title: Nte1p-mediated deacylation of phosphatidylcholine functionally interacts with Sec14p.
abstract: Deciphering the function of the essential yeast Sec14p protein has revealed a regulatory interface between cargo secretion from Golgi and lipid homeostasis. Abrogation of the CDP-choline (CDP-Cho) pathway for phosphatidylcholine (PC) synthesis allows for life in the absence of the otherwise essential Sec14p. Nte1p, the product of open reading frame YML059c, is an integral membrane phospholipase against CDP-Cho-derived PC producing intracellular glycerophosphocholine (GPCho) and free fatty acids. We monitored Nte1p activity through in vivo PC turnover measurements and observed that intracellular GPCho accumulation is decreased in a sec14(ts) strain shifted to 37 degrees C in 10 mm choline (Cho)-containing medium compared with a Sec14p-proficient strain. Overexpression of two Sec14p homologs Sfh2p and Sfh4p in sec14(ts) cells restored secretion and growth at the restrictive temperature but did not restore GPCho accumulation. Instead, newly synthesized PC was degraded by phospholipase D (Spo14p). Similar analysis performed in a sec14Delta background confirmed these observations. These results imply that the ability of Sfh2p and Sfh4p to restore secretion and growth is not through a shared function with Sec14p in the regulation of PC turnover via Nte1p. Furthermore, our analyses revealed a profound alteration of PC metabolism triggered by the absence of Sec14p: Nte1p unresponsiveness, Spo14p activation, and deregulation of Pct1p. Sfh2p- and Sfh4p-overexpressing cells coped with the absence of Sec14p by controlling the rate of phosphocholine formation, limiting the amount of Cho available for this reaction, and actively excreting Cho from the cell. Increased Sfh4p also significantly reduced the uptake of exogenous Cho. Beyond the new PC metabolic control features we ascribe to Sfh2p and Sfh4p we also describe a second role for Sec14p in mediating PC homeostasis. Sec14p acts as a positive regulator of Nte1p-mediated PC deacylation with the functional consequence of increased Nte1p activity increasing the permissive temperature for the growth of sec14(ts) cells.
authors.affiliation: Atlantic Research Centre, Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
authors.forename: J Pedro Fernández
authors.initials: JP
authors.lastname: Murray
authors.forename: Christopher R
authors.initials: CR
authors.lastname: McMaster
mesh_terms: D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002794:Choline / Q000737:chemistry; D017931:DNA Primers; D005227:Fatty Acids / Q000378:metabolism; D005821:Genetic Techniques; D005838:Genotype; D050356:Lipid Metabolism; D008954:Models, Biological; D016366:Open Reading Frames; D010713:Phosphatidylcholines / Q000737:chemistry*; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
chemical_list: D017931:DNA Primers; D005227:Fatty Acids; D010713:Phosphatidylcholines; D049294:Phospholipid Transfer Proteins; C483438:SEC14 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D002794:Choline