# Intracellular evaluation of protein droplet-forming capability using self-assembling peptide tags

## TL;DR

This paper introduces a new method to evaluate protein droplet formation inside living cells using self-assembling peptide tags.

## Contribution

A novel intracellular assay using self-assembling peptide tags to assess protein droplet-forming capability in living cells.

## Key findings

- The YK peptide tag system successfully induces protein oligomerization and droplet formation in cells.
- The co-chaperone Hsp70/Hsp90 organizing protein was found to have droplet-forming capability.
- An essential region for droplet formation in the co-chaperone was identified.

## Abstract

Protein droplet formation is a crucial process involved in transient cellular responses and pathogenic protein aggregations. Conventionally, the droplet-forming capability of target proteins has been evaluated through in vitro reconstitution studies, where purified proteins are dissolved in buffer solutions. However, such droplets are highly sensitive to environmental factors, including temperature, ionic strength, and molecular crowding. Therefore, in situ evaluation within living cells is highly desirable. Additionally, since droplet formation is typically initiated by nucleation involving dynamic protein oligomerization, simply expressing proteins in cells often fails to induce droplet formation, making intracellular evaluation challenging. In this study, we present an intracellular droplet-forming assay based on our peptide tag technique. This system employs short self-assembling YK peptide tags (7–15 residues), genetically fused to target proteins, to artificially induce oligomerization. Using this approach, we discover that the co-chaperone Hsp70/Hsp90 organizing protein possesses droplet-forming capability and identify the essential region required for its droplet formation.

Fusion with a self-assembling peptide provides a simple readout for identifying droplet-prone proteins in a living cellular context.