Yeast two-hybrid screen

Biological raw data are stored in public databanks (such as Genbank or EMBL for primary DNA sequences). The data can be submitted and accessed via the World Wide Web. Protein sequence databanks like trEMBL provide the most likely translation of all coding sequences in the EMBL databank. Sequence data are prominent, but also other data are stored, e.g.yeast two-hybrid screens, expression arrays, systematic gene-knock-out experiments, and metabolic pathways. 

PIAS (protein inhibitors of activated STATs) proteins were first discovered in yeast-two-hybrid screens as interacting molecules with STAT transcription factors. The mammalian family consists ofthe founding member PIAS3, which was described as a repressor of STAT3, and three additional members, PIAS1, PIASy (also known as PIAS4), and PIASx (also known as... 

We have previously shown that a 209 amino acid region (aa288-497, asymmetric localization domain) of Insc is necessary and sufficient for apical cortical localization and for mitotic spindle orientation along the apical-basal axis (Tio et al 1999). In a yeast two-hybrid screen we identified Partner of Inscuteable (Pins), a novel 658aa protein with multiple repeats of the Tetratricopeptide (TPR) motif. Affinity purification experiments using embryonic extracts demonstrate that Pins complexes with Insc in vivo. In vitro protein interaction assays demonstrates that Pins interacts with the Insc asymmetric localization domain (see Yu et al 2000). 

Bryant This has not been studied in Drosophila. Several Dig-like MAGUKs have been picked up in a yeast two-hybrid screen using as bait the Erb-B4 receptor (Garcia et al 2000), which is the only one of the mammalian EGF receptor family that has a C-terminus predicted to bind to PDZ domains. This interaction could be involved in controlling receptor localization. Potentially it is the same kind of interaction. 

The CSN is composed of eight subunits called CSNl to CSN8, which are highly conserved in eukaryotes, although only six of them occur in fission yeast. Two hybrid screens and biochemical methods such as far westerns, pull downs and coprecipitation defined a number of CSN subunit-subunit interactions. Figure 13.1 illustrates known subunit-subunit interactions. Initial insight into the architecture of CSN came from the first 2D electron microscopic analysis of purified CSN from human red blood cells [19] (see also Figure 13.2 below). 

Development of a Yeast Two-Hybrid Screen for Selection of Human Ras-Raf Protein Interaction Inhibitors... 

A yeast two-hybrid screening system was developed to screen for small molecules that inhibit the interaction of the Ras and the Raf proteins. Hyperpermeable yeast strains useful for high-throughput screening (HTS) for the two-hybrid system were created. Differential inhibition of the Ras-Raf vs the hsRPB4-hsRPB7 interaction allowed the identification of selective inhibitors. 

Young, K., Lin, S., Sun,L.,etal. (1998) Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen. Nat. Biotechnol. 16, 946-950. 

For years, it was thought that the cytosolic carboxyl termini as well as various intracellular loops of the 5-HT receptors bind to FRAPs. Efforts from our lab to identify FRAPs utilized yeast two-hybrid screens see Fig. 1 for an overview), phage display see Fig. 2 for an overview), and direct biochemical approaches (8-13). These studies led to the discovery of many 5-HT2A receptorinteracting proteins, including caveolin-1 (Cav-1), arrestin-2 (Arr-2), arrestin-3 (Arr-3), microtubule-associated protein-1A (MAP-1A), and postsynaptic density protein-95 (PSD-95) see Table 1). 

Kroeze WK, Willins DL, Roth BL. Proteins interacting with the third intracellular loop of human 5-HT2A serotonin receptors identified by yeast two-hybrid screening. Soc Neurosci Abstr 1999 25 1201. 

Buckholz RG, Simmons CA, Stuart JM, Weiner MP, Automation of yeast two-hybrid screening, J. Mol. Microbiol. Biotechnol., 1 135-140, 1999. 

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