Nucleolar targeting in an early-branching eukaryote suggests a general mechanism for ribosome protein sorting

The compartmentalised eukaryotic cell demands accurate targeting of proteins to the organelles in which they function, whether membrane-bound (like the nucleus) or non-membrane-bound (like the nucleolus). Nucleolar targeting relies on positively charged localisation signals and has received rejuvenated interest since the widespread recognition of liquid-liquid phase separation (LLPS) as a mechanism contributing to nucleolus formation. Here, we exploit a new genome-wide analysis of protein localisation in the early-branching eukaryote Trypanosome brucei to analyse general nucleolar protein properties. T. brucei nucleolar proteins have similar properties to those in common model eukaryotes, specifically basic amino acids. Using protein truncations and addition of candidate targeting sequences to proteins, we show both homopolymer runs and distributed basic amino acids give nucleolar partition, further aided by a nuclear localisation signal (NLS). These findings are consistent with phase separation models of nucleolar formation and physical protein properties being a major contributing mechanism for eukaryotic nucleolar targeting, conserved from the last eukaryotic common ancestor. Importantly, cytoplasmic ribosome proteins, unlike mitochondrial ribosome proteins, have more basic residues - pointing to adaptation of physicochemical properties to assist segregation.

Automated summary

By analyzing the protein localization properties of Trypanosome brucei, an early-branching eukaryote, this study reveals that nucleolar proteins in T. brucei share similar characteristics with those in common model eukaryotes, particularly basic amino acids. Experimental evidence showed that both homopolymer runs and distributed basic amino acids contribute to nucleolar targeting, along with a nuclear localization signal. These findings support the phase separation models of nucleolar formation and suggest a conserved mechanism for eukaryotic nucleolar targeting from the last eukaryotic common ancestor. Furthermore, the adaptation of physicochemical properties, such as an increase in basic residues, in cytoplasmic ribosome proteins may aid in their segregation.