Pathways for phospholipid deacylation in Saccharomyces cerevisiae and their impact on fatty acid trafficking and equilibrium

Abstract

The phospholipid (PL) class and species composition is a fundamental characteristic of membranes, essential to their proper function. The PL species distribution of cellular lipids is only partly achieved through de novo synthesis; PL degradation and PL remodeling are known to occur extensively and to be fundamental in the establishment of the steady state lipid composition. In the yeast Saccharomyces cerevisiae the pathways for PL deacylation, leading to PL degradation or PL remodeling, remained, until now, unidentified. The difficulty in the identification of the enzymatic activities involved lies, partly, in the fact that free fatty acids (FFA), lysophospholipids and glycerophosphodiesters (GPD), the direct products of PL deacylation, are rapidly metabolized and are therefore inaccessible to accurate experimental detection and quantification.In the case of FFA, metabolization is necessarily initiated by activation through conversion to acyl-CoA, a reaction catalyzed by acyl-CoA synthetases (ACS). The S. cerevisiae strain YB526, in which the genes coding for the ACS enzymes Faa1p, Faa2p, Faa3p and Faa4p have been deleted, is unable to activate, and therefore to metabolize, FFA. In consequence, FFA derived from lipid deacylation accumulate in this strain and can be quantified. An analysis of the effect that the interference with specific enzymatic activities and metabolic processes has on the FFA content of YB526 cells, reveals the role of those activities and processes in lipid deacylation and in the homeostasis of fatty acids (FA) and acyl-ester pools in general.This approach has revealed that phospholipases B are not involved in PL remodeling or other constitutive forms of PL deacylation. Autophagic degradation, in contrast, was identified as the quantitatively most prominent mechanism for PL deacylation. Despite the fact that autophagy constitutes a mechanism for bulk degradation, its role in the regulation of the cellular PL content appears to be not only quantitative, but also qualitative. Neutral lipid (NL) metabolism was identified as well as a central element in the regulation of PL species composition. NL metabolism as a whole, mediates FA trafficking to a quantitatively similar extent than autophagy. Within NL metabolism, the phospholipid:DAG acyltransferase (PDAT) Lro1p makes a substantial contribution to PL deacylation, and is suggested by our results as the most likely mediator in PL remodeling. The acyl-CoA dependant biosynthetic activities of NL metabolism appear to be involved as well in the establishment of PL species composition through a modulation of the substrate available for PL synthesis. Furthermore, we established that impaired NL synthesis leads to augmented autophagic degradation of PL, while impaired sequestration of substrate for autophagic degradation leads to enhanced NL synthesis.