Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

Vikas A Tillu,James Rae,Kerrie-Ann Mcmahon,Nicholas Ariotti,Robert G Parton,Ya Gao,Oleksiy Kovtun,Matthias Floetenmeyer,Natasha Chaudhary,Brett M Collins

doi:10.1038/s41467-021-21035-4

Abstract

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

Highlights

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins
We find that Cavin1 undergoes electrostatically driven self-association via its disordered regions that promotes liquid–liquid phase separation (LLPS) in vitro, that it can co-phase separate with CAV1, and this is dependent on specific sequence properties of the two proteins
Despite the fact that intrinsically disordered sequences are a prominent and highly conserved feature of all cavins, no previous studies have explicitly addressed their functional importance. We show that they are essential for caveola formation. They regulate the ability of Cavin1 to self-associate and undergo LLPS in vitro, where Cavin1 shows the classical properties of LLPS as demonstrated by phase separation that is sensitive to protein concentration, ionic strength, molecular crowding agents, and by the rapid exchange of protein in Cavin1 droplets as shown by Fluorescence recovery after photobleaching (FRAP)

Summary

Introduction

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin in vitro, assembly of Cavin oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin recruitment to caveolae in cells. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat. All cavin proteins share a highly characteristic domain architecture consisting of two core α-helical regions (HR1 and HR2) with relatively high sequence conservation11,12 These are connected by three intrinsically disordered regions (DR1, DR2, and DR3), that possess very little sequence homology but share the property of being enriched in negatively charged residues (Fig. 1a). The C-terminal α-helical HR2 region of Cavin is unique in the cavin family as it contains a stretch of repeated undecad sequences (11-mers) predicted to form a second coiled-coil structure termed UC1 (undecad of Cavin1)

Methods

Results

Conclusion