The phosphatidylcholine to phosphatidylethanolamine ratio of Saccharomyces cerevisiae varies with the growth phase.

Abstract

This study compares the effect of the growth phase on the phospholipid composition and the activity of several phospholipid biosynthetic enzymes in a wild-type yeast grown in fermentable (glucose) and non-fermentable (lactate) semi-synthetic and complete synthetic media. Several distinct differences as well as similarities were found. The cellular phosphatidylcholine: phosphatidylethanolamine (PC:PE) ratio was found to vary with the growth phase, with increases in PC levels at the expense of PE during the transition to stationary phase. The variation was most pronounced in semi-synthetic lactate medium, which is routinely used for the isolation of mitochondria, where the PC:PE ratio changed from 0.9 to 2.2 during this transition. Similar growth phase-dependent changes in PC and PE content were observed in isolated organelles such as mitochondria, mitochondria-associated membranes and microsomes. Phosphatidylinositol (PI) levels were much higher in cells grown on lactate compared to cells grown on glucose (20% vs. 5-10%). Irrespective of the medium, PI levels increased upon entering stationary phase. The activities of the phospholipid biosynthetic enzymes phosphatidylserine synthase and the phospholipid-N-methyltransferases were found to be maximal at the end of logarithmic growth and to decrease upon entering stationary phase in all media. Cells grown on lactate displayed a significantly higher phospholipid:protein ratio than cells grown on glucose. The results are discussed in terms of regulation of phospholipid biosynthesis and membrane biogenesis in response to growth phase and carbon source.

Full text

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Chapter 3
The phosphatidylcholine to phosphatidylethanolamine
ratio of Saccharomyces cerevisiae varies with the growth
phase
M.J.F.W. Janssen, M.C. Koorengevel, B. de Kruijff, A.I.P.M. de Kroon
Yeast 16 (2000), 641-650.
© 2000 John Wiley & Sons Limited, reproduced with permission.

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Chapter 3
40
Abstract
This study compares the effect of the growth phase on the phospholipid composition
and the activity of several phospholipid biosynthetic enzymes in a wild-type yeast grown in
fermentable (glucose) and non-fermentable (lactate) semi-synthetic and complete synthetic
media. Several distinct differences as well as similarities were found. The cellular
phosphatidylcholine (PC) to phosphatidylethanolamine (PE) ratio was found to vary with the
growth phase, with increases in PC levels at the expense of PE during the transition to
stationary phase. The variation was most pronounced in semi-synthetic lactate medium,
which is routinely used for the isolation of mitochondria, where the PC/PE ratio changed
from 0.9 to 2.2 during this transition. Similar growth phase dependent changes in PC and PE
content were observed in isolated organelles such as mitochondria, mitochondria-associated
membranes, and microsomes. Phosphatidylinositol (PI) levels were much higher in cells
grown on lactate compared to cells grown on glucose (20% versus 5-10%). Irrespective of the
medium, PI levels increased upon entering stationary phase. The activities of the
phospholipid biosynthetic enzymes phosphatidylserine synthase and the phospholipid-N-
methyltransferases were found to be maximal at the end of logarithmic growth and to
decrease upon entering stationary phase in all media. Cells grown on lactate displayed a
significantly higher phospholipid to protein ratio than cells grown on glucose. The results are
discussed in terms of regulation of phospholipid biosynthesis and membrane biogenesis in
response to growth phase and carbon source.
Introduction
The yeast Saccharomyces cerevisiae shares similar patterns of membrane
phospholipids and similar pathways of phospholipid metabolism with higher eukaryotes, with
a few exceptions (for a review see [Carman and Henry, 1989]). Two branches of
phospholipid biosynthesis diverge from the precursor CDP-diacylglycerol. Phosphatidyl-
inositol (PI) is formed from CDP-diacylglycerol and inositol, and the main biosynthetic route
leading to phosphatidylcholine (PC) in yeast starts with the conversion of CDP-
diacylglycerol and serine into phosphatidylserine (PS). PS is decarboxylated to phosphatidyl-
ethanolamine (PE) which is converted to PC by three sequential methylations. Alternatively,
PE and PC can be synthesized from phosphatidic acid (PA) via diacylglycerol and CDP-
ethanolamine or CDP-choline (Kennedy pathway), respectively. In yeast, the methylation of
PE by the phospholipid-N-methyltransferase (PNMT) enzymes is considered the primary
pathway of biosynthesis of PC when cells are grown in the absence of choline.
Many enzymes involved in the biosynthetic routes of phospholipids are coordinately
regulated at the transcriptional level in response to phospholipid precursors and growth phase
(for recent reviews, see [Carman and Zeimetz, 1996; Henry and Patton-Vogt, 1998; Carman

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Variation of the PC to PE ratio in yeast with growth phase
41
and Henry, 1999]. For example, the INO1 gene product which catalyzes the formation of
inositol, as well as the enzymes involved in the production of PC via CDP-diacylglycerol, are
repressed by inositol and choline, and the latter enzymes exhibit maximal activities in
exponential phase whereas the activities are reduced in stationary growth phase. In contrast,
the expression of PI synthase does not respond to inositol or growth phase, but is regulated by
the carbon source [Anderson and Lopes, 1996]. Furthermore, enzymatic activities can be
regulated by the membrane lipid composition and phosphorylation (e.g. PS synthase [Hromy
and Carman, 1986; Kinney and Carman, 1988]).
Most phospholipids are synthesized in the endoplasmic reticulum and closely related
membranes. The contribution of mitochondria to cellular phospholipid biosynthesis is
restricted to the formation of PE and cardiolipin (CL) [Daum and Vance, 1997]. This
necessitates efficient import of the phospholipids which are not synthesized in mitochondria
to ensure membrane growth and to maintain membrane lipid composition in the growing cell.
For studies on mitochondrial biogenesis mitochondria are usually isolated from yeast grown
on a non-fermentable carbon source such as lactate. In our research, which focuses on the
import of PC into mitochondria, we noticed that the phospholipid composition, more in
particular the PC/PE ratio, of these organelles was highly dependent on the moment of
harvest in the late log phase. Knowledge on the variation in phospholipid composition and
phospholipid biosynthetic enzymes under non-fermenting conditions is limited. It has been
reported that regulation of phospholipid biosynthesis and composition depends on the exact
growth conditions such as the carbon source [Anderson and Lopes, 1996; Gaynor et al.,
1991]. However, most studies on the regulation of phospholipid biosynthesis pertain to yeast
cells grown on glucose-based media only. A previous study on cellular phospholipid
biosynthesis and composition reported no significant growth phase dependent changes in the
PC/PE ratio under the culture conditions used [Homann et al., 1987]. However, in a more
recent study the PC and PE content were observed to vary with the growth phase [Jiranek et
al., 1998]. This prompted us to carry out a systematic investigation on the influence of both
the growth medium and the growth stage on the phospholipid composition of yeast cells and
derived subcellular fractions and on the activities of several phospholipid biosynthetic
enzymes in a wild-type yeast strain. It is documented for the first time that the PC/PE ratio in
yeast membranes varies with the growth phase.
Materials and methods
Materials
S-adenosyl-L-methionine was purchased from Sigma (St. Louis, MO). The
radiochemicals L-[3-3H]-serine and S-adenosyl-L-[methyl-3H]-methionine were obtained
from Amersham (Amersham, United Kingdom). CDP-diacylglycerol was from Doosan

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Chapter 3
42
(Korea). Yeast extract and yeast nitrogen base without amino acids were obtained from
Sigma and from Difco (Detroit, MI), respectively. Zymolyase was supplied by Seikagaku
(Japan). All other chemicals were analytical grade.
Growth conditions
The wild-type yeast strain Saccharomyces cerevisiae D273-10B (MATα) was grown
aerobically at 30˚C. Cells were precultured for 24 h in semi-synthetic lactate medium [Daum
et al., 1982]. 5 ml portions of the same preculture were used to inoculate 800 ml batches of
the following media: semi-synthetic lactate medium (SSL), semi-synthetic glucose medium
(SSG), complete synthetic lactate medium (CSL), and complete synthetic glucose medium
(CSG). Semi-synthetic media contained per liter: 3 g yeast extract, 1 g KH2PO4·3 H2O, 1 g
NH4Cl, 0.5 g CaCl2·2 H2O, 1.1 g MgSO4·7 H2O, 0.5 g NaCl, 0.3 ml of 1% (w/v) FeCl3.
Complete synthetic media (based on [Klig et al., 1985]) contained per liter: 6.7 g Difco yeast
nitrogen base without amino acids, 20 mg lysine, 10 mg arginine, 10 mg leucine, 30 mg
methionine, 10 mg adenine, 10 mg uracil. Lactate media contained per liter: 22 ml 90% lactic
acid and 1 g glucose [Daum et al., 1982]. Glucose media contained per liter: 20 g glucose and
1.1 ml lactic acid (to prevent the precipitation of calciumphosphate in SSG during
sterilization by autoclaving). The pH of all growth media was adjusted to 5.5 using KOH or
HCl before sterilization. Cell growth was monitored by measuring the OD600 (after dilution
to an OD600 of approximately 0.3) or by counting cell numbers using a hemocytometer.
Preparation of cell homogenates
Cells were harvested by centrifugation (10 min at 3600 x g) at the times indicated in
Figures 2-4 and resuspended in ice-cold water at a concentration of 0.1 g/ml (wet weight).
Cells were disrupted by vortexing twice for 1 min with glass beads (1.6 g/ml) with a 1 min
interval on ice. The broken cell suspension was stored at -80˚C until its use in enzyme assays
and phospholipid analysis.
Subcellular fractionation
Cells were harvested at the indicated times from cultures grown on semi-synthetic
lactate medium and spheroplasts were prepared using zymolyase as described previously
[Daum et al., 1982]. The isolation of mitochondria at pH 6.0 and further purification by
sucrose gradient centrifugation were based on a published procedure [Gaigg et al., 1995].
Mitochondrial outer membranes were isolated and purified based on [Mayer et al., 1995].
Microsomes were isolated as the 32,500 x g pellet of a 20,200 x g post-mitochondrial
supernatant. The isolation of mitochondria-associated membranes (MAM) was adapted from
a published procedure [Gaigg et al., 1995]. Full details of all fractionation procedures are
described elsewhere [De Kroon et al., 1999]. The fractions obtained were stored at -80˚C.

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Variation of the PC to PE ratio in yeast with growth phase
43
Enzyme assays
PS synthase specific activity (expressed as nmol serine incorporated per min per mg
protein) in the cell homogenate was determined in the presence of 4 mM Triton X-100, 0.6
mM MnCl2, 50 mM Tris-HCl, pH 8.0, and 0.2 mM CDP-diacylglycerol as described [Bae-
Lee and Carman, 1984], at a concentration of 0.1 mM L-[3-3H]-serine (28,000 dpm/nmol).
TLC analysis showed that over 95% of the lipid-incorporated label was present in PS and PE.
The combined specific activity of the PNMTs (expressed as nmol S-adenosyl-L-methionine
metabolized into chloroform soluble material per min per mg protein) was determined by
following the methylation of endogenous PE in the cell homogenate in the presence of 0.5
mM S-adenosyl-L-[methyl-3H]-methionine (10,000 dpm/nmol) and 50 mM Tris-HCl, pH 8.0
(based on [Gaynor and Carman, 1990]). Over 98% of the lipid-incorporated label was present
in monomethyl PE (PMME), dimethyl PE (PDME) and PC as was determined by TLC
analysis (not shown). In both enzyme assays cell homogenate samples corresponding to 40
µg of protein were incubated for 10 min at 30°C. Incubations were ended by adding 475 µl of
a mixture of chloroform, methanol and 0.5 M HCl (6:12:1, v/v/v) which was followed by
lipid extraction [Bligh and Dyer, 1959] and liquid scintillation counting of the dried lipid
extracts.
Phospholipid analysis of cell homogenates and subcellular fractions
Phospholipids were extracted according to the method of Bligh and Dyer [Bligh and
Dyer, 1959]. The phosphorus content of the organic phase obtained after extraction was
determined to yield the phospholipid phosphorus to protein ratio. Phosphorus was determined
by the method of Fiske and Subbarow [Fiske and Subbarow, 1925]. Protein concentrations
were measured using the BCA method (Pierce) with 0.1% (w/v) SDS added and bovine
serum albumin (BSA) as a standard. Phospholipid compositions were determined by two-
dimensional TLC analysis of lipid extracts containing 200-350 nmol of phospholipid
phosphorus as described [De Kroon et al., 1997].
Results
Growth characteristics
This study describes the influence of the growth condition (carbon source and
nutritional supplement) and the growth stage on the phospholipid composition and the
activities of several phospholipid biosynthetic enzymes in a wild-type yeast strain. Figure 1
shows the growth curves of the wild-type yeast strain D273-10B on both the semi-synthetic
and complete synthetic media with fermentable and non-fermentable carbon sources. The
semi-synthetic lactate medium was chosen because it is the medium routinely used to culture