Synapsin Condensation is Governed by Sequence-Encoded Molecular Grammars
Christian Pieter Hoffmann, Kiersten M. Ruff, Irina A. Edu, Min Kyung Shinn, Johannes Vincent Tromm, Matthew R. King, Avnika Pant, Hannes Ausserwӧger, Jennifer R. Morgan, Tuomas P. J. Knowles, Rohit V. Pappu, Dragomir Milovanović
Abstract
• Distinct biomolecular condensates formed by pre-synaptic proteins contain a unique bias of sequence grammars within their IDRs. • The IDR of synapsin-1, the essential protein for the clustering of synaptic vesicles, has a conserved compositional bias for Arg and blockiness of proline/polar residues. • Using sequence designs, we uncovered how conserved sequence features of the driver IDR of synapsin-1 affects condensation in vitro and in cells. • Synapsin-1 condensates are defined by a measurable interphase pH gradient. Multiple biomolecular condensates coexist at the pre- and post- synapse to enable vesicle dynamics and controlled neurotransmitter release in the brain. In pre-synapses, intrinsically disordered regions (IDRs) of synaptic proteins are drivers of condensation that enable clustering of synaptic vesicles (SVs). Using computational analysis, we show that the IDRs of SV proteins feature evolutionarily conserved non-random compositional biases and sequence patterns. Synapsin-1 is essential for condensation of SVs, and its C-terminal IDR has been shown to be a key driver of condensation. Focusing on this IDR, we dissected the contributions of two conserved features namely the segregation of polar and proline residues along the linear sequence, and the compositional preference for arginine over lysine. Scrambling the blocks of polar and proline residues weakens the driving forces for forming micron-scale condensates. However, the extent of clustering in subsaturated solutions remains equivalent to that of the wild-type synapsin-1. In contrast, substituting arginine with lysine significantly weakens both the driving forces for condensation and the extent of clustering in subsaturated solutions. Co-expression of the scrambled variant of synapsin-1 with synaptophysin results in a gain-of-function phenotype in cells, whereas arginine to lysine substitutions eliminate condensation in cells. We report an emergent consequence of synapsin-1 condensation, which is the generation of interphase pH gradients that is realized via differential partitioning of protons between coexisting phases. This pH gradient is likely to be directly relevant for vesicular ATPase functions and the loading of neurotransmitters. Our studies highlight how conserved IDR grammars serve as drivers of synapsin-1 condensation.