Hypo-osmotic stress activates Plc1p-dependent Ptdlns(4,5)P2 hydrolysis and InsP6 accumulation in yeast

Polyphosphoinositide-specific phospholipases (PICs) of the delta-subfamily are ubiquitous in eukaryotes, but an inability to control these enzymes physiologically has been a major obstacle to understanding their cellular function(s). Plc1p is similar to metazoan delta-PICs and is the only PIC in Saccharomyces cerevisiae. Genetic studies have implicated Plc1p in several cell functions, both nuclear and cytoplasmic. Here we show that a brief hypo-osmotic episode provokes rapid Plc1p-catalyzed hydrolysis of PtdIns(4,5)P-2 in intact yeast by a mechanism independent of extracellular Ca2+. Much of this PtdIns(4,5)P-2 hydrolysis occurs at the plasma membrane. The hydrolyzed PtdIns(4,5)P-2 is mainly derived from PtdIns4P made by the PtdIns 4-kinase Stt4p. PtdIns(4,5)P-2 hydrolysis occurs normally in mutants lacking Arg82p or Ipk1p, but they accumulate no InsP(6), showing that these enzymes normally convert the liberated Ins(1,4,5)P-3 rapidly and quantitatively to InsP6. We conclude that hypo-osmotic stress activates Plc1p-catalyzed PtdIns(4,5)P-2 at the yeast plasma membrane and the liberated Ins(1,4,5)P-3 is speedily converted to InsP6. This ability routinely to activate Plc1p-catalyed PtdIns(4,5)P-2 hydrolysis in vivo opens up new opportunities for molecular and genetic scrutiny of the regulation and functions of phosphoinositidases C of the delta-subfamily.