Marcel

through the mating factor signaling pathway when activated by the GPCR agonist [22]. In order to get efficient downstream coupling with the mating factor pathway kinases, the amino terminal domain of the Ga protein can be replaced by the same region of the human Ga protein [22,23]. The GPCRs expressed in this manner in yeast are useful for screening for agonists. Coupling can also be obtained with the natural yeast Ga protein [24,25].

The natural ligands of GPCRs can be large peptide ligands. In mammalian cells, these GPCRs can be activated by these peptides as well as their derivatives. Antagonists are identified by finding compounds that can displace these peptides. Short peptides can be coexpressed with GPCR in yeast to develop functional antagonist screens as well as for identifying ligands for orphan GPCRs [26].

Orphan GPCRs are those receptors that have been cloned by sequence homology to known GPCRs but whose function and natural ligand are not yet known. Pep-tide libraries have been expressed with secreted sequence tags that are secreted into the surface of the yeast where they come into contact with the GPCRs. Using these libraries, peptides that specifically interact with and activate the receptor in an autocrine manner can be identified.

GPCRs have been expressed in E. coli with limited success. However, E. coli has no naturally occurring GPCRs and there is no G-protein signaling pathway. Therefore both the G-protein and the GPCRs have been expressed in E. coli to obtain receptors that bind receptor agonists [27,28].

3. Functional Expression of Channels in S. cerevisiae

Potassium channels are important targets for cardiovascular, immunological, and neurological diseases. Functional screens for K+ channel openers and blockers involve expensive equipment and are technically difficult. Therefore simpler assays for developing high-throughput screens have been welcomed. A simple functional screen was developed in Saccharomyces using complementation of Trk potassium transporter knockouts [29] (Fig. 5). The inwardly rectifying potassium channel IRK1 has also been expressed in yeast to complement the Trk transporter defect. In the strain expressing the IRK1 channel, the ion channel with K+ channels

Figure 5 Functional expression of MinK potassium channel in S. cereviseae. MinK is a potassium channel found in the heart. It can complement the knockout of the potassium transporters TRK1 and TRK2 in yeast and allows the mutant yeast to grow in a low-potassium medium. Blocking of the MinK channel in yeast will prevent growth of the yeast in a low-potassium medium.

with K+ channels

Complement the K+ transporters

Complement the K+ transporters

Growth in the presence of a K+ channel opener m activity correlates well with the growth phenotype and with patch clamp experiments in Xenopus oocytes expressing these channels.

The influenza M2 channel has been expressed in S. cerevisiae [30]. The influenza M2 channel is a proton channel that is expressed in infected cells: its function is to increase the acidity of the milieu in which the virus sheds its capsid. When expressed in S. cerevisiae, the M2 proton channel increases the permeability of yeast membrane to ions resulting in loss of yeast cell viability. In order to develop a screen to find influenza M2 protein inhibitors, it was expressed from a galactose-inducible promoter (Fig. 6). The screen was designed to find compounds that permit growth and rescue the cells from the permeabilizing effects of M2 protein when the growth medium is supplemented with galactose.

Channel screens designed in S. cerevisiae have been useful for high-throughput screening. However, yeast is slow growing, and expression of channels in this microbe is difficult and time-consuming. E. coli provides an alternative for developing screens to find influenza virus M2 inhibitors. The influenza

Figure 6 Functional expression of the influenza virus M2 proton pump in S. cereviseae. The influenza M2 channel occurs as a tetramer and acts as a proton pump in virus infected cells. Expression of this channel in yeast increases membrane permeability and causes cell death. Blockers of the channel allow growth of yeast cells expressing M2.

Figure 6 Functional expression of the influenza virus M2 proton pump in S. cereviseae. The influenza M2 channel occurs as a tetramer and acts as a proton pump in virus infected cells. Expression of this channel in yeast increases membrane permeability and causes cell death. Blockers of the channel allow growth of yeast cells expressing M2.

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