Phosphatidylinositol-transfer protein and its homologues in yeast.

Yeast Sec14p acts as a phosphatidylinositol/phosphatidylcholine-transfer protein in vitro. In vivo, it is essential in promoting Golgi secretory function. Products of five genes named SFH1-SFH5 (Sec Fourteen Homologues 1-5) exhibit significant sequence homology to Sec14p and together they form the Sec14p family of lipid-transfer proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism and membrane trafficking.

Rhythms of the pineal N-acetyltransferase mRNA and melatonin concentrations during embryonic and post-embryonic development in chicken.

Development of a daily rhythmicity in transcription of a gene encoding a rate-limiting enzyme of melatonin biosynthesis, the arylalkylamine-N-acetyltransferase (AA-NAT) was studied by northern blot analysis in pineal glands of 16 and 19-day-old embryos and 1, 4, 8, 11, and 14-day-old chicks. In a parallel experiment, melatonin content in pineal glands and plasma was measured. A significant rhythm of AA-NAT expression was found at embryonic day (ED) 16, the earliest day assayed in this experiment. Expression was low during the daytime and a clear signal was found in the middle of the darktime. The intensity of the signal was increasing during the ontogeny. The nocturnal pineal melatonin concentrations were increasing over the studied period (from ED 19 until post-embryonic day 21). Midnight plasma melatonin concentrations increased from ED19 to PD 3 and oscillated around this value afterwards. Data show that rhythmic expression of AA-NAT mRNA starts very early in development of chicken and plays a major role in melatonin rhythm generation during embryonic development.

The yeast inositol-sensitive upstream activating sequence, UASINO, responds to nitrogen availability.

The INO1 gene of yeast is expressed in logarithmically growing, wild-type cells when inositol is absent from the medium. However, the INO1 gene is repressed when inositol is present during logarithmic growth and it is also repressed as cells enter stationary phase whether inositol is present or not. In this report, we demonstrate that transient nitrogen limitation also causes INO1 repression. The repression of INO1 in response to nitrogen limitation shares many features in common with repression in response to the presence of inositol. Specifically, the response to nitrogen limitation is dependent upon the presence of a functional OPI1 gene product, it requires ongoing phosphatidylcholine biosynthesis and it is mediated by the repeated element, UASINO, found in the promoter of INO1 and other co-regulated genes of phospholipid biosynthesis. Thus, we propose that repression of INO1 in response to inositol and in response to nitrogen limitation occurs via a common mechanism that is sensitive to the status of ongoing phospholipid metabolism.

Development of a transformation system for the multinuclear yeast Dipodascus (Endomyces) magnusii.

We have developed the first system for genetic transformation of the multinuclear yeast Dipodascus magnusii. The system is based on a dominant selectable marker and an autonomously replicating sequence. We have constructed a plasmid vector which contains a marker conferring resistance to zeocin and the segment of non-transcribed spacer of D. magnusii ribosomal DNA which supports the autonomous replication of plasmid DNA in yeast cells. Plasmid DNA has been transferred into D. magnusii cells by electroporation.

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.

Regulation of yeast phospholipid biosynthetic genes in phosphatidylserine decarboxylase mutants.

In the yeast Saccharomyces cerevisiae, the products of two genes (PSD1 and PSD2) are able to catalyze the decarboxylation of phosphatidylserine (PS) to produce phosphatidylethanolamine (PE) (C. J. Clancey, S. Chang, and W. Dowhan, J. Biol. Chem. 268:24580-24590, 1993; P. J. Trotter, J. Pedretti, and D. R. Voelker, J. Biol. Chem. 268:21416-21424, 1993; P.J. Trotter, and D. R. Voelker, J. Biol. Chem. 270:6062-6070, 1995). I report that the major mitochondrial PS decarboxylase gene (PSD1) is transcriptionally regulated by inositol in a manner similar to that reported for other coregulated phospholipid biosynthetic genes. The second PS decarboxylase gene (PSD2) is not regulated on a transcriptional level by inositol and/or ethanolamine. In yeast, phosphatidylcholine (PC) biosynthesis is required for the repression of the phospholipid biosynthetic genes, including the INO1 gene, in response to inositol. I show that the presence of a functional major mitochondrial PS decarboxylase encoded by the PSD1 gene is necessary for proper regulation of INO1 in response to inositol in the absence of ethanolamine. Disruption of the second PS decarboxylase gene (PSD2) does not affect the INO1 regulation. Analysis of phospholipid content of PS decarboxylase mutants suggests that the proportion of PC on total cellular phospholipids is not correlated to the cell's ability to repress INO1 in response to inositol. Rather, yeast cells are apparently able to monitor the flux through the phospholipid biosynthetic pathway and modify the transcription of phospholipid biosynthetic genes accordingly.

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.

The role of phosphatidylcholine biosynthesis in the regulation of the INO1 gene of yeast.

In yeast, as in other eukaryotes, phosphatidylcholine (PC) can be synthesized via methylation of phosphatidylethanolamine or from free choline via the CDP-choline pathway. In yeast, PC biosynthesis is required for the repression of the phospholipid biosynthetic genes, including the INO1 gene, in response to inositol. In this study, we analyzed the effect of mutations in genes encoding enzymes involved in PC biosynthesis on the transcriptional regulation of phospholipid biosynthetic genes. We report that repression of INO1 transcription in response to inositol is clearly dependent on ongoing PC biosynthesis, but it is independent of the route of synthesis. Our results also suggest that intermediates in the phosphatidylethanolamine methylation and CDP-choline pathways are not responsible for generating the regulatory signal that results in repression of INO1 and other coregulated genes of phospholipid biosynthesis. Furthermore, repression of INO1 is not tightly correlated to the proportion of PC in the total cellular phospholipids. Rather, we report that when the rate of synthesis of PC becomes growth limiting, the addition of inositol fails to repress the phospholipid biosynthetic genes, but when the rate of PC synthesis is sufficient to sustain normal growth, the addition of inositol to the growth medium has the effect of repressing INO1 and other phospholipid biosynthetic genes.

Mitochondrial DNA of Endomyces (Dipodascus) magnusii.

Mitochondrial DNA was isolated from a yeast-like microorganism, Endomyces (Dipodascus) magnusii. The mtDNA consisted of circular molecules 40.4 kb long. A restriction map was constructed using the cleavage data of seven endonucleases. The arrangement of several genes within the mitochondrial genome of E. magnusii was established by specific hybridization with probes prepared from the mtDNA of Saccharomyces cerevisiae.

Isolation of a dsRNA virus from Dipodascus (Endomyces) magnusii.

Virus-like particles (VLPs) of 40 nm diameter were isolated from the yeast-like fungus Dipodascus magnusii. These VLPs copurify with several linear double-stranded RNA molecules of different size. We have found some polymorphism in both the length and the number of these dsRNAs among six D. magnusii strains. Analysis of CsCl gradient-purified VLPs on PAGE/SDS electrophoresis showed one major protein component with an apparent molecular weight of 75 kDa.

A method for the efficient transfer of isolated mitochondria into yeast protoplasts.

When protoplasts isolated from non-respiring rho0 strains of S. cerevisiae are incubated with mitochondria from respiring rho+ strains in the presence of polyethylene glycol, a small fraction of the acceptor protoplasts acquires respiratory capacity. Pretreatment of the isolated mitochondria and protoplasts with Ca2+ ions enhanced the frequency of ensuing respiration-competent colonies by almost three orders of magnitude. When mitochondria were isolated from a strain exhibiting killer capacity as a cytoplasmic marker, in addition to the appropriate nuclear and mitochondrial markers, about one-third of the ensuing rho+ colonies were not killers. Possible mechanisms for the transfer of mitochondria into acceptor protoplasts were studied.