encoding HA-Vangl2 wild-type or the indicated Vangl2 mutantconstructs were incubated with glutathione beads bearing similar amounts ofGST or GST-μ1. The entire sample of bound HA-Vangl2 was evaluated byimmunoblotting with anti-HA antibody. (B) Yeast two-hybridanalyses recapitulated the results of the GST-pull down assay. Serialdilutions of the yeast colonies co-expressing the indicated constructs weredotted on the correspondent selective media. Pictures were taken after 3days of growth. (C) Purified MBP-Vangl2 C-terminus wild type,or the indicated mutant constructs were incubated with glutathione beadsbearing GST-μ1. The entire sample of bound MBP-Vangl2 was evaluated byimmunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.010
Mentions: The tyrosine-based sorting motif is known to interact with the μ subunit of theAP complexes (Bonifacino and Lippincott-Schwartz,2003). To test whether μ1-adaptin interacts with Vangl2 via the YYXXFmotif, we performed GST pull-down assays using purified GST-μ1 and lysates fromCOS7 cells transiently transfected with HA-Vangl2 wild-type or mutant constructs.HA-Vangl2 wild type specifically bound GST-μ1 (Figure 4A). The interaction between Vangl2 and GST-μ1 was severelyreduced when crucial residues of the YYXXF motif were mutated, whereas alaninesubstitutions of the adjacent dileucine amino acids had no effect (Figure 4A). Yeast two-hybrid analysis confirmedthat μ1-adaptin interacted with Vangl2 and mutation of the basolateral sortingmotif, including the restrictive F283A substitution, inhibited this interaction(Figure 4B). The less restrictive singletyrosine mutant, Vangl2 (Y280A), interacted weakly with μ1-adaptin whereasmutating both tyrosine residues blocked interaction (Figure 4B). To test whether the Vangl2 cytosolic domain directly interactswith μ1-adaptin, we purified GST-μ1 and MBP-tagged Vangl2 C-terminaldomain proteins. The MBP-Vangl2 C-terminal domain bound GST-μ1 whereas mutationof the YYXXF motif blocked this interaction (Figure4C), consistent with a direct and signal-dependent interaction. Theinteraction pattern correlated well with the Vangl2 mutant localization analysis intransfected cells (Figure 3B–V).10.7554/eLife.00160.010Figure 4.μ1-adaptin directly interacts with Vangl2 C-terminal cytosolicdomain in an YYXXF-motif dependent manner.

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