title: Cell cycle arrest and apoptosis, two alternative mechanisms for PMKT2 killer activity.
abstract: Pichia membranifaciens CYC 1086 secretes a unique 30kDa killer toxin (PMKT2) that inhibits a variety of spoilage yeasts and fungi of agronomical interest. The cytocidal effect of PMKT2 on Saccharomyces cerevisiae cells was studied. Metabolic events associated with the loss of S. cerevisiae viability caused by PMKT2 were qualitatively identical to those reported for K28 killer toxin activity, but different to those reported for PMKT. At higher doses, none of the cellular events accounting for the action of PMKT, the killer toxin secreted by P. membranifaciens CYC 1106, was observed for PMKT2. Potassium leakage, sodium influx and the decrease of intracellular pH were not among the primary effects of PMKT2. We report here that this protein is unable to form ion-permeable channels in liposome membranes, suggesting that channel formation is not the mechanism of cytotoxic action of PMKT2. Nevertheless, flow cytometry studies have revealed a cell cycle arrest at an early S-phase with an immature bud and pre-replicated 1n DNA content. By testing the sensitivity of cells arrested at different stages in the cell cycle, we hoped to identify the execution point for lethality more precisely. Cells arrested at the G1-phase by α-factor or arrested at G2-phase by the spindle poison methyl benzimidazol-2-yl-carbamate (MBC) were protected against the toxin. Cells released from the arrest in both cases were killed by PMKT2 at a similar rate. Nevertheless, cells released from MBC-arrest were able to grow for a short time, and then viability dropped rapidly. These findings suggest that cells released from G2-phase are initially able to divide, but die in the presence of PMKT2 after initiating the S-phase in a new cycle, adopting a terminal phenotype within that cycle. By contrast, low doses of PMKT and PMKT2 were able to generate the same cellular response. The evidence presented here shows that treating yeast with low doses of PMKT2 leads to the typical membranous, cytoplasmic, mitochondrial and nuclear markers of apoptosis, namely, the production of reactive oxygen species, DNA strand breaks, metacaspase activation and cytochrome c release.
authors.affiliation: Department of Microbiology, Complutense University of Madrid, Madrid, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Santos
authors.forename: Alejandro
authors.initials: A
authors.lastname: Alonso
authors.forename: Ignacio
authors.initials: I
authors.lastname: Belda
authors.forename: Domingo
authors.initials: D
authors.lastname: Marquina
mesh_terms: D017209:Apoptosis; D059447:Cell Cycle Checkpoints; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects*; D055646:Killer Factors, Yeast / Q000633:toxicity*; D050296:Microbial Viability / Q000187:drug effects; D010843:Pichia / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects*
chemical_list: D055646:Killer Factors, Yeast; C087906:killer toxin, Pichia