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Progress in Lipid Research 51 (2012) 350377

Contents lists available at SciVerse ScienceDirect

Progress in Lipid Research


journal homepage: www.elsevier.com/locate/plipres

Review

Acyl-CoA:diacylglycerol acyltransferase: Molecular biology, biochemistry


and biotechnology
Qin Liu a,1, Rodrigo M.P. Siloto a, Richard Lehner b, Scot J. Stone c, Randall J. Weselake a,
a

Agricultural Lipid Biotechnology Program, Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6H 2P5
Department of Pediatrics, Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Canada T6G 2S2
c
Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5
b

a r t i c l e

i n f o

Article history:
Available online 15 June 2012
Keywords:
Neutral lipids
Storage lipids
Triglyceride
Kennedy pathway
Obesity
Lipid biotechnology

a b s t r a c t
Triacylglycerol (TG) is a storage lipid which serves as an energy reservoir and a source of signalling molecules and substrates for membrane biogenesis. TG is essential for many physiological processes and its
metabolism is widely conserved in nature. Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20)
catalyzes the nal step in the sn-glycerol-3-phosphate pathway leading to TG. DGAT activity resides mainly
in two distinct membrane bound polypeptides, known as DGAT1 and DGAT2 which have been identied in
numerous organisms. In addition, a few other enzymes also hold DGAT activity, including the DGATrelated acyl-CoA:monoacylglycerol acyltransferases (MGAT). Progress on understanding structure/function in DGATs has been limited by the lack of detailed three-dimensional structural information due to
the hydrophobic properties of theses enzymes and difculties associated with purication. This review
examines several aspects of DGAT and MGAT genes and enzymes, including current knowledge on their
gene structure, expression pattern, biochemical properties, membrane topology, functional motifs and
subcellular localization. Recent progress in probing structural and functional aspects of DGAT1 and DGAT2,
using a combination of molecular and biochemical techniques, is emphasized. Biotechnological applications involving DGAT enzymes ranging from obesity therapeutics to oilseed engineering are also discussed.
2012 Elsevier Ltd. All rights reserved.

Contents
1.

2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
TG biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.
Role of DGAT in TG bioassembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Identification and molecular characterization of DGATs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Dgat1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Dgat2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
DGAT2-related enzymes in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
WS/DGAT in bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.
Soluble DGAT-related enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; ACBP, acyl-CoA binding protein; ARAT, acyl-CoA:retinol acyltransferase; ASO, antisense oligonucleotide;
AWAT, acyl-CoA:wax alcohol acyltransferase; BSA, bovine serum albumin; CE, cholesteryl esters; CPM, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin; DCR,
defective cuticle ridge; DG, diacylglycerol; DGAT, acyl-CoA:diacylglycerol acyltransferase; DGTA, diacylglycerol transacylase; EMS, ethyl methanesulfonate; ER, endoplasmic
reticulum; FA, fatty acid; G3P, sn-glycerol-3-phosphate; GPAT, acyl-CoA:sn-glycerol-3-phosphate acyltransferase; HMM, Hidden Markov Model; HTS, high throughput
screening; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPAT, acyl-CoA:lysophosphatidic acid acyltransferases; MAM, mitochondria-associated membranes;
MFAT, multifunctional O-acyltransferase; MG, monoacylglycerol; MGAT, acyl-CoA:monoacylglycerol acyltransferase; NBD, N,N0 -dimethyl-N(acetyl)-N0 -(7-nitrobenz-2-oxa1,3-diazol-4-yl) ethylene diamine; NEM, N-ethylmaleimide; PA, phosphatidic acid; PAP, phosphatidic acid phosphohydrolases; PC, phosphatidylcholine; PDAT,
phospholipid:diacylglycerol acyltransferase; PKA, protein kinase A; QTL, quantitative trait loci; SCD1, stearoyl-CoA desaturase 1; TG, triacylglycerol; TMD, transmembrane
domain; WS, wax ester synthase.
Corresponding author. Address: University of Alberta, Department of Agricultural, Food and Nutritional Science, 4-10 Ag/For Centre, Edmonton, AB, Canada T6G 2P5. Tel.:
+1 780 492 4401; fax: +1 780 492 4265.
E-mail addresses: qin.liu@ualberta.ca (Q. Liu), rodrigo.siloto@ales.ualberta.ca (R.M.P. Siloto), richard.lehner@ualberta.ca (R. Lehner), scot.stone@usask.ca (S.J. Stone),
randall.weselake@ualberta.ca (R.J. Weselake).
1
Present address: Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA.
0163-7827/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.plipres.2012.06.001

Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

3.
4.

5.

6.

7.

8.

9.

DGAT gene structure, ontogeny and expression pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Biochemical properties of DGATs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Differential biochemical properties of DGAT1 and DGAT2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Substrate selectivity properties of DGAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Modulators of DGAT activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Experimental approaches for detecting DGAT activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Topological characterization of DGAT1 and DGAT2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Membrane topology of DGAT1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Membrane topology of DGAT2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Functional motifs in DGAT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
Conserved and functional motifs in DGAT1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
Conserved and functional motifs in DGAT2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Subcellular localization of DGAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.
DGAT localization in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.
DGAT localization in animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.
DGAT localization in fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biotechnological applications of DGAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1.
DGAT as therapeutic targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2.
Use of DGAT in oilseed engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Closing comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction
Triacylglycerol (TG) is a non-polar acyl triester of glycerol and
fatty acids (FAs) [1]. As a major class of neutral lipid, the presence
of TG is widespread in eukaryotes including animals, plants and
fungi. In prokaryotes, TG accumulation is also a common feature
in species belonging to the actinomycetes group [2,3]. TG has multiple functions in living organisms, but serves principally as a reservoir of FAs for energy production. The synthesis and mobilization
of TG are essential cellular processes to maintain lipid and energy
homeostasis in all types of cells [46]. In mammals, TG is important for energy storage in adipose tissue. Also, it protects cells from
lipotoxicity, serves as a vehicle for energy (FAs) transport, provides
ligands for nuclear hormone receptors and signal transduction processes as well as substrates for the synthesis of lipids that maintain
the permeability barrier [710]. Excessive accumulation of TG in
human adipose and non-adipose tissues, however, is related to a
variety of pathological conditions such as obesity, type 2 diabetes,
coronary heart disease, hypertriglyceridemia and non-alcoholic
fatty liver disease [1113]. With increasing prevalence of these diseases, there is considerable interest in a better understanding of TG
metabolism, which is a potential target for pharmacological intervention. In the case of plants, TG is the main component of seed
oils functioning as an energy reservoir for the young seedling after
germination until the plant develops phototrophy [14]. In addition,
TG is required for pollen and seed development [15,16]. Vegetable
oils are of great value for human nutrition, consumed primarily for
food as a source of essential FAs [17,18]. Similarly, in fungi and
some bacteria, TG serves as energy storage compound also playing
an essential role in cell growth and development [2,3,15,1922]. In
recent years, because of the raised concern of developing renewable and environmentally-benign alternatives for crude oil, TGs
produced by plants and microorganisms have been regarded as potential resources for producing biofuels and chemical feedstocks
[17,18,23]. Therefore, while limiting TG production in humans
can represent potential treatment for metabolic-related disease,
enhancing tailor-made TG accumulation in plant seeds and microorganisms is a rapidly expanding area in biotechnology [24].
1.1. TG biosynthesis
TG biosynthesis has been extensively studied since the 1950s.
The complexity of TG synthesis is reected by the existence of mul-

tiple biosynthetic pathways described in different organisms and


tissues (Fig. 1). For instance, a monoacylglycerol (MG) pathway
mediated by acyl-CoA:monoacylglycerol acyltransferase (MGAT,
EC 2.3.1.22) was exclusively elucidated in animals [25,26], which
utilizes acyl-CoAs as the acyl donors and plays a predominant role
in dietary fat absorption in the small intestine [27,28]. Evidence for
this pathway has also been shown in plants [29]. Acyl-CoA-independent TG synthesis catalyzed by a phospholipid:diacylglycerol
acyltransferase (PDAT, EC 2.3.1.158) was demonstrated in plants
and yeast [3032]. Although PDAT activity has also recently been
found in the bacterium Streptomyces coelicolor, the prokaryote enzyme apparently does not share noticeable homology with its
eukaryotic counterpart [21]. Acyl-CoA-independent TG formation,
in mammals and plants, has also been reported to occur via transfer of an acyl chain between two molecules of diacylglycerol (DG)
to form TG and monoacylglycerol (MG) (molecules related to TG
biosynthesis are shown in Fig. 1A; pathways are compared in
Fig. 1B) [33,34]. Detailed reviews describing the biochemical pathways of TG synthesis can be found elsewhere [2,3,7,3540].
Although the previously-mentioned reactions lead to TG synthesis, the canonical pathway conserved in all organisms is known
as the Kennedy or sn-glycerol-3-phosphate (G3P) pathway [41,42].
The biosynthesis of TG starts with the acylation of G3P by acylCoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15)
producing lysophosphatidic acid (LPA). Acylation of LPA by acylCoA: lysophosphatidic acid acyltransferase (AGAPT, LPAAT or LPAT,
EC 2.3.1.51) generates phosphatidic acid (PA) which is then
dephosphorylated by lipins/PA phosphohydrolases (Lipin/PAP, EC
3.1.3.4), generating DG. The nal reaction of the TG biosynthetic
pathway is catalyzed by acyl-CoA:diacylglycerol acyltransferases
(DGAT or DAGAT, EC 2.3.1.20) which esteries DG producing TG
(Fig. 1B). In bacteria, a dual-function acyltransferase wax ester synthase (WS)/DGAT catalyzes the last acylation to produce TG, which
will be described in the next sections. In eukaryotes, these reactions occur mainly on the endoplasmic reticulum (ER) and the production of LPA and PA can also occur in mitochondria and on lipid
droplets [40,43]. Synthesis of TG has also been reported in the
cytosol and plant chloroplasts [4446]. In addition to the G3P,
dihydroxyacetone phosphate (DHAP) can be esteried to form
1-acyldihydroxyacetone-phosphate (acyl DHAP) which serves as
a precursor to synthesize LPA [47,48]. However, this DHAP

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Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

Fig. 1. Triacylglycerol (TG) biosynthesis in eukaryotes and prokaryotes [2,3,7,30,3439]. (A) Schematic representation of chemical structures of related substrates. LPA,
lysophosphatidic acid; PA, phosphatidic acid; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; G3P, sn-glycerol-3-phosphate; MG, sn-1-monoacylglycerol; DG, sn-1,2diacylglycerol; TG, triacylglycerol; (B) Pathways for TG synthesis in animals, plants, fungi and bacteria. Conserved sequential acylation is enclosed in shaded area. FA, fatty
acid, GPAT, acyl-CoA:sn-glycerol-3-phosphate acyltransferase; LPAT, acyl-CoA:lysophosphatidic acid acyltransferase; PAP, phosphatidate phosphatase; DGAT, acylCoA:diacylglycerol acyltransferase; MGAT, acyl-CoA:monoacylglycerol acyltransferase; DGTA, diacylglycerol transacylase; PDAT, phospholipid:diacylglycerol acyltransferase;
WS/DGAT, wax ester synthases/acyl-CoA:diacylglycerol acyltransferase.

pathway occurs only in mammals (Saccharomyces cerevisiae is an


exception), which was more commonly regarded as a pathway to
produce either lipid [48].
1.2. Role of DGAT in TG bioassembly
DGAT plays a key role in determining the carbon ux into TG.
For example, Stals et al. [49] observed that the rate of TG synthesis
in rat hepatocytes is controlled by the afnity of DGAT for acylCoA. A correlation between DGAT transcript level and oil accumulation has been reported in common oilseeds as well as in plant
species that accumulate unusual FAs [50]. Similarly, the level of
DGAT expression has been shown to be directly related to TG accumulation in mammals [9,51]. Recent studies describe the role of
DGAT in pheromone synthesis in insects [52] and also the characterization of protozoan multifunctional acyltransferases with
DGAT activity [53]. Detailed understanding of the molecular mechanisms of DGAT-mediated catalysis is crucial for the development
of novel strategies for manipulating TG synthesis in different

organisms. In recent years, considerable progress in DGAT function


has been achieved thanks to the identication of numerous genes
encoding DGAT across species. The reader is directed to insightful
reviews on topics including DGAT cloning, expression, biochemical
properties, tissue distribution and functional studies with emphasis on the role of DGAT in human diseases, energy homeostasis
[10,5456] and plant development [57,58]. In addition, regulatory
aspects of DGAT expression and activity have been reviewed elsewhere [54,55]. Despite this progress, advances in understanding
structure/function relationships of DGAT are limited by the hydrophobic nature of these acyltransferases and the difculties that
have been encountered in attempting to purify them to homogeneity in an active form [57,5961]. For example, extensive protein
degradation occurred during the solubilisation of tung tree DGAT1
from the inclusion bodies of Escherichia coli [60]. Also, recombinant
tung DGAT2 was puried after solubilisation with sodium dodecyl
sulfate but was not active [62]. In addition, a murine DGAT2
mutant without the presence of transmembrane domains (TMDs)
is still associated with membrane although it is active when

Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

353

Fig. 2. Dendrogram indicating the relatedness of protein sequences displaying DGAT activities was derived from the multiple sequence alignment. The organisms and
GenBank accession numbers for proteins used are as follows: Alcanivorax borkumensis, Ab, AbWS/DGAT, YP_694462.1; Ac, Ah, Arachis hypogaea, AhDGAT3, AY875644; A.
thaliana, At, AtDGAT1, NM_127503, AtDGAT2, NP_566952; AtWS/DGAT, AtDCR, AED93236, Brassica napus, Bn, BnDGAT1, AAD45536; Caenorhabditis elegans, Ce, CeDGAT1,
CAB07399, CeDGAT2a, CAB04533, CeDGAT2b, AAB04969; Homo sapiens, Hs, HsDGAT1, NP_036211 HsDGAT2, AAK84176, HsMGAT1, AAK84178, HsMGAT2, AAI03877,
HsMGAT3, AAO63579; Mus musculus, Mm, MmDGAT1, NP_034176, MmDGAT2, AAK84175, MmMGAT1, AAI06136, MmMGAT2, AAH52831; Mycobacterium tuberculosis, Mt,
MtWS/DGAT1, NP_217646.1; MtWS/DGAT2, NP_218251.1; MtWS/DGAT3, NP_217604.1; Ricinus communis, Rc, RcDGAT1, AAR11479, RcDGAT2, AAY16324; Streptomyces
avermitilis, Sa, SaWS/DGAT, NP_828432; S. cerevisiae, Sc, ScDGAT2, NP_014888; Schizosaccharomyces pombe, Sp, SpDGAT2, XP_001713160; Umbelopsis ramanniana, Ur,
UrDGAT2a, AAK84179, UrDGAT2b, AAK84180; Vernicia fordii (tung), Vf, VfDGAT1, ABC94471, VfDGAT2, ABC94474.

expressed in human cells [63]. These investigations suggest it may


be worth investigating the possibility of purifying an active DGAT2
truncation from either E. coli or insect cells. Even an enzyme truncation could set the stage for providing valuable structural insight
into DGAT action.
This review begins with a historical description of DGAT discovery in diverse organisms, and then focuses on recent progress in
probing low-resolution structural and functional aspects of two
widely distributed DGAT families, DGAT1 and DGAT2 (including
MGAT). Based on these investigations and on in silico analyses, a
general view of the similarities and divergences of DGAT in the
context of their roles in different organisms is developed. Finally,
biotechnological applications of DGAT in therapeutics and oilseed
engineering are discussed.
2. Identication and molecular characterization of DGATs
Although DGAT activity was rst reported in chicken liver in the
1950s [41,42], the encoding genes were not identied until recently [64,65]. With the extensive progress in genomics, bioinformatics, transgenics and protein purication, genes encoding two
major types of DGAT were characterized in eukaryotes, and designated DGAT1 and DGAT2. In addition to the nearly ubiquitous
occurrence of these genes in eukaryotes, a number of DGAT-related

genes have also been identied which encode MGATs, wax ester
(WS) synthases [66,67] and members of the BAHD2 acyltransferases which represent a large family of acyltransferases that utilize
CoA thioesters [68,69] (Fig. 2).
2.1. Dgat1
The rst DGAT gene was cloned from mouse in 1998 [64] based
on sequence homology between an expressed sequence tag and a
previously identied acyl-CoA:cholesterol acyltransferase (ACAT,
EC 2.3.1.26), a related enzyme that synthesizes cholesteryl esters
(CE) [70]. Functional expression of the mouse and human DGAT1
cDNA in insect cells showed that the encoded protein possessed
signicant DGAT activity but did not catalyze CE synthesis [64].
After this initial discovery, DGAT1 orthologs from a number of
other species, such as thale cress (Arabidopsis thaliana), were
quickly identied [7173]. DGAT1 was not found in the genome
of S. cerevisiae and most of yeast organisms, although the product
2
BAHD is named according to the rst four characterized members (BEAT or
benzylalcohol O-acetyltransferase from Clarkia breweri; AHCTs or anthocyanin Ohydroxycinnamoyltransferases from Petunia, Senecio, Gentiana, Perilla, and Lavandula;
HCBT or anthranilate N-hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus; DAT or deacetylvindoline 4-O-acetyltransferase from Catharanthus roseus)
[69].

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Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

of ARE2, one of the two ACAT orthologs in this species, appeared to


have weak DGAT activity [74]. Recent studies with Yarrowia lipolytica, a non-conventional yeast strain which can uptake numerous
hydrophobic compounds from the environment [75], indicated
that this organism possesses a gene (YlDGA2) homologous to
DGAT1 from plants [76]. This gene corresponds most closely to
ARE2 in S. cerevisiae.
Characterization of A. thaliana DGAT1 started with the identication of an ethyl methanesulfonate (EMS) mutant (AS11) with reduced DGAT activity, seed oil content and altered seed FA prole
[7173]. The residual DGAT activity in AS11, suggested the existence of other DGATs. This work resembles parallel studies in mice
in which DGAT1-decient mice (Dgat1/) had normal plasma TG
levels and abundant lipids in adipose tissue [9]. This nding indicated the possibility of an alternative mechanism(s) for TG synthesis, which was later attributed to a second family of DGAT genes
referred to as DGAT2.

2.2. Dgat2
DGAT2 was initially puried from the lipid body fraction of the
fungus Umbelopsis ramanniana, (formerly known as Mortierella
ramanniana) [65]. The gene encoding this polypeptide did not
share any homology with DGAT1 or ACAT1/2 and thus was classied
as DGAT2. In the same study, orthologs from S. cerevisiae and
Caenorhabditis elegans were also cloned and shown to encode proteins with DGAT activity. These results formed the basis for the
identication of members from this new DGAT gene family in
mouse and human [77]. Studies using gene targeting in mice suggested that DGAT2, more so than DGAT1, plays a predominant role
in TG biosynthesis in mammals [9,78]. While DGAT1 is virtually
absent in fungi, with the exception of Y. lipolytica as previously
described, DGAT2 is widely present in nature. In S. cerevisiae and
Y. lipolytica, DGAT2 (Dga1 in S. cerevisiae and DGA2 in Y. lipolytica)
have been conrmed to make a major contribution to TG synthesis
[19,79].
Molecular characterization of DGAT2 from plants has been challenging, probably because early attempts to functionally express a
DGAT2 cDNA from model plant A. thaliana were not successful. Synthesis of A. thaliana DGAT2 in yeast was blocked at the translational level, resulting in no protein accumulation [80]. This could
be a result from differences in codon usage between the yeast
and plants [8184]. However, functional studies indicated that A.
thaliana DGAT2 gene does not encode a functional enzyme in A. thaliana either. An insertion knockout in A. thaliana DGAT2 gene did
not produce signicant reduction in seed oil content, an effect that
was not compensated for by other DGAT-related enzymes [15].
Therefore, DGAT2 does not appear to play a substantial role in
TG synthesis in developing seeds of A. thaliana.
Despite these negative results in A. thaliana, functional plant
DGAT2s were characterized from species that produce oils enriched with unusual FAs [8587], such as castor bean (Ricinus communis), tung tree (Vernicia fordii) and ironweed (Vernonia
galamensis), which produce TGs enriched in ricinoleic (12-OH
18:1cisD9), a-eleostearic (18:3 cisD9, transD11, transD13) and
vernolic (12-epoxy, cisD9 octadecenoic) acids, respectively. These
plant DGAT2s were functionally characterized and found to have
a preference for substrates containing these unusual FAs. These
studies not only conrmed that, in certain plant species, DGAT2
plays a role in the biosynthesis of TG, but also highlighted the possible function of this enzyme in selecting substrates to be incorporated into TG. Further studies with plant DGAT2, including probing
potential protein interactions, and subcellular localization, will
help determine the molecular mechanisms involved in TG synthesis by DGAT1 and DGAT2.

2.3. DGAT2-related enzymes in mammals


In mammals, MGAT is mainly involved in dietary fat absorption
in the intestine [27,8891]. Three MGAT genes have been identied
and have signicant sequence similarity with mammalian DGAT2
genes, but not with DGAT1 genes, indicating that MGAT genes share
a common origin with DGAT2 (Fig. 2). Interestingly, human DGAT2
and MGAT2 genes are located in the same region of human chromosome 11 [90]. MGAT3, which shares a higher degree of sequence similarity with DGAT2 than with MGAT1 and 2, is only
found in higher mammals and humans, but not in rodents [92].
In addition to catalyzing DG synthesis, the three ER-associated
and acyl-CoA dependent MGAT isoforms (MGAT1, MGAT2 and
MGAT3) were also shown to possess DGAT activity when overexpressed in mammalian or insect cells [25,88,89,92].
Two isoforms of human acyl-CoA:wax alcohol acyltransferase
or multifunctional O-acyltransferase (designated AWAT or MFAT,
EC 2.3.1.75), which predominantly mediate the synthesis of wax
esters, have been identied [93,94]. AWAT was shown to exhibit
DGAT, MGAT and acyl-CoA:retinol acyltransferase (ARAT) activities
and has been proposed to play an essential role in lipid metabolism
in human skin. Since both MGAT and AWAT have been characterized in specic tissues in higher mammals, it is possible that other
DGAT-related genes could be expressed in certain tissues and/or organs. MGATs and AWATs share signicant sequence similarity to
DGAT2 [54]. Indeed, sequence analysis of both DGAT2 and MGAT2
gene sequences has provided evidence that MGAT and AWAT may
have originated from duplications of DGAT2 [95], but evolved to
catalyze the synthesis of distinct lipids during evolution. A soluble
acyl-CoA-dependent MGAT has been puried from developing peanut cotyledons [96]. Recently, it was demonstrated that peanut
oleosin is a bifunctional enzyme with MGAT and phospholipase
activities, indicating the possibility of identifying a microsomal
MGAT from peanut developing seed [97].
2.4. WS/DGAT in bacteria
None of the eukaryotic DGAT1 and DGAT2 enzymes share
homology with bacterial proteins. In some bacteria, TG formation
is catalyzed by a bifunctional membrane-associated enzyme that
possesses both WS and DGAT activities [3]. WS/DGAT was rst
identied in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, which can synthesize both wax ester and TG under
growth-limiting conditions [66]. Heterologous expression of Acinetobacter WS/DGAT in yeast was demonstrated to promote TG synthesis [98]. This WS/DGAT also exhibits MGAT activity in vitro
[67], suggesting that it has multiple roles in prokaryote lipid metabolism. Remarkably, this bifunctional enzyme is unrelated to the jojoba and mammalian WS or DGAT1, DGAT2 and PDAT from
eukaryotes. WS/DGAT homologues were also isolated and functionally characterized from Mycobacterium tuberculosis [99], Alcanivorax
borkumensis [100] and gram-positive bacteria Streptomyces avermitilis MA-4680 [101]. Based on this study, another WS/DGAT was
identied in A. thaliana [102] which catalyzes, predominantly, the
synthesis of wax esters. A mycoyltransferase from Mycobacterium
tuberculosis (named Ag85A) was recently found to have additional
DGAT activity detected by in vitro and in vivo experiments [103].
Ag85A is associated with the ability of M. tuberculosis to establish
and maintain a persistent tuberculosis infection.
2.5. Soluble DGAT-related enzymes
While DGAT and other related enzymes described before are
found to be associated predominantly with cellular membranes,
several other soluble enzymes catalyzing the synthesis of TG have
been also characterized. A DGAT with no homology to either

355

Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

Table 1
Acyl-CoA:diacylglycerol acyltransferases (DGAT) functionally characterized in recombinant organisms. The two-character code preceding each DGAT gene indicates the organisms
of origin are as follows: Ab, Alcanivorax borkumensis; Ac, Acinetobacter calcoaceticus At, Arabidopsis thaliana; Ah, Arachis hypogaea; Bn, Brassica napus; Ce, Caenorhabditis elegans; Ea,
Euonymus alatus; Ha, Helianthus annuus; Hs, Homo sapiens; Mm, Mus musculus; Mt, Mycobacterium tuberculosis; Nt, Nicotiana tabacum; Oe, Olea europaea; Pt, Phaeodactylum
tricornutum; Rc, Ricinus communis; Sa, Streptomyces avermitilis; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Tg, Toxoplasma gondii; Tm, Tropaeolum majus; Ur,
Umbelopsis ramanniana; Vf, Vernicia fordii; Vg, Vernonia galamensis; Zm, Zea mays. Adapted from Siloto et al. [24].
Gene

cDNA

Host used for expression and relevant genetic markers

Reference

AtDGAT1

Sf21 insect cells (Spodoptera frugiperda)


S. cerevisiae YMN5 (Slc1- D)
S. cerevisiae SCY 062
S. cerevisiae SCY059 (are1- D, are2- D), N. tabacum
A. thaliana AS11
S. cerevisiae H1266 (are2- D, dga1- D, lro1- D)
B. napus
P. pastoris
B. napus
S. cerevisiae H1266 (are2- D, dga1- D, lro1- D)
S. cerevisiae H1246 (are1- D, are2- D, dga1- D, lro1- D)
S. cerevisiae
S. cerevisiae SCY 062
S. cerevisiae H1246 (are1- D, are2- D, dga1- D, lro1- D
S. cerevisiae INVSc1
S. cerevisiae SCY1998 (dga1- D, lro1- D)
S. cerevisiae
S. cerevisiae SCY910 (are1- D, are2- D)
S. cerevisiae H1246 (are1- D, are2- D, dga1- D, lro1- D), A. thaliana, B. napus
Z. mays and S. cerevisiae (dga1- D, lro1- D)
S. cerevisiae SCY 062
McA-RH7777 cells (Rattus norvegicus)
S. cerevisiae 12501 (dga1-D)
Sf9 insect cells (S. frugiperda)
COS7-cells (Chlorocebus sabaeus)
H5 insect cells (Trichoplusia ni)
Sf9 insect cells (S. frugiperda)
Sf9 insect cells (S. frugiperda)
S. cerevisiae ScY2051 (are2- D, dga1- D, lro1-D)
S. cerevisiae 12501 (dga1- D)
Sf9 insect cells (S. frugiperda)
COS7-cells (C. sabaeus)
S. cerevisiae
A. thaliana and S. cerevisiae (dga1- D)
S. cerevisiae SCY1998 (dga1- D,lro1- D)
Sf9 insect cells (S. frugiperda)
S. pombe DKO (dga1- D,lro1- D)
Sf9 insect cells (S. frugiperda)
G. max
S. cerevisiae H1246 (are1- D, are2- D, dga1- D, lro1- D)
Sf9 insect cells (S. frugiperda)
Sf9 insect cells (S. frugiperda)
Sf9 insect cells (S. frugiperda)
AV-12 cells (M. auratus),COS-7 (C. sabaeus), Caco-2 cells (Homo sapiens)
S. cerevisiae SCY2056, (are2- D, dga1- D, lro1- D)
S. cerevisiae SCY2056, (are2- D, dga1- D, lro1- D)
Sf9 insect cells (S. frugiperda)
E.coli BL21 (DE3)
A. calcoaceticus
S. cerevisiae H1266 (are2- D, dga1- D, lro1- D)
E. coli BL21 (DE3)
E. coli BL21 (DE3)
S. cerevisiae H1266 (are2- D, dga1- D, lro1- D)
E. coli BL21 (DE3)
S. cerevisiae H1266 (are2- D, dga1- D, lro1- D)

[117]
[73]
[236]
[236]
[166]
[237]
[227]
[168]
[227]
[237]
[104]
[238]
[236]
[123]
[239]
[85]
[115]
[240]
[118]
[177]
[167]
[241]
[242]
[77]
[121]

BnDGAT1

DGAT1

EaDGAT1
EpDGAT1
HaDGAT1
NtDGAT1
PtDGAT1
VgDGAT1
VfDGAT1
RcDGAT1
TgDGAT1
TmDGAT1
ZmDGAT1
CeDGAT1
HsDGAT1

MmDGAT1
CeDGAT2
HsDGAT2

MmDGAT2
DGAT2

RcDGAT2
VfDGAT2
ScDGAT2
SpDGAT2

MGAT

AWAT

WS/DGAT

Soluble
DGAT

YlDGAT2
HmMGAT2
HmMGAT3
MmMGAT1
MmMGAT2
AWAT1
AWAT2
AbWS/DGAT
AcWS/DGAT
MtWS/DGATs
SaWS/DGAT
AtWS/DGAT
AhDGAT3
AtDCR

DGAT1 or DGAT2, but with 13% identity with the bacterial WS/
DGAT (Fig. 2), was identied in developing peanut (Arachis hypogaea) cotyledons (referred to as AhDGAT3) [44]. Homologous protein sequences of AhDGAT3 were also found in other plant species
such as A. thaliana, soybean (Glycine max) and rice (Oryza sativa)
[44,105]. Previously, a cytosolic 10S multi-enzyme complex from
the oleaginous yeast Rhodotorula glutinis was also found to possess
DGAT activity [106]. Cross-reactivity was observed between AhDGAT3 and antibodies raised against a R. glutinis DGAT peptide obtained from this multi-enzyme complex [44], suggesting that
these two proteins share sequence similarities. Another soluble

[65]
[77]
[93]
[242]
[77]
[143]
[86]
[124]
[85]
[65]
[125]
[65]
[228]
[79]
[92,94]
[89,92]
[25,88,92,94]
[25]
[93]
[93]
[94]
[100]
[66]
[67]
[99]
[101]
[102]
[44]
[108]

DGAT, known as DCR (Defective Cuticle Ridge), which belongs to


the BAHD family of acyltransferases, was also characterized in A.
thaliana [107]. DCR, which shares little sequence homology with
other plant DGAT1s, is involved in catalyzing the synthesis of TG
enriched in hydroxy FA, which may serve as a precursor for cutin
synthesis [108]. These studies demonstrated that TG synthesis
does not occur exclusively in membranes, but the process also occurs in the cytoplasm of some organisms. Soluble isoforms of other
acyltransferases involved in TG synthesis are well documented.
These include the plastidial GPATs from several plant species
[109], LPAT from A. thaliana [110] and R. glutinis [106], as well as

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Table 2
Structure of representative DGAT genes. Abbreviations for each gene indicating the organism of origin are described in Table 1.

DGAT1

DGAT2/MGAT

WS/DGAT
Soluble DGAT

Gene

ID

Chromosome

Sequence length (bp)

Number of exons

CeDGAT1
HsDGAT1
MmDGAT1
AtDGAT1
RcDGAT1

H19N07.4a
8694
13350
816464
8272569

5
8
15
2
N/A

2771
12321
9804
3531
7741

7
17
17
16
16

CeDGAT2a
CeDGAT2b
HsDGAT2
MmDGAT2
AtDGAT2
RcDGAT2
ScDGAT2
SpDGAT2
HsMGAT1
HsMGAT2
HsMGAT3
AbWS/DGAT

F59A1.10
W01A11.2
84649
67800
824315
8258757
854419
5802702
116255
610270
346606
4213840

5
5
11
7
3
N/A
15
3
2
11
7
N/A

1508
1385
32802
29051
2165
3450
1257
1487
38193
13398
5293
1374

2
5
8
8
8
9
1
4
6
6
7
1

SaWS/DGAT
AtWS/DGAT
AtDCR

1217410
833704
832459

N/A
5
5

1344
2751
3465

1
7
2

MGAT from developing peanut cotyledons, as noted earlier [96]. In


addition, PAP, which catalyzes the second-last in the Kennedy
pathway, is also present in the soluble fraction [111,112]. It has
been speculated that these soluble counterparts of TG-biosynthetic
enzymes may utilize different lipid pools and thus play different
roles in the regulation of cell function compared to membrane isoforms [106].
Currently, a relatively large collection of DGAT cDNAs, particularly from plants, is available which will facilitate more detailed
studies on enzyme function. Many of these genes, which are presented in Table 1, have been functionally characterized in recombinant systems (mainly in S. cerevisiae).

DGAT1 transcript is less abundant in liver leads to the hypothesis


that DGAT1 is more important for fat absorption in intestine and
basal level TG synthesis in adipose tissue where it is highly expressed [113]. In humans, DGAT2 is expressed in most tissues,
but at higher levels in hepatocytes and adipocytes [77]. In mice,

3. DGAT gene structure, ontogeny and expression pattern


DGAT1 and DGAT2 genes have no sequence homology and encode polypeptides with approximately 35% difference in length.
An evolutionary study indicated that DGAT1 and DGAT2 gene families originated from different ancestors, before the establishment
of eukaryotes [95]. It also suggested that DGAT1 and DGAT2 had a
functional convergence in eukaryotes which is reected in the
importance and ubiquitous occurrence of TG in all eukaryotes as
described at the beginning of this review.
The structures of many DGAT1 and DGAT2 genes have been discussed in previous reviews [24,54]. Table 2 summarizes the predicted genome locations and exon numbers for the DGAT-related
genes examined, illustrating the different nature and size for each
gene type. Generally, DGAT1 contains 1617 exons whereas DGAT2
contains at most 89 exons, except for C. elegans and fungi (Table
2). In these species, DGAT genes contain fewer exons. In addition,
comparisons of exonic and intronic structures indicate that C. elegans DGATs have an unrelated distribution of exons compared to
the DGAT orthologs from other animals and plants (Fig. 3). In animals, the exons of DGAT are grouped in the 30 portion of the gene
while in plants they are distributed throughout the whole sequence. Similar to DGAT2, MGAT genes also have 6 or 7 exons.
The expression pattern of DGAT1 and DGAT2 can differ substantially depending on the organism. In mammals, DGAT expression
was investigated in human and mouse. DGAT1 is ubiquitously expressed with the highest levels of expression in small intestine
and adipose tissue and the lowest in liver [54,113]. The only difference between the expression patterns of DGAT1 in human and mice
is the lower level of the transcript in mouse liver. The fact that

Fig. 3. Architecture of representative acyl-CoA:diacylglycerol acyltransferase


(DGAT) genes [24]. (A) DGAT1 from animals, (B) DGAT1 from plants, (C) DGAT2
from animals and (D) DGAT2 from plants. The genomic sequences of each DGAT are
represented by black bars. The arrows correspond to the exons and numbers
indicate the nucleotide positions. The two-character codes preceding each DGAT
gene indicate the organism of origin are as follows: At, Arabidopsis thaliana; Bt, Bos
taurus; Ce, Caenorhabditis elegans; Hs, Homo sapiens; Mm, Mus musculus; Mt,
Medicago truncatula; Os, Oryza sativa; Sc, Saccharomyces cerevisiae; Ss, Sus scrofa; Vf,
Vernicia fordii; Zm, Zea mays. Adpated from Siloto et al. [24].

357

Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

expression of DGAT2 occurs in a wider range of tissues than in humans. Modulating DGAT expression in muscle, adipose tissue and
pancreatic b-cells can affect insulin activity, adiposity and deterioration of tissue function [55].
Tissue expression of MGAT genes, which share signicant sequence similarity with mammalian DGAT2, has been studied in
mammals, as well. Much like DGAT1 and DGAT2, MGAT1 and MGAT2
are both expressed in adipose tissue as well as in other tissues
[92,93]. Notably, MGAT1 is highly expressed in stomach and kidney
and is not expressed in the intestine, while MGAT2 is predominantly expressed in the small intestine of mice/humans, but also
in the human liver. MGAT3 expression has been only detected in
the human gastrointestinal tract with the highest levels of expression occurring in the ileum [89]. Turkish et al. [93] showed that
MGAT3 is also expressed in the liver. Because MGAT transcripts
were found in other tissues in addition to intestine, where MGAT
plays a key role in dietary fat absorption, this indicates that MGAT
is involved in other aspects of lipid metabolism in other tissues in
mammals. Indeed, MGAT2 knock-out mice are protected from metabolic diseases induced by a high-fat diet, indicating MGAT2 plays
a key role in energy metabolism in response to dietary fat [28]. The
expression of MFAT/AWAT is predominant in the skin [93]. Using
in situ hybridization, Yen et al. [94] further demonstrated that MFAT/AWAT2 transcripts are restrained in sebaceous glands where
skin surface lipids (predominantly wax monoesters) are synthesized, conrming the previous nding by Cheng and Russell [114].
In plants, particularly oilseed crops, the expression pattern of
DGAT has been extensively studied, especially during seed development. Generally, expression of DGAT1 or DGAT2 is closely correlated with oil deposition in developing seeds. However, a quite
different scenario is found for the accumulation of the transcripts

with resulting polypeptide in a study in castor bean. While castor


bean DGAT1 transcripts reached maximum accumulation between
19 and 26 days after pollination, the corresponding protein was detected at a maximum rate between 47 and 54 days after pollination
[115]. A good correlation between enzyme activity and protein
accumulation in the microsomes, however, was observed. The facts
that accumulation of transcript greatly precedes detectable translation indicate that the RcDGAT gene is regulated at the posttranscriptional level. It is noted that the protein was detected using
an antibody raised against the C-terminal region of castor DGAT1
instead of epitope tag antibodies used in other studies, underscoring the importance of detecting the native protein. Unfortunately,
only few studies on DGAT expression pattern make use of this
technology [116]. More investigation is needed for unveiling how
plant DGAT1 is transcriptionally or posttranscriptionally regulated.
In contrast, more progress has been achieved in studies of DGAT
regulation in mammals [54,55].
While DGAT1 transcripts are always found during seed development, the relative level of DGAT2 expression can vary depending on
the species. For example, in developing seeds of soybean and A.
thaliana, DGAT2 has a very limited expression level [50]. Therefore,
DGAT1 likely plays a more substantial role in TG synthesis in these
common crops. On the other hand, in tung tree and castor bean,
DGAT2 is expressed at higher levels than DGAT1 in developing
seeds [85,86]. This is in agreement with biochemical evidence
showing that DGAT2s from these species exhibit a preference for
the unusual FAs produced by these plant species. DGAT transcripts
were also detected in other plant tissues [117,118] suggesting that
these enzymes may be related to other physiological processes in
addition to seed oil synthesis. For instance, DGAT1 has been shown
to be highly expressed during pollen development, presumably

Table 3
Expression pattern of DGAT genes in plants. A compilation of the results from many studies reporting plant DGAT transcript accumulation is displayed. All tissues analyzed in each
study are indicated (dap, days after pollination).
Organism

Gene

Tissues with the highest


expression

Tissues where expression was detected

Tissues where expression was


detected at low levels or not detected

Ref.

Arabidopsis thaliana

DGAT1
DGAT1

Cotyledonary stage embryos


Developing and germinating
seedlings
Young developing embryos

Flowers, petals, buds


Leaves, seedlings, owers, roots, leaves,
owers, developing siliques
Stems, leaves, early maturing seeds, young
developing embryos
Leaves, roots, young developing embryos
Roots, leaves, early maturing seeds
Roots, stems, leaves, early maturing seeds
Weak expression in stems, leaves, young
developing embryos
Roots, hypocotyls, cotyledons, young leaves,

Stems, leaves
Stems

[117]
[73]

Roots, young developing embryos

[50]

Early maturing seeds


Stems

Roots, early maturing seeds

[50]
[50]
[50]
[50]

Shoot tips, leaves, ower buds,


anthers, ovaries
Flower buds

[119]
[119]

[243]

Stems

[50]
[50]
[86]

Leaf, developing endosperm at 35 dap

[86]

Roots, leaves, petals

Leaves

[50]
[118]
[239]
[50]

Roots, leaves
Roots, stems, early maturing seeds

Leaves
Leaves

[50]
[50]
[50]
[85]
[85]

DGAT1

Euphorbia lagascae
Glycine max

DGAT2
DGAT1
DGAT1
DGAT2

Stems
Young developing embryos
Young developing embryos

Olea europaea

DGAT1

Embryos, mesocarps

DGAT2

Mesocarps

DGAT1

Late torpedo, early mesocarp, mid


mesocarp,
Young developing embryos
Young developing embryos

Ricinus communis

Stokesia laevis
Tropaeolum majus
Vernonia galamensis

Vernicia fordii

DGAT1
DGAT2
DGAT1
DGAT2

Developing endosperm at 25 dap

DGAT1
DGAT1
DGAT1
DGAT1

Young developing embryos


Developing embryos at 30 dap
20 dap seeds
Young developing embryos, early
maturing seeds
Young developing embryos
Young developing embryos
Young developing embryos
Flowers
Developing embryos

DGAT2
DGAT2
DGAT2
DGAT1
DGAT2

Roots, hypocotyls, cotyledons, shoot tips,


expanding leaves, young leaves, mature leaves,
embryos, anthers, ovaries
Young drupes, early and mid-torpedo, late
mesocarp, young anther, mature pollen
Roots, stems, leaves, early maturing seeds
Roots, leaves, early maturing seeds
Weak expression in leaf, developing
endosperm at 10, 15, 20, 25, 30 and 35 dap
Developing endosperm at 10, 15, 20, 25 and 30
dap
Roots, stems, leaves, early maturing seeds
Developing embryos at 815 and 2226 dap
Root, stem, leaf, fruit coat
Stems, roots
Stems, early maturing seeds
Leaves
Roots, stems, leaves, early maturing seeds
Developing embryos
Flowers

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accounting for TG accumulation in the pollen grain [15,116]. In


addition, DGAT expression was studied in olive (Olea europaea),
one of the few fruit crops that accumulate TG in the mesocarp.
During the last stages of olive drupe ripening, when TG is still
being synthesized, DGAT2 is up-regulated, while DGAT1 transcript
levels decline [119]. In seeds, however, DGAT1 seemed to be expressed at higher levels compared to DGAT2. These ndings suggest that DGAT1 and DGAT2 make different contributions to TG
accumulation in the seed and mesocarp of olive.
As for the soluble form of DGAT, computational analysis of the
microarray datasets showed that the gene encoding putative soluble DGAT from Arabidopsis (At1g48300) has a similar expression
pattern with DGAT1 with progressively higher transcript levels
during seed maturation [105]. Further investigation of the relationships between the enzyme activity and the expression level will be
needed to verify if DGAT3 is up-regulated post-transcriptionally or
posttranslationally, as well. A summary of DGAT expression in
plants is presented in Table 3.

observed to activate and/or inhibit overall in vitro DGAT activity


depending on the concentration of electrolyte used [57]. It has
been proposed that certain ions can alter the conformation of
DGAT1 and DGAT2 [61]. The differential sensitivity to magnesium
concentration, between DGAT1 and DGAT2, has been used as a basis for quantifying specically the in vitro activity of DGAT1 in
mammalian cells [122]. To date, no evidence has showed that
activities of DGAT1 or DGAT2 are regulated by magnesium in vivo.
A recent study examined the catalytic properties of three
MGATs, using both DG and MG as acyl acceptors [92]. Recombinant
human MGAT3, which shares a high sequence similarity with
DGAT2, displayed stronger DGAT activity compared to that of
MGAT1 and MGAT2. In addition, all the three MGAT isoforms preferred lauroyl (12:0)-CoA (12:0) and oleoyl (18:1cisD9)-CoA. As expected from the low sequence homology between MGAT3 and
DGAT1, MGAT3 was sensitive to detergent inhibition while DGAT1
was stimulated by the presence of detergent.
4.2. Substrate selectivity properties of DGAT

4. Biochemical properties of DGATs


The biochemical properties of DGAT have been conventionally
explored through in vitro enzyme assays which monitor the production of radiolabeled TG. In this assay, either radiolabeled acylCoA or sn-1,2-DG are used as substrates in reactions catalyzed by
cellular fractions, including microsomes [57,64]. Although membrane-associated DGAT activity has been known for many decades,
soluble DGAT forms were only recently identied. Therefore, earlier studies mainly focused on microsomal DGAT that unknowingly
at the time contained more than one type of DGAT polypeptide.
Therefore, it was impossible to assign substrate specicity to
DGAT1 or DGAT2 in assays conducted before the discovery of these
isoenzymes [57]. DGAT1 and DGAT2, however, have been functionally characterized at the molecular level leading to new molecular
tools for studying TG biosynthesis. For example, it is now possible
to express a specic DGAT cDNA in a mammalian or yeast cell devoid of endogenous DGAT activity, thus allowing for a more effective characterization of specic enzyme properties. As a result,
differences in the biochemical properties between DGAT1 and
DGAT2 can be investigated. In the case of plants, expression in vegetative tissues that usually have low or negligible DGAT activity
might represent a good alternative to yeast. For instance, functional expression of DGAT1 in tobacco leaves has been achieved
using a technically simple method [120].
4.1. Differential biochemical properties of DGAT1 and DGAT2
The different biochemical properties of DGAT1 and DGAT2 were
rst demonstrated in a study with mammalian orthologs of these
enzymes by taking advantage of DGAT1 knock-out mice, as reviewed by Yen et al. [54]. Although recombinant human or mouse
DGAT1 and DGAT2 have broad fatty acyl-CoA substrate specicity,
DGAT1 can utilize a broader range of acyl acceptors. For instance,
murine DGAT1 possesses additional in vitro enzyme activities such
as MGAT, wax synthase and acyl-CoA:retinol acyltransferase
(ARAT) activities [94,121]. DGAT2 only catalyzes TG synthesis, suggesting that murine DGAT1 has a less discriminating substrate
binding site than DGAT2 [54]. On the other hand, human and
mouse DGAT2 have a higher afnity for acyl-CoA and DG than
DGAT1 [54,77].
DGAT1 and DGAT2 have different sensitivities to the magnesium ions in in vitro assays [77]. DGAT1 is active under a broad
range of magnesium concentrations (0100 mM). DGAT2 is active
at low concentrations (520 mM) but is inhibited at higher concentrations (100 mM). Indeed, a number of electrolytes have been

Over the last few years, substantial attention has been devoted
to studying the substrate specicity of DGATs, especially those
from plants. A main motivation for conducting these studies was
to identify DGATs which could preferably incorporate desired acyl
groups into TG. A comparative study was conducted for DGAT1
versus DGAT2 from tung tree (V. fordii). Gene expression analysis
discussed in the previous section suggested that tung DGAT2, but
not DGAT1, preferentially incorporated a-eleostearic acid
[18:3cis(9,trans(11,13] into TG in seed oil bodies. Results from
in vitro enzyme assays using microsomal proteins indicated that
both DGATs have similar preferences for DG and acyl-CoA containing common FAs (palmitic [16:0], oleic [18:1cisD9], linoleic
[18:2cisD9,12], and a-linolenic acids [18:3cisD9,12,15]) [85]. Tung
DGAT2 had a lower activity than DGAT1 in vitro, which is in agreement with ndings based on enzyme assays with other plant and
mammalian orthologs of the enzymes. Through an in vivo experiment, however, it was shown that tung DGAT2 displays approximately vefold preference for production of trieleostearin than
DGAT1 by comparing the amount of TG synthesized by each isoform. Collectively, these results may suggest that DGAT catalysis
may involve other elements from the host, such as soluble cytoplasmic cofactors, absent in in vitro assays that normally utilize
microsomal membrane fractions. Discrepancies between results
from assay in vitro and in vivo studies on DGAT1 and DGAT2 have
been noticed in many studies. In rat hepatoma cells, for example,
DGAT2 overexpression led to considerably more TG accumulation
than did DGAT1 overexpression [78], although DGAT2 activity
was relatively lower in vitro [77]. It is worth noting, that in this
work, Shockey et al. [85] used an innovative approach to investigate the substrate preferences of tung DGAT in vivo. As a free FA,
a-eleostearic acid is considerably reactive and polymerizes quickly
upon exposure to air. Therefore, yeast cultures were grown in the
presence of tung oil, which contains trieleostearin, which is less
readily oxidized than the free FA. To enable the release of a-eleostearic acid from TG and subsequent import of the liberated a-eleostearic acid by yeast cells, the authors included a non-specic
lipase from Candida rugosa in the medium. By expressing tung tree
DGAT in a yeast knockout that is defective in TG synthesis, the
authors were able to determine substrate specicity for a single
DGAT. Similar strategies, based on exogenous FA supplementation
and the use of yeast knockouts, have been used by others [123].
Substrate preferences of plant DGAT2 for unusual fatty acyl
groups have also been reported for castor bean and Vernonia galamensis DGAT2 which display higher selectivity for substrates containing ricinoleic and vernolic acid, respectively [86,87,124]. When
compared with expression prole studies [50], these data provide

Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

359

Fig. 4. Kyte-Doolittle hydropathy plots of DGAT. Hydropathy plots from representative polypeptide sequences of DGAT were generated by the method of Kyte and Doolittle
[141] using a window size of 19. Polypeptides include DGAT1 from mouse (A), oleaginous yeast Yarrowia lipolytica (YlDga2p) (B), and tung tree (C) as well as DGAT2 from
mouse (D), baking yeast Saccharomyces cerevisiae (ScDga1p) (E), and tung tree (F). The most hydrophobic segments which may contain putative TMDs are indicated by roman
numerals.

support for the hypothesis that DGAT2 may play a role in the selective accumulation of unusual FAs in the seed oil of certain plants.
Certain substrate preferences of fungal DGAT2 orthologs have also
been observed. DGAT2 from U. ramanniana showed enhanced
activity towards medium-chain fatty acyl-CoAs (e.g. 12:0-CoA)
and enhanced preference for DG containing short- and mediumchain fatty acyl groups (6:0, 8:0 and 10:0) [65]. S. cerevisiae DGAT2
used 18:1-CoA or 16:0-CoA more efciently than 14:0-CoA, 18:0CoA (18:0), arachidonyl (20:4 cisD5,8,11,14)-CoA, or linoleoyl
(18:2 cisD9,12)-CoA [74]. In Saccharomyces pombe, DGAT2 was
found to prefer palmitoyl over oleoyl moieties [125]. Recently, in
Claviceps purpurea, which produces TGs rich in ricinoleic acid,
DGAT2 was demonstrated to prefer ricinoleoyl-CoA (as an acyl donor over 18:1-, 18:2- or a-18:3-CoA [126]. C. purpurea DGAT2
could be an attractive candidate gene for engineering of oilseed
crops to produce high levels of hydroxy FAs, which will be expanded upon later in the review.
4.3. Modulators of DGAT activity
Certain proteins and electrolytes have been shown to activate or
inhibit microsomal DGAT activity in a concentration-dependent

manner [57]. Microsomes used in most of these studies, however,


were isolated from sources which possibly contained both DGAT1
and DGAT2. Thus, our understanding of the molecular mechanism
by which activators or inhibitors exert their effects on specic
DGATs is rather limited. As previously indicated this has been overcome by studying individual DGAT enzymes in expression systems
with low or no endogenous DGAT activity. For example, human
embryonic kidney HEK293A cells transfected with DGAT cDNA
have been successfully used for DGAT1 inhibitor screening [127].
As well, using a quadruple knock-out yeast strain H1246 which
is devoid of TG biosynthesis [19], acyl-CoA binding protein (ACBP)
from Brassica napus has been shown to directly modulate DGAT1
utilization of acyl-CoAs depending on the ratio of acyl-CoA binding
protein to acyl-CoA in the reaction mixture. Specically, ACBP can
slightly stimulate DGAT1 activity when concentration of acyl-CoA
is higher than that of ACBP. Bovine serum albumin (BSA) had a similarly limited stimulatory effect on B. napus DGAT1 [128]. This data
on the effect of BSA differed from earlier reports in A. thaliana and
B. napus in which microsome were used and DGAT activity was increased by BSA [61,129].
Inhibition of DGAT2 activity by N-ethylmaleimide (NEM) was
demonstrated by previous reports for rat myotube tissue and the

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Table 4
Predicted TMDs in representative DGAT. The numbers between brackets denote the bibliographical reference for each algorithm.
DGAT

DGAT1

Program

MmDGAT1
YlDGAT1 (Dga2p)
VfDGAT1
MmDGAT2

DGAT2

ScDGAT2 (Dga1p)
VfDGAT2

HMMTOP [244]

TMHMM [245]

SVMtm [246]

SOSUI [247]

TOPCONS [248]

TMpred [249]

TMDs
Orientation*
TMDs
Orientation
TMDs
Orientation

9
IN
10
IN
10
IN

8
IN
8
IN
8
IN

8
N/A
9
N/A
9
N/A

6
N/A
5
N/A
8
N/A

9
IN
9
IN
9
IN

8
IN
10
OUT
9
IN

TMDs
Orientation
TMDs
Orientation
TMDs
Orientation

1
IN
4
IN
6
IN

1
IN
1
IN
3
IN

2
N/A
3
N/A
3
N/A

2
N/A
3
N/A
2
N/A

2
IN
3
IN
2
IN

2
IN
3
IN
6
IN

Orientation of N-terminus. Cytosol-IN.

fungus U. ramanniana [130,131]. Using the quadruple knock-out


yeast strain H1246, the mechanism by which S. cerevisiae DGAT2
is sensitive to thiol-modifying reagent NEM was also determined
[132]. NEM-mediated inhibition of S. cerevisiae DGAT2 is specic
to cysteine 314, which is in close proximity to a C-terminal conserved motif in DGAT2 that is important for its catalytic activity
[132] (discussed below).

4.4. Experimental approaches for detecting DGAT activity


As described above, the standard method to characterize the
biochemical properties of DGAT involves in vitro assays with
radio-labeled substrates including DG and acyl-CoA. DGAT activity
has also been measured in situ using permeabilized mammalian
cells to investigate the contribution of either DGAT1 or DGAT2 at
different sides of the ER membrane [122,133]. Recently, new
approaches have been developed for DGAT assays which avoid
radiolabeled substrates, such as the in vivo assay as detailed in tung
DGAT study above. Another example is the assay using fatty acylCoA labeled with nitrobenzoxadiazole uorescent probe N,N0 -dimethyl-N(acetyl)-N0 -(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene
diamine (NBD) to quantify activity in vitro [134]. In another study,
a thiol-reactive probe, 7-diethylamino-3-(4-maleimidophenyl)-4methylcoumarin, was used to quantify the formation of free CoASH
released from acyl-CoA in the in vitro assay [127]. This novel assay
has been successfully applied for both routine characterizations as
well as high throughput screening (HTS) assays.
Usually, in in vitro assays with plant and yeast microsomes, DG
is added in several molar excess compared to acyl-CoA. Despite of
these conditions, DGATs seem to prefer DG that is already present
in the membrane rather than exogenous DG [57]. This poses a limitation for in vitro DGAT assays, especially for experiments dealing
with selectivity for DG substrates. In some cases, it is possible to
remove endogenous DG from membranes by treating freeze-dried
microsomes with organic solvent followed by introduction of exogenous DG in the presence of phospholipid, thereby making DGAT
dependent on exogenous DG [17].
HTS LC/MS/-based assays have been also developed by several
groups to quantify DGAT activity [135,136]. Using either stable isotope-labeled FAs or an orthogonally pooled compound mixture of
DGAT inhibitor candidates, this HTS assay has been demonstrated
to be a valuable tool, for not only evaluating DGAT activities both
in vitro and in vivo, but for large-scale primary screening of DGAT1
inhibitors. Other biochemical and cell-based assays compatible with
HTS formats have been reviewed by Siloto and Weselake [137]. These
include scintillation proximity assays, in vivo activity-based selection and uorescence-based estimation methods [138140].

5. Topological characterization of DGAT1 and DGAT2


Understanding the topological pattern of DGAT1 and DGAT2 in
the membrane bilayer is essential for elucidating the catalytic
mechanism of these membrane-bound proteins. Generally, to
determine the topological organization of a membrane protein,
the hydrophobicity of the protein is rst predicted using a variety
of prediction programs based on the hydropathy plot method of
Kyte and Doolittle [141]. DGAT1s contain many hydrophobic segments which are generally believed to represent TMDs (Fig. 4
and Table 4). This type of analysis clearly indicates that DGAT1
and DGAT2 have different topologies which may relate to different
physiological roles for these enzymes forms in TG biosynthesis
[54]. Although orthologs of DGAT1 or DGAT2 have similar proles
of predicted TMDs, as suggested in Fig. 4, small divergences can be
observed by comparing the hydropathy proles, particularly for
DGAT2 [142]. For example, tung DGAT2 contains a higher number
of hydrophobic segments compared to murine and S. cerevisiae
DGAT2. Moreover, a 38 residue segment found exclusively in S.
cerevisiae DGAT2 resides in a hydrophilic region and is positioned
between the conserved motifs YFP and HPHG [143].

5.1. Membrane topology of DGAT1


DGAT1 polypeptides are predicted to have between 8 and 10
hydrophobic regions which are believed to form TMDs (Fig. 5).
To better compare the distribution of TMDs among different
DGAT sequences, the TMD organization of 55 DGAT1 polypeptides
from many different species was predicted and aligned by Geneious Pro 5.3.4 in which the Hidden Markov Model (HMM) technique was employed [144]. The resulting alignment indicated
that the positions of the hydrophobic stretches in most DGAT1
polypeptides were preserved [24]. The rst four and last three
TMDs are generally spaced by short polar stretches. Between
these groups are two or three hydrophobic regions separated by
a longer hydrophilic segment (Fig. 5). The membrane topology
has been experimentally determined for murine DGAT1 (overexpressed in HEK293T cells) using protease protection assays and
indirect immunouorescence in conjunction with selective permeabilization of cellular membranes [145]. Instead of the predicted eight TMDs, the authors detected only three regions of
the protein crossing the membrane bilayer. The hydrophilic Nterminus is oriented toward the cytosol and the rst TMD brings
the polypeptide to the lumen. The majority of the remaining
polypeptide does not face the cytosol, except for a small loop containing residue 240 that is anked by two TMDs, which in fact
were not detected by the prediction algorithms. Therefore, except

Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

361

Fig. 5. Alignment of transmembrane domains (TMDs) in DGAT1. The putative TMDs of DGAT1 polypeptides from 12 animal and 18 plant organisms were predicted and the
polypeptides were aligned. The identity of the alignment is graphed on the top using a window size of 6. The thick lines represent the sequence of each DGAT1 and the thin
lines represent the gaps generated by the alignment. The arrows denote the predicted TMDs. Note the higher level of positional conservation of the rst 4 and last 3 TMDs. The
gure was generated with Geneious Pro 5.3.4 (Biomatters) and optimized manually to t the publication format while preserving the relative positions of the TMDs and
conserved blocks. The TMDs were predicted using the Hidden Markov model (TMHMM).The organisms and GenBank accession numbers are described in the legend of Fig. 2
with the following additions: Aedes aegypti, Aa,AaDGAT1, XP_001658299; Drosophila melanogaster, Dm, DmDGAT1, AAL78365; Danio rerio, Dr, DrDGAT1, NP_956024;
Euonymus alatus, Ea, EaDGAT1, AAV31083; Glycine max, Gm, GmDGAT1, AAS78662; Jatropha curcas, Jc, JcDGAT1, ABB84383; Monodelphis domestica, Md, MdDGAT1,
XP_001371565; Medicago truncatula, Mt, MtDGAT1, ABN09107; Nicotiana tabacum, Nt, NtDGAT1, AAF19345; Nematostella vectensis, Nv, NvDGAT1, XP_001639351; Olea
europaea, Oe, OeDGAT1, AAS01606; Perilla frutescens, Pf, PfDGAT1, AAG23696; Physcomitrella patens, Pp, PpDGAT1, XP_001770929; Populus trichocarpa, Pt, PtDGAT1,
XP_002330510; Rattus norvegicus, Rn, RnDGAT1, BAC43739; Sus scrofa, Ss, SsDGAT1, NP_999216; Trichoplax adhaerens, Ta, TaDGAT1, XP_002112025; Toxoplasma gondii, Tg,
TgDGAT1, AAP94209; Tropaeolum majus, Tm, TmDGAT1, AAM03340; Vernonia galamensis, Vg, VgDGAT1, ABV21945; Vitis vinifera, Vv, VvDGAT1, CAN80418; Zea mays, Zm,
ZmDGAT1, ABV91586. Adpated from Siloto et al. [24].

for the N-terminus, most of the polypeptide constituting murine


DGAT1 is located in the ER lumen. The MBOAT conserved histidine, which likely contributes to the catalytic center, is oriented
towards the lumen, as well.
The topology of murine DGAT1 differs from the model proposed
for tung tree DGAT1. In the latter case, both termini were detected
facing the cytosol, as veried by immunohistochemical analysis of
tobacco BY-2 cells expressing the recombinant DGAT1 [85].
Although a detailed topology experiment was not conducted for
tung tree DGAT1, the presence of both termini on the same side
of the ER membrane suggests an even number of TMDs [85] (Table
4). It has been speculated that the different topological organization of mammalian versus plant DGAT1 may be due to a lower level
of sequence homology in certain regions [145]. Indeed, divergences
in the membrane topology between enzyme orthologs were also
observed for serine palmitoyltransferases [146] and DGAT2 (discussed below).

Other recent work indicated that DGAT1 could have two different topologies as a result of modulation of different physiological
states [133]. Taking advantage of the differential sensitivity of
DGAT1 to specic inhibitors, Wurie et al. [133] demonstrated that
approximately equal DGAT1 activities were detected in situ in both
cytosolic (overt) and luminal (latent) sides of the ER membrane
[133]. Also, loss of both cytosolic and luminal DGAT activities were
observed in microsomal vesicles prepared from liver of DGAT1
knockout mice, indicating that DGAT1 could have a dual topology.
Wurie et al. [133] proposed that the variance between experimental evidence from in situ and in vitro data could possibly result from
the inherent limitation of the methodology used for characterizing
topology in vitro. The strategy used by McFie et al. [145] was to
probe the accessibility of a protease to heterologously overexpressed DGAT1 containing FLAG and Myc epitope tags. Although this
approach could affect the proper insertion of the protein into
membrane, it is a generally accepted and widely used method for

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Fig. 6. Alignment of transmembrane domains (TMDs) in DGAT2 and MGAT. The putative TMDs of DGAT2 polypeptides from 13 fungus, 6 animal and 11 plant organisms as
well as 5 MGATs from animals, were predicted as described in Fig. 5 and the polypeptides were aligned. The identity of the alignment is graphed on the top using a window
size of 9. The arrows denote the predicted TMDs. Note that the rst two transmembrane domains are conserved in very similar positions based on the alignment. The thick
lines represent the sequence of each DGAT1 and the thin lines represent the gaps generated by the alignment. The gure was generated as described for Fig. 5. The organisms
and GenBank accession numbers are described in the legend of Fig. 2 with the following additions: Ajellomyces capsulatus, Ac, AcDGAT2, XP_001540241; Aspergillus oryzae, Ao,
AoDGAT2, XP_001822244; Branchiostoma oridae, Bf, BfDGAT2, XP_002208225; Bos taurus, Bt, BtDGAT2, CAD58968; Coccidioides immitis, Ci, CiDGAT2, XP_001240299;
Chlamydomonas reinhardtii, Cr, CrDGAT2, XP_001693189p; Dictyostelium discoideum, Dd, DdDGAT2, XP_635762; Laccaria bicolor, Lb, LbDGAT2, EDR14458; Medicago
truncatula, Mt, MtDGAT2, ACJ84867; Neosartorya scheri, Nf, NfDGAT2, XP_001261291; Oryza sativa, Os, OsDGAT2, NP_001057530; Ostreococcus tauri, Ot, OtDGAT2,
CAL58088; Penicillium marneffei, Pm, PmDGAT2, XP_002146410; Physcomitrella patens, Pp, PpDGAT2, XP_001777726; Populus trichocarpa, Pt, PtDGAT2, XP_002317635;
Talaromyces stipitatus, Ts, TsDGAT2, EED21737; Ustilago maydis, Um, UmDGAT2, XP_760084; Vitis vinifera, Vv, VvDGAT2, CAO68497; Zea mays, Zm, ZmDGAT2, ACG38122.
Adapted from Siloto et al. [24].

topological studies. On the other hand, topology models based on


activity assays may need to be evaluated with caution considering
the controversy related to changes of cytosolic and luminal DGAT
activities observed in reverse genetics studies. As mentioned earlier, loss of both cytosolic and luminal DGAT activities were observed in microsomal vesicles prepared from livers of DGAT1
knockout mice, supporting the fact that DGAT1 could have a dual

topology [133]. Increases in liver TG content, however, were observed with over-expression of DGAT1 or DGAT2, without noticeable difference in very-low-density lipoprotein (VLDL) TG levels
[147]. Luminal DGAT activities are widely considered to catalyze
the synthesis of TG for VLDL formation since the cytosolic TG droplet cannot be incorporated en bloc into VLDL [54,148,149].The phenomenon of dual topology in membrane proteins is not new [150]

Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

363

Fig. 7. Proposed topology model of murine and yeast DGAT2 (Dga1p) [142,143]. These two models showed that MmDGAT2 has two TMDs whereas ScDGAT2 contains four
TMDs. Both termini of two DGAT orientated toward the cytosol. Important functional motifs are represented by lled blue hexagons. Black-lled residues in ScDGAT2
represent regions where topology was established based on in silico analysis only. Topology of other regions was experimentally determined.

and, although it is tempting to speculate that this process might be


regulated by post-translational modications or alterations in protein multimerization, it is important to obtain direct evidence for
dual topology of DGAT1 in hepatocytes and other cell types.
5.2. Membrane topology of DGAT2
Hydropathy analyses indicated that DGAT2 polypeptides have
fewer hydrophobic regions than DGAT1. A hydrophobic region that
might represent one or two adjacent hydrophobic domains is conserved in the N-terminus (Fig. 6). This hydrophobic segment is
essential for the enzyme activity of S. cerevisiae DGAT2 [142] but
dispensable for murine DGAT2 [63]. The hydrophilic N-terminus
preceding the rst hydrophobic segment has quite variable length,
being much larger in polypeptides from fungi than those from animals and plants [24]. Although removal of N-terminus substantially decreases the activity of S. cerevisiae DGAT2, this region
seems to be dispensable for the enzyme activity [142]. Deletion
of the N-terminus of murine DGAT2 has no effect on DGAT activity
[151].
The membrane topology of DGAT2 was experimentally determined for murine DGAT2 [143]. In this protein, two adjacent TMDs
were located near the N-terminus with both N- and C-termini
residing in the cytosol (Fig. 7). Since the short loop connecting
these two TMDs is relatively hydrophobic, it is also possible this
extended hydrophobic stretch is embedded within the membrane
bilayer (as an alternative model). Under either condition, the bulk
of the C-terminal region is exposed to the cytosol and contains the
conserved motif HPHG (residues 161164 of murine DGAT2)
which appears to have an important role for the enzyme activity
as discussed in the next section. This model for DGAT2 topology
is consistent with the ndings observed for tung tree DGAT2,
where both termini are located in the cytosol [85]. Previously, a
model has been suggested in which cytosolic DGAT activities contribute to biosynthesis of TG destined for deposition into lipid

droplets in the cytosol [54]. The proposed topology model for murine DGAT2 indicates that DGAT2 could possibly be involved in the
synthesis of cytosolic TG deposition. A recent study using stableisotope labeled substrates combined with type-specic specic
DGAT inhibitors indicated that in mice, DGAT2 preferably incorporates endogenous FAs into exogenously added glycerol [152].
DGAT1, however, displayed a preference for incorporating exogenously added FAs to endogenous DG. It is then possible, that in
hepatocytes, DGAT1 is responsible mainly for the esterication of
dietary FAs while DGAT2 acts predominantly on the esterication
of de novo generated DG through the Kennedy pathway destined
for cytosolic lipid droplets. Despite these and other lines of evidence, it is still uncertain whether the topology of DGAT1 or
DGAT2 really inuences the destination of TG (cytosol versus
lumen).
Determination of the membrane topology for S. cerevisiae
DGAT2 [142] pointed to four TMDs. As represented in Fig. 7, the
conserved functional motif YFP, and the hydrophilic segment
exclusive to yeast DGAT2, reside in a long ER luminal loop following the rst TMD. The motif HPHG is embedded in the membrane.
These results indicate some similarities to the topology model of
murine DGAT2, but also reveal striking differences. Hydropathy
analysis of DGAT2 showed divergences between animal, plant
and fungal proteins (Fig. 4, Table 4). Because considerable differences have been detected in the topology of animal versus yeast
DGAT2, it would be valuable to explore in greater detail the topological organization of plant DGAT2.
Topological characterization of S. cerevisiae DGAT2 also indicated that active and/or binding sites of each DGAT are possibly located on the luminal side (see highlighted motif in Fig. 7), which
provides evidence for an acyl-CoA-dependent TG pathway in the
lumen. This hypothesis is supported by previous work in which
luminal DG and acyl-CoA was proposed to be generated by microsomal lipase and a microsomal carnitine acyltransferase, respectively [153,154]. The existence of this mechanism has been

364

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Fig. 8. Kyte-Doolittle hydropathy plots of MGATs. Hydropathy plots from representative polypeptide sequences of MGATs were generated by the method of Kyte and
Doolittle [141] using a window size of 19. Polypeptides include MGATs from humans MGAT1 (A), MGAT2 (B) and MGAT3 (C) and murine MGAT1 (D) and MGAT2 (E). The most
hydrophobic segments which may contain putative TMDs are indicated by roman numerals.

demonstrated in vivo in rat small intestine and with a cell-free


expression system.
Progress in understanding the topology of MGATs, however,
has lagged behind in comparison to DGAT. In silico analyses
(Figs. 6 and 8 and Table 5) showed that all MGAT family members
have similar hydropathy proles with at least one TMD, which is
similar to DGAT2. Prediction results also indicated that N-termini
of MGATs are oriented toward the cytosol. Considering the high
sequence homology between MGATs and DGAT2s, it is reasonable
to suggest that they might have similar topologies. But considering the differences already observed for DGAT2, it would not be

surprising if MGATs assume yet different conformations in the


membrane.
6. Functional motifs in DGAT
DGAT1 and DGAT2 are dissimilar proteins, not sharing detectable sequence homology, although catalyzing the same biochemical reaction. Sequence analysis of the corresponding amino acid
sequences of DGAT from a wide range of organisms clearly segregated DGAT1 and DGAT2 protein families [85,119]. An alignment
of DGAT1 and DGAT2 polypeptides from 70 organisms indicated

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Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377


Table 5
Predicted TMDs in MGATs. The numbers between brackets denote the bibliographical reference for each algorithm.
MGATs

HsMGAT1
HsMGAT2
HsMGAT3
MmMGAT1
MmMGAT2

Program
HMMTOP [244]

TMHMM [245]

SVMtm [246]

SOSUI [247]

TOPCONS [248]

TMpred [249]

TMDs
Orientation*
TMDs
Orientation
TMDs
Orientation

4
IN
1
IN
1
IN

1
IN
1
IN
1
IN

2
N/A
2
N/A
3
N/A

2
N/A
1
N/A
1
N/A

2
IN
2
IN
2
IN

4
IN
3
IN
5
OUT

TMDs
Orientation
TMDs
Orientation

4
IN
3
IN

1
IN
1
IN

2
N/A
4
N/A

2
N/A
1
N/A

2
IN
2
IN

3
IN
3
IN

Orientation of N-terminus. Cytosol-IN.

that one proline and one phenylalanine could be aligned between


members of DGAT1 and DGAT2 [155]. Indeed these and other residues can be aligned by optimizing the parameters such as the gap
opening and gap extension penalties in ClustalW. Hypotheses arising from these comparisons, however, must be interpreted with
caution and veried experimentally. Thus, conservation of functional motifs in DGAT enzymes can be obtained mainly through
alignments of individual DGAT families. In the absence of threedimensional structures of DGAT, these alignments would be helpful for formulating hypotheses regarding the catalytic mechanism
in this class of enzymes. In this section we examine functional motifs and residues that have been described for DGAT1 and DGAT2.
6.1. Conserved and functional motifs in DGAT1
DGAT1 belongs to a large protein family of membrane-bound Oacyltransferases (MBOAT) [156] whose members catalyze the
transfer of FAs onto hydroxy or thiol groups of lipids or proteins.
Other proteins such as ACAT1, ACAT2, LPAT and a protein-cysteine
N-palmitoyltransferase (skinny hedgehog, or sightless protein) are
also classied into this family [157,158]. The MBOAT family is
characterized by a long hydrophobic stretch in which a conserved
asparagine and histidine residues are proposed to be involved in
the active site of the enzyme (indicated in Fig. 10). Site-directed
mutagenesis studies indicated that the corresponding histidine
residues in human ACAT1, ACAT2 and murine DGAT1 are essential
for the activity of these enzymes [145,159,160]. It is possible that
motifs in DGAT1 that resemble catalytic sites from other acyltransferases might be remnants of ancient enzyme activities that eventually evolved to become DGAT1. To test this hypothesis, it would
be valuable to conduct mutagenesis in members of the DGAT1
family that possess multiple enzyme activities.
Alignments of DGAT1 polypeptides from many different organisms indicate about 7% identity [24,155]. The C-terminal portion of
DGAT1 is the most conserved region and is likely to contain the
catalytic pocket of the enzyme. The most variable region of DGAT1
is the hydrophilic N terminus that is composed of approximately
115 and 80 residues in plants and animals, respectively. The hydrophilic N-terminus is quite unique for each DGAT1 and might serve
distinct functions in different organisms. For instance, in plants
this region is encoded exclusively by the rst exon (Fig. 9), which
suggests that this segregated region of the gene evolved more rapidly than the exons encoding the rest of the protein. The only common feature of the N-termini of DGAT1 is a cluster of arginine
residues (Fig. 9). The role of these conserved arginines has not been
studied yet, but it is possible that they might assist in localizing
DGAT1 to the ER. Arginine-containing motifs are involved in the
subcellular localization of some membrane proteins [161]. For
example, in animals the thioredoxin-like transmembrane protein
TMX4 of the disulde isomerase family requires the di-arginine

motif RXR for ER localization [162]. The di-arginine motif in the


N-terminus of phospholamban promotes the retrieval of proteins
to the ER through COPI-mediated transport [163]. Also, in plants,
three congurations of arginine motifs (RR, RXR or RXXR) have
been shown to confer ER localization [164]. Some evidence from
these studies indicates that the mechanism of di-arginine signals
involve not only COPI-mediated retrieval, but also protein retention in the ER [161,164]. Although no experimental data veried
these hypotheses discussed above, the N-terminus of murine
DGAT1 appears to be involved in homotetramer formation as demonstrated by crosslinking assays [145].
Besides the MBOAT motif described above, Cao [155] identied
seven other conserved motifs in DGAT1 from more than fty
organisms. These motifs, represented in Fig. 10, are likely to contain the necessary residues important for DGAT activity. When
comparing the primary structure of these polypeptides, it is also
important to consider regions that are conserved in organisms
from different kingdoms. For example, the motifs PAHRXXXESPLSSDAIFXQ and SLFSXXSGFXN, preceding the rst hydrophobic segment, are conserved only in plants and animals,
respectively. Similarly, a serine residue corresponding to position
131 of A. thaliana DGAT1 is conserved only in plants. In addition,
the motif WVXRQ in plants corresponds to FL(L/I)(R/K)R in animals.
A long hydrophilic region located between the fourth and fth
hydrophobic domains, corresponding to positions 260278 of A.
thaliana DGAT1, shows signicant variability. In contrast, the conserved sequences PTLCYQXSYPR in plants and PTLCYEXXFPR in
animals, precede the fth hydrophobic region.
Different motifs have been thought to comprise the active site
of acyltransferases. A conserved histidine and an aspartic acid residue (HXXXXD) were found to be essential for the acyltransferase
activity of the bifunctional enzyme 2-acyl-glycerophosphoethanolamine acyltransferase/acylacyl carrier protein synthase (Aas, EC
2.3.1.40 and 6.2.1.20, respectively) [165]. The aspartic acid and histidine supposedly work as a charge relay mechanism to increase
the nucleophilicity of the hydroxy group from sn-1 glycerol-3phosphate facilitating the attack on the thioester bond of acylCoA. A similar mechanism has been proposed for DGAT enzymes
from prokaryotes, which have a similar motif (HHXXXDG) [99].
The motifs HXXXD and HXXXXD can be found adjacent to the
fourth and fth hydrophobic segment in DGAT1 from plants,
respectively (Fig. 10). These motifs are not present in DGAT1 from
animals and are not part of the active site of these enzymes.
A consensus sequence [N(S/A/G)R(L/V)(I/F/A)(I/L)EN(L/V)] was
identied by Jako et al. [166] in A. thaliana DGAT1, who proposed
that the invariant arginine and glutamic acid residues serve an
analogous function to the histidine and aspartic acid residues conserved in other plant DGAT1 polypeptides, respectively. It is noted
that this region is highly conserved in DGAT1 in the border of a
hydrophobic segment (Fig. 10).

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Fig. 9. Alignment of the hydrophilic N-terminus polypeptide sequence of DGAT1 from plants and animals. The positions corresponding to the cluster of arginines and the end
of the rst exon in plants are indicated. Accession numbers and abbreviations for the DGAT polypeptides can be found in the legend of Figs. 2 and 5.

Several other motifs in DGAT1 have been attributed to be


associated with specic functions, including a leucine zipper and
several phosphorylation sites. The leucine zipper was described

in DGAT1 proteins from several species and corresponds to ve


leucine residues consecutively separated by six amino acids
(L222, L229, L236, L243 and L250 of A. thaliana DGAT1)

Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

367

Fig. 10. Alignment of some conserved sites in DGAT1 polypeptides. The bar and horizontal arrows represent the A. thaliana DGAT1 polypeptide and the predicted TMDs,
respectively. The position of the MBOAT motif is indicated above. Motifs 17 correspond to the conserved regions described by Cao [155] as follows: Motif1 (GL block), Motif2
(KSR block), Motif3 (PTR block), Motif4 (QP block), Motif5 (LWLFFEFDRFYWWNWWNPPFSHP block), Motif6 (LQL block) and Motif7 (NGQPY block). The name of each block
corresponds to the order of conserved amino acids in each block rather than a conserved sequence. The vertical boxes contain the amino acid sequences for different DGAT
indicated on the left. The vertical arrows on these boxes indicate the position of relevant amino acids discussed on the text. From left to right the conserved motifs/residues
are RXXXE, HXXXD, HXXXXD, N and H conserved in the MOBAT family. Accession numbers and abbreviations for the DGAT polypeptides can be found in the legend of Figs. 2
and 5.

[115,117,167,168]. This leucine zipper is present in DGAT1 from


plants, but is not present in animals. It might mediate interactions
of DGAT1 subunit with other proteins or facilitate the binding of
modulatory proteins. Experimental evidences, however, are still
needed to unambiguously assign these functions.
Several studies also indicated the presence of multiple potential
phosphorylation sites in DGAT1 [115,117,168]. Experimental evidence from studies using rat hepatocytes and adipose tissue, suggested that DGAT activity can be regulated post-transcriptionally
by modulating phosphorylation state of the enzyme [54]. This is
probably not only because phosphorylation is a typical cellular
mechanism to control enzyme activity, but also because DGAT
utilizes DG, which has an important role in signalling cascades.
For instance, phosphorylation of residues situated close to the C1
domain of DG kinase (EC 2.7.1.107) regulates the afnity of this enzyme for DG [169]. Site-directed mutagenesis of a serine residue in
position 168 of castor bean DGAT1, which corresponds to a protein
kinase C site, resulted in a signicant decrease in enzyme activity
(Siloto et al., unpublished data). In addition, substitution of the
conserved serine 197 for an alanine in T. majus DGAT1 resulted

in increased enzyme activity [118]. This serine residue corresponds


to a putative SNF1-related kinase1 which is an important mediator
in sugar and hormone-signalling in plants [170]. Although these
lines of evidence suggest that phosphorylation could have a regulatory role for plant DGAT1 activity, direct experimental evidence
is still needed to prove this hypothesis. Deletion of the N-terminus
of murine DGAT1 removes 23 predicted protein kinase A sites
resulting in increased DGAT activity [145].
Potential substrate binding sites include regions where acylCoA and DG supposedly interact were also identied in several
DGAT1 polypeptides [73,118,168], for example, the putative diacylglycerol/phorbol ester binding motif HXXXXRHXXXP. This motif
present in some protein kinase C forms is apparently absent in
ACATs which utilize sterol as the acceptor for the acyl moiety. An
amino acid substitution close to this motif in T. majus DGAT1 (position 439) resulted in loss of enzyme activity [118], but it is not
clear whether it interfered with DG binding.
Acyl-CoA has been shown to associated with the hydrophilic Nterminus based on direct physical interaction with a recombinant
peptide [171173]. Since the last 20 amino residues preceding the

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rst hydrophobic segment compose the most conserved region of


the N-terminus, it is reasonable to assume that acyl-CoA interacts
with this segment. It has been demonstrated, however, that the
N-terminus is not only dispensable for the enzyme activity, but
its removal might actually increase the total activity [140,145].
Topology studies have shown that the N-terminus of mouse DGAT1
faces the cytosol whereas the active site is in the lumen. Therefore,
it is possible the association of acyl-CoA with the DGAT1 N-terminus represents an allosteric interaction instead of substrate binding. Enzymes that are regulated allosterically usually form
multimeric complexes and the N-terminus of DGAT1 assists the formation of multimers as demonstrated in DGAT1 from animals and
plants [172,174]. The N-terminus of murine DGAT1, particularly
residues 3784, is responsible for formation of tetramers but not dimers [145], indicating that other regions in DGAT1 are responsible
for dimerization. Since removal of the N-terminal region resulted
on increased activity of murine DGAT1, it was proposed that this
DGAT1 might be regulated by switching between an active dimeric
form and a tetrameric inactive or less active form [145].
Another proposed acyl-CoA binding site resides in the C-terminal portion corresponding to the motif FYXDWWN in ACATs [54].
Substitution of the tyrosine to alanine in this motif from yeast
ACAT1 resulted in decreased afnity for acyl-CoA [175]. In DGAT1,

the same motif is largely conserved and precedes the third last
hydrophobic segment. The variant FYQQWWN is found in DGAT1
from Y. lipolytica. In this motif, substitution of the conserved tyrosine in DGAT1 from T. majus (Y392A) resulted in decreased enzyme
activity while a double mutation in tyrosine and tryptophan
(Y392G/W395G) completely abolished enzyme activity [118].
Polymorphisms analysis in DGAT1 in natural populations has
also provided valuable information on the structure/function relationships of these enzymes. It is also helpful for conrming the role
of DGAT1 for TG biosynthesis in vivo in the original organisms. This
is the case of a non-conservative substitution of lysine to alanine in
DGAT1 from cattle, which is a quantitative trait locus (QTL) for
milk fat [176]. This lysine residue is conserved in most animals,
but not in plants. Another DGAT1 QTL was demonstrated for corn,
where the presence of a phenylalanine residue in position 469 of
corn DGAT1 increased the enzyme activity and overall seed oil content in corn [177]. This phenylalanine residue is conserved in most
DGAT1s from plants.
6.2. Conserved and functional motifs in DGAT2
DGAT2 polypeptides have fewer hydrophobic regions than
DGAT1. A hydrophobic region that might represent one or two

Fig. 11. Alignment of some conserved sites in DGAT2 polypeptides. The bar and horizontal arrows represent the U. ramanniana DGAT2a polypeptide and the predicted
transmembrane domains, respectively. Motifs 1 to 7 correspond to the conserved regions described by Cao [155] as follows: Motif1 (PH block), Motif2 (PR block), Motif3 (GGE
block), Motif4 (RGFA block), Motif5 (VPFG block), Motif6 (G block). The name of each block corresponds to the order of conserved amino acids in each block rather than a
conserved sequence. The vertical boxes contain the amino acid sequences for different DGAT indicated on the left. From left to right the conserved motifs/residues are YFP, (H/
E)PH(G/S), GGXXE, and RXGFX(K/R)XAXXXGXX(L/V)VPXXXFG(E/Q). Accession numbers and abbreviations for the DGAT polypeptides can be found in the legend of Figs. 2 and
6.

Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

adjacent hydrophobic domains is conserved in the N-terminal portion (Fig. 6). The hydrophilic N-terminus preceding the rst hydrophobic segment is variable in length, and is much larger in
polypeptides from fungi than those from animals and plants [24].
A comparison of 54 DGAT2 polypeptides indicated that these proteins are composed of an average of 344 amino acid residues [155].
DGAT2 polypeptide sequences across species were more diverse
than those for DGAT1. Cao [155] reported 4.7% of identity of protein sequences while we detected only 2.3% [24]. From an experimental perspective as discussed earlier, the higher divergence in
DGAT2 genes represents a hurdle for the identication of the ensuing cDNAs from organisms whose genomes that have not yet been
sequenced.
As demonstrated in Fig. 10, seven regions with remarkable sequence conservation have been located by Cao [155]. The most
conserved region in DGAT2 corresponds to the motif RXGFX(K/
R)XAXXXGXX(L/V)VPXXXFG(E/Q) located approximately 150 residues after the rst hydrophobic segment (Fig. 11). As described
in previous section, a cysteine 314 resides in close proximity to this
motif in S. cerevisiae DGAT2. Although replacement of this cysteine
to alanine did not affect enzyme activity, thiol-specic modication on this cysteine substantially inhibited ScDGAT2 activity.
These data indicate that attachment of thiol-specic group to cysteine 314 may interfere with the interaction of key amino acids in
this molecular signature, which eventually resulted in the loss of
enzyme activity [132]. Therefore, the motif mentioned above could
contribute to an important site for DGAT catalysis. Other conserved
regions include the motif GGXXE (motif 3 in Fig. 11) and a phenylalanine, arginine and proline in positions 164, 170 and 293, respectively, of U. ramanniana DGAT2a. In addition, the motif HPHG
(EPHS/G in plants) is conserved in sequences from animals and
fungi (Fig. 11). Preceding this motif, the most evident structural
divergence in DGAT2 is observed; a hydrophilic segment of
approximately 38 residues present in sequences from some fungi,
but absent in plants and animals. Though not essential for the enzyme activity, this region inuences the activity of S. cerevisiae
DGAT2 (positions 150187) [142], and might represent a specialized domain in this enzyme. These conserved motifs discussed
above have also been identied by Cao [155] using a large number
of DGAT2 polypeptides (Fig. 11).
The importance of several conserved regions in DGAT2 has been
determined. Stone et al. [143] demonstrated that the motif HPHG
conserved in both animal and fungi DGAT2 (Fig. 11) could be part
of the active site as it was essential for the activity of murine
DGAT2. Site-directed mutagenesis indicated that the second histidine residue in this motif appeared to play a predominant role in
enzyme function. Similarly, in S. cerevisiae, replacing the HPHG sequence by EPHS, conserved in plant orthologs, was detrimental to
enzymatic activity [142]. In addition, substitution of the second
histidine to alanine abolished enzyme activity. These results suggest that HPHG, and more specically the second histidine residue,
are essential for enzyme catalysis. Also, this motif could serve as a
candidate to explore possible important structural and functional
divergence between DGAT2 from animals, fungi and plants. Substitutions on another conserved motif YFP, located close to the TMDs
(Fig. 11), resulted in signicant decreases in activity in S. cerevisiae
DGAT2 [142].
The motif FLXLXXXn (n = non-polar residue) was proposed to
represent a neutral lipid binding domain of murine DGAT2 [143].
This motif was identied in several enzymes that bind to or catalytically act on neutral lipids, including TG hydrolase, hormonesensitive lipase, cholesterol ester transfer protein, and cholesterol
esterase [143,178,178]. Amino acid substitutions on the phenylalanine and rst leucine in this motif decreased DGAT activity, while
substitution of the second leucine abolished activity. This motif, located in the rst TMD of murine DGAT2, is present in most of

369

DGAT2 from vertebrates, but is only weakly conserved in plants


and fungal orthologs. Substitution of the corresponding phenylalanine and rst leucine in S. cerevisiae DGAT2 resulted in 50% of the
wild-type activity [142]. This same motif contains the putative
membrane lipid attachment segment LGVAC found in prokaryotes.
Substitution of the cysteine in this segment to a serine in murine
DGAT2 did not reduce DGAT activity, indicating that it is not essential for catalysis. In fact, substitution of all cysteine residues in S.
cerevisiae DGAT2 to alanine residues did not disrupt DGAT activity
as mentioned earlier [132]. Whether this site is involved in substrate binding, or not, will need to be determined.
7. Subcellular localization of DGAT
As described previously, the ER is regarded as the main site of
TG synthesis [57]. It is assumed that DGAT enzymes, like most
polytopic transmembrane proteins, are cotranslationally inserted
in the ER through a signal recognition particle (SRP)-dependent
mechanism. The SRP is a ribonucleoprotein complex that binds to
a hydrophobic domain in the nascent polypeptide emerging from
the ribosome, arresting the protein synthesis. The SRP binding domain in the nascent polypeptide is composed of a hydrophobic domain sequence that may be represent a transmembrane segment.
The translation resumes when the complex interacts with the
SRP translocon receptor in the ER membrane bilayer, allowing
the protein to be integrated into the lipid bilayer membrane
[179,180]. Considering that DGAT1 and DGAT2 have their N-terminus oriented towards the cytosol, the rst TMD of these enzymes
might serve as a type II signal anchor sequence. Indeed, a deletion
mutant of murine DGAT2 which lacks the entire N-terminus was
retained in the ER [151]. Similar studies on the translocation mechanism of DGAT1 have not been conducted so far. Instead, the specic subcellular localization patterns of DGAT1 and DGAT2 have
been studied in various organisms, which helped to elucidate the
role of DGAT in the spatial organization of TG synthesis.
7.1. DGAT localization in plants
Localization of plant DGAT enzymes has been studied for tung
tree DGAT1 and DGAT2. It was demonstrated that these enzymes
localize to the ER by a C-terminal ER retrieval motif /-X-X-K/R/
D/E-/, where / is any large hydrophobic amino acid residue [85].
The ER-retrieval sequence in tung tree DGAT1 is YYHDL, while
for DGAT2 the motif is LKLEI (Fig. 12). The motif YXHD is conserved
in the C-termini of DGAT1 from many organisms, but its role as an
ER-retrieval motif in animals was not observed [63]. Murine
DGAT2 mutant deleted with this region can still localize to the ER.
Epitope-tagged versions of tung tree DGAT1 and DGAT2 co-expressed in tobacco BY-2 cells were shown to be localized to different regions of the ER [85]. Taken together with the evidence from
the genetic and biochemical studies discussed previously, it was
proposed that tung tree DGAT1 and DGAT2 may have distinct
physiological roles and may interact with a different subset of proteins in the ER as discrete multiprotein complexes [85].
In plants, DGAT activity was also found in chloroplasts of leaves
[181], which can accumulate TG under conditions of stress or
senescence [182,183]. In addition, Kaup et al. [45] identied a
putative DGAT1 in the chloroplasts of senescing A. thaliana leaves
through immunoblotting. In peanut, DGAT activity was also present in cytosol [44]. Accumulation of TG in chloroplasts has been
also demonstrated in the green algae Chlamydomonas reinhardtii
[46]. In C. reinhardtii several enzymes involved in the TG biosynthetic pathway, including GPAT, LPAT and PDAT, but not DGAT
have been found associated with lipid droplets [184]. It is not yet
understood how these integral membrane proteins, which are nor-

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Fig. 12. Multiple sequence alignment showing conserved residues in the C-termini of DGAT. DGAT1 (A) and DGAT2 (B) polypeptides were aligned using Geneious (Biomatters
Ltd.). Accession numbers and abbreviations for the DGAT polypeptides can be found in the legend of Figs. 2, 5 and 6. The bars on top indicate the positions of the identied ER
retrieval motifs for tung tree DGAT1 (YYHDL) and tung tree DGAT2 (LKLEI).

mally associated with membrane bilayer, would be accommodated


in the phospholipid monolayer of lipid droplets. Also, the luminal
portion of these polypeptides would be in direct contact with the
neutral lipid core of lipid droplets.
7.2. DGAT localization in animals
In animals, DGAT activity can be detected in both in ER and
cytosolic lipid droplets. Fluorescence imaging in mammalian cells
showed that DGAT2, but not DGAT1, was associated with lipid
droplets [151,185]. The ER/lipid droplet dual localization was further investigated by detailed localization studies on murine DGAT1
and DGAT2 expressed in cultured mammalian cells [63,151]. It is
noteworthy that murine DGAT1 localizes predominantly in the
ER, while DGAT2 can be also found associated with lipid droplets
[63]. This is reected by the different morphology of lipid droplets
observed when expressing murine DGAT1 or DGAT2 in McArdle
RH7777 cells [78]. Expression of murine DGAT1 resulted in the production of much smaller lipid droplets compared to DGAT2.
The subcellular localization of murine DGAT2 has been studied
in detail. Under basal cell culture conditions, murine DGAT2 was
found in the ER and was enriched in mitochondria-associated
membranes (MAM). MAM is a distinct region of the ER-enriched
in lipid synthetic enzymes such as phosphatidylserine synthase
[186,187] and is in close proximity to the surface of lipid droplets
[188190]. By stimulating TG synthesis in cells with oleate, murine
DGAT2 was observed at or near the surface of lipid droplets and
also co-localized with mitochondria [151]. The N-terminal region
contains a positively-charged signal that appears to be important
for targeting to mitochondria [151], but it is still unclear why mur-

ine DGAT2 is associated with mitochondria. Since an isoform of


GPAT has also been identied in mitochondria [191], it is tempting
to speculate the existence of a TG biosynthetic pathway in this
organelle.
Subcellular localization using confocal microscopy cannot resolve between DGAT2 in ER membranes surrounding lipid droplets
and DGAT2 present on the lipid droplet surface itself [54,151]. Indeed, proteomic analyses of lipid droplets isolated from plants as
well as animal cells and tissues has not revealed DGATs associated
with this organelle [192198]. Murine DGAT2 mutant lacking its
TMDs was not present in the ER and was instead localized to mitochondria [63]. Surprisingly, it is still capable of promoting TG synthesis and storage in lipid droplets with reduced enzyme activity
[63]. Therefore, TMDs in murine DGAT may not play an essential
role in catalysis. Taken together, these ndings raised an interesting debate regarding the role of ER in lipid droplet formation.
Although a clearly dened lipid droplet-targeting motif has not
been identied yet, the interaction between murine DGAT2 and
the lipid droplet was found to be dependent on its C-terminus. In
the same study, a DGAT2 mutant lacking most of its C-terminus
still remained in the ER as mentioned in previous section, [63],
indicating that murine DGAT2 does not have a C-terminal ER-retrieval signal like tung tree DGAT2.
Similar to ndings in plants, both epitope-tagged DGAT1 and
DGAT2 from mice exist as multimeric protein complexes [63,145].
DGAT1 can form both dimers and tetramers. Tetramerization is
mediated by the N-terminus which is apparently involved in regulating DGAT1 activity [145,172,174]. Individual DGAT2 subunits
are also capable of interacting with each other to form a large protein complex [62,63]. When expressed in bacteria, tung tree DGAT2

Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

formed dimers [62]. The functional signicance of this highly


ordered supramolecular complex has not been determined. Interestingly, murine DGAT2 produced in a human Hela cells co-localized and physically interacted with stearoyl-CoA desaturase 1, an
ER enzyme that catalyzes the desaturation of acyl-CoAs to produce
monounsaturated fatty acids [199]. In mammals, DGAT2 could
potentially channel monounsaturated FAs into TG.
Subcellular localization of MGATs has also been recently investigated in mammalian cells. Immunocytohistochemical analyses
indicated that MGAT1, MGAT2 and MGAT3 localized to the ER
[63,88,200]. It would be valuable to further explore the differences
in primary structure of these enzymes to identify subcellular targeting signals in these polypeptides. Collectively, the studies previously mentioned demonstrate that it is still not clear if mammalian
DGAT1 and DGAT2 have distinct localization patterns in the ER as
found for tung tree DGATs.
7.3. DGAT localization in fungi
In fungi, DGAT activity has also been detected in the ER and lipid
droplets [201]. As previously described, DGAT2 was partially puried from the lipid body fraction of the fungus U. ramanniana [65].
Also, the corresponding DGAT2 has been suggested to localize in
yeast lipid droplets according to subcellular localization datasets
[202,203]. Several deletions within the S. cerevisiae DGAT2 polypeptide sequence were generated [142], but none prevented association of this enzyme with microsomal membranes. It has been
demonstrated that, in S. cerevisiae, DGAT2 transfers from the ER
membrane to the periphery of lipid droplets [204]. This study
showed that DGAT2 moves freely within the surface of lipid droplets and can also relocate back to the ER upon stimulation of lipolysis induced by cerulenin, a FA synthase inhibitor. Nevertheless, the
proposed topology model of S. cerevisiae DGAT2 with four TMDs
[142] cannot account for the bilateral movement of DGAT2 between
the ER and lipid droplets. It would be more easily accepted if the
protein assumed a hairpin structure with two adjacent TMDs as
proposed for murine DGAT2. A possible theory to t these observations is that yeast lipid droplets bud in the luminal side of the ER,
being surrounded by an additional ER membrane bilayer [204].
Conrmation of this hypothesis would require a careful re-evaluation of lipid droplet biogenesis in yeast. Since a DGAT1was only
identied in Y. lipolytica [76], it might be interesting to investigate
the localization of DGAT1 and DGAT2 in this organism, as well.
8. Biotechnological applications of DGAT
As previously discussed, considerable advances have been made
in the molecular and functional characterization of DGAT1 and
DGAT2 which have provided insight into the distinct roles of these
two different enzymes in TG metabolism. In addition, insights into
DGAT properties have further advanced the development of DGAT
applications in both therapeutics and oilseed engineering.
8.1. DGAT as therapeutic targets
Gene knock-out studies in mouse models have laid a foundation
to examine the relationship between DGAT and diseases related to
TG metabolism. Dgat1 knockout mice (Dgat1/), as described earlier, have reduced TG levels in tissues and do not lactate, but are fertile and still capable of TG synthesis. These lean mice were resistant
to diet-induced obesity because of increased energy expenditure
and increased physical activity [9,205]. Also, they have an enhanced
sensitivity to both insulin and leptin [206]. In addition, DGAT1 was
found to be required for hepatitis C virus core induced steatosis.
Therefore, DGAT1 inhibitor would also be possibly efcacious in

371

the treatment of steatosis infected by hepatitis C virus [207]. These


ndings sparked considerable interest in exploring DGAT1 as a
therapeutic target for treating obesity and diabetes in humans.
Numerous DGAT inhibitors have been characterized [208220]. Recently, a small DGAT1 selective inhibitor was reported to decrease
bodyweight and hepatic TG levels in a mouse model of diet-induced
obesity, which reproduced the phenotype of Dgat1/ mice [221].
King et al. [211] demonstrated that this potent and selective inhibitor could signicantly reduce serum TG levels in both genetic and
diet-induced mice models for hypertriglyceridemia. In addition, a
detailed characterization of the DGAT1 inhibitor T863 has been
undertaken [127]. The compound was specic for mammalian
DGAT1 and did not inhibit any other acyltransferases with DGATactivity, including DGAT2, MGAT2 or MGAT3. Treatment of mice
with T863 also recapitulated the phenotype observed in Dgat1/
mice, including altered intestinal fat absorption and lipid accumulation in enterocytes after a corn oil bolus. Obese mice fed a Western diet chronically (2 weeks) treated with T863 lost about 7% of
their weight without changes in food consumption. The weight loss
was accompanied by improvement in serum lipid levels, decreased
liver weight and improved insulin sensitivity. A similar inhibitory
effect on fat absorption was recently reported with another DGAT1
inhibitor [222]. Collectively, these compelling studies suggest that
it is feasible to use DGAT1 inhibition as a novel therapeutic strategy
to treat obesity, hepatic steatosis and hypertriglyceridemia.
In contrast to Dgat1knockout mice, Dgat2 knockout (Dgat2/)
mice were lipopenic and had skin abnormalities, leading to an early
death [78]. Since disruption of Dgat2 is lethal for mice, from a safety
perspective, inhibition of DGAT2 was initially less attractive as a potential therapy to treat TG-related disorders [54]. However, studies
using antisense technology (antisense oligonucleotides, ASO) to decrease Dgat2 expression indicated that this strategy could be promising. Treatment with Dgat2 gene-specic antisense oligonucleotide
(ASO) to knock down Dgat2 expression in mouse liver resulted in a
signicant decrease in hepatic DGAT2 activity in the diet-induced
mouse model of obesity [223]. Moreover, TG levels in both liver
and plasma were substantially reduced without other side effects
such as skin abnormalities [224]. Also, this down-regulation of
Dgat2 expression led to a remarkable decrease in body weight
and adipose tissue content, hepatic TG and insulin resistance induced by a high-fat diet [223,225]. Interestingly, in the rat model
of diet-induced non-alcoholic fatty liver disease, suppression of
Dgat2, but not Dgat1, with ASO treatment successfully improved
hepatic steatosis and insulin sensitivity [225]. Thus, DGAT2 inhibition also has a potential for treating hypertriglyceridemia, hepatic
steatosis and obesity. In addition, DGAT2 expression levels were
signicantly down-regulated in human psoriatic skin [226]. As previously described, DGAT2-decient mice had skin barrier abnormalities. Therefore, DGAT2 may also be a target for treating skinrelated diseases, although substantial work needs to be developed
in this area. Despite these ndings, high-resolution structural and
functional information on DGAT is still indispensable for understanding the mechanism by which inhibitors limit excessive TG
accumulation. Eventually, this would aid in designing novel treatments for human diseases.
Recently, MGAT2-decient mice (Mogat2/) were found to be
protected against high-fat diet-induced obesity, glucose intolerance, hypercholesterolemia and fatty livers [28]. Also, Mogat2/
mice had delayed intestinal fat absorption. Therefore, specic inhibition of MGAT2 could also be a useful strategy for treating metabolic-related diseases caused by excessive fat intake.
8.2. Use of DGAT in oilseed engineering
As crude oil resources decline, vegetable oils produced by oilbearing crops are gaining growing interest as sustainable replace-

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Table 6
Genetic engineering of oilseed plants through the use of DGAT [36]. Reprinted with permission of Elsevier Science Ltd.
Host species

Source species

Up-regulated gene

Phenotype

Ref.

B. napus
A. thaliana, N. tabacum
Glycine max
A. thaliana

B. napus, A. thaliana
A. thaliana
U. ramanniana
R. communis

Increased seed oil content


Increased seed oil content, seed weight
Increased seed oil content
Up to 30% hydroxy FAs

[227,230]
[166,236]
[228]
[124]

A. thaliana, B. napus
Glycine max

T. majus
V. galamensis

DGAT1
DGAT1
DGAT2
FAH12
DGAT2
DGAT1
DGAT1
DGAT2
EPX

Increased seed oil content


Up to 26% epoxy FA, increased seed weight, seed oil and protein content

[118]
[231]

ments for petroleum-derived chemical feedstocks for industrial


applications and for biofuels. Oilseed biotechnology can be applied
not only to increase overall vegetable oil production, but also to
produce tailored FA compositions in TG for specic applications.
DGATs are considered essential targets for metabolic engineering
of oilseeds as summarized in Table 6. The role of DGAT1 in oil synthesis is well established. For example, an EMS-induced mutation
of DGAT1 in A. thaliana (AS11) resulted in reduced seed oil content
and delayed seed maturation [73]. Similar ndings were obtained
from the A. thaliana mutant ABX45 [72]. Overexpression of DGAT1
orthologs from various plant sources including A. thaliana [166], B.
napus [227], and T. majus [118] have been successfully used for
boosting overall oil production in A. thaliana and B. napus. Lardizabal et al. [228] reported that over-expression of DGAT2 from the
fungus U. ramanniana also led to an increase in seed oil content
in soybean (Glycine max). Using eld trials in multiple locations,
Lardizabal et al. [228] demonstrated that an absolute increase in
oil of 1.5% is achievable through overexpression of the fungal
DGAT2. Similar experiments in corn resulted on oil increases up
to 0.9% by weight, by overexpressing a Neurospora crassa DGAT2
[229]. In canola, overexpression of B. napus or A. thaliana DGAT1 resulted in absolute increases in seed oil content of to 1.5% or greater
[227,230]. Studies of metabolic regulation through topdown control analysis indicated that DGAT might be more effective for
increasing seed oil content in canola, since in this crop a higher
metabolic control is exerted during glycerolipid assembly than
during FA synthesis [227]. In this case, overexpression of DGAT1
also reduced the penalty on seed oil content caused by drought
[227]. Although the increases in seed oil content are apparently
minor, it has been estimated that a 1% increase in soybean oil in
the United States translates to approximately $840 million increase for the crop value [228]. In Canada, it has been estimated
that 1% increase in canola seed oil content would result in a boost
of $90 million to the oilseed extraction and processing industry
(Canola Council of Canada).
One of the challenges faced in the metabolic engineering of
plants is that attempts to produce unusual FAs in oilseed crops often result in low accumulation of the end product. It has been proposed that endogenous enzymes from crops might incorporate
unusual FAs inefciently into TG. As previously discussed, plant
DGAT2 has been demonstrated to be selective for unusual FAs
[85,86], providing important tools for producing industrially useful
oils by co-expression of DGAT2 and an enzyme responsible for the
synthesis of an unusual FA. This hypothesis was tested by Burgal
et al. [124], by coexpressing a FA hydroxylase (FAH12) and DGAT2
from castor bean in A. thaliana. This resulted in a doubling of the
hydroxy FA content in the seed oil compared to expression of
FAH12 alone. Another example was demonstrated for the coexpression of an epoxygenase from Stokesia laevis (SlEPX) and
DGAT1 or DGAT2 from Vernonia galamensis in soybean [87,231].
Co-expression of V. galamensis DGAT1 or DGAT2 resulted in increases of approximately 12% or 21.9% vernolic acid in soybean
TG, respectively. These results indicate that DGAT1 is also useful

for engineering FA composition in oilseeds. Recently, Mavraganis


et al. [126] also reported that coexpression in yeast of FAH and ricinoleoyl-specic DGAT2 from C. purpurea produced elevated levels
of ricinoleic acid in the oil compared to expression of FAH alone.
This nding suggests that fungal DGAT2s also represent attractive
gene candidates for producing value-added novel oils in crops. Finally, expression of an enzyme with acetyl-CoA diacylglycerol acetyltransferase from Euonymus alatus in A. thaliana resulted in
substantial deposition of 3-acetyl-1,2-diacyl-sn-glycerols (acetylated TG) in seeds [232]. Acetylated TGs result in oils with much
lower viscosity that can be directly used in engines thus representing an attractive application as a biofuel. The acetyl-CoA diacylglycerol acetyltransferase belongs to the MBOAT acyltransferase
family and appears to be a specic type of DGAT that incorporates
exclusively acetyl groups at the sn-3 position [232]. It will be
invaluable to study the mechanisms involved in the substrate
selectivity of this enzyme, and how these relates to other DGATs.
In the long term, approaches to enhance the overall catalytic
efciency and modulate substrate selectivity of DGAT will be necessary to further raise the level of desired FA in the seed oil. Currently, approaches such as site directed mutagenesis and directed
evolution, are being explored as tools to address these objectives
[118,140,233]. Studies on the relative contribution of DGAT1 versus DGAT2 to seed oil accumulation, in many plant species, are still
underway. A detailed understanding of the catalytic mechanism
and regulation of DGAT is important for further progress in metabolic engineering of TG accumulation in oleaginous plants.
Considerable attention has been devoted recently to the production of TGs from algae. Among those, C. reinhardtii emerges as
a model for studying TG accumulation in this class of organisms
[234], although diatoms have also been investigated [123]. Genetic
and biochemical characterization of TG synthesis in C. reinhardtii
indicated the presence of a single gene encoding DGAT1 and ve
encoding DGAT2 (known as DGTT1 to DGTT5) [235]. This information is invaluable for the development of algae strains capable of
producing large amounts of TG.

9. Closing comments
Advances in molecular biology, biochemistry and biotechnological applications of DGAT-related proteins have facilitated a substantial increase in our understanding of the low-resolution
functional and structural relationships of these essential enzymes
involved in TG metabolism. While many enzymes possessing DGAT
activity were identied during the last fteen years, DGAT1 and
DGAT2 are the two major membrane-bound DGATs widely distributed in different organisms. As two unrelated polypeptides, catalyzing the same reaction, the simple picture of DGAT1 and
DGAT2 has recently been replaced by the more complex scenario
in which each enzyme seems to have unique functional roles
across species. Although the considerable advances have been
achieved in comparative analyses of these two enzymes, many

Q. Liu et al. / Progress in Lipid Research 51 (2012) 350377

additional important questions still remain largely unanswered.


For example, how are DGAT1 and DGAT2 regulated at the protein
level? Which proteins do they interact with? Do they interact with
other DGAT-related enzymes? Do they utilize the same substrate
pools? Many of the biological functions of DGAT1 and DGAT2 have
been proposed mostly on the basis of in vitro data using microsomes, and thus denitive evidence for their roles in vivo is lacking.
How different are their functions in vivo as two non-complementary enzymes? The foundation is also set for identifying the distinctiveness of the active sites of these two classes of enzymes.
Determination of a crystal and/or NMR structure of one of the
membrane enzymes will surely open the door to address the questions relating to their reaction mechanisms and other functions,
such as possible allosteric control by acyl-CoA. Although in silico
analysis of putative functional motifs has served as an informative
guide for mutagenesis for exploring low-resolution structural and
functional relationships, it appears that we are still a long way
from obtaining sufcient amounts of a puried DGAT in an active
form, which is a prerequisite for high-resolution structural determination. With the development of new assay approaches, particularly HTS LC/MS/-based assays, we can continue to make some
progress. These techniques could identify a series of key amino
acid residues, which would provide a basis for developing hypotheses regarding the catalytic mechanism. Many insights gained into
structure/function in DGAT will be useful where the enzyme is
considered as a target in combating obesity or for increasing the
oil content of biomass. Potent inhibitors for either DGAT1 or
DGAT2 can be designed accordingly. The time may come when
we can rationally design DGAT with desired substrate selectivity,
which will form a basis for a new generation of designer oil crops.
In conclusion, we await the discovery of three-dimensional structure of these membrane-bound enzymes which will validate the
ndings summarized here and allow us to comprehensively understand the molecular mechanism.
Acknowledgements
RJW acknowledges the support from Alberta Innovates BioSolutions, the Alberta Enterprise and Advanced Education Research
Capacity Program, AVAC Ltd., the Canada Foundation for Innovation (CFI), the Canada Research Chairs Program, and the Natural
Sciences and Engineering Research Council of Canada. SJS acknowledges the support from the Canadian Institutes of Health Research,
CFI, and the Heart and Stroke Foundation.
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