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

Background Protein complexes TAP (tandem-affinity purification)

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

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

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

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

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.

& 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

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

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

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

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

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

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

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.

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).

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.

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

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.

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.

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.