YEAST TWOHYBRID SYSTEM detection of proteinprotein interactions

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YEAST TWO-HYBRID SYSTEMdetection of
protein-protein interactions

• by
• Burcu Kaplan

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            Nature Reviews Molecular Cell Biology
2 55-63 (2001)
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INTRODUCTION

• many eukaryotic transcription activators
  have at least two distinct functional domains,
  one that directs binding to a promoter DNA
  sequence(Upstream Activating Sequence) and one
  that activates transcription
• This fact was illustrated by exchanging DNA
  binding domains and activation domains from one
  transcription factor to the next while retaining
  its function.
• It was shown that the activation domain of
  yeast GAL4 could be fused to the DNA-binding
  domain of E. coli LexA to create a functional
  transcription activator in yeast.

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INTRODUCTION
The two-hybrid technique exploits the fact that
the DNA-binding domain of GAL4 is incapable of
activating transcription unless physically, but
not necessary covalently associated with an
activating domain
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RATIONALE
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Ma and Ptashne

• GAL80 protein, normally a negative regulatory
  protein that interacts with GAL4, was converted
  into a transcriptional activator by fusing it to
  an activation domain (AD).
• The activation by this fusion protein, GAL80-AD,
  was dependent on the presence of a GAL4
  derivative bearing the GAL80 binding domain
  (C-terminal 30 amino acids) but lacking its own
  activation domain .

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Stanley Fields
Fields S., Song O.K. 1989. A novel genetic system
to detect protein-protein interactions. Nature
340, 245-246.
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Fields and Song (1989)

• SNF1 fused to the DNA-binding domain (DB)
• SNF4 fused to an activation domain (AD).
• Only after expression of these two chimeras, and
  subsequent interaction of SNF1 and SNF4, did they
  reconstitute a functional transcription factor
  that is able to induce reporter gene expression.
• These initial experiments confirmed that a
  transcriptional read-out could be used as a tool
  to study interactions between proteins not
  involved in the transcription process.

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Principle of the Two-hybrid system.
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Principle of the Two-hybrid system. (A), (B) Two
chimeras, one containing the DNA-binding domain
(DB blue circle) and one that contains an
activation domain (AD half blue circle), are
co-transfected into an appropriate host strain.
(C) If the fusion partners (yellow and
red)interact, the DB and AD are brought into
proximity and can activate transcription of
reporter genes (here LacZ).
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EXPERIMENTAL

• 1. a plasmid for synthesis of a protein which
  is brought to DNA due to its binding to a DNA
  binding domain. This is also known as a bait
  protein.2. at least one reporter gene that
  contains upstream binding sites for the bait.3.
  a plasmid for synthesis of a protein fused to an
  activation domain,prey protein

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Reporter Genes

• lacZ,blue colonies on ?- galactosidase media
• HIS3,growth on histidine media
• Multiple reporter

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Yeast strains

• haploids
• opposite mating types they will mate
• Deletions in GAL4 GAL80 genes, chromasomal DNA
• inhibit the basal expression of HIS3 with
  3-amino-triazole (AT), a known inhibitor of the
  HIS3 gene product.

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BAIT Plasmid

• DNA Binding domain
• cDNA encoding protein of interest
• insertion in a selectable plasmid Trp

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BAIT Plasmid
DNA Binding Domain
Bait (known ) protein
fusion
insertion into Plasmid ,Trp
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Transformation
Bait plasmid
Yeast cells I,-Trp,-Leu,-His
transformation
Growth on Trp media
selection
transformed baitcells
colonies
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PREY Plasmid

• Activation domain
• cDNA of interestie each ORF of a genome
• insertion in a selectable plasmid Leu

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PREY Plasmid
Activation Domain
cDNA
fusion
insertion into Plasmid ,Leu
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Transformation
Prey plasmid
Yeast cells II,-Trp,-Leu,-His
transformation
selection
Growth on Leu media
transformed preycells
colonies
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Determination of optimal AT concentration in a
pilot transformation using strong, weak and
intermediate interactors as positive and negative
controls. Cells are plated out on a AT gradient.
The optimal AT concentration (dotted double
arrow) is this which elimates false positives
(e.g. formed in case of bait empty library
plasmid but which still allows to detect weak
interactions).
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Controls

• Sequencing
• Western blot protein (bait) expression
• Reporter gene activityauto-activation
• Nuclear localization DB should contain a NLS
• Positive controlknown interaction

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TWO HYBRID ASSAY

• mate haploids on media lacking Leu Trp
• select colonies
• grow on media lacking His (reporter 1)
• select colonies
• grow on media with ?-galactosidase
• (reporter 2)

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MATING-Leu, -Trp media
Prey colonies
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growth
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2
1
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Bait colonies
1
2
3
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5
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Reporter Genes

• lacZ,blue colonies
• HIS3,growth on histidine media
• Multiple reporter
• Screen for growth on histidine media
• Pick colonies, transfer to ?- galactosidase media
• Select blue colonies

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TWO HYBRID ASSAY
Prey colonies
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Protein-protein interaction
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2
1
5
Bait colonies
1
2
3
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5
6
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Not finished yet.....

• Picking positive colonies
• Preparation of DNA from yeast
• Separating the two-hybrids
• Insert analysisAssay for specificity in two-
  hybrid
• Take the interaction outside the yeast system
• Confirmation of interaction
• Functional assays to test relevance of
  interaction

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Drawbacks

• Auto-activation
• the extensive use of chimeras folding ?
• S.cerevisiae as host
• extracellular proteins or proteins that contain
  strong(er) targeting signals.
• Bridging proteins
• Toxic proteins proteases
• artifactual partners

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Advantages over classical proteomics

• in vivo (exvivo)
• eukoryotic host
• Only the cDNA, full-length or even partial of the
  gene is needed.
• Weak transient interactions
• Semi-quantative affinities
• The corresponding gene is cloned

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NEW DEVELOPMENTS

• Improvements
• Reverse Hybrid System
• SRS system
• USPS system
• To higher eukaryotes Mammalian Two-hybrid
• Three-hybrid systems

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IMPROVEMENTS

• new yeast strain sensitive to weak interactions
  eliminates false positives HIS3, ADE2 and
  LacZ each under control of a different promoter
  (GAL1, GAL2, and GAL7 respectively) that respond
  to the same activator GAL4

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REVERSE HYBRID
Dissociations molecules prevent interaction A
counter selectable marker that is toxic under
particular conditions. Non-interacting hybrids
are alive
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The reverse two-hybrid system takes advantage of
counterselectable yeast reporter genes. URA3
expression sensitizes yeast cells to
5-fluoroorotic acid (5-Foa), CYH2 expression to
cycloheximide. Abrogation of the interaction
enables the cells to grow in the presence of
these drugs.
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SRS (Sos-recruitment)System
In cells that express a chimera of Sos-X (where X
is a target protein) along with a
membrane-localized version of a protein that
interacts with X (cDNA), the interaction between
X and its partner recruits Sos to the plasma
membrane, resulting in Ras activation
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SRS....

• Interactions that involve transcriptional
  activators / repressors not based on a
  transcriptional readout.
• interactions between proteins that require
  modification by cytoplasmic or membrane-associated
  enzymes.

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Three-hybrid systems

• Kinase three-hybrid system (tri-brid)
• Protein three-hybrid system
• peptide ligand three-hybrid system
• Peptide ligand and small ligand three-hybrid
  system

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Kinase three-hybrid system(tri-brid)
The protein target needs to be phosphorylated (P)
by a co-expressed kinase before interaction
occurs.
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Protein three-hybrid system
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peptide ligand three-hybrid system

• extracellular domain of a transmembrane receptor
• fusion to GAL4BD
• fusion to GAL4AD
• Expression of the native peptide ligand
• receptor dimerization
• transcription

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NEW APPLICATIONS OFYEAST TWO-HYBRID

• Interaction suppression
• A interacts with B
• Mutate A no interaction
• Screen for a mutation in B which restores
  interaction
• Protease Trap
• Trancription factor-protease site-domain

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Whole genome approaches

• Protein Linkage Maps
• two-hybrid
• binary interactions
• false-positives
• time-consuming
• TAP/MS Method

Gavin A, et al., Nature (2002) 415141-147
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TAP/MS Method for yeast proteome

• TAP( tandem affinity purificaiton) tag inserted
  at the 3 end of the genes.
• haploid cells
• expression of the tagged proteins
• TAP purification from total cellular lysates

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TAP purification

• high-affinity purification,
• mild elution using a sitespecific protease,
• affinity purification to obtain protein complexes
  with high efficiency and specificity

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protein complexes
PAGE
in gel trypsin digestion
MALDI-TOF-MS
Database search
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Cell cycle
Signaling
Transcription, chromatin structure
Protein and RNA transport
RNA metabolism
Protein metabolism
Cellular structure
Energy
Membrane traffic
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comparison

• 7 overlap with interactions seen by yeast two-
• hybrid assays
• compared with the YPD protein complexes, this
  study covers 56, whereas large-scale yeast
  two-hybrid approaches cover 10.
• The figure for the yeast two-hybrid data is 39
  compared with 56 in this study.
• To achieve the respective coverage, the yeast
  two-hybrid approaches required processing of 95
  of all yeast ORFs compared with 25 in this
  study.

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TAP/MS Y-II-H

• Characterization of
• Protein complexes

• Pairwise transient
• associations
• orientation

Complementary approches
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References

• Wim Van Criekinge,W.V., Beyaert, R. Yeast
  Two-Hybrid State of the Art, Biological
  Procedures Online 2, 1999
• Uetz et.al A comprehensive analysis of
  protein-protein interactions in Saccharomyces
  cerevisiae,Nature 403,2000
• Gavin et al., Functional organization of the
  yeast proteome by systematic analysis of protein
  complexes, Nature 415,2002

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THANKS FOR YOUR PATIENCE