Since their introduction, the interaction trap and other two-hybrid systems have been used to study protein-protein interactions. Despite their general use, little is known about the extent to which the degree of protein interaction determined by two-hybrid approaches parallels the degree of interaction determined by biochemical techniques. In this study, we used a set of lexAop-LEU2 and lexAop-lacZ reporters to calibrate the interaction trap. For the calibration, we used two sets of proteins, the Myc-Max-Mxi1 helix-loop-helix proteins, and wild-type and dimerization-defective versions of the lambda cI repressor. Our results indicate that the strength of interaction as predicted by the two-hybrid approach generally correlates with that determined in vitro, permitting discrimination of high-, intermediate-, and low-affinity interactions, but there was no single reporter for which the amount of gene expression linearly reflected affinity measured in vitro. However, some reporters showed thresholds and only responded to stronger interactions. Finally, some interactions were subject to directionality, and their apparent strength depended on the reporter used. Taken together, our results provide a cautionary framework for interpreting affinities from two-hybrid experiments.Biological systems depend on interactions between protein components. These interactions affect such diverse processes as the coordination of signal transduction by assembly of multisubunit complexes (57, 58), the regulation of apoptosis by the sequestration of Bax (54), and the control of gene expression through the selective association of transcription factors (19). Efforts to understand the functions of proteins often include identification and characterization of other cellular proteins with which they can interact. While some protein interactions are of high affinity and are easily detectable by physical techniques, a number of biologically important interactions, such as those of many enzymes with their substrates, are often relatively weak or transient and are not easily detectable by these methods.A number of approaches for studying protein association are in use, including cosedimentation through gradients, coimmunoprecipitation of purified proteins, assay of DNA binding activity for proteins that must dimerize to recognize a DNA site, and assay by two-hybrid systems (20) such as the interaction trap (31). In the last approach, a first protein (P1, or ''bait'') is fused to a known DNA-binding domain such as LexA (10) or GAL4 (41) and a second protein (P2) is fused to a transcriptional activation domain (AD). Coexpression of the two chimeric proteins in yeast cells in which the cognate binding site for the DNA-binding domain is located upstream of a reporter gene results in transcriptional activation of the reporter by the P2-fused AD if the chimeric proteins associate.Two-hybrid/interaction trap approaches have gained considerable popularity because they can detect novel interacting proteins that interact with a given bait by substituting...

Budding in Saccharomyces cerevisiae follows a genetically programmed pattern of cell division which can be regulated by external signals. On the basis of the known functional conservation between a number of mammalian oncogenes and antioncogenes with genes in the yeast budding pathway, we used enhancement of pseudohyphal budding in S. cerevisiae by human proteins expressed from a HeLa cDNA library as a morphological screen to identify candidate genes that coordinate cellular signaling and morphology. In this report, we describe the isolation and characterization of human enhancer of filamentation 1 (HEF1), an SH3-domaincontaining protein that is similar in structure to p130 cas , a recently identified docking protein that is a substrate for phosphorylation by a number of oncogenic tyrosine kinases. In contrast to p130 cas , the expression of HEF1 appears to be tissue specific. Further, whereas p130 cas is localized predominantly at focal adhesions, immunofluorescence indicates that HEF1 localizes to both the cell periphery and the cell nucleus and is differently localized in fibroblasts and epithelial cells, suggesting a more complex role in cell signalling. Through immunoprecipitation and two-hybrid analysis, we demonstrate a direct physical interaction between HEF1 and p130 cas , as well as an interaction of the SH3 domain of HEF1 with two discrete proline-rich regions of focal adhesion kinase. Finally, we demonstrate that as with p130 cas , transformation with the oncogene v-abl results in an increase in tyrosine phosphorylation on HEF1, mediated by a direct association between HEF1 and v-Abl. We anticipate that HEF1 may prove to be an important linking element between extracellular signalling and regulation of the cytoskeleton.

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Under conditions of environmental stress, prokaryotes and lower eukaryotes such as the yeast Saccharomyces cerevisiae selectively utilize particular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival. In S. cerevisiae, a complex of two such subunits, RPB4 and RPB7, preferentially associates with pol II during stationary phase; of these two subunits, RPB4 is specifically required for survival under nonoptimal growth conditions. Previously, we have shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus. Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in higher eukaryotes. In contrast to hsRPB7, hsRPB4 has diverged so that it no longer interacts with yeast RPB7, although it partially complements rpb4 ؊ phenotypes in yeast. However, hsRPB4 associates strongly and specifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II. hsRPB4 expression in humans parallels that of hsRPB7, supporting the idea that the two proteins may possess associated functions. Structure-function studies of hsRPB4-hsRPB7 are used to establish the interaction interface between the two proteins. This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequent structural and functional characterization of the pol II holoenzyme.Selective control of mRNA transcription in response to intracellular and extracellular signals occurs at multiple levels, with targets for regulation including gene-specific transcription factors, general transcription factors, and the RNA polymerase II holoenzyme (15,18,38,55,63). This last mechanism of regulation, involving modification of core RNA polymerase II (pol II) structural composition by altering incorporation of subunits or regulated phosphorylation, has been well documented in prokaryotes and in yeast (17,31,59,62,70). In higher eukaryotes, the majority of transcriptional control studies have focused on characterizing the expression and modification of gene-specific and general transcription factors. However, a growing body of work on mammalian transcriptional control has demonstrated that mammalian pol II is also subject to modification by phosphorylation of the largest subunit, presumably as a means of regulation (10,24,41,42,49). In contrast, the issue of subunit variation has not been actively investigated.Studies of eukaryotic pol II function have depended heavily on paradigms developed through detailed characterization of the yeast Saccharomyces cerevisiae pol II (reviewed in reference 70). Yeast pol II contains 12 subunits (RPB1-12), all of which have been cloned and sequenced and many of which have been subjected to genetic and biochemical functional analysis. Five of these subunits, the common subunits (RPB5, RPB6, RPB8, RPB10, and RPB12), are also incorporated into RNA polymeras...

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