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 covalently anchoring to the cell wall, consists of two components, Aga1p and Aga2p, whose syntheses are individually regulated. To facilitate the analysis of the protein-protein interaction on agglutination between a- and alpha-agglutinins, the construction of a yeast strain (MATa) with the functional protein prepared by genetic fusion of Aga1p- and Aga2p-encoding genes and by the expression system using the UPR-ICL promoter derived from the n-alkane-assimilating yeast, Candida tropicalis, which is functional under the condition of lower glucose concentration was tried and the agglutination ability of the constructed strain was evaluated with a yeast strain (MATa) which expressed AGalpha1 encoding alpha-agglutinin under the control of the same promoter. The genes were integrated into the yeast chromosomes. Cell agglutination between both (MATa) strains was observed microscopically when these two strains were mix-cultured to a glucose-decreased concentration. The agglutination was further confirmed by the sedimentation test and by the quantification using a filter. These results proved that the constructed Aga1p-Aga2p fusion protein was enoughly functional for the interaction with the Agalpha1 protein, and that this phenomenon occurred dependent on glucose concentration, but independent of the peptide pheromones secreted by the cells of the opposite mating types. Using this system, the role of two disulphide linkages between Aga1p and Aga2p on the binding activity between Aga2p and Aga1p was first evaluated. Under the treatment by the SH-compound (dithiothreitol), in which Agalpha2p is easily released into the medium from the intact cell surface, the Aga1p and Aga2p fusion protein was a good tool to 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 activity. The novel and simple system described here may further facilitate the study of molecular interaction in agglutination.
Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
W
W
Zou
M
M
Ueda
T
T
Murai
A
A
Tanaka
D000473:Alkanes / Q000378:metabolism; D002175:Candida / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion / Q000235:genetics* / Q000502:physiology; D004229:Dithiothreitol / Q000494:pharmacology; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D000072235:Mating Factor; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010675:Pheromones; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic
D000473:Alkanes; D008074:Lipoproteins; C095063:MFA2 protein, S cerevisiae; D010455:Peptides; D010675:Pheromones; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; D004229:Dithiothreitol