(Received for publication, September 18, 1996)
From the Department of Plant Sciences, University of California, Riverside, California 92521
Yeast (Saccharomyces cerevisiae) has been used extensively as a heterologous eukaryotic system to study the intracellular targeting of proteins to different organelles. The lipid bodies in yeast have not been previously subjected to such studies. These organelles are functionally equivalent to the subcellular storage oil bodies in plant seeds. A plant oil body has a matrix of oils (triacylglycerols) surrounded by a layer of phospholipids embedded with abundant structural proteins called oleosins. We tested whether plant oleosin could be correctly targeted to the lipid bodies in transformed yeast. The coding region of a maize (Zea mays L.) oleosin gene was incorporated into yeast high copy and low copy number plasmids in which its expression was under the control of GAL1 promoter. Yeast strains transformed with these plasmids produced oleosin when grown in a medium containing galactose but not glucose. The oleosin produced in yeast had a molecular mass slightly higher than that of the native protein in maize. Oleosin accumulated concomitantly with the storage lipids during growth of the transformed yeast, and it was not secreted. Subcellular fractionation of the cell extracts obtained by two different cell breakage procedures revealed that the oleosin was largely restricted to the lipid bodies. Oleosin apparently did not affect the lipid contents and composition of the transformed yeast lipid bodies but replaced some of the native proteins associated with the organelles. Immunocytochemistry of the transformed yeast cells showed that the oleosin was present mostly on the periphery of the lipid bodies. Oleosin isolated from maize or transformed yeast strain, alone or in the presence of phospholipids or SDS, did not bind to the yeast lipid bodies in vitro. We conclude that plant oleosin is correctly targeted to the lipid bodies in transformed yeast and that yeast may be used as a heterologous system to dissect the intracellular targeting signals in the oleosin.
Diverse organisms store lipids in subcellular particles as food reserves that will be mobilized during a forthcoming period of active metabolism. These lipid particles can be found in seeds, pollens, spores, and vegetative organs of plants (1). They are also present in the brown adipose (2) and other tissues of mammals (3), the eggs of some nematodes (4), and unicellular organisms such as yeast (5, 6), Euglena (7), and algae (8). Of all these subcellular storage lipid particles, those from plant seeds have been studied most extensively.
The seeds of many plant species store triacylglycerols
(TAGs)1 as food reserves for germination
and growth of the seedlings (1, 3). The TAGs constitute about 5-40%
of the total seed dry weight. They are present in small discrete
subcellular organelles called oil bodies (lipid bodies, oleosomes, and
spherosomes). The spherical organelles have diameters of about 0.6-2.0
µm, depending on the plant species in which they occur. Each oil body
contains a TAG matrix surrounded by a layer of phospholipids (PL)
embedded with proteins termed oleosins (9). Oleosins essentially cover the whole surface of the oil body and represent 1-4% of the total mass of the oil body; this percentage is related to the size of the
organelle. The Mr of oleosins range from 15,000 to 26,000, depending on the isoforms and plant species in which they
occur. Each oleosin molecule has the following three structural
portions (10-13): (a) an N-terminal amphipathic stretch
(20-60 residues) of undefined secondary structures residing on the
organelle surface; (b) a central hydrophobic domain (72 residues) of long antiparallel -structures penetrating into the
matrix; and (c) a C-terminal amphipathic
-helix (30-40
residues) locating on the organelle surface, interacting with the PL
layer. The three structural portions enable the protein to interact
with other molecules on the surface of an oil body. Oleosins form a
steric barrier on the surface of an oil body, preventing the PL layers
of adjacent oil bodies from contacting and coalescing. Maintenance of
the oil bodies as small entities provides a large surface area per unit
TAG and would facilitate lipase binding and lipolysis during
germination.
During seed maturation, oil bodies are synthesized on the rough ER by a budding process, which has been postulated on the basis of experimental evidence and consideration of the thermodynamics involved (1). TAGs are synthesized by enzymes in the ER and are sequestered, because of their hydrophobicity, between the two PL layers of the ER membrane, thereby forming a bud. PL synthesized by enzymes in the ER also diffuse to the surface of this budding TAG particle. Simultaneously, oleosin is synthesized on polyribosomes bound to the ER without appreciable co- or posttranslational processing. The newly synthesized oleosin moves to the budding TAG particle, presumably guided by the central hydrophobic domain. Alternatively, the oleosins being synthesized on polyribosomes are inserted directly into the budding particle. The budding particle, which has a TAG matrix surrounded by a layer of PL and oleosins, is released into the cytosol as a mature oil body.
Although it is well documented that oleosin is synthesized by polyribosomes bound to the ER, no cleavable N-terminal signal sequence in the protein has been identified (1). Apparently, oleosin does not enter the lumen of the ER, otherwise budding would occur at the luminal side of the ER and the newly formed oil body would enter the intracellular secretary pathway. The latter scenario occurs in the secretion of lipoproteins in mammals (14). The targeting signal in oleosin for the ER or budding oil body is unclear. At the N-terminal region of oleosin where a targeting signal could occur, there are no appreciable similarities in the length, the amino acid residues, and the charge distribution among oleosins of diverse plant species. Oleosin has a conserved central hydrophobic domain that could act as the targeting signal for the hydrophobic matrix of the oil bodies. This targeting signal would be equivalent to the internal hydrophobic targeting sequences in many membrane proteins of diverse organisms (15, 16). Whatever the targeting signal in the oleosin is, it appears to be universal, since maize (a monocotyledonous species) oleosin is correctly targeted to seed oil bodies in transformed Brassica (a dicotyledonous species) (17).
Yeast has been used extensively as a heterologous system to study the intracellular targeting of proteins (review, Ref. 18). Proteins from other eukaryotes have been tested to see whether they are correctly targeted to the appropriate subcellular locations in transformed yeast. These locations include the nuclei, mitochondria, peroxisomes, vacuoles, plasma membranes, and extracellular medium, etc. An obvious omission from this list of subcellular locations is the cytoplasmic lipid bodies. In yeast, the lipid bodies, of about 0.2-0.5 µm in diameter, contain about 50% TAGs and 50% steroid esters, which are accumulated as food reserves during the log phase and the late stage of growth (5, 6). These lipid bodies are similar to the seed oil bodies in function; whether they are also similar in structure is unknown. In this report, we show that plant seed oleosin is correctly targeted to the lipid bodies in yeast transformed with the plant oleosin gene.
Saccharomyces
cerevisiae DFY1-1A (MATa, ura3-53, Lys) and the
yeast/Escherichia coli shuttle vectors pQC5 and pQC6
(19) were kindly provided to us by Drs. Q. Chao and M. E. Etzler,
Section of Molecular and Cellular Biology, University of
California, Davis, CA. Plasmid pT7 (20) was obtained from Dr.
D. R. Gallie, Department of Biochemistry, University of
California, Riverside, CA.
A 594-base pair fragment in a cDNA clone, pL2± (21),
containing the complete coding sequence and 30 base pairs of the
5-untranslated sequence of maize 18-kDa oleosin, was amplified by
polymerase chain reaction. Using primers containing sequences for
creating specific restriction sites, EcoRI and
BamHI sites were added to the 5
and the 3
end of this
fragment, respectively. The blunt-ended EcoRI-BamHI fragment was then subcloned into
SmaI-cleaved plasmid pT7
(20) to create plasmid pL2T02
(Fig. 1).
The maize oleosin gene from plasmid pL2T02 was cut with EcoRI and ligated into the EcoRI site of the low copy number centromeric plasmid pQC5 (19) and the high copy number 2-µm plasmid pQC6 (19).
Yeast Culture Condition and TransformationYeast strain was
maintained on a agar medium of 1% yeast extract, 2% peptone, and 2%
glucose. Transformation was performed by the lithium acetate method
(22). Transformants were selected for uracil auxotrophy on
uracil-omitted synthetic complete medium (SCM ura) (22). In the
induction of the GAL1 promoter in the transformed yeast
strains, glucose (2%) in SCM ura
medium was replaced by galactose
(2%).
Yeast growth was monitored by reading the
A600 nm of the undiluted culture in a
spectrophotometer. The reading reached a maximum of 2.3 after 50 h
of growth (to be indicated as 100% in Fig. 4). The reading 2.3 was an
underestimated value, since the spectrophotometric reading became
nonlinear beyond 1.0. When the 50-h culture was diluted with culture
medium such that the reading of A600 nm was at
the range of 0.5-1.0, the calculated A600 nm
of the 50-h culture was about 4-6. All the
A600 nm readings described in the subsequent
sections of "Experimental Procedures" indicated those of the
undiluted cultures.
Northern Hybridization
Yeast RNA was prepared from mid-log
phase cells (A600 nm = 1.0) by a method using
glass beads for cell breakage (23). Electrophoresis was carried out
with a 1.5% (w/v) agarose, 2.2 M formaldehyde gel
(24), and 10 µg of RNA was applied to each lane. Following
electrophoresis, the RNA was transferred to a nylon membrane and
hybridized with a maize 18-kDa oleosin cDNA probe. The membrane was
washed as described (25). Plasmid pL2T01 containing the complete coding
sequence and 30 base pairs of the 5-untranslated sequence of the maize
oleosin gene was used as a template to make the DNA probe by a method
using random primers (24).
Yeast cells in 20 ml of culture medium were grown to an A600 nm of 2.0 and harvested by centrifugation at 3000 × g for 5 min. Cells were resuspended in TS buffer (50 mM Tris-HCl, pH 7.5, 1 M sorbitol) and heated at 100 °C for 10 min. The heated cells were centrifuged again and resuspended in new TS buffer. Acid-washed glass beads (425-600 microns) were added to the cell suspension. The cells were lysed by vortexing the suspension at a maximum speed for 10 min. The supernatant was collected as crude extract after the beads settled.
SDS-PAGE and ImmunoblottingUrea (8 M) and acrylamide (12.5%) SDS-PAGE and the pretreatment of samples were performed as described (26). Immunoblotting and the preparation of chicken antibodies raised against maize 18-kDa oleosin were as described (27).
Subcellular FractionationYeast cells harboring plasmid
pQC6-ole were grown in 400 ml of SCM ura medium containing 2%
galactose at 30 °C to an A600 nm of 2.0 (at
the late log phase). Cells were collected by centrifugation (3000 × g for 5 min), and the leftover medium was saved for
further analysis. The pelleted cells were resuspended in 60 ml of TS
buffer containing 3 mg/ml lyticase (ICN, Costa Mesa, CA) and incubated at 30 °C for 30 min. The resulting spheroplasts were harvested by
centrifugation (3000 × g for 5 min), washed in TS
buffer, and resuspended in 60 ml of 50 mM Tris-HCl, pH 7.5, 2 mM PMSF. The resuspension in a 250-ml flask was shaken
vigorously by hand and homogenized in an ice-cold glass tissue
homogenizer with 25 strokes, using a tight-fitting pestle. The
preparation was centrifuged at 1000 × g for 5 min to
remove cell debris. The supernatant was designated as the crude
homogenate. An aliquot of the crude homogenate was saved for further
use. Sorbitol was added to the crude homogenate to give a final
concentration of 1 M. The homogenate (15 ml each in two
tubes) was overlaid with 15 ml of TPS buffer (50 mM
Tris-HCl, pH 7.5, 2 mM PMSF, 0.5 M sorbitol),
and centrifuged at 100,000 × g for 2 h. A
floating layer consisting of lipid bodies was collected from the top of
the tube with a pipette. The leftover culture medium, the 100,000 × g supernatant, and the crude homogenate were subjected to
80% ammonium sulfate precipitation to concentrate the proteins in the
fractions. Each precipitated sample was resuspended in 300 µl of
Tricine buffer (0.15 M Tricine-KOH, pH 7.5, 1 mM MgCl2, 10 mM KCl, 1 mM EDTA, 2 mM dithiothreitol, 0.25 M sucrose). The 100,000 × g pellet was
resuspended in 100 µl of Tricine buffer. The lipid body preparation
was microcentrifuged at 14,000 × g for 15 min. The
floating layer was left undisturbed, and the solution below the layer
was removed with a fine needle. The microcentrifugation was performed
several times to concentrate the lipid bodies to a final volume of 100 µl.
An alternative method of subcellular fractionation was carried out. Yeast cells collected by centrifugation (3,000 × g for 5 min) were mixed with cold, acid-washed glass beads and homogenized with a mortar and pestle in a cold room. TPS buffer was added to make a suspension. The suspension was centrifuged (1,000 × g for 5 min) to yield a crude homogenate. The crude homogenate was subjected to a similar procedure of subcellular fractionation by centrifugation, as described in the preceding paragraph.
Preparation of Oleosin for in Vitro Binding TestMaize oleosin, in a 1:1:2 (w/w) mixture of three isoforms of 18, 17, and 16 kDa, was prepared from oil bodies isolated from maize kernel (9). The oil body fraction, in 0.15 M Tricine-KOH, pH 7.5, 1 mM MgCl2, 10 mM KCl, 1 mM EDTA, 2 mM dithiothreitol, 0.25 M sucrose, was mixed with an equal volume of diethyl ether to remove the neutral lipids. The ether extraction was repeated twice. The remaining suspension of oleosin and PL was sonicated and used (as preparations a and b under "Results"). An aliquot of the suspension was subjected to a procedure for PL removal using chloroform and methanol as described earlier (9). After removal of the PL, the remaining oleosin fraction was resuspended in either water or a solution containing 0.125 M Tris-HCl, pH 8.0, 1 mM EDTA, and 0.1% SDS, and the water suspension was sonicated and used (as preparations c and d, respectively, under "Results"). Sonication of each of the above suspensions was performed with a 4-mm diameter probe in a Braun-Sonic 2,000 ultrasonic generator (Freeport, IL) with a digital meter reading of 100 for three 20-s periods.
Oleosin synthesized in yeast strain transformed with pQC6-ole was prepared as follows. The transformed yeast cells were homogenized with glass beads, and the lipid bodies were isolated as described in the preceding section. The lipid body fraction was treated with diethyl ether to remove the neutral lipids as mentioned in the preceding paragraph. The resulting suspension of the 18-kDa oleosin, other proteins, and PL was sonicated and used (as preparation e under "Results"). An aliquot of the lipid body fraction was subjected to SDS-PAGE. After electrophoresis, the gel containing the oleosin was cut, and the protein in the gel was eluted into a solution containing 0.125 M Tris-HCl, pH 8.0, 1 mM EDTA, and 0.1% SDS. This suspension was used (as preparation f under "Results").
In Vitro Binding TestThe oleosin preparations were assayed
for their protein contents using the Bradford method (28). The amounts
of 18-kDa oleosin in the preparations were estimated by the amount of
this oleosin in comparison with those of other proteins of the
preparations in an SDS-PAGE gel. Each oleosin preparation was incubated
with the homogenate (1:50 v/v) of nontransformed yeast strain (prepared as described under "Subcellular Fractionation") by shaking in a
horizontal shaker at 200 rpm for 30 min in a cold room. The mixture was
then subjected to subcellular fractionation (described under
"Subcellular Fractionation"). In each mixture, the proportion of
18-kDa oleosin to homogenate was similar to that in the homogenate of
yeast strain transformed with pQC6-ole (to be described in Fig. 5),
except in oleosin preparation b, in which the oleosin:homogenate ratio
was five times higher.
Determination of Lipids
Yeast cells were collected by centrifugation from the culture at different stages of growth. The volume of the pelleted cells was considered as 1 volume. The sample was mixed with 5 volumes of TS buffer containing 2 µg/µl lyticase and incubated at 30 °C for 2 h. The resulting spheroplasts were harvested and lysed by osmotic breakage as described in the preceding section. The lysate was extracted immediately with an equal volume of diethyl ether. The ether fraction was collected, and the ether was evaporated under a stream of nitrogen gas. The acyl esters in the residual lipids were quantitated (29).
Isolated yeast lipid bodies were extracted with diethyl ether three times. The acyl esters of the extracted lipids were quantitated similarly (29). The lipids were resolved by TLC, using a plate coated with silica gel 60A (Whatman, Maidstone, United Kingdom). The plate was developed in hexane/diethyl ether/acetic acid (80:20:2, v/v/v), dried, and allowed to react with iodine for color development.
ImmunocytochemistryYeast strains nontransformed and transformed with pQC5-ole and pQC6-ole were grown to an A600 nm of 1.9. The culture was centrifuged at 3,000 × g for 5 min to pellet the cells. The pelleted cells were rinsed twice with water and resuspended in half-strength Karnovsky's solution at 4 °C (30). After 0.5 h and mild agitation, the solution was replaced with half-strength Karnovsky's solution, and the mixture was incubated for 12 h at 4 °C. The cells were washed twice with cold, 50 mM NaP buffer (pH 6.8). The preparation was rapidly dehydrated (in 1 h) in a series of ethanol solutions to 95% ethanol. The cells were infiltrated with L.R. White resin over 12 h and allowed to polymerize at 60 °C for 30 h.
Sections of 100 nm thickness were cut on a MT6000 Ultramicrotome (RMC Inc. Tucson, AZ) and picked up on nickel (300 mesh) grids. All subsequent procedures were carried out in 75-µl droplets in ceramic wells. Grids were submersed for 30 min in a solution of 150 mM NaP buffer, pH 6.8 (PBS), 0.1% Triton X-100, and 0.1% BSA. Grids were incubated for 1 h in anti-oleosin polyclonal chicken antibodies (27) diluted 1:500 with PBS, 0.1% Triton X-100, and 0.1% BSA. Grids were washed five times, each for 1 min, in PBS, 0.1% Triton X-100, and 0.1% BSA, and then incubated for 1.5 h in rabbit anti-chicken IgG conjugated with 12-nm colloidal gold particles (Jackson Immuno Research Laboratory, West Grove, PA) diluted 1:40 with PBS, 0.1% Triton X-100, and 0.1% BSA.
Grids were poststained for 15 min in 1% aqueous uranyl acetate and for 2 min in Reynold's lead citrate. Photographs were taken using Kodak 4489 Electron Microscope film (Eastman Kodak Co.) on a Philips 400 transmission electron microscope at 100 kV.
The coding region of a maize seed oleosin gene was incorporated into two yeast plasmids in which its expression was under the regulation of GAL1 promoter (Fig. 1). A low copy number centromeric plasmid (pQC5-ole) and a high copy number 2-µm plasmid (pQC6-ole) were constructed.
Untransformed yeast strain and yeast strains transformed with the above
two plasmids and with the same plasmids but harboring no maize oleosin
gene were grown in a medium containing galactose. Total RNAs were
extracted from these yeast strains at the mid-log phase of growth and
subjected to Northern blot hybridization using a
32P-labeled maize oleosin cDNA probe (Fig.
2). Oleosin mRNA, of about 0.8 kilobase pairs, was
present in yeast strains transformed with pQC5-ole and pQC6-ole and was
substantially more abundant in the latter strain. Yeast strain that was
not transformed or transformed with pQC5 or pQC6 (plasmid without
oleosin gene) did not contain oleosin mRNA. When yeast strain
transformed with pQC6-ole was grown in glucose instead of galactose, it
did not contain oleosin mRNA. These findings indicate that the
oleosin gene in yeast strain transformed with pQC6-ole was properly
expressed under the control of the GAL1 promoter.
The crude extracts of the above yeast strains were analyzed for their
contents of oleosin protein by SDS-PAGE and immunoblotting, using
antibodies against the maize oleosin (Fig. 3). The
preimmune IgY and antibody-containing IgY did not recognize any protein of the yeast strains not transformed or transformed with control plasmids (pQC5 or pQC6). The antibodies recognized a protein of 19 kDa
in yeast strains transformed with pQC5-ole and pQC6-ole, but they did
not recognize any other protein on the blot. This 19-kDa protein was
not recognized by the preimmune IgY. Although the 19-kDa protein in
yeast strain containing pQC6-ole was easily detectable, its presence in
yeast strain containing pQC5-ole was barely observable by the eye but
not observable after photography (Fig. 3). This 19-kDa protein should
be the oleosin derived from the maize gene in the plasmids. Apparently,
both oleosin mRNA and protein were stable in yeast strains
transformed with pQC5-ole and pQC6-ole.
Oleosin present in the two transformed yeast strains had an Mr of about 19,000, and no apparent breakdown products of oleosin of lower Mr were detected by immunoblotting (Fig. 3). In the preparation of crude extracts (shown in Fig. 3), the yeast cells were heated to 100 °C to prevent proteolysis and then homogenized. Breakdown products of oleosin were observed when the cells were treated with a commercial lyticase preparation and lysed by osmotic shock (to be described). Oleosin in transformed strains (Mr about 19,000) was larger than the native oleosin in maize (Mr about 18,000) (Fig. 3). The Mr of the maize oleosin deduced from the gene coding sequence was 18,615 (21). In maize, as well as in other plant species, oleosin synthesized in vivo does not undergo a co- or posttranslational processing that leads to an appreciable modification of the Mr. Nevertheless, oleosins isolated from different plant species all had their N termini blocked, as revealed in several reported failures of direct sequencing of the N termini (1). These findings show that in vivo modification of the protein in plant seeds did occur but did not lead to an appreciable change in Mr. Apparently, processing of newly synthesized oleosin mRNA or protein in transformed yeast is different from that in plants.
The yeast strain transformed with pQC6-ole produced a higher amount of oleosin, and we used this yeast strain to explore biochemically the expression of the oleosin gene and the subcellular location of the oleosin protein.
Oleosin Accumulated Concomitantly with Lipids in Transformed Yeast during GrowthYeast accumulates lipids (TAGs and steroid esters) as food reserves during growth. In the current study, yeast strains nontransformed and transformed with pQC6-ole or pQC6 grew in galactose and accumulated lipids by an indistinguishable pattern (data not shown). The accumulation of lipids in these strains lagged behind the increase in cell culture density (Fig. 4); this delay is expected for the accumulation of a food reserve. In the yeast strain transformed with pQC6-ole, the amount of oleosin per culture volume increased with time and appeared to follow that of the lipids (Fig. 4); the low sensitivity of the immunoblot assays prevents us from making a definite statement. Supporting evidence comes from the finding that the amount of oleosin per equal amounts of total cell proteins also increased with time (Fig. 4). One possibility is that oleosin was produced constitutively but did not accumulate (i.e. was degraded) in the absence of lipids. Further studies are required to examine this possibility. In all of the above yeast strains, cell growth was faster in a medium containing glucose than in one containing galactose (data not shown).
Subcellular Fractionation Revealed That Oleosin in Transformed Yeast Was Present Mostly in the Lipid BodiesWe explored the subcellular location of oleosin in the yeast strain transformed with pQC6-ole by fractionating the cell homogenate into various subcellular fractions and analyzing their oleosin contents. Cells at the late log phase (45 h, see Fig. 4) were harvested by low speed centrifugation. They were lysed by treatments with a lyticase preparation and followed by an osmotic shock. After cell breakage, the homogenate was centrifuged to yield fractions of 100,000 × g supernatant, 100,000 × g pellet, and floated lipid bodies. The crude homogenate, the culture medium (containing excreted proteins), and the various subcellular fractions were subjected to SDS-PAGE and immunoblotting with antibodies against the maize oleosin (Fig. 5). About 80% of the oleosin was recovered in the lipid body fraction, which contained substantially less than 1% of the proteins in the homogenate. The remaining oleosin was present in the 100,000 × g supernatant, and no oleosin was detected in the 100,000 × g pellet. Oleosin was absent in the medium, and thus the protein was not secreted. In this experiment, the oleosin was present as an intact form (Mr 19,000) and a degraded form (Mr 18,000). The degraded form apparently was produced not in vivo but in vitro during cell lysis (by ingredients in the lyticase preparation or internal proteases), because it was absent when we boiled the cells before cell breakage (Figs. 3 and 4) or lysed the cells rapidly using mechanical force (following paragraph).
We also studied the subcellular location of oleosin in the transformed yeast cells using an alternative procedure of cell breakage in which the oleosin was maintained intact. After being harvested by a low speed centrifugation, the cells were homogenized with glass beads using a mortar and pestle. The homogenate was subjected to a similar procedure of subcellular fractionation by centrifugation. As much as 80-90% of the oleosin could be recovered in the lipid body fraction, and the remaining oleosin was present in the supernatant and the pellet (Fig. 5). In this procedure of cell breakage, the percentage of oleosin recovered in the lipid body fraction varied, from 50 to 90%, apparently depending on the severity of the homogenization with the pestle. The advantage of using this procedure was that it generated no degraded oleosin.
The results obtained from subcellular fractionation using the two different procedures of cell breakage clearly show that oleosin in the cells of the yeast strain transformed with pQC6-ole was localized in the lipid bodies. Collaborative evidence comes from immunocytochemistry results (to be described).
Oleosin Did Not Alter the Lipids of the Transformed Yeast Lipid Bodies but Replaced Some of the Native Proteins Associated with the OrganellesLipid bodies isolated from yeast strains
nontransformed and transformed with pQC6-ole were subjected to lipid
extraction, and their lipid constituents were separated by TLC (Fig.
6). Two major neutral lipid constituents were resolved;
they were tentatively identified to be steroid esters and TAGs, as
reported earlier (5, 6). The lipid composition of the lipid bodies from
both yeast strains as resolved by TLC were indistinguishable (Fig. 6).
This finding, together with the identical patterns of cell growth and
lipid accumulation during culturing of the two yeast strains, shows
that the presence of oleosin in the yeast cell did not affect
appreciably the synthesis and accumulation of lipids in the lipid
bodies. Nevertheless, oleosin did affect the native yeast proteins in
the lipid bodies. The proteins in the lipid body fractions from the two
yeast strains were resolved by SDS-PAGE (Fig. 6). Equal amounts (acyl
ester bonds) of lipid bodies from the two yeast strains were loaded
onto the gel for a direct comparison. The proteins from these two
samples resolved in the gel would represent those from the same number
of lipid bodies, since the lipid bodies in both yeast strains appeared
to be of a similar size (next section). The organelles from the
nontransformed yeast strain contained several proteins, of
Mr in the range of 30,000-75,000 (Fig. 6). This
pattern of proteins associated with the lipid body fraction of yeast
was similar to that reported earlier (6). In the transformed yeast
strain, although the native proteins were still present, their amounts
were reduced to about half of those in the nontransformed yeast strain.
The loss of the native proteins apparently was compensated by the gain
of a similar amount of oleosin, and the protein-to-lipid ratios in the
lipid bodies from the two yeast strains remained about the same. Thus,
replacement of half of the native proteins with an equal amount of
oleosin did not affect appreciably the integrity and synthesis of the organelles.
Immunocytochemistry Showed That Oleosin Was Present Mostly on the Periphery of the Lipid Bodies in the Transformed Yeast
Immunocytochemistry was performed to locate oleosin in the
yeast strain transformed with pQC5-ole and pQC6-ole, and the
nontransformed yeast strain was used as a control. The lipid bodies in
the cells of these yeast strains were electron transparent, of
diameters about 0.2-0.5 µm (Fig. 7). They were
usually present in clusters and apparently did not coalesce. There were
interfacial materials on the periphery of the lipid bodies. In the
immunodetection of oleosin in the yeast strains, the nontransformed
strain showed no immunogold particles in the cytoplasm including the
lipid bodies, whereas yeast strains transformed with pQC5-ole and
pQC6-ole had numerous immunogold particles, which were concentrated on
the lipid bodies. There were more gold particles in the yeast strain transformed with pQC6-ole (high copy number plasmid) than in that with
pQC5-ole (low copy number). Enlarged views of the cells showed that
most of the immunogold particles were restricted to the periphery of
the lipid bodies (Fig. 8). Under the electron
microscopy, the vicinity of the lipid body surface was often sectioned,
and the tangential areas were exposed and heavily labeled. The
immunocytochemical observation complements the biochemical findings
that most oleosin was associated with the lipid bodies and,
specifically, was present on the periphery of the organelles.
By SDS-PAGE and immunoblotting (Fig. 3), we barely detected the oleosin in yeast strain transformed with the low copy number plasmid pQC5-ole. By immunocytochemistry, we were able to observe in this yeast strain numerous immunogold particles that were concentrated on the lipid bodies (Fig. 8). Apparently, immunocytochemistry was a more sensitive method of detection.
Oleosins Did Not Bind to Yeast Lipid Bodies in VitroWhether oleosin synthesized by transformed yeast strains bound to the lipid bodies in vivo merely because of its hydrophobicity was investigated. Maize oleosin was incubated with the homogenate of nontransformed yeast strain, and the mixture was then subjected to subcellular fractionation. Six different preparations of oleosin were used. (a) Oleosins in three isoforms, of 16, 17, and 18-kDa, together with the native PL, were obtained from isolated maize oil bodies and sonicated into a suspension; (b) same as a except the amounts of all components were five times higher; (c) same as a except the PL were removed by chloroform/methanol; (d) same as c except the oleosins were not sonicated but resuspended in 0.1% SDS; (e) yeast-synthesized oleosin, together with other proteins and PL, was obtained from lipid bodies isolated from pQC6-ole transformed yeast strain and sonicated into a suspension; and (f) yeast-synthesized oleosin was obtained from lipid bodies isolated from pQC6-ole transformed yeast strain by SDS-PAGE and resuspended in 0.1% SDS.
In each of the mixtures, the proportion of 18-kDa oleosin to homogenate
was similar to that in the homogenate of yeast strain transformed with
pQC6-ole (see Fig. 5), except for oleosin preparation b, in which the
proportion of oleosin to homogenate was 5 times higher. Fig.
9 shows results of the subcellular fractionation. In
each of the six mixtures, the in vitro applied oleosin was not found in the isolated lipid bodies. Instead, it was present in the
pellet fraction and, in most mixtures, also in the supernatants. Oleosin in the pellet should represent molecules that had aggregated, presumably because of their hydrophobicity. Oleosin in the supernatant should be in a non-aggregated form, of individual molecules alone or
associated with PL or other amphipathic molecules, such that it was not
pelleted. Oleosin in this form was present in the mixture during its
incubation with the homogenate and did not bind to the lipid bodies
in vitro. This observation reiterates that the oleosin
present on the lipid bodies in transformed yeast represents the
consequence of a specific in vivo targeting event.
Yeast transformed with a plant oleosin gene synthesizes oleosin,
which remains stable in the cell. In addition, the oleosin is correctly
targeted to the lipid bodies. The targeting appears to be a specific
in vivo event. In a transformed yeast strain that contains a
high amount of oleosin, we have demonstrated by biochemical means that
the oleosin is present mostly in the lipid bodies. This localization
does not appear to be a fortuitous association of the abundant foreign
hydrophobic protein with the lipid bodies because we have also shown by
immunocytochemistry that a transformed yeast strain containing a
minimal amount of oleosin has the oleosin present exclusively in the
lipid bodies. Oleosin is present only on the amphipathic surface of the
lipid bodies rather than on other amphipathic membranes. Also, oleosin
prepared in different forms, including solitary molecules and those
associated with PL and SDS, fails to bind to the yeast lipid bodies
in vitro. The targeting signal in oleosin is unknown,
although apparently it is not the C-terminal -helix (31, 32). What
is known is that oleosin is synthesized on bound polyribosomes without
appreciable co- or posttranslational processing (11). Whether the
targeting signal resides on the N terminus or the long central
anti-parallel
-stranded hydrophobic domain, or both, and what role
the ER plays in directing the oleosin to the budding oil body remain to
be elucidated. It is possible that the targeting signal and the
stability of the oleosin on the lipid body together contribute to its
stable association with the organelles.
It is tedious to study the targeting signal in oleosin by transforming plants with numerous modified oleosin genes. The current study offers the opportunity of using yeast as a heterologous system to analyze in detail the targeting signal in oleosin via in vitro mutagenesis of an oleosin gene. Plants and yeast share many intracellular targeting signals in proteins, although yeast does not recognize the targeting signal in plant storage protein for the vacuoles (19, 33). One should analyze extensively the targeting signals in the oleosins for the yeast lipid bodies and then test the validity of selected results with the tedious plant transformation system.
In plants, oleosin exists in two isoforms, which apparently occur as a heterodimer or heteromultimer on the surface of the oil bodies (26, 27). The current study shows that one oleosin isoform can be present alone on the organelle surface (i.e. the maize isoform 18-kDa oleosin used in the current study is present alone without its counterpart isoform 16-kDa oleosin). Thus, dimerization does not appear to play a role in targeting the oleosin to the yeast lipid bodies.
The plant oil bodies and the yeast lipid bodies are similar in their
apparent structure and function. Whether they possess some differences
remains to be explored. A plant oil body has a matrix of TAGs
surrounded by a layer of PL embedded with unique and abundant oleosins
(1). This organization allows the stable association of all the
molecules involved, such that numerous oil bodies of small sizes can be
maintained in the cytosol. On the basis of this concept, we visualize a
yeast lipid body to have the abundant hydrophobic TAGs and steroid
esters located in the matrix and the amphipathic PL and proteins (of
minimal but sufficient quantities (5, 6)) at the periphery. Unlike oleosins in plant oil bodies, the proteins in yeast lipid bodies are of
numerous molecular species (Fig. 6); whether these proteins serve as
the structural proteins is unknown. Isolated yeast lipid body fractions
contained diacylglycerol acyltransferase (34) and sterol
24 methyltransferase (6), which catalyze the last steps
of TAG and steroid ester synthesis, respectively. The activities of
these two enzymes in the lipid body fractions were high in terms of specific activities (on per mg of protein basis) but low in terms of
percentage of total cellular activities (because there were minimal
amounts of proteins in the lipid body fractions). Whether these enzymes
are authentic proteins of the lipid bodies needs to be elucidated. In
plants, diacylglycerol acyltransferase of a high specific activity but
of a low percentage of total cellular activity was reported to be
present in an isolated oil body fraction; it was subsequently shown to
be a contaminant from the ER (1, 3). Thus, we do not know whether in
yeast TAGs and steroid esters are synthesized in the ER, as has been
shown for the plant TAGs, or directly on the surface of existing lipid
bodies, as has been suggested on the basis of the high enzyme specific
activities. During utilization of the oil reserves in plants, lipase is
newly synthesized and binds to the oil bodies (35). In yeast, steroid ester hydrolase activity was detected in many subcellular fractions (36). Whether the activity belonged to one or more enzymes and which of
these enzymes, if any, mobilized the storage steroid esters remain to
be studied. Plant seeds contain several acyl hydrolases, which are
known not to be responsible for the mobilization of the storage oils
(1). Even less is known about the catabolism of TAGs in yeast lipid
bodies. The abundant information on the structure, function, and
ontogeny of plant oil bodies, both in concept and study techniques,
should be utilized for detailed studies of yeast lipid bodies.