Upload
vutu
View
238
Download
0
Embed Size (px)
Citation preview
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