Turnover of phosphatidylcholine in Saccharomyces cerevisiae. The role of the CDP-choline pathway.

The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae. In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37 degrees C. In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature. In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37 degrees C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift. These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway. Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.

Genetic regulation of phospholipid metabolism: yeast as a model eukaryote.

Baker's yeast, Saccharomyces cerevisiae, is an excellent and an increasingly important model for the study of fundamental questions in eukaryotic cell biology and genetic regulation. The fission yeast, Schizosaccharomyces pombe, although not as intensively studied as S. cerevisiae, also has many advantages as a model system. In this review, we discuss progress over the past several decades in biochemical and molecular genetic studies of the regulation of phospholipid metabolism in these two organisms and higher eukaryotes. In S. cerevisiae, following the recent completion of the yeast genome project, a very high percentage of the gene-enzyme relationships in phospholipid metabolism have been assigned and the remaining assignments are expected to be completed rapidly. Complex transcriptional regulation, sensitive to the availability of phospholipid precusors, as well as growth phase, coordinates the expression of the structural genes encoding these enzymes in S. cerevisiae. In this article, this regulation is described, the mechanism by which the cell senses the ongoing metabolic activity in the pathways for phospholipid biosynthesis is discussed, and a model is presented. Recent information relating to the role of phosphatidylcholine turnover in S. cerevisiae and its relationship to the secretory pathway, as well as to the regulation of phospholipid metabolism, is also presented. Similarities in the role of phospholipase D-mediated phosphatidylcholine turnover in the secretory process in yeast and mammals lend further credence to yeast as a model system.

GIT1, a gene encoding a novel transporter for glycerophosphoinositol in Saccharomyces cerevisiae.

Phosphatidylinositol catabolism in Saccharomyces cerevisiae cells cultured in media containing inositol results in the release of glycerophosphoinositol (GroPIns) into the medium. As the extracellular concentration of inositol decreases with growth, the released GroPIns is transported back into the cell. Exploiting the ability of the inositol auxotroph, ino1, to use exogenous GroPIns as an inositol source, we have isolated mutants (Git-) defective in the uptake and metabolism of GroPIns. One mutant was found to be affected in the gene encoding the transcription factor, SPT7. Mutants of the positive regulatory gene INO2, but not of its partner, INO4, also have the Git- phenotype. Another mutant was complemented by a single open reading frame (ORF) termed GIT1 (glycerophosphoinositol). This ORF consists of 1556 bp predicted to encode a polypeptide of 518 amino acids and 57.3 kD. The predicted Git1p has similarity to a variety of S. cerevisiae transporters, including a phosphate transporter (Pho84p), and both inositol transporters (Itr1p and Itr2p). Furthermore, Git1p contains a sugar transport motif and 12 potential membrane-spanning domains. Transport assays performed on a git1 mutant together with the above evidence indicate that the GIT1 gene encodes a permease involved in the uptake of GroPIns.

A role for phospholipase D (Pld1p) in growth, secretion, and regulation of membrane lipid synthesis in yeast.

The SEC14 gene encodes a phosphatidylinositol/phosphatidylcholine transfer protein essential for secretion and growth in yeast (1). Mutations (cki1, cct1, and cpt1) in the CDP-choline pathway for phosphatidylcholine synthesis suppress the sec14 growth defect (2), permitting sec14(ts) cki1, sec14(ts) cct1, and sec14(ts) cpt1 strains to grow at the sec14(ts) restrictive temperature. Previously, we reported that these double mutant strains also excrete the phospholipid metabolites, choline and inositol (3). We now report that these choline and inositol excretion phenotypes are eliminated when the SPO14 (PLD1) gene encoding phospholipase D1 is deleted. In contrast to sec14(ts) cki1 strains, sec14(ts) cki1 pld1 strains are not viable at the sec14(ts) restrictive temperature and exhibit a pattern of invertase secretion comparable with sec14(ts) strains. Thus, the PLD1 gene product appears to play an essential role in the suppression of the sec14(ts) defect by CDP-choline pathway mutations, indicating a role for phospholipase D1 in growth and secretion. Furthermore, sec14(ts) strains exhibit elevated Ca2+-independent, phophatidylinositol 4,5-bisphosphate-stimulated phospholipase D activity. We also propose that phospholipase D1-mediated phosphatidylcholine turnover generates a signal that activates transcription of INO1, the structural gene for inositol 1-phosphate synthase.

Role of the yeast phosphatidylinositol/phosphatidylcholine transfer protein (Sec14p) in phosphatidylcholine turnover and INO1 regulation.

In yeast, mutations in the CDP-choline pathway for phosphatidylcholine biosynthesis permit the cell to grow even when the SEC14 gene is completely deleted (Cleves, A., McGee, T., Whitters, E., Champion, K., Aitken, J., Dowhan, W., Goebl, M., and Bankaitis, V. (1991) Cell 64, 789-800). We report that strains carrying mutations in the CDP-choline pathway, such as cki1, exhibit a choline excretion phenotype due to production of choline during normal turnover of phosphatidylcholine. Cells carrying cki1 in combination with sec14(ts), a temperature-sensitive allele in the gene encoding the phosphatidylinositol/phosphatidylcholine transporter, have a dramatically increased choline excretion phenotype when grown at the sec14(ts)-restrictive temperature. We show that the increased choline excretion in sec14(ts) cki1 cells is due to increased turnover of phosphatidylcholine via a mechanism consistent with phospholipase D-mediated turnover. We propose that the elevated rate of phosphatidylcholine turnover in sec14(ts) cki1 cells provides the metabolic condition that permits the secretory pathway to function when Sec14p is inactivated. As phosphatidylcholine turnover increases in sec14(ts) cki1 cells shifted to the restrictive temperature, the INO1 gene (encoding inositol-1-phosphate synthase) is also derepressed, leading to an inositol excretion phenotype (Opi-). Misregulation of the INO1 gene has been observed in many strains with altered phospholipid metabolism, and the relationship between phosphatidylcholine turnover and regulation of INO1 and other co-regulated genes of phospholipid biosynthesis is discussed.

Effects of social experience on the corticosterone response of adult rats to pup cues.

Rat mothers whose pups have been removed show greater plasma corticosterone elevations when the pups are shocked and returned in a wire-mesh basket than when only the wire basket is returned. For mothers housed with an adult male or virgin female, this differential adrenocorticoid responsiveness between the shocked-pup and empty-basket conditions was not observed unless the adult partner was removed 18 hr before testing. Nonlactating adults housed with pups and mother for up to 2 weeks exhibited corticoid elevations equivalent to the shocked-pup and empty-basket conditions whether tested with the mother or 18 hr after her removal. These results indicate that the differential pituitary-adrenal responsiveness that female rats show to pup cues during lactation cannot be accounted for by the length of the mother's exposure to pups, and, further, that this relative responsiveness during lactation appears to be largely due to the suppression of adrenocorticoid reactivity to a nonsocial environmental disturbance.

The social behavior of a marmoset (Saguinus fuscicollis) group III. Spatial analysis of social structure.

The social behavior, and particularly the spacing patterns, of a marmoset (Saguinus fuscicollis) group in a semi-naturalistic enclosure were observed for 14 months. Data analysis revealed various changes as the group grew in size from four to eventually six members. Weekly mean distances between the adult pair supported a spatial measure for estrus for the female. Group dispersion, mean squared distance between all possible pairs, seemed to vary with the age composition of the group. Factor analysis of location correlation matrices by 4-week blocks resulted in age-related subgroupings. Each of the adult pair formed an independent subgroup except for behavioral estrus and early infant care periods when they formed one subgroup. The offspring, initially attached to the adults, gradually moved into juvenile/subadult subgroups. Increasing spatial independence was shown as an animal approached adulthood at 24 months of age.