Download A comprehensive analysis of protein

A comprehensive analysis of
protein-protein interactions in
Saccharomyces cerevisiae
Article by Peter Uetz, et.al.
Presented by Kerstin Obando
Background Information
• In April 1996 the genome sequence of
Saccharomyces cerevisiae, a budding yeast
was completed.
• Enabling an eukaryotic organism to be
analyzed on a genomic scale for the first
time.
A problem with two different
approaches to a solution
•
•
Problem: What are the roles of the 6,000
gene products and how do they interact to
create a eukaryotic organism?
Solution: Two-hybrid analysis will
produce additional information that can
place yeast ORF’s within a biological
context.
Two hybrid analysis
• Separates the coding sequences for the DNA-binding and activation
domains of a transcriptional activator and clones them into separate
vector molecules.
• The coding sequence of a candidate protein whose partners are sought
is then fused with the DNA-binding domain, this is known as the bait.
A library of coding sequences for proteins that might interact with the
‘bait’, called the prey, is made in fusion with the activation domain.
• If the bait and prey physically interact, the DNA-binding and
activation domains are closely juxtaposed and the reconstituted
transcriptional activator can mediate the switching on of the gene that
effects the color change.
Basically…
• They have two proteins that want to test to see if
they can physically associate.
• They attach each of them to separate fragments of
a third protein, called a transcription activator,
which has the ability to switch on genes.
• If the two proteins interact, then the two fragments
of the activator are reunited and a gene is turned
on that produces an easily monitored color change
in the yeast cells.
Protein array of
activation-domain hybrids
• An array of hybrid proteins was constructed to examine the
protein activity in a format that allowed the assay of every
predicted ORF. The hybrid array used in this study was a
set of yeast colonies derived from about 6,000 individual
transformants.
• The transformation event inserts one of the yeast ORFs
into a Gal4 transcription-activation domain vector to create
a hybrid protein.
• To enable rapid, large scale transformation, they generated
the ORFs as a set of PCR products with 70bp sequences at
their 5’ and 3’ ends that precisely matched sequences in the
activation domain vector pOAD.
More on the protein array…
• After transformation of a yeast two hybrid reporter
strain , they pooled two colonies from each
transformation plate to constitute a single array
element with the entire array contained on 16
microassay plates of 384 colonies each.
• Strains expressing different ‘bait’ molecules, 192
in all, were mated to each member of the array and
the positive interactions were identified on His3
deficient plates.
Results for this assay
• 87 of the 192 DNA-binding domain hybrids
screened were identified in a putative
protein-protein interaction, resulting in 281
interacting proteins.
• Exclusion of some known interactions
occurred due to unknown variability within
the data.
High-throughput screen of an
activation–domain library
• As an alternative method this team developed highthroughput screens based on a library made by pooling
transformants containing 6,000 potential ORFs fused to the
Gal4 activation domain
• In contrast to the array, the library screening method does
not keep cells expressing the 6,000 prey molecules
separate. Instead it pools them and each of the 6,000
strains expressing a different bait is mated with the pool.
Hybrid cells are selected and then screened for positive
interactions
More on the Library…
• The same PCR products and recipient plasmids
were used to generate two collections of
transformants, each consisting of 64 barcoded 96well plates.
• 87% were successfully cloned into both plasmids.
• After mating each DNA-binding domain to the
activation domain, they were transferred to
selective plates to select interacting pairs that
activated URA3 and lacZ reporter genes.
Results for the library
• 817 yeast ORFs (15%) were identified in a
putative protein-protein interaction,
resulting in 692 interacting protein pairs.
• Interactions identified
Independent experiment
286 (41%)
Multiple times in single exp.
186 (27%)
Only once
220 (32%)
Comparison of the approaches
• The two strategies yield different results. The array method is more
efficient, which is only partly attributable to the judicious choice of
baits. The library approach, while benefiting from much high
throughput, has the disadvantage that cells in the ‘prey’ pool compete
with one another during mating, so selecting against cell expressing
fusion products that retard either process. Thus, of the 12 ‘baits ‘ that
gave positive interactions with both screens 48 possible partners were
identified by the array approach, against only 14 in the library screen.
Visual Comparison
• Top: the array of
haploid transfomants
on leucine minus
plates.
• Bottom: Diploid
colonies after two
weeks of growth on a
tryptophan minus
plate.
Discussion
• Arrays of biomolecules possess unique advantages for the
handling and investigation of multiple samples. They
provide a fixed location for each element such that those
scoring positive in an assay are immediately identified;
they have the capacity to be comprehensive and of high
density.
• The high throughput library approach is reasonable to
employ in order to complete a screen of all encoded ORFs
of an organism, however the array approach, while more
time and labor intensive would probably provide more
positives.
Three types of Discoveries
1) Guilty by association - Interactions between
proteins of known and unknown function have
indeed allowed the role of the latter to be
inferred.
2) Unrecognized interactions have been identified
between proteins involved in the same biological
process.
3) The screen has provided clues for seeing how
individual biological events are integrated into
larger cellular process.
Quick Overview
• This study was interested in examining the
putative protein-protein interactions identified in
these screens in reference to there functional roles
according to the yeast protein database.
• 32% of the interactions found were between
proteins with no functional classification. This
observation indicates that there may be a
significant number of as yet undiscovered
pathways and/or complexes that can be identified
using systematic approaches.
Illustration of Interaction
• An example of the
data analysis software
that illustrates the
interactions revealed
and provides an
overview of the
mechanisms involved.
Questions?