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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

Juxtanuclear accumulated Vangl2 in Arfrp1 knockdown cells colocalizeswith Golgin 97 more than with GM130.(A)–(I) HeLa cells weretransfected with siRNA against Arfrp1 and re-transfected after 48 hr withplasmid encoding HA-Vangl2. After an additional 24 hr, cells wereimmunofluorescently labeled to evaluate coincident localization withGolgin 97 and GM130 (A–C), HA-Vangl2 andGM130 (D–F) and HA-Vangl2 and Golgin 97(G–I). Size bar = 10 μm.(J) Colocalization was quantified by analyzing theaverage value of the fraction of each marker's area that coincidedwith the other marker (mean ± SD; >15 cells each).DOI:http://dx.doi.org/10.7554/eLife.00160.005
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fig1s2: Juxtanuclear accumulated Vangl2 in Arfrp1 knockdown cells colocalizeswith Golgin 97 more than with GM130.(A)–(I) HeLa cells weretransfected with siRNA against Arfrp1 and re-transfected after 48 hr withplasmid encoding HA-Vangl2. After an additional 24 hr, cells wereimmunofluorescently labeled to evaluate coincident localization withGolgin 97 and GM130 (A–C), HA-Vangl2 andGM130 (D–F) and HA-Vangl2 and Golgin 97(G–I). Size bar = 10 μm.(J) Colocalization was quantified by analyzing theaverage value of the fraction of each marker's area that coincidedwith the other marker (mean ± SD; >15 cells each).DOI:http://dx.doi.org/10.7554/eLife.00160.005

Mentions: To identify the Arf proteins that regulate TGN export of Vangl2, we performed ansiRNA knockdown screen focusing on selected Golgi-localized Arf proteins in HeLacells stably expressing HA-Vangl2. The screen indicated that knockdown of Arf1 orArfrp1 caused a juxtanuclear accumulation of Vangl2 whereas knockdown of otherGolgi-localized Arfs did not affect Vangl2 trafficking. Arf1, which shares a 34%sequence identity with Arfrp1, plays a general role in regulating membranerecruitment of various vesicle coat proteins and lipid modification enzymes (Donaldson and Jackson, 2011). Arfrp1 is morespecifically localized at the TGN and has been shown to regulate TGN-to-plasmamembrane transport of E-cadherin and VSV-G (Shin etal., 2005; Zahn et al., 2008;Nishimoto-Morita et al., 2009). However,what Arfrp1 does to promote traffic has not been explored. We thus focused on Arfrp1and it’s role in the transport of PCP signaling proteins. The expression ofArfrp1 was efficiently reduced after siRNA treatment (Figure 1G) and knockdown of Arfrp1 caused a juxtanuclear accumulation ofVangl2 in a majority (65%) of the cells compared to mock treated cells (Figure 1A,D,H). Transport-arrested Vangl2colocalized with the TGN marker, Golgin 97 (Figure1A–F) but not the early endosomal marker EEA1, the late endosomalmarker Rab7 or the recycling endosomal marker Rab11 (Figure 1—figure supplement 1). Quantification ofcolocalization indicated that Vangl2 correlated more closely with the TGN marker,Golgin 97, than with the cis-Golgi marker, GM130 (Figure 1—figure supplement 2). Theseresults suggest that Arfrp1 regulates the export of Vangl2 from the TGN.10.7554/eLife.00160.003Figure 1.Knockdown of Arfrp1 leads to accumulation of Vangl2 at theTGN.


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)

Juxtanuclear accumulated Vangl2 in Arfrp1 knockdown cells colocalizeswith Golgin 97 more than with GM130.(A)–(I) HeLa cells weretransfected with siRNA against Arfrp1 and re-transfected after 48 hr withplasmid encoding HA-Vangl2. After an additional 24 hr, cells wereimmunofluorescently labeled to evaluate coincident localization withGolgin 97 and GM130 (A–C), HA-Vangl2 andGM130 (D–F) and HA-Vangl2 and Golgin 97(G–I). Size bar = 10 μm.(J) Colocalization was quantified by analyzing theaverage value of the fraction of each marker's area that coincidedwith the other marker (mean ± SD; >15 cells each).DOI:http://dx.doi.org/10.7554/eLife.00160.005
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1s2: Juxtanuclear accumulated Vangl2 in Arfrp1 knockdown cells colocalizeswith Golgin 97 more than with GM130.(A)–(I) HeLa cells weretransfected with siRNA against Arfrp1 and re-transfected after 48 hr withplasmid encoding HA-Vangl2. After an additional 24 hr, cells wereimmunofluorescently labeled to evaluate coincident localization withGolgin 97 and GM130 (A–C), HA-Vangl2 andGM130 (D–F) and HA-Vangl2 and Golgin 97(G–I). Size bar = 10 μm.(J) Colocalization was quantified by analyzing theaverage value of the fraction of each marker's area that coincidedwith the other marker (mean ± SD; >15 cells each).DOI:http://dx.doi.org/10.7554/eLife.00160.005
Mentions: To identify the Arf proteins that regulate TGN export of Vangl2, we performed ansiRNA knockdown screen focusing on selected Golgi-localized Arf proteins in HeLacells stably expressing HA-Vangl2. The screen indicated that knockdown of Arf1 orArfrp1 caused a juxtanuclear accumulation of Vangl2 whereas knockdown of otherGolgi-localized Arfs did not affect Vangl2 trafficking. Arf1, which shares a 34%sequence identity with Arfrp1, plays a general role in regulating membranerecruitment of various vesicle coat proteins and lipid modification enzymes (Donaldson and Jackson, 2011). Arfrp1 is morespecifically localized at the TGN and has been shown to regulate TGN-to-plasmamembrane transport of E-cadherin and VSV-G (Shin etal., 2005; Zahn et al., 2008;Nishimoto-Morita et al., 2009). However,what Arfrp1 does to promote traffic has not been explored. We thus focused on Arfrp1and it’s role in the transport of PCP signaling proteins. The expression ofArfrp1 was efficiently reduced after siRNA treatment (Figure 1G) and knockdown of Arfrp1 caused a juxtanuclear accumulation ofVangl2 in a majority (65%) of the cells compared to mock treated cells (Figure 1A,D,H). Transport-arrested Vangl2colocalized with the TGN marker, Golgin 97 (Figure1A–F) but not the early endosomal marker EEA1, the late endosomalmarker Rab7 or the recycling endosomal marker Rab11 (Figure 1—figure supplement 1). Quantification ofcolocalization indicated that Vangl2 correlated more closely with the TGN marker,Golgin 97, than with the cis-Golgi marker, GM130 (Figure 1—figure supplement 2). Theseresults suggest that Arfrp1 regulates the export of Vangl2 from the TGN.10.7554/eLife.00160.003Figure 1.Knockdown of Arfrp1 leads to accumulation of Vangl2 at theTGN.

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