Academic journal article Genetics

Genetically Engineered Transvestites Reveal Novel Mating Genes in Budding Yeast

Academic journal article Genetics

Genetically Engineered Transvestites Reveal Novel Mating Genes in Budding Yeast

Article excerpt

ABSTRACT Haploid budding yeast has two mating types, defined by the alleles of the MAT locus, MATa and MATa. Two haploid cells of opposite mating types mate by signaling to each other using reciprocal pheromones and receptors, polarizing and growing toward each other, and eventually fusing to form a single diploid cell. The pheromones and receptors are necessary and sufficient to define a mating type, but other mating-type-specific proteins make mating more efficient. We examined the role of these proteins by genetically engineering "transvestite" cells that swap the pheromone, pheromone receptor, and pheromone processing factors of one mating type for another. These cells mate with each other, but their mating is inefficient. By characterizing their mating defects and examining their transcriptomes, we found Afb1 (a-factor barrier), a novel MATa-specific protein that interferes with a-factor, the pheromone secreted by MATa cells. Strong pheromone secretion is essential for efficient mating, and the weak mating of transvestites can be improved by boosting their pheromone production. Synthetic biology can characterize the factors that control efficiency in biological processes. In yeast, selection for increased mating efficiency is likely to have continually boosted pheromone levels and the ability to discriminate between partners who make more and less pheromone. This discrimination comes at a cost: weak mating in situations where all potential partners make less pheromone.

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BIOLOGICAL processes are typically defined by the genes that are necessary and sufficient for function. However, in many cases, this minimal gene set does not encompass all the proteins involved in a process, and additional proteins promote biological efficiency. Finding these additional proteins may require the detection of subtle phenotypes, making it hard to know if all the genes involved in a process have been identified. One way to answer this question is to reengineer a pathway and ask whether the synthetic version fully mimics the natural function. Here, we show that this form of synthetic biology illuminates how cells of the budding yeast Saccharomyces cerevisiae mate efficiently.

Budding yeast can be stably maintained as haploids or diploids. Haploids mate when two cells of opposite mating types signal to each other using reciprocal pheromones and receptors, polarize and grow toward each other, and eventually fuse to form a single diploid. Yeast has two mating types, a and a (Figure 1A), determined by two alternative alleles at the MAT locus, MATa and MATa, which encode different transcription factors (Herskowitz 1988). These factors regulate the expression of mating-type-specific genes, many of which are involved with the production and detection of the pheromones that yeast cells use to signal to one another. The pheromones (a- and a-factor) are detected by G-proteincoupled receptors. MATa cells express a-factor (Betz and Duntze 1979), which is secreted through an ATP binding cassette transporter (Ste6) (McGrath and Varshavsky 1989) and the a-factor receptor (Ste2) (Blumer et al. 1988; Dohlman and Thorner 2001). MATa cells express a-factor (Kurjan and Herskowitz 1982; Singh et al. 1983) and the a-factor receptor (Ste3) (Hagen et al. 1986; Dohlman and Thorner 2001). Pheromone binding activates a signaling pathway that produces three responses: cell polarization, cell cycle arrest in G1, and increased transcription of pheromone response genes (Bardwell 2005).

Bender and Sprague (1989) used mutations that alter pheromone and receptor expression to show that a cell's mating type is determined by which pheromones and receptors it expresses. Although pheromone secretion and detection are the essential elements for mating, additional, mating-type-specific genes make mating more efficient. One of these is the MATa-specific a-factor protease, Bar1 (Sprague and Herskowitz 1981; MacKay et al. …

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