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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

Vangl2 Y279A Y280A is blocked at the TGN.COS7 cells were transfected with HA-Vangl2 wild type(A)–(C) or HA-Vangl2 (Y279A, Y280A)(D)–(F). After transfection for 24hr, cells were analyzed by immunofluorescence using anti-HA andanti-Golgin 97 antibody. Size bar = 10 μm.DOI:http://dx.doi.org/10.7554/eLife.00160.009
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fig3s2: Vangl2 Y279A Y280A is blocked at the TGN.COS7 cells were transfected with HA-Vangl2 wild type(A)–(C) or HA-Vangl2 (Y279A, Y280A)(D)–(F). After transfection for 24hr, cells were analyzed by immunofluorescence using anti-HA andanti-Golgin 97 antibody. Size bar = 10 μm.DOI:http://dx.doi.org/10.7554/eLife.00160.009

Mentions: The results from the affinity isolation suggest that AP-1 is an effector of Arfrp1,possibly cooperating to mediate TGN export of Vangl2. Consistent with thishypothesis, the C-terminal cytosolic domain of Vangl2 contains a conservedbasolateral-sorting motif (YXXF) which is known to interact with the AP complexes(Bonifacino and Lippincott-Schwartz, 2003)(red box, Figure 3A). Indeed, HA-Vangl2 islocalized basolaterally in MDCK cells (Kallay etal., 2006). To test whether this motif is important for the localization ofVangl2, we generated a series of HA-Vangl2 mutant constructs and examined theirlocalization. Strikingly, four Vangl2 mutants bearing mutations in the YXXF motif,including the single mutation (F283A), showed no detectable surface pattern (Figure 3E,H,K,Q). At high levels of expression,mutant Vangl2 was retained in the ER. However, at lower levels of expression, theseVangl2 mutant proteins accumulated in the juxtanuclear area which colocalized withthe TGN marker, Golgin 97 (Figure3E–M,Q–S). A Vangl2 YXXF double mutant (Y280A, F283A) andVangl2 looptail mutant (D255E) displayed quite distinctive localization to the TGNand ER, respectively (Figure 3—figuresupplement 1). A single tyrosine mutant, Vangl2 Y280A, was only partiallytransport defective (Figure 3N–P),whereas the double mutant Y279A Y280A resulted in a more complete arrest of mutantVangl2 at the TGN (Figure 3—figuresupplement 2). As a control, substituting alanine for both leucinesadjacent to the YXXF motif (green box, Figure3A) had no effect on Vangl2 localization (Figure 3T–V). These results suggest that TGN export of Vangl2depends on the conserved YYXXF motif in the C-terminal, cytosolic domain.10.7554/eLife.00160.007Figure 3.TGN export of Vangl2 depends on the conserved YYXXF sorting motif inthe C-terminal cytosolic domain.


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)

Vangl2 Y279A Y280A is blocked at the TGN.COS7 cells were transfected with HA-Vangl2 wild type(A)–(C) or HA-Vangl2 (Y279A, Y280A)(D)–(F). After transfection for 24hr, cells were analyzed by immunofluorescence using anti-HA andanti-Golgin 97 antibody. Size bar = 10 μm.DOI:http://dx.doi.org/10.7554/eLife.00160.009
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3s2: Vangl2 Y279A Y280A is blocked at the TGN.COS7 cells were transfected with HA-Vangl2 wild type(A)–(C) or HA-Vangl2 (Y279A, Y280A)(D)–(F). After transfection for 24hr, cells were analyzed by immunofluorescence using anti-HA andanti-Golgin 97 antibody. Size bar = 10 μm.DOI:http://dx.doi.org/10.7554/eLife.00160.009
Mentions: The results from the affinity isolation suggest that AP-1 is an effector of Arfrp1,possibly cooperating to mediate TGN export of Vangl2. Consistent with thishypothesis, the C-terminal cytosolic domain of Vangl2 contains a conservedbasolateral-sorting motif (YXXF) which is known to interact with the AP complexes(Bonifacino and Lippincott-Schwartz, 2003)(red box, Figure 3A). Indeed, HA-Vangl2 islocalized basolaterally in MDCK cells (Kallay etal., 2006). To test whether this motif is important for the localization ofVangl2, we generated a series of HA-Vangl2 mutant constructs and examined theirlocalization. Strikingly, four Vangl2 mutants bearing mutations in the YXXF motif,including the single mutation (F283A), showed no detectable surface pattern (Figure 3E,H,K,Q). At high levels of expression,mutant Vangl2 was retained in the ER. However, at lower levels of expression, theseVangl2 mutant proteins accumulated in the juxtanuclear area which colocalized withthe TGN marker, Golgin 97 (Figure3E–M,Q–S). A Vangl2 YXXF double mutant (Y280A, F283A) andVangl2 looptail mutant (D255E) displayed quite distinctive localization to the TGNand ER, respectively (Figure 3—figuresupplement 1). A single tyrosine mutant, Vangl2 Y280A, was only partiallytransport defective (Figure 3N–P),whereas the double mutant Y279A Y280A resulted in a more complete arrest of mutantVangl2 at the TGN (Figure 3—figuresupplement 2). As a control, substituting alanine for both leucinesadjacent to the YXXF motif (green box, Figure3A) had no effect on Vangl2 localization (Figure 3T–V). These results suggest that TGN export of Vangl2depends on the conserved YYXXF motif in the C-terminal, cytosolic domain.10.7554/eLife.00160.007Figure 3.TGN export of Vangl2 depends on the conserved YYXXF sorting motif inthe C-terminal cytosolic domain.

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