HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 46, 44100-44107, November 15, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/46/44100    most recent M206643200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Howe, A. G. Articles by McMaster, C. R. Search for Related Content PubMed PubMed Citation Articles by Howe, A. G. Articles by McMaster, C. R. Social Bookmarking                      What's this?

Cessation of Growth to Prevent Cell Death Due to Inhibition of Phosphatidylcholine Synthesis Is Impaired at 37 °C in Saccharomyces cerevisiae^*

Alicia G. Howe, Vanina Zaremberg, and Christopher R. McMaster

From the Departments of Pediatrics and Biochemistry and Molecular Biology, Atlantic Research Centre, IWK Health Centre, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada

Received for publication, July 3, 2002, and in revised form, August 26, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phosphatidylcholine is the most abundant phospholipid in eukaryotic cells, comprising 50% of total cellular phospholipid, and thus plays a major role in cellular and organellar biogenesis. In this study, we have used both nutritional deprivation as well as a conditional temperature sensitive allele of PCT1 (CTP:phosphocholine cytidylyltransferase) coupled with an inactivated phosphatidylethanolamine methylation pathway to determine how cells respond to inactivation of phosphatidylcholine synthesis. Metabolic studies determined that phosphatidylcholine biosynthesis decreased to negligible levels within 1 h upon shift to the nonpermissive temperature for the temperature-sensitive PCT1 allele. Phosphatidylcholine mass decreased to negligible levels upon removal of choline from the medium or growth at the nonpermissive temperature, with the levels of the other major phospholipids increasing slightly. Cell growth rate visibly slowed upon cessation of phosphatidylcholine synthesis. Cells remained viable for 7-8 h after phosphatidylcholine synthesis was prevented; however, at time points beyond 8 h, viability was significantly reduced but only if the cells had been previously grown at 37 °C and not 25 °C. The inhibition of phosphatidylcholine synthesis at 37 °C did not alter Golgi-derived vesicle transport to the vacuole as monitored by carboxypeptidase Y processing or to the plasma membrane as determined by invertase secretion. Immunofluorescence microscopy localized Pct1p to the nucleus and nuclear membrane. Pct1p activity is regulated by Sec14p, a cytoplasm/Golgi localized phosphatidylcholine/phosphatidylinositol binding protein that regulates Golgi-derived vesicle transport partially through its ligand-dependent regulation of PCT1 derived enzyme activity. Our nuclear localization of Pct1p indicates that the regulation of Pct1p by Sec14p is indirect.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phosphatidylcholine (PC)¹ is the most abundant phospholipid in eukaryotic cells comprising 50% of cellular phospholipid mass (1). In eukaryotic cells, PC can be synthesized de novo through either the CDP-choline or phosphatidylethanolamine (PE) methylation pathway. The CDP-choline pathway is found in all eukaryotic cell types and synthesizes PC by (i) phosphorylation of choline by choline kinase (CKI1) to produce phosphocholine and (ii) the rate-limiting transfer of a CMP moiety from CTP to phosphocholine by CTP:phosphocholine cytidylyltransferase (PCT1) to produce CDP-choline, followed by (iii) the transfer of phosphocholine from CDP-choline to diacylglycerol by a cholinephosphotransferase reaction (CPT1 or EPT1) to produce PC (2-8). The rate-limiting CTP:phosphocholine cytidylyltransferase is an amphitropic protein that exists in an inactive soluble form that requires translocation to membranes to become active, and this translocation event is believed to be the main form of regulation of this enzyme (9-12).

The PE methylation pathway for the synthesis of PC is found in mammalian hepatocytes and yeast cells (13-16), and PC is synthesized through this route by three successive methylations of the ethanolamine head group of PE (encoded by the CHO2 and OPI3 genes in yeast). The CHO2 gene product methylates PE once, and the OPI3 gene product transfers the final two methyl groups to form PC (15, 16). The contribution of the two pathways to PC synthesis in yeast is dependent on exogenous choline concentration with higher levels of choline favoring synthesis through the CDP-choline pathway (8).

As the major component of biological membranes, PC would be predicted to participate in organellar and cellular biogenesis as well as the formation of vesicles for the transport of proteins and lipids within cells. A role for PC in vesicle transport has been established through work on the Saccharomyces cerevisiae PC/phosphatidylinositol transfer protein Sec14p. Loss of function of Sec14p results in cell death through cessation of Golgi-derived vesicle transport. Inactivation of PC synthesis through the CDP-choline pathway completely rescues cell growth and vesicle transport defects in the absence of functional Sec14p (17-20). When bound to PC, Sec14p is an effective inhibitor of Pct1p (Fig. 1) and is believed to act as a PC sensor with Sec14p-mediated adjustment of the rate of PC synthesis and turnover as a requirement for the regulation of vesicle transport from the Golgi (21).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   PC biosynthesis in yeast. Yeast proteins known to perform particular enzymatic or regulatory steps are illustrated. Solid arrows represent metabolic steps, dotted arrows represent predicted activation events, and dotted lines represent predicted inhibitory events.

In this study, we constructed a yeast strain with an inactivated PE methylation pathway and a conditional temperature-sensitive allele of PCT1. This allowed us to simultaneously inactivate both routes for PC either by removing choline from the medium or by shifting cells to the nonpermissive temperature for function of the PCT1 temperature-sensitive allele, to address the role of decreased PC synthesis on cell growth and viability.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Yeast Strains, Media, and Materials-- Yeast and E. coli media were from Difco. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. AdvanTaq polymerase was a product of CLONTECH. Oligonucleotides were from Invitrogen. Lipids were purchased from Avanti Polar Lipids. [¹⁴C]Choline, [³H]methionine, and phosphorus-32 were purchased from American Radiolabeled Chemicals.

S. cerevisiae strain D319-8A ( leu2 his4 pct1^ts) (22) was mated with strain CTY410 (a his3-200 leu2-9 cho2::LEU2), and diploids were sporulated. Haploid strains that grew poorly on medium lacking choline at 25 °C and did not grow at 37 °C were isolated and mated twice with strain W303-1A (a ura3-1 his3-11 leu2-3,112 trp1-1 ade2-1) to make strain CMY134 ( ura his3 leu2 trp1 ade2 cho2::LEU2 pct1^ts). CMY134 was used to assess the role of PC in cell growth and viability. Strain CTY471 ( ura his3 leu2 trp1 ade2 pct1::URA3) was used for expression of wild type and tagged derivatives of PCT1 for metabolic and biochemical analyses. Yeasts were grown on rich yeast peptone dextrose (YPD) medium, or synthetic dextrose minimal medium supplemented as required for plasmid maintenance (23).

Recombinant DNA Techniques-- The PCT1 gene was amplified from isolated W303-1A yeast genomic DNA using AdvanTaq polymerase and TA cloned into pCR2.1 Topo (Invitrogen). The PCT1 gene was subsequently subcloned into the yeast low copy CEN/ARS shuttle vector pRS413 (24) to create plasmid pMM4. The pMM4 plasmid was digested with BlpI to liberate the majority of the PCT1 open reading frame, and the linearized plasmid DNA was transformed into W303-1A yeast to allow for gap repair of the PCT1 open reading frame from yeast genomic DNA to create plasmid pMM5. Plasmid pMM5 was isolated from yeast, amplified in DH5 E. coli, and sequenced in its entirety to ensure polymerase and gap repair fidelity.

An AgeI site was inserted at the most 3'-end of the PCT1 open reading frame using the Morph site-directed mutagenesis kit (5 Prime  3 Prime, Inc., Boulder, CO), and a 3-fold repeat of the HA epitope tag was inserted into this site by PCR amplification of the 3-fold hemagglutinin epitope repeat from plasmid pGTEP. The wild type and HA-tagged versions of PCT1 were subcloned into pRS413.

The potential temperature-sensitive allele of PCT1 (pct1^ts) was recovered from yeast strain CMY134 genomic DNA by digesting pRS413 containing the PCT1 gene with Blp1 to liberate the majority of the PCT1 open reading frame from the plasmid. The linearized plasmid DNA was transformed into CMY134 yeast to allow for gap repair of the PCT1 open reading frame from yeast genomic DNA into the plasmid. To ensure that the temperature-sensitive mutation was carried within the PCT1 gene isolated from strain CMY134, the PCT1 gene was recovered from strain CMY134 by gap repair and resubcloned into pRS413.

Membrane Preparation-- Total yeast cell membranes were prepared from midlog phase yeast as described (25).

Microscopy-- CTY471 (pct1::URA) containing pMM7 (Myc-tagged PCT1 under control of the inducible GAL1 promoter) or pESC-TRP vector control was grown in selective medium with galactose to log phase. Cultures were fixed by adding formaldehyde to the medium to a final concentration of 3.7% and incubating at room temperature for 2 h. Cells were washed twice with phosphate-buffered saline, and the cell wall was digested with zymolyase. The resulting spheroplasts were mounted on slides treated with polylysine and incubated successively in ice-cold methanol, ice-cold acetone and then rehydrated with room temperature phosphate-buffered saline.

Cells were incubated in blocking buffer (phosphate buffered saline plus 3% bovine serum albumin) in a humid chamber for 30 min. Mouse anti-c-Myc antibody (1:250 dilution in blocking buffer) was added in a humid chamber for 1 h. Slides were washed three times with blocking buffer and incubated with goat anti-mouse Texas Red-conjugated antibody (1:5000 dilution in blocking buffer) for 1 h in a humid chamber in the dark and then washed three times with blocking buffer. DAPI (1 µg/ml) was added to slides for 1 min, slides were washed three times with phosphate-buffered saline, and 20 µl of 90% glycerol, 10% phosphate-buffered saline was placed on the slides. Coverslips were added and sealed. Fluorescence microscopy was performed using a Zeiss axiophot microscope. Texas Red was visualized with Zeiss filter number 15, which excites at 546/560 nm and emits at 590 nm, whereas the UV filter was used to visualize DAPI-stained cells.

For actin staining, cultures were incubated with 0.66 µM AlexaFluor 568 Phalloidin (Molecular Probes, Inc., Eugene, OR) in phosphate-buffered saline for 1 h in the dark. Cells were washed four times with phosphate-buffered saline and mounted on polylysine-treated slides in 90% glycerol, 10% phosphate-buffered saline and visualized with Zeiss filter number 15.

Metabolic Labeling-- Logarithmic phase yeast cells were grown in synthetic minimal medium and labeled with [¹⁴C]choline or [³H]methionine for 1 h, or phosphorus-32 for 18 h. Lipids were extracted, lipid and lipid precursors were separated by thin layer chromatography, and radiolabel was quantitated by scintillation counting as described (25). All of the epitope-tagged versions of Pct1p used in this study were capable of reconstituting PC synthesis to levels similar to those observed for nontagged Pct1p as assessed by the rate of [¹⁴C]choline labeling of PC in a yeast strain containing a genetically inactivated PCT1 gene (CTY471  ura his3 leu2 trp1 ade2 pct1::URA3).

Vesicle Transport-- Exponential cultures of cells growing at 25 °C in supplemented minimal medium lacking methionine and cysteine were concentrated and resuspended in fresh medium and incubated at 25 °C for 1 h. Cells were then pulse-labeled for 10 min with [³⁵S]methionine/cysteine (Expre³⁵S³⁵S protein labeling mix; PerkinElmer Life Sciences) and then chased with the subsequent addition of methionine and cysteine at a final concentration of 0.5% each at 37 °C. Aliquots of cells (A₆₀₀ = 3) were taken at different times and transferred to tubes containing ice-cold 10 mM NaF/NaN₃ in 1 M sorbitol. Cells were disrupted, CPY was immunoprecipitated as described (28), proteins were resolved using 8% SDS-PAGE, and the gel was exposed to x-ray film for subsequent development.

To measure invertase secretion, yeast cells were grown to midlog phase at 25 °C in rich (YPD) medium containing the normal level of 2% glucose and centrifuged at 750 rpm for 5 min to pellet the cells. Pellets were washed twice with 5 ml of sterile water and resuspended in 5 ml of YPD containing only 0.1% glucose to induce invertase. These cultures were grown at 37 °C for 2 h, and invertase secretion was measured using the method described (26, 27). The invertase secretion index of each sample was determined by dividing external invertase () by total invertase (+).

Protein and Lipid Mass-- Protein was measured by the method of Lowry et al. (28). Lipid phosphorus was determined using the method of Ames and Dubin (29), and diacylglycerol mass was determined by the method of Priess et al. (30).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Metabolic Analysis of PC Synthesis-- Yeast strain CMY134 (cho2::LEU2 pct1^ts) or wild type yeast were maintained at 25 °C and while in log phase were labeled with [¹⁴C]choline for 1 h at 25 or 37 °C. As has been recently observed (31), there was an increase in PC synthesis upon shifting the wild type yeast to 37 °C; however, PC labeling was reduced to less than 5% of wild type in the pct1^ts-containing cells. Analysis of the metabolites of the CDP-choline pathway indicated a large rise in the labeling of phosphocholine accompanied the decrease in PC synthesis consistent with a block at the Pct1p step (Fig. 2).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   PC synthesis through the CDP-choline pathway. Wild type and CMY134 yeast (cho2::LEU2 pct1ts) were grown to midlog phase at 25 °C in synthetic minimal medium supplemented with 100 µM choline. Cells were centrifuged and washed with minimal medium, identical aliquots of the culture were grown at 25 or 37 °C, and cells were labeled with [14C]choline (10 µM; 1 × 105 dpm/nmol) for 1 h. Lipids and lipid precursors were extracted and separated by thin layer chromatography for quantitation by scintillation counting.

To ensure that the temperature sensitivity observed at the Pct1p step was due to a mutation within the PCT1 structural gene within strain CMY134 (and not a regulator of Pct1p), the PCT1 gene was recovered from CMY134 genomic DNA by allele rescue through transformation of CMY134 yeast with a BlpI digest of a CEN/ARS plasmid containing the wild type PCT1 gene. This digestion liberates the majority of the PCT1 open reading frame, and the linearized plasmid DNA was isolated by gel electrophoresis and transformed into CMY134 yeast to allow for allele rescue of the PCT1 open reading frame from yeast genomic DNA. The PCT1 gene recovered from CMY134 by allele rescue was subcloned into the low copy yeast vector pRS413 and transformed into the yeast CTY471 (pct1::URA3). The CTY471 strain is wild type except for genetic inactivation of its chromosomal PCT1 gene, and thus the ability of the PCT1 allele recovered from strain CMY134 can be compared with the wild type gene by monitoring the ability to synthesize PC from radiolabeled choline. The CTY471 yeasts were maintained at 25 °C, and the synthesis of PC from radiolabeled choline was monitored in CTY471 containing the PCT1 gene recovered from the genome of CMY134 or the wild type PCT1 gene for 1 h after a shift in the growth temperature from 25 to 37 °C. Upon shifting the yeast to 37 °C, the rate of PC synthesis in the cells containing the PCT1 allele rescued from the CMY134 genome was less than 2% that of the same yeast strain containing the wild type gene (Fig. 3A). This proves that it is the PCT1 gene in strain CMY134 that contains a mutation that renders the Pct1p enzyme activity temperature-sensitive.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   The PCT1 allele of strain CMY134 contains a temperature-sensitive mutation, and the PE methylation pathway is blocked. A, the PCT1 coding region within the genomic DNA of CMY134 was recovered into a centromeric plasmid by allele rescue. The wild type PCT1 gene and PCT1 gene recovered from CMY134 were transformed into yeast strain CTY471 ( ura his3 leu2 trp1 ade2 pct1::URA3), which contains a genetically inactivated PCT1 gene. The yeasts were grown at 25 °C to midlog phase in synthetic minimal medium supplemented as required for plasmid maintenance, and an aliquot of the culture was shifted to 37 °C. The cells were labeled with [14C]choline for 1 h, and lipids were extracted. Incorporation of labeled choline exclusively into PC was confirmed by separation by thin layer chromatography for subsequent quantitation by scintillation counting. B, PC synthesis through the PE methylation pathway in wild type and CMY134 yeast (cho2::LEU2 pct1ts) was measured in midlog phase at 30 °C in synthetic minimal medium supplemented with 100 µM choline. The culture was labeled with [3H]methionine for 1 h. Lipids were extracted, and incorporation of label into PC was determined by separation of lipids by thin layer chromatography for subsequent quantitation by scintillation counting.

To ensure that the cho2::LEU2 gene inactivation was preventing PC synthesis through the PE methylation pathway, CMY134 yeasts were labeled with [³H]methionine for 1 h and compared with wild type yeast. As expected, PC synthesis was reduced to 5% of wild type upon inactivation of the CHO2 gene (Fig. 3B).

The levels of yeast phospholipids were determined by labeling CMY134 cells, inoculated at the same stage of early log phase growth, to steady state with 32 phosphorous for 18 h. The relative levels of total phospholipid were drastically different depending on the growth temperature, the presence or absence of choline, and the presence or absence of a functional PCT1 gene (Fig. 4A). Cells grown at 25 °C in the presence of choline contained similar amounts of total phospholipid regardless of the presence of the plasmid-derived PCT1 gene due to the pct1^ts allele being functional at this temperature. The removal of choline from the medium at 25 °C reduced total recoverable phospholipid to approximately one-third that of cells grown in the presence of choline, indicating that cell growth, phospholipid synthesis, or the amount of phospholipid per cell was dramatically reduced. At the pct1^ts nonpermissive temperature of 37 °C, cells containing plasmid-derived PCT1 contained 40% of the level of phospholipid as the same strain grown at 25 °C, once again implying cell growth, phospholipid synthesis, or the amount of phospholipid per cell was dramatically reduced. The removal of choline at 37 °C reduced phospholipid levels in the PCT1-containing yeast a further 4-fold. Cells grown at 37 °C without a functional PCT1 gene resulted in the recovery of barely detectable levels of lipid phosphorus, implying that there were very few viable cells. Identical dpm from each of the strains from which phosphorus-32-labeled phospholipid was efficiently recovered were separated by two-dimensional thin layer chromatography to determine the relative levels of each phospholipid class. In each case, 40-45% of phospholipid was composed of PC when choline was present in the medium; however, upon the removal of choline, there was a total loss of PC (Fig. 4, B and C). Upon the removal of choline, the bulk of the phospholipid mass of PC was by in large replaced by two phospholipids, with PE increasing from 19-22% total phospholipid to 30-35% and phosphatidylserine increasing from 12-15 to 28-32%. Increases in the proportion of other phospholipids were observed in the absence of PC, with phosphatidylglycerol increasing slightly from 7-10 to 12-15% and phosphatidylinositol increasing from 5-10 to 15-18%.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Phospholipid steady state levels upon cessation of PC synthesis. A, the levels of yeast phospholipids were determined by labeling log phase cells inoculated at an initial absorbance of 0.1 at 600 nm with 32 phosphorous to steady state by continuous pulse for 18 h in synthetic minimal medium with or without 100 µM choline and containing the required supplements for growth and plasmid maintenance. B, to determine the relative levels of each phospholipid class, identical dpm from each strain were separated by two-dimensional thin layer chromatography on 20 × 20-cm silica gel plates using the solvent chloroform/methanol/water/ammonium hydroxide (70:30:2:1, v/v/v/v) in the first dimension and chloroform/methanol/water (65:25:4, v/v/v) in the second dimension and exposed to x-ray film. Representative results are shown. The identical labeling pattern was observed at the permissive temperature of 25 °C in the absence of the plasmid borne PCT1 gene (data not shown) as in its presence. C, the regions of each plate corresponding to the known mobility of the major phospholipids were scraped into scintillation vials, and radiolabel was determined. Data are the mean of two separate experiments performed in duplicate. PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine.

The glycerol backbone and fatty acyl chains required for the synthesis of PC through the CDP-choline pathway are obtained from diacylglycerol. We measured the mass of diacylglycerol in cells that could no longer consume this lipid due to inactivation of the CDP-choline pathway through choline deprivation, growth at the nonpermissive temperature for the pct1^ts allele, or both. At 2 h, there was very little change in the level of diacylglycerol mass under any of the growth conditions; however, after 18 h, the proportion of diacylglycerol compared with total phospholipid increased 3-4-fold at 37 °C but only when PC could not be made (due to choline deprivation and/or growth at the nonpermissive temperature for the pct1^ts allele) (Fig. 5). This increase in diacylglycerol mass was not observed when PC synthesis was prevented at 25 °C.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effect of cessation of PC synthesis on diacylglycerol mass. The levels of yeast diacylglycerol were determined after inoculating yeast in early log phase at an initial absorbance of 0.1 at 600 nm. Yeasts were subsequently grown at 25 or 37 °C for 2 or 18 h in synthetic minimal medium containing the required supplements for plasmid maintenance with or without 100 µM choline, lipids were extracted, and diacylglycerol and lipid phosphorus mass was determined as described under "Experimental Procedures." Data are the mean of two separate experiments performed in duplicate.

PC Synthesis and Cell Growth and Viability-- CMY134 (cho2::LEU2 pct1^ts) yeasts carrying the wild type yeast PCT1 gene on a low copy plasmid or empty vector were grown at 25 °C in the presence of choline to early log phase and then grown with or without choline at either 25 or 37 °C, and the cell growth rate was monitored (Fig. 6A). In the presence of choline, the cells containing the vector control ceased growth within 5 h, whereas the cells containing the PCT1 plasmid continued to grow through to early stationary phase at a rate similar to cells grown at 25 °C (the permissive temperature for the pct1^ts allele). Cells grown without choline did not continue to grow at either 25 or 37 °C with a more pronounced cessation of cell growth for cells grown at 37 °C regardless of the presence or absence of a functional Pct1p. The cessation of cell growth in the absence of choline was similar to that observed for cells grown in the presence of choline but without a functional Pct1p when grown at the nonpermissive temperature for the pct1^ts allele.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Effect of cessation of PC synthesis on cell growth and viability. CMY134 yeast (cho2::LEU2 pct1ts) transformed with a centromeric plasmid containing the wild type PCT1 gene or empty vector was grown to midlog phase at 25 °C in synthetic minimal medium supplemented as required for plasmid maintenance and with 100 µM choline. A, cells were subsequently grown at 25 or 37 °C with or without choline, and the growth rate was monitored by optical density at 600 nm (, PCT1 with choline at 25 °C; , PCT1 without choline at 25 °C; , vector with choline at 25 °C; , vector without choline at 25 °C; , PCT1 with choline at 37 °C; , PCT1 without choline at 37 °C; , vector with choline at 37 °C; , vector without choline at 37 °C). B, cells were removed at 8 or 23 h after shift to with or without choline after growth at 25 or 37 °C; an identical number of cells were serial diluted onto synthetic minimal medium plates supplemented as required for plasmid maintenance and with 100 µM choline; and growth recovery was monitored at 25 °C for 3 days.

We then tested whether cell growth inhibition in cells unable to synthesize PC was due to growth cessation or resulted in a loss of cell viability. Equivalent numbers of cells from CMY134 yeast containing the PCT1 gene or empty vector were serial diluted 8 or 23 h after with or without choline and/or growth at either 25 or 37 °C and then tested for growth on solid medium at 25 °C that was replete with choline. Cells maintained viability up to ~8 h, regardless of the choline content or growth temperature. However, at longer time points, cells that were unable to synthesize PC through the CDP-choline pathway due to genetic inactivation of the pct1^ts allele or choline deprivation resulted in a dramatic loss of cell viability but only for cells grown at 37 °C (Fig. 6B). Cells grown at 25 °C remained viable upon the removal of choline from the medium, although their PC mass was dramatically reduced and their growth rates had dropped to levels almost as low as those observed for cells grown at 37 °C.

Microscopic visualization of the above cells after 23 h under the indicated growth conditions resulted in the observation that the cells that were compromised for their ability to synthesize PC tended to contain a much larger proportion of small to medium size buds compared with cells that could readily synthesize PC (Fig. 7). In addition, the cells that were grown at 37 °C that were unable to restore growth upon return to the permissive temperature contained fewer cells, and those that were visible appeared to possess abnormal internal membranes. Staining these cells with DAPI or phalloidin did not reveal any gross differences in the localization of chromosomal DNA or cortical actin, respectively, when compared with cells that were able to restore cell growth upon return to choline-replete medium at the permissive growth temperature (data not shown).

View larger version (88K): [in this window] [in a new window]   Fig. 7.   Effect of cessation of cell growth on yeast morphology. CMY134 yeast (cho2::LEU2 pct1ts) transformed with a centromeric plasmid containing the wild type PCT1 gene or empty vector was grown to midlog phase at 25 °C in synthetic minimal medium with or without 100 µM choline and supplemented as required for plasmid maintenance. Cells were washed and subsequently grown at 25 or 37 °C with or without choline for 23 h and then visualized by Nomarski optics.

Ongoing PC Synthesis and Golgi-derived Vesicle Transport-- The appearance of what appear to be odd membrane configurations in the cells unable to synthesize PC, with the end result being cell death, implied that there might be alterations in cellular membrane transport. PC synthesis through the CDP-choline pathway is intimately associated with the regulation of vesicle transport through the action of Sec14p. Sec14p is a PC/phosphatidylinositol transfer protein whose essential cell growth and Golgi-derived vesicle transport defects can be rescued if the CDP-choline pathway for PC synthesis is inactivated (17-20). In addition, it has been observed that the PC-bound form of Sec14p inhibits the CDP-choline pathway by inhibiting Pct1p activity, although the precise mechanism for this inhibition has yet to be established (21). It is currently hypothesized that Sec14p acts as a sensor for the rate of PC synthesis through the CDP-choline pathway with increasing PC synthesis resulting in increased PC bound Sec14p and a titrating down of the rate of PC synthesis through Sec14p inhibition of Pct1p (32-34). The decreased PC synthesis results in less PC-bound Sec14p and thereby relieves the inhibition of PC synthesis (Fig. 1). Alterations in the rate of PC synthesis and turnover are believed to be major mechanisms regulating Sec14p-dependent Golgi-derived vesicle transport.

Our observation that the inhibition of PC synthesis resulted in rapid cell growth cessation and that this was coupled to the appearance of odd membranous structures in cells destined to die implied that decreased PC synthesis might alter membrane transport from the sites of PC synthesis in the endoplasmic reticulum/Golgi to other regions of the cell. These observations, along with the known role for ongoing PC synthesis in the regulation of Sec14p-derived Golgi vesicle transport, was the impetus that resulted in our measurement of the effect of cessation of ongoing PC synthesis on Golgi emanating vesicle transport. In CMY134 (cho2::LEU2 pct1^ts) yeast carrying the wild type yeast PCT1 gene on a low copy plasmid or empty vector, we monitored two separate Golgi-derived vesicle transport events, invertase secretion and the ability to process vacuole-destined CPY. After shifting cells to 37 °C (the nonpermissive temperature for ongoing PC synthesis), we observed no alterations in either invertase secretion or CPY transport from the endoplasmic reticulum (P1 form) to Golgi (P2 form) to vacuole (M form) (Fig. 8, A and B) despite a complete inability of cells to synthesize PC at the time points used (Figs. 2-4). Although ongoing PC synthesis is clearly linked to the requirement of Sec14p for cell viability and ongoing Golgi-derived vesicle transport, the cessation of PC synthesis itself does not inhibit vesicle transport.

View larger version (53K): [in this window] [in a new window]   Fig. 8.   Effect of cessation of PC synthesis on Golgi-derived vesicle transport. A, the indicated yeast strains were grown to midlog phase at 25 °C in rich (YPD; high choline levels) medium containing the normal level of 2% glucose and centrifuged at 2500 rpm for 5 min to pellet the cells. Pellets were washed twice with 5 ml of sterile water and resuspended in YPD containing 0.1% glucose for 2 h at 37 °C. Invertase activity was measured in the presence of Triton X-100 (total invertase), and its absence (extracellular invertase). The invertase secretion index is calculated as external invertase/total invertase. B, cells were grown at 25 °C to midlog phase in supplemented minimal medium lacking methionine and cysteine. Cells were pulse-labeled for 10 min with [35S]methionine/cysteine and then chased with the subsequent addition of methionine and cysteine at 37 °C for the indicated time points. Cells were disrupted, and CPY was immunoprecipitated from the supernatant using a polyclonal CPY antibody and protein A-Sepharose. The immunoprecipitated proteins were resolved by SDS-polyacrylamide gel electrophoresis, and the gel was exposed to x-ray film for subsequent development. Wild type and sec14ts cells are positive and negative controls for invertase secretion.

Subcellular Location of Pct1p-- Sec14p is a cytoplasmic/Golgi-localized protein that regulates PC synthesis by altering the activity of Pct1p; however, it is not known if this regulation is through a direct interaction between the two proteins or if an indirect pathway is involved. To delineate how Sec14p might alter Pct1p activity to allow for Sec14p to aid in sensing and regulating PC levels, we used subcellular fractionation and immunofluorescence to localize Pct1p. The yeast Pct1p protein was found mainly in the membrane fraction by Western blot (Fig. 9A). This observed protein distribution correlates well with previous analyses of the distribution of CTP:phosphocholine cytidylyltransferase enzyme activity within yeast subcellular fractions (8, 22). Immunofluorescence revealed that Pct1p localized to the yeast nucleus as determined by its colocalization with the DAPI stain for chromosomal DNA (Fig. 9B). No staining of yeast cells for Pct1p using the epitope-specific antibody was observed in vector control cells (data not shown). In yeast, the morphology of the cell as assessed by the size of the budding daughter cell is indicative of cell cycle stage, and at no stage were we able to observe Pct1p outside the nucleus.

View larger version (27K): [in this window] [in a new window]   Fig. 9.   Subcellular location of Pct1p. A, CTY471 (pct1::URA) yeast containing Pct1p with or without a 3× repeat of the HA epitope at its carboxyl terminus expressed under the control of its own promoter on a low copy CEN/ARS plasmid was grown to midlog phase at 30 °C, and the soluble (S) and membrane (M) fractions were prepared as described under "Experimental Procedures." Equal amounts of protein were separated by SDS-PAGE, and Western blots versus the HA epitope were performed. B, CTY471 (pct1::URA) containing pMM7 (Myc-tagged PCT1 under control of the inducible GAL1 promoter) or pESC-TRP1 vector controls were grown to midlog phase in galactose at 30 °C to induce Pct1p-Myc protein expression. Cultures were fixed by adding formaldehyde to the medium to a final concentration of 3.7%, washed with phosphate-buffered saline, and spheroplasted. Spheroplasts were mounted on slides treated with polylysine and incubated successively in ice-cold methanol, ice-cold acetone, and room temperature phosphate-buffered saline. Cells were incubated in blocking buffer (phosphate-buffered saline plus 3% bovine serum albumin) in a humid chamber for 30 min followed by mouse anti-c-Myc antibody in a humid chamber for 1 h, washed with blocking buffer, and incubated with goat anti-mouse Texas Red-conjugated antibody in the dark and washed three times with blocking buffer. DAPI (1 µg/ml) was added to slides for 1 min, slides were washed three times with phosphate-buffered saline and 20 µl of 90% glycerol, 10% phosphate-buffered saline was placed on the slides, and coverslips were added and sealed. Fluorescence microscopy was performed using a Zeiss axiophot microscope. Texas Red was visualized with Zeiss filter number 15 that excites at 546/560 nm and emits at 590 nm, whereas the UV filter was used to visualize DAPI staining of chromosomal DNA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PC is the major phospholipid in eukaryotic cells and plays a major role in cellular and organellar biogenesis (1, 17-21, 32-34). How cellular PC homeostasis and cellular integrity are maintained in response to changes in growth rate and cellular stresses and how cells sense PC levels and couple its metabolic regulation to cellular growth are very poorly understood. In this study, we constructed a yeast strain genetically deficient in both PC biosynthetic pathways. The PE methylation pathway contained a genetically inactivated CHO2 gene (cho2::LEU2), and thus the PE methyltransferase pathway was effectively turned off, whereas the CDP-choline pathway possessed a defect in the CTP:phosphocholine cytidylyltransferase gene (PCT1) that resulted in a temperature-sensitive allele (pct1^ts) whose encoded enzyme activity was inactivated when cells were grown at 37 °C (the nonpermissive temperature for pct1^ts-encoded protein). Thus, we were able to turn off PC synthesis by either removing choline from the medium or shifting cells to the nonpermissive temperature for the pct1^ts allele.

When choline was removed from the medium, yeast ceased growth within 8-10 h subsequent to choline removal at either 25 or 37 °C regardless of the presence of a functional plasmid-borne PCT1 gene. This is not surprising, since yeasts are unable to synthesize choline de novo. The cessation of PC synthesis by the elimination of choline from the medium resulted in growth cessation but not a loss in viability when the yeasts were grown at 25 °C. These results are consistent with previous data in which the PE methylation pathway was genetically inactivated and PC synthesis through the CDP-choline pathway was prevented by not including choline in the medium at the normal growth temperature of 30 °C for S. cerevisiae (35). We demonstrated that PC levels dropped to negligible levels in the yeast grown at 25 °C, and since yeast cannot synthesize choline de novo without an intact PE methylation pathway, this implies that the slowed growth rate was probably due to decreasing PC levels. The lack of cell death upon growth cessation due to decreased PC levels when grown at 25 or 30 °C indicates that yeast cells have a mechanism for preventing cell death (probably by halting cell growth) in response to a deficiency in PC biosynthesis. When the same yeast strain (cho2::LEU2 pct1^ts) was grown at 37 °C and PC synthesis was prevented by either choline deprivation or the absence of a functional Pct1p, yeast ceased growth at a rate slightly faster than that observed for cells grown at 25 °C. When assessed for viability subsequent to growth at 37 °C, there were very few viable cells. Cell death could be prevented at 37 °C if a functional plasmid-borne PCT1 gene and choline were both supplied, indicating that cell death was due to the decrease in PC mass at the elevated temperature. Thus, there is a selective inability for cells to cease growth and prevent cell death at 37 °C but not 25 °C (this study) or 30 °C (35) when PC synthesis is prevented. Consistent with the hypothesis that yeast cannot sense that PC synthesis through the CDP-choline pathway is off at 37 °C was our observation that there was a 3-4-fold increase in diacylglycerol mass at 37 °C when PC could not be made due to choline deprivation and/or growth at the nonpermissive temperature for the pct1^ts allele, and this increase was not observed at 37 °C when PC synthesis was restored due to the presence of plasmid-borne PCT1 or under any growth condition at 25 °C. This implies that cells are still attempting to supply diacylglycerol for consumption for PC synthesis by the CDP-choline pathway and is consistent with the premise that at 37 °C cells are unable to sense that PC synthesis has been turned off. Upon the cessation of PC synthesis, we detected an increase in the mass of each of the major phospholipid classes; however, each phospholipid type increased in a similar manner in cells that remained viable versus cells that died. This implies that substitution of PC with a different phospholipid type is not the mechanism that differentiates whether yeast cells remain viable or die.

Intuitively, one would predict that preventing the synthesis of PC (which supplies cells with 50% of its phospholipid) in the endoplasmic reticulum/Golgi would decrease vesicle transport from these organelles. To support this supposition, we observed what appeared to be odd membranous structures in yeast cells that possessed severe enough deficiencies in PC levels to ultimately lead to their death. However, our results clearly indicate that preventing PC synthesis did not alter the rate of endoplasmic reticulum to Golgi transport or Golgi-derived vesicle transport to either the plasma membrane or vacuole. A current biochemical and genetic model links PC synthesis with Sec14p, an essential PC/phosphatidylinositol-binding protein required for Golgi-derived vesicle transport (17-20). PC-bound Sec14p is an inhibitor of Pct1p activity (21) in vivo, and it is believed that regulation of PC synthesis and turnover by Sec14p is integral to Golgi function, with PC acting as a negative regulator of vesicle transport and diacylglycerol as a positive regulator (32-34). Thus, in our study, shutting off PC synthesis through the CDP-choline pathway in cells containing a deficient methylation pathway would decrease PC synthesis and eventually mass in the endoplasmic reticulum/Golgi, and the current hypothesis would predict that this would not be inhibitory to vesicle transport, which is exactly what we observed. In addition, if diacylglycerol promotes vesicle transport, then we should expect to see at least no change, or possibly an increase, in diacylglycerol mass due to an inability to consume diacylglycerol upon inactivation of the CDP-choline pathway. Diacylglycerol mass was unchanged under most of our growth conditions and was indeed found to rise 3-4-fold under conditions where PC synthesis was inhibited for 18 h at 37 °C. Our present results combined with previous data are consistent with a role for the diacylglycerol that is used for PC synthesis through the CDP-choline pathway as a positive regulator of Golgi-derived vesicle transport and the PC synthesized by this pathway as a negative regulator of Golgi vesicle transport. Although Sec14p regulates PC synthesis by altering Pct1p activity, the exact mechanism by which Sec14p regulates this activity is not known. Our immunofluorescence studies place Pct1p in the nucleus/nuclear membrane at all stages of the cell cycle, implying that Sec14p and Pct1p do not directly interact as Sec14p resides in the cytoplasm and on Golgi membranes (17). Therefore, there must be a signaling pathway that exists that allows Pct1p activity to be regulated by PC-bound Sec14p.

Recent work has revealed that at 37 °C PC synthesis is increased exclusively through the CDP-choline pathway in yeast and that this PC is used as a source for increased PC turnover via deacylation (31), although the phospholipase involved has yet to be isolated. We feel that the most likely interpretation of the combined data is either of the following. (i) Inhibiting PC synthesis through the CDP-choline pathway results in ongoing cell growth in the face of depleting PC levels; the heat-activated PC phospholipase is not responsive to the yeast cell growth-regulatory machinery; and its continued activity results in cell death at 37 °C. (ii) The increase in diacylglycerol mass that correlated with yeast cell death sends a signal to the cells that results in their death. The identity of this phospholipase and/or the sensing and signaling pathways that link PC and its upstream metabolite diacylglycerol with the cellular growth decision-making machinery are major requirements for a fuller understanding of the co-regulation of cellular PC homeostasis with cell survival.

	ACKNOWLEDGEMENTS

We thank Vytas Bankaitis, Satoshi Yamashita, Stephen Garrett, and Scott Emr for yeast strains, plasmids, and antibodies. We are indebted to Neale Ridgway, David Byers, Harold Cook, and the Dalhousie yeast genetics group for helpful discussions.

	FOOTNOTES

^* This work was supported by an operating grant from the National Sciences and Engineering Research Council, a senior clinical scholar award from the IWK Health Centre (to C. R. M.), and an IWK Health Centre graduate studentship (to A. G. H.).

To whom correspondence should be addressed. Tel.: 902-494-7066; Fax: 902-494-1394; E-mail: cmcmaste@is.dal.ca.

Published, JBC Papers in Press, August 27, 2002, DOI 10.1074/jbc.M206643200

	ABBREVIATIONS

The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; CPY, carboxypeptidase Y; YPD, yeast peptone dextrose; HA, hemagglutinin; DAPI, 4',6-diamidino-2-phenylindole.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				K. Stalberg, A. C. Neal, H. Ronne, and U. Stahl Identification of a novel GPCAT activity and a new pathway for phosphatidylcholine biosynthesis in S. cerevisiae J. Lipid Res., August 1, 2008; 49(8): 1794 - 1806. [Abstract] [Full Text] [PDF]

				A. G. Howe, G. D. Fairn, K. MacDonald, V. A. Bankaitis, and C. R. McMaster Regulation of Phosphoinositide Levels by the Phospholipid Transfer Protein Sec14p Controls Cdc42p/p21-Activated Kinase-Mediated Cell Cycle Progression at Cytokinesis Eukaryot. Cell, October 1, 2007; 6(10): 1814 - 1823. [Abstract] [Full Text] [PDF]

				R. Harrison, B. Papp, C. Pal, S. G. Oliver, and D. Delneri Plasticity of genetic interactions in metabolic networks of yeast PNAS, February 13, 2007; 104(7): 2307 - 2312. [Abstract] [Full Text] [PDF]

				H. A. Boumann, J. Gubbens, M. C. Koorengevel, C.-S. Oh, C. E. Martin, A. J.R. Heck, J. Patton-Vogt, S. A. Henry, B. de Kruijff, and A. I.P.M. de Kroon Depletion of Phosphatidylcholine in Yeast Induces Shortening and Increased Saturation of the Lipid Acyl Chains: Evidence for Regulation of Intrinsic Membrane Curvature in a Eukaryote Mol. Biol. Cell, February 1, 2006; 17(2): 1006 - 1017. [Abstract] [Full Text] [PDF]

				V. Zaremberg, C. Gajate, L. M. Cacharro, F. Mollinedo, and C. R. McMaster Cytotoxicity of an Anti-cancer Lysophospholipid through Selective Modification of Lipid Raft Composition J. Biol. Chem., November 11, 2005; 280(45): 38047 - 38058. [Abstract] [Full Text] [PDF]

				G. Pessi, J.-Y. Choi, J. M. Reynolds, D. R. Voelker, and C. B. Mamoun In Vivo Evidence for the Specificity of Plasmodium falciparum Phosphoethanolamine Methyltransferase and Its Coupling to the Kennedy Pathway J. Biol. Chem., April 1, 2005; 280(13): 12461 - 12466. [Abstract] [Full Text] [PDF]

				J. P. Fernandez Murray and C. R. McMaster Nte1p-mediated Deacylation of Phosphatidylcholine Functionally Interacts with Sec14p J. Biol. Chem., March 4, 2005; 280(9): 8544 - 8552. [Abstract] [Full Text] [PDF]