Measuring interactions of FERM domain-containing sorting Nexin proteins with endosomal lipids and cargo molecules

Endosomal recycling pathways regulate cellular homeostasis via the transport of internalized material back to the plasma membrane. Phox homology (PX) and band 4.1/ezrin/radixin/moesin (FERM) domain-containing proteins are a recently identified subfamily of PX proteins that are critical for the recycling of numerous transmembrane cargo molecules. The PX-FERM subfamily includes three endosome-associated proteins called sorting nexin (SNX) 17, SNX27, and SNX31. These are modular peripheral membrane proteins that act as central scaffolds mediating protein-lipid interactions, cargo binding, and regulatory protein recruitment. This chapter outlines the methodology employed to classify the PX-FERM family using combined bioinformatics and structure prediction tools. It further details the application of isothermal titration calorimetry and nuclear magnetic resonance spectroscopy to understand the mechanisms that underpin their endosomal membrane recruitment and subsequent recognition of NPxY/NxxY peptide sorting motifs, present in many cargo receptors and required for their trafficking. It is now increasingly recognized that the formation of a stable trafficking complex is dictated by a multitude of coordinated protein-protein and protein-lipid interactions, and the approaches highlighted here will be useful for future studies aimed at understanding these biomolecular interactions in greater detail.