Abstract
Wntless transports Wnt morphogens to the cell surface and is required for Wnt secretion and morphogenic gradients formation. Recycling of endocytosed Wntless requires the sorting nexin-3 (SNX3)-retromer-dependent endosome-to-Golgi transport pathway. Here we demonstrate the essential role of SNX3-retromer assembly for Wntless transport and report that SNX3 associates with an evolutionary conserved endosome-associated membrane re-modelling complex composed of MON2, DOPEY2 and the putative aminophospholipid translocase, ATP9A. In vivo suppression of Ce-mon-2, Ce-pad-1 or Ce-tat-5 (respective MON2, DOPEY2 and ATP9A orthologues) phenocopy a loss of SNX3-retromer function, leading to enhanced lysosomal degradation of Wntless and a Wnt phenotype. Perturbed Wnt signalling is also observed upon overexpression of an ATPase-inhibited TAT-5(E246Q) mutant, suggesting a role for phospholipid flippase activity during SNX3-retromer-mediated Wntless sorting. Together, these findings provide in vitro and in vivo mechanistic details to describe SNX3-retromer-mediated transport during Wnt secretion and the formation of Wnt-morphogenic gradients.
Highlights
SNX12 is a sorting nexin-3 (SNX3) homologue that associates with VPS35 (Fig. 1b); SNX11 is a closely related SNX-PX protein, having the highest degree of sequence similarity with SNX3/SNX12, but lacks the ability to bind to VPS35 (Fig. 1b)
By defining the mechanism of SNX3 binding to the retromer VPS35 subunit, we have established the essential role of this interaction for the in vivo retrieval of Wls and the maintenance of Wnt secretion in C. elegans
We have focused on the question of how the SNX3retromer couples to an evolutionary conserved membrane remodelling complex to elicit the formation of vesicular transport carriers enriched in the cargo protein Wls[14]
Summary
Upon reaching the plasma membrane, the fate of Wls differs from that of the secreted Wnt. Wls is internalised in an adaptor protein-2 (AP2) and clathrin-dependent manner[8,9,10]. Wls is internalised in an adaptor protein-2 (AP2) and clathrin-dependent manner[8,9,10] It is recognised and retrieved from the early endosome back to the Golgi by the sorting nexin-3 (SNX3) containing retromer complex (SNX3-retromer)[9,11,12,13,14,15]. A hetero-dimer of SNX-BAR proteins, SNX1 or SNX2, complexed to either SNX5, SNX6 or SNX32, forms a second protein assembly which is responsible for membrane deformation and tubular carrier formation[21,22,23,24]. We elucidate the central importance of SNX3 binding to retromer for Wnt secretion and, by employing quantitative proteomics combined with in vitro biochemical and in vivo genetic analyses in C. elegans, we reveal that an evolutionary conserved complex containing a putative aminophospholipid translocase (flippase) is required for SNX3-retromermediated trafficking of Wls and Wnt secretion
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