A protein interaction map for cell polarity development.

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

Genetic selection of peptide inhibitors of biological pathways.

Genetic selections were used to find peptides that inhibit biological pathways in budding yeast. The peptides were presented inside cells as peptamers, surface loops on a highly expressed and biologically inert carrier protein, a catalytically inactive derivative of staphylococcal nuclease. Peptamers that inhibited the pheromone signaling pathway, transcriptional silencing, and the spindle checkpoint were isolated. Putative targets for the inhibitors were identified by a combination of two-hybrid analysis and genetic dissection of the target pathways. This analysis identified Ydr517w as a component of the spindle checkpoint and reinforced earlier indications that Ste50 has both positive and negative roles in pheromone signaling. Analysis of transcript arrays showed that the peptamers were highly specific in their effects, which suggests that they may be useful reagents in organisms that lack sophisticated genetics as well as for identifying components of existing biological pathways that are potential targets for drug discovery.

Progress and variations in two-hybrid and three-hybrid technologies.

The original yeast two-hybrid system and its variants have proven to be effective tools for identification and analysis of protein-protein, protein-DNA and protein-RNA interactions. The two-hybrid assay is being applied to the entire complement of proteins of the yeast Saccharomyces cerevisiae to characterize the network of protein-protein interactions in the eukaryotic cell. The development of nontranscriptional cytosolic and membrane-associated two-hybrid methods has made it possible to detect and examine a number of protein-protein interactions in their normal cellular locations. Small-molecule hybrid systems have been developed which can be used to study protein-ligand interactions and to activate cellular processes by forcing protein associations.

The GCN4 leucine zipper can functionally substitute for the heat shock transcription factor's trimerization domain.

The heat shock transcription factor (HSF) is the only known sequence-specific, homotrimeric DNA-binding protein. HSF binds to a DNA recognition site called a heat shock element (HSE), which contains varying numbers of nGAAn units ("GAA boxes") arranged in inverted repeats. To investigate the role of trimerization on HSF's DNA-binding properties, we replaced the trimerization domain, which self-assembles to form a three-stranded alpha-helical coiled coil, with the GCN4 leucine zipper, which forms a two-stranded alpha-helical coiled coil. Surprisingly, this substitution did not effect the ability of HSF to function in vivo. Biochemical studies of an HSF-leucine zipper chimera in comparison to an HSF truncation show that the HSF-leucine zipper chimera, though dimeric in solution and dimeric when bound to a two-box HSE, forms a trimeric complex when bound to a three-box HSE. The ability to form trimers depends on the presence of three contiguous GAA boxes present in inverted repeats. The proximity of the leucine zippers due to the orientation of the binding sites suggests that the leucine zippers might be forming a three-stranded coiled coil and several experiments lend support to this model. The ability of the leucine zipper to change oligomeric states in context might explain why the leucine zipper can replace the trimerization domain of HSF in vivo.

Environment-sensitive labels in multiplex fluorescence analyses of protein-DNA complexes.

Fluorescein is widely used for protein labeling because of its high extinction coefficient and fluorescence emission quantum yield. However, its emission is readily quenched by various pathways. We exploit these properties of fluorescein to examine the self-association of a DNA binding protein and determine the amount of the protein in gel-shifted complexes with specific DNA. A construct (HSFDT385SH) of the heat shock transcription factor (HSF) was expressed that contains the DNA-binding and trimerization domains, residues 192-385 of HSF, with four additional COOH-terminal residues, GMLC, and then labeled at the COOH-terminal cysteine with fluorescein 5-maleimide to form HSFDT385-Fl. The fluorescence increase accompanying the formation of heterotrimers on titration of HSFDT385-Fl with HSFDT385SH) led to an estimate of 3 x 10(-16) M2 for the equilibrium constant for trimerization of HSFDT385SH. HSFDT385-Fl fluorescence also increased 1.7-fold on binding to specific DNA, but not to nonspecific DNA. The protein and DNA content of the several gel-shifted complexes of HSFDT385-Fl (lambdamaxem 532 nm) with specific DNA labeled noncovalently with the energy transfer heterodimer TOTAB (lambdamaxem 658 nm) were accurately determined by a two-color fluorescence emission assay with 488 nm excitation.

Stable fluorescent dye-DNA complexes in high sensitivity detection of protein-DNA interactions. Application to heat shock transcription factor.

The gel mobility-shift assay is an important tool for the study of protein-nucleic acid interactions. High detection sensitivity is typically attained by radioisotopic labeling of the target nucleic acid fragments. A novel fluorescence methodology offers significant advantages over this conventional approach. Ethidium, thiazole orange, and oxazole yellow homodimers form stable, highly fluorescent complexes with double-stranded DNA that can be detected in gels by a laser-excited, confocal, fluorescence scanning system with a sensitivity higher than that attainable with radioisotopic labeling. We describe here the use of these dyes in a gel-mobility assay to detect complexes of a truncation of the Kluyveromyces lactis heat shock transcription factor, containing the trimerization and DNA-binding domains (HSFDT), with target DNA. At an appropriate molar DNA base pair to dye ratio, the labeling of a DNA fragment with dimeric dye did not affect the binding to HSFDT. The detection of the fluorescent-dye labeled HSFDT-DNA complexes with the laser scanner achieves a spatial resolution far superior to that of conventional autoradiography and permits analysis of multimer protein-DNA complexes that are not resolved by traditional detection methods. We have used this technique to demonstrate that HSF forms multimeric complexes on DNA by addition of trimeric units. The latter conclusion is based on an analysis of the mobilities of the multiple HSFDT-DNA complexes and on a two-color mobility-shift fluorescence assay that uses a mutant of HSFDT engineered for site-specific labeling with fluorescein and target DNA labeled with an "energy transfer" dye, thiazole orange-thiazole blue heterodimer.