Chapter 1
Identification of PDZ Interactions by Yeast Two-Hybrid
Technique
Monica Castro-Cruz, Marta Monserrat-Gomez, Jean-Paul Borg,
Pascale Zimmermann, and Eric Bailly
Abstract
The yeast two-hybrid technique is a powerful method to detect direct protein–protein interactions. Due to
its accessibility, speed, and versatility, this technique is easy to set up in any laboratory and suitable for small
and large scale screenings. Here we describe the implementation of an array-based screening that allows for
the probing of the entire human PDZ ORFeome (or hPDZome) by yeast two-hybrid technique. With this
approach, one can rapidly identify the PDZ domains that are able to interact (up to K D in the high μmolar
range) with any candidate protein among a panel of 266 individual clones, thereby comprehensively
identifying its PDZ interactome.
Key words Yeast two-hybrid, Two-hybrid array, Protein–protein interaction, GAL4, Yeast strain,
PDZ interactions
1 Introduction
The yeast two-hybrid (Y2H) method was developed in the late
1980s and remains a straightforward genetic approach to detect
direct protein–protein interactions (PPIs) [1, 2]. It takes advantage
of the modular nature of transcription factors (TFs) like GAL4 to
render the expression of reporter genes dependent on the binding
properties of two partner proteins (Fig. 1). GAL4 comprises two
functionally independent domains, a DNA binding domain
(GAL4-BD) and a transcription activation domain (GAL4-AD)
that can be separately fused to the proteins of interest. If the
resulting hybrid components can interact through the two proteins
of interest, then a functional GAL4 is reconstituted allowing for the
transcription of the reporter/survival genes. In this setting, GAL4BD is commonly appended to one partner that serves as “bait” (X),
Jean-Paul Borg (ed.), PDZ Mediated Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2256,
https://doi.org/10.1007/978-1-0716-1166-1_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Monica Castro-Cruz and Marta Monserrat-Gomez contributed equally to this work.
1
Identification of PDZ Interactions by Yeast Two-Hybrid
Technique
Monica Castro-Cruz, Marta Monserrat-Gomez, Jean-Paul Borg,
Pascale Zimmermann, and Eric Bailly
Abstract
The yeast two-hybrid technique is a powerful method to detect direct protein–protein interactions. Due to
its accessibility, speed, and versatility, this technique is easy to set up in any laboratory and suitable for small
and large scale screenings. Here we describe the implementation of an array-based screening that allows for
the probing of the entire human PDZ ORFeome (or hPDZome) by yeast two-hybrid technique. With this
approach, one can rapidly identify the PDZ domains that are able to interact (up to K D in the high μmolar
range) with any candidate protein among a panel of 266 individual clones, thereby comprehensively
identifying its PDZ interactome.
Key words Yeast two-hybrid, Two-hybrid array, Protein–protein interaction, GAL4, Yeast strain,
PDZ interactions
1 Introduction
The yeast two-hybrid (Y2H) method was developed in the late
1980s and remains a straightforward genetic approach to detect
direct protein–protein interactions (PPIs) [1, 2]. It takes advantage
of the modular nature of transcription factors (TFs) like GAL4 to
render the expression of reporter genes dependent on the binding
properties of two partner proteins (Fig. 1). GAL4 comprises two
functionally independent domains, a DNA binding domain
(GAL4-BD) and a transcription activation domain (GAL4-AD)
that can be separately fused to the proteins of interest. If the
resulting hybrid components can interact through the two proteins
of interest, then a functional GAL4 is reconstituted allowing for the
transcription of the reporter/survival genes. In this setting, GAL4BD is commonly appended to one partner that serves as “bait” (X),
Jean-Paul Borg (ed.), PDZ Mediated Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2256,
https://doi.org/10.1007/978-1-0716-1166-1_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Monica Castro-Cruz and Marta Monserrat-Gomez contributed equally to this work.
1