No abstract

Saccharomyces cerevisiae a cells secrete an extracellular protein, called "barrier" activity, that acts as an antagonist of a factor, the peptide mating pheromone produced by mating-type a cells. We report here the DNA sequence of BAR], the structural gene for barrier activity. The deduced primary translation product of 587 amino acids has a putative signal peptide, nine potential asparagine-linked glycosylation sites, and marked sequence similarity of the first two-thirds of the protein with pepsin-like proteases. Barrier activity was abolished by in vitro mutation of an aspartic acid predicted from this sequence homology to be in the active site. Therefore, barrier protein is probably a protease that cleaves a factor. The sequence similarity suggests that the first two-thirds of the barrier protein is organized into two distinct structural domains like those of the pepsin-like proteases. However, the BAR) gene product has a third carboxyl-terminal domain of unknown function; deletion of at least 166 of the 191 amino acids of this region has no significant effect on barrier activity.

A hormone-like substance is secreted by alpha mating-type cells of heterothallic yeast strains. It induces in cells of the opposite mating type, a, a morphological change characteristic of the mating process. Secretion of this substance and mating ability have some common genetic determinants. In partially purified preparations, the substance has properties of an oligopeptide.

We have demonstrated and partially characterized the genetic control and pheromonal regulation of a soluble activity, produced only by mating-type a cells, that inhibits the action of the a mating pheromone, a-factor, on mating-type a cells. This activity was found to be associated with a heat-stable protein and to be secreted by MATa BARI, mata2 BARI, and matal mata2 BARI strains, but not by MATa BARI, MATalMATa BARI, matal BARI, or MATa barl strains, demonstrating that it is under the control of both the MATTa2 and the BARI genes. Secretion of this activity was also found to be stimulated to as much as five times the basal level by exposure of the cells to a-factor. This stimulation was maximal after 6 h at a pheromone concentration of approximately 2 U/ml. An assay for this activity was developed by using a refined, quantitative assay for a-factor. The pheromone activity of samples added to wells in an agar plate was related to the size of the halo of growth inhibition produced in a lawn of mutant cells that are abnormally sensitive. The a-factor-inhibiting activity was related to a reduction of the halo size when active samples were added to the lawn. Although the assay for a-factor was found to be relatively insensitive to pH over a range of several units, the a-factor-inhibiting activity displayed a sharp pH optimum at approximately 6.5. The properties of this activity have important implications concerning the role of the BARI gene product in recovery of mating-type a cells from cell division arrest by a-factor.