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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

Knockdown of μ1-adaptin or γ1-adaptin accumulates Vangl2 atthe TGN.(A) HeLa cells were mock transfected or transfected with siRNAagainst the indicated subunit of the AP-1 or AP-3 complex. At day 3 aftertransfection, total cell lysates were analyzed by immunoblotting withantibody against the indicated adaptin subunits or, as loading controls,p115 and tubulin. (B)–(J) HeLa cells weremock transfected (B–D) or transfected withsiRNAs against μ1-adaptin (E–G) orγ1-adaptin (H–J) and re-transfectedafter 48 hr with plasmid encoding HA-Vangl2. After an additional 24 hr,cells were analyzed by immunofluorescence. Size bar = 10 μM.(K) Quantification of the fraction of cells showingGolgi-accumulated Vangl2 (N = 3; >150 cells expressing lowerlevels of Vangl2 counted for each experiment).DOI:http://dx.doi.org/10.7554/eLife.00160.011
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fig5: Knockdown of μ1-adaptin or γ1-adaptin accumulates Vangl2 atthe TGN.(A) HeLa cells were mock transfected or transfected with siRNAagainst the indicated subunit of the AP-1 or AP-3 complex. At day 3 aftertransfection, total cell lysates were analyzed by immunoblotting withantibody against the indicated adaptin subunits or, as loading controls,p115 and tubulin. (B)–(J) HeLa cells weremock transfected (B–D) or transfected withsiRNAs against μ1-adaptin (E–G) orγ1-adaptin (H–J) and re-transfectedafter 48 hr with plasmid encoding HA-Vangl2. After an additional 24 hr,cells were analyzed by immunofluorescence. Size bar = 10 μM.(K) Quantification of the fraction of cells showingGolgi-accumulated Vangl2 (N = 3; >150 cells expressing lowerlevels of Vangl2 counted for each experiment).DOI:http://dx.doi.org/10.7554/eLife.00160.011

Mentions: To test whether AP-1 mediates TGN export of Vangl2, we knocked down the expression ofthe μ and γ subunits of AP-1 in HeLa cells transiently transfected withHA-Vangl2. Immunoblot analysis showed that the expression of μ1- andγ1-adaptins was significantly reduced after siRNA treatment (Figure 5A). As before, we focused on cellsexpressing lower levels of HA-Vangl2 and observed an accumulation of HA-Vangl2 in thejuxtanuclear area, colocalized with Golgin 97, with weak or no detectable surfacelabeling in over 60% of the treated cells (Figure5E–J and quantification in Figure5K). Around 20% of mock-treated cells displayed Golgi-localized Vangl2(Figure 5K) but retained strong surfacelabeling. As a control, knockdown of μ3-adaptin, which did not bind Vangl2 (notshown), or knockdown of δ3-adaptin had no significant effects on thelocalization of Vangl2 (Figure 5K). Theinteraction data and knockdown analysis suggest that AP-1 directly mediates TGNexport of Vangl2.10.7554/eLife.00160.011Figure 5.Knockdown of μ1-adaptin or γ1-adaptin accumulates Vangl2 atthe TGN.


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)

Knockdown of μ1-adaptin or γ1-adaptin accumulates Vangl2 atthe TGN.(A) HeLa cells were mock transfected or transfected with siRNAagainst the indicated subunit of the AP-1 or AP-3 complex. At day 3 aftertransfection, total cell lysates were analyzed by immunoblotting withantibody against the indicated adaptin subunits or, as loading controls,p115 and tubulin. (B)–(J) HeLa cells weremock transfected (B–D) or transfected withsiRNAs against μ1-adaptin (E–G) orγ1-adaptin (H–J) and re-transfectedafter 48 hr with plasmid encoding HA-Vangl2. After an additional 24 hr,cells were analyzed by immunofluorescence. Size bar = 10 μM.(K) Quantification of the fraction of cells showingGolgi-accumulated Vangl2 (N = 3; >150 cells expressing lowerlevels of Vangl2 counted for each experiment).DOI:http://dx.doi.org/10.7554/eLife.00160.011
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Knockdown of μ1-adaptin or γ1-adaptin accumulates Vangl2 atthe TGN.(A) HeLa cells were mock transfected or transfected with siRNAagainst the indicated subunit of the AP-1 or AP-3 complex. At day 3 aftertransfection, total cell lysates were analyzed by immunoblotting withantibody against the indicated adaptin subunits or, as loading controls,p115 and tubulin. (B)–(J) HeLa cells weremock transfected (B–D) or transfected withsiRNAs against μ1-adaptin (E–G) orγ1-adaptin (H–J) and re-transfectedafter 48 hr with plasmid encoding HA-Vangl2. After an additional 24 hr,cells were analyzed by immunofluorescence. Size bar = 10 μM.(K) Quantification of the fraction of cells showingGolgi-accumulated Vangl2 (N = 3; >150 cells expressing lowerlevels of Vangl2 counted for each experiment).DOI:http://dx.doi.org/10.7554/eLife.00160.011
Mentions: To test whether AP-1 mediates TGN export of Vangl2, we knocked down the expression ofthe μ and γ subunits of AP-1 in HeLa cells transiently transfected withHA-Vangl2. Immunoblot analysis showed that the expression of μ1- andγ1-adaptins was significantly reduced after siRNA treatment (Figure 5A). As before, we focused on cellsexpressing lower levels of HA-Vangl2 and observed an accumulation of HA-Vangl2 in thejuxtanuclear area, colocalized with Golgin 97, with weak or no detectable surfacelabeling in over 60% of the treated cells (Figure5E–J and quantification in Figure5K). Around 20% of mock-treated cells displayed Golgi-localized Vangl2(Figure 5K) but retained strong surfacelabeling. As a control, knockdown of μ3-adaptin, which did not bind Vangl2 (notshown), or knockdown of δ3-adaptin had no significant effects on thelocalization of Vangl2 (Figure 5K). Theinteraction data and knockdown analysis suggest that AP-1 directly mediates TGNexport of Vangl2.10.7554/eLife.00160.011Figure 5.Knockdown of μ1-adaptin or γ1-adaptin accumulates Vangl2 atthe TGN.

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