Master program Molecular and Cellular Life Sciences

Block 1: Intracellular membrane processes16 th October 2012

The yeast two-hybrid assay

Fulvio ReggioriDepartment of Cell Biology, UMC Utrecht

For what the yeast two-hybrid assay is used for?

1) To facilitate the identification of an interaction

between two proteins

2) To facilitate the characterization of this

interaction, e.g. identify the binding motifs

Why are the advantages of the yeast two-hybrid assay?

1) It exploits the rapid growth of yeast S. cerevisiae.

2) It exploits the uncomplicate and effective molecular biology of

yeast, e.g. easy to transform cells and to select transformed cells.

3) Very simple readouts

4) Possibility to screen for both interactors and binding point

mutants

The idea

Transcription factors have a DNA binding domain (DBD) and an

activation domain (AD)

The idea

Transcription factors have a DNA binding domain (DBD) and an

activation domain (AD)

The idea

Bait, protein of interest; Prey, other protein of interest or protein library

The idea

Bait, protein of interest; Prey, other protein of interest or protein library

The employed transcription factors

- Yeast Gal4, both its DBD and AD

- The DBD of LexA, a repressor enzyme, can also be used

- The AD of VP16, a trans-acting protein of the Herpes simplex

virus, can also be used

- The DBD of LexA can be used in combination with the AD of both

Gal4 and VP16

- The DBD of Gal4 can be used in combination with the AD of VP16

The reporter genes

These selected genes are put under the control of the promoter bound by the

selected DBD (Gal4 or LexA)

The reporter genesThese selected genes are put under the control of the promoter bound by the

selected DBD (Gal4 or LexA)

- HIS3, yeast gene involved in the synthesis of histidine

- ADE2, yeast gene involved in the synthesis of adenine

- URA3, yeast gene involved in the synthesis of uracyl

- LacZ, E. coli -galactosidase gene involved in the hydrolysis of -galactosides

into monosaccharides

Yeast auxotrophies and their exploitation for transformation

- Wild type yeast does not need amino acids and nucleotides for growth

- Lab wild type yeast are auxotroph for specific amino acids and nucleotides

For example: leu2 trp1 ura3 These cells do not grow if leucine,

triptophan and uracyl are not added to the medium.

Yeast auxotrophies and their exploitation for transformation

- Yeast vectors:

- Amp(r); ampicillin resistance gene

- Yeast HIS3 gene

- CEN; centromer = cromosom

- MCS; multi-cloning site

MCS

Yeast auxotrophies and their exploitation for transformation

- Yeast transformation with vectors (example leu2 trp1 ura3)

medium plus leucine, uracyland tryptophan

medium plus uracyland tryptophan but without

leucine

Yeast transformation for the yeast two-hybrid assay

- Yeast transformation with vectors (strain leu2 trp1 ura3)

Yeast transformation for the yeast two-hybrid assay

- Yeast transformation with vectors (strain leu2 trp1 ura3)

Selection of the double transformants on a medium without both leucine

and tryptophan

Yeast two-hybrid assay readouts

- Employed leu2 trp1 ura3 strains are also:

1) GAL4 promoter + HIS3

2) GAL4 promoter + ADE2

3) GAL4 promoter + LacZ

Yeast two-hybrid assay readouts

- Employed leu2 trp1 ura3 strains are also:

1) GAL4 promoter + HIS3

If the interaction is there, cells also grow on medium without histidine

2) GAL4 promoter + ADE2

If the interaction is there, cells also grow on medium without adenine

3) GAL4 promoter + LacZ

If the interaction is there, cells become blue medium containing X-gal

Why 3 readouts?

1) GAL4 promoter + HIS3

Weak interactions

2) GAL4 promoter + ADE2

Strong interactions

3) GAL4 promoter + LacZ

Quantification of the interactions by measuring the LacZ activity by an

enzymatic assay (hydrolysis of ortho-nitrophenyl-β-galactoside = ONPG)

Why 3 readouts?

1) GAL4 promoter + HIS3

Weak interactions

2) GAL4 promoter + ADE2

Strong interactions

3) GAL4 promoter + LacZ

Quantification of the interactions by measuring the LacZ activity by an

enzymatic assay (hydrolysis of ortho-nitrophenyl-β-galactoside = ONPG)

Why 3 readouts?

1) GAL4 promoter + HIS3

Weak interactions

2) GAL4 promoter + ADE2

Strong interactions

3) GAL4 promoter + LacZ

Quantification of the interactions by measuring the LacZ activity by an

enzymatic assay (hydrolysis of ortho-nitrophenyl-β-galactoside = ONPG)

Controls1) Grow control

2) Auto-activation controls

DBD-X + AD-empty

DBD-empty + AD-Y

-Trp-Leu -Trp-Leu

Controls

3) The yeast two-hybrid assay is an artificial system, interactions

have always to be proved in vivo by pull-down experiments!

1) Grow control

2) Auto-activation controls

DBD-X + AD-empty

DBD-empty + AD-Y

-Trp-Leu -Trp-Leu

Limitations

1) N-terminal tagging could make the analyzed protein non-functional

partial solutions: a) C-terminal tagging

b) Try both orientations, AD vs DBD

2) The binding occur in the nucleus, which could not have not the optimal

conditions for the binding

pH, ion concentration, membrane proximity…

3) Transmebrane proteins cannot be targeted to crhromosomes

Limitations

1) N-terminal tagging could make the analyzed protein non-functional

partial solutions: a) C-terminal tagging

b) Try both orientations, AD vs DBD

2) The binding occur in the nucleus, which could not have not the optimal

conditions for the binding

pH, ion concentration, membrane proximity…

3) Transmebrane proteins cannot be targeted to crhromosomes

4) If an interaction is not detected by the yeast two-hybrid assay, it

does not mean that is not occurring in vivo

Applications

1) Study of the binding between 2 proteins

2) Identification of the binding motifs

Truncations Point mutants

3) Screen to isolate binding partners

(this approach can also be used to identify the binding motifs)

Applications

Modifications of the yeast two-hybrid system

Reverse yeast two-hybrid (rY2H) assay ( for drug discovery)

Modifications of the yeast two-hybrid system

Yeast three-hybrid (Y3H) assay

Often, in state of a compound, a third

protein is used

Reichel and Johnsson (2005), Methods Enzymol, 399:757.

The yeast split-ubiquitin system

The yeast split-ubiquitin system

Additional advantages to the yeast split-ubiquitin system

1) Study of the binding between 2 proteins

2) Identification of the binding motifs

3) Screen to isolate binding partners

4) It can be used to study transmembrane proteins as well

5) The interaction can be studied in locus, in the right environment