Subcellular Localization of Secondary Lipid Metabolites Including Fragrance Volatiles in Carnation Petals

Pulse-chase labeling of carnation (Dianthus caryophyllus L. cv Improved White Sim) petals with [14C]acetate has provided evidence for a hydrophobic subcompartment of lipid-protein particles within the cytosol that resemble oil bodies, are formed by blebbing from membranes, and are enriched in lipid metabolites (including fragrance volatiles) derived from membrane fatty acids. Fractionation of the petals during pulse-chase labeling revealed that radiolabeled fatty acids appear first in microsomal membranes and subsequently in cytosolic lipid-protein particles, indicating that the particles originate from membranes. This interpretation is supported by the finding that the cytosolic lipid-protein particles contain phospholipid as well as the same fatty acids found in microsomal membranes. Radiolabeled polar lipid metabolites (methanol/water-soluble) were detectable in both in situ lipid-protein particles isolated from the cytosol and those generated in vitro from isolated radiolabeled microsomal membranes. The lipid-protein particles were also enriched in hexanal, trans-2-hexenal, 1-hexanol, 3-hexen-1-ol, and 2-hexanol, volatiles of carnation flower fragrance that are derived from membrane fatty acids through the lipoxygenase pathway. Therefore, secondary lipid metabolites, including components of fragrance, appear to be formed within membranes of petal tissue and are subsequently released from the membrane bilayers into the cytosol by blebbing of lipid-protein particles.     

Acyl chain and head group regulation of phospholipid catabolism in senescing carnation flowers

Microsomal membranes from the petals of senescing carnation (Dianthus caryophyllus L.) flowers contain phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol. These phospholipid classes decline essentially in parallel during natural senescence of the flower and when microsomal membranes isolated from young flowers are aged in vitro. However, measurements of changes in the endogenous molecular species composition of microsomal phospholipids during natural senescence of the flower petals and during in vitro aging of isolated membranes have indicated that the various molecular species of phospholipids have quite different susceptibilities to catabolism. Acyl chain composition and the nature of the head group are both determinants of their susceptibility to catabolism. As well, a comparison of the phospholipid catabolism data for naturally senesced membranes and for membranes aged in vitro suggests that the phospholipid composition of membranes is continuously altered during senescence by acyl chain desaturation and possibly retailoring so as to generate molecular species that are more prone to catabolism. The results collectively indicate that provision of particular molecular species of phospholipids with increased susceptibility to degradation contributes to enhanced phospholipid catabolism in the senescing carnation petal.     

Isolation and characterization of lipid in phloem sap of canola

Phloem isolated from canola (Brassica napus L.) stems was found to contain phospholipid, diacylglycerol, triacylglycerol, steryl and wax esters, and comparatively high concentrations of unesterified fatty acids. Indeed, the composition of phloem lipid was markedly different from that of microsomal membranes and cytosol isolated from both leaves and stems. Specifically, phloem lipid consisted predominantly of unesterified fatty acids and was enriched in medium-chain fatty acids, in particular, lauric, myristic and pentadecanoic acids. This unique composition also distinguished phloem lipid from that of well-characterized cytosolic lipid particles such as oil bodies found in plant cells. Moreover, levels of medium-chain fatty acids in the phloem increased when canola plants were stressed by exposure to sublethal doses of ultraviolet irradiation. Phloem levels of lauric acid, for example, increased by 11-fold upon treatment with sublethal ultraviolet irradiation. Spherical lipid particles were discernible in isolated phloem sap by electron microscopy, suggesting that the lipid in phloem is in the form of lipid particles. The presence of lipid in phloem may be reflective of long-distance lipid transport in plants, primarily in the form of free fatty acids.     

Immunopurification of H+-ATPase-containing lipid-protein particles from the cytosol of carnation petals

Lipid-protein particles originating from the plasma membrane were immunopurified from the cytosol of carnation petal cells ( Dianthus caryophyllus L. cv. Improved White Sim) using antibodies raised against the central hydrophilic domain of the H⁺-ATPase. The immunopurified particles are enriched in lipid metabolites, in particular free fatty acids and steryl/wax esters, by comparison with corresponding microsomal membranes, and the lipids of the particles are more saturated than those of microsomal membranes. Proteolytic catabolites of the H⁺-ATPase, a protein associated with the plasma membrane, but not the native H⁺-ATPase protein, are also present in the immunopurified cytosolic particles. Osmiophilic particles were discernible in the cytosol of carnation petal cells by transmission electron microscopy, and the association of H⁺-ATPase catabolites with a subpopulation of these particles was confirmed by immunogold labelling with H⁺-ATPase antiserum. Cross-reaction of the H⁺-ATPase antiserum with elements of the cytosol was also evident by immunofluorescent light microscopy. These observations collectively indicate that lipid-protein particles of plasma membrane origin are present in the cytosol of carnation petal cells and that their formation may serve as a means of removing lipid and protein metabolites from the plasma membrane which would otherwise destabilize its structure.     

Release of Photosynthetic Protein Catabolites by Blebbing from Thylakoids

Thylakoid proteins and their catabolites have been detected in lipid-protein particles isolated from the stroma of intact chloroplasts obtained from primary leaves of 2-week-old bean seedlings (Phaseolus vulgaris L. cv Kinghorn). The lipid-protein particles bear morphological resemblance to plastoglobuli seen in the chloroplasts of senescing leaves, but they are much smaller. They range from 10 to 320 nm in radius, are uniformly stained in thin sections visualized by transmission electron microscopy, and are discernible in the stroma of chloroplasts in corresponding thin-sectioned leaf tissue. The lipid-protein particles contain thylakoid lipids and are enriched in free fatty acids. Specifically, the free-to-esterified fatty acid ratio is about 1:1 in the particles compared to only 1:18 for corresponding thylakoid membranes. Western blot analyses indicate that these particles also contain thylakoid proteins and, in some cases, catabolites of these proteins including the CF1 [beta] and [gamma] subunits of ATPase, cytochrome f, and the 31- and 33-kD proteins of PSII. Lipid-protein particles with similar properties were generated in vitro from isolated, light-stressed thylakoids. Collectively, these data suggest that blebbing of lipid-protein particles may be a means of removing potentially destabilizing macromolecular catabolites from thylakoid membrane bilayers.     

Molecular composition and surface properties of storage lipid particles in wax bean (Phaseolus vulgaris)

Lipid particles have been isolated from seeds of wax bean (Phaseolus vulgaris), a species in which starch and protein rather than lipid are the major seed storage reserves. These lipid particles resemble oil bodies present in oil-rich seeds in that > 90% of their lipid is triacylglycerol. Moreover, this triacylglycerol is rapidly metabolized during seed germination indicating that it is a storage reserve. The phospholipid surfaces of oil bodies are known to be completely coated with oleosin which prevents their coalescence, particularly during desiccation of the developing seed. This would appear to be necessary since lipid is the major storage reserve in oil seeds, and there are very few alternate types of storage particles in the cytoplasm of oil seed endosperm to provide a buffer against coalescence of oil bodies by isolating them from one another. The present study indicates that the surfaces of lipid particles from wax bean are not completely coated with oleosin and feature regions of naked phospholipid. This finding has been interpreted as reflecting the fact that lipid particles in wax been seeds are less prone to coalescence than oil bodies of oil-rich seeds. This arises because the individual lipid particles are interspersed in situ among highly abundant protein bodies and starch grains and hence less likely to come in contact with one another, even during desiccation of the developing seed.     

Characterization of Thylakoid-Derived Lipid-Protein Particles Bearing the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Lipid-protein particles bearing the 55-kD ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39) large subunit (RLSU) and no detectable corresponding Rubisco small subunit (RSSU) were isolated from the stroma of intact chloroplasts by flotation centrifugation. Stromal RLSU-bearing particles appear to originate from thylakoids because they can also be generated in vitro by illumination of isolated thylakoids. Their formation in vitro is largely heat denaturable and is facilitated by light or ATP. RLSU-containing lipid-protein particles range from 0.05 to 0.10 [mu]m in radius, contain the same fatty acids as thylakoids, but have a 10- to 15-fold higher free-to-esterified fatty acid ratio than thylakoids. RLSU-bearing lipid-protein particles with no detectable RSSU were also immunopurified from the populations of both stromal lipid-protein particles and those generated in vitro from illuminated thylakoids. Protease shaving indicated that the RLSU is embedded in the lipid-protein particles and that there is also a protease-protected RLSU in thylakoids. These observations collectively indicate that the RLSU associated with thylakoids is released into the stroma by light-facilitated blebbing of lipid-protein particles. The release of RLSU-containing particles may in turn be coordinated with the assembly of Rubisco holoenzyme because chaperonin 60 is also associated with lipid-protein particles isolated from stroma.     

The lipoxygenase-dependent oxygenation of lipid body membranes is promoted by a patatin-type phospholipase in cucumber cotyledons

Oilseed germination is characterized by the mobilization of storage lipids as a carbon and energy source for embryonic growth. In addition to storage lipid degradation in germinating oilseeds via the direct action of a triacylglycerol lipase (TGL) on the storage lipids, a second degradation pathway that is dependent on a specific lipid body trilinoleate 13-lipoxygenase (13-LOX) has been proposed in several plant species. The activity of this specific 13-LOX leads first to the formation of ester lipid hydroperoxides. These hydroperoxy fatty acids are then preferentially cleaved off by a TGL and serve as a substrate for glyoxysomal β-oxidation. As a prerequisite for triacylglycerol (TAG) mobilization, a partial degradation of the phospholipid monolayer and/or membrane proteins of the oil body has been discussed. Evidence has now been found for both processes: partial degradation of the proteins caleosin and oleosin was observed and simultaneously a patatin-like protein together with transient phospholipase (PLase) activity could be detected at the oil body membranes during germination. Moreover, in vitro experiments with isolated oil bodies from mature seeds revealed that the formation of 13-LOX-derived lipid peroxides in lipid body membranes is increased after incubation with the purified recombinant patatin-like protein. These experiments suggest that in vivo the degradation of storage lipids in cucumber cotyledons is promoted by the activity of a specific oil body PLase, which leads to an increased decomposition of the oil body membrane by the 13-LOX and thereby TAGs may be better accessible to LOX and TGL.     

Co-association of cytochrome f catabolites and plastid-lipid-associated protein with chloroplast lipid particles

Distinguishable populations of lipid particles isolated from chloroplasts of yellow wax bean (Phaseolus vulgaris L. cv Kinghorn Wax) leaves have been found to contain plastid-lipid-associated protein (J. Pozueta-Romero, F. Rafia, G. Houlné, C. Cheniclet, J.P. Carde, M.-L. Schantz, R. Schantz [1997] Plant Physiol 115: 1185-1194). One population is comprised of plastoglobuli obtained from sonicated chloroplasts by flotation centrifugation. Higher density lipid-protein particles isolated from chloroplast stroma by ultrafiltration constitute a second population. Inasmuch as the stromal lipid-protein particles contain plastid-lipid-associated protein, but are distinguishable from plastoglobuli in terms of their lipid and protein composition, they appear to be plastoglobuli-like particles. Of particular interest is the finding that plastoglobuli and the higher density lipid-protein particles both contain catabolites of the thylakoid protein, cytochrome f. These observations support the view that there are distinguishable populations of plastoglobuli-like particles in chloroplasts. They further suggest that the formation of these particles may allow removal of protein catabolites from the thylakoid membrane that are destined for degradation as part of normal thylakoid turnover.     

Acceleration of membrane senescence in cut carnation flowers by treatment with ethylene

The lipid microviscosity of microsomal membranes from senescing cut carnation (Dianthus caryophyllus L. cv. White Sim) flowers rises with advancing senescence. The increase in membrane microviscosity is initiated within 3 to 4 days of cutting the flowers and coincides temporally with petal-inrolling denoting the climacteric-like rise in ethylene production. Treatment of young cut flowers with aminoethoxyvinylglycine prevented the appearance of petal-inrolling and delayed the rise in membrane microviscosity until day 9 after cutting. When freshly cut flowers or aminoethoxyvinylglycine-treated flowers were exposed to exogenous ethylene (1 microliter per liter), the microviscosity of microsomal membranes rose sharply within 24 hours, and inrolling of petals was clearly evident. Thus, treatment with ethylene accelerates membrane rigidification. Silver thiosulphate, a potent anti-ethylene agent, delayed the rise in microsomal membrane microviscosity even when the flowers were exposed to exogenous ethylene. Membrane rigidification in both naturally senescing and ethylene-treated flowers was accompanied by an increased sterol:phospholipid ratio reflecting the selective loss of membrane phospholipid that accompanies senescence. The results collectively indicate that the climacteric-like surge in ethylene production during senescence of carnation flowers facilitates physical changes in membrane lipids that presumably lead to loss of membrane function.     

Food vacuole-associated lipid bodies and heterogeneous lipid environments in the malaria parasite, Plasmodium falciparum

The malaria parasite Plasmodium falciparum induces a sixfold increase in the phospholipid content of infected erythrocytes during its intraerythrocytic growth. We have characterized the lipid environments in parasitized erythrocyte using the hydrophobic probe, Nile Red. Spectral imaging with a confocal microscope revealed heterogeneous lipid environments in parasite-infected erythrocytes. An insight into the nature of these environments was gained by comparing these spectra with those of triacylglycerol/phospholipid emulsions and phospholipid membranes. Using this approach, we identified a population of intensely stained particles of a few hundred nanometers in size that are closely associated with the digestive vacuole of the parasite and appear to be composed of neutral lipids. Electron microscopy and isolation of food vacuoles confirmed the size of these particles and their intimate association respectively. Lipid analysis suggests that these neutral lipid bodies are composed of di- and triacylgycerols and may represent storage organelles for lipid intermediates that are generated during digestion of phospholipids in the food vacuole. Mono-, di- and triacylglycerol suspensions promote beta-haematin formation, suggesting that these neutral lipid bodies, or their precursors, may also be involved in haem detoxification. We also characterized other compartments of the infected erythrocyte that were stained less intensely with the Nile Red probe. Both the erythrocyte membrane and the parasite membrane network exhibit red shifts compared with the neutral lipid bodies that are consistent with cholesterol-rich and cholesterol-poor membranes respectively. Ratiometric imaging revealed more subtle variations in the lipid environments within the parasite membrane network.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A₂. We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

Lipid composition of the isolated rat intestinal microvillus membrane

1. Rat intestinal microvillus plasma membranes were prepared from previously isolated brush borders and the lipid composition was analysed. 2. The molar ratio of cholesterol to phospholipid was greatest in the membranes and closely resembled that reported for myelin. 3. Unesterified cholesterol was the major neutral lipid. However, 30% of the neutral lipid fraction was accounted for by glycerides and fatty acid. 4. Five phospholipid components were identified and measured, including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine. Though phosphatidylethanolamine was the chief phospholipid, no plasmalogen was detected. 5. In contrast with other plasma membranes in the rat, the polar lipids of the microvillus membrane were rich in glycolipid. The cholesterol:polar lipid (phospholipid+glycolipid) ratio was about 1:3 for the microvillus membrane. Published data suggest that this ratio resembles that of the liver plasma membrane more closely than myelin or the erythrocyte membrane. 6. The fatty acid composition of membrane lipids was altered markedly by a single feeding of safflower oil. Membrane polar lipids did not contain significantly more saturated fatty acids than cellular polar lipids. Differences in the proportion of some fatty acids in membrane and cellular glycerides were noted. These differences may reflect the presence of specific membrane glycerides.     

Isolation and characterization of peanut spherosomes

Spherosomes of cotyledons of germinating peanuts (Arachis hypogea L.) were examined by electron microscopy and found to be particles about 1.0 to 2.0 μ in diameter bounded by a limiting membrane. Isolated spherosomes appear similar to spherosomes in situ. The isolated spherosomes are composed of 98.1% total lipids, 0.77% phospholipid and 1.27% protein by dry weight. The amounts of protein and phospholipid associated with the isolated spherosomes are sufficient to account for limiting membranes. Spherosomes amply account for the lipid in a peanut cotyledon. The activity of lipase and fatty acyl-Coenzyme A synthetase is not associated with the isolated spherosomes. This suggests that peanut spherosomes are principal sites of lipid storage but not of lipid degradation.     

Lipid conformation in crystalline bilayers and in crystals of transmembrane proteins

Dihedral torsion angles evaluated for the phospholipid molecules resolved in the X-ray structures of transmembrane proteins in crystals are compared with those of phospholipids in bilayer crystals, and with the phospholipid conformations in fluid membranes. Conformations of the lipid glycerol backbone in protein crystals are not restricted to the gauche C1-C2 rotamers found invariably in phospholipid bilayer crystals. Lipid headgroup conformations in protein crystals also do not conform solely to the bent-down conformation, with gauche-gauche configuration of the phospho-diester, that is characteristic of phospholipid bilayer membranes. This suggests that the lipids that are resolved in crystals of membrane proteins are not representative of the entire lipid-protein interface. Much of the chain configurational disorder of the membrane-bound lipids in crystals arises from energetically disallowed skew conformations. This indicates a configurational heterogeneity in the lipids at a single binding site: eclipsed conformations occur also in some glycerol backbone torsion angles and C-C torsion angles in the lipid headgroups. Stereochemical violations in the protein-bound lipids are evidenced by one-third of the ester carboxyl groups in non-planar configurations, and certain of the carboxyls in the cis configuration. Some of the lipid structures in protein crystals have the incorrect enantiomeric configuration of the glycerol backbone, and many of the branched methyl groups in structures of the phytanyl chains associated with bacteriorhodopsin crystals are in the incorrect S-configuration.     

Lipid composition of subcellular particles from sheep platelets. Location of phosphatidylethanolamine and phosphatidylserine in plasma membranes and platelet liposomes

The lipid composition of whole sheep platelets and their subcellular fractions was determined. The basic lipids show similar distributions in granules, microsomes, plasma membranes and whole platelets. Phospholipid (about 70% of total lipids) and cholesterol (25% of total lipids) are the principal lipid components. Free cholesterol represents about 98% of the total, whereas cholesteryl ester is a minor component. The phospholipid composition found in intact platelets and their subcellular particles is about: 35% phosphatidylethanolamine (PE), 30% phosphatidylcholine (PC), 20% sphingomyelin and 15% phosphatidylserine (PS). We also investigated aminophospholipid topology in intact platelet plasma membranes and platelet liposomes by using the nonpenetrating chemical probe trinitrobenzenesulfonic acid (TNBS), because they are the major components of total lipids. In intact platelets, PS is not accessible to TNBS during the initial 15 min of incubation, whereas 18% PE is labelled after 15 min. In contrast, in phospholipid extracted from platelets 80% PE and 67% PS react with TNBS within 5 min, while 27 and 25% PE and 15 and 19% PS from liposomes and isolated plasma membranes, respectively, were modified after 15 min of incubation. In view of this chemical modification, it is concluded that 22% of PE and less than 1% of PS are located on the external surface of intact platelet plasma membranes. The asymmetric orientation of aminophospholipids is similar between liposomes and isolated plasma membrane. PS (23 and 28%) and PE (34 and 31%) are scarcely represented outside the bilayer. The data found are consistent with the nonrandom phospholipid distribution of blood cell surface membranes.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A(2). We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

Topology and lipid selectivity of pulmonary surfactant protein SP-B in membranes: Answers from fluorescence

Contradictory results have been reported with respect to the depth of penetration and the orientation of pulmonary surfactant protein SP-B in phospholipid membranes and its relative selectivity to interact with anionic over zwitterionic phospholipid species. In the present study we have re-evaluated lipid-protein interactions of SP-B by analysing F?rster resonance energy transfer (FRET) efficiencies, obtained from time-resolved measurements, from the single tryptophan in SP-B to different fluorescently labelled phospholipids in matrix bilayers made of either pure phosphatidylcholine (POPC) or the full lipid extract obtained from purified surfactant. In the background of POPC membranes SP-B exhibits a certain level of selectivity for anionic fluorescent phospholipids over the corresponding zwitterionic analogues, but apparently no preference for phosphatidylglycerol over other anionic species such as phosphatidylserine. No selectivity was detected in membranes made of full surfactant lipids, indicating that specific lipid-protein binding sites could already be occupied by endogenous anionic phospholipids. Furthermore, we have analysed the fit of two different models of how SP-B could be orientated with respect to phospholipid membrane surfaces to the FRET data. The FRET results are consistent with topology models in which the protein has a superficial orientation, with no regions of exclusion by the protein to the access of phospholipids, both in POPC membranes and in membranes made of the whole surfactant lipid fraction. This discards a deep penetration of the protein into the core of bilayers and suggests that most hydrophobic segments of SP-B could participate in protein-protein instead of lipid-protein interactions.     

Characterization of the charged components and their topology on the surface of plant seed oil bodies.

Oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins called oleosins. Oil bodies isolated from maize (Zea mays L.) in a medium of pH 7.2 maintained their entities but aggregated when the pH was lowered to 6.8 and 6.2. Aggregation did not lead to coalescence and was reversible with an elevation of the pH. Further decrease of the pH from 6.2 to 5.0 retarded the aggregation. Aggregation at pH 7.2 was induced with 2 mM CaCl2 or MgCl2 but not with NaCl. Aggregation at pH 6.8 was prevented by 10 microM sodium dodecyl sulfate but not with NaCl. We conclude that oil bodies have a negatively charged surface at pH 7.2 and an isoelectric point of about 6.0. This conclusion is supported by isoelectrofocusing results and by theoretical calculation of the positive charges in the oleosins and the negative charges in phosphatidylserine, phosphatidylinositol, and free fatty acids. Apparently, lowering of the pH from 7.2 to 6.2 protonates the histidine residues in the oleosins, and neutralizes the oil bodies. Further decrease of the pH to 5.0 likely protonates the free fatty acids and produces positively charged organelles. Similar charge properties were observed in the oil bodies isolated from rape, flax, and sesame seeds. An analysis of the oleosin secondary structures reveals an N-terminal amphipathic domain, a central hydrophobic anti-parallel beta-strand domain (not found in any other known protein), and a C-terminal amphipathic alpha-helical domain. In the two amphipathic domains, the positively charged residues are orientated toward the interior facing the negative charged lipids, whereas the negatively charged residues are exposed to the exterior. The negatively charged surface is a major factor in maintaining the oil bodies as stable individual entities.