Yeast cell -
a system to study the action of a neurotoxic phospholipase A 2.
Uros Petrovic,
Igor Krizaj, Franc Gubensek
Biochemistry and Mol. Biology, Jozef Stefan Institute, Jamova 39, Ljubljana,
SI-1000, Slovenia (uros.petrovic@ijs.si)
Neurotoxic phospholipases A2 are the most toxic components in venoms of some snakes. Ammodytoxin is such molecule in the venom of the long-nosed viper (Vipera a. ammodytes). Although highly specific for neuronal cells, its cellular targets include also proteins that regulate basic cellular processes and which are expressed in virtually all types of cells. One of such targets is calmodulin, evolutionary highly conserved protein present in all eukaryotes. These facts prompted us to use Saccharomyces cerevisiae as a model to investigate the molecular basis of action of ammodytoxin. Although yeast and mammalian calmodulins are not identical (60% identity), mammalian calmodulin can functionally replace yeast calmodulin in S. cerevisiae. We demonstrated that ammodytoxin binds only vertebrate and not yeast calmodulin. Different yeast strains, one of them depending on human calmodulin gene, are used for heterologous expression of ammodytoxin inside the cells. This enables us to study both calmodulin-dependent and calmodulin-independent effects of ammodytoxin on yeast cells by monitoring their phenotype, such as changes in morphology and stress sensitivity, and alterations of their proteome and transcriptome in the presence of the toxin. These global approaches have enabled us to elucidate the mechanism of action of ammodytoxin in an eukaryotic cell. Therefore, the yeast cell appears to be a good model for studying the effect of neurotoxic phospholipase A2 molecules.