Mutagenesis and structural modeling implicate RME-8 IWN domains as conformational control points.

After endocytosis, transmembrane cargo is differentially sorted into degradative or recycling pathways. This process is facilitated by recruitment into physically distinct degradative or recycling microdomains on the limiting membrane of individual endosomes. Endosomal sorting complexes required for transport (ESCRT) mark the degradative microdomain, while the recycling domain is marked by the retromer complex and associated proteins RME-8 and SNX-1. The separation of endosomal microdomains is also controlled by RME-8 and SNX-1, at least in part via removal of degradative component HRS/HGRS-1 from the recycling microdomain. This activity is likely due to recruitment and activation of chaperone Hsc70 on the endosome by the RME-8 DNAJ domain. To better understand the mechanism of RME-8 function we performed a new phylogenetic analysis of RME-8 and identified new conserved sequence features. In a complementary approach, we performed structure-function analysis that identified the C-terminus as important for microdomain localization and likely substrate binding, while N-terminal sequences beyond the known single N-terminal PH-like domain are important for endosome recruitment. Random mutagenesis identified IWN4, and by analogy IWN3, to be important for the autoinhibitory DNAJ domain binding, with IWN3 playing a critical role in HRS uncoating activity. Combining AlphaFold structural predictions with in vivo mutation analysis of RME-8, we propose a model whereby SNX-1 and the IWN domains control the conformation of RME-8 and hence the productive exposure of the DNAJ domain. Furthermore, we propose that the activation of RME-8 is cyclical, with SNX-1 acting as an activator and a target of RME-8 uncoating activity.