Cell membranes composition is defined in ER and their restitution proceed by en bloc fusion of ER generated transport vesicles

doi:10.4236/health.2010.212214

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Vol.2, No.12, 1437-1447 (2010) Health

doi:10.4236/health.2010.212214

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

ABSTRACT

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

1. INTRODUCTION

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

1438

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. MATERIALS AND METHODS

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

[22].

2.2. Liver Perfusion Buffers

a) Buffered saline, with 10 mM potassium phosphate,

pH 6.8, b) buffered saline containing 0.5 mM MgCl2

and

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

Hepatocytes

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

A. Slomiany et al. / Health 2 (2010) 1437-1447

1439

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

[24].

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

Membranes

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

1440

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).

3. RESULTS

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,

24].

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

Cer

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

A. Slomiany et al. / Health 2 (2010) 1437-1447

1441

discernable.

Synthesis of ER transport vesicles in hepatocytes

200

400

600

800

1000

1200

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-

branes.

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

and

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

acylation.

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

cpm/100μl

A. Slomiany et al. / Health 2 (2010) 1437-1447

1442

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.

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,

(a)

(b)

(c)

(d)

(c)

(a)

(b)

(d)

A. Slomiany et al. / Health 2 (2010) 1437-1447

1443

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

[25-27].

Together, these results allowed us to hypothesize that

(a)

(b)

(c)

A. Slomiany et al. / Health 2 (2010) 1437-1447

1444

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.

4. DISCUSSION

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

1445

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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