Diverse effect of phosphatidylcholine biosynthetic genes on phospholipid homeostasis, cell autophagy and fungal developments in Metarhizium robertsii.

Phosphatidylcholine (PC) plays an important role in maintaining membrane integrity and functionality. In this study, two key genes (Mrpct and Mrpem) putatively involved in the cytidine diphosphate (CDP)-choline and phosphatidylethanolamine N-methyltransferase (PEMT) pathways for PC biosynthesis were characterized in the insect pathogenic fungus Metarhizium robertsii. The results indicated that disruption of Mrpct did not lead to any reduction of total PC content but impaired fungal virulence and increased cellular accumulation of triacylglycerol. Deletion of Mrpem reduced PC content and impaired fungal conidiation and infection structure differentiation but did not result in virulence defects. Lipidomic analysis revealed that deletion of Mrpct and Mrpem resulted in dissimilar effects on increase and decrease of PC moieties and other phospholipid species accumulations. Interestingly, we found that these two genes played opposite roles in activation of cell autophagy when the fungi were grown in a nutrient-rich medium. The connection between PC metabolism and autophagy was confirmed because PC content was drastically reduced in Mratg8Δ and that the addition of PC could rescue null mutant sporulation defect. The results of this study facilitate the understanding of PC metabolism on fungal physiology.

From yeast to humans - roles of the Kennedy pathway for phosphatidylcholine synthesis.

The major phospholipid present in most eukaryotic membranes is phosphatidylcholine (PC), comprising ~ 50% of phospholipid content. PC metabolic pathways are highly conserved from yeast to humans. The main pathway for the synthesis of PC is the Kennedy (CDP-choline) pathway. In this pathway, choline is converted to phosphocholine by choline kinase, phosphocholine is metabolized to CDP-choline by the rate-determining enzyme for this pathway, CTP:phosphocholine cytidylyltransferase, and cholinephosphotransferase condenses CDP-choline with diacylglycerol to produce PC. This Review discusses how PC synthesis via the Kennedy pathway is regulated, its role in cellular and biological processes, as well as diseases known to be associated with defects in PC synthesis. Finally, we present the first model for the making of a membrane via PC synthesis.

A Yeast Mutant Deleted of GPH1 Bears Defects in Lipid Metabolism.

In a previous study we demonstrated up-regulation of the yeast GPH1 gene under conditions of phosphatidylethanolamine (PE) depletion caused by deletion of the mitochondrial (M) phosphatidylserine decarboxylase 1 (PSD1) (Gsell et al., 2013, PLoS One. 8(10):e77380. doi: 10.1371/journal.pone.0077380). Gph1p has originally been identified as a glycogen phosphorylase catalyzing degradation of glycogen to glucose in the stationary growth phase of the yeast. Here we show that deletion of this gene also causes decreased levels of phosphatidylcholine (PC), triacylglycerols and steryl esters. Depletion of the two non-polar lipids in a Δgph1 strain leads to lack of lipid droplets, and decrease of the PC level results in instability of the plasma membrane. In vivo labeling experiments revealed that formation of PC via both pathways of biosynthesis, the cytidine diphosphate (CDP)-choline and the methylation route, is negatively affected by a Δgph1 mutation, although expression of genes involved is not down regulated. Altogether, Gph1p besides its function as a glycogen mobilizing enzyme appears to play a regulatory role in yeast lipid metabolism.

Autoradiographic detection of animal cell membrane mutants altered in phosphatidylcholine synthesis.

We have screened approximately 20,000 colonies of Chinese hamster ovary cells immobilized on filter paper [Esko, J.D. & Raetz, C.R.H. (1978) Proc Natl. Acad. Sci. USA 75, 1190-1193] for strains unable to incorporate [methyl-14C]-choline into trichloroacetic acid-precipitable phospholipid at 40 degrees C. Mutant 58, identified in this way, was specifically defective in choline incorporation, and other isolates were also blocked in thymidine and leucine incorporation into DNA and protein, respectively. Further analysis of mutant 58 revealed that the strain grew almost normally at 33 degrees C, the permissive temperature, but divided only once at 40 degrees C, the restrictive temperature. After a 20-hr incubation at 40 degrees C, the phosphatidyl-choline level dropped from 41% to 20% in the mutant whereas other phospholipids, including sphingomyelin, continued to accumulate. Wild-type cells contained approximately 50% phosphatidylcholine at both temperatures. Anion-exchange chromatography of the water-soluble choline metabolites extracted from mutant 58 revealed that phosphorylcholine accumulation increased from 6 nmol/mg of protein at 33 degrees C to 42 nmol/mg of protein at 40 degrees C whereas CDP-choline decreased from 0.42 nmol to less than 0.07 nmol per mg of protein. Phosphorylcholine also increased in wild-type cells shifted from 33 degrees C to 40 degrees C (from 1.8 nmol to 16 nmol per mg of protein), but the level of CDP-choline was not altered (from 0.52 nmol to 0.58 nmol per mg of protein). Enzymatic assays of extracts prepared from mutant and wild-type cells revealed a reduction of CTP: phosphorylcholine cytidylyltransferase (EC 2.7.7.15) activity (CDP-choline synthetase) in the mutant to 1/40th that in the wild type, and mixing experiments excluded the production of antagonists to CDP-choline synthesis in the mutant. Thus, the inability of the mutant to generate normal amounts of phosphatidylcholine in vivo was correlated with an enzymatic lesion in the biosynthesis of CDP-choline in vitro.