The pheromone a factor, secreted by Saccharomyces cerevisiae cells of the a mating type, serves to synchronize the opposite mating type (a cells) at GI as a prelude to fusion of the two cell types. We found that, in vitro, a factor inhibited the' membrane-bound adenylate cyclase of these cells in a dose-dependent manner. Moreover, one class (steS) of a cell mutants that grow normally at either 230 or 340C but that are unable to respond to a factor or to mate at the higher temperature possessed an adenylate cyclase activity that was not inhibited by a factor at 340C but was fully sensitive to inhibition at 230C. Furthermore, addition of cyclic AMP to a cell culture medium shortened the period of pheromone-induced GI arrest. We conclude that inhibition of adenylate cyclase activity by a factor may constitute, at least in part, the biochemical mode of action of the pheromone in vivo.Haploid cells of opposite mating type of Saccharomyces cerevisiae can fuse pairwise to form diploid cells. This process is triggered by the action of diffusible oligopeptide pheromones (reviewed in ref. 1). a Factor, the pheromone secreted by a haploids, elicits in a haploids the following developmentally specific events, in order of their appearance: an increase in adhesiveness (2); arrest of growth at the G1 stage of the cell cycle with concomitant cessation of nuclear DNA replication (3); and anisotropic cell wall synthesis (4) resulting eventually in the formation of morphologically abnormal cells if mating is precluded by the absence of a cells (5). Exposure of a cells to a factor thus synchronizes the culture as unbudded mononucleate cells primed for mating with their a counterparts.Although much is known about the a factor molecule, including confirmation of its primary structure (6) by solid-phase peptide synthesis (7), little work has been done to elucidate the biochemical mechanism of its action. By analogy with mammalian peptide hormones and neurotransmitters that act via cyclic AMP as an intracellular "second messenger" (8, 9), we decided to investigate the effect of cyclic AMP on the response of a cells to a factor in vio and the effect of a factor on yeast adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in vitro. We show that the a pheromone inhibits cyclic AMP production by the enzyme and that this may constitute at least part of its mode of action at the molecular level.MATERIALS AND METHODS Chemicals. All biochemicals were of the highest grade commercially available. Radiochemicals were purchased from New England Nuclear. All other chemicals were reagent grade. Purification of a factor and chemical synthesis of the entire molecule and partial segments have been described (7,10). Commercially prepared synthetic a factor (Peninsula Labs, San Carlos, CA) was also used, after extensive purification (7).Organisms and Growth Conditions. The standard laboratory yeast strains, X2180-1A (a), X2180-1B (a), and P2180A (a/at), and conditions for their cultivation have been described (11). The collection ...
