Establishment of a simple system to analyse the molecular interaction in the agglutination of Saccharomyces cerevisiae

Saccharomyces cerevisiae a-agglutinin, which is involved in mating and cova lently anchoring to the cell wall, consists of two components, Aga1p and Ag a2p, whose syntheses are individually regulated. To facilitate the analysis of the protein-protein interaction on agglutination between a- and alpha-a gglutinins, the construction of a yeast strain (MA Ta) with the functional protein prepared by genetic fusion of Aga1p- and Aga2p-encoding genes and b y the expression system using the UPR-ICL promoter derived from the n-alkan e-assimilating yeast, Candida tropicalis, which is functional under the con dition of lower glucose concentration was tried and the agglutination abili ty of the constructed strain was evaluated with a yeast strain (MA Ta) whic h expressed AG alpha 1 encoding alpha-agglutinin under the control of the s ame promoter. The genes were integrated into the yeast chromosomes. Cell ag glutination between both (MA Ta) strains was observed microscopically when these two strains were mix-cultured to a glucose-decreased concentration. T he agglutination was further confirmed by the sedimentation test and by the quantification using a filter. These results proved that the constructed A ga1p-Aga2p fusion protein was enoughly functional for the interaction with the Aged protein, and that this phenomenon occurred dependent on glucose co ncentration, but independent of the peptide pheromones secreted by the cell s of the opposite mating types, Using this system, the role of two disulphi de linkages between Aga1p and Aga2p on the binding activity between Aga2p a nd Aga1p was first evaluated. Under the treatment by the SH-compound (dithi othreitol), in which Ag alpha 2p is easily released into the medium from th e intact cell surface, the Aga1p and Aga2p fusion protein was a good tool t o make clear the role of the disulphide linkages. As a result, the linkages had a significant effect on not only the assembly but also the binding act ivity. The novel and simple system described here may further facilitate th e study of molecular interaction in agglutination. Copyright (C) 2000 John Wiley & Sons, Ltd.