Phospholipid remodeling and lipid signaling in the yeast, Saccharomyces cerevisiae - the role of phospholipases

Phospholipases are ubiquitous enzymes that catalyse the hydrolysis of acylester or phosphodiester bonds of glycerophospholipids. Several physiologically important functions of phospholipases in higher eukaryotes have been established, such as digestion of extracellular phospholipids (e.g. in the gut) and degradation of intracellular phospholipids during disintegration of effete organelle membranes. Phospholipases A2 , C and D take part in intracellular signal transduction systems by agonist induced generation of lipid second messengers. Phospholipases have been implicated in the generation of phospholipid molecular species via a deacylation-reacylation cycle or head group exchange, thus providing proper building blocks for the assembly of cellular membranes. Furthermore, phospholipases are components of insect and snake venoms and are essential for fungal infection, e.g. by Candida yeast. In the course of this project, the yeast Saccharomyces cerevisiae has been used as a model organism to study the role of specific phospholipases in some of the above mentioned cellular processes. Emphasis was on phospholipases B, firstly because very little is known about the function of these phospholipases which completely and irreversibly deacylate glycerophospholipids, and secondly because genes coding for three such phospholipases have been identified in the yeast genome. Mutants defective in one, two or three of the PLB genes, and cells overexpressing only a single phospholipase B were employed to study properties and function of each of the phospholipases B in vitro and in vivo. Comparison of the three phospholipases B revealed significant differences with respect to substrate selectivity and function in vivo. Phospholipase B1 (Plb1p) is responsible for the degradation of the major yeast phospholipid, phophatidylcholine. Plb2p degrades only extracellurar phospholipids and lysophospholipids, thus protecting (together with Plb1p) cells from the toxic effect of the latter and allows utilisation of the former as a source of fatty acids and other reusable portions of glycerophospholipids (e.g. inositol and choline). Plb3p degrades only phosphatidylserine and phosphatidylinositol; in contrast to the other two, this enzyme has low lysophospholipase activity and behaves like the Ca-dependent cytosolic phospholipase A2 of higher eukaryotes. Activity of phospholipases B controls levels and composition of the phospholipid matrix of cellular membranes, and the distribution of fatty acids between phospholipids and triacylglycerols (depot fat). Plb3p reduces the sensitivity of yeast cells against the toxic cation Al3+. Activity of the phospholipases B, which reside in the plasma membrane, is highly regulated at the level of transcription and at the level of enzyme. The latter regulation seems to involve as yet unidentified protein factors; strong regulation occurs by pH of the medium, di- and trivalent cations, ATP, and unesterified fatty acids generated during enzyme action. The phospholipases B are serine esterases and thus capable of catalysing transacylation reactions. This makes them potential candidates as biocatalysts that may be used for the production of taylored glycerophospholipids. Effective heterologous expression of a Plb1p-GST fusion protein has been achieved. The protein may thus be made available in sufficient amounts for application, e.g as a biocatalyst as mentioned above, or as a target to test for phospholipase B inhibitors that can be useful in the treatment of Candida infections that depend on virulent phospholipase B.