Vol.2, No.12, 1437-1447 (2010) Health
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Cell membranes composition is defined in ER and their
restitution proceed by en bloc fusion of ER generated
transport vesicles
Amalia Slomiany*, Bronislaw L. Slomiany
Research Center, University of Medicine and Dentistry of New Jersey, Newark, USA; *Corresponding Author: slomiabr@umdnj.edu
Received 28 September 2010; revised 19 October 2010; accepted 25 October 2010
The synthesis of endoplasmic reticulum (ER)-
derived transport vesicles is dictated by the
contents and derivation of the cellular cytosol.
The ER transport vesicles synthesized in the
presence of gastric epithelial cells cytosol are
destined for en bloc fusion with apical epithelial
membrane, whereas those generated in hepa-
tocytes-derived cytosol are destined for en bloc
fusion with basolateral membrane. Moreover,
during assembly of the dominant fraction of the
apical or basolateral transport vesicles, a sub-
stantial fraction of the vesicles is produced that
fuses with endosomes, and the vesicles with still
unknown destination that remain in cytosol. The
process of ER vesicles synthesis is blocked by
RNase treatment, whereas Golgi vesicles as-
sembly is not affected. The experiments indicate
that transport vesicles’ membrane composition
and fidelity of its construction is defined in ER.
The process involves synchronous membrane
lipid synthesis, cotranslational intercalation of
integral membrane proteins and containment of
the vesicular cargo.
Keywords: Membrane Biogenesis; Repair Fid elity;
Vesicular Transport; En Bloc Fusion
Numerous comprehensive reviews of cellular trans-
port provide detailed view of the proteins involved in
vesicles targeting and membrane fusion [1-8]. Neverthe-
less, the fundamental problem of membrane fusion still
remains a puzzle that has yet to be solved in any biolog-
ical system [9,10]. Thus far, the envisioned schemes do
not provide complete picture and explain how the diver-
sity of cellular membranous structures is maintained or
how the cell membranes are repaired and their compo-
nents restored [3,10,11]. In the proposed concepts of the
vesicular transport, the endoplasmic reticulum (ER) is
depicted as a protein synthetic and folding site apart
from its role in the vesicular membrane lipid synthesis
and membrane assembly that includes insertion of the
integral proteins dedicated for the specific vesicle and
destination site. In our view, the independent of each
other lipid assembly into the membrane and protein
translation and the capture of preassembled membranes’
proteins [2-5,12,13] is lacking precision that is critical to
retain cell characteristic features [2-4,14]. It is also not
plausible that preassembled or recycled vesicular carriers
could attain the degree of the reproducible fidelity that is
achieved when protein translation, membrane lipid syn-
thesis and vesicle formation occur in the synchrony and
is confined to a specific segment of ER. Moreover, the
lipid microenvironment that is required for the precise
function of membrane integral protein can only be re-
produced when the process involves simultaneous mem-
brane lipids synthesis, protein intercalation and en bloc
membrane replacement [14-17].
Our previously published studies of apical transport in
gastric mucosal epithelial cells demonstrated synchrony
in the assembly of transport vesicles membrane and
synthesis of apoprotein (apomucin) cargo [18,19]. Gas-
tric mucosal epithelium elaborates enormous quantity of
glycoprotein (mucin) and requires just as large synthetic
capacity to generate membranes for the vesicles to move
the cargo from ER to Golgi and to the apical cell mem-
brane. In the conducted studies by tracing labeled pro-
teins and lipids constituting the vesicular membrane and
secretory cargo we revealed that in ER newly synthe-
sized apomucin was packaged into vesicles generated
from newly synthesized membrane that contained apical
membrane-specific integral membrane proteins and li-
pids [15-21]. The newly ER-synthesized transporting
units fused with Golgi and underwent further processing
consisting of cargo glycosylation and vesicles’ mem-
brane destination-specific phosphatidylinositol- (PI) and
A. Slomiany et al. / Health 2 (2010) 1437-1447
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the ceramides (Cer) modification. PI was phosphorylated
to phosphatidylinositol 3- and 4-phosphates (PI3P and
PI4P) and Cer used for assembly of sphingomyelin (SM)
and glycosphingolipids (GSL) [15-17]. The vesicles
containing GPI-anchored mucin binding protein (MBP),
PI3P and mucin cargo fused with apical cell membrane
and the Golgi vesicle’s membrane incorporated en bloc
into apical epithelial continuum [15-17]. The en bloc
incorporation of the vesicular membrane into apical
membrane continuum was manifested by inclusion of
the labeled lipids that comprised the vesicle into the
apical membrane and was coupled with formation of
lyso-PI3P and generation of arachidonate [15]. Moreover,
the Golgi-modified vesicles that would not fuse with
apical membrane contained PI4P and portion displayed
affinity for endosomes [16,17].
Our studies presented here demonstrate the transfer of
ER-initiated, radiolabeled vesicular membranes to baso-
lateral site of the hepatocytes. The hepatocytes
ER-derived transport vesicles fuse with basolateral
membrane contain PIPs but their membranes are devoid
of glycosphingolipids and PI3P, and they do not fuse
with gastric epithelial apical membrane. Compositional
differences between lipids of ER membrane, apical and
basolateral cell membranes [18-20] and en bloc interca-
lation of lipids and membrane protein into specific site
of the cell membrane which is synchronized with apical
or basolateral secretion [15-17] support the concept that
transport vesicles are precisely fashioned in ER and de-
livered to the specific site for the precise membrane res-
titution and the cell function. The stereotypic view that
cellular vesicles capture protein, discharge it and return
for the next tour of transport [2] is insensitive to the
functional relevance of the precise intercalation of
membrane proteins within specific membrane lipids. The
precise intercalation of membrane-spanning proteins can
only be achieved during synchronized protein translation
and ER-membrane lipids biogenesis.
2.1. Materials
Radiolabeled precursors of phospholipids, glycolipids,
glycoproteins and other radiochemicals were purchased
from New England Nuclear (Boston, MA). Phospholipid
standards were from Avanti (Birmingham, AL) Matreya
(Pleasant Gap, PA) or prepared in our laboratory. Crea-
tine phosphokinase, phenylmethylsulfonyl fluoride
(PMSF), aprotinin, pepstatin, leupeptin, ATP, CTP, GTP,
fatty acyl CoA, glycerol 3-phosphate, RNase, and RNase
free sucrose were purchased from Sigma Chemicals (St.
Louis, MO). Polyacrylamide gel electrophoresis reagents
were from Bio-Rad (Rockville Centre, NY). All other
chemicals and reagents were purchased from J.T. Baker
Chemical Co. (Phillipsburg, PA) Fisher Scientific
(Springfield, NJ) and VWR Scientific (Piscataway, NJ).
The anti-rat albumin antibodies were purchased, and the
antimucin antibodies were prepared in our laboratory
2.2. Liver Perfusion Buffers
a) Buffered saline, with 10 mM potassium phosphate,
pH 6.8, b) buffered saline containing 0.5 mM MgCl2
0.5 mM MgS04, c) buffered saline (100 ml) containing
66 mg collagenase, 80 mg hyaluronidase, and 2 g of
albumin, d) MSB, pH 6.9 buffer consisting of 0.1 Pipes,
pH 6.9, 2.0 M glycerol, 1mM Mg acetate, 0.5 mM EG-
TA and mixture of protease inhibitors consisting of leu-
peptin, aprotinin and PMSF, e) MSB buffer containing
0.2 % Triton X100, f) 50 mM TRIS-HCl, pH 7.4 con-
taining 0.25 M sucrose, 10 mM MgCl2 1 mM DTT,10
mg/ml leupeptin and 2 mM PMSF.
2.3. Perfusion of Rat Liver and Isolation of
The abdomen of anestetized rat was open and liver
cannulated and perfused with 50 ml of ice cold Hanks
Balanced Salts (Sigma) without Ca2+containing 20 mg
collagenase, type IV, 20 mg hyaluronidase, 1 g defatted
albumin, and 3 g of heparin. After initial perfusion, the
liver was removed from animal’s abdomen, cut into
slices and incubated with cold Hanks solution. Thus
prepared slices were subjected to incubation in tissue
incubator in 95% 02 and 5% C02 for 40 min, at 37ºC. The
slices were broken up with rubber policemen, incubated
for additional 10 min and filtered through nylon mesh
that separated single cells from larger debris. The cells
were then centrifuged at 50xg for 2 min, washed twice
with the enzyme-free Hanks medium, twice with the
Minimum Essential Medium and counted in hemocyto-
meter. Thus prepared cells were used for preparation of
nuclei [24] subcellular organelles, cell cytosol [15,19,20]
and cellular membranes [15,16]. In the experiments
dedicated to the determination of lipid synthesis with
cell cytosol derived from gastric epithelial cells, or
RNase treated cytosol, the preparations of ER, Golgi,
endosomes or nuclei were additionally rinsed with PBS
and urea-PBS in order to remove the residual cytosolic
proteins. Thus prepared subcellular organelles and
membranes were used for experiments on transport ve-
sicles synthesis and fusion [15] and preparation of outer
nuclear membrane (ONM) and the Inner Nuclear Mem-
brane (INM) [24]. The synthesis of phosphatidylinosi-
tides, phospholipids and protein was determined using
radiolabeled [3H] inositol, [3H]arachidonate, [3H]choline,
[3H]serine, [3H]palmitate and [32P]ATP [15-20]. The
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synthesis of transport vesicles (ER, Golgi) was per-
formed in medium containing cytosol at concentration of
5 mg protein/ml of incubation mixture enriched with 50
mM ATP, 250 mM CTP, 50 mM GTP, 5 mM creatine
phosphate, 8.0 IU/ml creatine kinase, and where indi-
cated 25 mg/ml RNase, 10 mM UDP-Glc and 10 mM
palmitoyl CoA [15-20]. The same preparation of cytosol
was used in the experiments with nuclear membranes
2.4. Isolation and Separation of ER, Golgi,
Endosomes, Cellular and Outer and
Inner Nuclear Membranes
One volume of the isolated hepatocytes was homoge-
nized in 8 volumes of 10 mM potassium phosphate buf-
fer, pH 6.8, 1.3 M sucrose and 1mM MgCl2 to rupture at
least 80 % of cells [24]. The unbroken cells were re-
moved by centrifugation at 50xg for 3 min, and the ho-
mogenate centrifuged for 15 min at 1000g. The soluble
cellular material was saved for isolation of cellular or-
ganelles and cytosol while the nuclear pellet was
processed further and the Outer and Inner Nuclear
Membranes (ONM, INM) were collected [24].
The isolated on sucrose gradient hepatocytes membranes
were used for fusion experiments with Golgi-derived
transport vesicles. Following fusion, the apical portions
of the membrane were separated by dissolving the baso-
lateral membranes with aid of cold 0.2% Triton X100,
and recovering the apical fraction enriched in glycos-
phingolipids and glycoprotein through floatation on su-
crose gradient [17,23].
2.5. Prep aration of Transport-Active Cytosol
The viable hepatocytes (for hepatocytes cytosol) or
gastric mucosal epithelial cells (for gastric epithelial
cells cytosol) were homogenized for 10 sec at 600 rpm
in 3 volumes of buffer containing 0.25 M sucrose; 50
mM TRIS-HCl (pH 7.4); 25 mM magnesium acetate and
10 mM each of aprotinin, leupeptin, chemostatin; and 1
mM phenylmethylsulfonylfluoride. The homogenate was
centrifuged at 5,000 x g for 15 min, the supernatant di-
luted with 2 volumes of homogenization buffer, and cen-
trifuged at 10,000g for 20 min. The resulting supernatant
was then subjected to centrifugation at 100,000g for 1h.
Thus obtained soluble fraction was adjusted to 15 to 18
mg protein/ml; admixed with an ATP generating system
consisting of 40 mM ATP, 200 mM creatine phosphate,
2,000 units/ml creatine phosphokinase, and referred to as
transport active cytosol or active cytosol.
2.6. Preparation of Cellular Membranes
The cell membranes and subcellular organelles (ER,
Golgi) were recovered from the sediment resulting from
centrifugation at 100,000g. After removing supernatant,
the transport active cytosol, the residue was suspended
in buffer containing 0.2 M PIPES (pH 6.9), 2 M glycerol,
1 mM EGTA and 1 mM magnesium acetate and applied
on the top of discontinuous gradient of 2.0/1.5/1.3/1.0 M
sucrose and centrifuged at 100,000g for 16h. The cell
membranes were recovered from 1.0 M sucrose, Smooth
Endoplasmic Reticulum (SER) from 1.3 M sucrose,
RER from 1.5 M sucrose and Golgi from the top of the
2.0 M sucrose. Each sucrose-separated fraction was sub-
jected to further purification. The cell membranes were
washed with original Pipes buffer and centrifuged at
3,000 rpm for 2 min. To separate apical epithelial mem-
branes, the buffer was adjusted with 0.2% Triton X-100
and the mixture incubated at 4°C for 5 min [23]. This
treatment resulted in dissolving the membranes that were
free of glycosphingolipids and membrane glycoproteins.
The membranes enriched in glycosphingolipids and
glycoproteins were recovered by low speed centrifuga-
tion at 3,000 rpm for 2 min. Thus prepared fractions of
cell membranes were used in fusion experiments with
Golgi-derived transport vesicles. The endosomes were
isolated from the mitochondria-enriched fraction that
sedimented at 10,000g [17].
2.7. Generation and Purification of ER- and
Golgi-derived Transport Vesicles
ER- and Golgi-derived transport vesicles were gener-
ated in the presence of radiolabeled precursors according
to procedure described previously [15-20]. The ER or
Golgi membranes incubated with cytosol, ATP-gener-
ating system, UTP, CTP GTP, fatty acyl CoA and water
soluble cold or radiolabeled lipids precursors were incu-
bated for 30 min at 37°C, centrifuged over 0.3 M sucrose
and treated with stripping buffer at 2°C for 15 min fol-
lowed by centrifugation at 10,000g for 10min. to separate
transport vesicles from ER or Golgi membranes. The se-
parated from maternal membranes transport vesicles were
recovered from the supernatant resulting from centrifuga-
tion of the supernatant mixture at 150,000g for 60 min.
The crude fraction of the transport vesicles was suspended
in 55% sucrose, overlaid with 55-30% gradient and cen-
trifuged at 150,000g for 16 h. The purified transport ve-
sicles were recovered from the gradients as shown in
Figure 2.
2.8. Fusion Assays of Golgi-derived
Transport Vesicles with Cell
One volume of Golgi transport vesicles (1.3-1.5 pro-
tein/ml) was suspended in one volume of active cytosol
(15mg protein/ml and added to one volume of cell
A. Slomiany et al. / Health 2 (2010) 1437-1447
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membranes (8 mg protein/ml of whole cell membranes
or 2 mg protein/ml of apical epithelial membranes). The
reaction was allowed to proceed from 0-30 min at 4°C
(control) and at 37°C in the presence of ATP regenerat-
ing system consisting of 40 mM ATP, 200 mM creatine
phosphate, 2,000 units /ml of creatine phosphokinase, or
in the ATP depleting system containing 5 mM glucose
and 500 units/ml hexokinase. After incubation, the
membranes were recovered by centrifugation through
three volumes of 0.5 M sucrose at 3,000 rpm for 5 min.
The free vesicles were recovered from the supernatant
and used in fusion experiments with endosomes. The
cell membranes sedimented through sucrose were
washed with 25 mM Hepes-KOH buffer and treated for
5 min with 0.2 % Triton X-100 at 4°C. The soluble frac-
tion of the membrane was recovered in supernatant,
whereas apical the glycosphingolipids- and glycoprote-
in- containing membranes were in sediment. In the ex-
periments estimating en bloc fusion of transport vesicles
with the membrane, the associated but not fused vesicles
were released from the membrane by subjecting the
membrane fraction to treatment with 2 M urea for 30
min at 4°C and then the recovered membranes were cen-
trifuged through 0.5 M sucrose, washed and subjected to
lipid analysis.
2.9. Lipid Analysis
Preparations of ER, Golgi membranes, their transport
vesicles, the membranes recovered after incubation with
Golgi vesicles and the endosomes incubated with the
vesicles recovered after fusion with cell membranes
were subjected to lipid analysis. The lipid extracts and
high performance thin layer chromatography were per-
formed exactly as described in our previous studies
[15-20]. Formation of PIPs was ascertained by perform-
ing ER transport vesicles synthesis in the presence of
[3H]inositol or [3H]arachidonate followed by Golgi ve-
sicles formation either in the presence or the absence of
[32P] ATP [15]. Because the fusion of transport vesicles
was aided by PLA2-specific hydrolysis of the membrane
lipids, the resulting membranes were analyzed to dem-
onstrate release of the [3H]arachidonate and formation of
lysophosphatidylinositols (LPIs).
Our studies on homeostatic restitution of cellular and
subcellular membranes showed that vesicular intracellu-
lar transport is engaged in systematic and coordinated
replacement of lipids and proteins in the membranes of
the secretory, non-dividing epithelial cells, whereas res-
titution of lipids in the nuclear envelope biomembrane
proceeds without formation of transport vesicles [15-17,
Here, we concentrated on deciphering the assembly of
the biomembrane that delivers the ER products to Golgi
and then forms Golgi transport vesicles destined to baso-
lateral membrane of the hepatocytes. The biomembrane
of the hepatocytes-derived ER transport vesicles is syn-
thesized with the same phospholipids as gastric epithelial
ER vesicles, consists of phosphatidylcholine (PC), phos-
phatidylinositol (PI), phosphatidylethanolamine (PE) and
ceramide (Cer) and is free of sphingomyelin (SM) and
glycosphingolipids s (GSL) (Figure 1).
The assembly of the ER transport vesicles in hepato-
cytes and gastric epithelial mucosal cells was inhibited by
the treatment of the cell cytosol with RNase (Figure 2).
The attributes of the ER-initiated biomembrane and Golgi
transport vesicles derived from it are dictated by the cyto-
sol derivation (Figure 3). As demonstrated in Figure 3,
over 40% of Golgi transport vesicles that are initiated in
ER in the presence of hepatocytes-derived cytosol fuse
with basolateral membrane, only 11% with apical mem-
branes and remaining vesicles stay in the cytosol.
The experiments with ER transport vesicles generated
in the presence of gastric mucosal epithelial cell cytosol
demonstrate that 46% of the derived Golgi vesicles fuse
with apical membranes and only trace (1.5%) fuses with
basolateral membrane. In both systems about 50% of the
Golgi transport vesicles stay unbound in the cytosol.
Upon further incubation of the vesicles remaining in the
cytosol, prominent portion of the fraction (20%) fuses
with endosomes, whereas 24-27% of the transport ve-
sicles still remain free. Neither complete mixture of
Golgi transport vesicles prior reaction with cellular
membranes and endosomes, nor the portion remaining
after reaction
with those membranes displays any affinity for the
C /M/Acetic Acid/H
65/ 35/8/4
Figure 1. High Performance Thin Layer chromatography
(HPTLC) of the lipids extracted from the hepatocytes
ER-derived transport vesicles. The transport vesicle membrane
consisted of phosphatidylcholine (PC), phosphatidylinositol
(PI), phosphatidylethanolamine (PE) and ceramide (Cer). Nei-
ther sphingomyelin (SM) nor glycosphingolipids (GSL) were
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Synthesis of ER transport vesicles in hepatocytes
1 2 3 4 5 6 7 8 91011121314151617
Fraction number
RNAse (-)RNase (+)
Figure 2. Synthesis of ER-transport vesicles is inhibited by
treatment of the cytosol with RNase. The transport vesicles
were generated by the procedure described in Methods. The
vesicles that equilibrated between 45-50% sucrose (fraction
3-8) were recovered and their yield estimated by protein and
radioactivity determination.
Figure 3. Fusion of Golgi transport vesicles with cell mem-
branes is dictated by derivation of cytosol used for generation
of ER transport vesicles. The experiments were initiated with
ER-transport vesicles generated in the hepatocytes- or gastric
mucosal epithelial cells cytosol and continued through genera-
tion of Golgi transport vesicles. The latter were subjected to
fusion experiments with cold cell membranes, endosomes, ER,
intact nuclei (IN) Outer Nuclear Membrane (ONM) and Inner
Nuclear Membrane (INM). The experiments with fraction 4,
the unbound Golgi vesicles recovered from cytosol were not
fusing with ER, IN, ONM and INM are not presented.
association or fusion with ER, IN, ONM or INM. The
possibility that the remaining vesicles are specific for
fusion with mitochondria and lysosomes was not yet
explored. The results of the described experiments
demonstrate that RNA presence and derivation of the
cytosol are crucial at ER stage and determine ER trans-
port vesicles synthesis, composition and destination.
Moreover, the assembled ER products consist of assort-
ment of the vesicles that are destined to specific cell
membrane sites and cell organelles, but do not return to
ER or are involved in reconstitution of nuclear mem-
The results on Golgi-localized fine adjustments in the
ER-initiated biomembrane lipid composition of the gas-
tric and hepatocytes derived vesicles reveal the principle
differences in modifications of phospholipids that de-
termine destination-specific biomembrane (Figure 4).
As shown in Figure 4(a), ER-vesicles’-delivered PI is
subjected in Golgi to phosphorylation that affords three
PIP derivatives. Thus, Golgi transport vesicles that fuse
with gastric apical epithelial membrane contain PI and
PI3P, and upon fusion LPIP is produced in the apical
membranes (Figure 4(b)), the vesicles with affinity for
endosomes contain PI and PI4P (Figure 4(c)). The re-
maining vesicles (Figure 4(d)) contain PI, PI4P, and yet
unknown PIPs.
The Golgi transport vesicles generated from ER
transport vesicles assembled in the hepatocytes-derived
cytosol (Figure 5) contain PI and PIPs. The Golgi as-
sembled mixture of vesicles displays largely PI4P and
yet unidentified PIPs migrating on TLC between PI4P
PI, while PI3P represented minor fraction (Figure 5(a)).
Upon fusion, the basolateral membranes show presence
of PI, PI4P and PIP2 (Figure 5(b)), whereas the apical
membranes show presence of PI, PI3P and LPI3P (Fig-
ure 5(c)). The fraction of the vesicles which remains in
the cytosol after fusion with cellular membranes con-
tains mainly PI, PI4P and the fraction which co-migrates
in the area occupied by PI (Figure 5(d)). Since in all
experiments PI consisted of closely running doublet, it is
possible that this separation of PIs is due to specific fatty
The labeling of ER transport vesicles with [3H] ara-
chidonate and [3H] inositol allowed us to determine
whether basolateral fusion of transport vesicles proceeds
in similar fashion as apical and affords en bloc incorpo-
ration of the ER-assembled membrane. Results of these
experiments are shown in Figure 6. The panel (a) shows
TLC of the lipids extracted from basolateral membranes
following fusion with arachidonate- and inositol-labeled
Golgi transport vesicles. It demonstrates presence of
labeled phospholipids, which in chromatographic sepa-
ration utilizing solvent system consisting of ethyl ace-
tate/isooctane/acetic acid/water (90/50/20/100, by vol.),
appear as one fraction located at the origin of the plate
(1), the presence of free arachidonate (2) and arachido-
nate-containing glycerides (3). The panel (b) presents
separation of the phospholipids identified in panel (a) (1)
and shows presence of LPIP (1), PC (2) PIPs (3,4), PE
(5), PA (6) and yet unknown phospholipid identified in
position (7). Finally, panel (c) demonstrates spectrum of
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PIs that originates from [3H]inositol labeled PI of ER
transport vesicles that reached basolateral destination. It
Figure 4. Phosphorylation of ER transport vesicles PI in Golgi.
(a) Represents Golgi membranes after fusion with [3H] inositol
labeled ER transport vesicles. Four PI-derived phosphatidyli-
nositides were identified: 1-PI3P, 2-PI4P, 3-unknown PIP, and
4, -PI. (b) Golgi transport vesicles that fused with apical epi-
thelial membrane contained 1-PI3P, 4-PI, 5-LPI3P and 6-PIP2.
(c) Endosomal fraction following incubation with remaining
Golgi transport vesicles contained 2-PI4P, 4-PI, and traces of
PIP2. (d) Golgi transport vesicles that after incubation with
endosomes remained free in cytosol consisted of 2-PI4P, 4-PI,
and trace amount of 1,2- PIPs, 5-LPIPs and 6-PIP2. The sepa-
ration and quantitative analyses of polyphosphoinositides de-
picted in this figure and Figure 5 and 6 was performed in one
dimension on 1% ammonium oxalate impregnated 20x20 cm
thin layer chromatographic (TLC) plates developed in solvent
system consisting of chloroform/acetone/methanol/ glacial
acetic acid/water (40/15/13/12/8, by vol.) This procedure
yielded quantitative separation of PI, PIPs, and LPIP, and PIP2.
All TLC runs are compared with the elution profile of the [3H]
inositol labeled polyphosphoinositides identified in Golgi fol-
lowing fusion with ER-inositol labeled transport vesicles de-
picted in (a).
Figure 5. Phosphorylation of the hepatocytes ER transport
vesicles PI in Golgi. (a) Represents phosphatidylinositides in
Golgi-derived transport vesicles generated after fusion of [3H]
inositol labeled ER transport vesicles with Golgi and consists
of PIPs (1,2,3), PI (4), and PIP2 (6). (b) Golgi vesicles that
fused with basolateral membrane contained PI4P (2), PI (4)
and PIP2 (6). (c) The apical membrane fraction showed pres-
ence of PI3P (1), PI (4) and LPI3P (5). (d) The unbound ve-
sicles, which remained free in cytosol, contained traces of PI3P,
PI4P (2), PIP2 (6) and unidentified inositol-labeled lipid mi-
grating ahead of PIP2. The separation and identification of the
labeled PI lipids was as described for Figure 4.
demonstrates presence of LPIPs (1), PIP2 (2,3), PIPs
(4,5), PI (6) and yet unknown inositol-labeled lipid iden-
tified in position (7). Thus, the results presented above
provide evidence that basolateral fusion, just as apical,
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incorporates ER originated biomembrane targeted for
basolateral membrane of the hepatocytes. It results in en
bloc incorporation of the vesicular membrane (Figure 6)
Figure 6. Identification of [3H] arachidonate- and/or [3H] in-
ositol labeled lipids transferred via en bloc incorporated vesi-
cular membrane initiated in ER to hepatocytes basolateral
membrane. (a) Demonstrates fusion-induced release of arachi-
donate (2), the labeled phospholipids (1) and diacylglycerides
(3). (b) Depicts TLC separation of the phospholipids shown in
(A) position (1). The lipids consist of LPIP (1), PC (2), PIs (3),
PS (4), PE (5), PA (6) and unknown (7). (c) The [3H] inosi-
tol-labeled lipids identified in basolateral membranes follow-
ing en bloc fusion of ER-initiated transport vesicles consisted
of LPIP (1), PIP2 (2), PIP2 (3), PIPs (4,5), PI (6) and unknown
(7). The TLC solvent systems used are shown in each panel.
that is accompanied by generation of lysophospholipids
((1) in panel (b) and (c)) and arachidonate ((2) in panel
(a)), incorporation of phospholipids identified in panel
(b), and incorporation of the phosphorylation products of
ER-synthesized PI demonstrated in panel (c).
While the cytosol derivation impacts synthesis, com-
position and final destination of ER transport vesicles,
the Golgi-localized adjustments in spectra of transport
vesicles lipid modification are also destination sensitive.
In addition to PI phosphorylation, the synthesis of SM
and GSL is destination-specific. So much so, that in the
earlier studies we suggested that synthesis of the Cer
determined the quantity and destination of the transport
vesicles produced in gastric mucosal cells [15]. Indeed,
present study support that notion since the amount of
Cer in hepatocytes ER-transport vesicles is significantly
smaller (Figure 1) and apparently is attributed to higher
proportion of the vesicles with basolateral membrane
destination (Figure 3).
After basolateral, apical and endosomal fusion, the
significant amount of transport vesicles remaining in the
cytosol led us to further experimentation on the specific-
ity of transport in gastric mucosal epithelial cells and
hepatocytes. In all experiments with Golgi transport ve-
sicles derived from ER transport vesicles generated in
the hepatocytes- or gastric cells-derived cytosol the re-
maining radiolabeled transport vesicles (Figure 3, frac-
tion 4) in gastric cell cytosol or in hepatocytes cytosol
would not fuse with ER or IN, ONM or INM (not
shown). Since our earlier experiments with nuclear
membranes provided evidence that these membranes
biogenesis does not involve generation or release of
transport vesicles, and that their lipid composition is
different from Golgi transport vesicles, we concluded
that the remaining in cytosol vesicles do not provide
biomembrane for ER or nuclear membrane restitution. It
is quite possible that the fraction consist of transporters
dedicated for the mitochondrial or lysosomal membranes
renewal and cargo acquisition. Based on the above de-
scribed systematic fusion experiments with labeled ER-
and then Golgi-transport vesicles our strong contention
is that restitution of the organellar membranes does not
proceed through retrograde transport and re-utilization of
the ‘empty’ vesicles. The process is unidirectional and
that en bloc replacement of the ER-initiated membrane is
the quintessential asset for the accurate reproduction of
all cellular biomembranes.
Thus, from the studies presented here and the results
published earlier we conclude that vesicular transport is
dependent on the finely tuned synthesis of the
cell-specific proteins and lipids that assemble in ER into
the precise site-specific vesicular biomembranes [15-20].
Precise and explicit intercalation of the protein into the
membrane is only plausible when the translation of
mRNA and the synthesis of biomembrane proceed con-
comitantly and within the ER space capable to generate
specific lipids and translate specific mRNAs. Therefore,
the synthesis of ER transport vesicles is susceptible to
RNase treatment and the protein and lipids of a new
membrane determine the specific membrane maturation
in Golgi and control directional transport from Golgi to
specific sites of cell membrane and its organelles
Together, these results allowed us to hypothesize that
A. Slomiany et al. / Health 2 (2010) 1437-1447
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
the substrates in the in situ medium (the cell cytosol)
define the process of transport vesicles synthesis and
delivery to the next organelle, since depletion of mRNA
from either gastric or hepatocytes cytosol inhibits ve-
sicles synthesis that fuse with Golgi, and the process is
rescued with reinstatement of the initial cytosolic RNAs
[17]. The synthesis of the intracellular biomembrane and
the protein in ER must be initiated in a explicit site of
the ER organelle, the site that is capable to receive nuc-
lear transport of mRNAs to assemble appropriate quan-
tity of secretory and constitutive proteins, specifically
formulate biomembrane, and then deliver such packages
en bloc to Golgi and to final destination points.
As recent reviews of the advances in understanding of
the intracellular transport demonstrate, the investigations
focus on the role of numerous proteins present on trans-
port vesicles and their interaction with proteins project-
ing from the cellular membrane substrates
[1-6,8,14,25-29]. The sorting mechanisms presume that
for constitutive and regulated protein delivery individual
proteins or their domains precisely control cellular com-
partmental organization and the exquisite and precise
site-specific transfer of the products. In the illustrated
concepts, the entire emphasis is placed on the proteins
structure while the membrane lipids and the environment
that they create for the protein function is not considered.
It is assumed that the artificial lipid mixtures adequately
substitute the native lipids because the protein binding
through recognition of phospholipid-binding domain is
observed and the transport of the protein to its designat-
ed site is achieved [3,10,28]. At the same time, the
process allows to recover transporter and through retro-
grade movement use it for another round of ER products
delivery [29]. These theory-driven assumptions provide
illustrations of membranes with precisely intercalated
proteins where minute changes in protein structure may
impact process of fusion and membrane restitution,
whereas the supporting experiments determine only the
protein association with the membrane. Thus, it is ar-
guable whether such pseudo membranes depict factual
events and, whether mere closeness or even passive as-
sociation of two artificial membranes is recreated with
the fidelity that suffices the specific cargo delivery. It is
questionable whether the fundamental principle that
controls accuracy of the cell organelles and membranes
restitution is controlled by protein alone [1,2,9,28,29].
The decisive factor that argues against the principle role
of protein in cellular transport with the vesicles release
and use for another round of transport is the vesicles
synthetic complexity and the cellular need for metabolic
compensation and repair of the membranes [30-32].
In our opinion, that is based on numerous studies of
membrane biogenesis [15-20,24], the observed numer-
ous vesicles that are assembled in ER and delivered to
Golgi represent new cell-site specific transporters that
are produced simultaneously with their protein cargo,
and from Golgi are delivered to various sites for restitu-
tion and repair of the cell and its organelles [31,32].
Since, the 25 % of transport vesicles that remain in cy-
tosol after the major fractions fuse with apical, basola-
teral and endosomal membranes do not fuse with ER or
nuclear membranes, they may represent transporters
destined for mitochondria, lysosomes or other highly
selective sites of the cell which in our experiments were
not anticipated and presented [34,35]. The fact that radi-
olabeled lipids representing composition of these ve-
sicles could not be identified/incorporated into hepato-
cytes or gastric cells derived ER or nuclear membranes
argues against retrograde transport [3,6,10,33]. If the
retrograde movement was viable process, the labeled
lipids reflecting lipid composition of transport vesicles
would not be evident in the membranes recovered after
fusion, but instead be detectable in ER. In contrast, ER
synthesized membrane lipids assembled into membrane
of ER transport vesicles are carried to Golgi and from
Golgi to various sites of the cell where they incorporate
en bloc into membrane continuum.
As we have demonstrated earlier [15] and shown here
following basolateral fusion, the cold membrane sub-
strates contain exact mirror assemblies of the ve-
sicles-derived radiolabeled lipids. The vesicles enriched
in PI3P fuse with apical membrane, whereas the vesicles
enriched in PI4P fuse with basolateral membrane, or
display affinity for endosomes, or remain in cytosol and
presumably constitute portion of the vesicles that display
an affinity for other organelles. Moreover, the vesicular
fusion with Golgi, apical and basolateral membrane
(Figure 6) is accompanied with appearance of lyso-
phospholipids and arachidonate, which suggests in-
volvement of PLA2 in the membrane lysis and opening
of transport vesicles [15,18,36]. The evidence that in
Golgi the transport vesicles undergo progressive phos-
pholipid maturation that undoubtedly is dictated by their
destination and hence integral protein components,
demonstrate how the cell membranes’ specificity and
transport is maintained. This in our opinion is the
strongest argument against bidirectional transport and
the vesicles as mere micro-containers for the transport.
As we found out through the experiments described
here, the vesicles destination is decided in ER and dic-
tated by the cell cytosol components. Therefore, ER
transport vesicles produced in hepatocytes-derived cy-
tosol do not fuse with gastric apical membrane, and
conversely, the ER transport vesicles synthesized in gas-
A. Slomiany et al. / Health 2 (2010) 1437-1447
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
tric cell cytosol do not fuse with basolateral membrane
of the hepatocytes. By concentrating on membrane lipid
synthesis, the composition of the epithelial gastric and
the hepatocytic membrane and by following the fusion
of ER-initiated radiolabeled vesicles, we generated crit-
ical data that differentiate transport to the opposite do-
mains of the epithelial cells and demonstrated impact of
the cell cytosol on membrane composition and Golgi-
specific sorting.
Our study provides strong argument that Golgi sorting
is not primary but the consequential event regarding the
critical stages in development of transport vesicles and
the maintenance of membrane compositional accuracy.
By concentrating on the vesicular lipids we found out
that specific membrane composition in terms of lipids
and proteins are decisive in the process of vesicles fu-
sion with apical or basolateral segment of cell membrane,
and that synthesis of transport vesicle membrane is a
ER-cotranslational, area-specific process. At the same
time frame, the membrane integral proteins must be co-
translationally intercalated within actively synthesized
lipids and together they reproduce fragment of the
membrane that conforms to structural fidelity and de-
termines transport vesicles destination.
Our previous studies, work of others [5-7,18,19,31]
and the results presented here indicate that the restitution
of ER and the cell nucleus membranes is not consequen-
tial of the vesicular pathway, and is not maintained
through the retrograde vesicular transport. The lipids of
ER and nucleus are not reflecting the composition of the
apical cell membrane [9,23,29,32,34,36]. Also we could
not substantiate a popular explanation of the subcellular
membrane modification through Cer formation in plas-
ma membrane [37], or transferring of the individual li-
pids from cytosol into ER [10,11], or nuclear membrane
[22,34]. The incubation of ER, IN, ONM with cytosol
enriched with sphingolipid extracts from Golgi vesicles,
cell membrane rafts and caveolae, and apical cell mem-
brane was not facilitating lipid integration into the
membranes, and the lipids remained in cytosol [24]. Yet,
our studies on nuclei and the inner and outer nuclear/ER
membrane revealed feasible path associated with nuclear
membrane restitution that was associated with transport
of the cytosolic protein into the nucleus [24]. We have
demonstrated that restitution of nuclear membranes is
accomplished through the ability of endoplasmic reticu-
lum to synthesize membrane lipids and utilization of the
inositol phosphates (IPs) to generate PIPs and use them
to transport cytosolic protein to nucleus. Appearance of
labeled cytosolic protein and labeled PIPs in nucleus
allowed us to speculate that the lipid synthesis-induced
enlargement of ONM generates lateral movement of the
nuclear membrane between nuclear pores and thus ac-
complishes the transport of the cytosolic components
into the nucleus and nuclear components to the cytosol.
While we do not know whether nuclear membrane resti-
tution proceeds through en bloc formation of the entirely
new segments of the membrane, we are convinced that
the restitution of the nuclear/ER and cellular and orga-
nellar membranes is accomplished through specific
membrane lipid synthesis and lateral movement of new-
ly ER-generated membrane.
In summary, our results allowed us to speculate that
except ER/nuclear membrane, the principle, ER-initiated
and cytosol-controlled vesicular membrane synthesis
determines cellular transport and restitution. With this in
mind we need to determine whether external signals re-
leasing inositol phosphates (IPs) and provoking modifi-
cation of the cell membrane stimulate nuclear processes
that control transport of the sets of mRNA which will be
translated into gamut of membranes that are specifically
design to replace modified cell membrane, repair dam-
age, or replace missing fragments used to generate en-
dosomes, repair organelles affected by fission or deliver
organelle specific enzymes. Are these events that are
attributed to normal homeostatic repair and replacement
of organelles and plasma membranes operational when
singular protein translation is induced, inhibited or mu-
tated [30,31,33-37]? Will singular gene deletion or ge-
netic variation determine whether cell will lose its mem-
brane characteristic features and be rejected from its
environment as in cancer cells metastasis? If this were
the case, would the incorporation of proper mRNA into
cell cytosol restore normal cells characteristics? With
this, an important task for future studies is to assimilate
the concept that membrane restoration is achieved
through en bloc incorporation of the precisely synthe-
sized membrane segments in ER that predetermine apic-
al, basolateral or organellar sorting. Without early ER
coordination of the events, the complexity and diversity
of the membrane renewal process is incomprehensible.
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