Agglutination-dependent and -independent adhesin interactions during mating in Saccharomyces cerevisiae
Fungal adhesins are cell surface proteins that mediate adherence of fungal cells to host substrates and each other, which affects their access to nutrients, sexual conjugation, and survival in hosts. Most fungal adhesins receive two posttranslational modifications, glycophosphatidylinositol (GPI) anchor addition and O-glycosylation, and a subset of fungal adhesins contains highly conserved WCPL and CX 4 C domains of unknown function. This study was directed toward understanding the in vivo functional requirements of the posttranslational modifications and the WCPL/CX 4 C domains for fungal adhesins, using the mating system in Saccharomyces cerevisiae as a model.
Deletion of the signal sequence for GPI addition of the yeast a -agglutinin subunit, Aga1p, eliminated its activity, while deletions of different internal domains had varying effects on function. Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins. Aga1p and the α-agglutinin Agα1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2 , with phenotypes manifested only in MATα cells for single mutants but in both cell types when both genes are absent.
Besides the well-known agglutinin interaction between the a -agglutinin complex (Aga1p-Aga2p) and Agα1p, three more pairs of interactions were revealed by this study, including bilateral heterotypic interactions between Aga1p and Fig2p, and a weaker homotypic interaction between Fig2p and Fig2p, in cells of the opposite mating type. These four pairs of adhesin interactions are collectively required for maximum mating efficiency and normal zygote morphogenesis. GPI-less, epitope-tagged forms of Aga1p and Fig2p can be co-immunoprecipitated from the culture medium of mating cells in a manner dependent on the WCPL/CX 4 C domains in Aga1p. Using site-directed mutagenesis, the conserved residues in Aga1p that interact with Fig2p were identified.
To conclude, posttranslational modifications and the conserved WCPL/CX 4 C domains are necessary for the biogenesis and activity of Aga1p. Aga1p is involved in two distinct adhesive functions that are independent of each other, which raises the possibility for combinatorial interactions of this protein with its different adhesion receptors, Aga2p and Fig2p, a property previously unknown for fungal adhesins.