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. 2012 Jun;23(12):2352-61.
doi: 10.1091/mbc.E11-12-1059. Epub 2012 Apr 18.

Multiple repeat elements within the FAM21 tail link the WASH actin regulatory complex to the retromer

Da Jia  1 , Timothy S Gomez, Daniel D Billadeau, Michael K Rosen
Affiliations

Multiple repeat elements within the FAM21 tail link the WASH actin regulatory complex to the retromer

Da Jia et al. Mol Biol Cell. 2012 Jun.

Abstract

Wiskott-Aldrich syndrome protein (WASPs) control actin dynamics in cellular processes, including cell motility, receptor-mediated endocytosis, bacterial invasion, and vesicular trafficking. We demonstrated that WASH, a recently identified WASP family protein, colocalizes on endosomal subdomains with the cargo-selective complex (CSC) of the retromer, where it regulates retrograde sorting from endosomes in an actin-dependent manner. However, the mechanism of WASH recruitment to these retromer-enriched endosomal subdomains is unclear. Here we show that a component of the WASH regulatory complex (SHRC), FAM21, which contains 21 copies of a novel L-F-[D/E](3-10)-L-F motif, directly interacts with the retromer CSC protein VPS35. Endosomal localization of FAM21 is VPS35 dependent and relies on multivalency of FAM21 repeat elements. Using a combination of pull-down assays and isothermal calorimetry, we demonstrate that individual repeats can bind CSC, and binding affinity varies among different FAM21 repeats. A high-affinity repeat can be converted into a low-affinity one by mutation of a hydrophobic residue within the motif. These in vitro data mirror the localization of FAM21 to retromer-coated vesicles in cells. We propose that multivalency enables FAM21 to sense the density of retromer on membranes, allowing coordination of SHRC recruitment, and consequent actin polymerization, with retromer sorting domain organization/maturation.

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Figures

FIGURE 1:
FIGURE 1:
FAM21 directly interacts with retromer CSC. (A) Schematic representation of FAM21 constructs used. FAM21 contains 21 copies of the L-F-[D/E]3-10-L-F motif (LFa motif; black boxes). FN, FM, and FC contain repeats 1–6, 7–14, and 15–21, respectively. (B) GST-FAM21 pull-down of purified retromer CSC. Shown is a Coomassie blue–stained SDS–PAGE gel of input and bound samples for FN, FC, FM, and GST control. (C) GST-FM pull-down of retromer CSC with addition of increasing amounts of untagged FC protein. All input samples contained 1 nmol of GST-FM and 0.8 nmol of retromer CSC in the absence (lane 1) or presence of FC protein (lane 2, 1.5 nmol; lane 3, 10 nmol; lane 4, 20 nmol). Top, input samples separated by SDS–PAGE and stained with Coomassie blue. Bottom, diluted bound samples blotted against GST (loading control) and VPS29, respectively. (D) HeLa cells were transfected with shFAM21/HA-YFP-FAM21 rescue vectors expressing HA-YFP-FAM21 fusion proteins (amino acids 1–356 or R1-21), which were analyzed for association with VPS35 and WASH via immunoprecipitation.
FIGURE 2:
FIGURE 2:
Interaction between FAM21 and CSC requires the FAM21 motif and VPS35. (A) Schematic representation of FAM21 constructs used. (B) GST pull-down of retromer CSC using GST-FC and its derived fragments. Shown is a Coomassie blue–stained SDS–PAGE gel of input and bound samples for one FC subfragment that does not contain any repeats (noR), one containing six repeats (R17-20), one containing four repeats (R15-20), FC, and GST control. (C) GST-FC pull-down of retromer CSC or individual VPS35, VPS26, and VPS29 proteins. Shown is Coomassie blue–stained gel of input and bound samples from GST pull-down assays and purified retromer proteins. Uncomplexed VPS26 has some degradation products. VPS29 is an MBP fusion. Fusion tags of VPS35, VPS26, and VPS29 were proteolytically removed in retromer CSC. Asterisk, GST-FC.
FIGURE 3;
FIGURE 3;
Different FAM21 repeats show different efficiencies of CSC binding. (A) Schematic representation of FAM21 constructs used. (B) GST pull-down of retromer CSC using FC and its derived fragments. Shown is Coomassie blue–stained gel of input and bound samples from GST pull-down assays. Each input sample, except for GST control, contained 700 pmol of total LFa motifs and 800 pmol of retromer CSC.
FIGURE 4:
FIGURE 4:
Number of the FAM21 repeat dictates its endosomal localization. (A) Schematic representation of YFP-fused FAM21 truncation mutants and degree of early endosomal (EE) localization. (B) HeLa cells were transfected with control vector (YFP only) or various shFAM21/YFP-FAM21 dual suppression/rescue vectors. YFP-FAM21 fusion proteins (green) were analyzed for localization with EEA1 (red) via immunofluorescence.
FIGURE 5:
FIGURE 5:
Quality of FAM21 repeat also dictates endosomal localization. (A) Schematic representation of YFP-fused FAM21 truncation mutants and degree of early endosomal (EE) localization. (B) HeLa cells were transfected with control vector (YFP only) or various shFAM21/YFP-FAM21 dual suppression/rescue vectors and were analyzed as in Figure 4. (FC* contains the CapZ-binding motif.)
FIGURE 6:
FIGURE 6:
All three elements of the FAM21 motif contribute to binding the CSC. (A) Schematic representation of FAM21 constructs used and degree of retromer binding determined by GST pull-down. (B) GST pull-down of retromer CSC using FAM21 R21 and its mutants 1–5. FAM21 R20-21 is used as a positive control and GST as a negative control. Shown is Coomassie blue–stained gel of input and bound samples from GST pull-down assays. (C) GST pull-down of retromer CSC using FAM21 R21 and mutant 6. Shown is Coomassie blue–stained gel of input and bound samples from GST pull-down assays.
FIGURE 7:
FIGURE 7:
Model of SHRC interaction with retromer on membranes. (A) We propose that the CSC of the retromer is recruited to the endosomal membrane through mechanisms involving GTP-bound Rab 7 and cargo proteins that are destined to enter the retromer-mediated sorting pathway. When retromer accumulates to high density on the endomembrane, the SHRC is then recruited through cumulative binding of higher-affinity (*) and lower-affinity FAM21 repeats with VPS35 proteins, thus facilitating the cargo enrichment process and promoting subsequent F-actin–mediated maturation of the retromer subdomain, vesicle scission, and trafficking. (B) Bird's-eye view of the process discussed in A after initial recruitment of the SHRC via FAM21-mediated interactions with VPS35.
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