Lipid Metabolism and Membrane Biogenesis

394 Pages·2004·6.118 MB·English

by  Günther Daum (auth.), Günther Daum (eds.)

Introduction: Lipids: cellular glue… or are they
more than that?
Günther Daum
Some 40 years ago, there was a rumor in the research community that lipids are
the stuff you have to throw away before starting biochemical experiments. This
view has changed. Nowadays, it is widely accepted that lipids play an important
role in many biochemical and cell biological processes. They are involved in the
formation of biological membranes and therefore are important elements of organ-
elle biogenesis and function. This is not only true for bulk lipids such as the major
classes of glycerophospholipids, sterols, and sphingolipids, but also for less abun-
dant lipid species. Lipids with only minor concentrations in cells have more subtle
functions. For example, polyphosphoinositides, lysophospholipids, or ceramides
are essential for signaling processes. Certain classes of lipids, such as sphingolip-
ids, are also sensors of cellular stress (heat shock) and important components for
signal transduction. Moreover, it must not be forgotten that in many cases, lipids
are closely associated with membrane proteins. These lipids provide the appropri-
ate environment for membrane proteins to function as enzymes or transporters, or
to act as positive regulators of enzymes to gain optimum activity in membranes.
The lipid composition of vesicles involved in membrane traffic, recently, turned
out to be a most important parameter for protein targeting. For example, the pres-
ence of sterols and sphingolipids in secretory and endocytotic vesicles appears to
be essential. Membrane contact, as a mechanism of translocation of components
between subcellular compartments, may also depend on the lipid patterns of donor
and acceptor membranes. Finally, certain classes of lipids such as triacylglycerols
and steryl esters serve as lipid reserves, which can be mobilized during starvation
as a source of energy or as a source of fatty acids needed for the formation of
membrane phospholipids.
The world of lipid research has changed dramatically during the past few dec-
ades. Simple analytical methods such as thin-layer chromatography (TLC) or gas-
liquid chromatography (GLC), which are still valuable and most reliable methods,
have been complemented by more sophisticated techniques. For instance, mass
spectrometry, NMR methods, and fluorescence methods have become common in
lipid research laboratories. This approach led to a broader view of the variety of
lipid molecules and has provided insight into the occurrence of the different
classes of lipids even with low abundance in biological material. Biophysical
methods became valuable tools in which to study lipid properties in biological and
artificial membranes.
Genetic and molecular biological methods have been applied in order to under-
stand the pathways of lipid metabolism in detail and to learn more about the gene
products that are involved in these processes. The result of these studies is that
lipid metabolism must not be regarded only as an isolated process, but rather as a
part of total cellular metabolism, which is highly linked to other metabolic and cell

Topics in Current Genetics, Vol. 6
G. Daum (Ed.) Lipid metabolism and membrane biogenesis
© Springer-Verlag Berlin Heidelberg 2004
2 Günther Daum
biological pathways. Many regulatory phenomena caused by lipids, which have
not been recognized before, became evident through the molecular biological ap-
proach. Modern methods of cell biology, such as elaborate techniques of organelle
isolation and characterization as well as microscopic inspection, have provided in-
sight into localization of lipid-synthesizing enzymes and the dynamics of lipids
within a cell. Although we are only just beginning to understand mechanisms of
intracellular lipid transport and distribution, it is generally accepted that these
processes play an important role in the maintenance of cellular structure and func-
tion.
The complex view of a “lipidome”, which describes the large variety of lipid
classes and individual species in different organisms and organelles at different
stages of development, emerged from the combination of all the disciplines of
lipid research described above. However, this lipidome is far from being complete
at present. The field of lipid research, as many other fields of life science research,
is in a continuous flux of development and improvement to fill the gaps and find
the missing links, which are required to understanding cellular events with all their
facets.
The series of comprehensive reviews presented in this booklet summarizes the
state of the art in lipid research. The aim of this group of authors is to provide a
general overview of the field and to draw the reader’s attention to most recent in-
vestigations. Biochemical, cell biological and biophysical aspects of the four ma-
jor groups of lipids in eukaryotic cells, namely: glycerophospholipids, sterols,
sphingolipids, and storage lipids, will be reported and discussed. The experimental
systems that are addressed are mammalian, plant, and yeast cells, as they are the
most prominent and currently best-studied systems in lipid biochemistry, cell, and
molecular biology. In their contributions, C. R. McMaster and T. Jackson will re-
port on phospholipid synthesis in mammalian cells while R. Nebauer, R. Birner,
and G. Daum give an account about phospholipid biosynthesis in the eukaryotic
model Saccharomyces cerevisiae, whereas P. Moreau and J. J. Bessoule focus on
the dynamics of phospholipids in plant cells. Sterol research is addressed in the
contributions by G. C. Ness, who deals with the defects in cholesterol biosynthe-
sis, and M. -A. Hartmann who describes metabolism and functions of sterols in
plants, and N. D. Lees and M. Bard give their view of sterol biochemistry and
regulation in the yeast Saccharomyces cerevisiae. Aspects of lipid storage in the
form of triacylglycerols are reported by D. Cheng, C. C. Y. Chang, and T. Y.
Chang (Mammalian ACAT and DGAT gene family), J. A. Napier and F. Beau-
doin (Biosynthesis and compartmentation of triacylglycerol in higher plants), and
P. Oelkers and S. L. Sturley (Mechanisms and mediators of triglyceride synthesis
in eukaryotic cells). Finally, H. Le Stunff, S. Coursol, S. Milstien, and S. Spiegel
describe sphingosine-1-phosphate metabolism in mammalian cell signaling, and P.
Sperling, D. Warnecke and E. Heinz report on news from plant sphingolipids, and
Y. Hannun and A. Cowart address the problem of sphingolipids in baker’s yeast as
a rising foundation for eukaryotic sphingolipid-mediated cell regulation.
We, the authors of these contributions, hope that this review series will provide
basic information about lipids for all those who wish to get acquainted with this
field and provide access to the recent literature of the respective subjects. We also
Introduction: Lipids: cellular glue… or are they more than that? 3
wish, however, to share recent ideas of lipid research with experts in this field
through the manuscripts presented here and discuss challenging aspects and future
ideas related to current investigations. As the guest editor of this issue of Topics in
Current Genetics, I wish to thank all the authors for their contributions and for
their cooperative efforts in an internal peer reviewing process, which made it pos-
sible to provide this overview of recent developments in lipid research in eu-
karyotic cells.
1 Phospholipid synthesis in mammalian cells
Christopher R. McMaster and Trevor R. Jackson
Abstract
Phospholipids are the main components of biological membranes and as such act
as the major permeability barrier between cells and the extracellular space, as well
as defining the physical boundaries of intracellular organelles. Phospholipid types
are defined by in large by their head groups that in turn are the major determinants
of phospholipid function. Within a specific phospholipid type, heterogeneity also
exists by virtue of the fatty acids attached to each individual phospholipid as well
as the nature by which these fatty acids are attached to the lipid backbone. We
provide an overview of the pathways by which specific phospholipids are synthe-
sized in mammalian cells and present new discoveries covering the specific intra-
cellular sites of lipid synthesis, new factors affecting membrane synthesis, and
how alterations in the synthesis of specific phospholipids impact on signals that
affect various phenotypes including their regulation of cell growth.
1.1 Introduction
Phospholipids are a class of biological molecules that display a large diversity in
structure that in turn ascribes function to a particular type of phospholipid. The
major type of lipid present in mammalian cell membranes are glycerol backbone
based, referred to as glycerophospholipids. As the major component of mem-
branes, glycerophospholipids provide cellular and organellar permeability barriers.
However, an assessment of an organism’s glycerophospholipid composition re-
veals a plethora of different phospholipid types varying in the types of hydrophilic
head groups and hydrophobic fatty acids attached to the glycerol backbone. In all
mammalian cell types, the most abundant phospholipid is phosphatidylcholine
(PtdCho) that comprises about 40-50% of total cellular phospholipid. Next in
abundance is generally phosphatidylethanolamine (PtdEtn) making up 15-25% of
membrane phospholipid, followed by phosphatidylserine (PtdSer) and phosphati-
dylinositol (PtdIns) at about 5-10% each. The inositol head group of PtdIns can be
phosphorylated to form polyphosphorylated PtdIns that are present in very low
abundance, less than 1% of cellular lipid. The phospholipid composition of organ-
elles within a cell also varies quite dramatically with the most notable example be-
ing the almost exclusive synthesis and containment of phosphatidylglycerol
(PtdGro) and cardiolipin (CL) to mitochondrial membranes. Since only one or two
different types of phospholipids are required to form an effective lipid bilayer,

Topics in Current Genetics, Vol. 6
G. Daum (Ed.) Lipid metabolism and membrane biogenesis
© Springer-Verlag Berlin Heidelberg 2004
6 Christopher R. McMaster and Trevor R. Jackson
why do mammalian cells possess such complexity in their composition? The an-
swer lies in the distinct biological roles ascribed to individual phospholipid types.
As an organism becomes more complex, its phospholipid composition becomes
more varied to reflect the increased biological functions of its phospholipid com-
plement. Phospholipids serve as (i) second messenger molecules, (ii) membrane
receptors for the recruitment of specific proteins, (iii) chaperones to aid in protein
folding, and (iv) modulators of protein function. Indeed, about 30% of all proteins
are integral membrane proteins while another 30% are thought to function at a
membrane surface. Thus, altering the phospholipid composition of a particular cel-
lular membrane can have dramatic affects on the biology of the cell. In this re-
view, we describe the various pathways for glycerophospholipid synthesis, their
regulation, and the impact of altered phospholipid synthesis on cellular biologies.
1.2 Phospholipid biosynthetic pathways
1.2.1 Synthesis of the glycerol backbone
All glycerophospholipids share glycerol as the backbone to which head groups
and fatty acids are attached. Most cell types derive the glycerol backbone from the
glycolytic pathway (Fig. 1). The six carbon sugar glucose is metabolized to fruc-
tose-1,6-bisphosphate which is hydrolyzed to the three carbon sugars glyceralde-
hyde-3-phosphate and dihydroxyacetonephosphate. These three carbon sugars can
be interconverted by the enzyme triosephosphate isomerase with glyceraldehyde-
3-phosphate metabolism proceeding through glycolysis resulting in the production
of pyruvate for entrance into the respiratory pathway, and dihydroxyacetonephos-
phate being used for the synthesis of glycerol-3-phosphate for the production of
glycerophospholipids. Glycerol-3-phosphate can also be synthesized directly from
glycerol by glycerol kinase, however, this enzyme is found mainly in intestinal
cells and is used for phospholipid synthesis from products of digestion.
1.2.2 Transfer of the glycerol backbone to the membrane
The first committed step in glycerophospholipid synthesis is considered to be the
transfer of a fatty acid from fatty acyl CoA to the sn-1 position of glycerol-3-
phosphate by glycerol-3-phosphate acyltransferase (GPAT). The addition of the
hydrophobic fatty acid chain produces lysophosphatidic acid and allows for parti-
tioning of this molecule from its soluble precursors into cellular membranes (Fig.
1).
1 Phospholipid synthesis in mammalian cells 7

Fig. 1. Pathways for the synthesis of glycerol-3-phosphate for metabolism to phospholipids.
1.2.2.1 Glycerol-3-phosphate acyltransferases
Two isoforms of GPAT are believed to exist in mammalian cells. One isoform is
found in the outer mitochondrial membrane and the second GPAT activity is in
microsomal membranes (Saggerson et al. 1980). The mammalian microsomal
GPAT is an integral membrane protein that has yet to be purified and its encoding
cDNA has not been isolated. Based on the GPAT activity present in microsomal
enzymes this isoform effectively utilizes both saturated and unsaturated fatty acyl
CoA substrates and is inhibited by the sulfhydryl modifying agent N-
ethylmaleimide.
In contrast to the microsomal isoform, much more is known about the mito-
chondrial GPAT. The protein has been purified to homogeneity and both its
cDNA and gene have been isolated. The predicted protein sequence is similar to
the sole GPAT found in prokaryotes and has been demonstrated experimentally to
contain two membrane spanning helices with a cytoplasmic facing active site
(Gonzalez-Baro et al. 2001). The mitochondrial GPAT preferentially uses the
saturated palmitoyl CoA as its substrate. Mitochondrial GPAT activity can be ef-
fectively inhibited by arginine modifying agents and mutation of arginine 318 re-
sults in almost a complete ablation of enzyme activity implying this amino acid
residue participates in catalysis (Dircks et al. 1999; Lewin et al. 1999).
Transcription of mitochondrial GPAT is increased by feeding a high carbohy-
drate diet or an increase in insulin, and is decreased by starvation, decreased insu-
lin, or increased glucagon (Lewin et al. 2001). The increased expression of GPAT
8 Christopher R. McMaster and Trevor R. Jackson
due to a decreased cellular fat levels, or conversely increased carbohydrate levels,
is mediated by the sterol response element binding protein (SREBP) family of
transcription factors. SREBP1-c binds directly to the GPAT promoter to increase
the rate of GPAT transcription initiation and thus ensures that there is homeostasis
in the rate of glycerolipid synthesis by responding to cellular fat levels to appro-
priately alter the activity of the committed step in this process. Increased expres-
sion of mitochondrial GPAT in Chinese hamster ovary or human embryonic kid-
ney cell lines resulted in a shift in the incorporation of exogenous fatty acid
toward triacylglycerol and away from phospholipid implying mitochondrial GPAT
may play a role in shunting excess fatty acid away from membrane biogenesis and
into neutral lipid stores (Igal et al. 2001). Consistent with this hypothesis is the
several fold increase in mitochondrial GPAT expression upon differentiation of
pre-adipocytes into adipocytes.
Very recently, a knockout mouse containing a targeted disruption of the mito-
chondrial GPAT gene was generated (Hammond et al. 2002). These mice were vi-
able presumably due to the ability of the microsomal GPAT to provide the neces-
sary step in lipid synthesis required for cells to differentiate and proliferate.
Studies of the phenotypes of this mouse are still in its infancy but data to date in-
dicate that mice lacking mitochondrial GPAT have decreased triacylglycerol lev-
els in their liver and plasma. The saturated fatty acid palmitate was reduced by 20-
40% in liver triacylglycerol and at the sn-1 position of the major phospholipids.
This data is consistent with mitochondrial GPAT being the major pathway for the
flux of fatty acid into triacylglycerol, and the main regulator of the fatty acid com-
position at the sn-1 position of phospholipids.
1.2.2.2 Dihydroxyacetonephosphate acyltransferase
Dihydroxyacetonephosphate, the glycolytic precursor of glycerol-3-phosphate, can
also be directly fatty acylated to produce 1-acyl-dihydroxyacetonephosphate (Fig.
2) in peroxisomes (Nagan et al. 1998). The lipid product can be reduced by 1-acyl-
dihydroxyacetonephosphate reductase to produce lysophosphatidic acid for en-
trance into the glycerophospholipid biosynthetic pathway. However, this pathway
is thought to be a very minor contributor to net phospholipid synthesis, and instead
appears to be used mainly for the synthesis of phospholipids that have their fatty
acid attached through an ether linkage at the sn-1 position instead of the normal
ester bond. After synthesis of 1-acyl-dihydroxyacetonephosphate this lipid is then
acted on by the peroxisomal 1-alkyl-dihydroxyacetonephosphate synthase to con-
vert the ester linkage to an ether bond (Nagan et al. 1997). The 1-alkyl-
dihydroxyacetonephosphate is then enzymatically reduced to form 1-alkyl-
glycerol-3-phosphate for subsequent synthesis of sn-1 ether linked phospholipids
(Snyder 1999).
1 Phospholipid synthesis in mammalian cells 9
1.2.2.3 Rhizomelic chondrodysplasia punctata type 2 – a
dihydroxyacetonephosphate acyltransferase deficiency
The recent isolation of the human dihydroxyacetonephosphate acyltransferase
cDNA resulted in two interesting observations. Consistent with its peroxisomal
location the predicted amino acid sequence contained a type 1 peroxisome target-
ing sequence. Second, point mutations within the coding region of dihydroxyace-
tonephosphate acyltransferase were shown to be associated with decreased ether
lipid content in cells and these mutations were established to be the cause of rhi-
zomelic chondrodysplasia punctata type 2 (de Vet et al. 1998; Ofman et al. 1998).
Cells from patients with this autosomal recessive disorder have intact but malfunc-
tioning peroxisomes resulting in clinical abnormalities that include shortening of
the upper extremities, growth and mental retardation, and cataracts. Consistent
with the major role of dihydroxyacetonephosphate acyltransferase in the synthesis
of ether linked lipids was the observation that mutations in its immediate down-
stream enzyme in ether lipid synthesis, 1-alkyl-dihydroxyacetonephosphate syn-
thase, also results in rhizomelic chondrodysplasia punctata type 2. In a separate
but related study, a Chinese hamster ovary cell line with a specific defect in dihy-
droxyacetonephosphate acyltransferase activity was generated by mutagenesis and
these cells were found to be selectively defective in the synthesis of ether linked
versus ester linked lipids and possessed intact but malfunctioning peroxisomes
(Nagan et al. 1998). The combined observations to date imply that there is a spe-
cific function for ether lipid required for proper peroxisomal function with current
data favouring a generalized decrease in peroxisomal fitness as a major contribu-
tor to the clinical phenotypes of rhizomelic chondrodysplasia punctata type 2.
1.2.2.4 Lysophosphatidic acid acyltransferases
The addition of a fatty acid to the glycerol-3-phosphate backbone results in the
partitioning of the fatty acylated product into the lipid bilayer and is the genesis of
the synthesis of a biological membrane (Fig. 2). The lysophosphatidic acid pro-
duced is further fatty acylated at the sn-2 position by lysophosphatidic acid acyl-
transferase to produce phosphatidic acid (PtdOH). Two separate lysophosphatidic
acid acyltransferase cDNAs have been isolated, and although similar in amino
acid sequence, are derived from separate genes (Aguado and Campbell 1998;
Eberhardt et al. 1999; West et al. 1997). Both open reading frames predict proteins
with several membrane spanning helices and use a broad selection of fatty acyl
CoAs as substrates in vitro.
Lysophosphatidic acid acyltransferase activities are required to mediate the
transport of lipid and protein laden vesicles between cellular organelles. The
endophilin I protein is a lysophosphatidic acid acyltransferase whose enzymatic
activity uses arachidonoyl CoA as substrate and is required to mediate synaptic
vesicle invagination from the plasma membrane (Schmidt et al. 1999). Endophilin
I is a soluble protein that has no significant amino acid identity with the above in-
tegral membrane lysophosphatidic acid acyltransferases. A second lysophos-
phatidic acid acyltransferase implicated in membrane vesicle transport is
10 Christopher R. McMaster and Trevor R. Jackson

Fig. 2. The synthesis of phosphatidic acid.
CtBP/BARS whose lysophosphatidic acid acyltransferase activity promotes Golgi
membrane fission (Weigert et al. 1999). Like endophilin I, CtBP/BARS is soluble
and prefers unsaturated fatty acyl CoAs as substrates, but their amino acid se-
quences share limited similarity. It is proposed that converting lysophosphatidic
acid to PtdOH results in the production of a positive to negative membrane curva-
ture and thus contributes to membrane fission (Farsad et al. 2001). The metabolic
pathway from which the lysophosphatidic acid arises for use by either endophilin I
or CtBP/BARS has yet to be identified.
1.2.2.5 Congenital generalized lipodystrophy – a lysophosphatidic
acid acyltransferase deficiency
Mutations in the human gene for one of the known lysophosphatidic acid acyl-
transferases, AGAPT2, have been found to be the cause of the autosomal recessive
disorder congenital generalized lipodystrophy (Agarwal et al. 2002). This disorder
is characterized by a lack of adipose tissue, hypertriglyceridemia, insulin resis-
tance, hepatic steatosis, and early onset diabetes. The AGAPT2 gene product is
very highly expressed in adipose tissue and lack of function of the encoded protein
appears to cause congenital generalized lipodystrophy by preventing triacylglyc-
erol synthesis and storage in adipocytes.
1.2.3 Partitioning of phospholipid biosynthetic pathways
PtdOH is at a major branch point in the de novo synthesis of the major membrane
phospholipids. PtdOH can either be dephosphorylated to diacylglycerol (DAG) by
PtdOH phosphatase, or converted to CDP-diacylglycerol (CDP-DAG) by CDP-
DAG synthase (Fig. 3). DAG is utilized for the synthesis of PtdCho and PtdEtn
through the Kennedy pathways with these two lipids being used as substrates for
the synthesis of PS. CDP-DAG is used for the synthesis of PtdGro and PtdIns.
1 Phospholipid synthesis in mammalian cells 11

Fig. 3. Bifurcation from phosphatidic acid for the genesis of the major phospholipids of
mammalian cells.
PtdGro can be further metabolized to CL while PtdIns can be phosphorylated to
produce various polyphosphorylated PtdIns. PtdOH is also produced in various
membranes by phosphorylation mediated by DAG-kinase isoforms (Brose and
Rosenmund 2002).
1.2.3.1 PtdOH phosphatase
There are two main PtdOH phosphatase activities identified in mammalian cells
(Kai et al. 1997). Type 1 PtdOH phosphatase appears to be the main form respon-
sible for the synthesis of phospholipids. This enzyme has not been purified nor has