Localisation and function of phosphoinositide-specific phospholipase C in Sacchromyces cerevisiae

[摘要] Phosphoinositide-specific phospholipase C enzymes (PLCs) cleave the plasma membrane phospholipid PtdIns(4,5)P\(_2\) to generate two messengers, inositol (1,4,5) trisphosphate [Ins(1,4,5)P\(_3\)] and diacylglycerol (DAG). Ins(1,4,5)P\(_3\) is an important second messenger in animal cells. It releases calcium from intracellular stores by opening Ins(1,4,5)P\(_3\) receptors (InsP\(_3\)Rs) and is formed in response to activation of cell surface hormone and growth factor receptors. It is also important for its immediate phosphorylation to form higher phosphorylated inositol phosphates (InsPs), which are critical for various cell signaling functions. Sub-families of PLC enzymes exist in cells and the regulation of the \(\beta\) and \(\gamma\) type PLCs via G-protein coupled receptors and receptor tyrosine kinases respectively, is well understood. In contrast, the regulation of PLC-\(\delta\)s is still a mystery. This is particularly frustrating because these enzymes are the only isoforms of PLC found in fungi and plants, as well as animals, and thus are likely to perform an ancient and important function. Plc1p is the single PLC of the \(\delta\) family in the budding yeast Sacchromyces cerevisiae. It plays a key role in generating Ins(1,4,5)P\(_3\) in this fungi in response to stress and yet the yeast genome encodes no Ins(1,4,5)P\(_3\) receptor. This suggests that the functions of PLC-\(\delta\)s are mediated in a novel fashion, probably occurs via the higher inositol phosphates that derived upon rapid phosphorylation of Ins(1,4,5)P\(_3\) by a series of kinases (Arg82p, Ipk1p, Kcs1p and Vip1p). This study focuses on the localisation of GFP-Plc1p in order to gain insight into its function. Plc1p is present in both cytoplasm and nucleus. Plc1p is too large to diffuse through the nuclear pore complex, and thus relies on nuclear export signals (NES) and nuclear localisation signals (NLS) to facilitate its passage through the nuclear envelope. By creating mutants of Plc1p that are restricted to one of these compartments: GFP-Plc1p\(^{CAAX}\) for plasma membrane localisation, GFP-Plc1p\(^{NES}\) for nucleus and GFP-Plc1p\(^{PKI}\) for cytoplasm, I investigated which defects persist in yeast expressing these mutants as their sole Plc1p. My data suggest that these mutants do appear to display distinct subsets of phenotypes consistent with the idea of separate pools of inositol phosphates. I showed that both cytoplasmic and nuclear pools of Plc1p are important for function as neither PLC1\(^{NES}\) nor PLC1\(^{PKI}\) rescue the stress sensitivity of plc1\(\Delta\) mutants. Therefore, Plc1p are likely to shuttle in between different compartments to exert its diverse functions. It will be instructive to characterise inositol phosphate metabolism in these mutants to determine in which compartments basal and stimulated rises in inositol phosphates occur in yeast cells. In addition, I report the novel finding that Plc1p interacts with one (or more than one) of its metabolites. Plc1p appears to associate tightly with the plasma membrane in the absence of the inositol phosphate kinase Arg82p, most likely due to the absence of Arg82p-derived inositol polyphosphates, and such association is required for intact PH, X-Y and C2 domain – disruption in any of these domains would result in failure of such association. Hence, higher phosphorylated inositol phosphates seem to influence Plc1p’s localisation, and a physiological feedback cycle of regulation probably exists where Plc1p may be regulated by the products of its own catalytic activity. Future studies will seek to understand the role of this feedback cycle in stress signalling and physiology in yeast cells.

[发布机构] University:University of Birmingham;Department:School of Biosciences

[关键词] Q Science;QH Natural history;QH301 Biology