Functional organization of the yeast proteome by systematic analysis of protein complexes Anne-Claude Gavin, Markus Bosche, Roland Krause, Paola Grandi, Martina Marzioch, Andreas Bauer, Jorg Schultz, Jens M. Rick, Anne-Marie Michon, Cristina-Maria Cruciat, Marita Remor, Christian Hofert, Malgorzata Schelder, Miro Brajenovic, Heins Ruffner, Alejandro Merino, Karin Klein, Manuela Hudak, David Dickson, Tatjana Rudi, Volker Gnau, Angela Bauch, Sonja Bastuck, Bettina Huhse, Christina Leutwein, Marie-Anne Heurtier, Richard R. Copley, Angela Edelmann, Erich Querfurth, Vladimir Rybin, Gerard Drewes, Manfred Raida, Tewis Bouwmesster, Peer Bork, Bertrand Seraphin, Bernhard Kuster, Gitte Neubauer, and Giulio Superti-Furga (Nature 415. 2002) Anne-Claude Gavin et al.

Background Information Proteins rarely act alone Comprehensive protein interaction studies thus far: two-hybrid systems (ex vivo) protein chips (in vitro) GST pull-downs (in vivo) 3. Authors used tandem-affinity purification (TAP) and mass spectrometry Slide 2 At the biochemical level, proteins rarely act alone, but rather interact with one another, sometimes in complexes, to carry out specific cellular processes. Two hybrid systems: (+) pairwise/binary interactions between proteins; transient associations (-) characterization of protein complexes Protein chips: (-) it is in vitro TAP: The authors used a technique called tandem-affinity purification coupled with mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharyomeces cerevisiae, the budding or baker’s yeast.

Rationale for Using TAP/MS Method Fast purification with high yield In vivo Over-expression of proteins not required Prior knowledge of protein complex not required Purified complex can be used in a variety of studies Slide 3 Allows for fast purification with high yield of protein complexes under native, physiological conditions; therefore, this is an in vivo approach. In addition, the proteins under study do not have to be overexpressed, in fact it is preferred to maintain the expression of the fusion, target protein at natural levels. This is because protein overexpression can result in nonspecific or unnatural protein interactions. Prior knowledge of complex composition and function is not necessary. Since the purification procedure is gentle, the purified complex can be used protein identification, functional, or structural studies.

The Tandem Affinity Purification Tag target protein target protein

TAP Purification Strategy

TAP Purification Strategy

TAP Purification Strategy

TAP Purification Strategy … in detail PCR of the TAP cassette Transformation of yeast cells (homologous recombination) Selection of positive clones Large-scale cultivation Cell lysis Tandem affinity purification One-dimensional SDS-PAGE MALDI-TOF protein identification Bioinformatic data interpretation

The Experiment Processed 1,739 genes (1,143 human orthologues) Purified 589 protein assemblies Annotated 232 multiprotein complexes (98 known, 134 new) Proposed new/additional cellular roles for 344 proteins (231 new) Identified 1,440 distinct gene products

The Experiment …cont’d 9% of the 232 TAP complexes had no new component 2-83 components per TAP complex Assigned cellular roles to complexes according to YPD and literature studies Nine functional categories Slide 10 Of all 232 TAP complexes, only 9% had no new component. The size of the TAP complexes ranged from 2-83 components, with an average of 12 components per complex. Assigned cellular roles to complexes by computing functional assignments of the individual components according to YPD and by literature searching.

Some Statistics

A Higher-Order Map http://yeast.cellzome.com Slide 12 After the individual proteins of the complexes have been identified, the authors want to study the relationships between complexes to understand the integration and coordination of cellular processes. This is a protein complex network, it provides a functional description of the eukaryotic proteome at a higher level of organization. The complexes are linked to each other if they share components. The cellular roles of the complexes are color coded. Dark blue = transcription, DNA maintenance, chromatin structure. Violet = intermediate and energy metabolism. The website has a software package that allows you to navigate through this proteome map at both the complex and protein levels. http://yeast.cellzome.com

Linked Complexes Slide 13 This is an example of a complex linked to two other complexes by virtue of shared components. This illustrates the relationship between the protein and complex levels of organization.

Results of the Experiment Orthologues tend to interact with orthologues (53% vs 31%) Essential genes tend to interact with essential genes (44% vs 17%) Existence of “orthologous proteome” for eukaryotes? Slide 14 Orthologous proteins tend to interact with complexes enriched with other orthologues (mean 53%). Nonorthologous proteins have a lower tendency for such interactions (31%). The same goes for the interaction of essential genes. This suggests the existence of an “orthologous proteome” that may represent core functions for the eukaryotic cell.

Some Faults of the TAP Method “Super” unstable protein complexes TAP tag may interfere with complex Transient interactions Low stoichiometric complexes Physiology-specific interactions Slide 15 The TAP method may not be able to detect complexes where proteins do not interact with sufficient stability. The TAP tag may interfere with complex formation or protein localization and function. This method may also fail to detect transient interactions, low stoichiometric complexes, and those interactions occurring in specific physiological states which exponential growing cells do not exhibit.

Conclusions This TAP/MS method is the largest analysis of protein complexes Allows efficient identification of low-abundance proteins Allows purification of very large complexes Can complement other biochemical techniques Lower-order maps and higher-order maps provide crucial information Slide 16 Lower-order maps and higher-order maps provide crucial information for drug discovery efforts and may aid in the choice and evaluation of drug targets.

Great Summer Reading Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnol. 17, 1030-1032 (1999). Puig, O. et al. The tandem affinity purification (tap) method: a general procedure of protien complex purification. Methods 24, 218-229 (2001).