Abstract
Proteins are organized in large protein complexes that form an extensive network in the cell. They are the most versatile macromolecule in the cell and the interactions between each other are highly directed and essential for most cellular functions. The activity of protein complexes is in turn frequently regulated by
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post-translational modifications such as acetylation, phosphorylation, etc. Since protein complexes determine to a large extend the functional organization of a cell, their investigation is of high importance to achieve a better understanding of cellular pathways and biological function. The analysis of endogenously expressed protein complexes is particularly interesting and relevant as it represents the most authentic situation in the cell. Proteins assemble with their natural interaction partner and are modulated according to a genuine situation. The introduction of tandem affinity purification procedures made it possible to isolate endogenous protein complexes on a large scale directly from cells. The procedure consists of two subsequent purification steps which keep the protein interactions intact throughout the procedure. The technique is relatively fast, reproducible and yields in pure protein complexes. By proteomics approaches large protein-protein interaction networks of S. cerevisiae have been unravelled which indicated that proteins are organized into modules of protein complexes. These proteomics experiments provided very useful information about the constituents of different protein assemblies. The next step towards a comprehensive analysis of protein assemblies is to characterize these protein assemblies in more detail. It is important to determine the direct interactions between proteins, the overall topology of protein complexes, strong and weak interacting proteins, subtle differences between closely related protein complexes and how protein interactions are modulated. We set out to describe the local network of protein complexes that are involved in RNA metabolism in the cell in detail and focus in particular on the nuclear and cytoplasmic exosome and the Ski complex. The exosome plays a crucial role in the degradation of mRNA and also in the processing of diverse RNA such as rRNA, snRNA and snoRNA. The cytoplasmic exosome requires the Ski complex as a co-activator for the degradation of mRNA. These endogenous heterogeneous proteins assemblies are characterized by a combination of tandem affinity purification technique and multiplexed mass spectrometry approaches. (Quantitative) proteomics approaches identified all exosome proteins and yielded in a first generation phosphorylation map with a relative quantitation of exosome proteins and identified phosphorylation sites between the nuclear and cytoplasmic variant. A strong emphasis is placed on native (tandem) mass spectrometry as it offers unique possibilities to investigate structural features of protein assemblies. The requirement of just a low amount of protein complex for a comprehensive mass spectrometry analysis makes this technique ideal for the analysis of endogenously expressed protein assemblies. This technique allowed the investigation of the gas-phase structure of the exosome and determined strong and weak interacting components. Moreover this technique revealed a novel heterotetrameric stoichiometry for the Ski complex. In addition, acid disruption of the complex into monomeric proteins, dimeric and trimeric subcomplexes led to an improved model of the Ski assembly.
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