Tag Thy Neighbour: Nanometre-Scale Insights Into Kinetoplastid Parasites With Proximity Dependent Biotinylation

Abstract

Proximity labelling is a powerful and rapidly developing technology for exploring the interaction space and molecular environment of a protein of interest at the nanometre scale. In proximity labelling, a promiscuous biotinylating enzyme is genetically fused to the protein of interest, initiation of labelling then results in the biotinylating enzyme generating reactive biotin which covalently 'tags' nearby molecules. Importantly, this labelling takes place in vivo whilst the protein of interest continues to perform its normal functions in the cell. Due to its unique advantageous characteristics, proximity labelling is driving discoveries in an ever increasing range of organisms. Here, we highlight the applications of proximity labelling to the study of kinetoplastids, a group of eukaryotic protozoa that includes trypanosomes and Leishmania which can cause serious disease in humans and livestock. We first provide a general overview of the proximity labelling experimental workflow including key labelling enzymes used, proper experimental design with appropriate controls and robust statistical analysis to maximise the amount of reliable spatial information that is generated. We discuss studies employing proximity labelling in kinetoplastid parasites to illustrate how these key principles of experimental design are applied. Finally, we highlight emerging trends in the development of proximity labelling methodology.

Keywords: APEX2; BioID proximity labeling; BirA enzyme; biotinylation; kinetoplastid; leishmania; mass spectrometry - LC-MS/MS; proteomics; trypanosoma.

Figure 1

Proximity biotinylation workflow in kinetoplastids. Overview of the main experimental steps involved in a proximity biotinylation experiment. 1. Protein of interest is either tagged at the endogenous locus with a biotinylator such as BirA* or APEX2, or a tagged version is expressed from a different locus. 2. Adding exogenous biotin to the growth medium (BirA*) or biotin-phenol then H₂O₂ (APEX2) initiates in vivo proximity biotinylation. If performing XL-BioID, this may then be followed by an in vivo cross-linking step to increase capture of proximal proteins. A nuclear protein fused to BirA* is shown as an example. 3. The major advantage of proximity biotinylation is being able to completely lyse and solubilise cells with harsh lysis conditions such as radioimmunoprecipitation assay (RIPA) buffer. 4. Proximal proteins covalently tagged with biotin are enriched by incubating lysate with streptavidin beads. 5. Non-proximal proteins are washed off with a series of stringent bead washes. 6. Proteins remaining on beads are digested with trypsin and released peptides are desalted using C18. 7. Peptides are identified and quantified by quantitative mass spectrometry. Quantitation may be label free, TMT or SILAC based. 8. Enrichment of identified proteins vs. a control sample is calculated and statistical testing performed, e.g. with SAINTq or limma. Proteins significantly enriched in the sample vs. the spatial reference sample are classed as proximal proteins.

Figure 2

Overview of proximity biotinylation by BirA*. (A) Cartoon depicting a bait protein of interest (POI) fused to BirA*. Free biotin is converted into a reactive biotinyl-5’AMP which reacts with lysine residues in proteins within a 10 nm labelling radius. Signal accumulates over time due to dynamic changes in the protein complex. (B) Depiction of the exponential decay of reactive biotin levels as distance increases from the biotin ligase. (C) Example of using spatial or organellar control strains, in which a different protein is tagged with the biotin ligase and compared against the strain expressing the control protein. This allows for the computational removal of endogenously biotinylated proteins as well as labelled proteins that are not specific to complexes of interest.

Figure 3

Cartoon depiction of kinetoplastid targets explored using proximity biotinylation. Cutaway style drawing, showing stylised cellular location of (A) T. brucei and (B) Leishmania proximity biotinylation bait proteins. N.B. many of the represented proteins have dynamic localisations throughout the cell cycle.