Phosphatidic acid, a key intermediate in lipid metabolism

Authors


G. Daum, Institut für Biochemie und Lebensmittelchemie, Technische Universität Graz, Petersgasse 12/2, A-8010 Graz, Austria. Fax: + 43 316 873 6952, Tel.: + 43 316 873 6462, E-mail: f548daum@mbox.tu-graz.ac.at

Abstract

Phosphatidic acid (PtdOH) is a key intermediate in glycerolipid biosynthesis. Two different pathways are known for de novo formation of this compound, namely (a) the Gro3P (glycerol 3-phosphate) pathway, and (b) the GrnP (dihydroxyacetone phosphate) pathway. Whereas the former route of PtdOH synthesis is present in bacteria and all types of eukaryotes, the GrnP pathway is restricted to yeast and mammalian cells. In this review article, we describe the enzymes catalyzing de novo formation of PtdOH, their properties and their occurrence in different cell types and organelles. Much attention has recently been paid to the subcellular localization of enzymes involved in the biosynthesis of PtdOH. In all eukaryotic cells, microsomes (ER) harbour the complete set of enzymes catalyzing these pathways and are thus the usual organelle for PtdOH formation. In contrast, the contribution of mitochondria to PtdOH synthesis is restricted to certain enzymes and depends on the cell type. In addition, chloroplasts of plants, lipid particles of the yeast, and peroxisomes of mammalian cells are significantly involved in PtdOH biosynthesis. Redundant systems of acyltransferases, the interplay of organelles, regulation of the pathway on the compartmental level, and finally the contribution of alternative pathways (phosphorylation of diacylglycerol and cleavage of phospholipids by phospholipases) to PtdOH biosynthesis appear to be required for the balanced formation of this important lipid intermediate. Dysfunction of enzymes involved in PtdOH synthesis can result in severe defects of various cellular processes. In this context, the possible physiological role(s) of PtdOH and its related metabolites, lysophosphatidic acid and diacylglycerol, will be discussed.

Abbreviations
1-acyl-GroP AT

1-acyl-glycerol 3-phosphate acyltransferase

GrnP AT

dihydroxyacetonephosphate acyltransferase

Gro3P AT

glycerol 3-phosphate acyltransferase

PtdOH

phosphatidic acid

Introduction

Phosphatidic acid, a key intermediate in lipid metabolism

Phosphatidic acid (PtdOH) is an essential substrate for enzymes involved in the synthesis of glycerophospholipids and triacylglycerols. PtdOH enters the biosynthetic pathway of phospholipids through a CTP-dependent activation catalyzed by CDP-diacylglycerol synthase. This enzyme forms CDP-diacylglycerol, which serves as a direct precursor for phosphatidylinositol (PtdIns), phosphatidylglycerol (PtdGro) and cardiolipin (di-PtdGro). In yeast, phosphatidylserine (PtdSer) is also formed from CDP-diacylglycerol, whereas PtdSer of mammalian cells is synthesized by head-group exchange of phosphatidylethanolamine (PtdEtn) or phosphatidylcholine (PtdCho) [1]. PtdSer can be decarboxylated to PtdEtn which is finally converted to PtdCho by three methylation steps (Fig. 1).

Figure 1.

Figure 1.

Phosphatidic acid, a key intermediatein glycerophospholipid and triacylglycerol synthesis. The large arrows indicate reactions restricted to yeast. Di-PtdGro, cardiolipin.

PtdOH can also be dephosphorylated by phosphatidate phosphatase yielding diacylglycerol, which serves as a precursor for the formation of PtdEtn and PtdCho through the CDP-Etn and CDP-Cho pathway (Kennedy pathway), or for triacylglycerol synthesis through another step of acylation (see Fig. 1). Thus, all acylglycerol lipids are directly or indirectly derived from PtdOH (reviewed in [2,3]).

Pathways of phosphatidic acid biosynthesis

PtdOH can be synthesized via two different acylation pathways named after their respective precursor, namely (a) the Gro3P (glycerol 3-phosphate) pathway, and (b) the GrnP (dihydroxyacetone phosphate) pathway (Fig. 2). In the Gro3P pathway, the first step of acylation catalyzed by Gro3P acyltransferase (Gro3P AT) leads to the formation of 1-acylGro3P (also known as lyso-PtdOH). In the GrnP pathway, the first intermediate formed by GrnP acyltransferase (GrnP AT) is 1-acyl-GrnP. This compound is converted to lyso-PtdOH in an NADPH-dependent reaction catalyzed by 1-acyl-GrnP reductase. Lyso-PtdOH either formed through the Gro3P pathway or the GrnP pathway, respectively, is further acylated to PtdOH by 1-acylGro3P acyltransferase (1-acyl-Gro3P AT).

Figure 2.

Figure 2.

Formation of phosphatidic acid. 1-Acyl-GrnP red, 1-acyl-GrnP reductase.

Whereas the Gro3P pathway is the ‘universal’ route of PtdOH formation in prokaryotes, plants, yeast and mammalian cells, enzymes needed for the first two steps of PtdOH formation in the GrnP pathway, namely GrnP AT and 1-acyl-GrnP reductase, are only present in yeast and mammals [4–8]. The rate limiting reaction in PtdOH formation is the first step of acylation catalyzed either by Gro3P AT or GrnP AT [9]. As a consequence, the intermediates lyso-PtdOH and 1-acyl-GrnP do not accumulate under standard conditions. The physiological reason for this regulatory phenomenon may be that high amounts of lysolipids disturb membrane formation and integrity similar to detergents.

Besides de novo synthesis of PtdOH via the two acylation pathways described above, this lipid can be formed by alternative biosynthetic routes (see Fig. 2). Diacylglycerol derived from deacylation of triacylglycerol by triacylglycerol lipase, or by hydrolysis of glycerophospholipids through the action of phospholipase C can be phosphorylated by diacylglycerol kinase. Hydrolysis of glycerophospholipids catalyzed by phospholipase D results directly in the formation of PtdOH (see Fig. 2). These alternative pathways may be important to circumvent defects in the de novo synthesis of PtdOH.

In this review, our present knowledge about the synthesis of PtdOH in prokaryotes, plants, yeast and mammals will be summarized. Enzymes involved in PtdOH biosynthesis will be described with special emphasis on their localization within the cell and their substrate specificities. Furthermore, the physiological consequences caused by defects of enzymes involved in PtdOH synthesis will be discussed. Finally, the question as to the role of diacylglycerol, lyso-PtdOH and PtdOH in various cellular processes will be addressed.

Phosphatidic acid synthesis by acyltransferase reactions in prokaryotes

This section of the review will focus on acyltransferases involved in PtdOH biosynthesis of the Gram-negative prokaryote Escherichia coli. This microorganism has not only been intensively studied regarding its lipid metabolism but has also been widely used as a valuable tool to identify acyltransferases from eukaryotes. For this purpose, E. coli mutants with defects in acyltransferases were complemented with putative homologues of other cell types. This strategy led, for example, to the characterization of human 1-acyl-Gro3P AT [10] or Slc1p, an 1-acyl-Gro3P AT of the yeast [11].

In E. coli, formation of PtdOH occurs only via the Gro3P pathway [4,12]. The plsB gene encoding Gro3P AT [13] was demonstrated to catalyze acylation of Gro3P. Because deletion of plsB is lethal, it was concluded that only one Gro3P AT is present in E. coli. Most recently Heath and Rock [14] reported that a single missense mutation (A349T) in the plsB gene significantly reduces the activity of Gro3P AT and elevates the Km for Gro3P.

The plsB gene encodes a 83-kDa protein (Table 1). The enzymatic activity of Gro3P AT was found to be firmly associated with the inner membrane of E. coli with the active site facing the cytosol. Only a small part of this protein, if any, traverses the membrane bilayer [15,16].

Table 1. Genes and gene products involved in biosynthesis of phosphatidic acid in E. coli. Key references [13,16–18]. The GrnP pathway does not exist in E. coli
 Gro3P pathway
 Gro3P AT1-acyl-GroP AT
GeneplsBplsC
Gene product (m)83 kDa25 kDa
Subcellular localizationInner membraneInner membrane
Substrate specificitySaturated acyl-ACP or acyl-CoAUnsaturated acyl-ACP or acyl-CoA

The enzyme catalyzing the second acylation step during PtdOH formation in E. coli, 1-acyl-Gro3P AT, is encoded by the plsC gene [17] (Table 1). In contrast to Gro3P AT, 1-acyl-Gro3P AT has a molecular mass of only 25 kDa, although both enzymes catalyze essentially the same type of reaction. Because the Gro3P AT reaction is the rate limiting step in PtdOH biosynthesis, it has been speculated that the larger size of the enzyme may allow the binding of effectors necessary for balanced PtdOH production. An efficient regulation of this reaction is required to prevent accumulation of lyso-PtdOH which might disturb membrane integrity. Gro3P AT and 1-acyl-Gro3P AT of E. coli are also different with respect to their substrate specificity. Whereas Gro3P AT preferentially links saturated fatty acids to the sn-1 position of Gro3P, 1-acyl-Gro3P AT prefers utilization of unsaturated fatty acids (Table 1). Thus, the different substrate selectivity of the two acyltransferases contributes to the specific positional distribution of saturated and unsaturated fatty acids in glycerophospholipids.

It is not known at present whether acyl-CoAs, acyl carrier protein bound fatty acids (acyl-ACP) or both are the in vivo substrates for the E. coli acyltransferases [18]. Acyl-ACP may be the physiological substrate as indicated by measurements of the acyl-CoA and acyl-ACP pools in the microorganism, but both forms of fatty acyl derivatives can be utilized by the enzymes in vitro (reviewed in [16]).

Phosphatidic acid synthesis by acyltransferase reactions in plants

Similarly to bacteria, plants lack enzymatic activities of the GrnP pathway of PtdOH formation [4,12] and can thus synthesize PtdOH only from the precursor Gro3P. The scenario of PtdOH biosynthesis in plants (Fig. 3A), however, is more complex than in bacteria. Whereas in prokaryotes, which lack distinct organelles, only one enzyme exists for each acylation step, Gro3P AT and 1-acyl-Gro3P AT activities of plants are present in redundancy and localized in three different compartments. A soluble acylation system is present in chloroplasts, whereas the other two systems are membrane associated and localized to the ER and mitochondria. The mitochondrial Gro3P AT resides in the outer membrane of the organelle. The acylation machinery of the ER is associated with the cytosolic leaflet of the membrane. The three Gro3P ATs of plants are distinct proteins.

Figure 3.

Figure 3.

Localization of enzymes involved in phosphatidic acid biosynthesis in plants (A), yeast (B) and mammalian cells (C). Organelles: ER, endoplasmic reticulum; MITO, mitochondrion; CHL, chloroplast; LP, lipid particle; PX, peroxisomes. 1-Acyl-GrnP red, 1-acyl-GrnP reductase.

In chloroplasts, the final products of fatty acid synthesis, 16 : 0 and 18 : 0 acyl chains, are bound to ACP. Stearoyl-ACP can be desaturated to oleoyl-ACP by a stearoyl-ACP desaturase. These ACP-bound fatty acids can serve directly as substrates for PtdOH formation in chloroplasts. Alternatively, fatty acids produced in chloroplasts can be set free from ACP, converted to acyl-CoAs and exported from this compartment. These acyl-CoAs can serve as substrates for esterification of Gro3P in mitochondria or the ER. Irrespective of the site of PtdOH synthesis either a C16 or C18 fatty acid is linked to the sn-1 position of the glycerol moiety (Table 2) indicating that Gro3P ATs of plants have generally a weak substrate specificity. In some cases, however, this enzyme appears to be more specific. As an example, Yokoi et al. [19] reported that a Gro3P AT from chloroplasts of Arabidopsis thaliana plays an important role in determining the degree of desaturation in PtdGro with its preference for unsaturated fatty acids. This observation is of physiological importance because plants containing a large amount of unsaturated fatty acids in PtdGro of chloroplasts such as A. thaliana are resistant to cold, whereas other species like squash and rice, which contain lower amounts of unsaturated fatty acids in PtdGro, are rather sensitive to low temperatures. Moon et al. [20] reported that the presence of unsaturated fatty acids in PtdGro of thylakoid membranes protects the photosynthetic machinery against low temperature inhibition and is thus responsible for chilling tolerance. When rice leaves were transformed with the cDNA of the oleate-selective Gro3P AT of A. thaliana the level of the unsaturated fatty acid in PtdGro was significantly increased [19]. Remodelling of the phospholipid could be excluded because of the selective transformation of the Gro3P AT gene of A. thaliana into rice.

Table 2. Genes and gene products involved in biosynthesis of phosphatic acid in plants. The GrnP pathway does not exist in plants.
 Gro3P pathway 
OrganelleGro3P AT1-acyl-GroP ATReferences
Chloroplasts
 Gene product (m)51 kDa (pea)
44 kDa (squash)
50 kDa (Arabidopsis)
52 kDa (spinach)
 [22]
 Homology among species> 65%  
 LocalizationSolubleSoluble 
 Substrate specificitySaturated acyl-ACP (C16/C18)Unsaturated acyl-ACP (C16) 
Microsomes
 Gene product (m)3 Isoforms
70 kDa, 60 kDa, 54 kDa (avocado)
32 kDa (meadowfoam)
32 kDa (coconut)
[23–25]
 Homology among species 62% 
 LocalizationMembrane boundMembrane bound 
 Substrate specificitySaturated acyl-CoAs (C16/C18)C18 
Mitochondria
 Gene product (m)Not identifiedNot identified[22]
 LocalizationOuter membraneOuter membrane 
 Substrate specificitySaturated acyl-CoAs (C16/C18)C18 

Ferri and Toguri [21] reported that the stromal Gro3P AT of squash, a cold sensitive plant, used palmitoylCoA at a threefold higher rate than oleoyl-CoA when expressed in E. coli. In contrast, the stromal Gro3P AT of the chilling tolerant spinach preferred oleoyl-CoA over palmitoyl-CoA. Using chimeras of these two Gro3P ATs it was demonstrated that structural properties of the central part of the polypeptides contain the information that determines the substrate specificity. Thus, genetic engineering appears to be an appropriate possibility for manipulating the level of saturated fatty acids in the PtdGro of chloroplasts and thereby the chilling tolerance of plants.

The three 1-acyl-Gro3P ATs of plants which acylate the sn-2 position of 1-acyl-Gro3P are localized in the same subcellular sites as the Gro3P ATs (see Fig. 3A). In contrast to Gro3P ATs, however, 1-acyl-Gro3P ATs distinguish accurately between the length of fatty acid substrates. Whereas in general 1-acyl-Gro3P AT present in chloroplasts uses only C16 as a substrate, 1-acyl-Gro3P ATs bound to the membrane of the ER and the outer mitochondrial membrane utilize specifically C18 fatty acids (Table 2). Comparing structures of soluble and membrane-bound 1-acyl-Gro3P ATs of the same plant species may be an appropriate approach to understand their substrate specificities. Formation of specific PtdOH species, however, cannot be attributed to distinct organelles because remodelling may change the fatty acid composition of the primary acylation products.

Gro3P ATs from chloroplasts of pea, spinach and some other plants were purified and their amino acid sequences were determined. Homology between these soluble Gro3P ATs is more than 65% [22]. The molecular mass of most of these enzymes is around 50 kDa (Table 2). Mitochondrial Gro3P ATs from plant sources have not yet been isolated, and only one microsomal Gro3P AT from avocado mesocarp has been partially purified [23]. This enzyme exists as three isoforms with molecular masses of 70, 60 and 54 kDa.

Knutzon et al. [24] isolated a membrane-bound 1-acyl-Gro3P AT from coconut endosperm with an apparent molecular mass of 32 kDa (see Table 2). In contrast to the membrane bound 1-acyl-Gro3P ATs of most plants which prefer C18 fatty acyl-CoAs as a substrate, the 1-acyl-Gro3P AT from coconut was demonstrated to utilize preferentially lauryl-CoA.

1-acyl-Gro3P AT from meadowfoam [25] has a molecular mass of approximately 32 kDa, and is thus almost the same size as the 1-acyl-Gro3P AT from coconut. Amino acid sequences of the two enzymes exhibited 62% identity. Both polypeptides have four potential transmembrane spanning domains. Comparison of the hydropathy profiles of the two plant 1-acyl-Gro3P ATs with those of E. coli and Saccharomyces cerevisiae demonstrated high similarity. As 1-acyl-Gro3P AT from meadowfoam catalyzes the incorporation of oleic acid (18 : 1) as well as of erucic acid (22 : 1) into the sn-2 position of the glycerol backbone, this enzyme may be interesting for industrial oil production.

Another unusual specificity for very long chain fatty acids was observed with an 1-acyl-Gro3P AT of Limnanthes douglasii[26]. This enzyme, which has a molecular mass of 31.7 kDa and three predicted transmembrane spanning domains, catalyzes preferentially incorporation of erucic acid in the sn-2 position of Gro3P. Expression of this 1-acyl-Gro3P AT in a plsC-deficient E. coli strain resulted in a substrate specificity typical of the enzyme from L. douglasii.

In a more detailed study, Brown et al. [27] reported the presence of two genes encoding 1-acyl-Gro3P ATs with different expression patterns in L. douglasii. 1-Acyl-Gro3P AT1 encodes a protein similar to maize 1-acyl-Gro3P AT and Slc1p, an 1-acyl-Gro3P AT of yeast, but with weak homology to plsC of E. coli. 1-Acyl-Gro3P AT1 is a member of a small gene family and is more widely expressed than 1-acyl-Gro3P AT2, which is expressed from a single copy gene almost exclusively in developing seed. In contrast to 1-acyl-Gro3P AT1, 1-acyl-Gro3P AT2 does shows homology to the SLC1 gene of yeast and also to plsC of E. coli[27].

Phosphatidic acid synthesis by acyltransferase reactions in yeast

In yeast, PtdOH can be synthesized either by using Gro3P or GrnP as a precursor [7,8,28]. Yeast acyltransferases involved in the first step of acylation of Gro3P or GrnP have not yet been identified at a molecular level. In contrast, an enzyme encoded by the SLC1 gene (sphingolipid compensation) was identified as an 1-acyl-Gro3P AT due to its homology to the plsC gene of E. coli and by functional complementation of an E. coli mutant with a defect in this gene [11].

It has been a matter of debate for many years whether Gro3P AT and GrnP AT activities of the yeast are attributable to the same enzyme. Tillman and Bell [29] isolated a mutant which exhibited a reduced level of PtdOH formation through both the Gro3P pathway and the GrnP pathway. The authors concluded from this result that only one enzyme is responsible for the acylation of both substrates. In contrast, Racenis et al. [30] argued that acylation of the two precursors is catalyzed by different enzymes based on the observations that sensitivity against the sulfhydryl group reagent N-ethylmaleimide and the pH optima of the two activities were different. Both groups of authors, however, used only crude membrane fractions for their investigations and were thus not able to distinguish between distinct enzymes.

Christiansen [31] observed enrichment of Gro3P AT activity in lipid particles of the yeast, a compartment consisting of a hydrophobic core of triacylglycerols and steryl esters surrounded by a phospholipid monolayer with only few proteins embedded [32]. Studies in our laboratory employing elaborate techniques of yeast cell fractionation confirmed that the highest specific activity of Gro3P AT was present in the lipid particle fraction, although activity of this enzyme was also detected in microsomes [33,34] (Table 3; Fig. 3B). Using lipid particles of the gat1 mutant TTA1 [29] as an enzyme source it was demonstrated that this compartment contains only one protein catalyzing the first step of Gro3P acylation, namely the putative Gat1p. The Gro3P AT activity in the microsomal fraction of this mutant was significantly reduced, but a residual amount of PtdOH was still formed in this compartment. These results demonstrated that (a) Gat1p is not only present in the lipid particle fraction but is also the major acyltransferase in the ER, and (b) additional acyltransferase(s) contribute to PtdOH synthesis via the Gro3P pathway in the latter organelle. Other subcellular fractions of the yeast were practically devoid of Gro3P acylation.

Table 3. Genes and gene products involved in phosphatidic acid biosynthesis in the yeast S. cerevisiae. ADR, 1-acyl-GrnP reductase.
 Gro3P pathwayGrnP pathway
OrganelleGro3P AT1-acyl-GroP ATGrnP ATADRReferences
Lipid particles
 Genes/gene product (m)Putative Gro3P AT1SLC1 (34 kDa)Putative Gro3P AT1YIL124w (33 kDa)[11,34,35]
 LocalizationMembrane assoc.Membrane assoc. Membrane assoc. 
 Substrate specificity C18 : 1, C22 : 1,
C24 : 0
   
Microsomes
 Genes/gene product (m)Putative Gro3P AT1SLC1 (34 kDa)Putative Gro3P AT1YIL124w (33 kDa)[11,34,35]
 LocalizationMembrane assoc.Membrane assoc.Membrane assoc.Membrane assoc. 
 Substrate specificity C18 : 1, C22 : 1,
C24 : 0
   
Mitochondria
 Genes/gene product (m)Putative GrnP AT28

As only one Gro3P AT is present in yeast lipid particles the question of whether this enzyme can also catalyze acylation of the precursor GrnP was addressed. Whereas the lipid particle fraction of a wild-type strain was able to form 1-acyl-GrnP, lipid particles of the gat1 mutant were devoid of GrnP AT activity [28]. This result demonstrated that the putative Gat1p is capable of acylating both Gro3P and GrnP. In the microsomal fraction of the gat1 mutant the activity of GrnP AT was reduced to a similar level as that of Gro3P AT. The precursor usage of residual microsomal acyltransferases could not be determined.

Another important observation during these studies was that despite lacking significant Gro3P AT activity, yeast mitochondria can still acylate GrnP (Table 3; Fig. 3B). This result suggested that an additional acyltransferase is present in this organelle, which prefers GrnP as the precursor and is thus different from the putative Gat1p present in lipid particles and the ER [28]. Several attempts to purify the putative Gat1p and additional Gro3P ATs of yeast have failed so far.

The only acyltransferase involved in PtdOH formation of yeast that has been characterized at the molecular level so far is Slc1p (Table 3). As mentioned above, this protein was identified as an 1-acyl-Gro3P AT by its ability to complement the plsC deficiency in an E. coli mutant and by homology to this gene [11]. Slc1p has a molecular mass of approximately 34 kDa (Table 3) and is thus of a size similar to 1-acyl-Gro3P ATs of E. coli (25 kDa) and plants (32–33 kDa) (see Tables 1 and 2). Localization studies with an slc1 deletion strain demonstrated the presence of this enzyme in lipid particles, but similar to the putative Gat1p a portion of Slc1p was also detected in the ER (Table 3) [34]. Slc1p is the only enzyme catalyzing conversion of lyso-PtdOH to PtdOH in the lipid particle fraction, but residual 1-acyl-Gro3P AT activity was detected in the microsomal fraction of the slc1 deletion strain indicating the presence of additional 1-acyl-Gro3P ATs in this compartment (Fig. 3B).

In vitro, Slc1p of yeast uses 18 : 1, 22 : 1 and 24 : 0 acyl-CoAs as substrates (Table 3). Introduction of the SLC1 gene into plants (A. thaliana and Brassica napus) resulted in elevated 1-acyl-Gro3P AT activity in homogenates of the developing seed. In transformants, a substantial increase of the seed oil content and an increase in both the overall proportion and amount of very long chain fatty acids in triacylglycerol of the seed was observed [35]. Very long chain fatty acids were preferentially incorporated into the sn-2 position of triacylglycerol due to the presence of Slc1p in plant transformants.

Fatty acid substrate specificity of Gro3P AT from S. cerevisiae was not determined. Gro3P AT and 1-acyl-Gro3P AT of the filamentous fungus Mucor circinelloides were demonstrated to use various fatty acids as substrates although with different efficiency. The Gro3P AT of the fungus showed a preference for palmitoyl-CoA whereas 1-acyl-Gro3P AT used oleoyl- and linoleoyl-CoA as the preferred substrates [36]. Gro3P AT and 1-acyl-Gro3P AT present in the membranous fraction of the fungus Mortierella ramanniana exhibited in vitro a distinct preference toward oleoyl-CoA as a substrate as compared to palmitoyl-CoA [37].

Formation of PtdOH through the GrnP pathway requires the activity of both acyltransferases and also a reductase (see Fig. 2). The intermediate 1-acyl-GrnP is converted to lyso-PtdOH by an 1-acyl-GrnP reductase, an NADPH-dependent enzyme. The presence of such an activity in yeast was first reported by Racenis et al. [30]. Most recently, we were able to demonstrate that 1-acyl-GrnP reductase activity is distributed among the lipid particle fraction and the ER, in a manner similar to the acyltransferases Gat1p and Slc1p [28]. Thus, the entire set of enzymes involved in PtdOH formation through the GrnP pathway is present in these two subcellular compartments (see Fig. 3B). In contrast, mitochondria which harbour significant GrnP AT activity lack 1-acyl-GrnP reductase [28]. As a consequence, 1-acyl-GrnP produced in mitochondria has to be transported to a site of 1-acyl-GrnP reductase activity for further conversion to PtdOH.

Systematic studies of the most prominent proteins of lipid particles of the yeast in our laboratory led to the identification of an 1-acyl-GrnP reductase at the molecular level. This polypeptide has a molecular mass of 33 kDa and is the only enzyme catalyzing the reduction of 1-acyl-GrnP to lyso-PtdOH in lipid particles (Table 3). 1-Acyl-GrnP reductase activity present in microsomes of a respective deletion strain, however, was only decreased to 30% of wild-type level (K. Athenstaedt and G. Daum, unpublished results). This result demonstrates that (a) one 1-acyl-GrnP reductase of the yeast is dually located to lipid particles and the ER, and (b) additional enzymes for the conversion of 1-acyl-GrnP to lyso-PtdOH must be present in the microsomal fraction.

The knowledge about the subcellular localization of enzymes involved in the formation of PtdOH in yeast makes it possible to interpret some of the observations of Minskoff et al. [38] regarding regulation of PtdOH biosynthesis. These authors found that cellular 1-acylGro3P AT activity increased threefold, while GrnP AT activity increased up to ninefold in wild-type cells when shifted from glucose to a nonfermentable carbon source. Growth on nonfermentable carbon sources stimulated proliferation of mitochondria which contain an acyltransferase with a distinct preference for the precursor GrnP. Under these conditions, a higher contribution of the GrnP pathway versus the Gro3P pathway for PtdOH synthesis appears to occur. In addition, Minskoff et al. [38] observed that Gro3P AT, GrnP AT and 1-acyl-GrnP reductase activities increased in wild-type cells grown on glucose when they entered the stationary phase. This result can be explained by the fact that lipid particles accumulate at this stage; it may also indicate that lipid particles in vivo contribute significantly to PtdOH formation in yeast.

Phosphatidic acid synthesis by acyltransferase reactions in mammalian cells

Gro3P AT activity has been found in most mammalian tissues [9,39–41]. In a manner similar to yeast, both the Gro3P and the GrnP pathway can be used for PtdOH formation in mammalian cells. The first acylation step occurs in mitochondria, the ER and peroxisomes (Fig. 3C). Whereas in mitochondria and peroxisomes only Gro3P and GrnP, respectively, serve as precursors, both substrates can be acylated in microsomes [9].

As in plants, the mitochondrial Gro3P AT of mammalian cells is localized to the outer membrane of this organelle. Hesler et al. [42] demonstrated that the enzyme spans the transverse plane of the outer membrane. In contrast to the plant system, however, 1-acyl-Gro3P AT is not present in mammalian mitochondria at a significant level. Thus, mitochondrially formed lyso-PtdOH has to be transported to the ER for further conversion to PtdOH.

The two Gro3P AT isoforms present in mitochondria and in microsomes of mammalian cells are different in several respects. The mitochondrial Gro3P AT was found to be resistant to N-ethylmaleimide whereas the microsomal enzyme is sensitive to this inhibitor [9]. Another difference is the substrate specificity of the two isoenzymes [43]. The mitochondrial Gro3P AT has a preference for saturated fatty acyl-CoAs, whereas the microsomal Gro3P AT appears to utilize saturated and unsaturated fatty acyl-CoAs with similar efficiency (Table 4). When palmitoyl-CoA and oleoyl-CoA were added separately to the assay mixture both substrates were equally used by the microsomal Gro3P AT of type II cells isolated from adult rat lung [44]. When equal amounts of palmitoyl-CoA and oleoyl-CoA were added simultaneously to the in vitro assay system, however, the unsaturated fatty acid was incorporated much faster than the saturated fatty acid. Saturated and unsaturated acyl-CoAs were more or less equally distributed between the sn-1 and sn-2 position. It has to be considered, however, that these data were obtained in vitro and do not necessarily reflect the situation in vivo.

Table 4. Genes and gene products involved in phosphatidic acid biosynthesis in mammals. ADR, 1-acyl-GrnP reductase.
 Gro3P pathwayGrnP pathway 
OrganellesGro3P AT1-Acyl-GroP ATGrnP ATADRKey refs
Mitochondria     
 Genes/gene product (m)90 kDa (mouse liver)(Rat liver)[42,48,59]
 LocalizationOuter membrane  Outer membrane 
 Substrate specificitySaturated acyl-CoA    
Microsomes     
 Genes/gene product (m)Not identified1-acyl-GroP ATα (human); (32 kDa)GrnP ATPutative ADR[10,62,63]
  1-acyl-GroP ATβ (human); (31 kDa)   
 Homology among species70.5%    
 LocalizationMembrane boundMembrane bound Membrane assoc. 
 Substrate specificitySaturated or
unsaturated acyl-CoAs
    
Peroxisomes     
 Genes/gene product (m)GrnP AT
77 kDa (human)
67 kDa (guinea pig)
65 kDa (human placenta)
Putative ADR
appr. 75 kDa
(guinea pig)
[55–57,62]
 Homology among species  80%  
 Localization  Luminal side of membrane  
 Substrate specificity  Saturated acyl-CoAMembrane assoc. 

One might assume that preference of the mitochondrial Gro3P AT for certain acyl-CoAs contributes to the distinct fatty acid composition of glycerophospholipids and triacylglycerols. This is, however, not very likely because the microsomal system with its lower substrate specificity represents about 90% of total Gro3P AT activity of the cell under normal conditions [9]. Thus, the specific pattern of fatty acids in complex lipids is more likely derived from a remodelling process (reviewed in [45]),

Another difference between the two mammalian Gro3P AT isoforms is the response to hormonal and nutritional regulation. The mitochondrial Gro3P AT is more responsive to this type of regulation than the microsomal isoenzyme [46]. Hormones and nutrients, however, control both the activity of mitochondrial Gro3P AT and that of fatty acid synthase. Transcription of fatty acid synthase and Gro3P AT genes is coordinately regulated by nutrients (e.g. glucose) and hormones such as glucagon, insulin, glucocorticoids and thyroid hormone. As an example, insulin stimulates transcription of fatty acid synthase and mitochondrial Gro3P AT, whereas glucagon acts in the opposite way through the cis-acting elements within the promoters and their trans-acting factors (reviewed in [47]). Thus, the activity of Gro3P AT and the supply of the fatty acid substrate required for PtdOH synthesis are regulated at the same level.

Sul and coworkers [40,41,48,49] isolated a cDNA encoding a mouse liver Gro3P AT with a molecular mass of 90 kDa. Transfection of human cells with the respective ORF resulted in an increase of Gro3P AT activity in mitochondria but not in the microsomal fraction. This result indicated that Gro3P AT activity in the two compartments cannot be attributed to the same enzyme. This view was supported by experiments with rat hepatocytes using the inhibitor 5-amino-4-imidazole carboxamide riboside. In the presence of this reagent the mitochondrial Gro3P AT activity was decreased to 22–34% of the control whereas the microsomal Gro3P AT was not affected [50]. Identification of the microsomal Gro3P AT and cloning of the respective gene would contribute immensely to our understanding of regulation of PtdOH biosynthesis in mammals. Comparison of Gro3P ATs with higher or lower specificity for fatty acids would also allow correlation of the structural features of the enzymes with their ability to discriminate between saturated and unsaturated fatty acids.

GrnP acyltransferase (GrnP AT) is present in all animal tissues with the exception in erythrocytes. In mammalian cells, GrnP AT is obligatory for alkylglycerol and plasmalogen synthesis and has been detected in all ether lipid-containing organisms [51,52]. The question remains whether GrnP AT may also be involved in the biosynthesis of PtdOH because this enzyme is also present at high activity in tissues which harbour only a small amount of ether lipids, e.g. liver and adipose tissues. Thus, an active role of GrnP AT in the synthesis of nonether glycerolipids was envisaged [51]. In contrast, experiments with tissues from patients suffering from the so-called Zellweger syndrome, a peroxisomal disease which results in a dramatic decrease of the amount of ether lipids, demonstrated that nonperoxisomal acyltransferases do not acylate GrnP in vivo[53]. Furthermore, it was shown that the synthesis of diacyl species of glycerophospholipids in Zellweger tissues was normal indicating that the contribution of peroxisomal GrnP AT to the synthesis of nonether lipids is not essential.

Activity of GrnP AT has been detected in peroxisomes, the ER and also mitochondria of mammalian cells. GrnP AT from guinea pig liver peroxisomes was partially purified by Jones and Hajra [54] and later on by Webber and Hajra [55] using a modified protocol yielding a single protein with an apparent molecular mass of 67 kDa. Another peroxisomal GrnP AT from human placental membrane with an apparent molecular mass of 65 kDa was purified by Ofman and Wanders [56]. The highest activity of this GrnP AT was observed with palmitoyl-CoA as a substrate, whereas the activity with mono- or polyunsaturated acyl-CoAs was rather low. A peroxisomal GrnP AT from guinea pig liver cells was shown to catalyze also the reverse reaction of 1-acyl-GrnP to acyl-CoA and GrnP[54].

Thai et al. [57] isolated the cDNA of a human GrnP AT and characterized the corresponding protein with a molecular mass of 77 kDa (Table 4). This enzyme contains a C-terminal type I peroxisomal targeting signal (AKL) and three extended domains which may mediate interaction of the polypeptide with the peroxisomal membrane. Analysis of subcellular localization employing immunoelectron microscopy revealed that in rat liver the protein is exclusively associated with the luminal side of the peroxisomal membrane. Purification of this GrnP AT by sucrose density gradient centrifugation and anion-exchange chromatography, however, resulted in isolation of a heterooligomeric complex with three polypeptides of 76, 72 and 69 kDa. Amino acid sequence analysis revealed that the 72 and 69-kDa bands correspond to GrnP AT. The appearance of the 69-kDa form of GrnP AT is most likely due to modification of the enzyme by post-translational processes [57]. The 76-kDa protein of the complex was identified as 1-alkyl-GrnP synthase. Cross-linking experiments confirmed that GrnP AT and 1-alkyl-GrnP synthase interact with each other and form a heterotrimeric complex located on the luminal side of the peroxisomal membrane [58]. Similar to mitochondrial Gro3P AT, the peroxisomal GrnP AT of mammalian cells shows a preference for the saturated long chain acyl-CoAs, palmitoyl-CoA and stearoyl-CoA, and is rather insensitive towards N-ethylmaleimide (reviewed in [6]).

Besides the localization in peroxisomes, GrnP AT is also present in the ER and in mitochondria [59] (see Fig. 3C). The question remained whether the microsomal activity was due to nonspecific Gro3P AT. More recently it has been shown that at least two distinct microsomal acyltransferases exist which independently catalyze acylation of Gro3P and GrnP[51]. The low affinity for GrnP and the strong inhibitory effect of Gro3P suggests that mitochondrial GrnP AT activity is a side activity of mitochondrial Gro3P AT [59].

GrnP AT is highly conserved in mammals. As an example, human and mouse cDNAs specific for GrnP AT are approximately 80% homologous to each other. The human protein also shows homology to Gro3P ATs of several other species, e.g. rat, mouse, E. coli and Haemophilus influenzae. Distinct domains of these polypeptides comprising about 35–80 amino acid residues in length exhibit 78% homology and 45% identity.

Enzymes required for complete alkylglycerol and plasmalogen synthesis are not restricted to peroxisomes but were also found in the ER [60–62]. In one of the early steps of this pathway, 1-acyl-GrnP synthesized by GrnP AT has to be reduced in an NADPH-dependent reaction. This enzymatic step yielding lyso-PtdOH is catalyzed by 1-acyl-GrnP reductase (see Fig. 2). Localization studies in guinea-pig liver cells demonstrated the presence of acyl/alkyl GrnP reductase activity in peroxisomes, although a smaller portion of this enzyme was also detected in the ER [62] (Fig. 3C). Datta et al. [62] purified an acyl/alkyl GrnP reductase from guinea pig liver peroxisomes which has an apparent molecular mass of 60 kDa as estimated by SDS/PAGE and 75 kDa as determined by size-exclusion chromatography (Table 4). Amino acid analysis revealed that hydrophobic amino acids comprise 27% of the molecule; the amino acid sequence of the protein, however, was not determined. Due to the presence of 1-acyl-GrnP reductase activity in peroxisomes, lyso-PtdOH can be formed in this compartment.

It was only recently that the first mammalian 1-acyl-Gro3P ATs, two isoforms of human 1-acyl-Gro3P AT, were identified at the molecular level [10,63–65]. These two proteins, 1-acyl-Gro3P ATα and 1-acyl-Gro3P ATβ, whose genes are localized to different chromosomes (VI and IX), revealed 47.6% identity and 70.5% homology to each other. It was predicted from the DNA sequence that 1-acyl-Gro3P ATα is a 31.7-kDa protein (Table 4) containing seven potential hydrophobic regions. As the mature forms of the enzyme have molecular masses of 26 and 28 kDa, respectively, proteolytic processing of the polypeptide was envisaged. Sequence homology between the human 1-acyl-Gro3P ATα and 1-acyl-Gro3P ATs from other sources such as bacteria, yeast and plants is in the range 24.0–30.9%. Two conserved transmembrane regions of 1-acyl-Gro3P ATα were considered to form the active center of the polypeptide, which faces the cytosolic side of the ER and seems to be responsible for the selection of acyl chains regarding length and degree of unsaturation. Aguado and Campbell [64] demonstrated in vitro that the human 1-acyl-Gro3P ATα accepts fatty acids with 12–18 carbon atoms, depending on the degree of saturation. These authors also demonstrated that 1-acyl-Gro3P ATα is able to incorporate arachidonoyl-CoA into lyso-PtdOH. Such a substrate usage was considered to be of great relevance because arachidonic acid is the most important precursor for the formation of prostaglandins. In contrast, Stamps et al. [65] reported that 1-acyl-Gro3P ATα exhibits a distinct preference for oleoyl-CoA.

Human 1-acyl-Gro3P ATβ was identified by homology to a coconut 1-acyl-Gro3P AT [63]. A polypeptide with a molecular mass of 30.9 kDa was predicted from the nucleotide sequence of the ORF (Table 4). The putative protein has 33% identity to the 1-acyl-Gro3P AT of Coenorhabditis elegans, and 23% to 28% homology to 1-acyl-Gro3P ATs of prokaryotes, plants and yeast. Similar to GrnP AT and Gro3P AT, identical amino acid residues of all members of the 1-acyl-Gro3P AT family are restricted to certain domains. In contrast to 1-acyl-Gro3P ATα[64], 1-acyl-Gro3P ATβ was found to prefer arachidonoyl-CoA over palmitoyl-CoA as a substrate [63]. This result, however, has to be interpreted with caution as different assay systems were used for these studies.

Computational analysis predicted two potential transmembrane spanning helices for 1-acyl-Gro3P ATα and four for 1-acyl-Gro3P ATβ. Both enzymes are localized to the ER [64]. 1-Acyl-Gro3P ATβ is expressed in most tissues with the highest level in liver, where the highest GrnP AT activity was also observed. This result suggests that 1-acyl-Gro3P ATβ is an essential component of the phospholipid metabolic pathway in this tissue. It is not clear whether 1-acyl-Gro3P ATβ can modulate the intracellular amount of the second messenger lyso-PtdOH produced in activated cells, or whether its primary function is to generate PtdOH for the synthesis of phospholipids.

Interplay of organelles during biosynthesis of phosphatidic acid

Why are different organelles such as the ER, mitochondria, and in addition peroxisomes of mammalian cells, lipid particles of yeast and chloroplasts of plants involved in the biosynthesis of PtdOH? Having the two major functions of PtdOH in mind, namely as a precursor of glycerolipids and as a signalling molecule, several reasons for redundant biosynthetic systems of this lipid may be envisaged. One possible explanation for this redundancy is that different pools of PtdOH may serve as precursors for the synthesis of complex glycerol phospholipids and/or triacylglycerols in different organelles. Little is known, however, about regulatory mechanisms that channel PtdOH to form the various species of acylglycerolipids under specific physiological conditions. As an example, Hajra [6] suggested that membrane phospholipids synthesized via the Kennedy pathway or the CDP-diacylglycerol pathway (see Fig. 1) may be formed from PtdOH synthesized in the ER. On the other hand, triacylglycerol whose rate of synthesis strongly fluctuates under different physiological conditions, could be formed from PtdOH originating from mitochondria and/or peroxisomes. The fact that transcription of fatty acid synthase and Gro3P AT in mammalian cells is coordinately regulated by nutrients and hormones [47] may be regarded as an argument supporting this view. In contrast, however, transcription of fatty acid synthase [66] but not of Gro3P AT, GrnP AT and 1-acyl-Gro3P AT of the yeast is controlled by inositol [38].

As another hypothesis, acylating activities present in mitochondria, peroxisomes or lipid particles could provide a means of the cell to respond in a quick way to a need of PtdOH in addition to the ‘standard’ source from the ER [46]. This view is supported by the observation that Gro3P AT is the rate limiting enzyme for PtdOH formation, and mitochondrial Gro3P AT is more responsive to hormonal and nutritional regulations than the microsomal counterpart. The physiological control of these pathways and their relative importance in cellular glycerolipid metabolism remain to be established [6]. In contrast to the above mentioned hypothesis, which might be true for mammals and yeast, Wiberg et al. [67] demonstrated that in two transgenic laurate-producing oil seed rape cell lines, phospholipids and triacylglycerol are synthesized from the same diacylglycerol pools and thus most likely from a single cellular pool of PtdOH.

A possible function of mitochondrial and peroxisomal acylation reactions could be providing substrate cycles for acyl-CoAs by esterification of fatty acids to Gro3P or GrnP. This metabolic conversion may prevent acyl-CoAs from being subjected to β-oxidation that occurs in mitochondria and peroxisomes of animal cells. This view is supported by the finding that the peroxisomal GrnP AT of guinea-pig liver cells synthesizes 1-acyl-GrnP, and also catalyzes the reverse reaction, namely cleavage of 1-acyl-GrnP to GrnP and acyl-CoA [54]. GrnP AT present in mitochondria of S. cerevisiae[28], however, cannot be significantly involved in fatty acid protection because β-oxidation is restricted to peroxisomes in this microorganism [68].

As mentioned above, mitochondrial Gro3P AT and peroxisomal GrnP AT have a preference for saturated acyl-CoAs to be incorporated into the sn-1 position of the respective substrates. In contrast, Gro3P AT of the ER incorporates saturated, monounsaturated, and polyunsaturated acyl-CoAs with essentially the same efficiency [69]. Thus, mitochondrial and peroxisomal enzymes create a specific distribution of fatty acids in PtdOH, i.e. with a saturated fatty acid in sn-1 and an unsaturated fatty acid in sn-2 position of the glycerol backbone, which may be preserved during formation of complex glycerolipids. It has to be considered, however, that transacylases present in all types of mammalian cells (reviewed in [45]) may contribute significantly to the nonrandom distribution of fatty acids through a remodelling process.

Besides their role as precursors of acyl glycerolipids, lyso-PtdOH and PtdOH serve as second messengers [70,71]. To fulfill this function the presence of these components at distinct subcellular site(s) is required. The site of synthesis may be important to guarantee a balanced level of the regulatory pool of lyso-PtdOH and PtdOH. Which organelle(s) preferentially synthesize second messenger lyso-PtdOH and PtdOH is not known.

As a consequence of the occurrence of certain steps of PtdOH synthesis in different organelles, traffic of intermediates between subcellular compartments may be required. In prokaryotes and plants, intracellular transport of the intermediate lyso-PtdOH does not necessarily have to occur because both enzymes of the pathway, Gro3P AT and 1-acyl-Gro3P AT, are localized to the same compartments (see above). In mammals, lyso-PtdOH synthesized in the ER can be further acylated to PtdOH in the same compartment due to the presence of 1-acyl-Gro3P AT activity (Fig. 3C). Lyso-PtdOH, formed either by acylation of Gro3P in mitochondria or by acylation of GrnP and subsequent reduction in peroxisomes, must be transported to the ER for subsequent conversion to PtdOH. 1-Acyl-GrnP formed in peroxisomes may also be translocated to the ER to be converted to lyso-PtdOH because of the presence of 1-acyl-GrnP reductase in the latter compartment. Vice versa, 1-acyl-GrnP synthesized in the microsomal fraction could be further metabolized by 1-acyl-GrnP reductase present in peroxisomes. In yeast, all enzymes needed for the formation of PtdOH are present in the ER and in lipid particles. 1-Acyl-GrnP formed in mitochondria has to be transported to a site of 1-acyl-GrnP reductase and 1-acyl-Gro3P AT activities, i.e. the ER or lipid particles, for further conversion to PtdOH (Fig. 3B).

Both intermediates of the PtdOH biosynthetic pathway, lyso-PtdOH and 1-acyl-GrnP, are largely water-soluble, and simple diffusion from mitochondria or peroxisomes, respectively, to the site of further conversion to PtdOH in the ER is very likely. As an alternative, transport of lyso-PtdOH and 1-acyl-GrnP may be facilitated by carrier proteins or occur through vesicle flux. Haldar and Lipfert [72] found lyso-PtdOH in a post-mitochondrial supernatant of rat liver mitochondria when BSA was added to in vitro assays, whereas lyso-PtdOH sedimented with mitochondria in the absence of BSA. Vancura and Haldar [73] identified and purified a protein with the ability to bind lyso-PtdOH that also stimulates the conversion of mitochondrially synthesized lyso-PtdOH to PtdOH when microsomes were added to the assay mixture. The putative lyso-PtdOH carrier protein was found to be identical to liver fatty acid binding protein. Jolly et al. [74] demonstrated that rat liver fatty acid binding protein, which exists in two isoforms, can stimulate Gro3P AT but not 1-acyl-Gro3P AT in vitro. The two polypeptides also affected fatty acid remodelling in certain phospholipids in vitro. Isoform I preferentially enhanced oleate and palmitate incorporation into PtdEtn, while isoform II stimulated fatty acid remodelling in PtdCho, PtdSer and sphingomyelin. The role of liver fatty acid binding protein as a carrier for lyso-PtdOH in vivo, however, remains to be demonstrated.

Another possible mechanism to transport intermediates of PtdOH synthesis between the ER and mitochondria might be membrane contact. The so-called mitochondria-associated membrane which has been described for mammalian cells [75] and yeast [76] may be involved in such a process. Mitochondria-associated membrane is a subfraction of the ER that copurifies with mitochondria and exhibits high activities of certain lipid-synthesizing enzymes. Gro3P AT and 1-acyl-Gro3P AT, however, are not significantly enriched in the mitochondria-associated membrane fractions of yeast and mammalian cells. Nevertheless, mitochondria-associated membrane may serve as a mediator of translocation of these lipids between the ER and mitochondria through membrane contact.

Phenotype of mutant cells with defects in PtdOH biosynthesis

The prokaryote E. coli contains only one Gro3P AT and one 1-acyl-Gro3P AT. As a consequence, an E. coli strain lacking the plsB gene that encodes Gro3P AT cannot grow but is still viable for approximately 3 h at 42 °C [17]. This short survival time may be explained by the fact that diacylglycerol kinase (dgkA) phosphorylates endogenous diacylglycerol yielding a small amount of PtdOH, which serves as a precursor for ongoing phospholipid synthesis. On the E. coli genome the plsB gene is located proximal to the dgkA gene, but the open reading frames are not arranged in tandem [13]. Although plsB and dgkA do not have the same promoter, mutations in dgkR, a regulatory gene of dgkA, also affect the level of Gro3P AT activity [77]. Another reason for the short survival time of an E. coli plsBstrain might be the ability of 1-acyl-Gro3P AT to acylate Gro3P although with low efficiency. Similar observations were made with the 1-acyl-Gro3P AT of the yeast, the SLC1 gene product [34]. Deletion of the E. coli plsC gene that encodes 1-acyl-Gro3P AT results in lethality [17] because the other enzymes of this prokaryote are unable to substitute for the 1-acyl-Gro3P AT-catalyzed acylation of lyso-PtdOH. It was predicted that accumulation of lyso-PtdOH in a plsC strain causes lethality due to the detergent-like properties of the component, which may disturb membrane formation [17].

Plant cells contain three distinct sets of acyltransferases involved in PtdOH biosynthesis distributed among different organelles. Thus, dysfunction of one of these enzymes is unlikely to result in lethality regarding the role of PtdOH a as precursor of glycerolipids. Furthermore, it has been reported [67] that plant cells contain only one pool of diacylglycerol derived from PtdOH to which the different organelles may contribute equally. Nevertheless, different pools of PtdOH present in plant compartments might affect cellular processes other than lipid synthesis. Defects in acyltransferases with a more pronounced substrate specificity, such as the soluble 1-acyl-Gro3P AT of chloroplasts or the membrane bound 1-acyl-Gro3P ATs (see above), may cause changes in the cellular pattern of molecular species of PtdOH. Such an effect may result in formation of complex phospholipids with molecular properties different from those synthesized under standard conditions.

Similar to plants, yeast cells harbour at least two sets of acyltransferases involved in the formation of PtdOH. As a consequence, a mutant bearing a point mutation in the most prominent Gro3P AT, the putative Gat1p, a strain deleted of the SLC1 gene which encodes the major 1-acyl-Gro3P AT, or even a gat1 slc1 double mutant grow like wild-type. Gro3P ATs and 1-acyl-Gro3P ATs present in different organelles [34] thus provide a means for balanced growth of the cells even if one of the acylating enzymes is not functioning. In addition, formation of PtdOH by phosphorylation of diacylglycerol may serve as a salvage pathway, although the respective enzyme has never been characterized from yeast. Similar to acyltransferases involved in PtdOH formation, 1-acyl-GrnP reductase-catalyzed reduction of the intermediate 1-acyl-GrnP occurs in lipid particles and the ER. Due to the presence of redundant 1-acyl-GrnP reductase or another unidentified salvage pathway deletion of the major 1-acyl-GrnP reductase does not affect growth (K. Athenstaedt and G. Daum, unpublished results).

In mammalian cells, similar to cells of other eukaryotes, enzymes involved in the formation of PtdOH via Gro3P pathway occur in redundancy. As a consequence, lack of one of these polypeptides can be largely compensated by other isoenzymes. Furthermore, several diacylglycerol kinases with molecular masses of 80–90 kDa have been identified in mammalian cells [78–84] that can form at least a limited amount of PtdOH. In contrast to the function of diacylglycerol kinase in E. coli (see above), however, the major function of this enzyme in mammalian cells may not be biosynthesis of PtdOH, but attenuation of the level of diacylglycerol in signal transduction. In mammals, diacylglycerol serves as a second messenger and is generated by hydrolysis of PtdIns(4,5)P2 or other phospholipids through phospholipase C (reviewed in [85]).

Despite the general ability of mammalian cells to compensate for defects in single genes involved in PtdOH synthesis, certain specific defects caused by dysfunction of acyltransferases are known. A genetic disease due to a deficiency of acyltransferase(s) in human cells is the so-called Barth syndrome. Barth syndrome is an X-linked inheritable disorder characterized by short stature, cardioskeletal myopathy, neutropenia, abnormal mitochondria and respiratory-chain dysfunction [86–88]. It is often fatal in childhood due to cardiac failure or sepsis arising from acranulocytosis. The phenotype is quite variable. Recently, a gene mutated in patients afflicted with Barth syndrome was cloned and sequenced [86]. Due to alternative splicing, this gene encodes several proteins which were designated tafazzins. These tafazzins were shown to belong to a superfamily of acyltransferases involved in phospholipid synthesis in vivo.

In mammalian cells, the GrnP pathway is obligatory for alkylglycerol synthesis. Dysfunction of GrnP AT was demonstrated to cause several genetic diseases. This was first demonstrated with patients suffering from the so-called Zellweger cerebrohepatorenal syndrome. The level of GrnP AT activity in peroxisomes of Zellweger cerebrohepatorenal syndrome patients is only 10% of control cells. Nonperoxisomal acyltransferases in these cells failed to produce significant amounts of 1-acyl GrnP in vivo[53]. Thus it appears unlikely that the GrnP AT activity detected in the ER and mitochondria in vitro significantly contribute to acylglycerolipid synthesis.

Other human diseases associated with reduced GrnP AT activity are neonatal adrenoleukodystrophy, infantile Refsum disease and hyperpipecolic acidemia. Besides GrnP AT deficiency, these diseases exhibit multiple symptoms, such as an increase in the amounts of very long chain fatty acids (> C22), pipecolic acid and phytanic acid in serum and tissues, plasmalogen deficiency, and accumulation of intermediates of bile acid metabolism [89,90]. Dysfunction of GrnP AT is also observed in tissues of patients suffering from the genetic disorder rhizelomic chondrodysplasia punctata [89,90]. Rhizelomic chondrodysplasia punctata can be divided into three subgroups. Ofman et al. [91] demonstrated that all patients of type II disease bear mutations in the cDNA of GrnP AT thus pinpointing this disease to an enzymatic defect of the acyltransferase.

Physiological roles of diacylglycerol, PtdOH and lyso-PtdOH

As already mentioned in the Introduction, PtdOH plays a major role in the biosynthesis of membrane phospholipids and triacylglycerols (see Fig. 1). Conversion of PtdOH to diacylglycerol, however, is also important for regulatory aspects because diacylglycerol can serve as a lipid second messenger [85] and, as shown recently, as a mediator of vesicle formation [92]. The enzyme which catalyzes hydrolysis of PtdOH and generates 1,2-diacylglycerol and Pi is phosphatidate phosphatase (PAP). Phosphatidate phosphatases involved in glycerolipid synthesis exist in both soluble and membrane associated forms. The metabolic status of the cell affects the subcellular distribution of this enzyme which may in turn influence regulation of lipid metabolism. In mammals certain forms of phosphatidate phosphatase were characterized which appear to be involved in signal transduction (reviewed [93]).

Phosphatidate phosphatase of the prokaryote E. coli is a membrane bound polypeptide and has a molecular mass of 28 kDa. This enzyme is encoded by the pgpB gene. A pgpB mutant was originally characterized by a defect of phosphatidate phosphatase activity [94,95], but such a strain also exhibits defects in the activity of lyso-PtdOH phosphatase, diacylglycerol pyrophosphate phosphatase and phosphatidylglycerophosphate phosphatase.

In plants, phosphatidate phosphatase activities have been localized to plastids, microsomes and soluble fractions in a number of tissues [93]. Phosphatidate phosphatase from plastids is a membrane-bound polypeptide and exclusively located to the inner envelope membrane. Phosphatidate phosphatase localized to the microsomal fraction of plants was found to be Mg2+-dependent similar to its analogs in yeast and mammals. The majority of plant phosphatidate phosphatase (80% of total cellular activity) was found in the 40 000 g supernatant of the homogenate [96]. Both phosphatidate phosphatase or diacylglycerol acyltransferase of plants have been reported to catalyze the rate limiting steps in triacylglycerol formation [93]. Besides the important role for phosphatidate phosphatase in diacylglycerol synthesis, the enzyme is also involved in degradation and turnover of glycerophospholipids. As an example, the microsomal phosphatidate phosphatase of germinating seeds was shown to contribute to the degradation of membrane phospholipids [97].

In the yeast, phosphatidate phosphatases are localized to microsomes, mitochondria and cytosol (reviewed in [95]). The cytosolic phosphatidate phosphatase has an apparent molecular mass of 91 kDa and is activated by Mg2+, whereas N-ethylmaleimide, CDP-diacylglycerol, diacylglycerol and triacylglycerol inhibit this enzyme. Two forms of phosphatidate phosphatase are present in the ER (104 and 45 kDa). In contrast to the cytosolic phosphatidate phosphatase, the 104-kDa protein of microsomes is activated by CDP-diacylglycerol, cardiolipin and PtdIns. The occurrence of the 45-kDa protein is not only restricted to the ER, but the protein is also present in mitochondria. This enzyme, like its cytosolic counterpart is activated by Mg2+ and inhibited by the sulfhydryl group blocking reagent N-ethylmaleimide. At the genetic 1evel, the expression of yeast phosphatidate phosphatases is regulated by various factors [93]. As the most prominent example, supplementation of yeast cells with inositol leads to an increase of phosphatidate phosphatase activity [98]. The activity of phosphatidate phosphatase is augmented when cells enter the stationary phase of growth [98,99]. Detailed studies have revealed that inositol supplementation increased only the phosphatidate phosphatase activity associated with the 45-kDa protein, whereas both the 45-kDa and 104-kDa forms of phosphatidate phosphatase are induced in the stationary growth phase [100]. The latter alteration is paralleled by an increase in Gro3P AT, GrnP AT and 1-acyl-GrnP reductase activities in stationary cells [38] which, in summary, leads to an enhanced formation of triacylglycerol at the expense of phospholipids.

In mammalian cells, two types of phosphatidate phosphatases are known. One type is present in the ER and cytosol, the other type resides in the plasma membrane [93]. These two types of phosphatidate phosphatase are distinguished by their properties. The microsomal phosphatidate phosphatase PAP1, which is involved in glycerolipid metabolism, is Mg2+-dependent and can be selectively inactivated by N-ethylmaleimide. Glucagon, glucocorticoides, cAMP and growth hormones stimulate the enzyme activity whereas insulin decreases enzyme activity. On stimulation, the cytosolic phosphatidate phosphatase migrates to the ER to become functionally active, probably through the lipid environment of the membrane. Hopewell et al. [101] showed that binding of long-chain fatty acids and their CoA esters to the ER as well as the accumulation of PtdOH in this organelle act as signals for enhanced phosphatidate phosphatase association with the membrane, thereby increasing the synthesis of triacylglycerol (see Fig. 1). Microsomal phosphatidate phosphatase of mammalian cells has not yet been purified or identified at the molecular level.

In contrast to microsomal and cytosolic PAP1, the plasma membrane PAP2 of mammalian cells is Mg2+-independent and is not inactivated by N-ethylmaleimide. PAP2 appears to exist as several isoforms. Two human plasma membrane phosphatidate phosphatases have been purified and characterized by Kai et al. [102].

Diacylglycerol of mammalian cells is not only a precursor for glycerolipid synthesis, but it can also act as a second messenger. During ‘classical’ signal transduction the production of diacylglycerol occurs by phospholipase C-catalyzed breakdown of PtdIns(4,5)P2[85]. An alternative pathway of diacylglycerol formation comprises the enzymatic activity of phosphatidate phosphatases. This specific pool of diacylglycerol for signal transduction may be generated by the plasma membrane PAP2 [1].

Diacylglycerol has also been proposed to be essential for a functional protein secretory pathway [92]. Kearns et al. [103] demonstrated with the yeast S. cerevisiae that diacylglycerol promotes Golgi secretory function. The authors suggested that diacylglycerol may be derived from turnover of complex lipids through the action of Sec14p, the yeast PtdIns transfer proteins.

In contrast to the above mentioned hypothesis, it has also been proposed that PtdOH may be a mediator of protein secretion [92]. Henry and coworkers [104,105] showed that PtdOH derived from phospholipase D-catalyzed turnover of PtdCho, which is under control of Sec14p, promotes protein secretion in yeast. The molecular mechanism of this regulation, however, remains to be demonstrated. PtdOH generated upon a stimulatory signal by phospholipase D catalyzed breakdown of phospholipids can also act as a potent growth factor molecule, stimulate phospholipase C, protein and lipid kinases, mobilize Ca2+ flux, activate NADPH oxidase, induce hormone release, platelet aggregation and gene transcription, and change cytoskeletal dynamics [106] (reviewed in [71]). As a consequence, phosphatidate phosphatase may produce both the second messenger diacylglycerol through dephosphorylation of PtdOH and also terminate the signal of the second messenger PtdOH by the same reaction.

Lyso-PtdOH is a naturally occurring phospholipid with hormone and growth factor-like activities. Lyso-PtdOH acts as a signaling molecule that is rapidly produced and released by activated cells. Lyso-PtdOH added extracellularly binds to specific receptors of target cells and stimulates platelet aggregation, smooth muscle contraction, induction of neuronal shape changes and leads to cell proliferation [106] (reviewed in [70]). Exogenous lyso-PtdOH also stimulates GTP-dependent PtdIns hydrolysis and rapid breakdown of PtdCho in rat fibroblasts, and inhibits the activity of adenylate cyclase in a number of cell types, e.g. mouse, rat and human cells [107]. Lyso-PtdOH may also be involved in wound repair and blood clotting processes. In the serum, this lipid intermediate is present at physiologically active concentrations. Activated platelets secrete this substance and have thus been suggested as the primary source of serum lyso-PtdOH. The action of lyso-PtdOH is restricted to specific cell-types. Receptors for lyso-PtdOH have been, for example, detected on neuronal cells, brain homogenates, carcinoma cells, leukemic cells and fibroblasts, but not on rat liver membranes or human neutrophils [107].

Summary, conclusions and perspectives

Most enzymes involved in the synthesis of acylglycerolipids have been identified and characterized. Surprisingly, proteins involved in PtdOH biosynthesis, especially those of eukaryotic cells, belong to the small group of lipid-synthesizing enzymes still awaiting identification at the molecular level. Sequence homologies of acyltransferases which were already characterized in prokaryotes and, in some cases, in plants, yeast and mammals may help to fill the gaps. With the exception of acyltransferases present in plant chloroplasts, all enzymes involved in de novo biosynthesis of PtdOH identified so far by their function in vitro, are tightly bound to membranes. This property has made expression and purification of these proteins very difficult. In general, Gro3P ATs and GrnP ATs have higher molecular masses than 1-acyl-Gro3P ATs (see Tables 1–4). It was speculated that the larger size of Gro3P AT and GrnP AT allows binding of effectors thus providing a means to regulate the activity of these enzymes [16]. In contrast to Gro3P ATs and GrnP ATs, which catalyze the initial and rate limiting step in PtdOH synthesis, 1-acyl-Gro3P ATs appear to be subjected to fewer regulatory processes. Another difference between the acyltransferases is their substrate specificity. Whereas Gro3P ATs and GrnP ATs in general prefer saturated fatty acids as substrates, 1-acyl-Gro3P ATs preferentially catalyze incorporation of unsaturated fatty acids into the PtdOH molecule.

Comparison of the available sequences of membrane bound acyltransferases have revealed that proteins from bacteria, plants, yeast and mammals contain homologous regions. These homologies, however, are restricted to short stretches which may be the basis for the identification of additional acyltransferases by computational analysis. As an example, overall matches between human 1-acyl-Gro3P ATs and 1-acyl-Gro3P ATs from other organisms range from only 4% to 12% [10]. Gro3P ATs and 1-acyl-Gro3P ATs from different organisms, however, share a short amino acid sequence with an invariant His and Asp separated by four less well conserved residues in an HX4D configuration [108]. It has been demonstrated by site-directed mutagenesis that lack of the invariant His residue causes loss of acyltransferase activity. Thus, this amino acid appears to function in the active center of the enzyme to deprotonate the hydroxyl moiety of the acyl acceptor. Most recently, Lewin et al. [109] demonstrated the presence of four homologous blocks in acyltransferases. Site directed mutagenesis revealed that certain amino acids of blocks I, III and IV in the Gro3P AT of E. coli are required for enzymatic catalysis. Other residues in blocks II and III appear to be important for binding of the glycerol backbone. These domains are also present in 1-acyl-Gro3P ATs and GrnP ATs and may fulfill a similar role.

Whereas in prokaryotes only one Gro3P AT and one 1-acyl-Gro3P AT were detected, eukaryotic acyltransferases occur in redundancy. What is the reason for this redundancy? Is it to guarantee ongoing synthesis of acylglycerols when one acyltransferase becomes defective or is inhibited? We believe that the answer to this question is more complex. First, differences in the substrate specificity for acyl-CoAs being incorporated into PtdOH were observed with isoenzymes of Gro3P AT, GrnP AT and 1-acyl-Gro3P AT thus allowing generation of different species of this lipid. This observation, however, has to be interpreted with caution because these data were obtained in vitro. Different local concentrations of activated fatty acids in vivo may result in a different substrate utilization by acyltransferases.

Another reason for the redundancy of Gro3P ATs and 1-acyl-Gro3P ATs, which is probably more important, might be the formation of different cellular pools of lyso-PtdOH and PtdOH. These pools may provide specific species of PtdOH for the formation of complex glycerophospholipids or triacylglycerols in different organelles. As an example, PtdOH formed by the mitochondrial Gro3P AT of mammalian cells or the mitochondrial GrnP AT in yeast (see above) might be preferentially used for the synthesis of PtdGro and cardiolipin, whereas the pool of PtdOH formed by the microsomal system might be used primarily for synthesis of all other acylglycerolipids. As another hypothetical example, PtdOH formed in lipid particles of the yeast could be mainly used for synthesis of triacylglycerol, which is subsequently stored in the core of these particles, whereas ER-derived PtdOH might be preferentially used for the synthesis of glycerophospholipids. The hypothesis that PtdOH formed in subcellular compartments other than the ER is mainly used for the synthesis of triacylglycerol has been discussed by Hajra [6]. Contradictory evidence from Wiberg et al. [67] suggested that triacylglycerol and glycerophospholipids of plants are synthesized from the same pool of diacylglycerol. Different pools of PtdOH and lyso-PtdOH may, however, also be important to fulfill their regulatory functions. These pools are probably small and the enzymes responsible for their synthesis less abundant. As a consequence, these components may have escaped our attention so far.

The existence of different genes encoding proteins with the same or similar enzymatic activity also allows independent regulation of isoenzymes present in different subcellular compartments at the level of gene expression. An example, already mentioned in a previous section, is the mitochondrial Gro3P AT activity of rat liver cells that reveals hormone sensitivity and depends on the nutritional status whereas the microsomal Gro3P AT does not (reviewed in [47]). In combination with substrate specificities of Gro3P ATs and 1-acyl-Gro3P ATs, the transcriptional regulation may provide fine tuning of the initial steps of lipid biosynthesis.

Future studies will certainly focus on the identification of genes and gene products involved in PtdOH formation that have escaped characterization so far. Biochemical investigations will be required to isolate the proteins and characterize their enzymatic properties. A most challenging aspect will be the cell biology of PtdOH biosynthesis. Precise localization of the enzymes involved and determination of the subcellular origin of intermediate and end products of acyltransferases will be required. In vitro assays with isolated organelles will be used to demonstrate the localization of enzymes and the interaction and interplay of subcellular compartments during the process of PtdOH synthesis. Finally, the growing genome data bases will provide the information to search for homologues of enzymes involved in PtdOH formation. Thus, a combination of biochemical, cell biological, molecular biological and computational methods will hopefully lead, within a short time frame, to a complete overview of the biochemical reactions involved in the formation of the important lipid metabolite PtdOH.

Acknowledgements

Studies concerning the synthesis of phosphatidic acid in the yeast carried out in our laboratory were supported by the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich (project 11491 and SFB Biomembranes F 706), EUROFAN project BIO4-CT95–0080, and project 950080 of the Austrian Ministry of Science and Transportation.

Ancillary