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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

Subunits of AP-1 preferentially interact with the GTP-boundArfrp1.(A) Bovine brain cytosol was incubated with purified GDP-loadeddominant negative form (T31N) or GTPγS-loaded dominant active form(Q79L) of GST-Arfrp1. After incubation, the eluted fraction was resolved bySDS-PAGE and silver stained. Protein identification in the indicated gelslice performed by mass spectrometry revealed γ1-adaptin andserine/threonine-protein kinase (A-Raf) respectively.(B),(C). Bovine brain cytosol was incubatedwith purified GDP-loaded GST-Arfrp1 (wt) or GTPγS-loaded GST-Arfrp1(Q79L). After incubation, the entire sample of bound γ1-adaptin,μ1-adaptin and other indicated proteins was analyzed byimmunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.006
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fig2: Subunits of AP-1 preferentially interact with the GTP-boundArfrp1.(A) Bovine brain cytosol was incubated with purified GDP-loadeddominant negative form (T31N) or GTPγS-loaded dominant active form(Q79L) of GST-Arfrp1. After incubation, the eluted fraction was resolved bySDS-PAGE and silver stained. Protein identification in the indicated gelslice performed by mass spectrometry revealed γ1-adaptin andserine/threonine-protein kinase (A-Raf) respectively.(B),(C). Bovine brain cytosol was incubatedwith purified GDP-loaded GST-Arfrp1 (wt) or GTPγS-loaded GST-Arfrp1(Q79L). After incubation, the entire sample of bound γ1-adaptin,μ1-adaptin and other indicated proteins was analyzed byimmunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.006

Mentions: To elucidate the roles of Arfrp1 in TGN export of Vangl2, we sought to identify theeffectors of Arfrp1 using affinity chromatography. A similar approach documented thespecific interaction between the BBsome, which functions as a coat complex that sortsmembrane proteins to primary cilia, and Arl6 (Jinet al., 2010). Bovine brain cytosol was incubated with purified GST-taggedArfrp1 dominant negative (T31N) and dominant active (Q79L) mutant pre-loaded with GDPor GTPγS, respectively. After incubation, bound proteins were eluted andanalyzed by SDS-PAGE and silver staining. A series of protein bands were recovered inthe eluate of GTPγS-loaded GST-Arfrp1 (Q79L) immobilized on glutathione beads(Figure 2A). One of the bands wasidentified by mass spectrometry as the γ subunit of the adaptor complex 1(AP-1) (Figure 2A). Immunoblot analysisconfirmed that both γ1-adaptin and μ1-adaptin preferentially interactedwith the GTPγS-loaded Arfrp1 (Q79L), whereas EEA1, CRMP2 and dynamin II showedno binding or no GTP-dependent binding (Figure2B,C). Moreover, the δ subunit of AP-3 and the α subunit ofAP-2 showed no detectable binding (Figure 2C),suggesting the interactions between Arfrp1 and subunits of AP-1 are specific.10.7554/eLife.00160.006Figure 2.Subunits of AP-1 preferentially interact with the GTP-boundArfrp1.


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)

Subunits of AP-1 preferentially interact with the GTP-boundArfrp1.(A) Bovine brain cytosol was incubated with purified GDP-loadeddominant negative form (T31N) or GTPγS-loaded dominant active form(Q79L) of GST-Arfrp1. After incubation, the eluted fraction was resolved bySDS-PAGE and silver stained. Protein identification in the indicated gelslice performed by mass spectrometry revealed γ1-adaptin andserine/threonine-protein kinase (A-Raf) respectively.(B),(C). Bovine brain cytosol was incubatedwith purified GDP-loaded GST-Arfrp1 (wt) or GTPγS-loaded GST-Arfrp1(Q79L). After incubation, the entire sample of bound γ1-adaptin,μ1-adaptin and other indicated proteins was analyzed byimmunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.006
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Subunits of AP-1 preferentially interact with the GTP-boundArfrp1.(A) Bovine brain cytosol was incubated with purified GDP-loadeddominant negative form (T31N) or GTPγS-loaded dominant active form(Q79L) of GST-Arfrp1. After incubation, the eluted fraction was resolved bySDS-PAGE and silver stained. Protein identification in the indicated gelslice performed by mass spectrometry revealed γ1-adaptin andserine/threonine-protein kinase (A-Raf) respectively.(B),(C). Bovine brain cytosol was incubatedwith purified GDP-loaded GST-Arfrp1 (wt) or GTPγS-loaded GST-Arfrp1(Q79L). After incubation, the entire sample of bound γ1-adaptin,μ1-adaptin and other indicated proteins was analyzed byimmunoblot.DOI:http://dx.doi.org/10.7554/eLife.00160.006
Mentions: To elucidate the roles of Arfrp1 in TGN export of Vangl2, we sought to identify theeffectors of Arfrp1 using affinity chromatography. A similar approach documented thespecific interaction between the BBsome, which functions as a coat complex that sortsmembrane proteins to primary cilia, and Arl6 (Jinet al., 2010). Bovine brain cytosol was incubated with purified GST-taggedArfrp1 dominant negative (T31N) and dominant active (Q79L) mutant pre-loaded with GDPor GTPγS, respectively. After incubation, bound proteins were eluted andanalyzed by SDS-PAGE and silver staining. A series of protein bands were recovered inthe eluate of GTPγS-loaded GST-Arfrp1 (Q79L) immobilized on glutathione beads(Figure 2A). One of the bands wasidentified by mass spectrometry as the γ subunit of the adaptor complex 1(AP-1) (Figure 2A). Immunoblot analysisconfirmed that both γ1-adaptin and μ1-adaptin preferentially interactedwith the GTPγS-loaded Arfrp1 (Q79L), whereas EEA1, CRMP2 and dynamin II showedno binding or no GTP-dependent binding (Figure2B,C). Moreover, the δ subunit of AP-3 and the α subunit ofAP-2 showed no detectable binding (Figure 2C),suggesting the interactions between Arfrp1 and subunits of AP-1 are specific.10.7554/eLife.00160.006Figure 2.Subunits of AP-1 preferentially interact with the GTP-boundArfrp1.

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