We aim to characterise the functions of intrinsic disorder and phase separation during transcriptional control by ARF proteins in plants. ARFs have 3 conserved domains, of which the middle one is intrinsically disordered. We quantify disorder by spectroscopic approaches and characterise ARF phase separation in vitro by membrane osmometry. The biological relevance of phase separation is addressed by visualising fluorescently labelled ARF proteins with confocal microscopy. This will uncover the roles of intrinsic disorder/phase separation for transcription factor function.
A large proportion of the proteome is made up of intrinsically disordered proteins (or segments of proteins), especially in case of eukaryotic organisms. Intrinsically disordered proteins can exhibit a range of complex functions, like modulating allosteric interactions, forming DNA-protein condensates, tuning protein function, and transcriptional complex formation.
Intrinsic disorder is predicted to be particularly prominent in allosteric signaling proteins such as transcription factors. Transcription factors are required to initiate transcription of DNA in eukaryotes. In case of land plants, the small organic molecule auxin is involved as hormone in most developmental processes, which are mediated by changes in gene expression. These alterations in expression happen because auxin liberates ARF proteins from repressing dimeric interactions with auxin/indole-3-acetic acid proteins, as a result of ubiquitination and degradation of the repressor proteins. Liberation of ARF proteins activates them, leading to regulation of a defined set of target genes.
In general, ARF proteins consist of 3 domains: a DNA binding domain (DBD) located at the N-terminus, a middle region (MR), and a protein binding domain, which consists of a Phox and Bem1 (PB1) fold at the C-terminus. The middle region of this family of proteins is predicted to be intrinsically disordered. Indeed, limited proteolysis and NMR experiments performed in our lab show intrinsic disorder within ARF1. The functions of disorder of the middle region of ARF proteins are largely speculative and may be limited to Ångström-scale molecular properties only. For example, the middle region displays the highest divergence in amino acid composition, which is thought to be correlated to the respective ARF protein being a transcriptional activator or a repressor, respectively. Furthermore, a disordered middle region can serve as a focal signalling hub, because it may become structured upon interacting with cofactors or upon post-translational modifications, and/or aid in DNA binding affinity/specificity.
The Ångström-scale molecular properties of ARF proteins may translate to larger scale (micrometre) phenomena, like phase separation, possibly driven by disorder. The PB1 domain of ARF proteins mediates electrostatic interactions with other proteins, including the transcriptional co-repressors mentioned, and also causes homo- and hetero-oligomerisation of ARF proteins and thus this domain could also contribute to phase separation of ARFs. Phase separation may be a mechanism to spatially organize, to control monomeric concentration and to biochemically regulate the function of ARF proteins in the cell.
Aim of the project
We aim to characterise the potential roles of intrinsic disorder and phase separation for ARF proteins, we will take the following experimental approaches.
- Determination and quantification of intrinsic disorder in the middle region of ARF proteins.
- Characterisation of phase separation of ARF proteins in vitro.
- Visualisation of ARF protein phase separation in plant cells.
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