Ca²⁺-Calmodulin Promotes Survival of Pheromone-Induced Growth Arrest by Activation of Calcineurin and Ca²⁺-Calmodulin-Dependent Protein Kinase

Abstract

The cmd1-6 allele contains three mutations that block Ca 2؉ binding to calmodulin from Saccharomyces cerevisiae. We find that strains containing cmd1-6 lose viability during cell cycle arrest induced by the mating pheromone ␣-factor. The 50% lethal dose (LD 50 ) of ␣-factor for the calmodulin mutant is almost fivefold below the LD 50 for a wild-type strain. The calmodulin mutants are not more sensitive to ␣-factor, as measured by activation of a pheromone-responsive reporter gene. Two observations indicate that activation of the Ca 2؉ -calmodulin-dependent protein phosphatase calcineurin contributes to survival of pheromone-induced arrest. First, deletion of the gene encoding the calcineurin regulatory B subunit, CNB1, from a wild-type strain decreases the LD 50 of ␣-factor but has no further effect on a cmd1-6 strain. Second, a dominant constitutive calcineurin mutant partially restores the ability of the cmd1-6 strain to survive exposure to ␣-factor. Activation of the Ca 2؉ -calmodulin-dependent protein kinase (CaMK) also contributes to survival, thus revealing a new function for this enzyme. Deletion of the CMK1 and CMK2 genes, which encode CaMK, decreases the LD 50 of pheromone compared with that for a wild-type strain but again has no effect in a cmd1-6 strain. Furthermore, the LD 50 of ␣-factor for a mutant in which the calcineurin and CaMK genes have been deleted is the same as that for the calmodulin mutant. Finally, the CaMK and calcineurin pathways appear to be independent since the ability of constitutive calcineurin to rescue a cmd1-6 strain is not blocked by deletion of the CaMK genes.

Ca+2 Signal is Generated Only Once in the Mating Pheromone Response Pathway in Saccharomyces cerevisiae.

“…Other lines of research also suggest that Ca 2+ plays a regulatory role in the late stage of the pheromone response pathway. Mutants lacking a subunit of the Ca 2+ /calmodulin-dependent phosphoprotein phosphates calcineurin are defective in recovery (Cyert et al, 1991;Cyert and Thorner, 1992) and die in response to the pheromone, like the mid mutants (Moser et al, 1996;Withee et al, 1997). Mutants defective in either calmodulin itself or calmodulin-dependent protein kinase also lose viability after differentiating into shmoos (Moser et al, 1996).…”

“…Mutants lacking a subunit of the Ca 2+ /calmodulin-dependent phosphoprotein phosphates calcineurin are defective in recovery (Cyert et al, 1991;Cyert and Thorner, 1992) and die in response to the pheromone, like the mid mutants (Moser et al, 1996;Withee et al, 1997). Mutants defective in either calmodulin itself or calmodulin-dependent protein kinase also lose viability after differentiating into shmoos (Moser et al, 1996). An important point of these genetic and cell-biological data is that the early events seem to occur quite normally in all the mutants mentioned here, because the mutants can arrest in G1 and differentiate into shmoos just like their wild-type counterparts.…”

An Osmotically Induced Cytosolic Ca2+ Transient Activates Calcineurin Signaling to Mediate Ion Homeostasis and Salt Tolerance of Saccharomyces cerevisiae

“…Mating pheromone causes G 1 arrest and resumption of cell cycle progression by facilitating Ca ext 2ϩ influx, which results in calmodulin activation of calcineurin (1,(22)(23)(24)(25). Pheromone-induced Ca ext 2ϩ influx is mediated by Cch1p and Mid1p, which are plasma membrane proteins that by genetic criteria function as a single Ca 2ϩ uptake system (26 -29).…”

“…Ca 2ϩ -dependent yeast mating pheromone signaling has been characterized in some detail (1,(22)(23)(24)(25). Mating pheromone causes G 1 arrest and resumption of cell cycle progression by facilitating Ca ext 2ϩ influx, which results in calmodulin activation of calcineurin (1,(22)(23)(24)(25).…”

Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases

“…Besides strong promoters, inducible promoters allow carefully timed expression of proteins, especially those whose presence is toxic to cell growth. These promoters include GAL1-10 (induced by galactose), PHO5 (induced by low extracellular inorganic phosphate), and tandem heat shock HSE elements (induced by temperature elevation to 37°C) (57). It is worth noting that the GAL promoters are also exceptionally useful to turn off gene expression following a transfer to glucose medium.…”