Expression of the BAR1 gene in Saccharomyces cerevisiae: induction by the alpha mating pheromone of an activity associated with a secreted protein

Abstract

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.

Purification and characterization of the inducible a agglutinin of Saccharomyces cerevisiae.

“…Extraction and purifcation of a agglutinin S.cerevisiae Mat a cells carrying the bar 1-1 mutation (Manney, 1983;Chan and Otte, 1982) were grown in 10 1 of YPD medium (1 %) yeast extract, 2% bacto peptone, 2% glucose) to OD578 of 6.0. Then synthetic a factor (Bachem, Bubendorf, Switzerland) was added to a final concentration of 10 nM to induce a-agglutinin synthesis.…”

Common signal transduction system shared by STE2 and STE3 in haploid cells of Saccharomyces cerevisiae : autocrine cell-cycle arrest results from forced expression of STE2

“…Although the axand a-factors have distinctly different amino acid sequences, both molecules evoke seemingly identical responses in their respective target cells. These include: (i) changes in gene expression (Manney, 1983;Strazdis and MacKay, 1983;Hagen and Sprague, 1984;Stetler and Thorner, 1984;Nakayama et al, 1985); (ii) enhancement of adhesiveness to their mating partners (Sakai and Yanagishima, 1972;Fehrenbacher et al, 1978); (iii) cell-cycle arrest at GI phase (Buicking-Throm et al, 1973;Wilkinson and Pringle, 1974); and (iv) 'shmoo' formation, a morphological change which is recognized as an intermediate stage of the conjugation process (Hartwell, 1973).…”

The mating response cascade does not modulate changes in the steady‐state level of target mRNAs through control of mRNA stability

“…Once phosphorylated, Ste12 translocates into the nucleus and activates transcription by binding to pheromone response elements (PREs) within proximal promoters of responsive genes (Sengupta and Cochran, 1990). The mating response is attenuated in part by Ste12mediated transcription of the gene encoding the Bar1 periplasmic protease, which degrades the αfactor peptide (Manney, 1983), thus preventing continued activation of the GPCR (Ballensiefen and Schmitt, 1997;MacKay et al, 1988).…”