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I used the yeast two-hybrid system to identify potential binding partners of the most abundant glutamate transporter GLT-1. This system was developed by Fields and Song (1989), taking advantage of the domain structure of the yeast Saccharomyces cerevisiae transcriptional activator GAL4. In wild-type yeast, GAL4 is involved in regulating the transcription of genes encoding enzymes for utilisation of galactose (the

lac operon, including the lacZ gene). The amino-terminal domain of GAL4 (the GAL4 DNA binding domain) binds to specific DNA sequences upstream of the lacZ gene (upstream activation sequences, UAS). The GAL4 carboxy-terminal domain (the GAL4 activation domain) binds to other components of the transcription machinery to activate transcription. Both domains are essential for function: expression of either domain alone is not sufficient to drive transcription of the lacZ gene. These two domains, however, do not need to be physically linked, but require only to be in close proximity to activate transcription. This specific feature allows this transcription factor to be used in the yeast two-hybrid system, in which proteins encoding the two GAL4 domains fused to two different polypeptides of interest are expressed in yeast cells. If the polypeptides interact with each other then the GAL4 transcription factor will be reconstituted (i.e. the two domains are brought close to each other), and transcription of lacZ is resumed. The lacZ

gene encodes the enzyme p-galactosidase, which forms a blue reaction product from the substrate X-gal. The expression of the lacZ gene can therefore be used as a reporter for a positive interaction between the candidate polypeptides by the appearance of blue yeast colonies. A strain of yeast is required which has the endogenous GAL4 activator mutated out, so that it can be transformed with plasmids encoding the DNA-binding and activation domain fusion proteins. Many commercial systems now use a yeast strain which is also null for the transcription factor controlling the gene involved in the synthesis of the amino acid histidine (the HIS3 gene). This strain of yeast is engineered so that the reconstituted GAL4 transcription factor will also activate the HIS3 gene and allow growth on medium lacking histidine (synthetic dropout, SD-his). Expression of the

HIS3 gene is therefore used as another reporter of a positive interaction between the two polypeptides. This allows for two rounds of selection based on the expression of the two reporter genes; positive interactions will activate HIS3 allowing growth on SD-his, and expression of the lacZ gene will produce the blue product in a reaction with X-gal. Furthermore, mutations in the yeast strain (in this study, Y 190), enable selection for one or the other of the two plasmids expressing proteins encoding the DNA-binding and activation domains of GAL4 fused to their respective polypeptides, as follows. Y190 does not have functional LEU2 or TRPl genes, preventing growth on medium lacking leucine or tryptophan, respectively. These genes are instead encoded on either one of the (GAL4 DNA binding domain or activation domain) plasmids that will be expressed. In the system used in this study, the protein of interest (the GLT-1 amino terminal) was expressed as a fusion with the DNA-binding domain of GAL4 in the vector pPC97 (the “bait”), and the library proteins (among which I hope to find an interacting protein) were expressed as fusions with the activation domain in the vector pPC8 6.

The yeast two-hybrid system has many advantages over traditional techniques for investigating protein-protein interactions, such as immunoprécipitation and covalent

cross-linking. Perhaps the most important advantage is that the plasmid DNA encoding the binding partner can be easily isolated and sequenced to give the identity of the protein; no antibodies or protein purification are required. Furthermore, the interaction occurs inside a living cell (a yeast cell), and the proteins are therefore more likely to adopt their native conformation than in cell-free assays. Low affinity interactions can be detected; those with dissociation constants of up to 70 p,M have been detected (Yang et al. 1995).

Unfortunately, there are also a number of limitations of the system, which have to be circumvented or controlled for. Some 'bait' proteins may have inherent activation activity, a property that would prevent their use in the system. This can be checked by ensuring that the 'bait' protein will not activate the reporter genes when expressed alone. Similarly, some library fusion proteins may be able to bind DNA, and therefore activate the reporter genes in the absence of 'bait' fusion protein. This can be checked for by expressing the library fusion protein without the 'bait' protein. The eukaryotic cellular environment was listed above as an advantage of the system, but the fact that yeast cells are used, and often mammalian proteins are the subject of study, implies that this situation is still not optimal. Indeed, certain post-translational modifications such as phosphorylation, which could influence the interaction between two proteins, might not be carried out to the same extent in yeast as they would be in a mammalian cell. The over-expression of some fusion proteins can also be toxic to yeast, so the use of vectors with low copy numbers or weaker promoters might be necessary. For these reasons, if a positive interaction is isolated, and survives the “bait-only” and “library-only” controls, subsequent confirmation by biochemical means is required to confirm that the interaction occurs in vivo, and also to investigate the relevance of the interaction in the mammalian system.

In chapter 4 of this thesis I report the isolation of two proteins that interact with the glutamate transporter GLT-1 using the yeast-two hybrid system. An introduction to both protein families is given below in section 1.6 .

1.6 The imidazoline receptor family, the LÏM-domain containing protein family,