A mechanism for differential sorting of the planar cell polarity proteins Frizzled6 and Vangl2 at the trans-Golgi network

Abstract

In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical-basal axis, a process mediated by several conserved signaling receptors. Two PCP-signaling proteins, VANGL planar cell polarity protein 2 (Vangl2) and Frizzled6 (Fzd6), are located asymmetrically on opposite boundaries of the cell. Examining sorting of these two proteins at the -Golgi network (TGN), we demonstrated previously that the GTP-binding protein ADP-ribosylation factor-related protein 1 (Arfrp1) and the clathrin-associated adaptor protein complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled6 does not depend on Arfrp1 or AP-1. Here, to further investigate the TGN sorting process in mammalian cells, we reconstituted release of Vangl2 and Frizzled6 from the TGN into vesicles Immunoblotting of released vesicles indicated that Vangl2 and Frizzled6 exit the TGN in separate compartments. Knockdown analysis revealed that a clathrin adaptor, epsinR, regulates TGN export of Frizzled6 but not of Vangl2. Protein interaction analysis suggested that epsinR forms a stable complex with clathrin and that this complex interacts with a conserved polybasic motif in the Frizzled6 cytosolic domain to package Frizzled6 into transport vesicles. Moreover, we found that Frizzled6-epsinR binding dissociates epsinR from AP-1, which may separate these two cargo adaptors from each other to perform distinct cargo-sorting functions. Our results suggest that Vangl2 and Frizzled6 are packaged into separate vesicles that are regulated by different clathrin adaptors at the TGN, which may contribute to their asymmetric localizations.

Anin vitrovesicle formation assay to analyze protein sorting in the secretory transport pathway

“…When we performed the vesicle budding reaction in the absence of GTP and an ATPrS or in the presence of GMPPNP, the number of vesicles was greatly reduced ( Figure 1B, 1D). These analyses are consistent with a previous report (Ma et al, 2018) and are consistent with the slowly-sedimenting membranes in the budding reaction representing transport vesicles rather than fragments of the ER or Golgi.…”

“…An in vitro vesicle formation assay to reconstitute packaging of cargo proteins into vesicles from mammalian cells has been well-established (Kim et al, 2007;Ma et al, 2018;Merte et al, 2010;Niu et al, 2019). We sought to perform this assay in HEK293T cells on a large scale and then perform proteomic analysis on the isolated vesicles.…”

“…We have previously characterized reconstituted release of EGFR and Frizzled6 into transport vesicles (Ma et al, 2018;Mao et al, 2019). We thus performed this assay at a large scale using HEK293T cells transfected with plasmids encoding FLAG-tagged Frizzled6 or EGFR (the Frizzled6-FLAG group or the EGFR-FLAG group) ( Figure 6A).…”

“…The ENTH domain directly binds various SNAREs, including the Habc domain of Vti1B (Chidambaram et al, 2004;Chidambaram et al, 2008;Hirst et al, 2004;Miller et al, 2007;Wang et al, 2011). The C-terminal unfolded domain of epsinR interacts with the polybasic motif on Frizzled6 to package Frizzled6 into transport vesicles (Ma et al, 2018). We immunoisolated FLAG-tagged C-terminal unfolded domain of epsinR (epsinR ENTH -FLAG) and full length epsinR (epsinR FL -FLAG) from HEK293T cells.…”

“…We found that purified His-Vti1B Habc enhanced the efficiency of packaging of Frizzled6 into vesicles ( Figure 7D, compare lanes 2 and 3). Increase the concentration of His-Vti1B Habc in the vesicle formation assay increased the efficiency of vesicular release of Frizzled6 ( Figure 7E Next, we analyzed whether Vti1B is important for cell surface delivery of Frizzled6 in HeLa cells through a temperature shift approach (Ma et al, 2018). In this approach, we incubated HeLa cells at 20 °C in the presence of cycloheximide to accumulate newly-synthesized HA-Frizzled6 in the TGN.…”