Meet our scientists!
BackTopics Page
Old and defective proteins need to be degraded by cells. Proteins destined for degradation are first tagged with ubiquitin, a molecule that can be covalently attached to proteins. Specific types of ubiquitination direct proteins for degradation by proteasomes. Sayaka Yasuda, a senior scientist in the Protein Metabolism Project, studies proteasomes. She found that when cells are subjected to certain stresses, proteasomes congeal into liquid droplet structures in cells. Her work shows that stresses can increase amounts of denatured and defective proteins in cells. In these situations, proteasomes congeal with ubiquitinated substrates into liquid droplet foci, which likely increase the efficiency of protein degradation. Currently, she is working in Singapore, but hopefully she will return to Igakuken soon. She spoke to us about her work, published in Nature, 2020 Feb;578(7794):296-300.
How did you start this project?
I first learned imaging techniques when I was in graduate school. When I later joined the Protein Metabolism Project, I decided to use these techniques to image proteasomes in cells under a microscope in different conditions. Proteasomes are usually found diffusely within a cell, but I found that when cells are stressed, proteasomes congeal into punctate liquid droplet structures. That was the start of this work.
What are liquid droplets and why are they important?
They really look like small droplets of oil in water. Proteasome droplets contain ubiquitinated proteins, RAD23B (a molecule that shuttles ubiquitin to proteasomes and bridges their interaction), proteasomes, and P97 (a molecule required for proteasomal degradation). We think that protein degradation occurs in these droplets, and droplets increase the efficiency of proteasomal degradation by bringing all the components and targets for degradation together in a specific location.
Does protein degradation become more important when cells are under stress?
The primary targets that are degraded in our droplets are ribosomal proteins. When cells are subjected to hyperosmotic stress, ribosomal proteins aggregate, and we think that these aggregates are degraded in proteasome droplets. Droplets are transient structures that disperse once ubiquitinated proteins are degraded. They last longer than usual if we inhibit degradation by adding proteasome inhibitors and they disperse faster if we accelerate degradation. That’s why we think degradation occurs in droplets.
How is droplet formation beneficial to cells?
We thought that droplet formation should increase the likelihood that a cell would survive hyperosmotic stress, so we performed cell death assays. However, our RAD23B knockout lines increase cell death regardless of stress. So, while we think that droplet formation should improve protein degradation and increase cell survival, but we haven’t been able to prove that yet. Recently we’ve identified a sequence in RAD23B that is required for liquid droplet formation. We’re planning to put a mutation within this sequence to make RAD23B that is defective only for droplet formation. We’re planning to use this mutated protein to prove whether there is a significant survival benefit to droplet formation in cells.
What is the significance of your work and how does it differ from previous reports on liquid droplets?
Liquid droplet formation or liquid-liquid phase separation is a subject that is gaining a lot of attention these days. A relatively well-characterized mechanism for liquid phase separation is electrostatic interactions between proteins or RNAs. The idea is that electrostatic interactions cause proteins or RNAs to bind together and form a different phase, separate from other cellular components. Our results are distinct from previous results because we find that interactions between a particular domain of RAD23B and ubiquitin are responsible for phase separation, not just general electrostatic interactions. Since ubiquitination is a regulated post-translational modification, our results suggest that increases in ubiquitination regulate phase separation. This explains how proteasome droplets are initially formed, and also explains how they disperse when ubiquitinated proteins are degraded.