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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

Sorting signal-dependent binding of Arfrp1 to Vangl2 in cell lysates;Vangl2 binds Arfrp1 more efficiently than Arf1.(A) Cell lysates from COS7 cells transiently transfectedwith plasmids encoding HA-Vangl2 were incubated with glutathione beadsbearing similar amounts of GTPγS-loaded GST-Arf1 or GST-Arfrp1.After incubation, the entire sample of bound HA-Vangl2 was detected byimmunoblot. (B) Cell lysates from COS7 cells transientlytransfected with plasmids encoding Vangl2 wild type or the indicatedVangl2 mutant constructs were incubated with glutathione beads bearingsimilar amount of GTPγS-loaded Arfrp1. The entire sample of boundHA-Vangl2 was evaluated by immunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.013
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fig6s1: Sorting signal-dependent binding of Arfrp1 to Vangl2 in cell lysates;Vangl2 binds Arfrp1 more efficiently than Arf1.(A) Cell lysates from COS7 cells transiently transfectedwith plasmids encoding HA-Vangl2 were incubated with glutathione beadsbearing similar amounts of GTPγS-loaded GST-Arf1 or GST-Arfrp1.After incubation, the entire sample of bound HA-Vangl2 was detected byimmunoblot. (B) Cell lysates from COS7 cells transientlytransfected with plasmids encoding Vangl2 wild type or the indicatedVangl2 mutant constructs were incubated with glutathione beads bearingsimilar amount of GTPγS-loaded Arfrp1. The entire sample of boundHA-Vangl2 was evaluated by immunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.013

Mentions: Arf1 also mediates membrane recruitment of AP-1. A peptide containing themannose-6-phosphate receptor sorting signal stimulates Arf1-mediated membranerecruitment of AP-1 to liposomes (Zhu et al.,1998, 1999; Lee et al., 2008). We evaluated the effect of the Vangl2cytosolic domain on Arf1-mediated AP-1 recruitment using FLAG-tagged Arf1 and Arfrp1purified from mammalian cells. In contrast to incubations containingArfrp1-GTPγS, Vangl2 C-terminal domain did not stimulate AP-1 recruitment toliposomes in the presence of Arf1-GTPγS (Figure6F,G). This result suggests that the stimulation effect is specific forArfrp1 and indicates that Arfrp1- but not Arf1- associated AP-1 provides a preferredbinding site for the Vangl2 sorting signal. As expected, HA-Vangl2 from COS7 celllysates interacted with GST-Arfrp1 but weakly with GST-Arf1 (Figure 6—figure supplement 1A). The interaction betweenGST-Arfrp1 and HA-Vangl2 depended on the YYXXF motif (Figure 6—figure supplement 1B) suggesting that Arfrp1interacts with Vangl2 indirectly through the AP-1 complex.


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)

Sorting signal-dependent binding of Arfrp1 to Vangl2 in cell lysates;Vangl2 binds Arfrp1 more efficiently than Arf1.(A) Cell lysates from COS7 cells transiently transfectedwith plasmids encoding HA-Vangl2 were incubated with glutathione beadsbearing similar amounts of GTPγS-loaded GST-Arf1 or GST-Arfrp1.After incubation, the entire sample of bound HA-Vangl2 was detected byimmunoblot. (B) Cell lysates from COS7 cells transientlytransfected with plasmids encoding Vangl2 wild type or the indicatedVangl2 mutant constructs were incubated with glutathione beads bearingsimilar amount of GTPγS-loaded Arfrp1. The entire sample of boundHA-Vangl2 was evaluated by immunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.013
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539332&req=5

fig6s1: Sorting signal-dependent binding of Arfrp1 to Vangl2 in cell lysates;Vangl2 binds Arfrp1 more efficiently than Arf1.(A) Cell lysates from COS7 cells transiently transfectedwith plasmids encoding HA-Vangl2 were incubated with glutathione beadsbearing similar amounts of GTPγS-loaded GST-Arf1 or GST-Arfrp1.After incubation, the entire sample of bound HA-Vangl2 was detected byimmunoblot. (B) Cell lysates from COS7 cells transientlytransfected with plasmids encoding Vangl2 wild type or the indicatedVangl2 mutant constructs were incubated with glutathione beads bearingsimilar amount of GTPγS-loaded Arfrp1. The entire sample of boundHA-Vangl2 was evaluated by immunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.013
Mentions: Arf1 also mediates membrane recruitment of AP-1. A peptide containing themannose-6-phosphate receptor sorting signal stimulates Arf1-mediated membranerecruitment of AP-1 to liposomes (Zhu et al.,1998, 1999; Lee et al., 2008). We evaluated the effect of the Vangl2cytosolic domain on Arf1-mediated AP-1 recruitment using FLAG-tagged Arf1 and Arfrp1purified from mammalian cells. In contrast to incubations containingArfrp1-GTPγS, Vangl2 C-terminal domain did not stimulate AP-1 recruitment toliposomes in the presence of Arf1-GTPγS (Figure6F,G). This result suggests that the stimulation effect is specific forArfrp1 and indicates that Arfrp1- but not Arf1- associated AP-1 provides a preferredbinding site for the Vangl2 sorting signal. As expected, HA-Vangl2 from COS7 celllysates interacted with GST-Arfrp1 but weakly with GST-Arf1 (Figure 6—figure supplement 1A). The interaction betweenGST-Arfrp1 and HA-Vangl2 depended on the YYXXF motif (Figure 6—figure supplement 1B) suggesting that Arfrp1interacts with Vangl2 indirectly through the AP-1 complex.

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