Although disruption of an essential gene required for cell growth will yield nonviable spores, this method provides little information about what the encoded protein actually does in yeast cells. To learn more about how a specific gene contributes to cell growth and viability, investigators must be able to selectively inactivate the gene in a population of growing cells. One method for doing this employs a regulated promoter to selectively shut off transcription of an essential gene.

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A useful promoter for this purpose is the yeast GAL1 promoter, which is active in cells grown on galactose but completely inactive in cells grown on glucose. In this approach, the coding sequence of an essential gene (gene X) is ligated to the GAL1 promoter and inserted into a yeast shuttle vector (see Figure 6-15a). The recombinant vector is then introduced into haploid yeast cells in which gene X has been disrupted. Haploid cells that are transformed will grow on galactose medium because the normal copy of gene X on the vector is expressed in the presence of galactose. When the cells are transferred to a glucose-containing medium, however, gene X no longer is transcribed; as the cells divide, the amount of the encoded protein X declines, eventually reaching a state of depletion that mimics a loss-of-function mutation. The observed changes in the phenotype of these cells after the shift to glucose medium may suggest which cellular processes depend on the protein encoded by the essential gene X.

In an early application of this method, researchers explored the function of Hsp70 genes in yeast. Haploid cells with disruptions in all four redundant Hsp70 genes were nonviable unless the cells carried a vector containing a copy of a Hsp70 gene that could be expressed from the GAL1 promoter on galactose medium. On transfer to glucose medium, the vector-carrying cells eventually stopped growing because of insufficient Hsp70 activity. Careful examination of these dying cells revealed that their secretory proteins could no longer enter the endoplasmic reticulum (ER). This study provided the first evidence for the unexpected role of Hsp70 proteins in translocation of secretory proteins into the ER, a process examined in detail in Chapter 13.