Phase Separation of Disordered Protein in the Formation of Membrane-Less Organelles

Abstract

Intrinsically disordered proteins and regions (IDPs/IDRs), which do not have stable secondary and tertiary structure, are capable of adopting different structural states. Many IDPs/IDRs populate conformationally heterogeneous monomeric states or engage in discrete interactions with other proteins, leading to folding upon binding or retaining significant disorder in the bound state. Others are involved in large-scale association having different degrees of order, from more defined fibers, to variably networked gels and to disordered liquid demixed states or droplets. These latter have been suggested to provide the physical basis for cellular membrane-less organelles such as the nucleolus.We have studied the N-terminal disordered region of Ddx4, an RNA DEAD-box helicase that is essential for formation of a class of membrane-less organelles termed nuage or germ granules functioning in spermatogenesis. When expressed in HeLa cells, the protein forms spherical, micron-sized, liquid-like cellular organelles. In vitro, it phase separates to form droplets with similar morphological and dynamic properties to the organelles observed in cells. Phase separation is sensitive to salt, pointing to the importance of electrostatic interactions. The sequence features of the disordered N-terminus of Ddx4 that underlie phase separation include clustering of charged residues into blocks of net positive and negative charge, with over-representation of FG/GF pairs and RG/GR pairs within the positive blocks. Perturbations of these properties disrupt phase separation, pointing to multi-valent cation-pi interactions playing an important role. The transient sampling of multi-valent interactions in self-association of Ddx4 extends previous observations of dynamic multi-valent interactions in discrete complexes of IDPs/IDRs, such as for Sic1:Cdc4 binding. The insights obtained from these and ongoing biophysical studies of Ddx4 will be valuable for developing a general understanding of the biogenesis and disassembly of membrane-less cellular organelles.