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Published on: May 13, 2021
Nuclear immunophilin FKBP39 from Drosophila melanogaster drives spontaneous liquid-liquid phase separation
Aneta Tarczewska1, Krzysztof Wycisk1, Marek Orłowski1
1Department of Biochemistry, Molecular Biology and Biotechnology, Faculty of Chemistry, Wroclaw University of Science and Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wroclaw, Poland.
Abstract:
The FKBP39 from Drosophila melanogaster is a multifunctional regulatory immunophilin. It contains two globular domains linked by a highly charged disordered region. The N-terminal domain shows homology to the nucleoplasmin core domain, and the C-terminal domain is characteristic for the family of the FKBP immunophilin ligand binding domain. The specific partially disordered structure of the protein inspired us to investigate whether FKBP39 can drive spontaneous liquid-liquid phase separation (LLPS). Preliminary analyses using CatGranule and Pi-Pi contact predictors suggested a propensity for LLPS. Microscopy observations revealed that FKBP39 can self-concentrate to form liquid condensates. We also found that FKBP39 can lead to LLPS in the presence of RNA and peptides containing Arg-rich linear motifs derived from selected nuclear and nucleolar proteins. These heterotypic interactions have a stronger propensity for driving LLPS when compared to the interactions mediated by self-associating FKBP39 molecules. To investigate whether FKBP39 can drive LLPS in the cellular environment, we analysed it in fusion with YFP in COS-7 cells. The specific distribution and diffusion kinetics of FKBP39 examined by FRAP experiments provided evidence that immunophilin is an important driver of phase separation. The ability of FKBP39 to go into heterotypic interaction may be fundamental for ribosome subunits assembly.
Insights
Drosophila FKBP39 protein drives liquid-liquid phase separation (LLPS), forming condensates alone or with RNA and peptides. This immunophilin
Area of Science:
- Biochemistry and Molecular Biology
- Cell Biology
- Structural Biology
Background:
- FKBP39 is a multifunctional regulatory immunophilin from Drosophila melanogaster.
- It possesses a unique structure with two globular domains and a charged disordered region.
- Its partially disordered nature prompted investigation into its potential for liquid-liquid phase separation (LLPS).
Purpose of the Study:
- To investigate the capacity of FKBP39 to induce spontaneous liquid-liquid phase separation (LLPS).
- To explore FKBP39's role in heterotypic interactions with RNA and Arg-rich peptides.
- To assess FKBP39's LLPS-driving ability within a cellular context.
Main Methods:
- Computational prediction of LLPS propensity using CatGranule and Pi-Pi contact predictors.
- In vitro microscopy to observe FKBP39 self-condensation and heterotypic interactions.
- Fluorescence Recovery After Photobleaching (FRAP) experiments in COS-7 cells using FKBP39-YFP fusion proteins.
Main Results:
- FKBP39 demonstrates a propensity for LLPS, forming liquid condensates in vitro.
- FKBP39 facilitates LLPS with RNA and Arg-rich peptides, showing stronger heterotypic interactions.
- FRAP analysis confirmed FKBP39 as a driver of phase separation within COS-7 cells.
Conclusions:
- FKBP39 is a potent inducer of liquid-liquid phase separation (LLPS).
- Its ability to engage in heterotypic interactions enhances LLPS.
- FKBP39's phase separation capabilities may be crucial for ribosome subunit assembly.
