SWIP mediates retromer-independent membrane recruitment of the WASH complex

Abstract

The pentameric WASH complex facilitates endosomal protein sorting by activating Arp2/3, which in turn leads to the formation of F-actin patches specifically on the endosomal surface. It is generally accepted that WASH complex attaches to the endosomal membrane via the interaction of its subunit FAM21 with the retromer subunit VPS35. However, we observe the WASH complex and F-actin present on endosomes even in the absence of VPS35. We show that the WASH complex binds to the endosomal surface in both a retromer-dependent and a retromer-independent manner. The retromer-independent membrane anchor is directly mediated by the subunit SWIP. Furthermore, SWIP can interact with a number of phosphoinositide species. Of those, our data suggest that the interaction with phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) is crucial to the endosomal binding of SWIP. Overall, this study reveals a new role of the WASH complex subunit SWIP and highlights the WASH complex as an independent, self-sufficient trafficking regulator.

Structural basis for coupling of the WASH subunit FAM21 with the endosomal SNX27-Retromer complex

“…It is likely our data can be reconciled through the complex multivalent nature of FAM21 association with Retromer, such that although the VPS35 interaction with FAM21 reduces WASH interaction significantly (Figure 2G and 2H), and is required for a proportion of WASH recruitment to endosomal retrieval domains, the other minor sites in VPS29 retain an ability to engage FAM21 and may compensate for its loss. Further, FAM21 can interact with SNX27 and other endosomal proteins such as the Commander complex (25,49), and the population of WASH associated with endosomes in the asbence of Retromer as seen here (Figure 6A) and in other studies (50) provide an added level of potential compensation.…”

“…These studies also identified the 21 LFa repeats in the FAM21 tail and their importance for Retromer association (Jia et al, 2012). The C-terminal tail of FAM21 is sufficient for endosomal localisation (Harbour et al, 2012), and Retromer is important for endosomal recruitment of WASH (Harbour et al, 2010;Helfer et al, 2013;Jia et al, 2012;McGough et al, 2014b;Zavodszky et al, 2014), although WASH can also be recruited through Retromer-independent mechanisms via FAM21 and WASHC4/SWIP subunits respectively (Dostál et al, 2022). WASH depletion was also reported to result in exaggerated endosome-derived tubules (Derivery et al, 2009;Gomez & Billadeau, 2009;Ryder et al, 2013), although this is not universally observed (Buckley et al, 2016;Lee et al, 2016;McGough et al, 2014a;Miller et al, 2018).…”

“…These studies also identified the 21 LFa repeats in the FAM21 tail and their importance for Retromer association (26). The C-terminal tail of FAM21 is sufficient for endosomal localisation (13), and Retromer is important for endosomal recruitment of WASH (12, 26, 27, 35, 36), although WASH can also be recruited through Retromer-independent mechanisms via FAM21 and WASHC4/SWIP subunits respectively (50). WASH depletion was also reported to result in exaggerated endosome-derived tubules (9, 10, 69), although this is not universally observed (43, 46, 70, 71).…”

“…Retromer regulates the recruitment of a population of the WASH complex to endosomal membranes (8, 9), while binding of WASHC4 to membrane phosphoinositide lipids can promote Retromer-independent mechanism of endosomal association (21). Native immunoprecipitations provided the initital evidence for the WASH complex interacting with Retromer, while yeast two-hybrid assays suggested direct associations between VPS35, FAM21 and WASHC1 (8).…”