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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

Proposed model.(A) Model depicting Arfrp1- and AP-1-mediated TGN sorting ofVangl2. Arfrp1 is recruited to TGN membranes upon GTP binding, possiblymediated by a TGN localized GEF. Subsequently, GTP-bound Arfrp1 recruits AP-1to TGN membranes. GTP-bound Arfrp1 also promotes an open conformation of AP-1that directly interacts with the tyrosine motif on Vangl2 cytosolic domain,thereby enriching Vangl2 in budding vesicles. Binding of Vangl2 cytosolicdomain to AP-1, in turn, stabilizes the membrane association of AP-1 to allowsufficient time for AP-1 polymerization (possibly with clathrin as a coat outerlayer) and vesicle budding. This model is consistent with reports showing thattyrosine sorting motifs promote membrane recruitment of AP-1 mediated by Arf1(Crottet et al., 2002; Lee et al., 2008). (B) Theasymmetrically localized PCP signaling molecules, including Vangl2 and Frizzled6, are sorted by different sorting machineries for export from the TGN.Differential TGN sorting and polarized trafficking of these signaling receptorsmay contribute to their asymmetric distribution and the laterally polarizedorganization of epithelial cells.DOI:http://dx.doi.org/10.7554/eLife.00160.017
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fig10: Proposed model.(A) Model depicting Arfrp1- and AP-1-mediated TGN sorting ofVangl2. Arfrp1 is recruited to TGN membranes upon GTP binding, possiblymediated by a TGN localized GEF. Subsequently, GTP-bound Arfrp1 recruits AP-1to TGN membranes. GTP-bound Arfrp1 also promotes an open conformation of AP-1that directly interacts with the tyrosine motif on Vangl2 cytosolic domain,thereby enriching Vangl2 in budding vesicles. Binding of Vangl2 cytosolicdomain to AP-1, in turn, stabilizes the membrane association of AP-1 to allowsufficient time for AP-1 polymerization (possibly with clathrin as a coat outerlayer) and vesicle budding. This model is consistent with reports showing thattyrosine sorting motifs promote membrane recruitment of AP-1 mediated by Arf1(Crottet et al., 2002; Lee et al., 2008). (B) Theasymmetrically localized PCP signaling molecules, including Vangl2 and Frizzled6, are sorted by different sorting machineries for export from the TGN.Differential TGN sorting and polarized trafficking of these signaling receptorsmay contribute to their asymmetric distribution and the laterally polarizedorganization of epithelial cells.DOI:http://dx.doi.org/10.7554/eLife.00160.017

Mentions: Here we show that TGN export of Vangl2, a PCP signaling receptor, depends on anunexpected complex of a TGN-localized Arf GTP-binding protein, Arfrp1, and theGolgi-localized clathrin adaptor complex, AP-1. siRNA knockdown of Arfrp1 or of subunitsof AP-1 arrest Vangl2 traffic at the TGN as determined by co-localization of Vangl2 andthe TGN marker, Golgin 97. Further, we have identified a sorting signal within theC-terminal cytoplasmic domain of Vangl2, YYXXF, the Phe residue of which is crucial forVangl2 binding to AP-1 and traffic from the TGN to the cell surface. We propose a modelwherein the interaction of Arfrp1-GTP and AP-1 exposes a sorting recognition determinantof the AP-1 µ subunit that binds the sorting motif on Vangl2 (Figure 10A), and this binding in turn helps to stabilize AP-1assembly on membranes.10.7554/eLife.00160.017Figure 10.Proposed model.


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)

Proposed model.(A) Model depicting Arfrp1- and AP-1-mediated TGN sorting ofVangl2. Arfrp1 is recruited to TGN membranes upon GTP binding, possiblymediated by a TGN localized GEF. Subsequently, GTP-bound Arfrp1 recruits AP-1to TGN membranes. GTP-bound Arfrp1 also promotes an open conformation of AP-1that directly interacts with the tyrosine motif on Vangl2 cytosolic domain,thereby enriching Vangl2 in budding vesicles. Binding of Vangl2 cytosolicdomain to AP-1, in turn, stabilizes the membrane association of AP-1 to allowsufficient time for AP-1 polymerization (possibly with clathrin as a coat outerlayer) and vesicle budding. This model is consistent with reports showing thattyrosine sorting motifs promote membrane recruitment of AP-1 mediated by Arf1(Crottet et al., 2002; Lee et al., 2008). (B) Theasymmetrically localized PCP signaling molecules, including Vangl2 and Frizzled6, are sorted by different sorting machineries for export from the TGN.Differential TGN sorting and polarized trafficking of these signaling receptorsmay contribute to their asymmetric distribution and the laterally polarizedorganization of epithelial cells.DOI:http://dx.doi.org/10.7554/eLife.00160.017
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Related In: Results  -  Collection

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fig10: Proposed model.(A) Model depicting Arfrp1- and AP-1-mediated TGN sorting ofVangl2. Arfrp1 is recruited to TGN membranes upon GTP binding, possiblymediated by a TGN localized GEF. Subsequently, GTP-bound Arfrp1 recruits AP-1to TGN membranes. GTP-bound Arfrp1 also promotes an open conformation of AP-1that directly interacts with the tyrosine motif on Vangl2 cytosolic domain,thereby enriching Vangl2 in budding vesicles. Binding of Vangl2 cytosolicdomain to AP-1, in turn, stabilizes the membrane association of AP-1 to allowsufficient time for AP-1 polymerization (possibly with clathrin as a coat outerlayer) and vesicle budding. This model is consistent with reports showing thattyrosine sorting motifs promote membrane recruitment of AP-1 mediated by Arf1(Crottet et al., 2002; Lee et al., 2008). (B) Theasymmetrically localized PCP signaling molecules, including Vangl2 and Frizzled6, are sorted by different sorting machineries for export from the TGN.Differential TGN sorting and polarized trafficking of these signaling receptorsmay contribute to their asymmetric distribution and the laterally polarizedorganization of epithelial cells.DOI:http://dx.doi.org/10.7554/eLife.00160.017
Mentions: Here we show that TGN export of Vangl2, a PCP signaling receptor, depends on anunexpected complex of a TGN-localized Arf GTP-binding protein, Arfrp1, and theGolgi-localized clathrin adaptor complex, AP-1. siRNA knockdown of Arfrp1 or of subunitsof AP-1 arrest Vangl2 traffic at the TGN as determined by co-localization of Vangl2 andthe TGN marker, Golgin 97. Further, we have identified a sorting signal within theC-terminal cytoplasmic domain of Vangl2, YYXXF, the Phe residue of which is crucial forVangl2 binding to AP-1 and traffic from the TGN to the cell surface. We propose a modelwherein the interaction of Arfrp1-GTP and AP-1 exposes a sorting recognition determinantof the AP-1 µ subunit that binds the sorting motif on Vangl2 (Figure 10A), and this binding in turn helps to stabilize AP-1assembly on membranes.10.7554/eLife.00160.017Figure 10.Proposed model.

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