Developing a large-scale suppression screen for rescue of the prm1Δ mating defect in Saccharomyces cerevisiae

Abstract

Cell-cell membrane fusion is an important mechanism of eukaryotic cells for specialization and fertilization that is not fully understood. The yeast, Saccharomyces cerevisiae, can mate to form a diploid zygote by the fusion of two haploid cells, providing a model system in which to study membrane fusion. The transmembrane protein Prm1p has been shown to act at the plasma membrane fusion step of mating, but previous studies of its structure do not suggest a catalytic fusogen role, and 45% of bilateral prm1Δ mating pairs retain fusion function. Therefore Prm1p is likely not a primary fusogen. Here we propose and design a suppression screen in prm1Δ mating pairs that we expect to yield a true fusogen through rescue of the prm1Δ fusion defect. Protocols for isolation of a 2μ genomic tiling plasmid library and for transformation into MATa prm1Δ and MATα prm1Δ yeast cells expressing cytoplasmic GFP and DsRed have been developed. The genes FUS1, PUG1, and TMN2 were identified as partial suppressors by fluorescence transfer in preliminary experiments. PUG1 may rescue the prm1Δ phenotype by a role in organizing membrane lipid composition at the fusion site. Upon completion of an algorithm for automated fusion detection in a high content imaging system, the entire yeast genome will be screened. Such an overexpression screen is a powerful genetic tool to search for a fusogen and to perform future large-scale investigations.