The Tandem Affinity Purification (TAP) method is a powerful technique utilized in molecular biology to investigate protein-protein interactions. It involves the purification of protein complexes from biological samples followed by the identification of their components using mass spectrometry or other analytical methods. TAP is particularly valuable for studying the organization and dynamics of protein complexes within cells.
In the realm of protein-protein interaction studies, TAP serves several key purposes:
- Identification of Novel Protein Complexes: TAP enables researchers to isolate protein complexes from cells or tissues, allowing for the identification of novel protein-protein interactions. By purifying these complexes and analyzing their components, researchers can uncover new functional relationships between proteins.
- Identification of New Components within Protein Complexes: TAP can help identify previously unknown components within known protein complexes. By isolating a protein complex of interest and characterizing its composition, researchers can discover novel proteins that play roles in specific cellular processes or pathways.
- Identification of Interacting Partners of Target Proteins: TAP facilitates the identification of proteins that interact with a target protein of interest. By purifying the target protein along with its interacting partners, researchers can gain insights into the molecular mechanisms underlying protein function and regulation.
Since its introduction by Rigaut et al. in 1999, the TAP method has been widely adopted and successfully applied in various model organisms, including Escherichia coli, Drosophila melanogaster, and Homo sapiens. However, its application in plant systems was initially limited, with the first reports of plant protein complex isolation using TAP emerging in 2004.
Validating Interactions of Known Functional Proteins
In recent years, there has been a growing interest in applying TAP technology to study protein-protein interactions and protein complexes in plants. Researchers have successfully used TAP to analyze protein interactions and characterize protein complexes in plant species such as tobacco, Arabidopsis thaliana, and rice.
Rohila et al. utilized TAP in transient expression systems to isolate and characterize protein complexes in tobacco leaves. They successfully identified interacting partners of a target protein, demonstrating the utility of TAP in studying protein interactions in plants.
Similarly, Rubio et al. achieved success in Arabidopsis by expressing TAP-tagged proteins in stable transgenic systems. They utilized TAP to validate known components of a protein complex, highlighting the effectiveness of TAP in characterizing the composition of protein complexes in plant cells.
Furthermore, Sawa et al. utilized TAP technology to investigate protein-protein interactions involved in flowering time regulation in Arabidopsis. By employing TAP to isolate and purify protein complexes, they identified interacting partners of key regulatory proteins, shedding light on the molecular mechanisms controlling flowering time in plants.
Evaluation of alternative TAP tags in Arabidopsis cell suspension culture (Van Leene et al., 2008)
Identification of Novel Protein Complexes and Protein Interactions
Identifying novel protein complexes and protein interactions to elucidate molecular mechanisms underlying developmental processes and signaling pathways is a primary focus of proteomics and functional genomics research, and it's where the main functionality and advantages of Tandem Affinity Purification (TAP) technology lie.
TAP has been predominantly utilized in unraveling the molecular mechanisms of cell cycle regulation. Van Leene et al. pioneered the application of TAP to search for novel interactions among unknown proteins in plants, focusing on Arabidopsis cyclins. While over 100 key cell cycle genes have been identified in Arabidopsis, including at least 29 CDK genes and 52 cyclin-related genes, the interactions between CDK-cyclin complexes, between complexes and substrates, and between complexes and other molecules remain poorly understood. Using suspension-cultured Arabidopsis cells and a modified N-terminal TAP tag, they randomly selected six cell cycle proteins as TAP tag targets, identifying and confirming associations among 42 proteins, 28 of which were previously unknown. Subsequently, they utilized a GS-TAP tag to detect the interaction between UVH6 and a known transcription factor associated with the TFIIH complex, suggesting CDKD2 as a component of the TFIIH complex.
In the study of molecular mechanisms in plant hormone signaling, TAP technology has also been widely applied. Pauwels et al. isolated protein complexes from Arabidopsis cell suspensions treated with jasmonic acid (JA) using JAZ1-TAP tagged complexes. They identified a novel protein, NINJA (Novel Interactor of JAZ), and further analysis revealed NINJA as a transcriptional repressor involved in JA signaling. Additionally, Fernandez-Calvo et al. identified NINJA in complexes with MYC transcription factors, indicating its involvement in the JA signaling pathway.
Furthermore, TAP technology has been employed to identify membrane protein complexes in plants. Bassard et al. used GS tags with Arabidopsis cytochrome P450 proteins CYP73A5 and CYP98A3 as bait proteins, isolating 16 new interacting proteins from suspension cells under both induced and uninduced conditions, most of which were known ER membrane proteins, laying the foundation for elucidating the molecular pathways of lignin synthesis.
Moreover, TAP has been used to identify 14-3-3 protein complexes. Chang et al. conducted TAP experiments with Arabidopsis 14-3-3Ω as bait protein, revealing its interaction with at least 10 of its 12 subtypes and identifying 121 interacting proteins, the majority of which were previously unreported 14-3-3 interacting factors.
Beyond Arabidopsis, TAP technology has also been successfully applied in rice proteomics research. Rohila et al. studied protein kinases involved in various physiological processes such as stress signaling, pathogen resistance, ABA and ethylene signaling pathways, metabolism, and cell cycle regulation in rice. By utilizing TAP tags with 41 rice protein kinases, they purified 95% of the kinases and identified interactions with other rice proteins, some of which were consistent with findings in other species. Additionally, they identified a novel rice protein interacting with the highly conserved cell differentiation cycle CDC2 complex.
In the investigation of the molecular mechanisms of rice flowering regulation, Abe et al. used OsGI as bait protein and identified seven proteins interacting with OsGI, with one protein, dynamin, confirmed to interact via co-immunoprecipitation experiments.
Comparative Proteomics
Tandem Affinity Purification (TAP) has emerged as a powerful tool for comparative proteomics studies in plants, enabling researchers to investigate protein complexes and interactions across different plant species and tissues. For example, in a study focused on Arabidopsis thaliana, TAP was employed to isolate and identify positive regulators of the transcription factor DREB2A, a key player in the plant's response to drought stress. This exemplifies TAP's utility in making inter-species and inter-tissue comparisons of protein complexes and interactions, shedding light on the molecular mechanisms underlying stress responses in plants.
Post-Translational Modifications (PTMs) Analysis
TAP combined with mass spectrometry has significantly contributed to our understanding of post-translational modifications (PTMs) within protein complexes. For instance, a study in Arabidopsis thaliana utilized TAP to identify the phosphorylation status of Pol II, a major plant RNA polymerase. This analysis provided valuable insights into the regulatory roles of phosphorylation within the Pol II protein complex, highlighting the importance of PTMs in modulating protein function and activity in plants.
Subcellular Protein Localization
TAP has played a pivotal role in deciphering subcellular protein localization dynamics in plants. By identifying interacting partners of proteins localized to specific cellular compartments, researchers can elucidate the spatial and temporal dynamics of protein complexes. For example, a study investigating the autophagy process in plants utilized TAP to explore the subcellular localization and dynamics of a protein complex involved in autophagy regulation, providing valuable insights into the molecular mechanisms underlying this essential cellular process.
Integrating TAP with Transcriptomics and Metabolomics
The integration of TAP with other functional genomics approaches, such as transcriptomics and metabolomics, has enabled comprehensive studies of plant biology. By combining TAP data with transcriptome profiling, researchers can unravel the molecular mechanisms underlying key developmental processes and signaling pathways. For instance, a study combining TAP with transcriptome analysis investigated the molecular mechanisms of maize kernel development, providing a holistic understanding of the regulatory networks governing this critical developmental process in plants.
Systems Biology Perspective
From a systems biology perspective, TAP data can be integrated into computational models to elucidate intricate regulatory networks in plants. By incorporating experimental data obtained via TAP into computational models, researchers can predict the behavior of biological systems and identify key regulatory nodes for further investigation. For example, TAP was employed to study SUMOylation, a critical PTM, in plants, and the generated data was integrated into a systems biology model to unravel the regulatory network of the SUMOylation process, illustrating the power of TAP in elucidating complex regulatory mechanisms in plants.
Reference
- Van Leene, Jelle, et al. "Boosting tandem affinity purification of plant protein complexes." Trends in plant science 13.10 (2008): 517-520.
