The functional characterization of a novel protein, Liat1.
The Ligand of Ate1 (Liat1) was discovered in our lab by its interaction with Arginyl tRNA-protein transferase-1 (Ate1), a component of the N-degron pathway of protein degradation. However, its functional significance remains unknown. This study is the first functional characterization of Liat1. Here, we found that the N-terminal half of Liat1 is intrinsically disordered. Intrinsically disordered proteins tend to drive a phenomenon called liquid-liquid phase separation (LLPS). This involves the reversible conversion of a homogenous solution into separate phases of distinct density through the condensation of one or more of its biomolecular components. This process results in the formation of membrane-lacking subcellular organelles that organize cellular components and enhance biochemical reactions. Using a combination of immunocytochemistry, bimolecular fluorescence complementation, and biochemical techniques, we found that Liat1 undergoes LLPS in the dense fibrillar compartment (DFC) of the nucleolus. Mutational analysis revealed that a low complexity, poly-lysine region contained in the N-terminal intrinsically disordered region of Liat1 is required for its self-interaction and nucleolar phase separation. In addition to Ate1, Liat1 interacts with Jmjd6, a bifunctional enzyme with demethylase and lysl-hydroxylase activity. We show that Jmjd6 modifies Liat1 and prevents its nucleolar targeting through in a manner that requires its hydroxylation activity. In efforts to identify the Liat1 amino acids modified by Jmjd6, we expressed Jmjd6 in bacterial cells and established an in vitro assay to monitor it lysyl-hydroxylation under purified conditions. In efforts to characterize the nucleolar function of Liat1, we monitored its behavior in response to specific stress conditions. Specifically we found that Liat1 remains in the core of the nucleolus when rDNA transcription is inhibited; conditions which disrupt the overall structure of the nucleolus. Furthermore, we found that Liat1 is upregulated and localized to the cytosol during viral infection. These studies reveal distinct behaviors of Liat1 in response to specific forms of cell stress. Finally we found that Liat1 interacts with small ribosomal subunit proteins and that cells lacking Liat1 have defects in rRNA processing. Collectively, our data indicates that Liat1 participates in LLPS in the dense fibrillary component of the nucleolus in a manner regulated by Jmjd6; and suggest that it plays a significant role in rRNA processing and/or assembly as well as in various cellular stress responses.