1. Functional organization of the yeast proteome by systematic analysis of protein complexes
Presented by Nathalie Kirshman and Xinyi Ma

2. Background
Protein complexes
TAP (tandem-affinity purification)

3. Background – protein complexes
At the biochemical level, proteins rarely act alone; rather, they interact with others to perform cellular tasks
Protein complexes:
a unity of protein component
Component
Protein complex is a unity of protein component, and has its cellular function

4. Significance – protein complex
A higher-order map
contains fundamental biological information
offers the context for a more reasoned and informed approach to drug discovery

5. Background - TAP
A purification technique for studying protein-protein interaction.

6. Background - TAP
A purification technique for studying protein-protein interaction.
A fusion protein =
TAP tag + tagged protein
3 parts: CBP+TEV+Protein A
Entry point

7. In this study… Backer’s yeast, S. cerevisiae
A model system relevant to human biology
Process 1,739 genes, and purify 589 protein assemblies
Define 232 distinct multiprotein complexes
Propose new cellular roles for 344 protein
Most important point of the study: find the interaction b/w proteins, and assign the functional role of proteins.

8. & Using TAP and mass spectrometry to analyze protein complexes
Figure 1
Assess the approach
Statistic results during organization of purified assemblies into complexes
Figure 2
Using “reverse” analysis to reveal high composition integrity
(specific example)
Figure 3
Investigating the relationships between complexes
Figure 4
Parallel analysis of human and yeast complexes by comparison of orthologous proteins
Figure 5

9. SDS-PAGE: separate purified protein assemblies into protein components
1,739 genes  1,548 can be tagged by TAP  1,167 in detectable level  589 successful purified tagged proteins
Technical bias against protein below 15k
SDS-PAGE: separate purified protein assemblies into protein components
MALDI-TOF: a mass spectrometry, identify protein components

10. Efficiency of the approach
Of the 589, 78% presented associated partners  efficient for the large scale identification
Possible causes of remaining 22%:
May not form sufficiently stable or soluble complexes
TAP tag may interfere with complex assembly or protein localization and function

11. Statistic results during organization of purified assemblies into complexes
The approach can provide deep insights of protein components and complexes:
location, coverage of components, novelties, number of components, distribution and orthologues distribution

12. Using “reverse” analysis to reveal high composition integrity
A specific example of Polyadenylation Machinery

13. Using “reverse” analysis to reveal high composition integrity
A specific example of Polyadenylation Machinery

14. Using Pta1 as the entry point, we identified 12 known and 7 new components
The composition of the complex can be validated by “reverse analysis”
Use other component
as entry point

15. Investigating the Relationships Between Complexes
Complexes that shared components were linked, creating a plot with a network of components.
Complexes that were at least 50% orthologues are shown at double the size.
The different colors represent different cellular roles.
Ie. orange = mRNA metabolism, red = cell cycle, etc.

16. Investigating the Relationships Between Complexes
This is zoomed in to a small area of the previous picture.
It is an example of a complex (yeast TAP-C212) that is linked to two other complexes (yeast TAP C77 and TAP C110).

17. Parallel Analysis of Human and Yeast Complexes by Comparison of Orthologous Proteins
A comparison was done between the Arp2/3 complex in yeast and in the Arp2/3 complex humans.
The orthologous pairs are indicated by arrows.
This demonstrates that there is a conservation of complex composition between yeast and humans.

18. Parallel Analysis of Human and Yeast Complexes by Comparison of Orthologous Proteins

19. Critique
The TAP tag can interfere with complex assembly and protein localization/function, so they were unable to purify and identify interactions with 22% the purified tagged proteins.
They mentioned that there is a bias between proteins that are smaller than 15K.

20. Summary
Overall, this method is efficient for the retrieval and identification of cellular protein complexes.
This method also facilitated the study of complex relationships.
There are some limitations with the approach, including the TAP tag’s interference.

21. Further Reading
<- Discusses the usage of the two-hybrid system to determine protein-protein interactions.
<- Discusses the usage of proteome chips to analyze protein interactions and activity.