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Although cloned viral ORFeomes are particularly well suited for genome-wide interaction mapping due to the limited size of viral genomes, only a few such studies have been published. Here, we summarize virus interaction mapping projects involving vaccinia virus, hepatitis C virus (HCV), potato virus A (PVA), pea seed-borne mosaic virus (PSbMV), and bacteriophage T7, as well as some projects in progress. The studies reported suggest that virus-specific coding and replication strategies must be taken into account to yield accurate numbers of protein interactions. In particular, the number of false negatives can be significant for RNA viruses expressing precursor polyproteins (because interactions between full-length mature proteins are often not detected due to incorrect processing) and for viruses replicating in the cytoplasm whose transcripts have not been selected for splicing signals. In conclusion, the studies on viral protein interaction maps suggest that cloned pathogen ORFeomes will contribute to a holistic picture of the pathogenesis of infectious diseases and are ideal starting points for new approaches in systems biology. Both viral ORFeome and interaction mapping projects are being documented on our Web site (http://itgmv1.fzk.de/www/itg/uetz/virus/).

The biological significance of protein interactions, their method of generation and reliability is briefly reviewed. Protein interaction networks adopt a scale-free topology that explains their error tolerance or vulnerability, depending on whether hubs or peripheral proteins are attacked. Networks also allow the prediction of protein function from their interaction partners and therefore, the formulation of analytical hypotheses. Comparative network analysis predicts interactions for distantly related species based on conserved interactions, even if sequences are only weakly conserved. Finally, the medical relevance of protein interaction analysis is discussed and the necessity for data integration is emphasized.

In yeast Saccharomyces cerevisiae the G protein betagamma subunits (Ste4/Ste18) have long been known to transmit the signal required for mating. Here we demonstrate that GTPase-deficient mutants of Galpha (Gpa1) directly activate the mating response pathway. We also show that signaling by activated Gpa1 requires direct coupling to an RNA binding protein Scp160. These findings suggest an additional role for Gpa1 and reveal Scp160 as a component of the mating response pathway in yeast.

All members of the regulator of G protein signaling (RGS) family contain a conserved core domain that can accelerate G protein GTPase activity. The RGS in yeast, Sst2, can inhibit a G protein signal leading to mating. In addition, some RGS proteins contain an N-terminal domain of unknown function. Here we use complementary whole genome analysis methods to investigate the function of the N-terminal Sst2 domain. To identify a signaling pathway regulated by N-Sst2, we performed genome-wide transcription profiling of cells expressing this fragment alone and found differences in 53 transcripts. Of these, 40 are induced by N-Sst2, and nearly all contain a stress response element (STRE) in the promoter region. To identify components of a signaling pathway leading from N-Sst2 to STREs, we performed a genome-wide two-hybrid analysis using N-Sst2 as bait and found 17 interacting proteins. To identify the functionally relevant interacting proteins, we analyzed all of the available gene deletion mutants and found three (vps36 Delta, pep12 Delta, and tlg2 Delta) that induce STRE and also repress pheromone-dependent transcription. We selected VPS36 for further characterization. A vps36 Delta mutation diminishes signaling by pheromone as well as by downstream components including the G protein, effector kinase (Ste11), and transcription factor (Ste12). Conversely, overexpression of Vps36 enhances the pheromone response in sst2 Delta cells but not in wild type. These findings indicate that Vps36 and Sst2 have opposite and opposing effects on the pheromone and stress response pathways, with Vps36 acting downstream of the G protein and independently of Sst2 RGS activity.

The two-hybrid system is a genetic method for detecting protein-protein interactions. The assay can be applied to random libraries or arrays of colonies that express defined pairs of proteins. Arrays enable the testing of all possible protein pairs for interactions in a systematic fashion. The array format makes a large number of individual assays comparable and thus greatly simplifies the identification of false positives. Two-hybrid arrays have been used to study interactions among the proteins of yeast, hepatitis C virus, vaccinia virus, Drosophila, Caenorhabditis elegans, mouse and other species, and have already identified thousands of interactions.

A two-hybrid screen against an activation domain array of Saccharomyces cerevisiae proteins was carried out for 31 yeast proteasome proteins. Fifty-five putative interactions were identified: 21 between components of the proteasome complex and 34 between proteasome proteins and other proteins. Many of these latter interactions involved either proteins of the ubiquitin pathway, cell cycle proteins, protein kinases or a translation initiation factor subunit. The role of eleven proteins associated with proteasome function by these screens was analyzed by examining the corresponding deletion strains for temperature sensitivity and canavanine sensitivity and for the stability of a ubiquitin-beta-galactosidase fusion protein. These assays additionally implicated three proteins, Bim1, Ump1, and YKL171W, in proteasome function. This study demonstrates the utility of genome-wide two-hybrid assays as an entry point for the further analysis of a large protein complex.

Large-scale two-hybrid screens have generated a wealth of information describing potential protein--protein interactions. When compiled with data from systematic localizations of proteins, mutant screens and other functional tests, a network of interactions among proteins and between proteins and other components of eukaryotic cells can be deduced. These networks can be viewed as maps of the cell, depicting potential signaling pathways and interactive complexes. Most importantly, they provide potential clues to the function of previously uncharacterized proteins. Focusing on recent experiments, we explore these protein-interaction studies and the maps derived from such efforts.

A global analysis of 2,709 published interactions between proteins of the yeast Saccharomyces cerevisiae has been performed, enabling the establishment of a single large network of 2,358 interactions among 1,548 proteins. Proteins of known function and cellular location tend to cluster together, with 63% of the interactions occurring between proteins with a common functional assignment and 76% occurring between proteins found in the same subcellular compartment. Possible functions can be assigned to a protein based on the known functions of its interacting partners. This approach correctly predicts a functional category for 72% of the 1,393 characterized proteins with at least one partner of known function, and has been applied to predict functions for 364 previously uncharacterized proteins.

The increasing rate at which complete genome sequences become available necessitates rapid and robust methods for investigating the functions of their encoded proteins. Efforts have been made to study protein function by systematically screening large sets of proteins using the two-hybrid method. Analyses of the complete proteomes of baceriophage T7, the mammalian viruses hepatitis C and vaccinia, as well as of several protein complexes including RNA splicing proteins and RNA polymerase III from yeast, have been undertaken. Saccharomyces cerevisiae has been studied extensively by two-hybrid methods, with more than 2500 protein-protein interactions described. Systematic studies on metazoan proteomes are, however, still in their infancy.

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.

Ld proteins (formins) are encoded by the limb deformity (ld) gene and define a family of related gene products regulating establishment of embryonic polarity. In this study we establish that chicken and murine Ld proteins interact directly with Src family kinases (c-Src and c-Fyn). Specific binding is mediated by the proline-rich domain present in Ld proteins and the ligand binding surface of the Src SH3 domain. Co-immunoprecipitation of Ld and c-Src proteins from transfected cells shows that these proteins associate in vivo. Immunolocalization and biochemical fractionation of fibroblasts confirms the predominant nuclear localization of Ld proteins, but unexpectedly identifies a population of Ld proteins associated to cellular membranes. This population of Ld proteins co-localizes with membrane-associated c-Src proteins at both plasma and perinuclear membranes. These studies indicate that the morphoregulatory Ld proteins interact with signal transduction cascades by association to membrane-bound Src family kinases.