Vivian L. MacKay

The STE4 and STE18 genes of yeast encode potential β and γ subunits of the mating factor receptor-coupled G protein

The STE4 and STE18 genes are required for haploid yeast cell mating. Sequencing of the cloned genes revealed that the STE4 polypeptide shows extensive homology to the beta subunits of mammalian G proteins, while the STE18 polypeptide shows weak similarity to the gamma subunit of transducin. Null mutations in either gene can suppress the haploid-specific cell-cycle arrest caused by mutations in the SCG1 gene (previously shown to encode a protein with similarity to the alpha subunit of G proteins). We propose that the products of the STE4 and STE18 genes comprise the beta and gamma subunits of a G protein complex coupled to the mating pheromone receptors. The genetic data suggest pheromone-receptor binding leads to the dissociation of the alpha subunit from beta gamma (as shown for mammalian G proteins), and the free beta gamma element initiates the pheromone response.

A functionally compromised intermediate in extrathymic CD8+ T cell deletion

We have established a model system for analyzing the induction of self-tolerance among mature peripheral T cells in V beta 5 TCR Tg mice. Both CD4+V beta 5+ and CD8+ V beta 5+ cells undergo a superantigen-driven chronic deletion in the periphery of I-E mice. Prior to their disappearance, CD4+ transgene-expressing cells are activated and then rendered anergic to further stimulation through their TCRs. This scenario differs strikingly in the CD8+ cellular compartment, which is characterized by a distinct population of CD8loV beta 5lo cells localized to the blood and spleen. CD8lo cells are small, express the surface phenotype of memory cells, and rapidly incorporate BrdU in vivo. The kinetics of their appearance and disappearance in adult thymectomized mice, the rapid chasing of BrdU from labeled cells, and their in vivo cortisone sensitivity all suggest CD8lo cells are slated for deletion. Furthermore, their functional incompetence can be documented in vitro in the absence of internucleosomal DNA fragmentation. Thus, we have identified an intermediate population of T cells targeted for peripheral deletion that, although functionally compromised, has not yet undergone programmed cell death.

Characterization of the Bar Proteinase, An Extracellular Enzyme from the Yeast Saccharomyces Cerevisiae

Haploid S. cerevisiae cells of the a mating type constitutively secrete an extracellular proteinase that cleaves the peptide mating pheromone (α-factor) secreted by mating-type α cells. DNA sequence analysis of the BAR1 gene that encodes Bar proteinase demonstrated that the primary translation product of 587 amino acids has strong homology to two-domain aspartic proteinases such as pepsin, chymosin, and others, but contains a unique third domain that is not homologous to these enzymes. When produced by wild-type yeast cells, the Bar enzyme exists as a heterogeneous, heavily glycosylated protein with apparent molecular weight >200,000 Da. By producing the proteinase in mutant yeast strains that are defective in glycosylation, we have been able to purify and characterize a homogeneous species. In this paper, we will describe some of the enzyme’s physical properties and substrate requirements, as well as present data indicating that the third domain is required for secretion of the proteinase to the culture medium.