Biomolecular condensation of hairpin proteins coordinates cytoplasmic clients to spatially distinct microdomains of the ER membrane (360G-Wellcome-218651_Z_19_Z)

The endoplasmic reticulum (ER), as a single continuous membrane network, coordinates a variety of biological processes across the entire cell, providing a platform for the spatiotemporal segregation of cytoplasmic biochemistry – a crucial feature for cell survival. Despite this, our understanding of how the ER corrals most cytoplasmic clients to specific sites on its membrane remains slim. ER structure is partially governed by a series of hairpin proteins known to stabilise specific membrane curvatures. The Reticulons (RTNs) and Receptor Expression-Enhancing Proteins (REEPs) are two such families of protein. We have shown that RTN/REEPs cluster into ER microdomains and contain cytosolic-facing intrinsically disordered regions which enable the formation of biomolecular condensates, potentially tethering specific cytosolic proteins to the ER membrane. Thus, we propose the novel and testable hypothesis that membrane curvature-stabilising RTN/REEPs drive regional functional specificities at ER microdomains. To test this, we will generate a comprehensive spatial map of RTN/REEP-microdomains using single-molecule localisation microscopy, with a custom optical-tweezer configuration used to quantify precise membrane curvature preferences for each hairpin. Using two orthogonal screens we aim to identify a complement of cytosolic proteins capable of co-condensing with each RTN/REEP, performing appropriate bioassays to assess the functional consequences of destabilising hairpin:cytoplasmic-partner interactions.