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

Area of Science:

• Cell Biology
• Molecular Biology
• Protein Dynamics

Background:

• Transmembrane cargo is sorted into degradative or recycling pathways after endocytosis.
• Distinct endosomal microdomains, marked by ESCRT (degradative) or retromer/RME-8/SNX-1 (recycling) complexes, facilitate this sorting.
• RME-8 and SNX-1 control microdomain separation, partly by removing HRS/HGRS-1 from recycling domains.

Purpose of the Study:

• To investigate the functional mechanism of RME-8 in endosomal microdomain organization.
• To identify conserved sequence features and structure-function relationships of RME-8.
• To propose a model for RME-8 activation and its role in cargo sorting.

Main Methods:

• Phylogenetic analysis of RME-8.
• Structure-function analysis using in vivo mutation studies.
• Random mutagenesis to identify key functional domains.
• AlphaFold structural predictions integrated with experimental data.

Main Results:

• Identified conserved sequence features in RME-8.
• Determined the C-terminus is crucial for microdomain localization and substrate binding.
• N-terminal sequences are important for endosome recruitment.
• Identified IWN domains as critical for DNAJ domain interaction and HRS uncoating.
• Proposed a model where SNX-1 and IWN domains regulate RME-8 conformation and DNAJ domain exposure.

Conclusions:

• RME-8, regulated by SNX-1 and its IWN domains, controls endosomal microdomain organization.
• RME-8 activation is a cyclical process involving SNX-1 as both an activator and a target.
• This mechanism is vital for efficient endosomal cargo sorting and recycling.