Yeast-two-hybrid methods

Whether we discuss silk, proteins embedded in membranes, or soluble complexes of cytosolic proteins, we must ask questions about interactions. A first step is to identify interactions720-730 among proteins either in vitro or in living cells.731 Proteomic methods, which include the yeast two-hybrid method (Box 29-F), are widely used for this purpose. It is possible to identify large sets of interacting proteins, to identify disease states, to observe effects of drugs, and to compare metabolism among species. 

Figure 1 The classic yeast two-hybrid method and derivatives, (a) Schematic diagram of the yeast two-hybrid approach, describing an interaction between protein X and protein Y. Protein X is fused to a transcription factor DNA-binding domain (the "bait" construct), and protein Y is fused to a transcription factor activation domain (the "prey" construct), (b) High-throughput applications of the yeast two-hybrid method use mating of haploid strains carrying bait and prey, respectively. Hybrids can be mated in arrayed formats (as shown) or as libraries, (c) The reverse two-hybrid method uses a counter-selectable marker to indicate loss of protein interaction because of disruption by an inhibitor protein/small molecule ("/" illustrated in the diagram) or mutation(s) in proteins X and/or Y.

We see two major appearing frontiers for new kinds of molecular data. The first is proteomics (See Chapter 4 of volume 2) and metabolomics. With a combination of 2D gel, mass spectrometry, protein microarray and yeast-two-hybrid methods, a large amount of protein sequence, expression, and interaction data will be produced on a cell-wide level. On the one hand, bioinformatics has to address the challenge of interpreting these data. On the other hand, especially the protein interaction data will provide an interesting basis for probing deeper into the details of regulatory networks. Such data are collected in special protein interaction databases such as DIP [9,10] and BIND [11],... 

Figure 3 Schematic illustrating the reverse yeast two-hybrid method for PPI detection, (a) In the absence of any inhibitors, the reconstituted transcription factor leads to the expression of a cytotoxic product, and cell death, (b) In the presence of an inhibitor that targets the PPI, transcription is inhibited, and the cell survives.

The experiments described above indicate that technology is available to couple SPR with mass spectrometry. These methods should be useful for protein-protein interaction mapping. For example, immobilized proteins can be used as hooks for fishing binding partners from complex protein mixtures under native conditions. The coupling of techniques can lead not only to the rapid identification of interacting proteins but will also provide information on the kinetic parameters of the interaction. This approach should serve as an excellent complement to the use of in vivo techniques such as the yeast two-hybrid system. 

Bai, C., and Elledge, S. J. (1997). Gene identification using the yeast two-hybrid system. Methods Enzymol. 283, 141-156. 

It should be emphasized that the nature of all presented protocols is very general and, thus, their application for a comprehensive characterization of your favorite multiprotein complex (YFMPC) in yeast might require only minor modifications. The logical sequence of all required steps is schematically shown in Fig. 2.1. The initial large-scale Ni affinity isolation of eIF3 followed by mass spectrometry (MS) of its subunit composition has already been described (Asano et al, 2002), and methods for identification of protein-protein interactions such as yeast two-hybrid (Y2H) and in vitro glutathione-S-transferase (GST) pull-down analysis are presented in volume 429. This chapter focuses on a description of the small-scale one-step in vivo affinity purification techniques that were used to determine the effects of deletions and... 

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). 

For large-scale analysis, this strategy has been further modified for use in the array method (Ito et al., 2000 Uetz et al., 2000 Walhout et al., 2000). Such analyses in Sachromyces cerevisiae and Caenorhabditis elegans have already reported many potential interactions that need to be confirmed by further biological experimentation. Finally, another modification of the yeast two-hybrid system has been recently reported, termed reverse two-hybrid (Vidal and Endoh, 1999). This technique is being used to identify compounds and peptides that can disrupt protein-protein interactions and thus have an effect on various signaling pathways. 

Traditionally protein-protein interactions studies have been performed in vitro after isolation and purification of individual proteins. While some in vivo or in situ protein-protein interaction studies can be performed by traditional methods using microinjection of purified proteins into oocytes, technical complexities limit the number of proteins that can be studied. Furthermore, many putative proteins of interest, predicted by genomic analysis, are not characterized and cannot be used in such studies. Some of the limitations posed by traditional methods have been overcome by use of yeast two-hybrid systems. These systems allow studies of many recombinant test proteins... 

Interpretation of results of these studies is still difficult. Results of two-hybrid methods become more useful if they can be coordinated with other approaches. For example, computational methods can predict interactions from genome sequences alone. 11/0 More than 45,000 interactions have been predicted among yeast proteins. Reliable identification of such motifs as DNA-binding domains and Ca2+- binding domains can complement two-hybrid analysis.11 The yeast genome is predicted to contain 162 coiled-coil sequences and at least 213 unique interactions between them.0 Examination of sequences of protein families in the Protein Data Bank (PDB) led to prediction of 8151 interactions of 664 types between protein families in yeast.P... 

Kamada, S., H. Kusano, H. Fujita, M. Ohtsu, R.C. Koya, N. Kuzumaki, and Y. Tsujimoto. 1998. A cloning method for caspase substrates that uses the yeast two-hybrid system cloning of the antiapoptotic gene gelsolin. Proc Natl Acad Sci USA. 95 8532—7. 

Examples of such protein-protein interaction selection systems are phage display (Smith, 1985 Winter et al., 1994), display on other viruses (Kasahara et al., 1994), bacterial surface display (Georgiou et al., 1993 Daugherty et al., 1999), yeast display (Kieke et al., 1997 Boder and Wittrup, 1997), the yeast two hybrid system (Fields and Song, 1989 Chein et al., 1991), and protein-fragment complementation assays (Pelletier et al., 1998). These methods all contain a necessary in vivo step, which has a number of disadvantages that will be discussed in the following sections. 

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