Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.

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Functional organization of the yeast proteome by systematic analysis of protein complexes

Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry. Through systematic tagging of open reading frames (ORFs), the majority of complexes were purified several times, suggesting screen saturation. The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.

http://llama.mshri.on.ca/courses/Biophysics205/Papers/Gavin_2006.pdf

Proteome survey reveals modularity of the yeast cell machinery

https://www.uab.edu/proteomics/pdf_files/2006/Puig_et_al_Methods.pdf

The tandem affinity purification (TAP) method: a general procedure of protein complex purification.

Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control

In eukaryotes, seven Sm proteins bind to the U1, U2, U4 and U5 spliceosomal snRNAs while seven Smlike proteins (Lsm2p–Lsm8p) are associated with U6 snRNA Another yeast Sm-like protein, Lsm1p, does not interact with U6 snRNA Surprisingly, using the tandem affinity purification (TAP) method, we identified Lsm1p among the subunits associated with Lsm3p Coprecipitation experiments demonstrated that Lsm1p, together with Lsm2p–Lsm7p, forms a new sevensubunit complex We purified the two related Sm-like protein complexes and identified the proteins recovered in the purified preparations by mass spectrometry This confirmed the association of the Lsm2p–Lsm8p complex with U6 snRNA In contrast, the Lsm1p–Lsm7p complex is associated with Pat1p and Xrn1p exoribonuclease, suggesting a role in mRNA degradation Deletions of LSM1, 6, 7 and PAT1 genes increased the half-life of reporter mRNAs Interestingly, accumulating mRNAs were capped, suggesting a block in mRNA decay at the decapping step These results indicate the involvement of a new conserved Sm-like protein complex and a new factor, Pat1p, in mRNA degradation and suggest a physical connection between decapping and exonuclease trimming

A Sm-like protein complex that participates in mRNA degradation.

Bacteria respond to nutritional stresses by producing an intracellular alarmone, guanosine 5'-(tri)diphosphate, 3'-diphosphate [(p)ppGpp], which triggers the stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, upon fatty acid or carbon starvation, SpoT enzyme activity switches from (p)ppGpp degradation to (p)ppGpp synthesis, but the signal and mechanism for this response remain totally unknown. Here, we characterize for the first time a physical interaction between SpoT and acyl carrier protein (ACP) using affinity co-purifications and two-hybrid in E. coli. ACP, as a central cofactor in fatty acid synthesis, may be an ideal candidate as a mediator signalling starvation to SpoT. Accordingly, we show that the ACP/SpoT interaction is specific of SpoT and ACP functions because ACP does not interact with the homologous RelA protein and because SpoT does not interact with a non-functional ACP. Using truncated SpoT fusion proteins, we demonstrate further that ACP binds the central TGS domain of SpoT, consistent with a role in regulation. The behaviours of SpoT point mutants that do not interact with ACP reveal modifications of the balance between the two opposite SpoT catalytic activities thereby changing (p)ppGpp levels. More importantly, these mutants fail to trigger (p)ppGpp accumulation in response to fatty acid synthesis inhibition, supporting the hypothesis that the ACP/SpoT interaction may be involved in SpoT-dependent stress response. This leads us to propose a model in which ACP carries information describing the status of cellular fatty acid metabolism, which in turn can trigger the conformational switch in SpoT leading to (p)ppGpp accumulation.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2006.05442.x

Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism.

The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli.

Most gram-negative bacteria in search of nutrients and space in which to multiply release toxins that selectively kill competing microorganisms while causing no harm to the producing cell. The highly specific protection function is conferred on producing cells by immunity proteins produced from the

Emmanuelle Bouveret

Papers

The tandem affinity purification (TAP) method: a general procedure of protein complex purification.

A Sm-like protein complex that participates in mRNA degradation.

Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism.

The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli.

Colicin Import into Escherichia coli Cells