We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing over 23 million double mutants, identifying ~550,000 negative and ~350,000 positive genetic interactions. This comprehensive network maps genetic interactions for essential gene pairs, highlighting essential genes as densely connected hubs. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections among gene pairs, rather than shared functionality. The global network illustrates how coherent sets of genetic interactions connect protein complex and pathway modules to map a functional wiring diagram of the cell.

A detection system for interactions between membrane proteins in vivo is described. The system is based on split-ubiquitin

The type IIa Na ؉ -dependent inorganic phosphate (Na/ P i ) cotransporter is localized in the apical membrane of proximal tubular cells and is regulated by an endocytotic pathway. Because molecular processes such as apical sorting, internalization, or subsequent degradation might be assisted by associated proteins, a yeast twohybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed. Most of the potential proteins found belonged to proteins with multiple PDZ modules and were either identical/related to PDZK1 or identical to NHERF-1. Yeast trap truncation assays confined the peptide-protein association to the C-terminal amino acid residues TRL of type IIa cotransporter and to single PDZ domains of each identified protein, respectively. The specificity of these interactions were confirmed in yeast by testing other apical localized transmembraneous proteins. Moreover, the type IIa protein was recovered in vitro by glutathione S-transferase-fused PDZ proteins from isolated renal brush border membranes or from type IIa-expressing oocytes. Further, these PDZ proteins are immunohistochemically detected either in the microvilli or in the subapical compartment of proximal tubular cells. Our results suggest that the type IIa Na/P i cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, parathyroid hormone controlled endocytosis or the lysosomal sorting of internalized type IIa cotransporter.In kidney, reabsorption of filtered inorganic phosphate (P i ) takes place along the proximal tubules and is controlled by a variety of hormones (e.g. parathyroid hormone, PTH) 1 and other factors (e.g. dietary intake of P i ) (1, 2). Three structurally unrelated sodium-dependent phosphate (Na/P i ) cotransporter families have been identified (1, 3). By immunohistochemistry, it was apparent that members of the type I and the type IIa Na/P i cotransporters are located in the apical membrane of proximal tubular cells (4, 5). Targeted inactivation of the type IIa Na/P i cotransporter gene (npt2) provided strong evidence that ϳ70% of Na-dependent P i transport across the brush border membrane is mediated by the type IIa Na/P i cotransporter (6). Furthermore, the type IIa cotransporter represents the major target for the many factors described to regulate proximal tubular P i reabsorption (2). Additionally, reduced proximal P i -reabsorption, as observed in X-linked hypophosphatemia, is due to a decreased abundance of the type IIa Na/P i cotransporter (7).According to the current mechanistic view, inhibition of proximal tubular Pi-reabsorption, such as by PTH or by a diet of high P i content (acutely given), is achieved by a removal of type IIa cotransporters (2) from the apical membrane. Results obtained from in vivo (rats) and in vitro (OK cells) studies indicated that internalized type IIa Na/P i cotransporters are subjected to degradation in the lysosomes (8, 9). Besides Na/P i cotransport, the activity of the brush border Na/H exchanger, NHE-3, is regulated ...

Acetylation of Saccharomyces cerevisiae histone H3 on K56 by the histone acetyltransferase (HAT) Rtt109 is important for repairing replication-associated lesions. Rtt109 purifies from yeast in complex with the histone chaperone Vps75, which stabilizes the HAT in vivo. A whole-genome screen to identify genes whose deletions have synthetic genetic interactions with rtt109⌬ suggests Rtt109 has functions in addition to DNA repair. We show that in addition to its known H3-K56 acetylation activity, Rtt109 is also an H3-K9 HAT, and we show that Rtt109 and Gcn5 are the only H3-K9 HATs in vivo. Rtt109's H3-K9 acetylation activity in vitro is enhanced strongly by Vps75. Another histone chaperone, Asf1, and Vps75 are both required for acetylation of lysine 9 on H3 (H3-K9ac) in vivo by Rtt109, whereas H3-K56ac in vivo requires only Asf1. Asf1 also physically interacts with the nuclear Hat1/Hat2/Hif1 complex that acetylates H4-K5 and H4-K12. We suggest Asf1 is capable of assembling into chromatin H3-H4 dimers diacetylated on both H4-K5/12 and H3-K9/56.

Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein-protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.

We carried out a large-scale screen to identify interactions between integral membrane proteins of Saccharomyces cerevisiae by using a modified split-ubiquitin technique. Among 705 proteins annotated as integral membrane, we identified 1,985 putative interactions involving 536 proteins. To ascribe confidence levels to the interactions, we used a support vector machine algorithm to classify interactions based on the assay results and protein data derived from the literature. Previously identified and computationally supported interactions were used to train the support vector machine, which identified 131 interactions of highest confidence, 209 of the next highest confidence, 468 of the next highest, and the remaining 1,085 of low confidence. This study provides numerous putative interactions among a class of proteins that have been difficult to analyze on a high-throughput basis by other approaches. The results identify potential previously undescribed components of established biological processes and roles for integral membrane proteins of ascribed functions.Saccharomyces cerevisiae ͉ split-ubiquitin ͉ support vector machine S ystematic studies of protein interactions in yeast have provided insights into the functions of many of the proteins encoded by this single-celled eukaryote. However, the roles of many integral membrane proteins remain poorly understood. Biochemical purifications require detergents to isolate proteins away from lipid molecules, and the large-scale nature of the affinity precipitation͞mass spectrometry projects (1, 2) precluded adjusting the detergents for individual integral membrane proteins. Two-hybrid assays (3, 4) require that the two proteins localize to the nucleus; integral membrane proteins, targeted to an aqueous nuclear environment, may aggregate or misfold.To increase the representation of integral membrane proteins in the protein-protein interaction network of Saccharomyces cerevisiae, we examined pair-wise interactions among 705 integral membrane proteins by the split-ubiquitin membrane yeast two-hybrid system (5). This modified form of the split-ubiquitin assay (6-8) is one of several hybrid protein approaches that detect interactions occurring at membranes. The split-ubiquitin membrane yeast two-hybrid system allows direct identification of yeast transformants that encode a pair of interacting proteins by use of a transcriptional reporter.Analyses of previous large-scale interaction data sets revealed significant numbers of false negatives and false positives (9). False negatives may represent interactions unsuitable for detection by a particular technique and, thus, may not be easily remedied. False positives can potentially be identified by a failure to be validated through additional experiments. However, the large-scale nature of this study and the difficulties associated with biochemical analysis of integral membrane proteins preclude confirmation of these results by alternative experimental approaches. Therefore, we used a learning algorithm, the support vector machi...

Interphase chromatin is arranged into topologically separated domains comprising gene expression and replication units through genomic sequence elements, so-called MAR or SAR regions (for matrix- or scaffold-associating regions). S/MAR regions are located near the boundaries of actively transcribed genes and were shown to influence their activity. We show that scaffold attachment factor B (SAF-B), which specifically binds to S/MAR regions, interacts with RNA polymerase II (RNA pol II) and a subset of serine-/arginine-rich RNA processing factors (SR proteins). SAF-B localized to the nucleus in a speckled pattern that coincided with the distribution of the SR protein SC35. Furthermore, we show that overexpressed SAF-B induced an increase of the 10S splice product using an E1A reporter gene and repressed the activity of an S/MAR flanked CAT reporter gene construct in vivo . This indicates an association of SAF-B with SR proteins and components of the transcription machinery. Our results describe the coupling of a chromatin organizing S/MAR element with transcription and pre-mRNA processing components and we propose that SAF-B serves as a molecular base to assemble a 'transcriptosome complex' in the vicinity of actively transcribed genes.

High‐throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome‐scale interaction mapping. Here, we report Barcode Fusion Genetics‐Yeast Two‐Hybrid (BFG‐Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG‐Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein‐pair barcodes that can be quantified via next‐generation sequencing. We applied BFG‐Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG‐Y2H increases the efficiency of protein matrix screening, with quality that is on par with state‐of‐the‐art Y2H methods.