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					                             LIPID-ASSOCIATED PROTEINS IN PLANTS


Denis J Murphy
Biotechnology Unit, School of Applied Sciences,
University of Glamorgan, CF37 1DL, United Kingdom


In this paper, the newly emerging roles of lipid assemblies and their associated proteins in non-storage
processes in cells will be examined. An expanded version of this review will appear in the forthcoming
treatise on plant lipids (1). Lipid-associated proteins are a relatively newly discovered class of proteins
that are specifically associated with macromolecular lipid assemblies, other than bilayer membranes, in
the cells of a wide range of organisms from eubacteria to mammals. The most common type of non-
bilayer lipid assembly in cells is a spherical organelle known as a lipid body or lipid droplet. These
organelles, which are typically 0.5 – 2µm in diameter, are made up of a neutral lipid core surrounded
by an annulus made up of a phospholipid monolayer and a specific population of proteins. Lipid bodies
(often called oil bodies in plants) have tended to be regarded as mere storage sites for carbon and
energy. However, recent progress in elucidating the functions of intra and extra-cellular lipid bodies,
and especially their associated proteins, have revealed hitherto unsuspected dynamic roles for these
organelles in processes, such as lipid import/export and in the subcellular trafficking of both lipids and

Lipid-associated proteins have been particularly well-characterised in plants, most notably in lipid-
storing tissues like seeds and fruits where they are exemplified by the oleosins. Oleosins are tightly
associated with storage lipid bodies in many, but not all, oil-accumulating plant tissues. Although
oleosin genes appear to be ubiquitous components of the genomes of true plants, i.e. the Plantae, the
levels of oleosin protein accumulation can vary enormously between different species. Proteins similar
to seed oleosins may also be present within the cytosol of in the cells of certain types of pollen grain
that store lipid, for example entomophilous pollen like that of many Brassicaceae, including
Arabidopsis thaliana (2). A separate class of oleosin-like proteins, or oleo-pollenins, has also been
found in floral tissues, including the tapetum and on the external surfaces of pollen grains. So far
reports of these proteins have been restricted to the Brassicaceae and it is not known whether similar
proteins are found in other plants (3). Oleosins and oleosin-like proteins appear to be structural proteins
with no discernible enzymatic motifs.

During recent years, several additional classes of lipid-binding proteins have also been described in
plants, including caleosins, steroleosins and protein kinases. Unlike oleosins, caleosins are not only
associated with lipid bodies: they have also been found on ER membranes. Since caleosins have a
single putative membrane-spanning domain, as well as calcium binding and protein kinase domains,
they may have a role in signalling as well as in oil-body assembly and mobilisation. Steroleosins are of
unknown function, but may be analogous to the sterol-binding proteins that are principal components
of fungal lipid bodies. The plastidial lipid-associated proteins (PLAPs) were originally found in the
specialised plastids, called chromoplasts, that are found in non-green pigmented tissues like coloured
flower petals and fruits. Inside the chromoplasts, lipidic pigments, and their associated PLAPs such as
carotenoids are stored in a range of differently shaped structures, from long, thin fibrils to classic
globular droplets similar to storage oil bodies.


Caleosins were so named because they contain a conserved EF-hand, calcium      -binding domain and
because they were initially believed to be similar to oleosins in being uniquely associated with oil
bodies in seeds (4,5). Similar proteins had been discovered earlier as gene products expressed in
developing and germinating seeds of rice in response to abscisic acid or osmotic stress (6). Similar
proteins or the genes encoding them have now been found in a wide range of plants from maize, rice
and barley to soya and sesame. A caleosin-like sequence is also present in the genome of the single-
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                             LIPID-ASSOCIATED PROTEINS IN PLANTS

celled alga, Auxenochlorella protothecoides, which indicates that caleosins are probably ubiquitous in
plants and algae. This is in contrast with oleosins, which are only found in true plants (so far they have
not been reported in algae). Caleosin-like sequences are also present in at least two fungi, namely the
lipid-accumulating fungus, Neurospora crassa, and the cereal pathogen, Magnaporthe grisea.
Immunodetection assays indicated that the caleosin protein from rice, OsEFA27, was associated with a
cell membrane fraction (6) although the caleosin from sesame was reported to be exclusively
associated with lipid bodies (4). More recently, we have reported evidence from immunofluorescence
microscopy that caleosins in rapeseed were localised both on lipid bodies and in specific domains of
the ER that may be associated with vesicle trafficking (5). Very recent findings have emphasised that,
although there are many intriguing similarities between caleosins and oleosins, there also important
differences. In particular, analysis of caleosins from Arabidopsis thaliana shows that they are members
of a large family of as many as 9 genes. Although one or more caleosin isoforms are tightly bound to
lipid bodies during seed development, other isoforms accumulate in vegetative tissues and are probably
integral membrane proteins of the ER (5,7,8).

The key structural features of caleosins are an N-terminal region with a single Ca 2+-binding EF hand
domain, a central hydrophobic region able to form a single bilayer span, and a C-terminal region with
several putative protein kinase phosphorylation sites, as shown in Figure 1. So far, only two caleosin
proteins have been shown experimentally to bind calcium (6,9). Caleosins lack an N-terminal signal
peptide, but do have a central, hydrophobic region of more than 30 residues with the potential to form a
transmembrane helix and amphipathic ß-sheets (6). This hydrophobic domain is much shorter than the
analogous 70-residue domain of oleosins. Like oleosins, caleosins contain a proline-rich region with
the potential to form a “proline knot” motif of the type that appears to be so important in the lipid-body
targeting of oleosins (10). In addition to the hydrophobic and proline-rich domains, caleosins also
possess an immediately adjacent potential amphipathic α-helical doma in, which may play a role in
their binding both to bilayer membranes and to lipid bodies. These properties have been used as the
basis of structural models of the different forms of caleosins (3).

Highly resolving Tricine-based SDS-PAGE gels have enabled us to distinguish physically between the
caleosin isoforms from Arabidopsis thaliana that bind respectively to lipid-bodies and the ER
membrane (5,7). A 25kDa isoform is only synthesised during the mid-late stage of seed development
and is exclusively located on the surfaces of lipid-bodies. This 25kDa isoform persists after seed
desiccation and dispersal, along with oleosin, as a major lipid-associated protein and is then mobilised
concurrently with the storage lipid bodies after seed germination. In contrast, the 27kDa caleosin
isoform is ER-associated and appears to be present in many tissues including roots, stems, young
leaves and seeds. Using sections of rapeseed root tip cells, immunoblotting and immunolocalisation
studies revealed that caleosin co-localised with the ER marker BiP and also with membranes labelled
for α-TIP, a marker for protein storage vacuoles (5). Parallel experiments indicated that
immunodetectable oleosin is expressed in rapeseed root tip cells, and that caleosin is associated with it
on what appeared to be lipid bodies. The presence of lipid bodies has been reported in root tips or root
caps of rice, pea and maize and garden cress and these data indicate that root lipid bodies contain two
proteins previously believed to be specific for seed lipid bodies, i.e., caleosin and oleosin. It has also
been reported that lipid bodies in root tip cells from garden cress concentrate calcium (11), which
would be consistent with the presence of caleosin on their surfaces.

From the known primary structures of caleosins and the presence of conserved functional motifs, like
EF hands and kinase domains, one can speculate about their possible functions in plants. For example,
the Ca 2+- binding status of caleosins, and perhaps their phosphorylation status, could well modulate
aspects of their function. Caleosins may be involved in processes such as membrane and lipid-body
fusion. Ca 2+-mediated fusion has been shown to be involved in the maturation of microlipid bodies
released from the ER to produce the large cytosolic lipid bodies found in milk secreting cells of
mammary glands (3). Likewise, in seeds and other storage-lipid accumulating plant tissues, nascent
lipid-bodies are probably released as small droplets from the ER and then undergo several rounds of
fusion to produce the mature 0.4-2µm diameter lipid bodies characteristic of such tissues. We have also
observed that the lipid-body caleosins persisted throughout seed desiccation, dormancy and for at least
the first six days of post-germinative development. It is likely that lipid bodies need to dock with
glyoxysomes to facilitate the concerted lipolysis, fatty acid oxidation and gluconeogenesis that occur
during storage lipid mobilisation. Once again, caleosins may play a role in this lipid trafficking process.

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                             LIPID-ASSOCIATED PROTEINS IN PLANTS

The microsomal caleosins found in very young embryos and in other plant tissues, such as roots and
leaves, may be involved in other membrane-fission and/or fusion events relating to trafficking between
the ER and storage or transport vesicles. The association of caleosins with either ER membranes or
lipid bodies may be regulated by their binding of Ca 2+ in a similar manner to the Ca 2+-mediated
association of lipocortin-1 with plasma membranes in human cell lines (12). The emerging dynamic
role of lipid bodies in the cellular metabolism of most organisms requires a mechanism for trafficking
of components between the lipid bodies and other organelles such as the ER, glyoxysomes and plasma
membrane. This will involve protein mediators to regulate appropriate targeting of fusion/fission of the
lipid bodies. The role, if any, of caleosins in such processes in plant (and perhaps in fungi) promises to
be a fascinating topic for future investigations.

Plastid lipid-associated proteins (PLAPs)

These proteins are localised exclusively in plastids and, like caleosins but unlike oleosins, they are not
restricted to the Plantae but are also found in unicellular photosynthetic eukaryotes (algae) and
homologues have even been found in cyanobacteria. Originally regarded as having a
storage/stabilisation role for lipidic pigments in chromoplasts, PLAPs are now recognised as having
several additional role in plastids. For example, PLAPs are implicated in thylakoid membrane
assembly/turnover as well as participating in various stress responses. PLAPs were first described 1976
when it was observed that the pigment-containing fibrillar lipoprotein assemblies of nasturtium flower
petal chromoplasts had a distinctive protein composition dominated by a polypeptide of about 30kDa
(13). Similar lipid -associated proteins of 35-38kDa have also been reported from higher plant
chromoplasts and triacylglycerol/carotenoid globules of the alga, Dunaliella bardawil. The algal
protein was localised on the surface of plastoglobuli and, as with oleosin in seeds, its cleavage by
trypsin led to coalescence of the globules, which suggested that the function of this PLAP might be to
stabilise these plastid lipid bodies. In 1994, the gene encoding the 32kDa bell pepper protein was
named fibrillin in view of the fibrillar nature of the chromoplast lipoprotein structures from which it
was derived (14). However, it is now clear that the plant protein is associated, not only with fibrils and
globules in various plastid types, but also with thylakoid membranes (15,16). Therefore, a more general
term such as plastid lipid-associated protein (PLAP) is probably more appropriate (16).

It is now clear that PLAPs belong to a large class of homologous proteins found throughout oxygenic
photosynthetic organisms. The discovery of a PLAP homologue in the cyanobacterium, Synechocystis,
indicates the probable ancient origin of this protein in the endosymbiotic precursors of plastids (17). In
addition to forming the major protein component of triacylglycerol /carotenoid-rich fibrils and globules
in chromoplasts, PLAPs are present in other plastid types such as elaioplasts and chloroplasts (17,18).
The PLAPs of elaioplasts are located on globular lipid bodies that resemble the triacylglycerol
/carotenoid globules of chromoplasts, except that their lipid components are mainly sterol esters and
fatty aldhydes (16). By way of contrast, the PLAPs of chloroplasts are associated both with
plastoglobuli and thylakoid membranes (15,18,19). Indeed, it has been reported that plastids from
Brassica rapa can contain up to three distinct PLAP isoforms, each of which is associated with
globules containing a different mixture of neutral lipids (20).

While the presence of PLAPs on neutral lipid bodies, including fibrils and globules, can be rationalised
as providing a stabilising surface structure (14), their apparently ubiquitous distribution in plant tissues
and their association with thylakoid membranes is more difficult to explain. A possible clue has come
from studies of the induction of PLAP gene expression in response to various environmental factors,
including drought stress, wounding or application of exogenous ABA. Since one of the primary
responses of plants to such stresses is often a re-arrangement of their photosynthetic membranes, it has
been proposed that PLAP has a general role in the formation/disassembly/turnover of plastid membrane
complexes (21). More recently, it was found that the PLAP homologue from potato was associated
with photosystem II, which is one of the major multi-subunit pigment-protein complexes of thylakoid
membranes (22). Antisense-mediated reduction of PLAP accumulation in transgenic potato plants led
to reduced photosynthetic efficiency and stunted growth, which reinforces the view that PLAPs play
important role(s) in both plastid membranes and lipid bodies. It is likely that there are several classes of
PLAP-like proteins in plants. Some of these may be expressed ubiquitously while other, like the ChrC
protein of cucumber (23) may have a more restricted pattern of expression, e.g. in response to stress.

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                             LIPID-ASSOCIATED PROTEINS IN PLANTS

Those PLAPs that are expressed ubiquitously in plants are of particular interest since they appear to
associate strongly both with the monolayer surfaces of plastid lipid bodies and fibrils and with the
bilayer membrane of the thylakoids. Analysis of the conserved regions of PLPs does not reveal any
obvious homology with other plant lipid-body proteins, such as oleosins or caleosins, although there
are some interesting motifs in PLAPs that may be significant in their lipid -binding properties. For
example, in the middle of the protein there are two non-polar regions of 16 and 22 residues
respectively, each of which is flanked by relatively polar regions, which could potentially form
transbilayer or monolayer-associating domains. Further structural studies are required to elucidate the
mechanism(s) of lipid binding, and hence the biological functions of PLAPs. Finally, like seed lipid
bodies, it is now emerging that plastid lipid assemblies probably contain several minor protein
components in addition to the dominant oleosin or PLAP classes (24). In view of their lower
abundance, these minor proteins are less likely to have structural roles, but may well be involved in
other aspects of lipid-body function or possibly in more generalised intra plastidial lipid trafficking. It
is possible that some of these PLAPs may play a role in the conversion of thylakoid membranes into
the triacylglycerol-rich globules found in senescing leaves (25) and following exposure to a wide range
of stresses, including ozone exposure, fungal infection, chilling, freezing and thawing (3).

Minor lipid-associated proteins in plants

Oleosins tend to be very abundant when they occur on storage oil bodies and caleosins are moderately
abundant, i.e. caleosin bands can be readily discerned on SDS-PAGE gels of many oil-body extracts.
However, there are also several relatively minor lipid-associated polypeptides that appear to be
enzymes rather than structural proteins. It is still not known whether these are specific oil-body
proteins or mere contaminants. Potential candidates include enzymes involved in triacylglycerol
biosynthesis and, for many years, there have been reports of the presence of such enzymes on lipid
bodies. This is mirrored by the well-documented findings of both triacylglycerol and sterol ester
biosynthetic enzymes on fungal lipid bodies (see below). The apparent presence of triacylglycerol
biosynthetic enzymes on seed lipid bodies may actually be due to the existence of membranous
appendages of the ER remaining attached to the lipid bodies following their budding off from the ER
proper. This has been proposed from ultrastructural studies (26,27) and would explain why membrane
bilayer proteins can be associated with a non-bilayer structure like an oil body. The membranous
appendages may facilitate re-fusion of oil bodies with the ER for the further metabolism of oil-body
triacylglycerol, e.g. by desaturases or transacylases, as has been reported during sunflower seed
development (28,29). Similar lipid-body appendages have been described in animal cells (1,3).

More recently, the apparently specific binding of a sterol-binding dehydrogenase to oil bodies has been
reported in sesame (30). This protein, tentatively named steroleosin, is similar in sequence to a protein
encoded by a family of 8 genes in Arabidopsis. It is not known whether all steroleosin-like proteins in
plants are associated with oil bodies or whether, like caleosins, some isoforms associate instead with
other cellular components. Although steroleosins are relatively minor components of oil bodies and are
not yet confirmed as being ubiquitous in plants, their discovery is interesting in view of the finding that
another sterol-binding protein, a sterol ∆24-methyltransferase, is the major protein associated with lipid
bodies in yeast (31). The importance of this class of enzyme in plants is shown by reports that sterol
∆24-methyltransferases in tobacco and rapeseed control the flux of carbon into sterols in seeds (32) and
modulate growth in Arabidopsis (33). Another potentially relevant finding is the recent report of the
presence of a sterol dehydrogenase on the surfaces of mammalian lipid bodies, with the implication
that these organelles might play important roles in sterol metabolism and other aspects of lipid
transport and membrane trafficking (34).

A further putative oil-body protein recently described from developing sesame seeds is a calcium-
dependent protein kinase of 55kDa (35). This oil-body-associated polypeptide was capable of calcium-
dependent autophosphorylation in vitro and cross-reacted with an antibody to a soybean protein kinase.
The detection of similar proteins in oil bodies of other plant species suggests that these lipid-associated
kinases may be ubiquitous in oilseeds. It can be speculated that these kinases, which are specifically
active during seed development, may be involved in oil-body ontogeny. A possible substrate would be
the relatively abundant lipid-associated isoforms of caleosin that are also present in developing seeds
and which possess several conserved phosphorylation sites.

16th International Plant Lipid Symposium, Budapest, June 2004
                             LIPID-ASSOCIATED PROTEINS IN PLANTS

In some species, seed oil bodies have been found to contain proteins that are associated with the
mobilisation of the storage triacylglycerol that occurs after germination. An example is the
lipoxygenase that is reportedly associated with oil bodies in cucumber seeds (36). Using GST-fusion
constructs, it was shown that this protein can be targeted both to oil bodies and to liposomes and that
the targeting required the presence of an N-terminal beta-barrel domain (37). Lipoxygenases are only
active with polyunsaturated acyl substrates but many seeds do not store such fatty acids, and it is
therefore unlikely that these proteins are present on storage oil bodies in all oilseeds. However, another
class of cucumber oil-body protein described by the same group could well be ubiquitous in plants.
This is a patatin-like protein that has phospholipase A2 activity that was transiently expressed and
associated with oil bodies coincidentally with lipid mobilisation (37). Although the authors posit a role
for this protein in storage lipid mobilisation, it could also have a signalling function. Phospholipases
A2 have been shown to have such roles in both plants and animals (38). Interestingly, a similar patatin-
like protein was recently reported in the proteome of lipid bodies from human CHO K2 cells (39).
Patatins are the major family of storage proteins in potato tubers and, like some seed storage proteins,
they appear to be derived from a group of enzymes that had esterase activities. If this is the case, then it
may be misleading to refer to the motif common to the plant and animal lipid-body proteins as a patatin
domain. Rather, these are all proteins with esterase-like domains, some of which have secondarily lost
their enzymatic activity and become storage proteins in some higher plants.

During the past few years, it has become increasingly evident that lipid bodies in many cells may be far
more dynamic that was previously assumed according to the stereotypical view that these lipids were
simply rather inert carbon stores. Our changing view of the nature and function of these hitherto
misunderstood organelles has emerged largely thanks to recent progress in the characterisation of the
various classes of lipid -associated proteins that have been described here. Ironically, the first class of
these proteins to be studied in detail, the oleosins, appears to be solely involved in lipid storage and
mobilisation, especially in seeds. However, even in the case of oleosins there are hints that they may
sometimes be present in non-storage, meristematic tissues of shoots and roots, where they may have
other novel functions (3,8). Over the past five years, much of the progress in elucidating the non-
storage roles of intracellular lipid bodies has come from studies in animal systems. Here, new and
exciting discoveries are being made at a rapid rate. For example, the PAT family of lipid-associated
proteins are now known to be present throughout the Metazoa and are also found in slime molds
(40,41); caveolins are true lipid -body proteins (42,43); lipid bodies contain other proteins associated
with lipid metabolism and trafficking (39) and lipid-body proteins retain their targeting properties in
ectopic systems (44). It is also becoming apparent that lipid-associated proteins may be implicated in a
host of serious human diseases and pathologies that include hepatitis C, Parkinson’s disease, CHILD
syndrome, retinopathy and even skin irritation (1).

For the first time, improved imaging techniques, such as multi-photon, laser-assisted confocal
microscopy, coupled with the use of more powerful reporter reagents, like high-output fluorophores,
have allowed for the real-time analysis of the behaviour of lipid bodies and their associated proteins in
living cells (45). This has enabled investigators to begin to dissect out the various populations of lipid
bodies; some in rapid flux in cells while others are less dynamic (46). This kind of direct real-time
observational study forms a vital link with other more “snapshot” approaches and is beginning to allow
us to assert with some confidence that lipid bodies are much more than mere storage entities. There is,
therefore, an emerging consensus that cytosolic lipid bodies in animals and fungi are complex
multifunctional organelles that participate in a host of cellular processes including membrane
trafficking, lipid-based signalling, sterol homeostasis, (34,39,45).


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Figure 1
Primary sequences and domain organisation of caleosins from plants and fungi.
A, domain organisation of a typical caleosin isoform that shows the major putative functional regions, namely the calcium -
binding EF hand, membrane spanning region (Memb), proline-rich motif (Pro), tyrosine kinase site (Tyr) and three casein kinase
II phosphorylation sites (CK).
B, comparison of the 23 amino acid sequences of caleosins from plants and fungi that have been described to date. CLO1-9, the
nine caleosin -like sequences from Arabidopsis; BARLEY1-3, three barley sequences; RICE1, 2, EFA27, three rice sequences;
FAGUS, one sequence from fig; SOYA, one sequence from soya; SESAME, one sequence from sesame; NEUROSPORA, one
sequence from the fungus Neurospora crassa ; 1-, 2-MAGNAPORTHE, two sequence from the fungus Magnaporthe grisea,
AUXENOCHLORELLA, one sequence from the microalga Auxenochlorella protothecoides. Data were obtained from BLAST
searches and aligned using the PRODOM database.

  1                                                                                     245

                         EF hand Memb Pro Tyr               2CK           CK

16th International Plant Lipid Symposium, Budapest, June 2004
                           LIPID-ASSOCIATED PROTEINS IN PLANTS


16th International Plant Lipid Symposium, Budapest, June 2004