In Vivo Proximity Biotinylation for Protein Interaction Studies in Paramecium tetraurelia.

Abstract

Paramecium is a major model organism for the study of molecular mechanisms involved in programmed genomic rearrangements and transposon elimination during meiotic reproduction. Many details of this process remain enigmatic due to the complexity of the involved molecular machinery and transient protein-protein interactions. Here, we present a method for time-resolved labeling of proteins with biotin located close to a target protein of interest. The method can easily be adapted to different targets since the biotinylation is achieved by injection of a DNA encoding a target protein fused to an engineered biotin ligase. The fusion construct is expressed from the promoter of the endogenous gene, enabling a stage-specific expression profile similar to that of the native protein. Notably, the covalently attached biotin moiety allows for the selective enrichment of the labeled proteins using streptavidin-coated beads. Furthermore, with immunofluorescence staining, we show that efficient labeling requires supplementation of the culture with biotin. The plug-and-play engineered expression vector, combined with the efficient injection procedure demonstrated here, allows for the rapid generation of Paramecium lines expressing a modified protein. The biotinylation procedure demonstrated here can be followed up with mass spectrometry for the identification of the enriched proteins that contain the interacting partners of the target protein. Proximity biotinylation has the potential to simplify and accelerate the discovery of protein-protein interactions in Paramecium and ramp up the efforts to understand its genome editing machinery.