Chapter 21
Biotin-Based Proximity Labeling of Protein Complexes
in Planta
Madiha Khan, Rajagopal Subramaniam, and Darrell Desveaux
Abstract
Proteome networks are a crucial facet of biological systems that mediate cellular functions and responses to
the environment. However, a main limitation of traditional approaches to study protein interactions, such
as yeast-2-hybrid and affinity purification-coupled with mass spectrometry (AP-MS), is their restricted
ability to identify interactions for membrane-bound and/or insoluble protein complexes. These types of
interactions include many of the protein complexes that mediate the perception and response to cellular
stimuli and are therefore of great research interest. Proximity-dependent biotinylation (PDB) coupled to
mass spectrometry provides a powerful approach to survey proximal protein interactions in living cells,
including membrane bound and insoluble complexes. One PDB method, BioID, translationally fuses a
promiscuous biotin ligase to a bait protein of interest, allowing covalent biotinylation of proximal proteins
(within ~10 nm). Modified proteins can be purified from cells without the need to maintain protein
interactions, and subsequently identified by mass spectrometry. Although BioID has revolutionized the
study of proteomes in numerous organisms, its application to plant systems has only recently been realized.
In this chapter, we outline a protocol for BioID in tissues of the model plant Arabidopsis thaliana.
Key words BioID, In planta, Arabidopsis thaliana, Plant, Protein complexes, Affinity purification,
Mass spectrometry, Proximal proteins, Biotinylation, Protein–protein interaction
1 Introduction
Numerous tools have been developed to map the composition and
organization of protein complexes in cellular systems. Among the
most recent are proximity-dependent biotinylation (PDB)
approaches that covalently biotinylate proteins that are in the vicinity of a bait protein of interest. These approaches have numerous
advantages over traditional approaches, such as yeast two-hybrid
and AP-MS, including the ability to study membrane-associated
and insoluble proteomes [1]. BioID is a PDB approach that fuses a
mutated E. coli biotin ligase, BirA* (BirA R118G) to a bait protein
of interest to promiscuously biotinylate neighboring proteins
[2]. BirA* releases reactive bioAMP molecules which covalently
react with primary amines of proteins (e.g., exposed lysine residues)
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_21, © Springer Science+Business Media, LLC, part of Springer Nature 2021
425
Biotin-Based Proximity Labeling of Protein Complexes
in Planta
Madiha Khan, Rajagopal Subramaniam, and Darrell Desveaux
Abstract
Proteome networks are a crucial facet of biological systems that mediate cellular functions and responses to
the environment. However, a main limitation of traditional approaches to study protein interactions, such
as yeast-2-hybrid and affinity purification-coupled with mass spectrometry (AP-MS), is their restricted
ability to identify interactions for membrane-bound and/or insoluble protein complexes. These types of
interactions include many of the protein complexes that mediate the perception and response to cellular
stimuli and are therefore of great research interest. Proximity-dependent biotinylation (PDB) coupled to
mass spectrometry provides a powerful approach to survey proximal protein interactions in living cells,
including membrane bound and insoluble complexes. One PDB method, BioID, translationally fuses a
promiscuous biotin ligase to a bait protein of interest, allowing covalent biotinylation of proximal proteins
(within ~10 nm). Modified proteins can be purified from cells without the need to maintain protein
interactions, and subsequently identified by mass spectrometry. Although BioID has revolutionized the
study of proteomes in numerous organisms, its application to plant systems has only recently been realized.
In this chapter, we outline a protocol for BioID in tissues of the model plant Arabidopsis thaliana.
Key words BioID, In planta, Arabidopsis thaliana, Plant, Protein complexes, Affinity purification,
Mass spectrometry, Proximal proteins, Biotinylation, Protein–protein interaction
1 Introduction
Numerous tools have been developed to map the composition and
organization of protein complexes in cellular systems. Among the
most recent are proximity-dependent biotinylation (PDB)
approaches that covalently biotinylate proteins that are in the vicinity of a bait protein of interest. These approaches have numerous
advantages over traditional approaches, such as yeast two-hybrid
and AP-MS, including the ability to study membrane-associated
and insoluble proteomes [1]. BioID is a PDB approach that fuses a
mutated E. coli biotin ligase, BirA* (BirA R118G) to a bait protein
of interest to promiscuously biotinylate neighboring proteins
[2]. BirA* releases reactive bioAMP molecules which covalently
react with primary amines of proteins (e.g., exposed lysine residues)
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_21, © Springer Science+Business Media, LLC, part of Springer Nature 2021
425