SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

Abstract

Coat complexes coordinate cargo recognition through cargo adaptors with biogenesis of transport carriers during integral membrane protein trafficking. Here, we combine biochemical, structural, and cellular analyses to establish the mechanistic basis through which SNX27–Retromer, a major endosomal cargo adaptor, couples to the membrane remodeling endosomal SNX-BAR sorting complex for promoting exit 1 (ESCPE-1). In showing that the SNX27 FERM (4.1/ezrin/radixin/moesin) domain directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the SNX1/SNX2 subunits of ESCPE-1, we propose a handover model where SNX27–Retromer captured cargo proteins are transferred into ESCPE-1 transport carriers to promote endosome-to-plasma membrane recycling. By revealing that assembly of the SNX27:Retromer:ESCPE-1 coat evolved in a stepwise manner during early metazoan evolution, likely reflecting the increasing complexity of endosome-to-plasma membrane recycling from the ancestral opisthokont to modern animals, we provide further evidence of the functional diversification of yeast pentameric Retromer in the recycling of hundreds of integral membrane proteins in metazoans.

Native holdup (nHU) to measure binding affinities from cell extracts

“…We also detected the association of SNX27 and the heterodimeric SNX1/2-SNX5/6 SNX-BAR complex with the strongest affinity measured with SNX1. A short fragment of SNX1/2 was reported to interact with the FERM domain of SNX27 with comparable affinity to the one obtained from nHU 30 . We identified the actin-regulatory Wiskott Aldrich Syndrome protein and scar homologue (WASH) complex as the partner of SNX27 with the strongest affinities measured with WASH2 and FAM21 31 .…”

An evolving understanding of sorting signals for endosomal retrieval

“…A key advance in the field is the recent observation that SNX-BARs directly interact with SNX27 (Yong et al, 2021c;Chandra et al, 2021;Simonetti et al, 2022). The interaction is mediated by the Asp-Leu-Phe (DLF) motifs located within the N-termini of SNX1/SNX2 and the FERM domain of SNX27 (Yong et al, 2021c;Chandra et al, 2021;Simonetti et al, 2022). Both SNX1 and SNX2 harbor more than one DLF motif, which could be used to promote and stabilize the formation of vesicular/tubular structures.…”

“…In the first model (concurrent model), we suggest that the formation of the SNX27-SNX-BAR-retromer supercomplex aids its membrane recruitment to different types of cargoes, which contain an SBM, PDZbm, or both (Yong et al, 2021c) (Figure 2A). In the second model (sequential model), Cullen & colleagues proposed that cargoes possessing a PDZbm are first recognized by SNX27-retromer and then ''handed over'' to SNX-BAR-decorated tubulovesicular carrier due to the SNX-BAR-SNX27 interaction (Simonetti et al, 2022) (Figure 2B). Tubule maturation and fission may be further aided by actin polymerization and recruitment of motor proteins (Figures 2A and 2B).…”

An interaction between β’-COP and the ArfGAP, Glo3, maintains post-Golgi cargo recycling

“…We note AlphaFold successfully predicts interactions between folded domains and short peptides in other trafficking proteins exhibiting similar affinities. One example is the SNX27 FERM interaction with DxF motifs 46,47 ; AlphaFold models and the experimentally determined X-ray structure are very similar 48 . Overall, the systematic mutagenesis approach combined with biophysical experiments provided the most compelling evidence for identifying where Glo3 binds b'-COP on the C-terminal propeller (Figure 3D).…”