A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae
TL;DR: Examination of large-scale yeast two-hybrid screens reveals interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes.
Abstract: Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.
Citations
More filters
TL;DR: Developments in this field are reviewed, including such concepts as the small-world effect, degree distributions, clustering, network correlations, random graph models, models of network growth and preferential attachment, and dynamical processes taking place on networks.
Abstract: Inspired by empirical studies of networked systems such as the Internet, social networks, and biological networks, researchers have in recent years developed a variety of techniques and models to help us understand or predict the behavior of these systems. Here we review developments in this field, including such concepts as the small-world effect, degree distributions, clustering, network correlations, random graph models, models of network growth and preferential attachment, and dynamical processes taking place on networks.
17,647 citations
Cites background from "A comprehensive analysis of protein..."
...Interaction networks have been studied by a number of authors [206, 212, 274, 376, 394]....
[...]
TL;DR: The major concepts and results recently achieved in the study of the structure and dynamics of complex networks are reviewed, and the relevant applications of these ideas in many different disciplines are summarized, ranging from nonlinear science to biology, from statistical mechanics to medicine and engineering.
9,441 citations
TL;DR: This work states that rapid advances in network biology indicate that cellular networks are governed by universal laws and offer a new conceptual framework that could potentially revolutionize the view of biology and disease pathologies in the twenty-first century.
Abstract: A key aim of postgenomic biomedical research is to systematically catalogue all molecules and their interactions within a living cell. There is a clear need to understand how these molecules and the interactions between them determine the function of this enormously complex machinery, both in isolation and when surrounded by other cells. Rapid advances in network biology indicate that cellular networks are governed by universal laws and offer a new conceptual framework that could potentially revolutionize our view of biology and disease pathologies in the twenty-first century.
7,475 citations
Cites background from "A comprehensive analysis of protein..."
...2 , which shows the protein interaction map of the yeast Saccharomyces cerevisiae as predicted by systematic two-hybrid screen...
[...]
TL;DR: It is demonstrated that the phenotypic consequence of a single gene deletion in the yeast Saccharomyces cerevisiae is affected to a large extent by the topological position of its protein product in the complex hierarchical web of molecular interactions.
Abstract: The most highly connected proteins in the cell are the most important for its survival. Proteins are traditionally identified on the basis of their individual actions as catalysts, signalling molecules, or building blocks in cells and microorganisms. But our post-genomic view is expanding the protein's role into an element in a network of protein–protein interactions as well, in which it has a contextual or cellular function within functional modules1,2. Here we provide quantitative support for this idea by demonstrating that the phenotypic consequence of a single gene deletion in the yeast Saccharomyces cerevisiae is affected to a large extent by the topological position of its protein product in the complex hierarchical web of molecular interactions.
5,115 citations
TL;DR: The analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions, which contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.
Abstract: 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.
4,895 citations
Cites background from "A comprehensive analysis of protein..."
...The only comprehensive protein-interaction studies are based on ex vivo and in vitro systems, such as two-hybrid system...
[...]
References
More filters
TL;DR: A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score.
88,255 citations
"A comprehensive analysis of protein..." refers methods in this paper
...The resulting sequences were compared with the yeast sequence database using Blast2 (ref...
[...]
TL;DR: The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars1, although not for plasmids containing a 2 microns origin replication.
Abstract: Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D13-1A with plasmid YRp7 were studied in detail with CsCl. The optimum incubation time was 1 h, and the optimum cell concentration was 5 x 10(7) cells per ml. The optimum concentration of Cs+ was 1.0 M. Transformation efficiency increased with increasing concentrations of plasmid DNA. Polyethylene glycol was absolutely required. Heat pulse and various polyamines or basic proteins stimulated the uptake of plasmid DNA. Besides circular DNA, linear plasmid DNA was also taken up by Cs+-treated yeast cells, although the uptake efficiency was considerably reduced. The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars1, although not for plasmids containing a 2 microns origin replication. Images
6,673 citations
TL;DR: A novel genetic system to study protein-protein interactions between two proteins by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae, which may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.
Abstract: Protein-protein interactions between two proteins have generally been studied using biochemical techniques such as crosslinking, co-immunoprecipitation and co-fractionation by chromatography. We have generated a novel genetic system to study these interactions by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. This protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization. It consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG); and a C-terminal domain containing acidic regions, which is necessary to activate transcription. We have generated a system of two hybrid proteins containing parts of GAL4: the GAL4 DNA-binding domain fused to a protein 'X' and a GAL4 activating region fused to a protein 'Y'. If X and Y can form a protein-protein complex and reconstitute proximity of the GAL4 domains, transcription of a gene regulated by UASG occurs. We have tested this system using two yeast proteins that are known to interact--SNF1 and SNF4. High transcriptional activity is obtained only when both hybrids are present in a cell. This system may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.
6,529 citations
"A comprehensive analysis of protein..." refers methods in this paper
...We chose to use the yeast two-hybrid system because it can identify pairs of proteins that physically associate with one anothe...
[...]
[...]
Université catholique de Louvain1, McGill University2, Stanford University3, Pierre-and-Marie-Curie University4, Ludwig Maximilian University of Munich5, Centre national de la recherche scientifique6, École Normale Supérieure7, Washington University in St. Louis8, John Radcliffe Hospital9, Max Planck Society10, University of Basel11, University of Manchester12
TL;DR: The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration and provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history.
Abstract: The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.
4,254 citations
"A comprehensive analysis of protein..." refers methods in this paper
...[a] In the YPD database, proteins have been classi®ed into at least one category, and 32% (1,109) have been placed in more than one category....
[...]