WASH and WAVE actin regulators of the Wiskott-Aldrich syndrome protein (WASP) family are controlled by analogous structurally related complexes

Abstract

We recently showed that the Wiskott-Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.

Fig. 1.

WASH functions within a multiprotein complex. (A) Silver stained 10% SDS-PAGE of the bovine WASH complex eluting from the final gel filtration column during its purification. (B) Jurkat T cell lysates were immunoprecipitated with control rabbit IgG (NRS) or the indicated WASH complex antibodies and immunoblotted as indicated. (C) HeLa cells were suppressed, lysed and immunoblotted as indicated.

Fig. 2.

Recombinant WASH complex is inhibited. (A) Silver stained 4–20% SDS-PAGE gel of the purified recombinant human WASH complex. All components are full length except FAM21 (residues 1–356 only). (B) Pyrene-actin assembly assays containing 10 nM Arp2/3 complex and 4 μM actin (5% pyrene labeled) in KMEI-15G buffer (control, black) plus 100 nM WASH-VCA (red), recombinant SHRC (green), or denatured recombinant SHRC (by prior heating at 60° for 10 min, blue).

Fig. 3.

SHRC organization is analogous to that of the WAVE complex. HeLa cells were transfected with various shWASH/HA-YFP-WASH (A) or shCCDC53/HA-YFP-CCDC53 (B) reconstitution vectors, and mutant HA-YFP-fusion proteins were analyzed for integration into the WASH complex via immunoprecipitation. Lysates were immunoblotted to verify suppression.

Fig. 4.

FAM21 binds to CAPZ and inhibits its capping activity. (A) HeLa cells were transfected with various shFAM21/HA-YFP-FAM21 reconstitution vectors, and mutant HA-YFP-fusion proteins were analyzed for association with WASH and CAPZα via immunoprecipitation. (B) Inhibition of CAPZ capping activity by FAM21, monitored by pyrene-actin polymerization assay using VopL. FAM21 C1 fragment (residues 938–1067), C2 (residues 1010–1067), and C2* (residues 938–1067 with R1031D).

Fig. 5.

The WRC and SHRC are structurally similar. Two-dimensional class averages of the recombinant SHRC (A) and miniWRC (B). The three highest ranked (most similar) classes out of 10 of each complex are shown. Number of particles in each class is listed. Cartoon models of SHRC (C) and WRC (D) assemblies.